AU2007201813A1 - Probiotic bacterium: Lactobacillus fermentum - Google Patents

Description

S&F Ref: 752294AUD1 k e,

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT Name and Address of Applicant Actual Inventor(s): Address for Service: Invention Title: Probiomics Limited, an Australian company, ACN 084 464 193, of Suite G09, 1 Central Avenue, Australian Technology Park, Eveleigh, New South Wales, 1430, Australia Patricia Lynne Conway Spruson Ferguson St Martins Tower Level 31 Market Street Sydney NSW 2000 (CCN 3710000177) Probiotic bacterium: Lactobacillus fermentum The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845c(766323 1) -1-

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SProbiotic bacterium: Lactobacillus fermentum TECHNICAL FIELD The present invention relates to variants of the bacterium Lactobacillusfermentum, formulations of the variant and components thereof, and their use in preventing and/or treating disease in mammals and promoting mammalian health.

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BACKGROUND

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CAny discussion of the prior art throughout the specification should in no way be O considered as an admission that such prior art is widely known or forms part of common 10 general knowledge in the field.

Indigcnous bacteria play a major role in preventing certain bacterial and fungal diseases. They do this by a process of bacterial antagonism, preventing other microbes from establishing a presence in the body. Mechanisms involved in this activity include direct competition for nutrients, alterations in the acidity of an area making it hostile for other microbes, producing inhibitory metabolites and anti-microbial chemicals and by preventing other microbes from attaching to host surfaces.

Indigenous flora also influences the immune system. Animals reared in genn-free environments exhibit underdeveloped and relatively undifferentiated lymphoid tissues, low levels of immune proteins and a primary response to infection rather than a pronounced secondary response.

These microbes play an important role in the health of the host, by producing essential vitamins and nutrients required by the colonocytes, assisting with the degradation of complex nutrients, protecting the host from invasion by pathogens and stimulating the immune system. In addition, it has been proposed that the microbes in the bowel contribute to mineral absorption and lipid metabolism because of the lowered pH as a result of the short chain fatty acids produced by the microbes. These short chain fatty acids are regulators of colon physiology and play an important role in maintaining normal bowel function.

The upper gastrointestinal tract contains only bacteria swallowed with the saliva and food. As a result of the high acidity of gastric juices, few organisms, mostly lactobacilli, can be cultured from the normal stomach. The upper small intestine contains relatively sparse numbers of lactobacilli and enterococci. The flora of the gastrointestinal tract gradually changes until it becomes similar to that found in the Scolon, predominantly Bacteroides, Bifidobacteriun, Fusobacterium, Lactobacillus and SEubacterium.

This normal flora of the body is a complex ecosystem, which is regulated by the diet, microbial interactions and host factors such as the motility of the gut and intestinal secretions. External factors such as stress, dietary changes and medications can affect the normal flora and alter the types of organisms present or their metabolism. Lfthe Sbalance is disrupted it can be detrimental to the host and can cause disease.

Sometimes, however, the balance is lost and invading pathogens are successful in penetrating the body's defences causing infections or triggering inappropriate immune responses. Similarly the balance between host and indigenous flora can be compromised CI leading to infection by normally dormant organisms, e.g. episodes of thrush (Candida albicans infection) or Clostridium diicile infection following the use of antibiotics.

Disappointingly, the "magic bullets" that antibiotics apparently offered have lost some of their impact. In the last 30 years, an ever-increasing problem of antibiotic resistance has becomne a major public health issue. Today, some antibiotics are all but useless against certain microbes, while some bacteria are resistant to almost all known antibiotic medications.

Further, evidence is accumulating that the indigenous microbes of the large bowel may be implicated in irritable bowel syndrome (IBS), a condition characterized by changes in bowel habit, disordered defaecation and distension.

Clearly alternative and perhaps complementary methods of treatment are required.

It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

SUMMARY OF THE INVENTION A new probiotic bacterial variant of Lactobacillusfermentum having surprising advantageous features over pre-existing strains of this bacterium as well as other probiotic microorganisms has been isolated. It is particularly useful in the prevention and/or treatment of gastrointestinal disorders due to its advantageous ability to colonise the gastrointestinal tract. Further, this variant has advantageous immunomodulatory effects, which occur both locally in the gut and at mucosal sites throughout the body via the common mucosal immune system. As a result of the commonality of the mucosal system the variant can also be used in the prevention and/or treatment of other disorders S-3-

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Sofmucosal surfaces and conditions resulting from mucosal surface disturbances or Sdisturbances in the immune system or status of a subject.

According to a first aspect, there is provided a novel Lactobacillusfermentnn variant having the following characteristics: it is gram positive, facultative rod, c it ferments ribose, galactose, glucose, fructose, mannose, maltose, lactose, 00 melibiose, sucrose, trehalose, raffinose, L-arabinose and mannitol, it survives at pH 1.5 for at least four hours with a loss in growth of no more than log3, it propagates in the presence of 0.5% bile salts at 37 C resulting in an optical C density increase of 0.5 to 0.8, it is stable with storage in gelatin capsules at 25 C, it adheres to Peyer's Patches at greater than log5' cfu per mg tissue, inhibiting pathogens both by a direct antagonistic effect and by triggering an immune modulation; or a component thereof.

Preferably, the Lactobacillus fermentun variant strain is VRI 003 (accession number NM02/31074).

Preferably, the component is a cell fragment, extract, secretion or purified component.

According.to a second aspect of the invention, there is provided a composition comprising a Lactobacillusfermentum variant or a component thereof according to the first aspect and a pharmaceutically acceptable carrier.

Preferably, the composition comprises strain VRI 003 or a component thereof. In a preferred embodiment, the composition comprises live cells of the Lactobacillus fermentum variant. However, it will be clear to the.skilled addressee that the compostion may also contain dead cells of the Lactobaoillus variant. In one embodiment, the composition comprises a component that is a cell fragment, extract, secretion or a purified component.

Procedures for preparing such fragments, extracts, secretions or purified components are well known. For example, the cells can be sonicated or the cell wall itself can be physically disrupted and/or the cell contents collected. Whole cells can be S-4- O washed to extract surface components and these extracts can then be fractionated if required.

<r Preferably, the Lactobacillusfermentum is combined with other compounds such as prebiotics, non-digestible dietary components, dietary fibre or pharmaceutically active compounds. More preferably, the prebiotic comprises or consists of imnlin, a resistant Cc starch, an oligosaccharide, a gum or a beta-glucan. Even more preferably, the prebiotic 00 is an unmodified high amylose maize starch or a beta-glucan.

The composition may be prepared as a tablet, capsule, powder, gel, paste, liquid formulation, dietary supplement or a food product and the like. Preferably, the composition is prepared in a tablet or capsule form C1 According to a third aspect, the invention provides a method for the prevention and/or treatment of a gastrointestinal disorder or a symptom thereof, comprising the step of administering a Lactobacillusfermentum variant or a component thereof according to the first aspect, or a composition according to the second aspect, to a subject in need of such treatment.

Preferably the Lactobacillusfermentum is the variant VRI 003.

The terms "subject" and "individual" are used interchangeably in this specification and in the context of the present invention include within their scope any mammal which can develop, or already has, a gastrointestinal disorder and/or disorders ofmucosal surfaces and/or conditions resulting from mucosal surface disturbances or disturbances in the immune system of whatever cause. The preferred subjects for administration of the treatment of the present invention are humans, domestic pets and farm animals.

Preferably, the gastrointestinal disorder is irritable bowel syndrome (IBS), inflammatory bowel disease, Crohn's disease and/or symptoms thereof, such as for example diarrhoea, bloating, flatulence, abdominal cramping, abdominal pain, or constipation. The gastrointestinal disorder may be caused by pathogenic organisms that may be bacterial, viral or protozoan. However, it may also be caused merely by colonisation of the gastrointestinal tract with inappropriate organisms or by inflammatory and/or autoimmune mechanisms.

Preferably, the disturbance or disorder is the result of colonisation of the subject's gastrointestinal tract by a pathogen. Preferably, the disturbance or disorder is the result of a high pathogen load as herein defined. More preferably the pathogen is a bacteria, virus or protozoa. Most preferably, the pathogen is Salmonella, E. coli, Helicobacter,

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C1 Vibrio, Pseudomonas, Clostridium, bacteroides; or a virus such as Norvalks or Q, rotavirus, or a protozoan such as Cryptosporidium, Entamoeba, Giardia and Dientamoeba.

In the context of the present invention, a "high pathogen load" may occur when a subject has been subjected to attack by a pathogen in an amount not within the Cm normal range of their everyday exposure; been subjected to attack by a virulent F 00 pathogen; been subjected to attack by a pathogen at a time when the subject's O resistance is lowered, for example, at a time when the immune system is depleted and/or when the subject's other natural defence mechanisms are not functioning normally. The subject's resistance may be low due to, for example, stress or antibiotic teatment.

In a fourth aspect, the invention provides a method for preventing and/or treating a disorder of a mucosal surface or a symptom thereof, comprising the step of administering a Lactobacillusfermentum variant or a component thereof according to the first aspect, or a composition according to the second aspect to a subject requiring such treatment.

Preferably, the disorder of the mucosal surface is eczema roseaca or atopic dermatitis.

In a fifth aspect, the invention provides a method of stimulating IL-12 production in a subject, comprising the step of administering an effective amount of a Lactobacillus fermentum variant or a component tberof according to the first aspect, or a coniposition according to the second aspect, to a subject in need of such treatment.

In a sixth aspect, the invention provides a method of up-regulating IFN-y comprising the step of administering an effective amount of a Lactobacillusfernentunm variant or a component thereof according to the first aspect, or a composition according to the second aspect, to a subj ect in need of such treatment.

In a seventh aspect, the invention provides a method of inducing a Th 1-type response in a subject, comprising the step of administering an effective amount of a SLactobacillusfermentum variant or a component thereof according to the first aspect, or a composition according to the second aspect, to a subject in need of such treatment.

In an eighth aspect, the invention provides a method of inhibiting the growth of a pathogen, comprising the step of administering an effective amount of a Lactobacillus fermentun variant or a component thereof according to the first aspect, or a composition according to the second aspect, to a subject in need of such treatment.

