WO2015154140A1

WO2015154140A1 - Methods and compositions for delivering a probiotic

Info

Publication number: WO2015154140A1
Application number: PCT/AU2015/050154
Authority: WO
Inventors: Ambrosios Kambouris
Original assignee: Kambouris Shares Pty Ltd
Priority date: 2014-04-09
Filing date: 2015-04-03
Publication date: 2015-10-15
Also published as: AU2015245942A1

Abstract

The present invention provides a method for delivering a probiotic organism to an animal in need thereof, the method comprising the steps of: (i) administering the probiotic organism orally to the animal, and (ii) administering an effervescent agent orally to the animal to cause the generation of an effervescence product within the alimentary tract of the animal, wherein the probiotic organism and effervescence product co-locate at a site in the alimentary tract of the animal such that viability and/or a physiologic effect of the probiotic is improved relative to a method whereby there is no co-location of an effervescence product and the probiotic organism. The method may be used for treating a condition associated with the level or activity of probiotic bacteria in the alimentary tract, such as a digestive condition, an immune function condition, a wellness condition, or an allergic condition.

Description

METHODS AND COMPOSITIONS FOR DELIVERING A PROBIOTIC

FIELD OF THE INVENTION

The present invention is concerned with the delivery of probiotic organisms to animals, including humans.

BACKGROUND TO THE INVENTION

It has been long recognized that certain microorganisms of the gastrointestinal tract are beneficial to the health of animals. Many studies have shown the beneficial effects of ingesting certain microorganisms. Such benefits include relief of diarrhoea, constipation and a reduction of risk in diseases such as osteoporosis, atherosclerosis, dyslipidaemia, cardiovascular disease, obesity and type 2 diabetes.

Accordingly, the prior art provides many compositions which aim to deliver beneficial microorganism directly to the gastrointestinal tract. In that context, the organisms and products containing the organisms are typically referred to as "probiotics".

Probiotics may be administered to animals (including humans) having a need (real, suspected or perceived) for supplementation of gut microflora. Commercially available products such as Inner Balance™ (Swisse), iFlora Multi-Probiotic™ (Sedona Labs), Culturelle Kid's Probiotic™ (Vature) and Protexin™ (a veterninary product of International Animal Health Products, Australia) aim to deliver an efficacious number of beneficial microorganisms to the gastrointestinal tract of an animal. It is generally accepted in the art that literally billions of organisms should be administered to confer maximal health benefit to a subject. Probiotic numbers are typically cited using the unit "colony forming units" (cfu), the value of which is representative of only viable cells. Such viable cells can be dormant and require reactivation which can occur in the gastrointestinal tract. For optimizing health, between 1 billion and 10 billion cfu taken several times in the course of a week is recommended.

Probiotics taken as a dietary supplement must be viable, and therefore be protected from exposure to heat, moisture or air during storage. Furthermore, conditions within the gut may be generally hostile to certain microorganisms, and indeed it is known that many organisms do not survive the passage through the gastrointestinal tract.

l Survival of probiotics has been estimated at between 20% and 40% of all viable organisms present in a composition. Even for those organisms that survive passage, there is a question as to whether they can effectively interact with the gut wall. The prior art suggests that the co-administration of prebiotics with probiotics may assist in improving the viability and physiological activity of probiotics. Prebiotics are non-digestible carbohydrates used for food by probiotic organisms. These carbohydrates support a favourable environment that will benefit the host by assisting in the growth or activity of one or a limited number of bacteria in the colon.

Other approaches have focussed on the physical protection of the organisms using microencapsulation techniques.

Probiotic beverages can be negatively affected by the presence of a preservative (such agents being bacteriostatic or bacteriocidal) such that any living organisms may become non-viable. Furthermore, the presence of sugars in a beverage may lead to fermentation reactions being catalysed by probiotic organisms leading to undesirable changes in beverage composition. A further problem in the art is the palatability or commercial attractiveness of the probiotic compositions of the prior art. Existing beverages are not acceptable to many consumers, and especially children.

It is an aspect of the present invention to overcome or alleviate a problem of the prior art to provide methods and compositions for improving the numbers of viable probiotic organisms that can be delivered to a target site within the gastrointestinal tract and/or improve the activity of a probiotic organism at a target site.

The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application. SUMMARY OF THE INVENTION

In a first aspect the present invention provides a method for delivering a probiotic organism to an animal in need thereof, the method comprising the steps of:

(i) administering the probiotic organism orally to the animal, and

(ii) administering an effervescent agent orally to the animal to cause the generation of an effervescence product within the alimentary tract of the animal,

wherein the probiotic organism and effervescence product co-locate at a site in the alimentary tract of the animal such that viability and/or a physiologic effect of the probiotic is improved relative to a method whereby there is no co-location of an effervescence product and the probiotic organism.

