Probiotic composition based on the enterococcus strain and used as a treatment means and method for the production thereof



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In a fourth aspect the present invention provides a Vitamin Bl2 supplement comprising Propionibacterium jenseMii 702 together with a delivery agent. It will be understood that delivery agents suitable for use in the supplement include pharmaceutically acceptable delivery agents.
The supplement may be provided in the form of a capsule, tablet or powder (loose or in capsules), granule or paste. The supplement also can be prepared as an oral spray. In another embodiment it is provided in the form of a vitamin B12 fortified food product, such as a breakfast cereal, soy milk product, or vegetarian burger patty.
In a fifth aspect the present invention provides a method for preventing, treating or ameliorating the adverse effects of Vitamin B12 deficiency in a host which method comprises administering a supplement or food of the invention to the host. The host may be a human or other animal.
In a sixth aspect the invention provides a growth promoter for animal feed comprising Propiofaibacterium jensenii 702 together with a suitable carrier, excipient or diluent.
Feeding animals with propionibacteria can improve their growth, vitamin supply and inhibition of harmful intestinal bacteria without side effects associated with antibiotics or chemotherapeutic growth promoters.
In a seventh aspect the present invention provides a method of improving growth and/or vitamin supply and/or inhibiting harmful intestinal bacteria in a host animal comprising administering an effective dose of the growth promoter of the sixth aspect to the host animal. It will be understood that this aspect of the invention also embraces a method for

the manufacture of a growth promoter of the sixth aspect of the invention for use in a method of improving growth and/or vitamin supply and/or inhibiting harmful intestinal bacteria in a host animal comprising administering an effective dose of the growth promoter of the sixth aspect to the host animal.


In an eighth aspect the invention provides a method of preventing food spoilage and/or extending the shelf life of food products comprising preparing the food by including a fermentation step involving use of Propionibacterium jensenii 702.
Fermented foods have a long consumption history and often have better quality and function than the original food materials.
Dairy starters, including lactic acid bacteria and propionibacteria, can restrict the growth of spoilage and pathogenic organisms due to their ability to produce inhibitory metabolites in fermented products. Inhibitors include broad-spectrum antagonists, organic acids, diacetyl, and hydrogen peroxide. Some starters also produce bacteriocins or bactericidal proteins active against species that usually are related closely to the producer culture.
Propionibacteria can produce propionic acid, propionates, diacetyl and bacteriocins. Propionic acid demonstrates broader inhibition than acetic and lactic acids. Propionic acid and its sodium and calcium salts are effective antimycotics and are added to many baked products to inhibit molds. The propionates also inhibit Gram-negative bacteria but are ineffective against Gram-positive species. Diacetyl has inhibitory effects on yeast, lactic acid bacteria, Gram- positive species and Gram-negative cultures. It has also been reported that Propionibacterium freuderzreichii subsp. shermanii JS has been used with Lactobacillus rhamnosus LC 705 in fermented milks and bread to improve the shelf life of the products. The mixed culture was found to inhibit spoilage yeasts, molds and Bacillus sp.
In a ninth aspect the present invention provides a method of preventing food spoilage and/or extending the shelf life of food products comprising preparing the food by including a fermentation step involving use of Propionibacteriumjetisenii 702 in mixed culture with another food fermentation microorganism.
In a tenth aspect the invention provides a food prepared using Propionibacterium jenseni 702 or parts thereof.
In one embodiment the food is a cultured milk product such as Propioni-acidophilus milk.
In an eleventh aspect there is provided a method of preparing a food comprising using Propionibacteriurra jensenii 702 or parts thereof in the preparation.
Probiotic foods include'fermented'probiotic food and'non-fermented'probiotic food.'Fermented'probiotic food contains live probiotic microorganisms which have grown in the food and produced a fermented product.'Non-fermented'probiotic food contains probiotic microorganisms or their components which have been added to the food without

noticeably changing the organoleptic properties of the food.'Fermented'probiotic foods include probiotic dairy foods, cheese, butter, cream, yoghurt and drinking yoghurt and some frozen foods. The foods may be liquid. The raw materials used in'fermented'probiotic foods include milk, soy milk, grain, legumes, fruit products and vegetable products.