In a ninth aspect, the invention provides a method of modulating indigenous p microbes of the gastrointestinal tract, comprising the step of administering an effective amount of a Lactobacillusfermentum variant or a component thereof according to the Sfirst aspect, or a composition according to the second aspect, to a subject in need of such treatment.

c The required dosage amount will vary according to the severity of the condition to 00 be treated, the cause of the condition, age of the subject and other standard clinical O parameters which can be easily determined by routine procedures within the skill set of those skilled in the art.

The Lactobacillusfermenitum variant, or a component thereof or the composition comprising said variant, may be administered by any known means but preferably it is administered orally.

The Lactobacillusfermentum variant, or the composition comprising said variant or a component thereof, may be administered in conjunction with one or more other pharmaceutically active agents. The variant, or the composition comprising said variant, may be administered simultaneously (co-administered) with the other treatments or it may be administered sequentially in any order.

It is preferred that the Lactobacillusfermentum variant or a component thereof or a composition comprising it, be administered daily. It can be administered several times per day, or it may be administered infrequently (for example every second or third day), depending on the progress of treatment of the condition in question, its cause and severity. These parameters can also be easily determined by those skilled in the art.

In the context of the present invention, the term "stable" includes within its scope a loss in viability of up to 25% per six months' storage at 25 C.

In the context of the present invention, the term "component" as it applies to components of the Lfermentum of the invention includes but is not limited to cell fragments, extracts, secretions and purified agents derived from the bacterium.

Unless the context clearly requires otherwise, throughout the description and the claims, the words 'comprise', 'comprising', and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".

13RIEF JDFSCRIM'ON OF Mil FEGURES Figure 1. Effect on different concentrations of bilo salts on growth and viability of Lfernentum VRI 003 1iigure 2. The rffect of low pH on viability of ViRI 003 culture in Nitro.

Figure 3. Adhesion of Lctctobacillus spp to mouse Peyer' s patches 0C) Figures 4 and 5. A two-dimensional analys'is of the cell wall proteins exjtracted from Lfermnentunz VRI 003 cells compared to the cell walls of Lfernentwn LMG, previously shown not to adhere to the Peyer' s patches, shows that the VRI 003 upregulates two cell wall proteins and downregulates the expression of a number of (71 other cell wall proteins, (Figure 4) when compared to Lfermentwn LMG (Figure Figure 6. Effect of Lfenientum VRI 003 stiimulation on cytokine production by macrophages.

Figure 7. Enhancement of cytokine levels in the Peyer's patches following oral dosing of Lferenturn VRI 003.

Figure 8. Effect of oral administration ofLfertnenturn VRI 003 on cytokine levels in the spleen following oral dosing of Lferrenturn VIRI 003.

Figure 9. Stability of Lfeinentum VRI 003 in Size I gelatin capsules combined with low water activity resistant starch and stofed in foil-foil blister packs at 4C, and Figure 10. Changes in bacteria in faecal material from LBS subject #4 during an 8week trial. icrobial profiles monitored during the baseline, placebo, washout and synbiotic treatment periods. Results etkpressed as colony forming units (cfiu) per tnL.

Figure 11. Changes in bacteria in faecal material from lBS subject #5 during an 8week* trial. M icrobial profiles monitored during the baseline, placebo, washout and synbiotic: treatment periods. Results expressed as colony forming units (cfu) per mL.

Figure 12. Changes in bacteria in faecal material from LBS subject #9 during an 8week trial. Microbial profiles monitorod during the baseline, placebo, washout and synbiotic treatmeat periods. Results expressed as colony forming units (cf4) per mL.

Figure 13. Changes in bacteria in faecal material fr-om lM subject #2 during ani 8week trial. Microbial profiles moniitored during the baseline, placebo, washout and synbiotie treatment periods. Results expressed as colony forming units (cfa) per mL.

Figure 14. Changes in bacteria in faecal material from IBS subject during an S..

week trial, Microbial profiles monitored during the baseline, placebo, washout* and synbiotic treatment periods. Results expressed as colony fomiing units (cfu) per rnL.

Figure 15. Changes in bacteria in faecal material from IBS subject #7 during an Sweek trial. Microbial profiles monitored during the baselineQ, placebo, washout and synbiotic treatment periods, Results expressed as colony fouming units (cfu) per nffL.

00 Figure 16. Changes in bacteria in faecal material fiomJIBS subject 116 during at 8we-ek trial. Microbial profiles monitored during the baseline, placebo, washout and synbiotic treatment periods. Results expressed as colony forming units (cfu) per niL.

c-i DETAILED)DESCRIPTION OF TIE INVEi NTION Although there have been various attempts to use probiotics in the prevention and treatment of many disorders, the evidence for the use of probioti~s in the treatment of gastrointestinal disorders Bas been vry variable. It has now been found that a Lactobazcillusfernentunz variant having the following characteristics: it is a Gram positive, facultative rod, it ferments ribose, galactose, glucose, fructose, mannose, maltose, lactose, melibiose, sucrose, trehalo~e, raffinose, L-arabinose and mannitol, it survives at pH 1.5 for at least four hours with a loss in growth of no more than log 3 it propagates in the presence of 0.5% bile salts at 37 C resulting in an optical density increase of 0.5 to 0.8, it is stable with storage in gelatin capsules at 25 C, it adheres to Peyer's patches at greater than log5 cfu per mg tissue, inhibiting pathogens both by a direct antagonistic effect and by triggering ana imrmune modulation; or components thereof, is/are highly effective in the prevention and/or treatment of a gastrointestinal disorder and/or symptoms thereof. Preferably, the variant is Lactobacillusfrmzentuni VIRI 003. The present invention also provides a method for the pievention and/or treatment of a gastrointestinal disorder comprising the administration of a Lacro1,acillusferrnenturn vaiant or a component thereof, or a composition comprising the variant to a subject. The variant can be combined with other agents, for -9example, a prebiotic, a non-digestible dietary component, dietary fibre or a pharmaceutically active compound such as aspirin and statins.

The methods and compositions of the present invention have been developed for human and veterinary applications in the treatment of gastrointestinal disorders, but as a result of the commonality of the mucosal system, the treatments may be applied to other disorders of mucosal surfaces and conditions resulting from mucosal surface 00 disturbances or disturbances in the immune system or status of a subject. Whether used for treatment of humans or domestic animals, the underlying principles are the same and advantageously the treatments of the present invention may be used irrespective of the 10 cause of the conditions described above.

C Typically, the effective daily dosage is in the range of about 108-1012 bacteria and frequency of administration is once or twice daily. For long-term intake the amount may, for example, be below the above-mentioned range; and in other circumstances, it can be used at an amount above the range.

The Lactobacillusfermentum variant can be formulated by known means, using conventional pharmaceutically acceptable carriers, excipients, solvents or adjuvants.

Such procedures and ingredients are well known and are amply described in standard texts and manuals, for example "Remington: The Science and Practice of Pharmacy", 1995, Mack Publishing Co. Easton, PA 18042, USA, which is incorporated herein by reference.

The Lactobacillusfermentum variant or components thereof may also be formulated into a food product by the usual well-known means.

The composition comprising the bacterium includes viable bacteria, wet bacteria, dried bacteria or components of the bacterium including but not restricted to cell fragments, extracts, secretions and purified components.

The food or drink product of the invention contains at least one of the bacterium, a material containing the same and a processed product thereof as the effective ingredient.

A composition can be formulated to be suitable for oral administration in a variety of ways, for example in the form of a tablet, a capsule, a liquid, a dietary supplement, a paste, a gel, a food product and the like. Other formulations will be readily apparent to one skilled in the art.

The bacterium can be used in food or drink products or can be used in combination with other food materials and food components appropriately in conventional manners.

SPreferably, the composition is prepared as a dairy or dairy-based food product with or Swithout other components that are routinely used in the production of such dairy products.

The compositions of the present invention may also include known antioxidants, buffering agents, and other agents such as coloring agents, flavorings, vitamins or cminerals. Thickening agents may be added to the compositions such as corn starch, guar 00 gum, xanthum gum and the like. Preferred additional components of a therapeutic composition of this invention can include prebiotics such as inulin, non-digestible dietary components, dietary fibre, pharmaceutically acceptable compounds and other nutrients. Dietary or supplementary enzymes such as lactase, amylase, glucanase, c catalase, and the like enzymes can also be included. Preferred prebiotics include unmodified high amylose maize starch or beta-glucan.

The bacterium is combined with a carrier which is physiologically compatible with the gastrointestinal tissue or mucosal surface of the species to which it is administered.

Carriers can be comprised of solid-based, dry materials for formulation into tablet, capsule or powdered form; or the carrier can be comprised of liquid or gel-based materials for formulations into liquid or gel forms. The specific type of carrier, as well as the final fornulation depends, ir part, upon the selected route(s) of administration.

Typical carriers for dry formulations include, but are not limited to: trehalose, malto-dextrin, rice flour, micro-crystalline cellulose (MCC) magnesium sterate, inositol, FOS, GOS, dextrose, sucrose, and like carriers, Dry formulations powders) may be added to supplement commercially available foods liquid formulas, dairy products, or water). Similarly, the specific type of formulation depends upon the route of administration.

Suitable liquid or gel-based carriers include but are not limited to: water and physiological salt solutions; urea; alcohols and derivatives methanol, ethanol, propanol, butanol); glycols ethylene glycol, propylene glycol, and the like).

Preferably, water-based carriers possess a neutral pH value pH Preservatives may also be included within the carrier including methylpaiaben, propylparaben, benzyl alcohol and ethylene diamine tetraacetate salt. The composition of the carrier can be varied so long as it does not interfere significantly with the pharmacological activity of the active ingredients.