In one embodiment, the effervescent agent consists of, or comprises, a chemical compound capable of releasing a gas upon exposure to moisture.

In one embodiment, the effervescent agent consists of, or comprises, an alkali metal carbonate salt, optionally in combination with an organic acid.

In one embodiment, the effervescence product is a gas

In one embodiment, the gas is carbon dioxide.

In one embodiment, the probiotic organism is a dried preparation. In one embodiment, the step of administering the probiotic substance occurs substantially contemporaneously with the step of administering the effervescent agent.

In one embodiment, the method comprises the further step of administering a prebiotic compound.

In another aspect the present invention provides a dosage unit comprising or consisting of a dried preparation of a probiotic organism and an effervescent agent.

In a further aspect the present invention provides a beverage comprising an effervescent agent, a probiotic, and optionally an effervescence product. Yet a further aspect of the present invention provides a method for treating a condition associated with the level or activity of probiotic bacteria in the alimentary tract, the method comprising

(i) administering the probiotic organism orally to an animal in need thereof, and (ii) administering an effervescent agent orally to the animal to cause the generation of an effervescence product within the alimentary tract of the animal,

In one embodiment, the condition associated with the level or activity of probiotic bacteria in the alimentary tract is a digestive condition, an immune function condition, a wellness condition, or an allergic condition.

In one embodiment, the digestive condition is selected from the group consisting of abdominal bloating, constipation, diarrhoea, and flatulence.

In one embodiment, the wellness condition is fatigue.

In another aspect, the present invention provides a beverage comprising an effervescent agent, a probiotic, and optionally an effervescence product.

In one embodiment, the beverage is based on a consumer beverage. In one embodiment, the beverage is based is based on a fruit juice, a vegetable juice, or a flavoured beverage.

A further aspect of the present invention provides a beverage container comprising

(i) a first region comprising a substantially aqueous liquid, and

(ii) a second region comprising a probiotic substance and an effervescent agent, wherein the container is configured such that the aqueous liquid contacts the probiotic substance and effervescent agent in response to an action of a user of the container.

Further provided by the present invention is a kit of parts comprising a probiotic organism and an effervescent agent. In one embodiment, the kit comprises instructions for the user to (i) contact the probiotic organism and effervescent agent with an aqueous liquid, and to then (ii) ingest the liquid. In one embodiment, the kit comprises a container.

In one embodiment, the kit comprises a prebiotic.

DETAILED DESCRIPTION OF THE INVENTION

After considering this description it will be apparent to one skilled in the art how the invention is implemented in various alternative embodiments and alternative applications. However, although various embodiments of the present invention will be described herein, it is understood that these embodiments are presented by way of example only, and not limitation. As such, this description of various alternative embodiments should not be construed to limit the scope or breadth of the present invention. Furthermore, statements of advantages or other aspects apply to specific exemplary embodiments, and not necessarily to all embodiments covered by the claims. Throughout the description and the claims of this specification the word "comprise" and variations of the word, such as "comprising" and "comprises" is not intended to exclude other additives, components, integers or steps.

Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may.

In a first aspect, the present invention provides a method for delivering a probiotic organism to an animal in need thereof, the method comprising the steps of

(i) administering the probiotic organism orally to the animal, and

(ii) administering an effervescent agent orally to the animal to cause the generation of an effervescence product within the alimentary tract of the animal, wherein the probiotic organism and effervescence product co-locate at a site in the alimentary tract of the animal such that viability and/or a physiologic effect of the probiotic is improved relative to a method whereby there is no co-location of an effervescence product and the probiotic organism. Without wishing to be limited by theory in any way, Applicant proposes that effervescence has a protective role for probiotic organisms during passage through the gut. Greater survival of the microorganisms allows for the delivery of greater numbers of viable cells to any one of more of the stomach, the duodenum, the small intestine, the cecum, the large intestine or the rectum.

A further advantage of greater survival is that the numbers of priobiotic organsims administered may be lower to provide a given number of viable bacteria. This may allow for the use of smaller dosage units, and also lower the cost of production given the need for a lower cfu for a given organism.

Furthermore, and separate to the issue of survival, the effervescence may assist in one or more of the many physiological activities that living probiotic organisms can engage in such as interacting with the mucosal cells of the gut wall, releasing beneficial compounds, sequestering or metabolizing undesirable compounds and the like.

