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

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88. WO2004001022 - 31.12.2003


IP Class 4 Digits: A61K; A23K; C12N; A23L; C12P
IP Class: A23K1/00; C12N1/20; A23L1/29; C12P1/04; A23L1/00; A61K39/05
E Class: A61K35/74; A23K1/00C2B; C12N1/20; A23L1/03M; A23L1/30M; C12R1/01; A23L1/30B; A61K39/04; C07K14/35
Application Number: WO2003AU00775 (20030620)
Priority Number: AU2002PS03124 (20020621); AU2002PS03152 (20020624); AU20020952793 (20021118)
Family: WO2004001022
Cited Document(s): US5639659; EP1060745; EP1308506


Probiotic Propionibacterium jensenii 702

Technical Field

The present invention relates to probiotic Propionibacterium strains and their use in the preparation of probiotic supplements and foods. The invention relates to the provision of Vitamin B12, propionic acid, folacin and bacteriocins by probiotic strains, stimulation of bifidobacteria growth, production of favourable effects on the lipid metabolism and on the immune system of hosts through immunostimulation, immunomodulation or use of a probiotic strain as an adjuvant, reduction of homocysteine and ss glucuronidase and the prevention, treatment or amelioration of conditions associated with a need for these activities.
The probiotic bacteria of the invention can be used in humans or other animals. In at least some applications, the bacteria can be used dead and parts rather than whole cells may be used. The present invention also relates to the preparation of vaccines for use in protecting patients from infectious diseases, in particular tuberculosis.
Background Art


Probiotics, which are introduced into the gastrointestinal tract, can influence gastrointestinal microflora and play a beneficial role in the host. The term'probiotic'is derived from the Greek meaning'for life'. It was first used to describe substances secreted by one microorganism which stimulated the growth of another microorganism (Lilly and Stillwell, 1965). In 1992, Havenaar suggested that probiotics be defined as'mono-or mixed cultures of live microorganisms which, when applied to animal or man, beneficially affect the host by improving the properties of the indigenous microflora (Havenaar et al. , 1992).
Havenaar's definition was the first that applied the term probiotics to both humans and animals. In consideration of the current applications and proven effects of probiotics, Salminen et al (1999) proposed a new definition: Probiotics are microbial cell preparations or components of microbial cells that have a beneficial effect on the health and well-being of the host. This definition includes microbial cells (viable or non-viable) and parts of cells as probiotics, but not metabolites such as antibiotics. This definition also indicates the application of probiotics is not restricted to use in foods.
Recently some evidence has indicated that dairy Propionibacterium strains can have beneficial effects, including the production of propionic acid, folacin, bacteriocins, and vitamin B12 ; stimulation of bifidobacteria growth, and favourable effects on the lipid metabolism and the immune system of hosts. The use of Propionibacteriun2 strains enhances the nutritional and therapeutic qualities of the food, extends the shelf life of the product,

inhibits the growth of pathogenic and harmful microflora and contributes better organoleptic properties to the product.

Dairy propionibacteria consist of four species in the genus Propionibacterium : P. freuderaf-eichii ; P. acidopropionici ; P. jensenii ; and P. tl7, oeiiii. All four dairy propionibacteria species are found to live in foods, which include any fresh, uncooked food, such as vegetables ; fruits, nuts, germinated seeds, beans, dairy products and certain fermented items.
Propionibacteria are widely used as starter cultures in the food industry.
Propionibacterium species are traditionally used during the manufacture of Swiss or Swiss- type cheeses, producing'eyes'and the typical sweet flavour of Swiss cheeses.
Propionibacterium species are also used in vegetable fermentation, as food and feed additives, in infant foods and for the biosynthesis of propionic acid and vitamin Bon.
Where the probiotic effect depends on a viable organism, a probiotic strain cannot affect its host unless its population reaches a certain minimum level around 106 to 108 cfu/g of intestinal contents. Such probiotic bacteria, which are delivered orally, have to survive the upper gastrointestinal tract, then reach and persist in the lower part of the gut to exert their beneficial effects on the host.
The low pH of the stomach and the antimicrobial action of pepsin are known to provide an effective barrier against entry of bacteria into the intestinal tract. The pH of the stomach ranges from 2.5 to 3.5, but can reach as low as pHl. 5, or as high as pH6 or above after food intake. The type of food affects the emptying of the stomach. Normally, food remains in the stomach between two and four hours, however, liquids empty from the stomach within about 20 minutes. Extensive in vitro tests have been used to select gastric transit tolerant, including acid tolerant, lactic acid bacteria (Charteris et al, 1998; Clark et al,

