CLIAMS:1. A probiotic composition comprising a combination of Lactobacillus casei (ATCC SD5213) and Lactobacillus fermentum (NCDC-141), wherein said Lactobacillus casei and said Lactobacillus fermentum are combined in a ratio of 1:1.
2. The probiotic composition as claimed in claim 1, wherein the said Lactobacillus casei and said Lactobacillus fermentum are in the form of live bacteria.
3. The probiotic composition according to claim 1, wherein said Lactobacillus casei and said Lactobacillus fermentum are present in a dosage from about 106 cfu to 109 cfu per serve.
4. The probiotic composition as claimed in any of the preceding claims, wherein said composition helps in immunomodulation.
5. The probiotic composition as claimed in any of the preceding claims, wherein said composition enhances cellular defenses by up-regulating the expression of beta defensins and claudin-1 and down regulating the expression of claudin-2.
6. The composition as claimed in claim 1, wherein said composition is probiotic supplement, food, feed or nutritional supplement, food product or pharmaceutical composition.
,TagSPECI:Field of the invention
The present invention relates to a synergistic probiotic combination for immunomodulation. More specifically the present invention relates to a combination of two probiotic strains, L. casei ATCC SD5213 and L. fermentum NCDC-141.

Background of the invention
Probiotics are gaining importance in view of their extended health benefits. Intake of probiotics has been proven to be effective in the prevention and/or management gastrointestinal disorders including acute gastroenteritis, antibiotic-associated diarrhea and intestinal inflammatory disorders such as Crohn's disease, pouchitis, etc. Many in vitro and in vivo studies have demonstrated the health effects of probiotics and their combinations by various immunomodulatory mechanisms.

Miriam Schlee et.al., (Infection and Immunity, 2007, 75(5), 2399–2407) reported the use of probiotics that induces hBD2 expression in infected cell culture model and demonstrated that E. coli Nissle 1917 induces intestinal epithelial cells to block adherence and inhibit invasion of various pathogenic strains. The authors have demonstrated that the probiotic effect of E. coli Nissle 1917 is due to induction of hBD2 in cell culture in a time and dose dependent manner. E. coli Nissle 1917 in contrast to many other E. coli strains induces hBD2 expression through NF-kB.

In another study, M. Schlee, et.al., (Clinical and Experimental Immunology, 2008, 151, 528–535) demonstrates that a bacterial mixture VSL#3 which is a mixture of eight different bacterial strains could strengthen intestinal barrier functions through the up regulation of hBD2. In this study the VSL#3 described is a lyophilized mixture of eight Gram positive bacteria namely, B. longum, B. infantis, B. breve, L. acidophilus, L. casei, L. delbrueckii ssp. bulgaricus, L. plantarum and Strptococcus salivarius ssp. thermophilus. For hBD2 expression detection, the Caco-2 cells were incubated with different concentration of different probiotic preparations and expression profiles were measured by quantitative real-time PCR using the same set of primers as used in the present invention. The result shows that the maximal amount of hBD-2 mRNA was expressed after 6 hours of incubation. The highest induction of hBD-2 was elicited by the probiotic cocktailVSL#3, followed by L. fermentum and P. pentosaceus, whereas L. acidophilus PZ 1129 and L. paracasei had only a weak effect. The housekeeping gene expression (GAPDH) remained stable at all time-points. The induction of hBD-2 was also shown to be dose dependent. At the lowest concentration of 1 x 106 bacteria per ml, hBD-2 expression remained unaffected. Only the VSL#3 combination exerted an inducing effect at the concentration of 1 x 107 cells/ml. P. pentosaceus, L. fermentum and VSL#3 induced hBD-2 mRNA significantly at concentrations of 1 x 108 cells/ml, which was enhanced further at the highest bacteria cell number tested.

WO 2012/062781 and US 2013/0028877, discloses a non-replicating heat treated La1 (NCC533) strain to induce defensin expression strongly. Among these, there is family of beta defensins including hBD1 and hBD2. It was found that heat treated L. johnsonii (La1, NCC533) up regulates hBD1 more strongly than live counterpart. It is further disclosed that probiotic microorganisms may be selected from the group consisting of bifidobacteria, lactobacilli, propionibacteria or combinations thereof.