Increasing the interaction frequency between the cell lining the gut and probiotics using an effervescence product such as C0₂, which is preferably in the form of a dissolved gas at a partial pressure higher than would other normally be found in the lumen of the gastrointestinal tract. This effect may also be caused by any other effervescence products produced, such as a sodium acid salt (such as sodium citrate) that is produced in an effervescence reaction. As a conjugate base of a weak acid, sodium citrate may provide a buffering activity in the gastrointestinal tract thereby stabilizing pH and allowing for greater interaction between probiotic and mucosal cells. The effervescence may also provide for an increase the hydrophobicity of the probiotic cell and optimization of the probiotics surface charge, resulting in increased attachment of the probiotic to the gastrointestinal tract lining with consequential improvement in probiotic survival and stimulation of the immune system. By attaching more rapidly to the gut mucosa cells, the probiotic microorganisms survive in higher numbers and are more effective in producing a health benefit to the consumer. It has long been known that probiotic action is potentiated where an ability to adhere to intestinal surfaces is evident. Attachment to mucosa prolongs the time probiotics can influence the gastrointestinal immune system and microflora of the host. Thus the ability to adhere to intestinal surfaces is thought to correspond to the efficacy of the probiotic strain.

The present methods provides for improved immune system function. The intestinal tract is cleansed by probiotics as they attach under the layer of debris about the intestinal walls, thereby dislodging the accumulated decaying material. Facilitation of probiotic-mucosa interactions by the effervescence reaction assists in this attachment.

Attachment of probiotics to the intestinal walls further assists the immune system by producing a mildly acidic environment that slows the growth of disease-causing bacteria. Additional benefits of probiotics come from the fact that they stimulate B-Lymphocyte and related antibody production. They produce large pools of extra antibodies capable of protecting and defending against infection.

Probiotic organisms also assist in inactivating or killing viruses, parasites, yeast and fungi. They also stimulate the body's own production of alpha-interferon, a key regulator of our immune response. Increased survival rates of probiotic organisms as provided by the present methods may potentiate the aforementioned benefits.

A further advantage of the present method is that the effervescent agent may block histamine in the stomach to reduce the amount of hydrochloric produced. This allows greater numbers of probiotics to transverse the upper gastrointestinal tract where stomach acid and bile kills a large proportion of the organisms

The buffering effect of effervescent solutions increases pH of the stomach so that contents of the stomach may be more quickly passed to the small intestines. This again reduces exposure of the probiotics to acids and bile that reduce the viability of the probiotics.

The effervescent reaction may further alter the para-cellular pathway of the intestines and reduces the thickness of the mucus layer lining the intestinal wall. This combined with the surface charge altering and hydrophobicity of the probiotic increasing during the effervescent reaction would cause attraction and attachment or interaction through physical proximity. Such an increased in cellular interaction would stimulate the immune cells such as macrophages and denteric cells found in the gut mucosa, as discussed elsewhere herein. As used herein, the term "probiotic" includes any microorganism known or predicted to be beneficial to animal health when delivered in sufficient numbers. Exemplary organisms include bacteria belonging to the order Lactobacillales, including but not limited to those of the genus Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Enterococcus, Oenococcus, Sporolactobacilius, or Teragenococcus; or a yeast belonging to the order Saccharomyces. The probiotic organism may be of the transient type which do not colonise and grow within the gastrointestinal tract of the subject, or the non-transient type which undergo some replication in the gut. The effervescent agent may be an elemental species, a combination of elementary species, a compound or a combination of compounds. The agent typically consists of, or comprises, a chemical compound capable of releasing a gas upon exposure to moisture. Again without wishing to be limited by theory it is proposed that the release of gas (typically in the form of bubbles) acts by physical (as distinct from chemical) means to improve the viability and/or activity of the probiotic organisms. For example, bubbling may facilitate the mixing of the probiotic organisms with the contents of the gastrointestinal tract (such as semi-digested food). This mixing may shield the organisms from direct contact with stomach acid to at least some extent, thereby improving survival of the organisms through the stomach. Chemical compounds useful as an effervescent agent include alkali metal carbonate salts (such as sodium bicarbonate or sodium carbonate). Such salts are typically used in conjunction with, or formulated with a soluble, food grade organic acid such as citric acid, tartaric acid, malic acid, fumaric acid or adipic acid. A typical effervescent reaction can involve citric acid and sodium bicarbonate as follows;

3NaHC0₃(aq) + ₃C_{6 5}0₇(aq) -> 3H₂0(aq) + 3C0₂(g) + NasCeHsOy (aq)

This reaction suggests that 1 mole of sodium bicarbonate will produce 1 mole C0₂ gas. So if 252 mg sodium bicarbonate reacts completely, there will be 132 mg C0₂ gas, or 67.2 ml C0₂ gas (since STP; standard temperature and pressure, 1 mol of C0₂ is equal to 22.4 L). From this equation, it can be seen that the amount of C0₂ produced is contingent on the amount of bicarbonate present in the presence of adequate amounts of citric acid and moisture.

Bicarbonate in typical effervescent mixtures can range from 5% to 45% by weight. The amount used would depends on the speed required for dissolution to occur. Speed of dissolution is also altered by the formulation into a powder (rapid), granules (slower) and tablets (slower again).