1993; Chou and Weimer, 1999). There has been, however, only limited research into the in vitro acid tolerance of propionibacteria and this was with two Propionibacterium freudenreichii strains (Jan et al 2000; Mantere-Alhonen, 1983).

Another barrier probiotic bacteria must survive is the small intestine. The adverse conditions of the small intestine include the presence of bile salts and pancreatin. The transit time for food through the small intestine is generally between one and four hours. Bile salt- resistant lactic acid bacteria can be selected by testing their ability to survive in the presence of bile salt and their growth in selective medium with various levels of bile (Gilliland et al,

1984; lbrahimand Bezkorovainy, 1993; Clark and Martin, 1994; Chung et al, 1999). A concentration of 0.15-0. 3% bile salt has been reported as a suitable concentration to select probiotics for human use. No report has been found concerning the small intestinal transit tolerance of Propionibacterium strains.

After surviving passage through the upper gastrointestinal tract, probiotic bacteria need to adhere to the gut epithelium in order to colonize and persist in the gastrointestinal

tract. The complexity of the intestinal mucosa and its microflora make it very difficult to study bacterial adhesion iii vivo. Living probiotic microorganisms can provide nutritional benefits either during the preparation of fermented probiotic foods or in the digestive tract of hosts. After fermentation, the texture and flavour of raw materials can be significantly improved; the adverse effects of some components in foods can be reduced, such as food intolerance and allergies caused by some oligosaccharides and proteins; levels of amino acids and vitamins can be increased to improve the nutritive value of the food; and sugars and other spoilage promoting components of foods are removed, which leads to longer shelf-life and ensures food safety. Evidence has been presented that the bioavailability of calcium, zinc, iron, manganese, copper and phosphorus is increased in fermented yoghurt compared to in milk. Studies have also demonstrated an increase in riboflavin and niacin in yoghurt, vitamin B6 in cheddar cheese, vitamin B12 in cottage cheese and folic acid in a variety of products including yoghurt, cottage cheese, Cheddar cheese and sour cream. The enzymatic hydrolysis of probiotic microorganisms has also been shown to enhance the bioavailability of protein and fat. Bacterial protease can increase the production of free amino acids which can benefit the nutritional status of the host especially if the host has an endogenous protease deficiency.

Consumption of foods containing viable probiotics is reported to produce health benefits which include (1) alleviation of intestinal disorders such as constipation and diarrhoea caused by infection by pathogenic organisms, antibiotics, or chemotherapy; (2) stimulation and modulation of the immune system; (3) anti-tumor effects due to inactivation or inhibition of carcinogenic compounds in the gastrointestinal tract by reduction of intestinal bacterial enzyme activities such as 0-glucuronidase, azoreductase, and nitroreductase; (4) reduced production of toxic end products such as ammonia, phenols and other metabolites of protein known to influence liver cirrhosis (5) reduction in serum cholesterol and blood pressure; (6) maintenance of mucosal integrity; (7) alleviation of symptoms of lactose intolerance; (8) prevention of vaginitis.
Non-viable probiotics and cell wall components of some probiotic strains also have beneficial effects on hosts and are less likely to cause safety concerns than intact cells.
Propiovibacterium strains contain uniquely high concentrations of vitamin B12 (Hettinga and Reinbold, 1972a) and their vitamin B, 2 production has been considered the most efficient (Hettinga and Reinbold, 1972b). Propionibacterium strains are often used for vitamin B 12 enrichment of cultured milk products and fermented beverages. Inclusion of P. freudenreichEi subsp. shermanii results in an increase of vitamin B12 in Kefir, Streptococci cultured milks, Tvorog cheese, Domiati cheese, and Zabady cheese. Inclusion of P. freudenreichii subsp. shernianii increases folic acid content in cultured milk products and fermented beverages, including Kefir and Streptococci cultured milks.

