DESC:Field of the Invention

The present invention provides a probiotic composition comprising a pharmaceutically effective amount of probiotic ingredient selected from Bifidobacterium bifidum, Lactobacillus plantarum, Lactobacillus acidophilus, Bacillus clausii and Saccharomyces boulardii. The invention further provides the use of probiotic composition for treating or preventing a metabolic syndrome or gastrointestinal disease or disorder selected from hyperlipidemia, diabetes, insulin resistance, obesity, obesity-related disorders, irritable bowel syndrome or inflammatory bowel disease.

Background of the Invention

The metabolic syndrome is a collection of disorders that are linked to obesity, diabetes, cardiovascular disease. Inflammation is more common in people with metabolic syndrome, and they are more susceptible to various conditions such polycystic ovarian syndrome, fatty liver, cholesterol gallstones, asthma, poor sleep, and some malignancies. The frequency of metabolic syndrome is quickly increasing over the world, notably in emerging countries.
A large number of studies have reported that the intestinal flora plays an extremely important role in the physiological metabolism of human body. The structural imbalance of intestinal flora is associated with a variety of diseases, including metabolic diseases (obesity, hyperlipidemia, diabetes, etc.), gastrointestinal diseases (irritable bowel syndrome and inflammatory bowel disease, etc.). The occurrence of metabolic syndrome is also closely related to the imbalance of intestinal flora. The commonly used intestinal flora regulating preparations include probiotics and prebiotics, etc.
Probiotics are lactic acid bacteria, which are found in milk or milk products, living or decaying plants, as well as in human and animal intestines. Lactic acid bacteria are a diverse group of Gram-positive, non-motile, microaerophilic or anaerobic bacteria that ferment sugar to produce acids, including lactic acid, and include the genera Bifidobacterium: Enterococcus, Lactobacillus: Lactococcus, Leuconostoc, and Pediococcus, among others. However, the most promising probiotic strains include the members of the Genera-lactobacillus, bifidobacterium and enterococcus. The goal of metabolic syndrome treatment is to reduce and eliminate risk factors. Drug therapy is the most common therapeutic approach.
US20160151434A1 discloses Lactobacillus plantarum strains, a composition thereof or a culture thereof. EP2318513A1 discloses probiotic, anti-inflammatory strains of Bifidobacterium longum, uses for treating disease, and for preparing human or pet food or pharmaceutical composition. It further discloses bacterial polysaccharide composition obtained from the strains, their use for the treatment of disease, and pharmaceutical compositions comprising such polysaccharide. EP0975227A1 discloses a composition including an isolated Bacillus species, spores or an extracellular product of B. coagulans, suitable for topical application, for inhibiting growth of yeast, fungus, bacteria or Herpes simplex virus. US20140205581A1 discloses a composition containing Saccharomyces cerevisiae var boulardii and the enzyme superoxide dismutase. WO2011092261A1 provides probiotic composition for use in the treatment of bowel inflammation. CN103977014A discloses a medication for treating metabolic syndrome that contains Akebia saponin and curcumin and can prevent and treat metabolic syndrome by regulating the metabolic processes of proteins, lipids and carbohydrates of patients. CN103596949A describes a novel compound molecular structure which has a therapeutic effect on diabetes and metabolic syndrome. CN103446139A describes a pharmacological combination for treating metabolic syndrome that includes puerarin, cinnamic acid, and berberine hydrochloride, and which improves insulin resistance while also lowering blood glucose, blood pressure, blood lipids, and improving abdominal obesity. US2020/0261515A1 discloses a probiotic composition and uses thereof, especially to a probiotic composition for alleviating metabolic syndrome and uses thereof. It further discloses a probiotic composition consisting of Bifidobacterium adolescentis CCFM8630, Lactobacillus reuteri CCFM8631 and Lactobacillus rhamnosus CCFM1044. US2020/0323929A1 discloses a probiotic composition comprising one or more of the following: Lactobacillus rhamnosus GG, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillu paracasei, Lactobacillus plantarum, Lactobacillus salivarius, Bifidobacterium infantis, and Bifidobacterium longum Bifidobacterium bifidum. US2020/0405786A1 discloses probiotic formulations for the treatment and alleviation of metabolic and oxidative stress, inflammation and neurodegeneration, wherein the formulations comprise or consist of any combination of the three probiotic strains: Lactobacillus fermentum (NCIMB 5221), Lactobacillus plantarum (NCIMB 8826) and Bifidobacteria longum spp. infantis (NCIMB 702255). US2021/0077547A1 discloses a spore-based probiotic composition that comprises at least one viable probiotic microorganism having a biological or therapeutic effect on microbiome in humans. One exemplary composition contains five different strains of Bacillus spp.
In accordance to the Joint Food and Culture Organization (FAQ) and the world health organization (WHO) guidelines, probiotics organisms must have the ability to stay alive and they should be resistant to the exposure to the bile in the human gut. On the top of these features they must be safe and effective and they must sustain their efficacy for the entire period of the shelf life of the product. As we begin to appreciate the practical significance of microbes on the human physiology and disease, new therapeutic preparations are needed for treating metabolic syndrome. Accordingly, the inventors of the present invention have come up with a probiotic formulation which is excellent in acid resistance and therefore, it is also viable in the stomach and it may have higher intestinal delivery. The probiotic formulation is excellent in bile acid resistance and therefore it is superior to intestinal fix. Further the probiotic formulation is excellent for the treatment of metabolic syndrome.
Further, there are no studies available in art on the efficacy of probiotic formulation comprising selected probiotic strains in the treatment of metabolic syndrome. For the reasons stated in this specification, which will become apparent to those skilled in the art upon reading and understanding the specification, there is a need for an intervention that facilitates improved and optimized therapeutic response in the treatment of metabolic syndrome.