The effervescent agent may simply be a gas (such as carbon dioxide) which is in aqueous solution. The gas can be caused to bubble causing effervescence by, for example, by decreasing the pressure under which the solution is kept. For example, a sealed vessel may contain water having an amount of carbon dioxide dissolved therein. The sealed nature of the vessel maintains the gas in solution (at least to some extent). When the seal is broken (when the lid is removed, for example) the water effervesces due to release of pressure in the bottle. At that point, a dried preparation of probiotic bacteria may be added to the water upon which the effervescing water (along with the probiotic bacteria) is consumed.

Given the benefit of the present specification, the skilled person appreciates that gases can be created in a liquid by various means alternative to those described above. All such means known to the skilled person are included in the ambit of this invention.

Given the benefit of the present specification, it will be apparent to the skilled person that many means exist to facilitate the co-localization off the effervescent agent and probiotic organism at a target site in the gastrointestinal tract. For example, timing of the administration of the two components (more of which is discussed elsewhere herein), the use of tablet coverings to delay the release of the probiotic organisms until a target site in the gastrointestinal tract is reached, means for delaying or facilitating gastric emptying, and like are all potentially useful. In one embodiment, the probiotic organism is administered as a dried preparation which releases viable organisms upon exposure to moisture. Exemplary processes for the production of dried probiotic preparation include spray drying and lyophilisation.

The prior art provides suitable dry dosage forms of probiotics, many of which enable higher bacterial survival. An advantage is that the low water-activity of lyophilized bacterial cells preserve viability in both storage and after administration. Further, by selection of a tablet forming matrix, the entrapped bacteria may be protected against the low pH environment in the stomach. Other parameters that may be modulated to improve bacterial survival in tablets include compression force, matrix forming excipients (such as hydroxypropyl methylcellulose phthalate, HPMCP) or other swelling agents, which may in turn affect the tensile strength of the tablet. The tensile strength of a probiotic organism tablets may increase in HPMCP content. Tablets manufactured with high compression force show a protracted disintegration time and higher bacterial cell viability. The skilled person will be enabled to alter the aforementioned parameters with the aim of providing for an acceptably short disintegration time while still providing for acceptable survival rates. Indeed, given the protective function of the effervescence reaction of the present invention, survival may be less of a consideration with the more important parameter being disintegration time.

Disintegration time may be especially important where the dosage unit is intended to be dissolved in water (or any other beverage) before ingestion. In such instances matrix forming excipients and high compression forces may well be avoided in formulation, with fine granules or even powder formulations being preferred.

The skilled person is familiar with methods of producing effervescent granules, powders and tablets. Given the advantage of using a dried preparation, dry methods such as slugging, direct compression and roller are preferred. The avoidance of avoiding methods requiring water may generally be avoided given the possible negative effect on cell viability and the possibility of reacting with the effervescent agent. However the use of organic solvents may avoid the problems by the use of aqueous liquids in formulation given their ability to evaporate comprehensively, and general inability to initiate an effervescence reaction. It is to be appreciated that the co-localization can be achieved in circumstances whereby the effervescent agent is administered before, contemporaneously with, or after administration of the probiotic organism.

As one example, the effervescent agent (in solid form) is administered firstly, followed by the administration of the probiotic organism. In these circumstances, the delay between the two administrations cannot be so long that there is no co-localization of the effervescent agent and probiotic bacteria. Preferably, the delay in administration is no more than about 1 minute, but in some circumstances may be less than about 2, 3, 4,5, 6, 7, 8, 9 or 10 minutes. As another example, the effervescent agent may be dissolved in a glass of water to commence the effervescence reaction, with the effervescing solution being ingested, and the reaction continuing in the gastrointestinal tract of the subject. Thus, the subject ingests an effervescent agent and an effervescence product (such as carbon dioxide), with the agent being converted to the product within the subject.

In a further example, the probiotic in dried form is first administered, followed by the effervescent agent in solid form. In that circumstance, contact of both the agent and organism with moisture in the gastrointestinal tract will trigger the effervescence reaction, and also the release of viable microorganisms. Water may be swallowed either before or after either or both administration steps to facilitate the effervescence reaction and release of organisms.

In another example, a dried preparation of probiotic organism and a effervescent agent are maintained separately until both are contact with water in a vessel. Upon the commencement of the effervescence reaction, the water is ingested by the subject. The effervescence reaction continues in the stomach, and the probiotic organisms are released into the stomach.

