FIELD OF INVENTION

The present invention relates to a probiotic synergistic composition comprising two or more Lactobacillus species, wherein the composition is useful for generating, improving or reinforcing homeostasis and/or immunity in a subject.

BACKGROUND OF THE INVENTION

As a prerequisite for a microorganism to be considered as a probiotix;, it should have the ability to adhere to the various biological surfaces. Adhesion plays an important role in the modulation of local and systemic immunological activities and helps microbes form communities on the surface. It also promotes the competition of these microorganisms with the autochthonous microflora, pathogen exclusion, enhanced healing of damaged mucosa and prolonged transient colonization which is important for probiotic activity [Sarah L, Jos V, and Sigrid C. J. D K. Genes and Molecules of Lactobacilli Supporting Probiotic Action. Microbiology and Molecular biology reviews, Dec. 2008, Vol. 72, No. 4. p. 728-764].

Competition of commensal and probiotic bacteria with pathogens for adhesion and colonization is one of the important protective mechanisms of gastrointestinal tract. For pathogenic bacteria, the adhesion to epithelium is a critical step, since it allows the release of enzymes and toxins initiating necrotic processes directly into the target cell, thereby facilitating the invasion. To cause infection, pathogenic bacteria, after penetrating intestinal mucus, must adhere to enterocytes. Similar to gut, mouth is another prime target where we harbor a large diversity of bacteria. The biofilm formation on the teeth begins by the adhesion of certain microbial species, to the acquired pellicle. After the primary colonisers are attached, further accumulation of bacteria continues through the attachment of new species and the growth of the attached bacteria, causing a condition of dental plaque [Howard K. K, Xuesong H, Renate L, Maxwell H. A,and Wenyuan Shi. Interspecies Interactions within Oral Microbial Communities. Microbiology and Molecular biology reviews, Dec. 2007, Vol. 71. No. 4. p. 653-670].

US5709857 describes pharmaceutical, veterinary or alimentary compositions for the treatment or prophylaxis of gastrointestinal pathologies in which it is desirable to administer lactobacilli. The composition as described comprises at least one of the CNCM 1-1390, CNCM 1-1391, CNCM 1-1392 and CNCM 1-1447 strains.

US5965128 describes specific strains of probiotic E. coli, their isolation, characteristics and methods of use to prevent or treat E. coli 0157:H7 carriage by a ruminant animal and a method for preventing the carriage of E. coli 0157:H7 by a ruminant. The method as described is especially useful for treating young ruminants, such as bovine calves, at an early age, before exposure to E. coli 0157:H7 which may be present in the environment. The method is also useful to prevent animals shipped to a feedlot from being contaminated at the feedlot.

US6468525 describes a probiotic formulation, useful as a food supplement and a material for reestablishing beneficial bacteria to the body's intestinal tract. The composition 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.

US6797266 describes a probiotic compositions comprising Lactobacillus casei strain K.E01 having ATCC accession number PTA-3945. The disclosed probiotic compositions are useful in inhibiting enteric pathogen diseases in animals and in maintaining animal health.

US6696057 describes a fermented probiotic composition for the treatment of gastrointestinal disorders, and hyperlipidemia. The composition comprises a mixture of Lactobacillus bulgaricus and Streptococcus thermophilus lactic acid bacteria; and a carbohydrate containing media.

US7807440 describes a probiotic bacterial strain belonging to the genus Lactobacillus species selected from the group comprising Lactobacillus plantarum, Lactobacillus crispatus, and Lactobacillus gasseri. Further, the document describes its use as a medicament, a composition comprising the strain, the composition, e.g., being a food product or a pharmaceutical composition, a hygiene product, a biological pure culture of the strain, and a novel food.

t
US7901925 describes a probiotic composition comprising a strain of Lactobacillus delbrueckii, ssp. Bulgaricus and methods for using the composition for treatments of diseased states and boosting immune response.

