FIELD OF THE INVENTION
The present invention relates to a unique combination of prebiotics, probiotics and metabolites (processed probiotics) in maintaining or improving gastrointestinal and immune health which has faster and enhanced activities than the currently available marketed probiotic formulations. The synergistic combination can be used in various types of food and presented in tablet, capsule and sachet forms. Consumers can also take it on its own, or added into drinks for their convenience.
BACKGROUND OF THE INVENTION
The term probiotic is often categorized as nutraceutical or functional food. However, there is a difference between these.
Nutraceuticals are "naturally derived bioactive compounds that are found in foods, dietary supplements and herbal products, and have health promoting, disease preventing, or medicinal products" (Ref: Nutraceuticals Institute). It may be prepared and marketed in the food-format of granules, powder, tablet, capsule, liquid or gel and may be packed in sachet, ampoule, bottle and so on and to be taken as measured unit quantities.
Functional foods are foods which contain an ingredient that gives that food health-promoting properties over and above its usual nutritional value, for example, probiotic yoghurts.
The term "probiotic" refers a live microbial food supplement, which beneficially affects the host animal by improving its intestinal microbial balance. Lactobacillus acidophilus

and Bifidobacterium spp. constitute a major part of the natural microflora of the human intestine, and when present in sufficient numbers, create a healthy equilibrium between beneficial and potentially harmful microflora in the gut. Once in the intestinal tract, they flourish to the point where potentially harmful bacteria that come along cannot grow due to the competition from these beneficial microorganisms. These microorganisms also play a role in inhibiting the growth of pathogenic organisms through production of organic acids and bacteriocins, and by deconjugation of bile salts.
Probiotics exert their action by:
• Modulation of host immune response like enhanced antibody production, enhanced natural killer cell activity, Modulation of dendritic cell phenotype and function, Modulation of NF-kB and AP-1 pathway, Altered cytokine release Induction of regulatory T cells, Induction of PPAR-g, Modulation of apoptosis Inhibition of proteasome activity.
• Enhanced epithelial barrier function by enhanced tight junction protein phosphorylation, Up regulation of mucous production, enhanced epithelial cell glycosylation, increased slgA.
« Production Anti-microbial effects by Decreased luminal pH Stimulation of defensin, Secretion of anti-microbial peptides, Inhibition of pathogenic bacterial invasion, Blockade of bacterial adhesion to epithelial cells. (Ref: Current opinions in pharmacology, probiotics and nutraceuticals: non-medicinal treatment of gastrointestinal diseases. Jan 2006).

The prevalence of these organisms in the intestines may be reduced with age, dietary changes, antibiotic consumption and/or stress, and their absence or low viability may cause varying degrees of digestive problems and weakening of the immune system.
"Prebiotics" are non-digestive carbohydrates they act as a nutrient source for the good probiotic bacteria. This encourages the probiotic bacteria to grow in a favorable environment, which in turn reduces the chances that harmful microorganisms may start to grow in our digestive tract. It beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria that can improve the host health in the colon. Currently, two soluble but non-digestible dietary ingredients are of great interest in the food industry because of their unique physicochemical and functional properties. Inulin is naturally present in significant quantities in vegetables (e. g., chicory, artichokes, asparagus, salsify, leeks, onions, garlic, even Wheat) and is made up of linear chains of fructose molecules connected by [3 (2-1) linkages with 30-600 of polymerization. Oligofructose is a natural constituent of inulin, except it has 2-70 of polymerization and a slightly higher sweetness index level. Inulin and oligofructose, when ingested, enter the large intestine almost quantitatively and are not hydrolyzed into their monosaccharide moieties in the upper intestinal tract. As a result, they can be recognized as efficient stimulators or prebiotics in the diet.
From our modern diet to stress, age, exposure to environmental toxins, excessive hygiene, the overuse of antibiotics (both as medicine and in our food supply) as well as the frequent use of medicines like birth control, acid suppressants (Proton Pump Inhibitors), antacids, steroids, hormone replacement therapy and NSATDs, many of us have damaged the very system that is in place to protect and sustain us.