In another example, the effervescent agent and probiotic organisms are formulated into a dosage unit. The single dosage unit is either swallowed, or contacted with water in a vessel. In one embodiment, the method comprises the further step of administering a prebiotic compound. Different probiotic bacteria prefer a different prebiotics as a food source. For example, Bifida bacteria (found mostly in the colon), prefers GOS, a natural probiotic originally found in breast milk. Prebiotic substances such as lactulose and certain fructose containing compounds, pass directly into the cecum without undergoing digestion in the small intestines, thus improving the survival of probiotics crossing the upper part of the gastrointestinal tract. This function of prebiotics enhances the effects of probiotics in the large bowel. The prebiotic may, in one embodiment, be co-formulated into a single dosage unit with either or both of the effervescent agent and the probiotic organism.

In another aspect, the present invention provides a dosage unit comprising a dried preparation of a probiotic organism and an effervescent agent. This form of the invention provides a highly convenient means of delivering the probiotic organisms to a target site in the gut. For example, the dosage unit may be dropped into a glass of water, effervesce and be consumed. Alternatively, the dosage unit may be swallowed, with effervescence occurring due to moisture in the stomach or water subsequently consumed. The single dosage unit may be a capsule or a pellet comprising the probiotic organism and an effervescent agent. Alternatively, it may be a sachet containing dried and powered probiotic organism and a powdered effervescent agent. Having the benefit of the present specification, the skilled person is capable of optimizing any aspect of the method, or any substance used in the method, or any dosage unit to optimize the survival or positive effect of a probiotic organism in the gastrointestinal tract. For example, the species and amount of probiotic may be the subject of routine experimentation

In optimizing the present methods to potentiate probiotic organism survival, the skilled person may implement existing methods for assessing survival. Simulated gastric juices can be used to assess the effect of condition within the gastrointestinal tract on the viability of probiotic organisms. Simulated gastric juice may be formulated using common compounds such as glucose (3.5 g liter^"1), NaCI (2.05 g liter^"1), KH₂P0₄ (0.60 g liter^"1), CaCI₂ (0.1 1 g liter^"1), and KCI (0.37 g liter^"1), adjusted to pH 2.0 using 1 M HCI. Porcine bile (0.05 g liter^"1), lysozyme (0.1 g liter^"1), and pepsin (13.3 mg liter^"1) are added as stock solutions prior to analysis.

It is routine for the skilled person to perform a viability count (in cfu) for bacteria exposed to a simulated gastric juice. Bacterial plating methods using nutrient media are very well known and for the purpose of brevity will not be explained in this specification.

In optimising the present methods to potentiate probiotic cell adhesion to the intestinal mucosa, the skilled person is familiar with methods for determining bacterial adhesion by vivo and in vitro methodologies. Some methods are directed to interactions with the mucus covering the epithelial cells. The mucus covering the epithelial cells is the initial surface that ingested micro-organisms confront in the human gut and is considered an important site for bacterial adhesion and colonisation. Mucus is continually subjected to degradation, conversely new mucin glycoproteins (the major components of mucus) are constantly secreted. Thus, bacteria able to adhere to mucus but unable to reach the epithelial cells might be dislodged from the mucosal surface with the degraded mucin and washed away with the luminal contents. This may partly explain the transient pattern of colonisation characteristic for most probiotic bacteria. On the basis of these remarks, an in vitro evaluation of the bacterial adhesion to human intestinal mucus provides a good model for studying the ability of probiotics to adhere to intestinal surfaces. Mucus for optimizing the present methods may be obtained from the faeces of human subjects, and extracted by ethanol precipitation. The quantitation of the bacterial adhesion to the intestinal mucus may be determined according to an adhesion assay. For example, a solid phase mucus layer is prepared by incubating the clarified mucus suspension on polystyrene microtitre plate wells. The immobilised mucus is then covered with radioactively labelled bacteria, the plates incubated, and the wells washed to remove any unbound bacteria. To release and lyse the adhered bacteria NaOH is added to each well and the plates further incubated. The lysate is removed from the wells, mixed with scintillation liquid and the radioactivity measured by liquid scintillation counting. The proportion of adhered bacteria was assessed as the percentage of radioactivity recovered from the wells as compared to the radiolabeled bacterial suspension.

It is proposed that a further advantage of the invention is that effervescence assists adhesion of probiotic cells and colonisation of area of the gastrointestinal tract including the mouth, the oesophagus, the small intestine, the ascending colon, the transverse colon, the descending colon and the rectum. The skilled person is enabled to assess such adhesion using methods similar to those discussed above. Prior art compositions are not capable, or not as capable of ensuring adhesion and colonization non-gastric regions of the alimentary tract. Many regions of the tract benefit from adhesion and/or colonization of probiotic bacteria.