Summary of the Invention
Accordingly, the present invention provides an oral probiotic composition comprising at least two probiotic ingredients and a prebiotic for treating or preventing metabolic syndrome or gastrointestinal disease or disorder. The invention further provides methods of formulating probiotic formulations. In particular, the present invention provides one or more of the following embodiments:
[1] A probiotic composition comprising a therapeutically effective amount of probiotic ingredient selected from Bifidobacterium bifidum, Lactobacillus plantarum, Lactobacillus acidophilus, Bacillus clausii and Saccharomyces boulardii.
[2] The probiotic composition according to [1] further comprises one or more excipients.
[3] The probiotic composition according to [2], wherein one or more excipients are selected from additives, carriers, bioavailability enhancers, side-effect suppressing components, diluents, buffers, flavouring agents, binders, preservatives, additive sweetener, prebiotic or other ingredients.
[4] The probiotic composition according to [3], wherein the prebiotic is selected from fructooligosaccharides(FOS), galacto-oligosaccharides (GOS) or xylooligosaccharides (XOS).
[5] The probiotic formulation according to [1] to [4] further comprises other agents selected from tolbutamide, glibenclamide, glipizide, chlorpropamide, gliclazide, metformin or herbal therapy.
[6] The probiotic formulation according to [5], wherein the other agent is metformin or herbal therapy.
[7] The probiotic formulation according to [1] to [6], wherein the formulation is in the dosage form selected from gelatine capsule, HPMC capsule, sachet or tablet.
[8] The probiotic formulation according to [7], wherein the dosage form is capsule.
[9] A probiotic formulation comprising the following ingredients:
Culture Probiotic capsules Placebo capsules
Bifidobacterium bifidum TBB037 0.5 Billion 0
Lactobacillus plantarum TLP017 0.25 Billion 0
Lactobacillus acidophilus TLA076 0.5 Billion 0
Bacillus clausii TLBC034 0.25 Billion 0
Saccharomyces boulardii RVSB01 0.5 Billion 0
Fructooligosaccharide (FOS) 125 mg 125 mg
Excipients qs 325 mg

[10] The probiotic composition according to [1] for use in treating or preventing metabolic syndrome selected from hyperlipidemia, diabetes, insulin resistance, obesity and obesity-related disorders.
[11] A probiotic composition according to [1] for use in treating or preventing gastrointestinal disease or disorder selected from irritable bowel syndrome or inflammatory bowel disease.
[12] A method for delaying or inhibiting the onset of type 2 diabetes in a pre- diabetic subject, the said method comprising administering to the subject a probiotic composition according to [1].
[13] A method of lowering blood glucose level, the said method comprising administering to the subject a probiotic composition according to [1].
[14] A method of controlling a lipid level, the said method comprising administering to the subject a probiotic composition according to [1].
[15] A method of lowering the blood pressure, the said method comprising administering to the subject a probiotic composition according to [1].
Preferably, the invention provides one or more of the following embodiments:
[16] An oral probiotic composition comprising a mixture of at least two probiotic ingredients selected from Bifidobacterium bifidum, Lactobacillus plantarum, Lactobacillus acidophilus, Bacillus clausii and Saccharomyces boulardii; a prebiotic; and optionally at least one excipient wherein the formulation is in the form of powder, granules, solution, suspension in an aqueous liquid or a non-aqueous liquid, or an oil-in-water (o/w) liquid emulsion or a water-in-oil (w/o) liquid emulsion.
[17] The oral probiotic composition according to [16], wherein said probiotic composition comprises a mixture of living microbes comprising Bifidobacterium bifidum, Lactobacillus plantarum, Lactobacillus acidophilus, Bacillus clausii and Saccharomyces boulardii.
[18] The oral probiotic composition according to [17], wherein the prebiotic is selected from fructooligosaccharides(FOS), galacto-oligosaccharides (GOS) or xylooligosaccharides (XOS), and present in the range of about 1 mg to about 1g.
[19] The oral probiotic formulation according to [18] , wherein the amount of fructooligosaccharides (FOS) is present in the range of about 1 mg to about 1 g preferably about 5 mg to about 500 mg.
[20] The oral probiotic composition according to [16], wherein the excipient is selected from additive, carrier, bioavailability enhancer, side effect suppressing component, diluent, buffer, flavouring agent, binder, preservative and additive sweetner.
[21] The oral probiotic composition according to [16], wherein the amount of each microbial strain is present in the range of about 1×102 cfu to about 1×1011 cfu.
[22] The oral probiotic composition according to [16], wherein the said probiotic composition is co-administered with at least one other therapeutic agent selected from tolbutamide, glibenclamide, glipizide, chlorpropamide, gliclazide, metformin or herbal therapy by simultaneous or sequential administration.
[23] The oral probiotic composition according to [16], wherein the said composition is present in dosage forms selected from gelatin capsule, HPMC capsule, sachet or tablet.
[24] The oral probiotic composition according to [16], wherein the said composition contains not more than 0.1% to 10% impurities when stored for at least one day to 2 years at ambient or refrigerated conditions.
[25] The oral probiotic composition according to [16], for use in treating or preventing metabolic syndrome selected from hyperlipidaemia, diabetes, insulin resistance, obesity or obesity-related disorders; gastrointestinal disease or disorder selected from irritable bowel syndrome or inflammatory bowel disease; for delaying or inhibiting the onset of type 2 diabetes; lowering blood glucose or blood pressure or controlling lipid level.
[26] The oral probiotic composition according to [16], wherein dosage form is capsule, which comprises:
Bifidobacterium bifidum present in an amount of about 0.5×109 cfu;
Lactobacillus plantarum present in an amount of about 0.25×109 cfu;
Lactobacillus acidophilus present in an amount of about 0.5×109 cfu;
Bacillus clausii present in an amount of about 0.25×109 cfu;
Saccharomyces boulardii present in an amount of about 0.5×109 cfu, and fructooligosaccharides (FOS) present in the amount of about 125 mg to about 500 mg, wherein the said composition contains not more than 0.1% to 10% impurities when stored for up to 2 years at ambient or refrigerated conditions.
[27] The oral probiotic composition according to [26], wherein the dose of capsule is 450 mg.
[28] The oral probiotic composition according to [26] and [27] wherein the capsule of 450 mg is given for three months.
[29] The probiotic composition according to [26] further comprises other agents selected from tolbutamide, glibenclamide, glipizide, chlorpropamide, gliclazide, metformin or herbal therapy.