Prior art discussed many combinations of probiotic strains and their importance in management of diseases via various cellular and immunomodulatory mechanisms. Given the intricate and unpredictable etiology of human disease and continuous challenges posed by human immune system in terms of less or no response to probiotic drugs administered, always there has been a continuous effort for identifying the most effective probiotic strains that are effective in the management of disease in vivo.

Object of invention
It is an object of the present invention to overcome the drawbacks of the prior art.
It is another object of the present invention to provide a synergistic blend of probiotic strains with enhanced efficacy as compared to individual strains in treating the disease.
It is yet another object of the present invention to provide a probiotic combination which provides enhanced and efficient immunomodulatory activity.
It is a further object to the present invention to provide a probiotic combination which effects up regulation of claudin-1 expression accompanied by down regulation of claudin-2.

Summary of the present invention
Accordingly to one aspect of the present invention there is provided a probiotic composition comprising a combination of Lactobacillus casei (ATCC SD5213) and Lactobacillus fermentum (NCDC-141); said Lactobacillus casei and said Lactobacillus fermentum is combined in a ratio of 1:1.

Brief Description of the Accompanying Drawings
Figure 1 illustrates the effect of probiotics on expression of ß-defensin (antimicrobial peptide) in Caco-2 cells.
Figure 2 illustrates the effect of different concentration of probiotic blends on expression of ß-defensin (antimicrobial peptide) in Caco-2 cells.
Figure 3 illustrates the effect of probiotics on expression of claudin 1 and cluadin 2 (membrane integrity) from Caco-2 cells.
Figure 4 illustrates effect of different concentrations of probiotic blend on expression of claudin-1 and cluadin-2 (membrane integrity) in Caco-2 cells.
Figure 5 illustrates the effect of different blends of probiotics on expression of ß-defensin from Caco-2 cells.

Detailed description of the invention
The present invention relates to a synergistic probiotic composition for immunomodulation. The present invention provides a synergistic blend of probiotic strains with enhanced efficacy as compared to individual strains observed in terms of increased antimicrobial peptides production and expression of genes involved in maintaining membrane integrity in the state of disease.
The present inventors have found that a combination of two specific probiotic strains, i.e.,
L.casei ATCC SD5213 and L.fermentum NCDC-141 when combined in a specific ratio showed a synergistic immunomodulatory effect on intestinal infectious model. The said synergistic probiotic combination of present invention was evaluated for its immunomodulatory effects on Salmonella typhi infected Caco-2 cells (intestinal epithelial cells) (an intestinal infection model). It was observed that the probiotic combination showed enhancement of antimicrobial peptide (ß-defensin) expression.

It is known that, the intestinal cells shows a disturbance in barrier integrity characterized by dysregulation in expression of markers of membrane integrity i.e. claudin-1 and claudin-2 post infection. Claudin-1 and claudin-2 are amongst claudin family of proteins that make important components of the tight junctions between cells and thus limit the flow of pathogens in the body. The present inventors have found that the selected probiotic combination shows synergistic effect in modulating membrane integrity by simultaneously up regulating claudin-1 expression accompanied by down regulation of claudin-2 expression levels. It was surprisingly found that the immunomodulatory effects of probiotics were found to be strain specific and also combination specific.

Accordingly the present invention provides a probiotic composition comprising a combination of Lactobacillus casei (ATCC SD5213) and Lactobacillus fermentum (NCDC-141) where Lactobacillus casei (ATCC SD5213) and Lactobacillus fermentum (NCDC-141) is combined in a ratio of 1:1.

The bacterial strains in the probiotic composition of the present invention can be in the form of live bacteria only.

The bacterial strains Lactobacillus casei (ATCC SD5213) and Lactobacillus fermentum (NCDC-141) can be present in an amount from about 1:100 to 100:1 (106 cfu to 109 cfu per day).

The probiotic composition of the present invention is effective is treatment for various health conditions but not limited to such as metabolic disorders, infections of upper and lower gastrointestinal tract, irritable bowel syndrome, inflammatory conditions like Colitis , Enteritis and Inflammatory bowel disease. The typical therapeutically effective dose of the probiotic composition ranges from 106 cfu to 109 cfu per serve

The probiotic composition of the present invention can be provided in the form of powder, tablets, capsules, and food formats.