The administration of the combination of probiotic and effervescent agent may be by way of addition of same to any beverage, such as a dairy-based drink (including milk), a soft drink (or soda), mineral water, fruit juice, vegetable juice, alcoholic drink and the like. Accordingly, in a further aspect, the present invention provides a beverage comprising an effervescent agent, a probiotic, and optionally an effervescence product. Where the beverage is already carbonated, the addition of the effervescent agent may enhance the survival and functioning of the probiotics within the gastrointestinal tract. An advantage of the present invention that beverages having a greater consumer acceptability may be prepared. Probiotic foods & beverages are dominant segments in the global probiotic market, estimated to grow at 6.8% p. a. and reach USD 37.9 billion in 2018. The ability to add a dried probiotic in combination with effervescent agent(s) to an already consumer acceptable beverage (such as a fruit juice, vegetable juice, soda, soft drink, mineral water, alcoholic drink, dairy-based drink, cordial and the like) expands significantly the options by which a consumer may ingest probiotic organisms.. Such advantage is afforded by the provision of a dried probiotic and effervescent agent which may be added to a beverage. Thus, a beverage may already comprise a preservative which may damage a probiotic organism during storage. Addition of the dried probiotic just before consumption would negate any negative effect of a preservative. Moreover, a beverage containing a sugar and probiotic may ferment over time, thereby altering the composition in a negative manner. For example, bitter metabolites may be generated or the pH adversely affected. The addition of the probiotic just before consumption will avoid negative alterations that may otherwise occur where the probiotic organisms are stored with a fermentable energy source. In other embodiments, a consumer acceptable beverage may be generated de novo by including a flavouring agent, sweetener, sugar, aroma agent or any other beverage ingredient or additive in combination with the dried probiotic and effervescent agent. Thus, the consumer adds the combination to water and is immediately provided with an effervescing and flavoursome beverage having the health benefits of probiotics. In that regard, reference is made to the apple flavoured composition described in Example 2 herein.

In another aspect the present invention provides a beverage container comprising:

(i) a first region comprising a substantially aqueous liquid, and

(ii) a second region comprising a probiotic substance and an effervescent agent, wherein the container is configured such that (a) the aqueous liquid contacts (b) the probiotic substance and effervescent agent in response to an action of a user of the container.

A useful beverage container may comprise a blister pack (the second region) which is rupturable such that the contents (an effervescent agent and a probiotic organism) are dropped into a vessel (the first region) disposed underneath and containing an aqueous liquid such as water.

In one embodiment the blister pack is characterized by a rupturable base and a cover of a formable material, the base and cover defining at least one pocket which accommodates a probiotic organism and an effervescent agent. The blister pack is adapted be sealingly fitted over the neck portion of a container. In use, the vessel includes water and the user applies a force to the cover sufficient to rupture the rupturable base such that the probiotic organism and effervescent agent are caused to drop into the container thereby contacting the aqueous liquid.

In one embodiment the opening is associated with a neck portion of said container, said base extending across the opening such that an outer perimeter thereof is fixed to the neck and acts as an anti-tamper seal.

In one embodiment the base ruptures when a minimum amount of force is applied on said cover, causing said element to fall into the container body.

In one embodiment the seal between the outer perimeter of the base and the container neck is such that rupture of said base to allow for said element to be added does not cause said seal to break. In one embodiment the seal is such that the blister pack can be manually detached from the neck of the container.

In one embodiment the blister pack includes tabs which, when pulled, break said seal. In one embodiment the container further includes a lid that is removably attachable from said neck such that when the lid is attached it envelops said blister pack.

In one embodiment the lid includes a means of sealing said opening when the blister pack has been removed.

In one embodiment the base is of a lower burst strength than said cover. In one embodiment the base is of aluminum.

In one embodiment the cover is of thermoformable or coldformable plastic.

In one embodiment the rupturable base is porous and adapted to diffuse oxygen through said blister pack.

A particularly suitable container is described in published international patent application WO 2012/0451 16, the content of which is incorporated herein by reference in its entire. In another aspect the present invention provides a kit of parts comprising a probiotic organism and an effervescent agent. Optionally the kit further comprises a prebiotic substance. In one embodiment, the kit provides comprises instructions for the user to (i) contact the probiotic organism and effervescent agent with an aqueous liquid, and to then (ii) ingest the liquid.

In one embodiment, the kit provides instructions for the user to ingest the probiotic organism and the effervescent agent. Preferably, the instructions are for the user to ingest the probiotic organism and the effervescent agent contemporaneously.

The kit comprises a container, into which an aqueous liquid may be disposed, and into which a probiotic and an effervescent agent may be added. The container may comprise instructions as described above.

The kit may further comprise a prebiotic substance.

The present invention will be now more fully described by reference to the following non- limiting examples.

EXAMPLES

Example 1 : Dosage Unit A dosage unit of the present invention was formulated by Ferngrove Pharmaceuticals (South Granville, NSW, Australia). Tables 1 and 2 show formulation details of a preferred dosage unit. The dosage unit contained 3 strains of probiotic (L Acidophilus, L Casei and B bifidum). Sodium bicarbonate and citric acid were used as the effervescent agent (with carbon dioxide being generated, in use, as an effervescence product). Excipients including inulin, lactose and colloidal silica were added, to provide a 4g dosage unit presented as a granular powder.