Each of these probiotic known in the art is limited in its application and effectiveness. It is desired for generating, improving or reinforcing homeostasis and/or immunity in a subject in an effective and cost efficient manner using a single probiotic composition. Therefore, a new composition and method for generating, improving or reinforcing homeostasis and/or immunity is needed that is effective in reducing symptoms, reasonable in cost to the patient, does not exhibit significant adverse side effects, and which may be beneficial in reducing the risk of other side effects on health such as developing cancer. Of course, it is also desirable that the composition be easy to manufacture and deliver to the patient. Thus, there is a need to provide a probiotic composition exhibiting enhanced probiotic properties in terms of reducing or preventing infection, enhancing immunity, and/or improving health and vitality of a subject.

SUMMARY OF THE INVENTION

An aspect of the present invention relates to a probiotic synergistic composition comprising two or more Lactobacillus species selected from the group consisting of Lactobacillus helviticus strain NCIM 2126, Lactobacillus fermentum strain NCDC 141, Lactobacillus rhamnosus strain NCDC 24, and Lactobacillus paraplantarum strain NCDC 321, wherein the presence of more than one Lactobacillus species in the composition exhibits improved adhesion of to a biological surface.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

The following drawings form part of the present specification and are included to further illustrate aspects of the present invention. The invention may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

Figure 1 shows blending of probiotic cultures for adhesion on polystyrene surfaces at 24 hrs.

Figure 2 shows blending of probiotic cultures for adhesion on saliva coated surfaces at 2 hrs.

DETAILED DESCRIPTION OF THE INVENTION

Those skilled in the art will be aware that the invention described herein is subject to variations and modifications other than those specifically described. It is to be understood that the invention described herein includes all such variations and modifications. The invention also includes all such steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

Definition

For convenience, before further description of the present invention, certain terms employed in the specification, examples and appended claims are collected here. These definitions should be read in light of the remainder of the disclosure and understood as by a person of skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art.

The articles "a", "an" and "the" are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

The term "plurality" means more than one.

The terms "at least two," "more than one" and "plurality" ere. used interchangeably.

The terms "comprise" and "comprising" are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as "consists of only."

Throughout this specification, unless the context requires otherwise the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.

The term "including" is used to mean "including but not limited to." "Including" and "including but not limited to" are used interchangeably.

The term "Colony forming unit (CFU)" used in the present invention refers to a measure of viable and/or bacterial cell present in the probiotic compositions as disclosed in the present invention. For convenience the results are given as CFU/mL (colony-forming units per milliliter) for liquids and CFU/g (totohy-forming units per gram) for solids.

The term "probiotic" means a live microorganism that survives passage through the gastrointestinal tract and has a beneficial effect on the subject.

The terms "probiotic composition(s)," "composition(s) comprising blend of microorganism", "composition(s) comprising mixture of microorganisms" "composition(s) comprising blend of Lactobacillus strains", "composition(s) comprising mixture of Lactobacillus strains" and "composition(s)" are used interchangeably.

The term "subject" means a living organism, including a plant, a microbe, a human, an animal (domestic, agricultural, or exotic), etc.

The term "treating" is art recognized and includes preventing a disease, disorder or condition from occurring in a patient which may be predisposed to the disease, disorder and/or condition but has not yet been diagnosed as having it; inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder or condition, e.g., causing regression of the disease, disorder and/or condition. Treating the disease or condition includes ameliorating at least one symptom of the particular disease or condition, even if the underlying pathophysiology is not affected.

The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products, compositions, and methods are clearly within the scope of the invention, as described herein.

The present invention provides a probiotic synergistic composition for generating, improving and/or reinforcing homeostasis or immunity in a subject, wherein the composition comprises at least two 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 La&tobacillus species to a biological surface. The present invention further provides a process for preparing a composition comprising at least two Lactobacillus species selected from the 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.

The present invention also provides dietary supplements and pharmaceutical preparations comprising at least two 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.

To obtain the probiotic synergistic composition as disclosed in the present invention it is very important to select suitable strains of microorganisms to prepare a blend or combination of the different strains of the Lactobacillus species that can be used in the probiotic composition. As a blend they can have more effective probiotic attributes, adhesion being one such attributes which is of prime importance to have health benefiting effect.