The specific effects of a broken microbiome can be poor nutrient absorption and digestion, as well as poor mental, metabolic and immune function. Everything from our energy levels, to mood, mental state, and weight stems from the balance of good flora inside our Gastrointentinal tract. Lifestyle factors that are detrimental to our intestinal flora often don't discriminate - they wipe out all types of our good bacteria.
To be effective, probiotic supplementation should deliver a variety of strains to properly counter these indiscriminate effects. Each strain performs a different function within the intestinal tract and offers a different set of benefits to the individual. Integrating both probiotics, prebiotics and metabolites (processed probiotics) which in synergy affects the host beneficially by improving the survival and implantation of live microbial dietary supplements in the gastrointestinal tract.
Users therefore gain similar benefits from taking a single product rather than taking two separate ones, and research (probiotics, prebiotics and immunomodulation of gut Mucosal Defenses: homeostasis and immunopathology. Hardy et al, Nutrients V.5 (6) 2013)) even suggests that combination of two can even have more beneficial effects on human health than taking prebiotics or probiotics alone.
Metabolites (processed probiotics) are biologically active substances derived from the products of probiotics that benefit human health", and they are mainly composed of probiotic bacteria cell and its metabolites. Processed probiotics include not only organic acids, but also unique components found in cell walls like peptides, polysaccharides and fatty acids. Processed probiotics can change the ecology of intestinal microflora, more importantly, they can quickly take action to improve the intestinal / immune system due to their small molecular size.

Any discussion of the prior art throughout the specification should be in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.
Some of the closed prior art with respect to the present inventions are described below to differentiate the present invention with the existing arts.
921/CHENP/2006 discloses a method for preparing liquid probiotic composition, comprising bacteria having at least a basal biologic activity, wherein said bacteria have been selected according to at least one selection pressure, wherein the composition preferably includes an auto lysate (complete substances for maintaining bacteria) and wherein the composition is substantially free from substances suitable for bacterial growth but not similarly suitable for mammals, and particularly not suitable for human beings. Peptone and buffering salts, particularly phosphates, may not be harmful in small doses, but they are not specifically suitable for human beings and free from substances generated by bacteria.
2381/DELNP/2006 discloses a stabilized dry bacterial composition comprising greater than 10% dried bacterial concentrate having a concentration of bacteria of at least 1 x 10s cfu/g are provided. The compositions have improved stability. Also provided are packaged bacterial compositions, unit-dose compositions and methods of manufacturing the compositions of the present invention.
2511/MUMNP/2008 discloses a probiotic composition or premix comprising live or regeneratable 5 probiotic microorganisms, yeast walls and/or deactivated yeasts, and, optionally a nutritional supplement comprising in particular vitamins and/or trace elements

and/or amino acids and/or other additives intended for animal nutrition. It also 10 relates to granules comprising said composition and intended for animal nutrition, and also to a method for preparing said granules.
1327/DELNP/2009 discloses a stabilized dry bacterial composition comprising greater than 10% dried bacterial concentrate having a concentration of bacteria of at least 1 x 108 cfu/g are provided. The compositions have improved stability. Also provided are packaged bacterial compositions, unit-dose compositions and methods of manufacturing the compositions of the present invention.
3468/CHE/2011 discloses a probiotic synergistic composition for generating, improving or reinforcing homeostasis and/or immunity in a subject, wherein the composition comprises two or more Lactobacillus species selected from a group consisting of Lactobacillus helviticus, Lactobacillus fermentum, Lactobacillus rhamnosus and Lactobacillus paraplantarum, wherein the presence of more than one Lactobacillus species exhibits improved adhesion of the Lactobacillus species to a biological surface.
2599/KOLNP/2012 discloses a composition comprising probiotic bacteria for the treatment of pathologies associated with alterations of the immune system. In particular, this invention relates to the use of selected probiotic bacteria for the preparation of a composition for the treatment of allergies, such as atopic dermatitis.
4042/DELNP/2014 discloses a packaged probiotic composition which is useful for treating or preventing bacterial colonization in wounds and tissue. A characteristic feature of the invention is that the probiotic composition contains a probiotic microorganism which is in