-11- The methods of the present invention comprise administration of the Lactobacillus fermentum variant or components thereof a composition comprising the variant or components thereof, to a human or animal to treat and/or prevent a gastrointestinal disorder or a disorder of a mucosal surface and conditions resulting from mucosal surface disturbances or disturbances in the immune system or status of a subject and/or M the symptoms associated with such disorders, for example, irritable bowel syndrome 00 (IBS), inflammatory bowel disease and/or symptoms thereof, such astor example C diarrhoea, bloating, flatulence, abdominal cramping, abdominal pain, or constipation, and eczema or rosacea. This treatment can result in a modulation of the indigenous 10 microbes of the gastrointestinal tract. Administration is preferably carried out using a C tablet, a capsule, a gel, a liquid formulation, a powder, a paste, a dietary supplement or a food product and the like formulation, all formulated to contain a therapeutic composition of the present invention by use of methods well-known within the art.

Preferred embodiments of the invention will now be described by way of example only.

EXAMPLES

Example 1: Origin and Identification The VRI 003 variant was isolated from a healthy subject. In a series of laboratory experiments, the VRI 003 variant was found to adhere to the gastrointestinal epithelial tissue. It was also shown to have a demonstrable effect on human gastrointestinal pathogens, and was resistant to bile acids. The VRI 003 variant also survived in a low pH environment and was resistant to pepsin and to nutrient limited conditions.

The bacterial variant VRJ 003 can be cultured on Rogosa agar (Oxoid) Plates were incubated at 37 C in an anaerobic chamber for 24 hours. The strain was purified by successive transfers on MRS agar (Oxoid) plates incubated at 37 C in an anaerobic chamber for 24 hours and the final culture stored at -70 0 C in 20% glycerol by subculturing in MRS broth at 37 C for 24 hours in anaerobic conditions prior to the addition of glycerol to the culture broth.

Variant VRI 003 was a catalase negative, Gram positive rod which produced gas when grown anaerobically on brain heart infusion (BHI) (Oxoid) broth containing glucose. It was broadly identified therefore as an heterofermentative lactobacillus and confirmed to be L. fermentum strain according to the API carbohydrate kit (50 CHL kit; Biomerieux; supplier data base and database with a certainty of 99.9%. In particular, -12- 10, 11, 12, 13, 25, 28, 29, 30, 31, 32 and 25 axe utiized by strain VRlo003.

The cell is a short rod, Gram posiltive and consistent with the description of the morphology of 1actobzcilusferinentum.

(71 Lactobcilusferienztin strain VRI 003 was deposited under the provisions of the Budapest Treaty, at the Australian Government Analytical Laboratories, PO BOX 385 Pymble 2073, NSW, Australia, on 27 August 2002 and the deposit was allocated the 00 accession number NMO2/3 1074.

Example 2: Characteiristics and Description of Strain VRI 003 Colony morphology When grownuon MRS (Oxoid) agar in an anaerobic chamber at 37 C, the colonies are approximately Imm in diameter, shiny, dome shaped opaque and when touched with a loop show a stickiness. This stickiness may be due to the presence of an extracellular polymer. Incubation of similar plates in aerobic conditions at 37 C yields.

some colonies of rough appearance and irregular edge. Subculftuing of these rough colonies onto MRS agar and incubation at 37 C in the anaerobic chamber yields opaque.

dome shaped, shiny opaque colonies that are sicky to touch.

(Ri) Growth in Broth The culture of variant VRI 003 in MRS (Oxoid) broth, or in various other broths, results in a viscous broth when grown at 370C in anaerobic conditions..

(ill) Resistancc to bile acids As shown in Figure 1, strain NWR 003 grows in the presence of 0.51% and 0. 15% bile salts wvhen acids were added to MRS broth (Oxoid) and the strain incubated anaerobically at 37 C for 8 hours.

(iv) Suxrvival in low p11 envirounient VRI 003 from glycerol stock was passaged twice in MRS broth before use for the assay.

On day of assay the cultures were harvested from the NMS broth by centxifigation at 4000xg and pellet was diluted in Clark and Lu bs buffer adjusted to pHs 1.5 and respectively to give an optical density of 0.4 (corresponding to 1 x 108 CFU/mL). The survival of the organism was determined by'serially diluting aliquots talcen hourly over 4 hours and spreading 100 microlitre aliquots on MRS agar plates that were then incubated at 37C anaerobically for 24 h. As shown in Figure'-2, Lfermetziurn strain VRI 003 showed better survival than Lferinan turn strain VRI 002 whicha decreased from log 8.75 cfu per nil at pH 1. 5 to log 4.03 cfu/mIn over 4 hours.

13 Carbohydrate UtilizatioA Using the API 5OCHL1 carbohydrate utilization kits to study which carbohydrates the Lfermentw'n VRJ 003 could utilize, some differences with Lfermnentum strain VPJ 002 were notcd with time of incubation. After 6 hours incubation, VRI 003 was able to utilize the following sugars, as defined by medium turning yellow, Ribose 00 Galactose Glucose Fructose Mannose CK1 Maltose Lactose Melibiose Sucrose Trehalose Raffinose.

VRI 002 was able to utilise all the above at this lime, except mannose.

Aftr 6 hours incubation, both strains were able to slightly utilise Gluco Na Te, By 18 hours' incubation, a change was observed in the mannose-containing area of the strip from which it *vas inferred that VRI 002 was utilizing mannose, but relatively slowly.

By. 24 hours' incubation, it was clear that VRI 003 was utilizing the sugars L- Arabiaoso and Mannitol. VRI 002, in addition to these two started utilizing extra sugars, namely, rhantnose and N-Acetyl-Glucosamine.

Afler 48 hours' incubation, VrRI-003 strain had utiliseci all the Gluco Na Te, while the VI-002 strain had only very slight utilisation.

(vi) Adhesion of Lfe~rnentunt YRI 003 in vitro adhesion to Peyer's patches Radioactively labeled Lferthentum VRI 003 was incubated for 30 mins with Peyer's patches in vitro and level of adhesion was quantified. As shownm in Figure 3, V)Rj 003 exhibited enhanced binding to Peyer's patch cells compared to other strains found in the lab (LA I1-L.acidophilus 1, LA 2-L.acidopius 2, L.casei and VRI-002). As the Peyer's Patches act as the sampling site for the Immune system in. the gut. This enhanced 14- 00 adhesion of Lfernentum strain VRI 003 to the Peyer's Patches will lead to an enhanced inununostimulatory effect of this strain.

Protein mediated attachment of Lfermentnrn 003 to Peyer's Patches.

order to characterize the mechanism of attachnaeiit to the Peyer's Patches, the L fernienturn cells were pretreated prior to the adhesion assay with proteinase eoazyoes and nietxaperiodate. to detemaine whether polysaccharide of proteins were involved in the adhesion. It was shown that the treatment with metaperiodate increased adhesion and therefore without being bound by theory, one oan conclude that the extracellular polysaccharides were not involved in adhesion and, in fact, reduced attachment. The treatment of the, Lferinentum cells with proteinase enzymes dramatically decreased attachment, thus implicating cell wall proteins in the attachment to the Peyer's Patches.

Furthermore, cell surface extracts could significantly block binding when added to the tissue prior to the addition of the whole Lfermcntuwn cell s, confrming the presence of a moiety with affinity for the Poyer's Patches on the cell wall surface of the Lfertnentum VRI 003.

Table L.Effcct of chemical and enzymatic treatments of Lfernentio VIII 003 and Peyer's patch tissues on the adherence.

Treatjat PBS only Froteinase K Trypsin Feriodate Iodate Buffer Cell surface proteins (5 jig/mil) Cell surface proteins (50 jgg/ml) Cell surface proteins (t00jig/mI) Adhesion Lidex Lfermnzetwn VR[ 003 Peyer's patches 100110.13 100±8105 33.48±L5.67w 120.7610.27 48.69±5.22 60.81-+0.38 224.08±21.73* 108.59±2.80 100±0.84 48,15±10O.23* 46.14±7.91* 42.23:L6.37* 64, 24±6.60 69,54+3.69 100±12.50

ND)

ND

ND

aAdhesion index expressed as a percentage of the n-umber of adhering bacteria per mg wet weight of Peyer's patches. Adhesion to control tissues represents 100% adhesion.

15 Data are expressed as means standard errors means. Eiter the Lfernwurn

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RJ 003 or the Peyer's patches were pre-treated with the vaxious treatments prior to'bejg included in the adhesion assay..

A two-dimensional analysis of the cell wal proteins extracted from the L ferinentum VRI 003 cells and compared to the cells walls of Lfennentuzn LMG, previously shown not to adhere to the Peyer's Patches, shows that the VRI 003 00 upregulates two cell wall proteins and downregulates the expression of a number of other cell wall proteins (Figure when compared to Ljcrmnentuyn [MG (Figure Adhesioti to human intestinal jnucosa Subjects taing the Lfermeneum as a freeze-dried* powder in a gelatin capsule (log C1 10- 11 per day for 14 days) were endoscoped and a biopsy taken from the wall of the( small intestine. The biopsy was washed and homogenized prior to being serially diluted and plated on MRS agar for isolation of adhering lactobacillus. The isolated lactobacillus were purified and proven to bae frinentum VRI 003 using VF[ 003 specific antibodies.

(vii) Effect of Lfirmeztuni YR[ 003 on immunity parameters Direct effects of Lfernentu::t VRI 003 on mancrophages Bone marrow derived macrophages were generated and stimnulated with different concentrations of Lfermentian VRI 003 for 4 hours. Lfennentum VRI 003 were either given -at a Jai.gh concetration (multiplicity of infection (MOI- 260; Conc. 1) or a low concentration (MOI -26, Conc. 2).

As can be seen firom Figure 6, there is a concentration response-dependent cytok-ine release by the macrophages. On day I post stimulation, the mnacrophages that were stimulated with the higher concentration of either Lferinenturn VRI 002 or L fernien turn V1RI 003 produced significantly greater IL- 12 compared to the macrophages that had received the lower concentration. The levels of IL, 10 produced by the macrophages in response to stimulation by Lfeinen-irnz VRT 003 was very low compared with the levels of IL-12 produced.

This is as expected as IL-12 and l-10 are produced mutually exclusive of the other by macrophages. The level of IL- 12 produced by nmacrophages is believed to play a sigaificant role in determining the immune response directed against an antigen. The presence of JL-12 in the local eavironment of the gastrointestinal tract dendlitic cells prime T cells towards a ThlI type response. This data suggests that a higher doses L -16fermentuni VRJ 003 has the ability to skew the immune system towards a Thi response, and that this is done more effectively than is done by the Lfernwntwin VRJ 002.