Although research has been conducted on the benefits of probiotic adherence, there has not been much emphasis paid on a blend or combination of two probiotic strains for their ability to adhere. Blends or combination of probiotic bacteria has been studied with respect to many health benefiting properties but adherence as a mechanism to exert these property is yet to be explored. This area also imposes challenges as not all probiotic bacteria have positive symbiotic effects on growth and probiotic attributes. Therefore, proper selection of different combination of bacteria becomes a challenge. Lactic acid bacteria are well documented for their ability to adhere and inhibit attachment of pathogens thereby reducing colonization and preventing infection. The use of different strains of Lactobacillus as a blend or combination for improved adherence capability is not documented. Lactobacillus rhamnosus GG is a widely used commercial strain (Valio Ltd., Finland) with clinical evidence of its probiotic activities and its adhesion was tested in a number of experiments carried out by different groups.

For the purpose of the present invention, four different strains of Lactobacillus species have been used in the probiotic synergistic composition of the present invention. The composition comprises (1) Lactobacillus helviticus strain NCIM 2126 procured from National Collection of Industrial Microorganism, National Chemical Laboratory (NCL), Dr. Homi Bhabha Road, Pune - 411008, Maharashtra, (2) Lactobacillus fermentum strain NCDC 141 procured from National Collection of Dairy Culture, Dairy Microbiology Divisions, National Dairy Research Institute, Karnal-132001, Haryana, India, (3) Lactobacillus rhamnosus strain NCDC 24 procured from National Collection of Dairy Culture, Dairy Microbiology Divisions, National Dairy Research Institute, Karnal-132001, Haryana, India and (4) Lactobacillus paraplantarum strain NCDC 321 procured from National Collection of Dairy Culture, Dairy Microbiology Divisions, National Dairy Research Institute, Karnal-132 001, Haryana, India.

The selected Lactobacillus species have a particular beneficial impact on humans and animals, both on their gastrointestinal tract and their immune system. They have a particular impact on intestinal pathogens such as Salmonella typhimurium, Escherichia coli enteropathogens, Shigella dysenterieae, and other pathogenic enterobacteria known for infection in human. These selected species also have impact on parasites such as helminthes (Toxocara canis), protozoa {Cryptosporidium spp, Giardia lambia, Entamoeba histolytica, Toxoplasma gondii, Dientamoeba fragilis) or yeasts {Candida spp.).

The probiotic synergistic of the present invention can be used in any therapy either for preventing colonization by harmful organisms; or for reestablishing gastrointestinal flora after antibiotic treatment. When the composition is ingested at a high concentration by humans or animals, the Lactobacillus species present in the composition colonizes the intestine creating the necessary environment for a useful and homogeneous flora.

Administration of the composition of the present invention to the human or animal can also act against the deleterious effects of antibiotics as well as complete the recovery of humans and animals, avoiding recycling of intestinal bacteria in the case of diarrheas.

Further, the probiotic synergistic composition of the present invention can be incorporated into food products thus providing novel enriched functional food product exhibiting not only nutritional benefits but also other advantages leading in better health conditions. The Lactobacillus species in the composition colonizes the intestine thereby positively modifying the intestinal flora and improving the immune system function and, accordingly, the general health of the organigm

The compositions of the present invention additionally comprises any suitable adjuvants, excipients, additives, carriers, additional therapeutic agents, bioavailability enhancers, side-effect suppressing components, diluents, buffers, flavoring agents, binders, preservatives or other ingredients that do not preclude the efficacy of the composition.

Combinations of the four Lactobacillus species of the present invention showed synergisms in terms of enhanced adherence of the lactobacillus cells to a biological surface than the individual Lactobacillus species. The combination of these Lactobacillus species exhibits all the prerequisite probiotic attributes thus making an attractive composition for use as a food product, a food or nutritional supplement for improved and better health.

The probiotic synergistic composition comprising the four Lactobacillus species viz. Lactobacillus helviticus, Lactobacillus fermenium, Lactobacillus rhamnosus and Lactobacillus paraplantarum may be compounded with foods such as, but not limited to dairy products, grains, breads, meats, fruits, vegetables, rice and the like. The compositions of the present invention can be compounded with additional ingredients. For example, the composition can be mixed with carbohydrates lipids, polypeptides, fatty acids, phytochemicals, and combinations thereof. Carbohydrates that may be used in accordance with the teachings of the present invention include monosaccharides, disaccharides, oligosaccharides and polysaccharides such as, but not limited to trehalose, maltose, sucrose, dextrose, lactose, inulin, ribose, malt dextrin and the like.