Probiotic foods may contain one or several strains of microorganisms. The food may be prepared by adding the one or more microorganisms to the already prepared food.
Alternatively, the one or more microorganisms may be added to the food during preparation.
This may result in growth of the microorganism (s) within the food and resultant effects on the properties of the food. The microorganism (s) may participate in fermentation of raw materials in the preparation. The microorganism (s) may provide partial or complete fermentation of raw materials in the preparation. The microorganisms used in this aspect may only provide fermentative functions or may provide fermentation and probiotic functions.
The food may be prepared using one or more microorganisms and then have further microorganisms added.
Lactic acid bacteria and probiotic cultures for commercial food production, and starter cultures for fermentation can be purchased from international companies such as Christian Hansen Pty Ltd (Bayswater, Australia) and Gist-Brocades Australia Pty Ltd (Moorebank, Australia). Other research organisations such as the CSIRO Starter Culture Collection (Highett, Australia) and the Australian Starter Culture Research Centre (Werribee, Australia), have good lactic acid bacteria and potential probiotic collections but these organisms are only available on a small scale.
In a twelfth aspect the invention provides a method of treating gastrointestinal disease and/or lactose intolerance in a patient comprising administering a food or supplement of the invention to the patient. In one embodiment the patient is an infant. The patient could also be a non-human animal. It will be understood that this aspect of the invention also embraces a method for the manufacture of a food or supplement of the invention for use in a method of treating gastrointestinal disease and/or lactose intolerance in a patient comprising administering a food or supplement of the invention to the patient.
Propionibacterium strains are used in infant foods in the treatment of gastrointestinal diseases and lactose intolerance. Propioni-acidophilus milk prepared using Lactobacillus acidophilus and P. freudenreic7aii subsp. shermayaii was shown to be more efficacious than acidophilus milk in the treatment of staphylococcal infection of the intestine of infants.
Various cultured milk products have been found to be effective in the treatment of lactose- intolerant infants. The enzyme B-galactosidase, required for lactose hydrolysis, has been found to be present in Propionibacterium at higher concentration than those observed in lactic acid bacteria. It has been recommended that propionic acid bacteria be used for the manufacture of cultured milk products to treat lactose intolerant patients.

In a thirteenth aspect the present invention provides a method for enhancing the growth of bifidobacteria in the gut of a host comprising administering Propionibacterium jensenii 702 or a food or supplement of the invention to the host. The host may be a human or other animal. It will be understood that this aspect of the invention also embraces a method for the manufacture of a food or supplement of the invention for use in a method for enhancing the growth of bifidobacteria in the gut of a host comprising administering Propionibacterium jensenii 702 or a food or supplement of the invention to the host.


Propionibacterium has been shown to be able to enhance the growth of bifidobacteria in vitro. A bifidogenic growth stimulator was found to be present in the cell-free filtrate of Propionibacterium freudenreichii 7025 culture and in the methanol extract fraction of the cells (Kaneko et al. , 1994). This water soluble stimulator is stable to heat and proteolytic enzymes, and is different from vitamin Bi2 and organic acids. This stimulator enhanced the growth of Bifidobacterium bifidum, B. longum, B. breve and B. adolescentis strains. The stimulation effects on bifidobacteria of Propioyzibacteriun a was also tested in healthy human subjects (Bougle et al. , 1999), and the results confirmed the bifidogenic properties of Propiofzi. bacterium fr-eudenreichii.
In a fourteenth aspect there is provided a mixed culture of Propionibacterium jensenii 702 with one or more other probiotic bacteria.
Mixed cultures containing propionibacteria and other probiotic bacteria result in a number of beneficial effects. Mixed cultures of propionibacteria and lactic acid bacteria are widely used in fermentation. When propionibacteria are added with lactic acid bacteria in vegetable fermentation, there are increases in folacin, vitamin B12, propionic and acetic acid contents, inhibition of harmful and pathogenic microorganisms and extension of the shelf-life of the products. Furthermore, mixed cultures of B. longum and P. freudenreichii yield better antimicrobial activities against M. Iuteus, Pseudomo7zas sp., and S. aureus than B. longum alone.
In addition probotic bacteria and formulations thereof of the invention can be combined with other agents such as anti oxidants and other molecules which provide beneficial effect to the probiotic.
In a fifteenth aspect the present invention provides a method for affecting the lipid metabolism of a host comprising administering Propionibacterium jensenii 702 or a food or supplement of the invention to the host. The host may be a human or other animal. It will be understood that this aspect of the invention also embraces a method for the manufacture of a food or supplement of the invention for use in a method for affecting the lipid metabolism of a host comprising administering Propionibacterium jensenii 702 or a food or supplement of the invention to the host.