When added to water contained in a vessel, the dosage unit effervesced to provide a pleasant tasting beverage. Example 2: Clinical Study

A study to examine the effectiveness of effervescent probiotic compositions was carried out by a registered Australian Health Practitioner.

This study was predicated at least in part on accepted dogma that gut health is important not only to disorders of the gut per se, but also in broader health considerations such as general wellness including fatigue and allergies.

Medically and scientifically, gut tissue represent almost half of the body's total immune system. It is now accepted that the normal flora of the gut plays a large role in overall gut health. Probiotics have thus been accepted as a nutritional tool for improving gut health , and also general health. Poor gut health is also associated with poor immune functionality.

Symptoms associated with poor gut health include flatulence, bloating, irregular bowel movements, irregular faecal morphology (diarrhoea, constipation), fatigue, skin allergies, changes in digestive functions etc. Patience with such gastrointestinal symptoms, often consult traditional and/or alternative health practitioners.

Treatment of poor gut health, often involves changes to a patient's diet and furthermore, supplementation with probiotics.

To test the effectiveness of effervescent probiotics, a clinical research protocol was set up using patients of a clinic who suffered from simple gastrointestinal symptoms such as those mentioned above. A questionnaire was completed by the treating practitioner relating to patient gut symptoms. In particular, abdominal bloating, incidence and severity of flatulence, bowel movements (regular or not), appetite scale, allergies, constipation/diarrhoea, and fatigue. Patients suffering from gut symptoms were asked to volunteer in the trials and were asked to take a white powder after dissolving it in water or juice. The powders given to the patients was white and tasteless and contained two probiotic strains. These were: Lactobacillus rhamnosus, and Bifidobacterium lactis (See table below, probiotic formulation). Ingredient Amount w/w%

(grams)

Base - dried probiotic 3.55 88.75

Flavour MTP-008553 0.16 4.00

Apple

Citric Acid 0.14 3.50

Sucrulose 0.15 3.75

TOTAL 4.00 100

Table: Probiotic mixture composition. Base as supplied either contained effervescent chemicals or no effervescent chemicals. The total 4 grams was added to water, and the composition consumed while effervescing. Patients were administered either effervescent probiotics or probiotic powder that did not have effervescence. Both doses supplied 5 billion active colony forming units (CFU) each time it was consumed, as determined by Ferngrove Pharmaceuticals (Sydney Australia; suppliers of the probiotic powders).

A total of 30 patients were examined in these clinical trials. Half of the patients were adminsitered non-effervescent probiotics and the remaining half were administered effervescent probiotics. The probiotic dose was given once daily on an empty stomach before breakfast and the patient was asked to revisit the Health Practitioner every two weeks for reassessment and re-evaluation of the gastrointestinal symptoms initially recorded on the questionnaire. Clinical trials were concluded after 3 months of probiotic treatment. Clinical Effectiveness of effervescent probiotics

Questionnaire responses were analysed and symptoms which had responded positively with the probiotic treatment were tabulated (table 2). To compare the effectiveness of effervescence when used with probiotics, a score was given initially to each symptom for the following symptoms: Abdominal bloating, constipation, diarrhoea, bowel regularity and fatigue (Table 2).

The initial severity of the symptoms was always given a score of 10 and this reduced subjectively as perceived by the patient. A score of 0 meant that the gastrointestinal symptom had completely resolved. The recorded score was an average of the score given by each participant with that symptom. Not all patients had all symptoms at the commencement of the study. Finally, to determine the effectiveness of effervescence in the relief of gastrointestinal symptoms, the scores for each symptom at two weekly interviews was added and divided by the number of symptoms being examined. A lower score represented an improvement of gastrointestinal health.

From the table below, it appears that both effervescent and non-effervescent probiotic powders helped improve the symptoms associated with an unhealthy gut. Interestingly however, effervescence in the same probiotic formulation helped relieve the symptoms quicker (week 2) and more effectively (week 12)

Table: Effect of Effervescence on the effectiveness of probiotic powders in relieving gastrointestinal symptoms Example 3: Beverage Container

Reference is now made to Figs. 1 to 5 which show diagrammatically a beverage container of the present invention 10 adapted to deposit a tablet comprising probiotic organisms and an effervescent agent into a beverage, whereby the user unscrews the lid 12 from the beverage container 10, which may have a typical tamper evident sealing ring on the lid (not shown). With the lid 12 removed, the user is presented with the blister pack 14, which contains a dried probiotic organism preparation, sodium bicarbonate and tartaric acid all co-formulated into a readily dissolvable tablet 16. The blister pack has instructions printed on the cover 18 instructing the consumer to press the tablet 16 into the beverage 28.