direct contact with water during storage of the probiotic composition. The invention furthermore pertains to methods of using and producing the probiotic composition.
7705/DELNP/2015 discloses a targeted delivery system for the oral delivery of probiotics or therapeutic agent for various indications including and not limited to active and prophylaxis treatment of infection antibiotic associated diarrhea irritable bowel syndrome Crohn s disease intestinal flora replacement supplemental flora treatments for patients taking antibiotics and for restoration of balance and signaling between the intestinal microbiome and the intestinal cells in patients under treatment of metabolic syndrome manifestations specifically diabetes insulin resistance obesity hyperlipidemia and hypertension. This invention restores altered probiotic organism imbalances that are characteristic of said diseases among others as well as defines a platform technology development for site specific delivery of probiotic organisms in the GI tract of a mammal most specifically the ileum and/or right colon of a human subject.
US 6,468,525 discloses a probiotic formulation, useful as a food supplement and a material for reestablishing beneficial bacteria to the body's intestinal tract, comprises a mixture of beneficial probiotic microflora comprising lactobacillus acidophilus, bifidobacterium bifidum, lactobacillus salivarius, bifidobacterium infantis and bifidobacterium longum, fructooligosaccharides, L-glutamine and N-acetyl glucosamine.
The above prior arts disclose either the formulation with probiotics and prebiotics individually; none of the prior art discloses about the formulation which comprises the combination of probiotics, prebiotics and metabolites optionally in combination with other excipients.

Drawback of the existing commercialized formulations:
Formulations currently available in Indian market are only one probiotic or mixture of Probiotics or Combination of mixture of probiotics with prebiotics (Symbiotic). Only probiotics, when we take orally, it will neither colonize in intestine nor grow well. But in presence of prebiotics (i.e. symbiotic) as intestinal flora condition changed it not only allow probiotics to colonize better but also using this prebiotic, probiotic grow faster.
Drawback of Symbiotics:
1) Intestinal flora condition change to a certain extent but not completely, as desired.
2) Probiotics use prebiotics as substrate and make necessary components for growth, so growth rate is faster but still not fastest.
3) Once probiotic grows, then only, effect is seen. Therefore, overall treatment duration is longer which ultimately leads to increased treatment cost.
It would be desirable and necessary to prepare a formulation comprising a combination of probiotics, prebiotics and metabolites (processed probiotic) optionally with other excipient to achieve direct, significantly enhanced, and with better effectiveness. Faster and increased effect will lead to reduced frequency and as a result lesser treatment cost. Since, the formulation is an integrated and improved combination of probiotics, prebiotics and metabolites (optionally with other excipients), it could prove to be even better in maintaining and improving gastrointestinal and overall immune health, particularly catering to the needs of malnourished children.

SUMMARY OF THE INVENTION
Accordant with the present invention an integrated probiotic-prebiotic formulation consisting the following ingredients:
• Probiotics comprising Bifidobacterium bifidum, Bifidobacterium lactis and Lactobacillus acidophilus
• Prebiotics comprising Lactosucrose, Fructooligosaccharides, Glucans-30 and Dextrin
• Metabolites {Processed Probiotics) and pharmaceutically acceptable excipients
Li another aspect of the invention wherein an integrated probiotic-prebiotic formulation consisting the following ingredients as given below:
• the concentration of prebiotics ranges from 54 to 60 weight percent
• the concentration of probiotics ranges from 5 to 10 weight percent
• the concentration of metabolites ranges from 5 to 10 weight percent and
• the concentration of metabolites ranges from 25 to 30 weight percent.
Yet another objective of the invention is to develop the above said formulation in the form of capsules for enhancing human immunity and to reduce risk of allergies.
In an aspect, the present invention provides an integrated probiotic-prebiotic formulation in the form of capsule for strengthening human intestines consisting of the following ingredients:
• Probiotics comprising Lactobacillus acidophilus, Lactobacillus Casei, Lactobacillus Sporogenes
• Prebiotics comprising Lactosucrose, Fructooligosaccharides, Maltodextrin
• Processed probiotics (metabolites) and pharmaceutically acceptable excipients.