Propionibacteria can affect the lipid metabolism of their hosts. Mice fed with a diet containing propionibacteria showed a reduced lipid serum concentration. The hypolipemic effect is believed to be due to lower intestinal absorption or higher lipid catabolism because of the presence of the propionibacteria. In another study, various strains of Propionibacterium freudenreichii were tested for cholesterol uptake in brain-heart infusion (BHI) medium supplemented with pleuropneumonia-like organism (PPLO) serum fraction as the cholesterol source. The results indicated that Propionibacterium freudenreichii reduced the cholesterol content in the medium by 50% after 10-14 days of incubation at 32 C.


Furthermore, 70% of cholesterol removed by propionibacteria from the medium can be recovered by solvent extraction from washed propionibacteria cells. The uptake of cholesterol by the propionibacteria did not require strictly anaerobic conditions and was not affected by the presence of bile acids.
In a sixteenth aspect the present invention provides a method for affecting the immune system of a host and alleviating associated disease conditions which method comprises administering Propiotiibacteriuinjensenii 702 or a food or supplement of the invention to the host. The immune system of the host can be affected by way of immune stimulation, immune modulation or through Propionibacterium jensenii 702 or a food or supplement of the invention functioning as an adjuvant. Also Propionibacteriumjensenii 702 or a food or supplement of the invention can provide alleviation of allergy. Further, dead Propionibacterium jensenii 702 or parts thereof can be used for affecting the immune system of a host. The host can be a human or other animal. It will be understood that this aspect of the invention also embraces a method for the manufacture of a food or supplement of the invention for use in a method for affecting the immune system of a host which method comprises administering Propionibacterium jensenii 702 or a food or supplement of the invention to the host.
Propionibacterium can affect the immune system of the host. The injection of Propionibacteriurz avidum KP-40 into mice decreased the colonization of a lymphosarcoma in liver tissue (Pulverer et al., 1994). Propioizibacteriuttzfreudenreichii subsp. sherniaiiii JS has been found to affect the proliferative activity of B and T lymphocytes. An increase in the phagocytic activity of peritoneal macrophages and in carbon-clearance activity was also observed in mice fed with P. acidopropionici. Dead propionibacterium have been use as immune stimulants in horses.
In a seventeenth aspect the invention provides a method for protecting a probiotic microorganism during passage through the gastrointestinal tract of a host which method comprises administering the probiotic in the presence of a food including a soy or cereal based product. The host may be a human or other animal. In one embodiment the soy based product is soy milk. In another embodiment the cereal based product is a cereal beverage.

In an eighteenth aspect the invention provides a method for treating, preventing or ameliorating the adverse effects of high homocysteine in a host which method comprises administering a food or supplement of the invention to the host. The host may be a human or other animal. It will be understood that this aspect of the invention also embraces a method for the manufacture of a food or supplement of the invention for use in a method for treating, preventing or ameliorating the adverse effects of high homocysteine in a host which method comprises administering a food or supplement of the invention to the host.