Effect of Lfermentum VRI 003 on Immune parameters i vivo- BALB/c mice were orally administered with I x 10 efuimouse/day of L fertnentum VRI 003. The inice were sacrificed at different timiepoints. Spleens and Peyer's Patches were excised and immune parameters measured.

00 BALM/ mice were orally administered with I x 10 CFU/monse/day of.L fermentunt VRI 003 for 5 days. Control mice received PBS. 'The mice were sacrificed on day 6. Spleens and Peyer's patches were excised and immune parameters measured.

As shown in Figure 7, oral feeding of Lfennenturn VRI 003 for 5 days leads to signxificantly enhanced IFN-y and IL- 12 by the Peyer' s patches compared to the control group and to the other lab strain used in the experiment (Another strain of Lfementunz -LF Figure 8 shows that feeding Lfernentupt VRI-003 did not seem to affec~t cytokine levels in the spleen. IL-12 and IFN-y levels off-fermentuni VRI 003 fed mice were comparable to control imice (which had been fed PBS). Therefore oral feeding of L fenentwn VRI 003 in normal mice in the absence of an active-infection stimulates mucosal immunity, and primes it for mounting optimal responses against gut pathogens.

(viii) Stability of freeze-dried powder o.fLfermeistum MR 003 Figure 9 shows that VRI 003 is particularly stable in gelatin capsules when combined with low wvater activity resistant starch and stored at 25C and W~hile the capacity to colonize the human gastrointestinal tract is not a prerequisite for the active function of a probiotic; strain in the digestive tract, it is a desirable characteristic., If the probiotic strain can adhere to the gastrointestinal tract epithelium, it can colonise, i.e. establish and growv within the tract, and continually produce metabolifes that may mediate the beneficial effect. The variant VRI 003 colonises the htuman digestive tract when orally administered to a range of humans.

Antagonistic effects.

From in vitro pathogen inhibition studies it was apparent that the strain produces metabolites that inihibit the growth of a range of p .otential pathogen;, both Gram negative and Gram positive species including numerous strains of Escfzerichia coi, Sathnonella typhinturium, Clc'stridium perringen, Closliidiunz dij9icile, Enterococcusfaecium, Enterococcrusfaecais, Candida alblean4- and Staphylocc=c~ aureus..

17- C1 Without wishing to be bound by any particular mechanism of action, for a Q. probiotic strain to effectively protect the subject from pathogens of the gastrointestinal tract, it may need to produce some metabolites inhibitory to the growth of the pathogen.

c This inhibitory effect on growth is referred to as an antagonistic effect and the antagonistic metabolites can be classified as low or high molecular weight compounds.

Three different methods have been used to evaluate the antagonistic capacity of strain 00 VRI 003 against a range of human pathogens, namely a direct plate co-culture assay, O broth culturing in spent culture fluid from the Lactobacillus and an animal challenge model.

The direct plate method was used for screening a large number ofpathogens by using a point inocula of the Lactobacillus.that are overlaid with the pathogens and the size of the zone of growth inhibition of the pathogen measured after incubation. To quantify further the antagonistic effect detected using this method, the VRI 003 was cocultured with the pathogen in liquid medium and the number of viable pathogen cells enumerated after 24 hours. The size of the zone was used to quantify the extent of inhibition.

The inhibitory activity of strain VRI 003 was also examined to determine whether low and high molecular weight metabolites of the lactobacillus mediated the growth inhibitory effects detected. The bacterial spent culture fluid ofVRI 003 was collected and one part dialysed to retain only compounds with a molecular weight greater than 8,000. The growth of selected human pathogens was studied in the absence and presence of the dialysis retentates and the non-dialysed spent culture fluids. While the non-dialysed filter sterilised spent culture fluid prevented growth of the pathogens, the dialysates exhibited bacteriostatic as well as bacteriocidal effects.

From in vitro pathogen inhibition studies it was apparent that the strain produces metabolites that inhibit the growth of a range of potential pathogens, both Gram negative and Gram positive species. The variant VRI 003 produced both low and high molecular metabolites that could inhibit pathogens and protect in animal challenge studies. The oral administration of the strain to mice challenged with Salmonella prevents the observed weight loss that occurs in control mice only receiving the Salmonella and no Lactobacillus.

-18- Example 3: Culture and Formulations Growth of the culture Lactobacillusfermentum variant VRI 003 is grown in a fermentation vessel at 37°C. The vessel is then cooled and the fermentation broth concentrated, preferably by centrifugation. The collected culture is dried, preferably by freeze-drying and subsequently milled. The milled material is then blended with the major excipient to 00 give the desired level of microbes per gram of dry material. The level to be used is Sdependant on the application (range up to log 11 per gram). The standardised material is then used in the formulation by mixing all ingredients in a blender (preferably a Vblender).

(ii) Formulations Formulation A: High amylase maize based (symbiotic formulation) Lactobacillus fermentum VRI 003 Hi-maize 958 (or 1043) 170mg Stearic acid up to Silica dioxide up to Formulation B: Microcrystalline cellulose (MCC) based Lactobacilltusfernentwn VRI 003 100mg Avicell Ph 112 (or equivalent) 170mg Stearic acid up to 4.5 mg Silica dioxide up to 4.5 mg Formulation C: Either the High amylase maize base or the MCC based as described in A and B, with colloidal silica (up to 4.5 mg) instead of silica dioxide or with silica dioxide as well (up to 4.5 mg).

One of the desired characteristics for VRI 003 is that it remains viable and has the capacity to grow within the human gastrointestinal tract after dosage. As outlined above this characteristic was one of the selection criteria for a desirable strain. However, this is not necessarily essential for the desired beneficial effects.

An additional important factor in this regard is that even though the strain has the capacity to survive the various conditions within the tract, the strain must retain viability and desired strain characteristics when grown on a large scale and dispensed in a product form. All results presented above were based on VRI 003 cells harvested directly from actively growing laboratory cultures. The following is an analysis of the viability and -19-

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strain characteristics of VRI 003 after large scale culture and freeze-drying, as well as after encapsidation in gelatin capsules.

(iii) Viability of freeze-dried VRI 003.

The viability of VRI 003 after large-scale production and freeze-drying was determined by analysing the colony forming units per gram (CFU/ g) of dried material.

CcrThe dried powder was examined both before and after encapsulation in the gelatin 00 capsules. The number of viable cells was determined for ten individual 1 g samples of the powder. The contents often capsules were also individually analysed.

VRI 003 maintained high levels of viability through production and encapsulation.

The dried powder contained 5.6 x 1010 cfu/g and the contents of the capsules contained C' 4.15 x 1010 CFU/g; results expressed as the mean where n When the capsules were stored in foil/foil packaging, a 1.5 log, or less, loss of viability was noted with storage at 30C and 25C for 6 months.

(iv) Variant characteristics The dried powder and capsule contents were suspended it phosphate buffered saline (0.1M, pH 7.2) to yield a 100-fold dilution. The variant characteristics outlined above were tested on this bacterial suspension- For all tests, an actively growing culture of VRI 003 was included as the internal control. As detailed in the following no marked loss of strain characteristics was noted for the freeze-dried powder or for the capsule contents.

Further, the powder and capsule contents showed similar adhesion characteristics to those of laboratory grown control culture.

Example 4: Effect of administratiou of Lactobacillus fermentumVRT, VRI 003, with or without a prebiotic, on symptoms of irritable bowel syndrome (IBS) and gastrointestinal flora profile Experimental design Participants Seven patients with IBS (2 male and 5 female) participated in the study after.

giving written informed consent (HREC 99264). All patients had not taken antibiotics three months prior to commencement of the study. All patients were examined by a physician prior to participation in the trial for presentation of IBS diagnostic criteria and absence of organic disease. All patients were symptomatic at the time of the study.

C<N Trial design The single-blinded placebo trial extended for a duration of 8 weeks, consisting of Sthree weeks placebo treatment followed by two weeks washout (no treatment) and c, concluding with 3 weeks synbiotic treatment, ie, treatment with a formulation containing VRI 003 variant and a prebiotic. A smaller group of patients was treated with a c formulation containing VRI 003 alone, 00 Patients' diets were not controlled in the study, however they were advised to O refrain from consuming fermented milk products yoghurt and sour cream). Fresh faecal samples were requested from the patients on day one of the trial to form the baseline, and then on days 21, 35 and 56 (before and after each treatment period). An initial questionnaire was completed by the patients on the first day of the trial and thereafter weekly questionnaires were completed by the patients for each week of the trial.

Typical treatment protocol was as follows (provided for the symbiotic formulation but a similar protocol was used for a formulation with VRI 003 alone).

Capsules and carbohydrates Empty capsules were manually filled. Assembly of capsules was performed in a laminar flow hood and materials were sterilized for 15 min under UV prior to use.

Placebo capsules contained microcrystalline cellulose NF XVIH (Mingtai Chemical Company Ltd, Taiwan), while probiotic capsules consisted of Lactobacilus fermentum (VRI003) as a freeze-dried powder (1.67X10 t cfu.gmn 1 DSM Moorebank, Sydney). Assembled capsules were placed into 120 mL specimen jars with two sachets of silica gel.

Maltodextrin (Fieldstar, Goodman Fielder, Sydney) and resistant starch (Culture Pro T 958, Penfold Australia) were utilized as the placebo and prebiotic carbohydrates respectively. Carbohydrates were placed under UV for 15 min in a laminar flow hood, before samples were weighed out (20 g) and placed into 70 mL specimenjars which were stored in the clean room.

Questionnaires An initial questionnaire was used to establish the patients' previous and present symptoms prior to commencement of the trial. Patients were asked to rate the severity of their symptoms, including diarrhoea, constipation, alternating diarrhoea and 21 constipation, flatulence, bloating and crampy abdon-inal pain-, on a scale of 0 (absent) through to 10 (extreme). Other questions relating to causes and relief of gut disturbances and food allergies ware also queried, Patients were required to complete a weekly cpuestionnaire at the end of each week for the duration of the trial which again asked them to rate the severity of their M symptoms and also the nature of any gut disturbances. in addition, questions regarding 00 the consumption and apparent effects of the capsules and carbohydrate were also queriea.