Yet another objective of the present invention is to develop the above said formulation consisting the following ingredients as follows: 50 to 55 weight percent prebiotics, 7 to 12 weight percent probiotics, 0.5 to 2 weight percent metabolites and 35 to 40 weight percent excipients.
Li another aspect, the present invention provides an integrated probiotic-prebiotic sachet formulation for strengthening human intestines consisting the following ingredients:
• probiotics comprising Lactobacillus acidophilus, Lactobacillus Casei and Lactobacillus Sporogenes
• prebiotics comprising Lactosucrose, Fructooligosaccharides and dextrin
• metabolites and pharmaceutical^ acceptable excipients
In yet another aspect, the present invention provides an integrated probiotic-prebiotic sachet formulation described above consisting of 20 to 25 weight percent prebiotics, 5 to 10 weight percent probiotics, 0.1 to 1 weight percent metabolites and 65 to 70 weight percent excipients.
A formulation as claimed in any one of the preceding claims wherein the excipients are selected from the group consisting of corn starch, magnesium stearate, microcrystalline cellulose, silicon dioxide, sugar alcohols, potato starch, vitamin C and the fruit flavor either alone or in combination thereof.
BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES
In order that the invention may be readily understood and put into practical effect, reference now will be made to exemplary embodiments as illustrated with reference to the

accompanying figures, wherein like reference numbers refer to identical or functionally similar elements throughout the separate views. The figures together with a detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present invention, where:
Figure 1 depicts schematic representation of new probiotic formulation for unique synergistic effect.
Figure 2 depicts the Immuno-stimulating activity of process probiotics/ metabolites
Figure 3 depicts the immune enhancement
Grpl: Bifidobacterium bifidum , Bifidobacterium lactis, Lactobacillus acidophilus
Grp3: Grpl + Lactosurcose, Fructo oligosaccharides, Glucans-30 & Dextrin
Grp4: Grp3 +L. plantarum metabolites
Grr5: Grp3 +Lac. delbrueckii metabolites
GrplO: Grp3 +L. plantarum & Lac. delbrueckii metabolites
Figure 4 depicts the intestinal enhancement
Grp2: Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus sporogenes
Grp6: Grp2+Lactosurcose, Fructooligosaccharides & Maltodextrin
Grp7: Grp6+L. plantarum metabolites
Grr8; Grp6+ Lac. delbrueckii metabolites
Grp9: Grp6 +L. plantarum & Lac. delbrueckii metabolites

In an aspect, the present invention provides an integrated probiotic-prebiotic formulation in the form of capsule for strengthening human intestines consisting of the following ingredients:
• Probiotics comprising Lactobacillus acidophilus, Lactobacillus Casei, Lactobacillus Sporogenes
• Prebiotics comprising Lactosucrose, Fructooligosaccharides, Maltodextrin
• Processed probiotics (metabolites) and pharmaceutically acceptable excipients.
Yet another objective of the present invention is to develop the above said formulation consisting the following ingredients as follows: 50 to 55 weight percent prebiotics, 7 to 12 weight percent probiotics, 0.5 to 2 weight percent metabolites and 35 to 40 weight percent excipients.
In another aspect, the present invention provides an integrated probiotic-prebiotic sachet formulation for strengthening human intestines, consisting of the following ingredients:
• probiotics comprising Lactobacillus acidophilus, Lactobacillus Casei and Lactobacillus Sporogenes
• prebiotics comprising Lactosucrose, Fructooligosaccharides and dextrin
• metabolites and pharmaceutically acceptable excipients
In yet another aspect, the present invention provides an integrated probiotic-prebiotic sachet formulation described above consisting of 20 to 25 weight percent prebiotics, 5 to 10 weight percent probiotics, 0.1 to 1 weight percent metabolites and 65 to 70 weight percent excipients.