The probiotic composition of the present invention can comprise of other excipient such as prebiotics, dietary fibers, vitamins minerals, herbal extracts, fats proteins etc as recommended by regulatory authorities like FSSAI.

The present composition is formulated by combining 5×107 cfu (~ 10mg) of L.casei ATCC 5213 and 5×107 cfu (~ 10mg) of L.fermentum NCDC141

The present invention is now illustrated by way of non-limiting examples.

Example-1
Preparation of microbes: Individual bacteria ( L. casei ATCC 5213, L. fermentum NCDC 141, L. rhamnosus ATCC SD 5675 and L. rhamnosus NCDC 24 ) were propagated by culturing in MRS broth (10g/L peptone, 10g/L beef extract,5g/L yeast extract, 20g/L glucose, 2g/L ammonium citrate, 5g/L sodium acetate, 0.1g/L magnesium sulphate, 0.05g/L manganese sulphate and 2g/L dipotassium phosphate) at 37°C for 24hrs. Bacterial aliquot (108 cfu) of individual probiotics and their combinations (net count 108 cfu) were inoculated in 1ml of DMEM (Dulbecco’s modified Eagle medium from Sigma) containing 1% FBS and were then used for treatment of intestinal cells (Caco-2 cells).

Effect of probiotic bacteria on expression of genes involved in membrane integrity and antimicrobial peptide production:
For evaluation of the effect of probiotics on expression of genes involved in membrane integrity and anti-microbial peptide production, Caco-2 cells (1×105cells/well) were pretreated with live S.typhi (107 cells/well) for 4 hours, and then treated with different live probiotics or their combinations (as shown in Tables 2, 3 and 4; 108 cells/well) for 4 hours. Cells incubated without S.typhi and probiotics were used as control. After the incubation, cells were washed with phosphate basal solution for thrice and harvested using trizol reagent (Invitrogen) for isolation of RNA. Later the cDNA was prepared using ABI high capacity cDNA kit. Briefly 1µg of RNA was taken along with 2.5 µl of random hexamers and made up to 15µl with nuclease free water then kept at 65ºC for 5 min. After incubation these samples were added to master mix (2.5µl of 25mM dNTPS, 2.5 µl of 10X RT buffer, 0.625µl of RNase inhibitor, 0.875 µl of RT enzyme and 3.5 µl of nuclease free water) and kept for cDNA synthesis by PCR. After cDNA synthesis, sample (cDNA) was used for evaluation of expression of genes by real time PCR using primers as tabulated in table: 1.
Gene expression analysis:
Evaluation of expression of genes by real time PCR using primers as tabulated below

Table 1: Details of primers used for expression studies

PCR method: cDNA of control and treatment samples were subjected to real time expression study by using SYBR green I mix (ABI system) along with respective primers. Using the protocols mentioned below. And expression was quantitated using ABI Prism 7500 Sequence Detection System (Applied Biosystems).
Human beta defensin-2(hBD-2): Initial denaturation at 95°C for 10 min was followed by 45 cycles of 95°C for 15s, the primer-specific annealing temperature for 5s, and elongation at 72°C for 15s followed by annealing temperature was set at 62°C. Amplification using these primers resulted in a 174-bp fragment. At the end of each run melting curve profiles were produced by cooling the sample to 65°C for 15s and then heating slowly at 0.20°C/s to 95°C with continuous measurement of fluorescence to confirm amplification of specific transcripts. For the housekeeping gene, GAPDH (forward primer CCACTCCTCCACCTTTGAC and reverse primer ACCCTGTTGCTGTAGCCA) were used.
Membrane integrity gene (claudin-1 and 2) expression: Initial denaturation at 95°C for 10 min was followed by 40 cycles of 95°C for 15s, the primer-specific annealing temperature for 5s, and elongation at 72°C for 15s followed by annealing temperature of both Claudin-1 and 2 were set at 60°C. Amplification using these primers resulted in a 130-bp (claudin-1) and 110bp (claudin-2) fragments. At the end of each run melting curve profiles were produced by cooling the sample to 65°C for 15s and then heating 100% to 95°C with continuous measurement of fluorescence to confirm amplification of specific transcripts.
The expression of various probiotic strains individually as well as in combination. The probiotic combination of the present invention (L. casei ATCC SD5213 and L. fermentum NCDC-141) showed a highest enhancement of antimicrobial peptide (ß-defensin) expression. It was also observed that addition of another strain (L. rhamnosus Howaru) to the present combination (L casei and L. fermentum NCDC-141) negated the synergism. Further to select most efficacious concentration, different ratios of the selected blend was evaluated for ability to stimulate ß-defensin production by infected Caco-2 cell model. L. fermentum NCDC-141 and L. casei ATCC SD5213 were evaluated in ratios (final conc. 108 cfu/well) i.e. 1:1, 1:10, 10:1, 1:100 and 100:1 respectively.
Figure 1 and Table-2 demonstrate the effects of probiotics or their combination on gene expression (ß defensin-2) in Caco-2 cells treated with S.typhi. The data is represented as fold change in expression with respect to control (untreated Caco-2 cells)