Vitamin Bi2 production by propionibacteria has been correlated with cellular yields.

The vitamin B 12 production level is affected by many factors, including the presence of cobalt, inclusion of different media supplements, dissolved oxygen concentration, and the pH of the fermentation broth (Hettinga and Reinbold, 1972b, Quesada-Chanto et al. , 1994b, Berry and Bullerman, 1966, Quesada-Chanto et al., 1998).
Vitamin B12 is essential for the growth and health of human beings. It is required for the production of red blood cells and myelin surrounding nerves, and acts as a coenzyme in the metabolism of fatty acids, carbohydrates and proteins. Natural synthesis of vitamin B12 occurs only in microorganisms. Microbial synthesis of vitamin Bl2 does occur in the human colon, but it is not absorbed. Therefore, human beings appear to be entirely dependent on a dietary intake of vitamin B12 to maintain adequate serum levels and body store.
Vitamin B12 is a term used in nutritional literature, however, in most biochemical and chemical texts, cobalamin is used rather than vitamin B12. The term'vitamin B12'now is used as a generic name for cobalamins which exhibit anti-pernicious anaemia activity.
Vitamin B12 deficiency is reported to be one of the risk factors for heart disease, multiple sclerosis, stroke, breast cancer, Alzheimer's disease, some psychiatric syndromes and accelerated aging (Choi, 1999, Wynn and Wynn, 1998, Gariballa, 2000, Delva, 1997).
The prevalence of vitamin B12 deficiency varies with the age and general health of the population, the normal serum vitamin B12 threshold, and other criteria required for diagnosis.
A more prevalent, mild and preclinical cobalamin deficiency has been recently recognised.
Preclinical deficiency is the state in which metabolic evidence of insufficiency exists in a person who has no clinical symptoms, including megaloblastic anaemia (Carmel, 2000).
Vitamin B 12 deficiency may be caused by either hereditary factors or acquired factors.
Most vitamin B12 deficiency is associated with inadequate nutritional intake. Animal products, such as meat, fish, egg, and dairy products contain adequate amounts of vitamin B, 2 for the human body. Vegetarian diets generally have levels of vitamin B12 lower than the RDI.
People on vegetarian diets and their infants tend to have a low level of vitamin B 12 and develop vitamin B12 deficiency. Studies show that low serum vitamin B12 levels are found not only in strict vegetarians but also lactovegetarians and lacto-ovovegetarians. Vitamin Bl2 deficiency is a real hazard in unsupplemented or unfortified vegan and vegetarian diets. Many processed vegetarian foods are fortified with vitamin Buzz however these foods are not available to some vegetarians because of ethical or economic reasons. Vitamin B12 deficiency has been shown to result in retardation of growth and psychomotor development in children.
Therefore, particular care should be taken with children on vegetarian diets to ensure adequate vitamin B12 intake occurs.
Another cause of vitamin B 12 deficiency is food-vitamin B12 malabsorption.

Laboratory rats have been used to study the effects of vitamin B, 2 deficiency. Vitamin Bl2 deficiency causes growth retardation, energy metabolism depression and an increase in urinary methylmalonic acid (MMA) excretion in rats. Vitamin B12 deficiency also affects testicular tissue in rats.

Treatment for vitamin B12 deficiency varies according to the cause of the deficiency.
The two most frequent treatments for vitamin B12 deficiency are parenteral injection and oral therapy.
It has been found that large numbers of patients do not seek medical advice in the early stages of vitamin B12 deficiency. Vitamin B12 food supplements and common food fortification with vitamin B12 could prevent the occurrence of vitamin B12 deficiency in some patients, reduce the problems of deficiency and reduce health care costs.