In one embodiment of the present invention the disaccharide is trehalose dihydrate, the oligosaccharide is fructo-oligosaccharide and the polysaccharide is malt dextrin.

Suitable lipids include, but are not limited to soy,bean oil, olive oil, palm kernel oil, peanut oil, walnut oil, cannola oil and the like. Suitable polypeptides include whey protein, egg albumin, gelatin, milk proteins, and other animal and plant proteins. Finally, phytochemicals as used herein include such compounds as polyphenols, saponins, flavanoids, monoterpenes, allyl sulfides, lycopenes, carotenoids, polyactetylenes, silymarin, glycyrrhizin catechins and others.

The composition of the present invention can be taken orally as a bolus in the form of a gelatin capsule, pressed tablet, or gel cap. The composition can also be taken orally in the form of a liquid beverage. The liquid beverage may contain other ingredients such as, but not limited to flavor enhancers, sweeteners, viscosity enhancers and other food additives. The present invention can also be taken together with other foods either separately or compounded therewith.

The probiotic synergistic composition of the present invention can be administered to any animal in need of thereof including, but not limited to mammals, birds, reptiles and fish. Typical applications include administering the probiotic composition of the present invention to humans, horses, swine (pigs), cows, sheep, dogs, cats, rabbits, chickens, turkeys, pheasants, quail, parakeets, parrots, and other wild and domesticated animals.

The probiotic synergistic composition of the present invention can be used to inhibit or treat enteric pathogen-associated diseases when administered to an animal in need thereof using the methods described in the present specification. Enteric pathogen diseases include those diseases caused by pathogens such as diarrhea, irritable bowel syndrome and intestinal hemorrhages. Examples of enteric pathogens associated with these diseases include, but not limited to enteropathogenic Escherichia coli (EPEC), enterotoxigeneic E. coli (ETEC), Salmonella enteriditis, Yersina pseudotuberculosis and Listeria monocytogenes.

It is theorized by the present inventor, not offered as a limitation that the inhibition and treatment of the enteric pathogen diseases is accomplishe'l by the probiotic composition of the present invention through a competitive binding process. In other words the probiotic Lactobacilli species of the present invention compete with enteric pathogens for binding sites on the intestinal mucosa. Because the probiotic Lactobacilli of the present invention have a higher affinity and avidity for these binding sites than the enteric pathogens, the probiotic Lactobacilli of the present invention displace the enteric pathogens into the intestinal milieu where they are harmlessly flushed from the intestines by normal metabolic processes.
In yet another embodiment of the present invention, there is provided a probiotic synergistic composition for generating, improving or reinforcing homeostasis and/or immunity in a subject, wherein the composition comprises at least two Lactobacillus species at a concentration ranging from 1x-108 cfu/ml to 5 x 108 cfu/ml, wherein the Lactobacillus species is selected from the group consisting of Lactobacillus helviticus, Lactobacillus fermentum, Lactobacillus rhamnosus and Lactobacillus paraplantarum.

In yet another embodiment of the present invention, there is provided a probiotic synergistic composition for generating, improving or reinforcing homeostasis and/or immunity in a subject, wherein the composition comprises at least two Lactobacillus species at a concentration ranging from 1 x 108 cfu/ml to 5 x 108 cfu/ml, wherein the Lactobacillus species is selected from the group consisting of Lactobacillus helviticus strain NCIM 2126, Lactobacillus fermentum strain NCDC 141, Lactobacillus rhamnosus strain NCDC 24, and Lactobacillusparaplamarum strain NCDC 321.

In an embodiment of the present invention, there is provided a probiotic synergistic composition for generating, improving or reinforcing homeostasis and/or immunity in a subject, wherein the composition comprises Lactobacillus helviticus at a concentration ranging from 1 x 108 cfu/ml to 5 x 108 cfu/ml and Lactobacillus rhamnosus at a concentration ranging from 1 x 108 cfu/ml to 5 x 108 cfu/ml.