Table-2
Sample ß-defensin
S.typhi control 0.556308253
Lactobacillus casei (ATCC SD5213) 0.817010399
Lactobacillus rhamnosus (ATCC SD5675) 0.553749421
Lactobacillus fermentum (NCDC-141) 0.540903231
Lactobacillus rhamnosus (NCDC 24). 0.576689053
Lactobacillus casei (ATCC SD5213) + Lactobacillus rhamnosus (ATCC SD5675) 0.945273797
Lactobacillus casei (ATCC SD5213) + Lactobacillus fermentum (NCDC-141) 5.085697978
Lactobacillus casei (ATCC SD5213) + Lactobacillus rhamnosus (NCDC 24). 0.533307496
Lactobacillus rhamnosus (ATCC SD5675) + Lactobacillus fermentum (NCDC-141) 0.215041754
Lactobacillus rhamnosus (NCDC24) + Lactobacillus rhamnosus (ATCC SD5675) 0.858107392
Lactobacillus fermentum (NCDC-141) + Lactobacillus rhamnosus (NCDC24) 0.802389679
Lactobacillus casei (ATCC SD5213) + Lactobacillus rhamnosus (NCDC24) + Lactobacillus rhamnosus (ATCC SD5675) 0.617713533
Lactobacillus casei (ATCC SD5213) + Lactobacillus fermentum (NCDC-141) + Lactobacillus rhamnosus (ATCC SD5675) 0.619867552
Lactobacillus rhamnosus(ATCC SD5675) +Lactobacillus fermentum (NCDC-141)+Lactobacillus rhamnosus (NCDC24) 0.498349807

Lactobacillus fermentum (NCDC-141- ATCC 11976), Lactobacillus rhamnosus (NCDC 24-ATCC8014) were procured from NCDC (National collection of dairy cultures, Karnal) and L. casei ATCC SD5213, L. rhamnosus ATCC SD5675 were procured from ATCC.

From table 2 it is evident that only the combination of specific strains Lactobacillus casei (ATCC SD5213) and Lactobacillus fermentum NCDC-141, ATCC) provides unexpected up regulation of ß-defensin. Further the effect was found to be strain specific.

To validate the strain specific synergistic activity, the present inventors had evaluated L. casei (NCDC 017) and L. fermentum (NCDC 605) strains and it was observed that the combination evaluated didn’t show the same synergy as observed with L. casei (ATCC SD5213) and L. fermentum (NCDC141) (Fig 5). As evident from Figure 5, even when the two strains are combined with the respective second set of L. casei and L. fermentum, no synergy was observed. This indicates the synergy between the two strains being claimed is strain specific.

Further probiotic strains L. fermentum NCDC-141 and L. casei ATCC SD5213 were combined in varying ratio to stimulate ß-defensin production in S.typhi infected Caco-2 cells. Figure 2 demonstrates the synergistic effect of the two strains in defensin production only when used in a specific ratio i.e. 1:1.