Preparation of faecal material Faecal inoculumi was collected from subjects with LBS whom had not taken antibiotics for three months prior to comnmencement of the.Wral, Faeces were collected .into a clean plastic container and transferred into the anaerobic chamber upon arrival, One part faeces was homogenized with three parts half-strongth Wilkin-Ohaigrens (WC) broth (Oxoid, CM643) in a Seward stomacher bag.

Enumeration of bacteria A ten-fold serial dilution was performed in half-strength Wilkin-Chaigens (WC) broth (Oxoid, CM643) in sterile microcentrifuge tubes, after which 10 JIL aliquots were drop-plated in triplicate onto various media (table in order to assess the numbers of major microbial groups present in the faecal samples. Media were prepared according to the manufacturer's recommendations. Samples plated onto R.CA plates were first heated to 90*C for 10 mini.

-22 00 Table 1 Bacteria populations enumerated in fermentation.

Medlium Microorganisms Dilutions Incabation plated Nutrient Agar (NA; CM3') Total aerobes 104to 1 02, 24 hr MacConkey Agar (MfAC; Enterobact eria 10 -4o 02, 24 hr CM7') WC +I Blood' Agar (CM619') Total anaerobes 1-tO I10' Ant) 2 ,.48 hr WC 4- Blood' T Antibiotic Gram negative 10-4 to Ant) 2 48hr supplement 2 (CM6 19*) anaerobes Ro6gosa Agar (CM627) Lactobacill 10 A to 10-" A-nt2, 48 hr Raffinose Bifibacterium Agar Bifidobacteria 10-4 to 10-3 Ant) 2 48hr (RB; Hartemink, 1995) Reinforced Clostridial Agar Clostridia 1 0- to 10,5 Ant) 2 72br (RCA;- CM115 l) Oxoid 1Oxoid defibrinated blood (50 mL per 1 L agar) 2 Oxoid G-N anaenobo selective s~ipplernont SR1 08B (Hi) Effects of administration of Lferrn7m VRI 003 alone or in combination with a prebiotic Suzmmary results Table 2: Effects ofLfernentim strain VRJ 003 on the severity of symptoms of IBS whien administered alone or together with a prebiotic (high amylose maize starch) (resistant stach Results expressed as which corresponds to no symptoms to up to "+"where the more severe symptoms are presented as S~mptoms Baseline Placebo VRI 003+RS VRI 003 alone Diarrhoea Bloating II! Administration of VRI 003 was effective even when administered alone.

However, the combination of'VRT 003 with high ainylose maize starch elevated the levels of VI 003 bacteria and decreased levels of certain bacteria such as Salmonella and Clostridiumn When compared to the probiotic alone or the beta-giucan alone.

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Example 5: Changes in the faecal microbial profiles using material from IBS subjects during the trial Results have been obtained for changes in the total aerobes, enteric bacteria, total anaerobes, Gram negative anaerobes, lactobacilli, clostridia abd bifidobactexia during the trial. Time points displayed on the graph are representative of the faecal microbal profile c at the start of the trial (baseline), end of placebo treatment, end of washout period and 0O end of synbiotic treatment. In regard to the results, subjects were separated into the subgroups, diarrhoea, constipation and alternating diarrhoea and constipation predominant, according to the symptoms they normally experience with the condition.

This sub grouping allows subjects to be appropriately assessed due to the wide

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I variability in symptoms experienced by those with the condition (Akehurst and Kaltenthaler, 2001). One subject, however, did not fit into either of the three subgroups and has been placed into a separate group for flatulence, bloating and crampy abdominal pain predominant Diarrhoea predominant subjects Changes in the faecal microbial profile of diarrhoea predominant subjects are shown in figure 10 (subject figure 11 (subject 5) and figure 12 (subject 9).

Subject 4 (figure 10): Total aerobes remained at a constant level of 1x10 s log efu.mLt except during the washout period, in which total aerobes increased by one log.

Numbers of enteric bacteria which were initially detected at lxl0 s log cfu.mL t decreased by one and a half log by the end of the synbiotic treatment period, though enteric bacteria began to display a widening disparity compared to numbers of total aerobes. at the end of the washout period. Both total anaerobes and Gram negative anaerobes, which began at 5x10 8 log cfu.mL t displayed an increase of one log by the end of the synbiotic treatment period. Lactobacilli were initially detected at Ixl10 log cfu.mL 1 and exhibited a decrease of two log at the end of the placebo and synbiotic treatment periods, which peaked again in between, during the washout period. Clostridia remained undetectable throughout the whole trial, while numbers of bifidobacteria, which began at 5x10 7 log cfu.mL", increased by one log during the synbiotic treatment period.

Subject 5 (figure 11): Total aerobes, which were initially detected at 5x10 5 log cfu.mLL 1 increased by one log during the washout and synbiotic treatment periods.

Enteric bacteria remained at levels similar to those of the total aerobes (5x105 log -24- C1 cf.mLL') during all time points, except at the end of the placebo treatment period, in which numbers of enteric bacteria decreased by two log. Numbers of total anaerobes and Gram negative anaerobes continued throughout the trial at a constant level of 1x109 log c1 cfu.mL though decreased by one to one and a half log at the end of the placebo treatment period. Lactobacilli, which began at 1x10 6 log cfu.mL' 1 decreased by one log at the end of the placebo treatment period but increased by three log at the end of the 0 washout period, only to decrease again by one log at the end of the synbiotic treatment 0 period. Clostridia was detected at the beginning of the trial at 1x10l log cfu.mL' and coremained undetectable throughout the rest of the trial. Bifidobacteria, which began at 5x10 8 log cfu.mL-t, decreased -by one log at the end of the placebo and synbiotic treatment period, but increased by one log in between during the washout period.

Subject 9 (figure 12): Total aerobes and enteric bacteria were detected at 1x10 7 log cfu.mL- 1 at the baseline and proceeded to decrease slowly by one and a half log by the end of the synbiotic treatment period. Total anaerobes and gram negative anaerobes were detected at 5x10 s log cfu.mL at the baseline, increased by one log at the end of the placebo treatment period and remained at a steady level for the rest of the study.

Lactobacilli were detected at 5x10 5 log cfu.mL' at the baseline, however became undetectable during the placebo treatment period. Lactobacilli were detected again at 5x10 3 log ef.mL 1 at the end of the washout period and increased by one log at the end of the symbiotic treatment period. Clostridia were absent for the duration of the trial.

Bifidobacteria began at 5x10 7 log cfu.mL'', decreased by one a half log at the end of the placebo treatment period, however increased again to 5K108 log cfu.mL' by the end of the washout period and remained at this level till the end of the synbiotic treatment period.

Constipation predominant subjects Changes in the faecal microbial profile of constipation predominant subjects are shown in figure 13 (subject 2) and figure 14 (subject 3).

Subject 2 (figure 13): Total aerobes and enteric bacteria were initially detected at Ixl07 log cfu.mLt and remained at similar levels throughout the trial, except at the end of the placebo treatment period, where numbers of enteric bacteria increased by one log.

Numbers of total anaerobes and Gram negative anaerobes remained at similar levels of log cfu.mL' throughout the trial, with an increase of one log at the end of the placebo treatment period, which decreased by half a log by the end of the synbiotic O treatment period. Lactobacilli, which began at 1x10 3 log cfu.mL', increased by one log Sat the end of the placebo treatment period, but became uidetectable at the end of washout period and increased again by three log by the end of the synbiotic treatment period. Clostridia was present only at the beginning of the trial at lx10 3 log cfu.mL and remained undetectable throughout the rest of the trial. Bifidobacteria were initially detected at Ixl07 log cfu.ml' and decreased by one and a half log at the end of the 0placebo treatment period, and then became undetectable during the rest of the trial.

Subject 3 (figure 14): Total aerobes and enteric bacteria remained at similar C, levels of 5x10 4 log cfiumL 1 for the duration of the trial and increased by two log at the end of the placebo treatment period, though decreased by one log by the end of the 1 synbiotic treatment period. Total anaerobes and Gram negative anaerobes began at xl10 7 log cfu.mLt and continued a steady increase of two log by the end of the synbiotic treatment period. Lactobacilli were detected at the beginning of the trial at xl10 4 log cfu.mL 1 but then became undetectable at the end of the placebo treatment period.

However, lactobacilli were shown to increase by four and a half log at the end of the washout period and decrease again by half a log at the end of the synbiotic treatment period. Clostridia were only detected at the end of the placebo treatment period at x 104 log cfu.mL t and remained undetectable for the rest of the trial Bifidobacteria, which began at 1x10 7 log cfu.mL' was not able to be detected at the end of the placebo treatment period, though became detectable again by the end of the washout period and increased by four and a half log by the end of the synbiotic treatment period.

Alternating diarrhoea and constipation predominant subject Changes in the faecal microbial profile of an altemating diarrhoea and constipation predominant subject is shown in figure 15 (subject 7).

Subject 7 (figure 15): Total aerobes and enteric bacteria which were initially detected at 5x1 0 log cfu.mL" 1 increased by two log at the end of the washout period, however decreased by one log at the end of the synbiotic treatment period. Total anaerobes and Gram negative anaerobes began at 5x10 8 log cfu.mL 1 and remained at a steady level throughout the study, until the synbiotic treatment period, where total anaerobes decreased by one log while Gram negative anaerobes decreased by two log.

Lactobacilli were not detected for most part of the study, except by the end of the synbiotic treatment period, at 5x10 3 log cfu.mL' l In contrast, clostridia was only detected at the beginning of the trial at 1x10 3 log cfu.mL" 1 Bifidobacteria were initially .26 C1detected at 5xi 0 6 log cfu.mIA, however becamne undetectable during the placebo treatment period, only to be detected again dluring the washout per iod at 5x10 Ch og cfu.mL-1 and increased by half a log at the end of the synbiotio treatment period.