In an embodiment of the present invention, there is provided a probiotic synergistic composition for generating, improving or reinforcing homeostasis and/or immunity in a subject, wherein the composition comprises Lactobacillus helviticus at a concentration ranging from 1 x 108 cfu/ml to 5 x 108 cfu/ml and Lactobacillus paraplantarum at a concentration ranging from 1 x 108 cfu/ml to 5 x 108 cfu/ml.

In another embodiment of the present invention, there is provided a probiotic synergistic composition for generating, improving or reinforcing homeostasis and/or immunity in a subject, wherein the composition comprises Lactobacillus fermentum at a concentration ranging from 1 x 108 cfu/ml to 5 x 108 cfu/ml and Lactobacillus rhamnosus at a concentration ranging from 1 x 108 cfu/ml to 5 x 108 cfu/ml.

In another embodiment of the present invention, there is provided a probiotic synergistic composition for generating, improving or reinforcing homeostasis and/or immunity in a subject, wherein the composition comprises Lactobacillus paraplantarum at a concentration ranging from 1 x 108 cfu/ml to 5 x 108 cfu/ml and Lactobacillus rhamnosus at a concentration ranging from 1 x 108 cfu/ml to 5 x 108 cfu/ml.

In another embodiment of the present invention, there is provided a probiotic synergistic composition for generating, improving or reinforcing homeostasis and/or immunity in a subject, wherein the composition comprises Lactobacillus paraplantarum at a concentration ranging from 1 x 108 cfu/ml to 5 x 108 cfu/ml, Lactobacillus helviticus at a concentration ranging from 1 x 108 cfu/ml to 5 x 108 cfu/ml and Lactobacillus rhamnosus at a concentration ranging from 1 x 108 cfu/ml to 5 x 108 cfu/ml.

In another embodiment of the present invention, there is provided a probiotic synergistic composition for generating, improving or reinforcing homeostasis and/or immunity in a subject, wherein the composition comprises Lactobacillus fermentum at a concentration ranging from 1x10 cfu/ml to 5 x 10 cfu/ml, Lactobacillus helviticus at a concentration ranging from 1 x 108 cfu/ml to 5 x 108 cfu/ml and Lactobacillus rhamnosus at aconcentration ranging from 1x10 cfu/ml to 5 x 10 cfu/ml In yet another embodiment of the present invention, 'here is provided a probiotic synergistic composition for generating, improving or reinforcing homeostasis and/or immunity in a subject, wherein the composition comprises Lactobacillus fermentum at a concentration ranging from 1 x 108 cfu/ml to 5 x 108 cfu/ml, Lactobacillus helviticus at a concentration ranging from 1 x 108 cfu/ml to 5 x 108 cfu/ml and Lactobacillus paraplantarum at a concentration ranging from 1 x 108 cfu/ml to 5 x 108 cfu/ml In still yet another embodiment of the present invention, there is provided a probiotic synergistic composition for generating, improving or reinforcing homeostasis and/or immunity in a subject, wherein the composition comprises Lactobacillus fermentum at a concentration ranging from 1 x 108 cfu/ml to 5 x 108 cfu/ml, Lactobacillus rhamnosus at a concentration ranging from 1 x 108 cfu/ml to 5 x 108 cfu/ml and Lactobacillus paraplantarum at a concentration ranging from 1 x 10 cfu/ml to 5 x 10 cfu/ml In still another embodiment of the present invention, there is provided a probiotic synergistic composition for generating, improving or reinforcing homeostasis and/or immunity in a subject, wherein the composition comprises at least two Lactobacillus species selected from the group consisting of Lactobacillus helviticus, Lactobacillus fermentum, Lactobacillus rhamnosus and Lactobacillus paraplantarum, wherein the composition optionally comprises a carrier.

The probiotic synergistic composition disclosed in the present invention further comprises addetives such as dietary fibers, vitamins and minerals.

Examples of the carrier that can be used in the probiotic composition as disclosed in the present invention includes but not limited to dietary fibers, inulines, carbohydrates, proteins, lipids, phytochemicals, glycosylated proteins, a pharmaceutically acceptable carrier and combinations thereof.

The probiotic synergistic composition disclosed in the present invention can be used as a food supplement in the food product such as bread, beverage, cheese, yoghurt, juice, health bars, spreads, biscuits and cereals.

Another embodiment of the present invention relates to the probiotic synergistic composition disclosed in the present invention that can be formulated in encapsulated or coated form.