A formulation as claimed in any one of the preceding claims wherein the excipients are selected from the group consisting of corn starch, magnesium stearate, microcrystalline cellulose, silicon dioxide, sugar alcohols, potato starch, vitamin C and the fruit flavor either alone or in combination thereof.
Various strains oflactobacillus and Bifidobacterim to be used in the current invention are described here. The genus Lactobacillus includes various Gram positive facultative anaerobic or microaerophilic rod-shaped bacteria. They are a major part of the lactic acid bacteria (LAB) group including Lactobacillus, Lactococciis, Enterococcus, Oenococcus Pediococcus, Streptococcus and Leuconostoc species that can convert hexose sugars to lactic acid thus producing an acid environment which inhibits the growth of several species of harmful bacteria. The present invention includes but not limited to Lactobacillus acidophilus, L. casei, L. paracasei, L. rhamnosus, L. delbrueckii subsp. bulgaricus, L. brevis, L. johnsonii, L. plantantm and L. fermentum and Bifidobacterium infantis, B. adolescentis, B. animalis subsp animalis, B. animalis subsp lactis, B. bifidum, B. longum, B. breve. These probiotics are grown using the traditional method involving either synthetic or semi synthetic media.
Process probiotics (metabolites) can either be obtained by sonication or heat treatment of dead cells of lactic acid bacteria. In the present invention, an advanced method for preparing the dead cells is reported. The bacteria (which includes different species of probiotics may or may not be limited to lactobacillus strains) were heat treated at 100°C-120°C for 30 minutes to 1 hour. This suspension was cooled immediately. The method has clear advantages over sonication as it is easily scalable. The dead bacterial cells obtained by the above process is known as processed probiotics or metabolites.

The present disclosure herein beiow provided examples for better understanding of the instant disclosure. However, a person skilled in the art would be aware that the illustrations provided herein are only prospective in nature and can be extrapolated under the purview of the instant disclosure.
EXAMPLE 1
Preparation of Metabolites (Processed Probiotics)
Lactobacillus/ selected probiotic was seeded in MRS (de Man, Rogosa, Sharpe) broth. It was cultured for 24 hours at 32°C. After growth, the culture was centrifuged at 4200 x g, 4°C for 10 minutes. The resulting precipitate containing cells was solubilized in physiological saline. Then, the resulting cells are resuspended in sterile-deionized water, and heated at 100°C for 30 minutes. After heat treatment, the suspension was cooled immediately and subsequently lyophilized.
EXAMPLE 2
Experiment to demonstrate the immune-stimulating activity of processed probiotics
Many lactic acid bacteria and bifida bacteria possess "immune-stimulating activity". However, the strength of the immune-stimulating activity varies greatly depending on the specific bacterial strains. Processed probiotics were characterized for immune-stimulating activity. Spleen cells of mice were stimulated with various processed probiotics. The immune-stimulating activity was evaluated by measuring interleukin-12 (IL-12) production induced by each substance. The result is shown in Figure 2. This indicates that process L. plantarnm & L. delbrueckii showed comparatively better immune-stimulating activity with decreased EL -12 production. Hence, these metabolites were used for further study to establish final formulation for gastro-intestinal, immune and anti-allergic applications.

EXAMPLE 3
Immune Enhancement experiment details
HT29 cells were grown in RPMI1640 with 10% fetal calf serum, and divided into seven groups: control (only cell), TNF-a, group -1 (Bifidobacterium bifidum, Bifidobacterium lactis, Lactobacillus acidophilus), group-3 (Grpl + Lactosurcose, Fructo oligosaccharides, Glucans-30 & Dextrin), group-4 (Grp3 + L. plantarum metabolites), group-5 (Grp3 +Lac. delbrueckii metabolites) & group 10 (Grp3 + L. plantarum &Lac. delbrueckii metabolites). After the cells were grown to confluence in single layer, cells were washed three times with PBS pH 7.4, to remove culture medium and non-adherent cells. The different formulations in culture medium were transferred into individual wells respectively. TNF-a (10 ng/ml) was added into each well of groups except control. After 1 hour, the supematants were collected and centrifuged followed by measurement of IL-8 after 3 hours. The results are shown in figure 3. This Indicates Formulation of group 4 is the best for Immune enhancement.
EXAMPLE 4
Intestinal Enhancement experiment details
HT29 cells were grown in RPMI1640 with 10% fetal calf serum, and divided into seven groups: control (only cell), TNF-a, group -2 (Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus sporogenes), group-6 (Grp2+Lactosurcose, Fructooligosaccharides & Maltodextrin), group-7 (Grp6+L. plantarum metabolite), group-8 (Grp6+ Lac. delbrueckii metabolites) and group 9 (Grp6 + L. plantanim & Lac. delbrueckii metabolites). The cells were grown to confluence in single layer and washed three times with PBS pH 7.4, to remove culture medium and non-adherent cells. The different formulations in culture medium were transferred into individual wells respectively. After one hour, TNF-a (10 ng/ml) was added into each well of groups except control. After 3 hours, the supematants