Brief Description of the Drawing
The invention may be more completely understood in consideration of the following description of the invention in connection with the accompanying drawing, in which: Figure 1 shows the outline of the clinical study comprising enrolment, allocation of subjects, follow-up and analysis.

Detailed Description of the Invention
The methods and products of the present disclosure have been described in preferred embodiments. It will be apparent that one of ordinary skill in the art can change or appropriately modify and combine the methods described herein to implement and apply the present invention without departing from the content, spirit and scope of the disclosure. All such modifications or changes are within the scope of the present invention.
According to one aspect, there is provided a probiotic composition comprising a pharmaceutically effective amount of one or more probiotic ingredients selected from Bifidobacterium bifidum, Lactobacillus plantarum, Lactobacillus acidophilus, Bacillus clausii and Saccharomyces boulardii.
Wherein, the term “therapeutically effective amount” refers to the effective amount of the compound (active ingredient or excipient) that may be incorporated into a pharmaceutical formulation of the invention and administered to a patient without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained. Each carrier, excipient must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. An “effective amount” can be provided in one or more administrations. The exact amount required will vary depending on factors such as the identity and number of individual probiotic strains employed, the subject being treated, the nature of the disease(s) or condition(s) suffered by the subject that is to be treated and the age and general health of the subject, and the form in which the composition is administered. For any given case, an appropriate “effective amount” may be determined by one of ordinary skill in the art.
As used herein the term “active Ingredient” refers to probiotic ingredient selected from Bifidobacterium bifidum, Lactobacillus plantarum, Lactobacillus acidophilus, Bacillus clausii and Saccharomyces boulardii.
In an embodiment the present invention provides an oral probiotic composition comprising a mixture of at least two probiotic ingredients selected from Bifidobacterium bifidum, Lactobacillus plantarum, Lactobacillus acidophilus, Bacillus clausii and Saccharomyces boulardii; a prebiotic and optionally at least one excipient.
Wherein, Bifidobacterium bifidum belongs to the genus Bifidobacterium, a member of the Bifidobacteriaceae family. The genus Bifidobacterium is comprised of 48 different taxa, 40 of which have been isolated from the gastrointestinal tract (GIT) contents of mammals, birds, or insects, while the remaining eight from sewage and fermented milk. B. bifidum is an essential bacteria found in the human intestine. When it is low or absent all together in the human intestine, it is an indication of being in an unhealthy state. In a particular embodiment, the present invention includes all species of genus Bifidobacterium, while B. bifidum TBB-037 is a preferred species, which is isolated from Infants. In another particular embodiment, the present invention provides probiotic formulation comprising B. bifidum TBB-037 in combination with one or more other probiotic ingredients and one or more excipients.
Wherein, Lactobacillus plantarum is a Gram positive lactic acid bacterium commonly found in fermented food and in the gastro intestinal tract and is commonly used in the food industry as a potential starter probiotic. Among the lactic acid bacteria, L. plantarum attracted many researchers because of its wide applications in the medical field with antioxidant, anticancer, anti-inflammatory, antiproliferative, anti-obesity and antidiabetic properties. In a particular embodiment, the present invention includes all species of genus Lactobacillus, while L. plantarum TLP-017 is a preferred species, which is isolated from human. In another particular embodiment, the present invention provides probiotic formulation comprising L. plantarum TLP-017 in combination with one or more other active ingredient(s) and one or more excipients.
Wherein, Lactobacillus acidophilus is a species of Gram-positive bacteria in the genus Lactobacillus. L. acidophilus is a commercially significant bacterial probiotic, originally isolated from the human gastrointestinal tract. Acidophilus is sometimes used to prevent or treat several health conditions, including diabetes, acne, IBS or lactose intolerance. In a particular embodiment, the present invention includes all species of genus Lactobacillus, while L. acidophilus TLA-076 is a preferred species, which is isolated from dairy. In another particular embodiment, the present invention provides probiotic formulation comprising L. acidophilus TLA-076 in combination with one or more other active ingredient(s) and one or more excipients.
Wherein, Bacillus clausii is a commonly used Bacillus spp. Probiotic. Bacillus clausii was recommended as an adjunct treatment with oral rehydration solution for acute viral diarrhea. B. clausii may also be considered for prevention of antibiotic-associated diarrhea, Clostridium difficile-induced diarrhea, and as adjunct treatment of Helicobacter pylori. In a particular embodiment, the present invention includes all species of genus Bacillus, while B. clausii TBA-034 is a preferred species, which is isolated from soil. In another particular embodiment, the present invention provides probiotic formulation comprising B. clausii TBA-034 in combination with one or more other active ingredient(s) and optionally one or more excipients.
Wherein, Saccharomyces boulardii is a commonly used Saccharomyces. S. boulardii is used for treating and preventing diarrhea, including infectious types such as rotaviral diarrhea in children, diarrhea caused by gastrointestinal (GI), irritable bowel syndrome (IBS), inflammatory bowel syndrome, etc. S. boulardii is also called a "probiotic," a friendly organism that helps to fight off disease-causing organisms in the gut such as bacteria and yeast. In a particular embodiment, the present invention includes all species of genus Saccharomyces, while S. boulardii TSB-009 is a preferred species, and is isolated from fermented fruits. In another particular embodiment, the present invention provides probiotic formulation comprising S. boulardii TSB-009 in combination with one or more other active ingredient(s) and one or more excipients and one or more excipients. The amounts of individual microorganisms to be administered to subjects or to be included in compositions disclosed herein will depend on a variety of factors including the identity and number of individual strains employed, the condition or disease to be treated or against which the composition is designed to be used, and the form in which a composition is administered. For any given case, appropriate amounts may be determined by one of ordinary skill in the art using only routine experimentation. By way of example only, the amount of each microbial strain present in a single dose of a composition disclosed herein may be from about 1×102 cfu to about 1×1011 cfu, and may be about 1×103 cfu, about 2.5×103 cfu, about 5×103 cfu, about 7.5×103 cfu, 1×104 cfu, about 2.5×104 cfu, about 5×104 cfu, about 7.5×104 cfu, about 1×105 cfu, about 2.5×105 cfu, about 5×105 cfu, about 7.5×105 cfu, about 1×106 cfu, about 2.5×106 cfu, about 5×106 cfu, about 7.5×106 cfu, about 1×107 cfu, about 2.5×107 cfu, about 5×107 cfu, about 7.5×107 cfu, about 1×108 cfu, about 2.5×108 cfu, about 5×108 cfu, about 7.5×108 cfu, about 1×109 cfu, about 2.5×109 cfu, about 5×109 cfu, about 7.5×109 cfu, about 1×1010 cfu, about 2.5×1010 cfu, about 5×1010 cfu, about 7.5×1010 cfu, and about 1×1011 cfu. In one embodiment, the microbial strain present in a single dose of a composition may be from about 1×109 cfu, about 2.5×109 cfu, about 5×109 cfu, about 7.5×109 cfu, about 1×1010 cfu, about 2.5×1010 cfu, about 5×1010 cfu, about 7.5×1010 cfu, and about 1×1011 cfu, more preferably from about 1×109 cfu, about 2.5×109 cfu, about 5×109 cfu, about 7.5×109 cfu. In a preferred embodiment, the microbial strain present in a single dose of a composition is 2.5×109 cfu/g.