Another embodiment of the present invention relates to the probiotic synergistic composition disclosed in the present invention, wherein the composition is in the form of tablets, sucking tablets, sweets, chewing gums, capsules, cream, gel, ointment, lotion, melting strips and sprays.

Another embodiment of the present invention relates to the probiotic synergistic composition disclosed in the present invention, wherein the composition can be used for treatment of human or an animal.

Another embodiment of the present invention provides a process for preparing the synergistic composition as claimed in claim 1, wherein said process comprises mixing the individual Lactobacillus species selected from a group consisting of Lactobacillus helviticus strain NCIM 2126, Lactobacillus fermentum strain NCDC 141, Lactobacillus rhamnosus strain NCDC 24, and Lactobacillus paraplantarum strain NCDC 321 at a concentration ranging from 1 x 108 cfu/ml to 5 x 108 cfu/ml and optionally adding a carrier to obtain the composition.

The probiotic synergistic composition disclosed in the present invention can also be prepared by the methods known in the art.

The property of adhesion of individual Lactobacillus species as well as a combination of various Lactobacillus species on polystyrene surfaces and saliva coated surfaces was studied. The combination of various Lactobacillus species showed enhanced ability to adhere to the surface as compared to the individual Lactobacillus species. However, it is to be noted that not all combination of Lactobacillus species showed enhancement in adherence. There was only certain combination of Lactobacillus species which showed better adhesion to a surface as compared to individual Lactobacillus species. In certain cases, the combination of Lactobacillus species had less ability to adhere to a surface than individual species.

The gastrointestinal microflora has been shown to play a number of vital roles in maintaining gastrointestinal tract function and overall physiological health. For example, the growth and metabolism of the many individual bacterial species inhabiting the gastrointestinal tract depend primarily upon the substrates available to them, most of which are derived from the diet [ Gibson G. R. et al., 1995. Gastroenterology 106: 975-982; Christi, S. U. et al, 1992. Gut 33: 1234-1238]. These findings have led to attempts to modify the structure and metabolic activities of the community through diet, primarily with probiotics, which are live microbial food supplements. Probiotic microorganisms are live microorganisms that confer a benefit when administered in adequate amounts to confer a health benefit on the host, often by inhibiting the growth of other biological organisms in the same corporeal environment. The best known probiotics are the lactic acid-producing bacteria (i.e., Lactobacilli) and Bifidobacteria, which are widely utilized in yoghurts and other dairy products. These probiotic organisms are non-pathogenic and non-toxigenic, retain viability during storage, and survive passage through the stomach and small intestine. Since probiotics do not permanently colonize the host, they need to be ingested regularly for any health promoting properties to persist. Commercial probiotic preparations are generally comprised of mixtures of Lactobacilli and Bifidobacteria, although yeast such as Saccharomyces has also been utilized.

The probiotic synergistic composition comprises harvested cells. The harvested cells can be prepared by routine culturing in a standard medium which can optionally be substituted with further nutrients. The cells are generally harvested by centrifugation and washed using a standard diluent such as Maximum Recovery Diluent (MRD). The cells can then be resuspended in MRD or other suitable type of diluent or medium. The suitable carrier is then optionally added to the cells.

The Lactobacillus species, Lactobacillus helviticus, Lactobacillus fermentum, Lactobacillus rhamnosus and Lactobacillus paraplantarum were routinely cultured separately in suitable culture medium supplemented with carbon source and nitrogen source under suitable culturing conditions. The cells were cultured until a specific time period. The cells were harvested by centrifugation, washed in a standard diluents and re-suspended in the diluent. Subsequently 5 x 107 cfu/ml of each individual Lactobacillus species was taken to prepare the composition. A suitable carrier was also added to the composition while mixing.

Combinations of the four Lactobacillus strains of the present invention showed better adherence to a surface than the individual Lactobacillus strains. The combination of these Lactobacillus strains have all the prerequisite probiotic attributes thus making an attractive composition for use as a food product or a food supplement for health benefiting effects.