Other., flatulence, bloating and crampy abdominal pain predominant subject Changes in the faecal microbial profile of a flatulence, bloating and crampy abdominal pain predomiinant subject is sho~rn in figure 16 (subject 6).

00 Subject 6 (figure 16): Total aerobes and enteric bacteria were initially detected at 105 log cfti.mU' and remained level until the synbiotic treatment period, which saw a two log increase in total aerobes and enteric bacteria. Total anacrobds and Gram negative anaerobes were detected at Wx03 log cfu.mL 4 at the baseline and increased by one log at the end of the placebo treatment period, after which they remained at this level for the rest of the study. Lactobacilli remained at a relatively stable level of 1004 log cfu.tl for the duration of the study. Clostridia was not detected during the study, except at the end of the synbiotic treatment .period at 5x1&3 log clix,iL7'. Bifidobacteria began at 1xI 0' log cftim~l and decreased by three log by the end of the placebo treatment p eod, only to increase again to W07& log cfU.rnIL 4 during the washout period and finally decreased by three log at the end of the synbiotic; treatment period.

Example 6: Changes in the severity of symytomg of IBS subjects during the trial Results have been obtained for the changes in the severity of symptoms including diarrhoea, constipation, alternating diarrhoea and constipation., flatulence, bloating and cranipy abdominal pain. Each symptom is graded on a scale of 0 to 10, with 0 indicating absence of the symptom and 10 indicating extreme severity of the symptom. Results are displayed on a scale of I to 5, with 1 indicating mild severity of the symptom and indicating extreme severity of the symptom. Changes in the severity of symptoms were monitored every week in all subjects, however, the results will display only time points representing the baseline (begInImng of the trial), placebo treatment period, washout period and synbiotic treatment period. Subjects were again separated into subgroups, diar-rhoea-, constipation- and alternating diarrhoea and constipation predominant in accordance with the symptoms they nonmally experience with the condition.

Diarrhoea predominant subjects Changes in the severity of Symptoms for diarrhoea predominant subjects are shown in table 3 (subject table 4 (subject 5) and table 5 (subject 9).

-27- N ~Subject 4 (table The severity of diarrhoea at the baseline wvas extremely hidgh, however this was reduced to half the severity by the end of the placebo treatment period, 'which further decreased down to low severity by the end of the synbiotic treatment period. Flatulence and bloating were at mediumn severity at the baseline and eventually reduced to low severity by the end of the synabiotic treatment period.

Crampy abdominal pain was also low at the start of the baseline and further reduced 00 in severity down to 1 by the end of the synbiotic treatment period.

Table 3 Changes in the severity of symptomns of subject #4 during an 8-week trial.

Severity of syniptorns were mionitored during the baseline, placebo, washout and synbiotic treatment periods. Results expressed as (low severity) to (extreme severity) Symptom Baseline Placebo Washout Syiibiotic Diarrhoea Constipatiou Flatulence +4 Bloating Crampy abdominal pawn____ Subject 5 (table The severity of ciunhoea at the baseline was extremnely high and only decreased slightly to 4 by the placebo treatment period, after which it reniaed at high severity. Flatulence was low at the baseline, absent during the placebo treatment period, but increased again during the wvashout and synbiotic treatincnt periods by 1 and 2 respectively. The severity of crampy Abdominal pain remained at the same level of 3 throughout the trial, though decreased a little by 1 during the washout period. Symptoms of flatulence and bloating remained absent throughout the duration of the trial.

-28- Table 4 Changes in tle severity of symptoms of subject #5 during an 8 week trial.

Severity of symiptotus were monitored during the baseline, Placebo, washout and synbiotic treatment periods. Results expressed as (low severity) to (extreme severity).

Symptom BaseJijie )Placebo Washout 'Sy~biotic iiarrhoea Constipation- .1?iatulence Bloating Crainpy abdominal pain Subject 9 (table Diarrhoea. began at mid range at the baseline and+ continued at this severity up until the synbiotio treatment period, where it had reduced by 2 down to low severity. Constipation was absent during the trial, apart ftom the placebo treatment period, in which it had increased by 1. Flatulence was also absent during the trial, however increased by 3 during the washout period. Bloating, which was at medium severity at the baseline, reduced by I duaring the placebo treatment period, However, ths increased again by 1 during the washout period and then dropped down to low severity during the probiozic treatment period. Crampy abdoniinal pain was absent throughout the duration of the trial.

Table 5 Changes in the severity of symptoms of subject #9 dutring an 8 week trial.

Severity of symptoms were moaitored during the baseine, placebo, washout and synbiotic treatment periods. Results expressed as (low severity) to (extreme sevenity).

29 Coustipation Predominant su~bjects Changes in the severity of symaptoms for constipation predominant subjects are shown in table 6 (subject 2) and table 7 (subject 3).

(71 Subject 2 (table Diarrhoea, which was absent at the baseline and for the most part of the trial, increased by 1 durinig the synlbiotic treatment period. Constipation, ___flatulencte and bloating remained at high severity at the baseline and during the placebo and washout periods, though decreased by I dur-ing the syribiotic treatment period. ICraxnpy abdominal pain was at high severity at thie baseline, and during the placebo treatuient period, however reduced by 3 down to low severity durig tho washout and syulbiotic treatment period.

Table 6 Changes in the severity of symptoms of subject #2 durinig an 8 week trial.

Severity of symptoms wore monitored during tie baseline, placebo, washout and synbiotic treatment periods. Results expressed as (low severity) to +I +I (extremec severity).

Symiptomn Baseline Placebo Washout Synbiotic Diarrhoea Constipation FlWatulence I Bloating Crampy abdominal pait I t Subject 3 (table Constipation began at low severity at the baseline and continued at this level diu-ing the placebo* treatment period, howerver became ab-sent during- the washout and synrbiotic treatment periods. Flatulence began at very high severity at the baseline, -reduced down to 3 during the placebo treatment period and further reduced down to low severity during the washout and synbiotic treatment periods. Bloating also began at ver-y high se'vority at the baseline and was also able to reduce in severity durig the placebo, washout and synbiotic treatment periods to 4, 3 and 3 respectively.

Crainpy abdominal pain began at highi severity at the baseline and reduced to low severity during the placebo treatniett period, after whioli it became absent for the remainder of the trial. Diarrhoea was absent for the duration of the tinal.

Table 7 Changes ini the severity of symptoms of subject #3 during an 8 week trial.

Severity of symptoms were monitored during the baseline, placebo, washout and synbiotic treatmient periods. Results expressed as (low severity) to (extreme severity).

Symnptom Baseline Placebo Washout Syftbiotic Diarrhoea- 00 Constipation Flatulence B~loating T T Crampy CI abdomnal pain Alternatinig diarrhoea and constipation predoinuant subject Changes in the severity of symptoms for an 1ternating diarrhoea and constipation predominant subject is shown in table 8 (subject 7).

Subject 7 (table Diarrhoea started at low severity at the baselie but increased to 2 during thec washout and synbiotic treatment periods. Constipation, which, was at low severity at the baseline, decreased by 1 during the placebo and washout periods, but increased to 3 during the synbiotic treatment period. Flatulence began at low severity at the baseline and remained low for the most part of the trial, however it increased to 3 during the synbiotic. treatment period. Bloating began at low severity (2) and decreased to 1 during the placebo treatment period, however continued to increase during the washout and synbiotic treatment periods to 3 and 4 respectively. Cramnpy abdominal pain was absent during the trial, except during the synbiotic treatment period, when it had increased to miediumn severity -31- Table 8 -Changes in the severity of symptoms of subject #7 during an 8-week trial.

Severity of symTptoms wvere monitored durinig the baseline, placebo, washout and synbiotic treatment periods. Results expressed as (low severity) to (xrm severity).

Symiptomn Baseline Placebo Washout Synbiotic Diarrhoea 00 Constipation Flatulence Bloating Crampy CI abdominal pain Other: flatitlextce, bloating and cranipy abdominal pain predominant subject (Thanges in the severity of symptoms for a flatulence, bloating and crainpy abdominal pain predominant subject is shown in table 9 (subject 6).

Subject 6 (table Diarrhoea was absent for most of the trial, except during the synbiotic treatment period ih which it had increased by 1 to low severity. Flatulence began at medium sevenity at the baseline and reduced to low severity during the placebo and.washout periods, however increased up to medium severity once again during the synbiotic treatment period. Bloating also began at medium severity at the baseline and became absen~t during the placebo treatment period, however it had also increased again during the washout and synbiotic treatment periods to 1 and 3 respectively, Crampy abdoinal pain began at low severity at the baseline, was absent during the placebo treatment period but increased by 1 and 3 during the washout and synbiotio treatment periods respectively. Constipation remained absent for the duration of the trial.

-3 2ri Table 9 Changes in the severity of symptoms of subject #46 during an S week trial.

Severity of symptoms were monitored during the baseline, placebo, washout and synbiotic treatment periods. Results expressed as (low severity) to (extreme severity).

FSymptom Baseline Placebo' Washout Synbiotic Diarrhoea Constipation Flatulence Bloating Crampy abdominal paini____ Discussion Correlations between changes in the faeeal microbial proffles ustug materinl fromt M~S subjects and severity of symptows durine the trial The clinical trial lasted for a total duration of 8 weeks, the first 3 weeks involved a placebo treatrnent consisting of maltodextrin capsules and cellulose. This was followed by a 2 week washout period in which no treatment was tak-en. Thie last 3 weeks involved a symbiotic treatment consisting of VIRI 003 capsules and RS. Faccal samples were collected at the beginning of' the trial to establish a baseline, after which furwther samples were taken before and after each treatment period. In addition,weekty questionnaires were also completed by each patient to monitor changes in their severity of symptomns via a scoring system graded from 0 to 10, with 0 being the absence of the symptom and being the most severe. Results were then converted to a scale of I to 5 for convenience and shown for the baseline, placebo, washout and symbiotic treatment periods.