In a nineteenth aspect the invention provides a method for treating, preventing or ameliorating the adverse effects of P-glucuronidase in a host which method comprises administering a food or supplement of the invention to the host. The host may be a human or other animal. It will be understood that this aspect of the invention also embraces a method for the manufacture of a food or supplement of the invention for use in a method for treating, preventing or ameliorating the adverse effects of (3-glucuronidase in a host which method comprises administering a food or supplement of the invention to the host.
Steroids and some carcinogenic compounds are metabolised in the liver and conjugated with glucuronic acid before secretion via bile into the small intestine. Bacterial - glucuronidase can hydrolyse the glucuronide releasing the apparently harmful compounds (Gadelle et al. , 1985, Fujisawa and Mori, 1996, Nanno et al. , 1986). Strains of Propiofzibacteriufn isolated from human faeces have been found to be (3-glucuronidase positive in vitro (Nanno et al. , 1986). Interestingly, in an in vivo study, the glucuronidase activity in the faeces of mice was reported to be the same or even reduced after the mice were fed with dairy propionibacteria strains, including strains of P. freudenreiclzii and P. acidipropionici (Perez-Chaia et al. , 1999). It has been suggested that the Propionibacterium strains have different glucuronidase activity depending upon it growing in an in vitro or an in vivo environment.
In a twentieth aspect the invention provides a method for reducing the risk of cardiovascular disease in a host which method comprises administering a food or supplement of the invention to the host. The host may be a human or other animal. It will be understood that this aspect of the invention also embraces a method for the manufacture of a food or supplement of the invention for use in a method for reducing the risk of cardiovascular disease in a host which method comprises administering a food or supplement of the invention to the host.
Probiotic Propionibacteria sp. may produce beneficial effects on serum cholesterol.
Somkuti and Johnson (1990) demonstrated ira vitro that P. freudenreichii could uptake cholesterol from a growth medium. There have been a number of ill vivo studies on the cholesterol lowering effects of probiotics and fermented milks. Controversy exists due to the

results being varied and the experimental design often having faults (Taylor and Williams, 1998). Cardiovascular disease (CVD) is one of the world's major killers, being responsible for at least one third of deaths annually (WHO, 2001). High cholesterol is one of the major risk factors for CVD. Recently it has also been realised that high homocysteine levels can also increase the risk of CVD (Ozkan et al. , 2002; van der Griend et al. , 2000). There is an inverse relationship between Vitamin B12 levels and homocysteine. P. jensenii 702 can produce high levels of vitamin Bi2. Therefore the beneficial effects of P. jensenii 702 could extend beyond improving vitamin B, 2 levels in the host, to reducing the risk of CVD by lowering homocysteine levels, and exerting a positive effect on serum lipids.


In a twenty first aspect the present invention provides a method for preventing, treating or ameliorating the adverse effects of an immune imbalance in a host which method comprises administering P. jefzsenii 702, a supplement or food of the invention to the host. The host may be a human or other animal. It will be understood that this aspect of the invention also embraces a method for the manufacture of a food or supplement of the invention for use in a method for preventing, treating or ameliorating the adverse effects of an immune imbalance in a host which method comprises administering a supplement or food of the invention to the host.
In a twenty second aspect the present invention provides a method for modulating the immune response in a host which method comprises administering P. jensenii 702, a supplement or food of the invention to the host. The host may be a human or other animal. It will be understood that this aspect of the invention also embraces a method for the manufacture of a food or supplement of the invention for use in a method for enhancing the immune response in a host which method comprises administering a supplement or food of the invention to the host.
The immune modulation may comprise immune stimulation in which P. jensenii 702, the supplement or food functions as an adjuvant to a particular antigen or antigens in a vaccine. Alternatively P. jensenii 702, the supplement or food may act as a general immune stimulant providing overall stimulation to the recipient's immune system.
In a twenty third aspect the present invention provides a mucosal vaccine for tuberculosis comprising an antigen comprising at least one cellular or secreted protein of Mycobacterium tuberculosis or at least one immunologically active part thereof together with a mucosal adjuvant.
Typically the vaccine is an oral vaccine. The vaccine may alternatively be administered via another mucosal surface. An example is nasal administration. It will be understood that the adjuvant chosen is suited to the route of administration; for instance for an oral vaccine an effective oral adjuvant is used.