Experiments were conducted using various probiotic strains individually and in combination to obtain the expression profiles of Claudin 1 and Claudin 2 in the Caco-2 cells treated with the probiotics.

Figure 3 and Table 3 demonstrates the synergistic effect of the selected probiotic combination in modulating the expression of Claudin -1 and Claudin-2. The data indicated that the selected combination is effective in modulating membrane integrity by simultaneously up regulating claudin-1 expression accompanied by down regulation of claudin-2 expression levels and the immunomodulatory effects of probiotics was found to be strain specific and also combination specific. Table-3 represents the data as fold change in expression with respect to control (untreated Caco-2 cells).

Table-3
Sample Claudin-1 Claudin-2

S.typhi control 0.46277282 1.444943767
L. casei (ATCC SD5213) 2.133909862 2.286450118
L. rhamnosus (ATCC SD5675) 3.1214796 1.981249078
L. fermentum (NCDC-141) 0.96176745 0.969211344
L. rhamnosus (ATCC SD5675) + L. fermentum (NCDC-141) 1.710742014 0.256644653
L. casei (ATCC SD5213) + L. fermentum (NCDC-141) 3.419177141 0.768648998
L. casei (ATCC SD5213) + L. rhamnosus (ATCC SD5675) 1.101544285 0.208756047

The inventors of the present invention also evaluated the effect of different concentrations of two probiotic strains in combination on expression profiles of claudin-1 and claudin-2 in S.typhi infected cells. L. fermentum NCDC-141 and L. casei ATCC SD5213 were evaluated in ratios (final concentration 108 cfu) i.e. 1:1, 1:10, 10:1, 1:100 and 100:1 respectively.

Figure 4 demonstrates that only combination containing bacterial strains in the ratio 1:1 showed a significant effect by simultaneously up regulating the expression of claudin-1 and down regulating claudin-2 expression. However, other ratios showed little effect. Table-4 shows results of different concentration of probiotics (in combination) analyzed for gene expression profiles (ß defensin-2, claudin-1 and claudin-2) in Caco-2 cells treated with S.typhi (infected model). Data represented as fold change in expression with respect to control (untreated Caco-2 cells).

Table-4: probiotic combination in different ratio (L. casei ATCC SD5213 and L. fermentum NCDC-141)
Sample name Fold change of ß defensin-2 Fold change of Claudin-2 Fold change of Claudin-1

S.typhi control 0.060502 2.007557 0.280589
L. casei + L. fermentum (1:1) 2.966157 0.59388 1.934196
L. casei + L. fermentum (1:10) 0.204801 0.998559 0.267079
L. casei + L. fermentum (1:100) 0.280026 0.706638 0.247988
L. casei + L. fermentum (10:1) 0.10712 0.837613 0.200514
L. casei + L. fermentum (100:1) 0.194193 1.323573 0.322046

Example 2:
The present combination is formulated as tablet, capsule, and Buttermilk / Dahi.
Preparation of Tablet: Probiotic bacterial combination (5×107 cfu (~ 100mg) of L. casei ATCC 5213 and 5×107 cfu (~ 100mg) of L. fermentum NCDC141) mixed with tablet formulation (5 kg) comprising of fillers, disintegrates, lubricants, glidants, binders and other excipients like of vitamins (A, B, C and E), minerals, herbal extracts, flavor and stabilizers and then cut into tablets of size 500mg each.
Preparation of capsule: Probiotic bacterial combination (5×107 cfu (~ 100mg) of L.casei ATCC 5213 and 5×107 cfu (~ 100mg) of L.fermentum NCDC141) mixed with components (2.5Kg) including microcrystalline cellulose, citric acid, stearic acid, and excipients like vitamins, minerals and flavors. The mixture was then dispensed into 250mg capsule covered with gelatinous coat.
Preparation of Dahi/Butter milk: Probiotic combination (5×107 cfu (~ 10mg) of L.casei ATCC 5213 and 5×107 cfu (~ 10mg) of L. fermentum NCDC141) was inoculated in 100ml of sterilized milk in the presence of Dahi cultures (108 cfu) like Lactococcus lactis ssp diacetylactis and lactococcus lactis ssp cremoris and incubated at 30°C for 8 hours.