The Lactobacillus dietary supplement may be compounded with foods such as, but not limited to dairy products, grains, breads, meats, fruits, vegetables, rice and the like. The form the Lactobacillus dietary supplement of the present invention assumes is not important and is non-limiting. The compositions of the present invention can be compounded with additional ingredients. For example, the composition can be mixed with a carbohydrate selected from the group consisting of trehalose, glucose, sucrose, fructose, maltose and combinations thereof. Proteins such as albumin and/or whey can be added to the compositions of the present invention. The composition of the present invention can be taken orally as a bolus in the form of a gelatin capsule, pressed tablet, or gel cap. The composition can also be taken orally in the form of a liquid beverage. The liquid beverage may contain other ingredients such as, but not limited to flavor enhancers, sweeteners, viscosity enhancers and other food additives. The present invention can also be taken together with other foods either separately or compounded therewith.

The probiotic synergistic composition of the present invention can be administered to any animal in need of thereof including, but not limited to mammals, birds, reptiles and fish. Typical applications include administering the probiotic composition of the present invention to humans, horses, swine (pigs), cows, sheep, dogs, cats, rabbits, chicken's, turkeys, pheasants, quail, parakeets, parrots, and other wild and domesticated animals.

The probiotic synergistic composition of the present invention can be used to inhibit or treat enteric pathogen-associated diseases when administered to an animal in need thereof using the methods described in the present specification. Enteric pathogen diseases include those diseases caused by pathogens such as diarrhea, irritable bowel syndrome and intestinal hemorrhages. Examples of enteric pathogens associated with these diseases include, but not limited to enteropathogenic Escherichia coli (EPEC), enterotoxigeneic E. coli (ETEC), Salmonella enteriditis, Yersina pseudotuberculosis and Listeria monocytogenes.

It is theorized by the present inventor, and not offered as a limitation, that the inhibition and treatment of the enteric pathogen diseases is accomplished by the probiotic composition of the present invention through a competitive binding process. That is, the probiotic Lactobacilli species of the present invention compete with enteric pathogens for binding sites on the intestinal mucosa. Because the probiotic Lactobacilli of the present invention have a higher affinity and avidity for these binding sites than the enteric pathogens, the probiotic Lactobacilli of the present invention displace the enteric pathogens into the intestinal milieu where they are harmlessly flushed from the intestines by normal metabolic processes.
The advantages of the synergistic composition of the present invention include but are not limited to use in treatment of enteric pathogen-associated diseases such as diarrhea, irritable bowel syndrome and intestinal hemorrhages. Examples of enteric pathogens associated with these diseases include, but not limited to enteropathogenic Escherichia coli (EPEC), enterotoxigeneic E. coli (ETEC), Salmonella enteriditis, Yersina pseudotuberculosis and Listeria monocytogenes. The composition of the present invention can be formulated with food products such as milk-based food products, grain-based food product, starch-based food products and nutritional beverages which is easy to consume.

Although the subject matter has been described in considerable detail with reference to certain preferred embodiments thereof, other embodiments are possible. As such, the spirit and scope of the appended claims should not be limited to the description of the preferred embodiment contained therein.

EXAMPLES

The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure.

Example 1

10 CFU/ml was taken for adherence assay of individual Lactobacillus species viz. Lactobacillus helviticus strain NCIM 2126, Lactobacillus fermentum strain NCDC 141, Lactobacillus rhamnosus strain NCDC 24, and Lactobacillus paraplantarum strain NCDC 321.

For the adherence assay of combination of two Lactobacillus species, 5x 107 CFU/ml of individual strain was taken to make the final cell count in the combination to be 108 CFU/ml. In a blend containing three strains 3.3 x 10' CFU/ml of each strain was used whereas, when there were four strains in a blend 2.5 x 107 CFU/ml of each strain was added in the blend. The cells were incubated in 96-well polystyrene coated plates and saliva coated plates for 24 hours and 2 hours, respectively. After incubation, the medium containing non-adhered cells were aspirated and the wells were washed two times with physiological saline. After washing, 50 ul of absolute ethanol was added to each well for fixing and incubated for 10 minutes at room temperature. Then, ethanol was removed and the plate was air dried followed by addition of 100 \\\ of 2% crystal violet and incubated for another 15 minutes. The stain was removed and the wells were again washed two times with physiological saline to remove excess stain. The quantitative analysis of adhesion was performed by adding 200 ul absolute ethanol to each well and incubating on a dancing shaker for 10 minutes to completely dissolve the stain. Then, the optical density was measured at 492 nm by an ELISA plate reader.