When force is exerted onto the cover 18, preferably by a thumb or some other manual means, it pushes against the tablet 16, forcing the rupturable base 20 to break as illustrated in Figure 2. Once sufficient force is exerted to push the table 16 completely through the base 20, the tablet 16 falls into the container 10 as illustrated in Figure 5.

The cover 18 is therefore indented into the neck of the container, however because of the durability of the material and the seal to the container neck 22 the cover 18 does not rupture or come away from the container 10, but remains sealed. This allows the user to twist or shake the container 10 to mix the beverage 28 with the tablet 16 without having to be cautious that the liquid may spill and allows the tablet 16 to be at least partially dissolved into the beverage 28 before consumption. Fig. 3 shows rupture of the rupturable base 20, allowing the tablet 16 to enter the container.

Fig. 4 shows the tablet 16 dissolving in the beverage. The bicarbonate and tartaric acid commence an effervescence reaction with the beverage to release viable probiotic organisms into the beverage.

When the user is ready to drink the beverage, the rupturable base is completely removed from the container (Fig. 5).

It will be appreciated that in the description of exemplary embodiments of the invention herein, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof, for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects. This method of disclosure, however, is not to be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment. Thus, the claims following the Detailed Description are hereby expressly incorporated into this Detailed Description, with each claim standing on its own as a separate embodiment of this invention.

Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those skilled in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination.

In the description provided herein, numerous specific details are set forth. However, it is understood that embodiments of the invention may be practiced without these specific details. In other instances, well-known methods, structures and techniques have not been shown in detail in order not to obscure an understanding of this description.

Thus, while there has been described what are believed to be the preferred embodiments of the invention, those skilled in the art will recognize that other and further modifications may be made thereto without departing from the spirit of the invention, and it is intended to claim all such changes and modifications as falling within the scope of the invention.

Claims

CLAIMS:

1. A method for delivering a probiotic organism to an animal in need thereof, the method comprising the steps of

(i) administering the probiotic organism orally to the animal, and (ii) administering an effervescent agent orally to the animal to cause the generation of an effervescence product within the alimentary tract of the animal, wherein the probiotic organism and effervescence product co-locate at a site in the alimentary tract of the animal such that viability and/or a physiologic effect of the probiotic is improved relative to a method whereby there is no co-location of an effervescence product and the probiotic organism.

2. The method of claim 1 wherein the effervescent agent consists of, or comprises, a chemical compound capable of releasing a gas upon exposure to moisture.

3. The method of claim 1 or claim 2 wherein the effervescent agent consists of, or comprises, an alkali metal carbonate salt, optionally in combination with an organic acid.

4. The method of claim 1 or claim 2 wherein the effervescence product is a gas

5. The method of claim 2 wherein the gas is carbon dioxide.

6. The method of any one of claims 1 to 3 wherein the probiotic organism is a dried preparation.

7. The method of any one of claims 1 to 4 wherein the step of administering the probiotic substance occurs substantially contemporaneously with the step of administering the effervescent agent.

8. The method of any one of claims 1 to 6 comprising the further step of administering a prebiotic compound.

9. A method for treating a condition associated with the level or activity of probiotic bacteria in the alimentary tract, the method comprising

(i) administering the probiotic organism orally to an animal in need thereof, and (ii) administering an effervescent agent orally to the animal to cause the generation of an effervescence product within the alimentary tract of the animal, wherein the probiotic organism and effervescence product co-locate at a site in the alimentary tract of the animal such that viability and/or a physiologic effect of the probiotic is improved relative to a method whereby there is no co-location of an effervescence product and the probiotic organism.

10. The method of claim 9 wherein the condition associated with the level or activity of probiotic bacteria in the alimentary tract is a digestive condition, an immune function condition, a wellness condition, or an allergic condition.

1 1. The method of claim 10 wherein the digestive condition is selected from the group consisting of abdominal bloating, constipation, diarrhoea, and flatulence.

12. The method of claim 10 wherein the wellness condition is fatigue.

13. A dosage unit comprising or consisting of a dried preparation of a probiotic organism and an effervescent agent.

14. A beverage comprising an effervescent agent, a probiotic, and optionally an effervescence product.

15. The beverage of claim 14 that is based on a consumer beverage.

16. The beverage of claim 14 or claim 15 that is based on a fruit juice, a vegetable juice, or , a flavoured beverage.

17. A beverage container comprising (i) a first region comprising a substantially aqueous liquid, and

18. A kit of parts comprising a probiotic organism and an effervescent agent.

19. The kit of claim 16 comprising instructions for the user to (i) contact the probiotic organism and effervescent agent with an aqueous liquid, and to then (ii) ingest the liquid.

20. The kit of claim 16 or claim 17 comprising a container.

21. The kit of any one of claims 16 to 18 comprising a prebiotic.