The probiotic composition of the present invention may also include one or more excipients, for example alternative sweeteners, which are alternatives to sucrose and other mono- and disaccharides that provide sweetness. The examples of sweeteners include, but are not limited to, saccharin, aspartame, neotame, advantame, acesulfame-potassium, sucralose, stevia or fructooligosaccharides (FOS). In another embodiment, the present invention provides that the the prebiotic is selected from fructooligosaccharides (FOS), galacto-oligosaccharides (GOS) or xylooligosaccharides (XOS), and present in the range of about 1 mg to about 1g. In a preferred embodiment, the sweetener is FOS. In accordance with particular embodiments of the invention, at least one sweetener may be administered or be present in a composition in an amount of from about 1 mg to about 1 g, or more typically between about 5 mg to about 500 mg. Alternatively, the composition may comprise about 10 mg, 100 mg, 125 mg, 150 mg, 300 mg or 500 mg, preferably 125 mg.
Compositions of the disclosure may further comprise any suitable excipients, including for example additives, carriers, bioavailability enhancers, side-effect suppressing components, diluents, buffers, flavouring agents, binders, preservatives or other ingredients that are not detrimental to the efficacy of the composition. Suitable excipients can be found in standard pharmaceutical texts, such as those described in Remington’s Pharmaceutical Handbook (Mack Publishing Co., NY, USA). In particular, the excipients include, for example maltodextrin, magnesium stearate, silicon dioxide and the like.
According to some embodiments, the present invention provides the probiotic formulation having the components as shown in Table 1 (TFPL-01 Formulation).
Culture Strain no* Probiotic capsules Placebo capsules
Bifidobacterium bifidum TBB037 0.5 Billion 0
Lactobacillus plantarum TLP017 0.25 Billion 0
Lactobacillus acidophilus TLA076 0.5 Billion 0
Bacillus clausii TLBC034 0.25 Billion 0
Saccharomyces boulardii RVSB01 0.5 Billion 0
Fructooligosaccharide (FOS) - 125 mg 125 mg
Excipients - qs 325 mg
* Lab Identification Number
In one particular embodiment, the probiotic formulation of the present invention is stable. The formulation of the present invention contains not more than 10%, not more than 9%, not more than 8%, not more than 7%, not more than 10% , not more than 6%, not more than 5%, not more than 4%, not more than 3%, not more than 2%, not more than 1%, not more than 0.5%, and not more than 0.1% of the total impurities when stored for at least one day, at least two days, at least three days, at least four days, at least five days, at least six days, at least seven days, for at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 6 weeks, at least 8 weeks, for at least 3 months, at least 6 months, for at least 1 year, or at least 2 years at ambient or refrigerated conditions.
Compositions suitable for oral administration may be presented as discrete units (i.e. dosage forms) such as gelatine or HPMC capsules, sachets or tablets, each containing a predetermined amount of each component of the composition as a powder, granules, as a solution or a suspension in an aqueous liquid or a non-aqueous liquid, or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
Another embodiment of the present invention provides an oral probiotic composition wherein dosage form is capsule, which comprises:
Bifidobacterium bifidum present in an amount of about 0.5×109 cfu;
Lactobacillus plantarum present in an amount of about 0.25×109 cfu;
Lactobacillus acidophilus present in an amount of about 0.5×109 cfu;
Bacillus clausii is present in an amount of about 0.25×109 cfu;
Saccharomyces boulardii present in an amount of about 0.5×109 cfu and
fructooligosaccharides (FOS) present in the amount of about 125 mg to about 500 mg wherein the said composition contains not more than 0.1% to 10% impurities when stored for up to 2 years at ambient or refrigerated conditions.
When the composition is formulated as capsules, the components of the composition may be formulated with one or more pharmaceutically acceptable carriers such as starch, lactose, microcrystalline cellulose and/or silicon dioxide. Additional ingredients may include lubricants such as magnesium stearate and/or calcium stearate. The capsules may optionally be coated, for example, with a film coating or an enteric coating and/or may be formulated so as to provide slow or controlled release of the composition therein.
Tablets may be prepared by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the components of the composition in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant (for example magnesium stearate or calcium stearate), inert diluent or a surface active/dispersing agent. Moulded tablets may be made by moulding a mixture of the powdered composition moistened with an inert liquid diluent, in a suitable machine. The tablets may optionally be coated, for example, with a film coating or an enteric coating and/or may be formulated so as to provide slow or controlled release of the composition therein. The compositions may be provided to the user in a powder form. For oral administration, the composition may then be mixed with a suitable volume of an aqueous medium, typically with agitation, to dissolve the components, or produce a suspension, suitable for ingestion. Thus, the compositions may be provided to a user in a powder form, which powder may then be added by the user to any type of aqueous medium (for example water or fruit juice) and consumed thereafter. Alternatively, the composition may be provided as a beverage, pre-mixed with an aqueous medium such as water. In another embodiment the compositions may be added in powder form by the user to any type to a food product (for example, yoghurt) and consumed thereafter. In another embodiment, the compositions may simply be consumed as a powder in the absence of a drink or additional food product.