All vaccines have one aim, this being to"prime the immune system to swiftly destroy specific disease-causing agents, or pathogens, before the agents can multiply enough to cause symptoms" (Langridge, 2000).

The world's biggest killer from a single infectious agent is tuberculosis (TB) (WHO,

1992). Tuberculosis is a disease that primarily affects the lungs, and is caused by the bacterium Mycobacteriunn tuberculosis. Currently there are 2 billion people infected with the bacterium (Agger & Andersen, 2001). This equates to one third of the world's population.

The majority of these cases are in the developing world where health care is at a minimum.
Three million people die annually from the disease that has a fatality rate of 50% if left untreated (Collins & Kaufmann, 2001). The vaccine currently available for TB, the BCG, has varying rates of effectiveness ranging from 0-80% in different populations (Guerin, 1997), and to date has been largely ineffective in preventing the proliferation of this disease. It has been estimated that the development of a new vaccine with only 50% efficacy could prevent 9 million deaths from tuberculosis by the year 2030 (Murray & Salomon, 1998).
M. tuberculosis is a highly successful pathogen that has evolved numerous mechanisms to evade the immune response of the host (Flynn, 1999). The human immune response to TB is unique and complicated, involving a strong T helper 1, cell mediated response. The challenges of the bacterium itself, combined with attempting to stimulate the correct aspects of the immune system, has made years of attempts to produce a vaccine significantly better than the BCG, futile.
Tuberculosis, in its active form, causes symptoms such as a persistent cough, chest pain, fever, weight loss, night sweats and in advanced cases bloody sputum (Vaccine Weekly, editorial, 2001a). Tuberculosis is primarily transmitted through the air suspended in tiny droplets of sputum via coughing, sneezing, talking and laughing (Miller & Schieffelbein,

1998). However repeated exposure over a prolonged period of time, in conjunction with the

intensity of exposure, is necessary for transmission and establishment of disease in the host (NIAID, 1999; Miller & Schieffelbein, 1998).

With proper antibiotic treatment, there is a 90% cure rate (NIAID, 1999; Collins & Kaufmann, 2001). However there are problems with this method of treatment. Administering treatment requires supervision to ensure compliance, it is relatively expensive, has many side effects and a cure requires more than six months treatment (Broker, 1999). A recent study has shown that resistance has been developed in a multitude of countries to the four main drugs used to treat TB, isoniazid, rifampin, ethambutol and streptomycin (Mustafa, 2002). This means that infection with multi-drug resistant tuberculosis (MDR-TB) strains cannot be effectively treated with any of the available drugs, and the death rate of MDR-TB patients is 40-60% (NIAID, 1999).
Mycobacteriu a tuberculosis can infect a host but reside dormant inside the body without exhibiting symptoms of active disease. This is known as a latent tuberculosis infection. The danger with latent infections is that they can reactivate at any time, usually when the host is in a weakened state, particularly when co-infected with auto-immune diseases such as HIV/AIDS or in the elderly (NIAID, 1996; Tsuyuguchi, 1996). More than 50% of people with a HIV/TB co-infection will develop active tuberculosis. When the immune system is weakened the bacteria breaks out of the macrophages, where it was residing in a dormant state, and enters the blood stream to cause disease. If left untreated, 50% of those with active disease will die (NIAID, 1996). The majority of tuberculosis cases occur in the world's poorest nations (Kaufmann & Hess, 2000), where health care is at a minimum and malnutrition and HIV are commonplace.
The most popular initiative since BCG to combat tuberculosis, and the ensuing proliferation of resistant stains of TB, is WHO's directly observed therapy short-course (DOTS) (Collins & Kaufmann, 2001). This strategy aims to increase patient compliance to taking the full course of standardised short-course chemotherapy (Gleissberg, 1999). This in turn aims to eliminate the conditions that select out antibiotic resistant strains of M. tuberculosis by ensuring the full course of medication is taken. However this strategy, is likely to be as ineffective as BCG at controlling the problem. Statistics show that the success rate of such therapy is only 62% in certain African countries (Collins and Kaufmann, 2001).
Consequently there is a general agreement that TB will not be eradicated from the world through chemotherapeutic regimes alone (Hess & Kaufmann, 1999).
BCG is the most widely used vaccine in the world (Kaufmann and Hess, 2000). To date over 3 billion doses have been administered (Collins, 2000). A controlled, clinical trial beginning in 1968 in India involving 265,000 subjects, showed that participants in the placebo group had marginally better immunity to TB than those that received BCG (Tripathy, 1987).
BCG was created at the start of last century and consists of a live attenuated strain of