As a surprising property, it was observed that the influence of co-culture of different Lactobacillus can be synergistic or antagonistic. The bacteria can either enhance or inhibit the growth of another bacterium. With proper selection of at least two Lactobacillus species, one can enhance the ability of these strains together in a composition to have better probiotic attributes as compared to individual strains.

Figure 1 shows the adhesion of individual Lactobacillus and combination of Lactobacillus species on polystyrene surface at 24 hours. When adhesion was observed on polystyrene surfaces, the blends of (L. fermentum + L .rhamnosus; L. rhamnosus and L. helviticus) showed enhanced adhesion when compared to individual cultures. Where as in the other blends containing two probiotic strains there significant differences in adhesion were not observed. In the probiotic blend where three strains were used (L. fermentum, L .rhamnosus and L. paraplantarum) blend showed increase in the adherence as compared to individual strains. Significant increase in adhesion was not observed when a blend of four strains was studied for their adhesion as compared to adhesion of individual strains.

Figure 2 shows the adhesion of individual Lactobacillus and combination of Lactobacillus species on saliva coated surface at 2 hours. Similarly when adherence of blends on saliva coated surfaces was tested, there was increase in adhesion in certain blends as compared to individual strains. On saliva coated surfaces two different blends of (L. rhamnosis + L. paraplantarum; L. rhamnosis + L. fermentum; L, rhamnosis + L.helviticus and L. paraplantarum + L.helviticus) showed enhanced adherence ability than individual strains. Similar to the results observed on polystyrene surfaces except for blend of (L. helviticus, L. rhamnosus and L. paraplantarum) all other blends containing three different strains showed increase in the adherence as compared to individual strains.

As a surprising property, it was observed that the influence of co-culture of different Lactobacillus can be synergistic or antagonistic. The bacteria can either enhance or inhibit the growth of another bacterium. With proper selection of at least two Lactobacillus species, one can enhance the ability of these strains together in a composition to have better probiotic attributes as compared to individual strains.

I/we claim;

1. A probiotic synergistic composition comprising two or more Lactobacillus species selected from the group consisting of Lactobacillus helviticus strain NCIM 2126, Lactobacillus fermentum strain NCDC 141, Lactobacillus rhamnosus strain NCDC 24, and Lactobacillus paraplantarum strain NCDC 321, wherein the presence of more than one Lactobacillus species in the composition exhibits improved adhesion of to a biological surface.

2. The synergistic composition as claimed in claim 1, wherein said composition comprises Lactobacillus species at a concentration ranging from 1 x 108 cfu/ml to 5xl08cfu/ml.

3. The synergistic composition as claimed in claim 1, wherein said composition optionally comprises a carrier selected from a group consisting of dietary fibers, inulines, carbohydrates, proteins, lipids, phytochemicals, glycosylated proteins, a pharmaceutically acceptable carrier and combinations thereof.

4. The synergistic composition as claimed in claim 1, wherein said composition further comprises vitamins and minerals.

5. The synergistic composition as claimed in claim 1, wherein said composition is a food supplement or a food product.

6. The synergistic composition as claimed in claim 5, wherein said food product is selected from the group consisting of bread, beverage, cheese, yoghurt, juice, health bars, spreads, biscuits and cereals.

7. The synergistic composition as claimed in claim 1, wherein said composition is encapsulated or coated.

8. The synergistic composition as claimed in claim 1, wherein said composition is in the form of tablets, sucking tablets, sweets, chewing gums, capsules, cream, gel, ointment, lotion, melting strips and sprays.

9. A process for preparing the synergistic composition as claimed in claim 1, wherein said process comprises mixing the individual Lactobacillus species selected from a group consisting of Lactobacillus helviticus strain NCIM 2126, Lactobacillus fermentum strain NCDC 141, Lactobacillus rhamnosus strain NCDC 24, and Lactobacillus paraplantarum strain NCDC 321 at a concentration ranging from 1 x 108 cfu/ml to 5 x 108 cfu/ml and optionally adding a carrier to obtain the composition.