The probiotic microorganisms may be conveniently incorporated in a variety of food and/or beverage products, nutraceutical products, probiotic supplements, food additives, pharmaceuticals and over-the-counter formulations. The food or food additive may be a solid form such as a powder, or a liquid form. Specific examples of the types of beverages or foods include, but are not limited to water-based, milk-based, yoghurt-based, other dairy-based, milk-substitute based such as soy milk or oat milk, or juice-based beverages, water, soft drinks, carbonated drinks, and nutritional beverages, (including a concentrated stock solution of a beverage and a dry powder for preparation of such a beverage); baked products such as crackers, breads, muffins, rolls, bagels, biscuits, cereals, bars such as muesli bars, health food bars and the like, dressings, sauces, custards, yoghurts, puddings, pre-packaged frozen meals, soups and confectioneries.
Further, the present invention provides one or more of the following embodiments.
According to one embodiment, there is provided a probiotic composition for use in treating or preventing metabolic syndrome selected from hyperlipidemia, diabetes, insulin resistance, obesity and obesity-related disorders wherein the said probiotic composition comprises one or more probiotic ingredients selected from Bifidobacterium bifidum, Lactobacillus plantarum, Lactobacillus acidophilus, Bacillus clausii and Saccharomyces boulardii.
In another embodiment, there is provided a probiotic composition for use in treating or preventing gastrointestinal disease or disorder selected from irritable bowel syndrome or inflammatory bowel disease, wherein the said probiotic composition comprises one or more probiotic ingredients selected from Bifidobacterium bifidum, Lactobacillus plantarum, Lactobacillus acidophilus, Bacillus clausii and Saccharomyces boulardii.
In another embodiment, the present invention provides a method for treating or preventing metabolic syndrome selected from obesity, hyperlipidemia or diabetes in a subject, the said method comprising administering to the subject a therapeutically effective amount of a probiotic composition comprising one or more probiotic ingredients selected from Bifidobacterium bifidum, Lactobacillus plantarum, Lactobacillus acidophilus, Bacillus clausii and Saccharomyces boulardii.
In another embodiment, the present invention provides a method for treating or preventing gastrointestinal diseases selected from irritable bowel syndrome or inflammatory bowel disease in a subject, the said method comprising administering to the subject a probiotic composition comprising therapeutically effective amount of one or more probiotic microorganisms selected from Bifidobacterium bifidum, Lactobacillus plantarum, Lactobacillus acidophilus, Bacillus clausii and Saccharomyces boulardii.
According to another embodiment, the present invention provides a method for delaying or inhibiting the onset of type 2 diabetes in a pre-diabetic subject, the method comprising administering to the subject a probiotic composition comprising therapeutically effective amount of one or more probiotic microorganisms selected from Bifidobacterium bifidum, Lactobacillus plantarum, Lactobacillus acidophilus, Bacillus clausii and Saccharomyces boulardii.
In another embodiment, the present invention provides a method of lowering blood glucose level, said method comprising administering to the subject a probiotic composition comprising therapeutically effective amount of one or more probiotic microorganisms selected from Bifidobacterium bifidum, Lactobacillus plantarum, Lactobacillus acidophilus, Bacillus clausii and Saccharomyces boulardii.
In another embodiment, the present invention provides a method of controlling a lipid level, said method comprising administering to the subject a probiotic composition comprising therapeutically effective amount of one or more probiotic microorganisms selected from Bifidobacterium bifidum, Lactobacillus plantarum, Lactobacillus acidophilus, Bacillus clausii and Saccharomyces boulardii.
In another embodiment, the present invention provides a method of lowering the blood pressure, said method comprising administering to the subject a probiotic composition comprising therapeutically effective amount of one or more probiotic microorganisms selected from Bifidobacterium bifidum, Lactobacillus plantarum, Lactobacillus acidophilus, Bacillus clausii and Saccharomyces boulardii.
As used herein the term “subject” refers to any mammal, including, but not limited to, livestock and other farm animals (such as cattle, goats, sheep, horses, pigs and chickens), performance animals (such as racehorses), companion animals (such as cats and dogs), laboratory test animals and humans. Typically the subject is a human.
As used herein the terms “treating” refers to any and all applications which remedy, or otherwise hinder, retard, or reverse the progression of, a disease or disorder or at least one symptom of a disease or disorder, including reducing the severity of a disease or disorder. Thus, treatment does not necessarily imply that a subject is treated until complete recovery from a disease or disorder. Similarly, the terms “preventing” refers to any and all applications that prevent the establishment of a disease or disorder or otherwise delay the onset of a disease or disorder.
The compositions, uses and methods of the present disclosure may be employed as an adjunct to other therapies or treatments for diabetes, type 2 diabetes, metabolic syndrome, insulin resistance, obesity and obesity-related disorders. Accordingly compositions and methods disclosed herein may be co-administered with other agents that may facilitate a desired therapeutic outcome, for example sulfonylureas such as such as tolbutamide, glibenclamide, glipizide, chlorpropamide, gliclazide, metformin or herbal therapy. By “co- administered” is meant simultaneous administration in the same formulation or in two different formulations via the same or different routes or sequential administration by the same or different routes. By “sequential” administration is meant a time difference of from seconds, minutes, hours or days between the administration of the agents, compositions or treatments. Sequential administration may be in any order. Similarly, methods and compositions of the present disclosure may be employed in conjunction with lifestyle changes by the subject, such as a healthy diet and adequate exercise.
In one embodiment, the probiotic formulation decreases both fasting as well as postprandial glucose levels by 16.3% and 12.8%, respectively. In another embodiment, co- administration of probiotics with herbal therapy for three months has the following effect on blood glucose levels: reduced HbA1c levels by about 9.9%, increased C-peptide levels by 3.4%, reduced HOMA IR by 2.6% in probiotic group, increased beta cell activity by 41% in probiotics group, and increased Insulin sensitivity by 2.8%.
In another embodiment, co-administration of probiotics with herbal therapy for three months reduced cholesterol level by about 13%, triglyceride levels by 17.8%, increased HDL cholesterol levels by about 7.4%.
In yet another embodiment, the effect of probiotic formulation therapy on kidney function may involve monitored urea, creatinine and uric acid levels in plasma before and at the end of the therapy. With the present formulation, a significant reduction in urea, uric acid, creatinine was observed. In yet another embodiment, significant variations in enzyme markers of liver such as bilirubin, SGOT and SGPT were recorded.