In one embodiment the oral adjuvant is a bacterial vector. In a preferred embodiment the oral adjuvant is P. jensenii 702, or part thereof.


The antigen is typically selected from the group consisting of whole M. tuberculosis cells, disrupted M. tuberculosis cells or parts thereof, and soluble M. tuberculosis proteins which are either secreted or cellular or a combination of secreted and cellular soluble proteins, collected from actively multiplying M tuberculosis cultures. The antigen can be provided as a short term culture filtrate. The antigen can be a combination of whole cell sonicate and short term culture filtrate. Antigen preparations comprising soluble proteins can be fractionated into different size ranges. For example, an initial size separation into fractions of < 20kDa, 20- 30kDa, 30-35kDa, 35-65kDa, and > 65kDa can be employed to determine the fraction or fractions which provide a suitable response. These size fractions can be further refined.
Combinations of selected fractions of secreted and cellular proteins can be utilised with the proportion of each fraction optimised to produce the desired immune response.
In one embodiment the antigen comprises an effective amount of a combination of secreted and cellular soluble proteins present at an effective ratio.
The vaccine is administered at an effective dose as determined in accordance with routine practice in this field. An effective dose is sufficient to produce immunity but not large enough to induce tolerance.
The number and timing of administrations is also determined in accordance with routine practice in this field.
The vaccine may be formulated in accordance with standard procedures for the preparation of mucosal vaccines. The vaccine may, for instance, be formulated as a liquid, tablet, powder, as part of a food or in an aerosol.
The vaccine may provide a systemic or a mucosal immune response or both in the host to Mycobacterium tuberculosis.
In a twenty fourth aspect the present invention provides a method for producing an immune response in a patient to Mycobacterium tuberculosis which method comprises delivering a vaccine of the twenty third aspect to a patient via a mucosal surface.
In a twenty fifth aspect the present invention provides a method for vaccinating a patient against Mycobacterium tuberculosis which method comprises delivering a vaccine of the twenty third aspect to a patient via a mucosal surface.
In a twenty sixth aspect the present invention provides a method for protecting a patient against Mycobacterimn tuberculosis infection which method comprises delivering a vaccine of the twenty third aspect to a patient via a mucosal surface.
In a twenty seventh aspect the present invention provides a method for protecting a patient against Mycobacterium tuberculosis reactivation which method comprises delivering a vaccine of the twenty third aspect to a patient via a mucosal surface.

In a twenty eighth aspect the present invention provides a method for protecting a patient against tuberculosis which method comprises delivering a vaccine of the twenty third aspect to a patient via a mucosal surface.


In a twenty ninth aspect the present invention provides the use of an antigen comprising at least one cellular or secreted protein of Mycobacterium tuberculosis or at least one immunologically active part thereof in the manufacture of a vaccine for use in a method for producing an immune response in a patient to Mycobacterium tuberculosis which method comprises delivering a vaccine of the twenty third aspect to a patient via a mucosal surface.
In a thirtieth aspect the present invention provides the use of an antigen comprising at least one cellular or secreted protein of Mycobacterium tuberculosis or at least one immunologically active part thereof in the manufacture of a vaccine for use in a method for vaccinating a patient against Mycobacterium tuberculosis which method comprises delivering a vaccine of the twenty third aspect to a patient via a mucosal surface.
In a thirty first aspect the present invention provides the use of an antigen comprising at least one cellular or secreted protein of Mycobacterium tuberculosis or at least one immunologically active part thereof in the manufacture of a vaccine for use in a method for protecting a patient against Mycobacterium tuberculosis infection which method comprises delivering a vaccine of the twenty third aspect to a patient via a mucosal surface.
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