Mycobacterium bovis, the strain of mycobacterium that causes tuberculosis in cattle. This strain is closely related to M. tuberculosis with a greater than 90% DNA homology, hence it was expected that it would prime an excellent immune response to infection with M. tuberculosis (Andersen, 2001). Despite this the BCG vaccine has not been the solution to tuberculosis it was anticipated to be when it was created (Ravn et al, 1997). Recent research has shown that the BCG vaccine actually lacks vital proteins with good antigenic properties that have now been identified as playing a pivotal role in the immune response to TB (Ravn et al, 1997).

It is thought that the lack of certain essential antigens in BCG is due to the occurrence of genetic deletions during the attenuation process (Elhay & Andersen, 1997). BCG was created by attenuating M. bovis through serial passage on bile-containing agar plates (Kaufmann & Hess, 2000). These valuable proteins, now lost, include the intensively researched esat-6, which is strongly recognised in tuberculosis patients (Collins, 2000).
Recent field trials have shown protection levels from BCG to be very low and in some cases undetectable (Andersen, 2001).
Brandt et al (2002) confirmed the long held hypothesis that exposure to certain environmental mycobacteria stimulates an immune response that controls the multiplication of BCG, and hence prevents a full vaccine-induced immune response from developing (Brandt et al, 2002). Although exposure to environmental bacteria does prime the immune system and provides a certain level of protection from other mycobacteria, the protection is not sufficient to significantly reduce the growth of M. tuberculosis in animal models (Brandt et al, 2002).
This study concluded that"BCG, as a live vaccine, is particularly sensitive to the influence of preexisting immune responses to antigens shared between M. avium and BCG" (Brandt et al, 2002), and hence can account, in part for the ineffectiveness of the BCG vaccine. In addition to this BCG is also a hindrance to the detection and early treatment of the disease by producing false positives on the main tool for diagnosis, the PPD skin test (Fatkenheuer et al, 1999).
Despite BCG's clearly evident downfalls and inadequacies, the synthesis of new prototype vaccines has yet to produce immune responses in animal models that are significantly more effective than BCG (Andersen, 1994). The major reason why BCG has continued to be administered around the world is the advantage of the vaccine being safe, easy to produce and cheap, at only a few cents per shot (Orme, 1999).
The above discussion of background art is included to explain the context of the present invention. It is not to be taken as an admission that any of the documents or other material referred to was published, known or part of the common general knowledge in Australia at the priority date of any one of the claims of this specification.

Throughout the description and claims of this specification, the word"comprise"and variations of that word, such as"comprising"and"comprises"are not intended to exclude other additives, steps or integers.

Description of the Invention

In a first aspect the present invention provides a novel isolated Propionibacterium strain, Propionibacterium jenseyiii 702. Propionibacterium jefasenii 702 was deposited under the provisions of the Budapest Treaty at the Australian Government Analytical Laboratories, 1 Suakin Street Pymble NSW 2073 Australia under accession number NM02/29517 on 12 June 2002.

In a second aspect the invention provides a formulation comprising Propionibacteriurn jensenii 702 together with a culture medium, storage medium excipient, carrier or diluent.
In a third aspect the present invention provides use of Propionibacterium jensenii 702 as a probiotic strain. The strain can be used as a probiotic in humans or in other animals. The bacterium can be used alive or dead and parts of the bacterium can be used.

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