Experimental
The present invention is illustrated below by reference to the following examples. However, one skilled in the art will appreciate that specific methods and results discussed are merely illustrative of the invention, as innumerable variations, modifications, applications, and extensions of these embodiments and principles can be made without departing from the spirit and scope of the invention.

Example 1: Preparation of Pure Culture
Bifidobacterium bifidum, Lactobacillus plantarum, Lactobacillus acidophilus, Bacillus clausii and Saccharomyces boulardii isolated from infants, healthy human, dairy,soil, fermented fruits respectively. Samples were aseptically collected in a pre-sterilized container and immediately bought to the laboratory. 5g of sample added to the 45 mL of Man Rogosa Sharpe (MRS) broth, incubated at 37°C for 24 hours. Then 0.1 mL aliquots of the sample of suitable dilutions were plated onto MRS agar. The plates were incubated at 37°C from 24 hours. Isolated distinct colonies were further subcultured on freshly prepared MRS agar plates to obtain pure culture. Preliminary identification of the isolates was performed by microscopic observations. The isolates were selected and screened by biochemical tests.

Example 2: Clinical Trial of Probiotic Formulation of the Present Invention
2.1 Study design
The study design included following steps:
Selection of patients with metabolic syndrome meeting inclusion criteria and those consented to participate in the study.
Measurement of anthropometric and biochemical parameters once before and at selected intervals during the course of the study.
Administration of “TFPL-01” in defined dose daily for a period of 3 months.
Measurement of parameters at the end of the study.
Analysis of the data.
Registration and selection of subjects for study
In the context of the present invention, the tests for the exposure of TFPL-01 of the present invention were conducted in total of 60 subjects with metabolic syndrome were randomly selected after taking written informed consent based on harmonized criteria (2009) from the weekend diabetic clinic of Health Center, Jiwaji University. All the subjects have already consumed the herbal supplement for the treatment of metabolic syndrome. Figure 1 shows the number of subjects enrolled and drop outs during the course of study. The patients' baseline characteristics are summarized in (Table 2). The following criteria were employed for selecting the subjects for the study.
The subjects with metabolic syndrome who agreed to remain exclusively on ‘Probiotic therapy TFPL-01’ for a minimum of three months were only registered for the study. Before starting the study, all participating subjects were given a verbal explanation about the objectives of the study, nature of probiotic formulation going to be administered, i.e., “TFPL-01”, rationale and duration of therapy in the local language. Anthropometric measurements, weight, height and waist were recorded at the beginning and at the end of the study period.
Dose and duration of Supplement
Probiotic capsules BD 450 mg/capsule (2 capsules/day) given for three months. Details of the probiotics used in the study are given in Table1.
Biochemical parameters monitored
The fasting and postprandial plasma glucose, glycosylated hemoglobin (HbA1c), C- peptide, lipid profile, kidney and liver function test were analyzed. Measurements were made before and at the end of the study. Insulin resistance (IR), Insulin sensitivity and ß- cell activity were calculated by using HOMA2 calculator.
Statistical Analysis
Statistical analysis was carried out using a paired t test (Sigma stat 3.5). A p-value *p<0.05, **p<0.001 was considered statistically significant.
Table 2: Demographic details of subjects administered with ‘TFPL-01 Therapy’
Measure Placebo TFPL-01
Sex (n) (Male/Female) n=30 (18/12) n=30 (16/14)
Age (years) 50.64±2.4 58.10±1.5
BMI (kg/m2) 26.2±4.4 25.75±0.5
SBP (mm Hg) 131.0±8.1 134.7±1.2
DBP(mm Hg) 81.3±6.3 82.8±1.1
FBG (mg/dl) 142.2±1.1 152.40±7.7
HbA1c (%) (mmol/mol) 7.5±0.2 6.70±0.3

2.2 Results
A. Effect of ‘TFPL-01’ on Blood Glucose Levels
Table 3 shows the fasting and post prandial blood glucose levels before and after three months of therapy.
A significant decrease (p<0.001) was recorded in both fasting as well as postprandial glucose levels (16.3% and 12.8% respectively) and glycosylated haemoglobin (9.9%) at the end of three months therapy. Co-administration of probiotics with herbal therapy for three months reduced HbA1c levels by about 9.9 % while C-peptide levels were increased by 3.4% in subjects co-administered with probiotics and herbal therapy. HOMA IR reduced by 2.6% in probiotic group. Beta cell activity was increased by 41% in probiotics group respectively. Insulin sensitivity was increased by 2.8%.
Table 3
Parameters Placebo group Probiotic group
BS AS BS AS
Fasting 142.2 ± 31.1 126.0 ±24.8 ** 152.4 ± 30.5 127.53 ±23.8**†
P.P 180.8 ±37.1 166.4 ± 32.2** 206.2 ±46.3 179.9 ±44.1 **
HbA1C 6.7 ±1.3 6.4 ±1.23 ** 7.5 ±1.5 6.8 ±1.3 ** †
C- Peptide 1.91±0.3 1.96±0.3 2.06 ±0.2 2.13±0.3
HOMA 2 IR 1.6±0.3 1.6±0.2 1.8±0.2 1.7±0.3
HOMA 2 % S 63.8±14.0 64.7±13.8 57.1±7.7 58.6±7.9
HOMA 2 % B 56.5±20.3 69.5±19.0 51.8±16.1 73.4±20.1
Note: Values are given in Mean±SD, BS: Before supplement, AS: After supplement. *(P<0.05), ** (P< 0.001) Significance difference with in the groups by paired t test, (P<0.05), ‡ (P<0.001) Significance difference between the groups by student t test

B. Effects of ‘TFPL-01’ on Lipid Levels
Co-administration of probiotics with herbal therapy for three months reduced cholesterol level by about 13% while triglyceride levels was reduced by 17.8%. HDL cholesterol levels were increased by about 7.4% after co-administration of probiotics with herbal therapy. Probiotic supplementation reduced the LDL cholesterol level by 17.9%. VLDL cholesterol levels were reduced by 17.8% in subjects co-administered with probiotics and herbal therapy. Changes are statistically significant within and between the groups as shown in below Table 4.
Table 4
Parameters Probiotic group Placebo group
BS AS BS AS
Total Cholesterol 176.7 ± 22.5 152.9±25.6 **† 172.1±37.56 155.3±30.13 **
Triglycerides 156.9±28.5 129.6±27.7 **† 162.4±29.4 143.8±29.1 **
HDL- Cholesterol 40.2±1.6 43.2±1.4 **‡ 41.2±1.5 42.6±1.2 **
LDL-Cholesterol 103.8±20.96 85.2±25.42** 98.3±36.3 83.9±29.9 **
VLDL-Cholesterol 31.4±5.7 25.9±5.5 **† 32.5±5.9 28.8±5.8 **
TC/HDL ratio 4.3±0.5 3.5±0.6 4.2±0.9 3.6±0.7
LDL/HDL ratio 2.6±0.5 1.9 ±0.6 2.4 ±0.8 1.9±0.7
Note: Values are given in Mean±SD, BS: Before supplement, AS: After supplement. *(P<0.05) ** (P<0.001). Significance difference within the groups by paired t test, † (P<0.05), ‡ (P< 0.001) Significance difference between the groups by student test

On probiotic supplementation mean waist circumference was reduced by 1.4% and BMI by 1.5%. It was also observed that probiotic supplementation reduced systolic and diastolic blood pressure by 6.9 % and 3.9%, respectively as shown in Table 5.
Table 5: Effects of ‘TFPL-01’ on blood pressure and physiological parameter
Parameters Probiotic group Placebo group
BS AS BS AS
BMI 25.7±3.3 25.3±3.0** 26.2±4.4 25.9±4.2
Waist 37.2 ±2.3 36.7±2.4* 35.6±2.7 35.1±3.1*
Systolic blood pressure 134.8±9.4 125.4 ±7.7**† 131±8.1 127±8.3**
Diastolic blood pressure 84.8 ±3.9 79.0 ±3.5**† 81.3 ±6.3* 79.4 ±6.6
Note: Values are given in Mean±SD, BS: Before supplement, AS: After supplement. *(P<0.05) ** (P<0.001) Significance difference within the groups by paired t test, † (P<0.05), ‡ (P<0.001) Significance difference between the groups by student test