were collected and centrifuged for measurement, followed by measurement of IL-8. The results are shown in figure 4. This indicates that formulation of group 7 is best suited for Intestinal enhancement.
Advantages of the invention
Only probiotics, when we take orally, it may or may not colonize in intestine completely and may also not grow. But in presence of prebiotics, as intestinal flora condition changes, it not only allow probiotics to colonize but also using these prebiotics, probiotic grow faster. The advantage of our new formulation is:
1) Improve intestinal flora condition, so that better colonization is achieved.
2) The metabolites can be used as instant food for probiotics, so faster growth is expected as compared to symbiotic.
3) Heat treated probiotics also provide instant immunity and formulations are more stable.

We claim:
1. An improved probiotic formulation consisting the following ingredients:
• Probiotics comprising Bifidobacterium bifidum, Bifidobacterium lactis and Lactobacillus acidophilus
• Prebiotics comprising Lactosucrose, Fructooligosaccharides, Glucans-30 and Dextrin
• Metabolites (Processed Probiotics) and pharmaceutically acceptable excipients
2. An improved probiotic formulation according to claim 1, wherein
• the concentration of prebiotics ranges from 54 to 60 weight percent
• the concentration of probiotics ranges from 5 to 10 weight percent
• the concentration of metabolites ranges from 5 to 10 weight percent and
• the concentration of metabolites ranges from 25 to 30 weight percent.

3. An improved probiotic formulation as claimed in any of the preceding claims, wherein the formulation is in the form of capsule for enhancing human immunity and to reduce risk of allergies.
4. An improved probiotic formulation consisting the following ingredients:

• Probiotics comprising Lactobacillus acidophilus, Lactobacillus Casei, Lactobacillus Sporogenes
• Prebiotics comprising Lactosucrose, Fructooligosaccharides, Maltodextrin
• Processed probiotics (metabolites) and pharmaceutically acceptable excipients

5. An improved probiotic formulation consisting of 50 to 55 weight percent prebiotics, 7 to 12 weight percent probiotics, 0.5 to 2 weight percent metabolites and 35 to 40 weight percent excipients
6. An improved probiotic formulation as claimed in claim 4 & 5, wherein the formulation is in the form of capsule for healthy human intestines.

7. An improved probiotic sachet formulation sachet formulation consisting the following
ingredients:
• probiotics comprising Lactobacillus acidophilus, Lactobacillus Casei and Lactobacillus Sporogenes
• prebiotics comprising Lactosucrose, Fructooli go saccharides and dextrin
• metabolites and pharmaceutical^ acceptable excipients

8. An improved probiotic formulation consisting of 20 to 25 weight percent prebiotics, 5 to 10 weight percent probiotics, 0.1 to 1 weight percent metabolites and 65 to 70 weight percent excipients
9. An improved probiotic formulation as claimed in claim 7 & 8, wherein the formulation is for healthy human intestines.
10. An improved probiotic formulation as claimed in any one of the preceding claims wherein the excipients are selected from the group consisting of corn starch, magnesium stearate, microcrystalline cellulose, silicon dioxide, sugar alcohols, potato starch, vitamin C and the fruit flavor either alone or in combination thereof.