Patients were grouped into 3 subgroups according to their predominance in symptoms, as shown by Akehurst er al and Weston et at (AIkehurst and Kaltenthaler, 2001; Weston, et at., 1993). Separating patients into'either diarrhoea, constipation or alternating diarrhoea and constipation predominant subgroups allows change-s itx particular symnptomns to be assessed accordingly, due to wide variability among different patients in the: symptoms they normally experience with the condition. One subject in particular displayed symptomsunlike those e-xperienced by any of the other patients -33- Q in the 3 subgroups, and was thereby grouped into an others group for flatulence, bloating k and crampy abdominal pain predominant symptoms.

Diarrhoea predominant subjects Subject 4 exhibited a general decrease in the severity of all symptoms (table 3) by the end of the synbiotic treatment period, in particular diarrhoea, which started off at c high severity at the baseline and then proceeded to low severity by the end of the O synbiotic treatment period. A possible correlation with this symptom may be seen in the slow decrease of enteric bacteria (figure 10) from Ixl10 log cfu.mL- 1 at the baseline, down to 5x10 3 log cfu.mL'' at the end of the synbiotic treatment period. Numbers of lactobacilli do not seem to correlate vith any of the symptoms displayed by subject 4, C1 however bifidobacteria were observed to increase by one log during the synbiotic treatment period, which correlates with previous discussion that bifidobacteria are able to utilize RS. However, it is unlikely that the increase in bifidobacteria resulted in the decrease of diarrhoea, as reduction in the severity of this symptom began during the placebo period, so it may be that in this subject, enteric bacteria may have had more of an effect on the severity of diarrhoea.

Subject 5 did not appear to display an overall reduction in the severity of any symptoms (table 4) during the trial. However, it may be noted that during the placebo treatment period, absence of flatulence was observed, which may be related to a two log decrease in the numbers of enteric bacteria (figure 11). The general overview of this subject would indicate that the syabiotic treatment did not appear to promote any beneficial effects on the individual, but rather the placebo treatment may have had some .beneficial effect on the severity of symptoms. Numbers of lactobacilli increased by three log during the washout period, though decreased one log by the end of the synbiotic treatment period. The large increase in lactobacilli during the washout period may also relate to the decrease in crampy abdominal pain exhibited by the subject during this time, which then increased in severity by the end of the synbiotic treatment period, along with a minor decrease in lactobacilli during this period. The observation of an increase in lactobacilli associated with a decrease in crampy abdominal pain may also relate to the finding by Nobaek et al, which observed that administration ofLactobacillus plantamrn decreased pain and flatulence in patients with IBS (Nobaek, et al., 2000). A one log increase in bifidobacteria was also observed during the washout period, which also correlates with an increase in lactobacilli, as bifidobacteria are hypothesized to -34indirectly promote the growth of lactobacilli. Although symptoms did not appear to improve during the synbiotic treatment period in this subject, comments in the weekly questionnaires indicate that symptoms stayed the same during the treatment, rather than increase in severity.

Subject 9 exhibited an overall improvement in the severity of all symptoms (table by the end of the trial, particularly diarrhoea and bloating, which had reduced from oC medium severity down to low severity by the end of the synbiotic treatment period. The decrease in these symptoms may be related to a two log decrease in enteric bacteria from N the baseline to the end of the synbiotic treatment period. Bifidobacteria were also observed to increase by three log after theplacebo treatment period. However, this c increase began during the washout period, when symptoms still remained at medium severity so numbers ofbifidobacteria may not have had an effect on the lowering of symptoms during the synbiotic treatment period. Numbers oflactobacilli were also observed to increase (one log) by the end of the synbiotic treatment period, which may be correlated with the decrease in severity of symptoms. The increase in bifidobacteria and lactobacilli may also be associated with the administration of RS, which has been shown to promote the growth bifidobacteria and thereby lactobacilli (Brown, et al., 2000).

Constipation predominant subjects Subject 2 did not appear to display an improvement in most symptoms (table 6), apart from crampy abdominal pain, which greatly decreased in severity during the washout and synbiotic treatment period. Results from the faecal microbial profile (figure 13) show that during the synbiotic treatment period, bifidobacteria were unable to be detected and a decrease (one log) in total aerobes and enteric bacteria were observed during this time. This would indicate that the decrease in enteric bacteria may reduce symptoms of crampy abdominal pain, however it can only be speculated that the absence ofbifidobacteria may be related also to a decrease in the symptom, as bifidobacteria are perceived to be beneficial in the human gut. The faecal microbial profile of subject 2 also shows that lactobacilli were not promoted to higher numbers during the synbiotic treatment period. This may be due to low starting numbers of the lactobacilli in the gut of the subject, which may require a longer time period to stimulate to higher numbers.

Although the subject did show an overall improvement in symptoms, the weekly

O

O questionnaires indicate that symptoms that were experienced during the trial remained Ssteady and did not increase in severity.

Subject 3 displayed an overall general improvement in the severity of symptoms .(table particularly constipation and crampy abdominal pain, which subsided by the end of the washout and synbiotic treatment period. The improvement in these symptoms c may be correlated with the large increase (four log) in the number ofbifidobacteria (figure 5) at the end of the synbiotic treatment period. The increase in bifidobacteria indicates that the administration ofRS during the synbiotic treatment period has rC stimulated the growth ofbifidobacteria. Administration of dietary fibre RS) has also been shown to reduce symptoms of constipation through faecal bulkifig (Lambert, et N al., 1991; Phillips, et al., 1995). Although lactobacilli numbers remained low compared to the bifidobacteria during the trial, it is possible that if the trial extended for a longer period of time, lactobacilli may increase in numbers to a larger extent. Enteric bacteria also decreased (one log) by the end of the synbiotic treatment period and though only a minor decrease, it may have also contributed to the decrease in the severity of these symptoms.

Alternating diarrhoea and constipation predominant subject Subject 7 displays improvement in all symptoms (table 6) during the placebo treatment period, whilst exhibiting an increase in the severity of symptoms during the synbiotic treatment period. A gradual increase in the numbers of enteric bacteria (figure were observed during the trial, which suggests that an increase in enteric bacteria may be related to an increase in symptoms such as bloating and diarrhoea. An important note to be considered however, is that administration ofprebiotics, such as RS, are likely to increase symptoms ofbloating and flatulence, even in healthy subjects (Cummings et al., 2001; Munster et al., 1994), so an increase in these symptoms experienced by an IBS subject is not unlikely. Bifidobacteria were also observed to increase in numbers during the washout period, while not being able to detected during the placebo treatment period, implying that an increase in bifidobacteria may perhaps be related to an increase in severity of these symptoms. Bifidobacteria were also observed to be detected in very high numbers on RB media and it has been noted that other species of bacteria apart from bifidobacteria may be detected on these plates, such as lactobacilli species (Hartemink et al.,'1996). Thus biochemical tests may have to be performed on the colonies growing on RB media to confirm that they were all bifidobacteria species.

-36- C\Z Although symptoms appeared to increase in severity during the synbiotic treatment p_ period, comments from the weekly questionnaire indicate that experience of these symptoms were normal in relation to the baseline.

Other: flatulence, bloating and crampy abdominal pain predominant subject Changes in the severity of symptoms observed for subject 6 (table show that Cc this subject experienced a decrease in the severity of symptoms during the placebo 00 treatment period, while the synbiotic treatment period appeared to have an adverse effect Son symptoms. Numbers ofbifidobacteria, and to a lesser extent, lactobacilli, were observed to decrease (figure 16) during the placebo treatment period. Bifidobacteria decreased in numbers by three log during the placebo treatment period, corresponding Swith a decrease in flatulence and absence of bloating and crampy abdominal pain. It can only be suggested however that bifidobacteria may be related to an increase in these symptoms as they are viewed to be beneficial bacteria. It should also be taken into account that administration of prebiotics, particularly RS, has been shown to increase symptoms of bloating and flatulence (Cummings, et al., 2001; Munster, et al., 1994), which were symptoms displayed by subject 6 during administration ofRS during the synbiotic treatment period. A general improvement in the severity of symptoms during the placebo treatment period may also be attributed to the possible bulking effect caused by cellulose ard thereby ease of defaecation. Weekly questionnaires also indicate that the subject consumed a small amount of alcohol during the washout period, which may have aggravated an increase in symptoms. Furthermore, it was noted this subject experienced stress during the synbiotic treatment period, which may have also increased severity of symptoms, as observed by others (Lidbeck and Nord, 1994; Longstreth and Wolde-Tsadik, 1993).

Trial results of the faecal microbial profiles indicate a general decrease in the number of enteric bacteria correlated with a decrease in the severity of symptoms. An increase in the number of lactobacilli has also been associated with an improvement in some symptoms, such as a reduction in flatulence (subject However, lactobacilli do not seem to exert a very prominent effect in terms of an improvement in symptoms. rn comparison, increases in bifidobacteria may appear to have some correlation with symptom severity, though it may be a mixed one. An increase in bifidobacteria may be observed to be related to a decrease in constipation and crampy abdominal pain (subject however increases in bifidobacteria may also be associated with an increase in -37flatulence and crampy abdominal pain (subject Perhaps different species.of Sbifidobacteria may be involved in increases or decreases in symptom severity, or it may be that different patients are affected by colonization of different gut microorganisms which affect the levels of bifidobacteria in the gastrointestinal tract.

The trial has provided preliminary results which demonsirate favourable changes in the faecal microbial profiles and severity of symptoms of IBS patients while being OQ administered a synbiotic formulation containing VRI 003 and RS, or VRI003 alone.

Example 7: Treatment of eczema SA 21-year old female patient presented with medically-diagnosed severe eczema manifested by a widespread rash all over the body and dilated capillaries on the cheeks.

C1 The skin is very dry and feels tight due to dryness. Treatment at presentation included cortisone creams and moistuxiser daily.

Treatment with L. fermentum VRI-003 commenced with 3 capsules in the morning with food and 3 capsules in the evening with food daily each capsule containing 4.2 x 1010 cfu. Moisturiser and cortisone creams were applied as usual. Following 2 weeks of oral administration of Lfermentum, the severity of eczema was reduced by 2 units using a visual analog scale (VAS; scale 1-10 with 1 low and 10=severe), redness of the skin has been reduced, allergy has been reduced by 2 units using VAS, texture of the skin has slightly improved. Itchiness has also been reduced and there was less flaking observed.