C. Effect of ‘TFPL-01’ on Biomarkers of Toxicity:
Effect of probiotics therapy on kidney function was monitored by estimating urea, creatinine and uric acid levels in plasma before and at the end of the therapy. The data presented in Table 6 showed significant reductions in urea (28.5±1.6 to 24.6±1.7), uric acid (from 5.6±0.3 to 5.4±0.3 mg/dl), creatinine (from 0.9±0.07 to 0.8±0.06 mg/dl) and Significant variations in enzyme markers of liver viz., bilirubin (from 0.75±0.09 to 0.63±0.08), SGOT (from 35.2±9.3 to 29.2±7.17 IU/L) and SGPT (from 34.07±3.8 to 26.41±2.6 IU/L) were recorded.
Table 6
Parameters Placebo group Probiotic group
BS AS BS AS
Urea 28.5±1.6 24.6±1.7 ** 26.85±2.8 22.9±2.3**
Uric acid 5.6±0.3 5.4±0.3 ** 5.9±1 5.2±1**‡
Creatinine 0.9±0.07 0.8±0.06 0.9±0.4 0.8±0.3*
BUN 7.62 ±0.75 6.8 ±0.7 * 12.3 ±1.7 10.55 ± 1.5*†
Bilirubin 0.75 ±0.09 0.633 ± 0.08** 0.52 ± 0.05 0.40 ± 0.04**
ALP 101.8 ± 8.3 91.33 ± 7.0** 117.3 ±5.8 104.8 ±5.7**
ALT 34.07 ±3.8 26.41 ±2.6** 35.44 ± 3.7 25.8 ±2.9**
AST 35.2± 9.3 29.2 ± 7.1** 30.4 ± 3.8 24.95 ±3.5**
GGT 20.48 ±2.4 20.82 ±2.3** 31.45 ±4.6 27.08 ±4.4**
Note: values are given in Mean±SD, BS: Before supplement, AS: After supplement. *(P<0.05), ** (P<0.001) Significance difference with in the groups by paired t test † (P<0.05), ‡ (P<0.001) Significance difference between the groups by student t test
,CLAIMS:We Claim:

1. An oral probiotic composition comprising a mixture of at least two probiotic ingredients selected from Bifidobacterium bifidum, Lactobacillus plantarum, Lactobacillus acidophilus, Bacillus clausii and Saccharomyces boulardii; a prebiotic; and optionally at least one excipient wherein the composition is in the form of powder, granules, solution, suspension in an aqueous liquid or a non-aqueous liquid, or an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.

2. The oral probiotic composition as claimed in claim 1, wherein said probiotic composition comprises a mixture of living microbes comprising Bifidobacterium bifidum, Lactobacillus plantarum, Lactobacillus acidophilus, Bacillus clausii and Saccharomyces boulardii.

3. The oral probiotic composition as claimed in claim 1, wherein the prebiotic is selected from fructooligosaccharides (FOS), galacto-oligosaccharides (GOS) or xylooligosaccharides (XOS), and present in the range of about 1 mg to about 1g.

4. The oral probiotic composition as claimed in claim 1, wherein the excipient is selected from additive, carrier, bioavailability enhancer, diluent, buffer, flavouring agent, binder, preservative and additive sweetner.

5. The oral probiotic composition as claimed in claim 1 or 2, wherein the amount of each microbial strain is present in the range of about 1×102 to about 1×1011 cfu.

6. The oral probiotic composition as claimed in claim 1, wherein the said probiotic composition is co-administered with at least one other therapeutic agent selected from tolbutamide, glibenclamide, glipizide, chlorpropamide, gliclazide, metformin or herbal therapy by simultaneous or sequential administration.

7. The oral probiotic composition as claimed in any of the preceding claims, wherein the said composition is present in dosage forms selected from gelatin capsule, HPMC capsule, sachet or tablet.

8. The oral probiotic composition as claimed in any of the preceding claims, wherein the said composition contains not more than 0.1% to 10% impurities when stored for at least one day to 2 years at ambient or refrigerated conditions.

9. The probiotic composition as claimed in claim 1, for use in treating or preventing metabolic syndrome selected from hyperlipidaemia, diabetes, obesity or obesity-related disorders; irritable bowel syndrome or inflammatory bowel disease; lowering blood glucose or blood pressure or controlling lipid level.

10. The oral probiotic composition as claimed in claim 1, wherein dosage form is capsule, which comprises Bifidobacterium bifidum present in an amount of about 0.5×109 cfu; Lactobacillus plantarum present in an amount of about 0.25×109 cfu; Lactobacillus acidophilus present in an amount of about 0.5×109 cfu; Bacillus clausii present in an amount of about 0.25×109 cfu; Saccharomyces boulardii present in an amount of about 0.5×109 cfu, and fructooligosaccharides (FOS) present in the amount of about 125 mg to about 500 mg, wherein the said composition contains not more than 0.1% to 10% impurities.

Dated this 21st day of February, 2022

(POOJA)
(IN/PA-1838)
APPLICANT’S AGENT
LEVINNOV IP CONSULTANTS PRIVATE LIMITED