Overall, there has been an improvement on the patient's quality of life.

Example 8: Eczema patient with bowel disturbances A 33-year old male patient presented with medically-diagnosed eczema in the form of inflamed, itchy and dry skin and open sores. Previous treatment included steroid ointments. After an appendectomy, the subject also experienced bowel problems.

Treatment with L. fermentum VRI 003 commenced with 3 capsules in the morning with food and 3 capsules in the evening with food on a daily basis. Each capsule contains 4.2 x 01 0 cfu. After 2 weeks of taking L.fermentim VRI 003, the subject reported reduction of itchiness associated with his eczema. He likewise experienced an improvement in his gastrointestinal health i.e. visits to the toilet have been considerably reduced.

-38- Example 9. Croha's, disease whit chronic fatigue and poor absorption/availablity of minerals and vitaminxs.

A 3 5-40 year old female with Crohn's disease for more than 10 years consu med 3 capsules each morning and night with food (each capsule contained 4 x 1 0 "cfu ofl fermentum VRI 003) and after one week symptoms of diarrhoea, cramps, abdominal pain, constant fatitgue and fluid retentio.D decreased dramatically. Stools became formed.

00 Iron and vitamin B12 levels were excellent, thus conlitming that the presence of L fornzent ur 003 in the intestine improved absorption and/or bioavailability of iron and vitaniinBl2, Example 10: Effects: of probiotic L.f.orniantam VRI-003 on Irritable Rowel Syndrome (113S) Patients A randomized, multicentre, cross-over trial was conducted on IBS patients to see the effects of probiotic L. fennentn VRI 003 in reducing severity of gastrointestinal distubances seen in EBS. The st-Ldy was carried out for a total of 18 weeks (2 weeks follow up after completion of the study) wherein subjects received an active and placebo treatment. Details of the probiotic treatmeat schedule is as follows: WEK TREATW{NT 0-2 Baseline 4 weeks Probiotic/Placebo 6 weeks Wash-out Period (No Treatment) 4 weeks- Probiotic/Placebo This study targeted subjects belonging to the 17-74 years old age group that satisfied the lBS Rome Criteria which was verilied by a medical consultanr.The study, however, excluded patients experienucing gastrointestinal infections and any other type of organic diseases Liver disease, etc). Volunteers were also strongly advised not to take in any other kind of probiotics whilst participating in the study.

P'atient History: A 54 year old, white, female who had LBS since her teenage years.

Active IBS symptoms include diarrhoea and bloating especially in the evening. Subject I's symptoms aggravate when she eats food high in sugar content such as cakes, pastries, and chocolate.

-39- Treatineni: I cap* sule of L. ferynentuni VRI 003 was taken in the morning and in the evening with food, Each csuecontained 3x 10 10 VBI-0031 capsule, Sytnptazns Assessinent: Takcing I capsule of L, ferienturn VRI-003 in the morning and.

in the evening- resulted to regular bowel movement. Faeces became firmer and consistent and there. was reduction in the intensity of bloating. However, L. fermentum VRI-003 did not minimize the severity of the symptoms when sugar n" ch food was taken. in the diet nor -did it worsen die symptoms.

Example 11. Benefits of Lfervnenturn VRI 003 for Rosacea symptoms A mate 45-55 years old with active lBS and Rosacea on the face had bcen treated for-years by his dermatologist for the redness and lesions that are manifestations of the ClRosacea condition. While being treated for the IB S with Lfermentwn VRI 003 (log I1I cfu per day in a gelatincapsule) the patient experienced an extreme reduction in the redniess and lesions on his face due, to the Rosacea and was able to stop usage of all antibiotic creams and steroid creams. His face condition was maintained in remissions while takting the Lfermentum 003.

Although the invention has been described with reference to these specific examples, it will be appreciated by those skilled in the art that the invention may be embodied in many other forms.

Claims (21)

1. A Lctcobacillusferrnentun variant having the following chaxaoten stics: it is a Gram positive, facultative rod, it ferments ribose, galactose, glucose, fructose, rnaaflose, maltose, lactose, melibiose, suicrose, trehialose, raffinose, L-arabinose and mannitol, it survives at pH 1.5 for up to 4 hours with a loss in growthi of no more than. log 3, it propagates in the presence of 0.5% bile salts at 37 C resulting in an increase in optical density of 0.5 to 0.8, it is stable when stored in gelatin capsu les at 25 C, it adheres to Peyer's Patches at greater than log 5 cfu per mng tissue inhibiting pathogens both by a direct antagonistic effect and by triggering an immixune modulation; -or a component thereof. A Lactobacllluvferme:tum variant according to claim 1, wherein said variant is VRI 003 (accssion no. NM02/3 1074).

3. A component of A Lactobacillusferrnentun variant according to claimn 1, wherein said component is a cell fragment, extract, secretion or purified component.

4. A composition comprising a Laciobacillusfernenhum variant according to claims 1 or 2, or a component of a Lactobacillusfermentum variant accordingto claim I or 3 and a pharmaceutically acceptable carrier. A composition according to claim 4, further comiprising a prebiotic, a non- digestible dietary component, dietary fibre or a pharmaceutically active compound.

6. A composition according to claim 5, wherein the prebiotic is inulin, a resistant starch, an eligosaceharide, a gum or a beta-glucan.

7. A composition according to claim 6, wherein said prebiotic. is unmodified high arnylose starch or beta-glucan.

8. A composition according to any one of claims,4 to 7, wherein said composition comprises live cells of the Lactobacillusfenientum variant, -41

9. A composition accordintg to any one of claims 4 to 7, wherein said composition comprises dead cells of the Lactobacillusfermenlin variant. (7110. A composition according to any one of claims 4 to 9; in the form of a tablet, capsule, powder, pasle, gel, liquid fotmulation, dietary supplement or food product. ri11. A comp osition according to claim 10, wherein said composition is in the form of a tablet. A composition according to claim 10, wherein said composition is in the form of 00 a food product.

13. A composition according to claim 12, wherein said food product is a dairy or r- dairy-based food product.

14. A composition according to claim 10, wherein said composition is in the form of a liquid formulation. A composition according to claim 10, wherein said composition is in the form of a dietary supplement.

16. A method for preventing and/or treating a gastrointe stinal disorder or symptoms thereof, comprising the step of administering a Lactobacillusfermenrurn variai according to claim 1 or 2, a component of a Lactobacllusferncentun variant according to claim I or 3, or a composition according to any one of claims 4 to to a subject in need of such treatmient.

17. A method according to claim 16, whereih' said composition is administered orally. 12. A method according to claim 16 or 17, wherein said gastrointestinal disorder is* irritable bowel syndrome, inflammaatory bowel disease, diarrhoea, bloating, flatulence, abdominal cramping, abdominal pain, constipation or Crohn's disease.

19. A method accoyding. to any one of claims 16 to 18, wherein said gastrointestinal disorder is ca'used by a pathogen. A method according to claim 19, wherein said gastrointestinal disorder is caused by a bacterium, virus or protozoa.

21. A method according to claim 20, wherein said bacterium is Salmonella, Escherichia coi, Li-eficobacter, Vibrio or Pseudomonas.

22. A method according to claim 20, wherein said virus is a. Norwalks or rotavis. -42 A method according to claim 2Q. wherein said protozoa is Clyptosporidiunz, Entxuoeba, Giardia or LDientamnoeba.

24. A method for preventing and/or treating a disorder of a iucosal sufface and/or symptoms; thereof, comprising the step of administering an effective amountoa *Lactobacillwferrtnelltu,, variant according to claim 1 to 2, a component of a *lactobacillusfetnzentunz variant according to claim 1 or 3, or a composition according to aniy one of claims 4 to 15, to a subject in need of such treatment. A method according to claim 24, wherein said disorder is eczema, atopic 00 dermatitis or rosacea,

26. Use of a Lactobacillusfermentuni variant according to claim I or a component of a Lactobacilligfermentuni variant according to clafim 1 or 3, or a composition according to any one of claims 4 to. 15, for the manufacture of a medicament for the treatment and/or prevention of a gastrointestinal disorder, a disorder of a mucosal surface or symptoms thereof,

27. A method of stimulating M1-12 production in a subject, comprising the step of administering a lactobacillusfernengan variaat according to claim 1 to 2, a component of a LactobacilIusfertnentucm variant according to claimn I or 3, or a composition according to any bne of el ainig 4 to 15, to a subject in need of such treatment.

28. A method of up-regulating IFN-yr comprising the step of administering a Latctol'acfflusfetrmentum variant according to claim 1 to 2. a component of a Lactobacilusfcrinenfunt variant according to claim I or 3, or a composition according to any one of claims 4 to 15, to a subject in need of such treatment

29. A method of inducing a Th 1 -type response in a subject, comprising the step of administering a Lactobacillusfementurn variant according- to claim I to-27-a. component of a~acobacilhw~fennenhizn variant according to claim 1 or 3, or a composition according to any one of claims 4 to 15, to a subject in need of such treatment. A method of inhibiting the growth of a pathogen, comprising the step of administering a Lactobacillayfennetztur variant according to claim 1 to 2, a component of a Lactobacillu.7fennentum variant according to claim .I or 3, or a composition according to any one of claims 4 to. 15, to a subject in need of such treatment. 43

31. A method of modulating indigenous microbes of the gastrointestinal tract, comprising the step of administering aLactobaciltrennum variant according to claim~ 1 to a component of a Lactobacilhisfermentuin variant -aoiding to claim 1 or 3, or a composition according to any one of claims 4 to to a subject in nepd of such treatment.

32. Use of a Lactobacillusfermentum variant according to claim I or 2, a ___component of a Lacto1bacillusfermentum variant according to claimInlor -3,or a 00 composition according to any one of claims, 4 to 15, for the manufacture of a medicament for stimulating IL-I12 production, up-re~gulatiog IFN-y, inducit~g a Tb 1 -type response, inhlibiting the growth of a pathogen, or modulating indigenous microbes of th~e gastrointestinal tract, in a subject. Dated 24 April, 2007 Probiomnics Limited Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON