WO2005060937A1

WO2005060937A1 - Compressed tablets comprising viable probiotic microorganisms

Info

Publication number: WO2005060937A1
Application number: PCT/DK2004/000913
Authority: WO
Inventors: Karin Lund Nielsen; Svend Laulund; Inge Knap; Mette Winning
Original assignee: Chr. Hansen A/S
Priority date: 2003-12-23
Filing date: 2004-12-22
Publication date: 2005-07-07

Abstract

The present invention relates a probiotic composition in the form of a compressed tablet containing dried, viable, probiotically active organisms. The tablet formulation comprises a stabilizing agent which facilitates the manufacture of tablets, that contain probiotically active organisms, by direct compression of the formulation into tablets. A process for the preparation of a probiotic composition in the form of a compressed tablet comprising viable probiotically active organisms is provided. The invention also describes the use of the probiotic composition in the form of a compressed tablet to prepare a medicament for treating or preventing of diseases or improving the general health status of a mammal.

Description

COMPRESSED TABLETS COMPRISING VIABLE PROBIOTIC MICROORGANISMS

FIELD OF THE INVENTION

The present invention relates to the field of formulations containing probiotically active organisms. In particular a formulation and a process is provided which facilitates the manufacture of tablets, that contain probiotically active organisms, by direct compression into tablets.

TECHNICAL BACKGROUND OF THE INVENTION

Probiotics constitute a class of microorganisms defined as live microbial organisms that beneficially affect animal or human hosts. The beneficial effects include improvement of the microbial balance of the intestinal micro flora and the improvement of the properties of the indigenous microflora. The beneficial effects of probiotics may be mediated by a direct antagonistic effect against specific groups of undesired organisms, resulting in a decrease of their numbers, by an effect on the metabolism of such groups of organisms or by a general stimulatory effect on the immune system of animal or human hosts. Probiotics may suppress undesired intestinal organisms by producing antibacterial compounds and/or by successful competition for nutrients and/or adhesion sites in the gastrointestinal tract. Additionally, they may alter microbial metabolism by increasing or decreasing enzyme activity or they may stimulate the immune system by increasing antibody levels or increasing macrophage activity. Probiotics may even express antitumour activity or facilitate lowering of blood cholesterol levels, Fuller (1989); Elmer (2001 )).

Probiotic microorganisms have been identified among microorganisms classified as yeasts, fungi and bacteria. For instance, the yeast Saccharomyces boulardii and the bacterium Lactobacillus rhamnosus strain GG have demonstrated their efficacy in clinical tests for the prevention of gastrointestinal disorders of antimicrobial-associated diarrhea and also filamentous fungi e.g. Aspergillus oryzae have been assigned a probiotic effect (Elmer (2001); Salminen (2001 ); Andersson (2001); Jouany (1998))

Lactic acid bacteria are in general recognised as being useful as probiotics or "probiotically active" organisms, i.e. organisms that may beneficially affect animal or human hosts. As used herein the term "lactic acid bacteria" refers to gram-positive, microaerophilic or anaerobic bacteria which ferment sugars with the production of acids including lactic acid as the predominantly produced acid, acetic acid, formic acid and propionic acid. The industrially most useful lactic acid bacteria are found among Lactobacillus, Streptococci and Bifidobacterium species (Fuller (1989)).

Compressed tablets seem to be the dominant dosage form for self-administration of pharmaceutical compositions. Tablets can be produced at a high speed and consequently at low prices (Laulund (1994)) and consumers are used to and frequently prefer this dosage form. Thus there is a need for formulations and a method which allows the manufacture of compressed tablets containing viable probiotically active organisms.

To provide health benefits, the suggested minimal concentration for probiotic bacteria is 10⁶ CFU/g of a product (Shah (2000)). However, during compression into tablets the tablet formulation is subjected to relatively high pressure for a short period of time. Many probiotically active organisms, including the most interesting probiotic lactic bacteria, are highly sensitive to the pressure caused when probiotically active organisms are formed into tablets by direct compression (Laulund (1994))

Although prior art provides several examples of probiotic containing powders, capsules, and extruded granula, the quantity of literature describing production methods, that ensure a minimum reduction of the number of viable lactic acid bacterial in tablet preparations is very limited and obsolete (Laulund (1994)).

EP 1 213 347 describes a method to produce probiotic containing dry granules by extrusion while ensuring a minimal loss of viability. In EP 1 213 347 it is contemplated that the extruded granules may be formed into tablets in a process subsequent to the extrusion, but no examples were provided.

The prior art further provides WO 00/07571 which discloses a formulation in the form of a sodium bicarbonate powder or an effervescent tablet containing lyophilised microorganisms and buffering substances. The buffering substances were added to the probiotic organisms after the organisms were freeze-dried and thus did not act as a stabilizing agent in the sense of the present invention.

The present invention is based on the surprising discovery that an aqueous suspension of probiotically active organisms can be formulated with a stabilizing agent so that the subsequent drying and further processing results in a formulation which can be formed into tablets by direct compression in a tablet press while retaining sufficient amounts of probiotically active organisms.

SUMMARY OF THE INVENTION The present invention provides in a first aspect a probiotic composition in the form of a compressed tablet comprising dried, viable, probiotically active organisms and a stabilizing agent comprising both an antioxidant, a bulking agent and a gelatinizer.

In a second aspect, the invention provides a process for the probiotic composition in the form of a compressed tablet comprising viable probiotically active organisms comprising the following steps: (a) culturing one or more cultures of probiotically active organisms to obtain a concentrated aqueous suspension of the cells, said suspension containing at least 10⁵ CFU per g; (b) mixing into the concentrated aqueous suspension of the cells a stabilizing agent comprising both an antioxidant, a bulking agent and a gelatinizer; (c) reducing the water content of the mixture to obtain a dry composition; (d) optionally reducing the size of the material of step (c) by crushing, grinding or milling to obtain a granulate; (e) optionally blending the granulate of step (d) with one or more pharmaceutically acceptable binders filling agents, lubricants, diluents, disintegrating agents, colouring agents, flavouring agents, flow-inducing agents, melting agents or other excipients and optionally further dry blending the granulate of step (d) with a component having a buffering capacity at a pH in the range of from 2 to about 3.9 to form a directly compressible formulation; and (f) compressing the granulate of step (d) or (e) for a short period of time in a tablet press to form a compressed tablet of the desired shape.

In a third aspect, the invention provides a method for reducing the occurrence of infections with pathogens, the method comprising the step of administering a tabletted composition comprising dried, viable, probiotically active organisms and a stabilizing agent comprising a combination of an antioxidant, a bulking agent and a gelatinizer.

In a further aspect, the invention describes the use of the probiotic composition for the preparation of a medicament for treating or preventing diseases or improving the general health status of a mammal.

DETAILED DISCLOSURE OF THE INVENTION

It is a primary result of the present invention that the inventor is able to disclose a generally useful probiotic composition in the form of a compressed tablet comprising viable, probiotically active organisms, and a method by which the probiotic tablet dosage form can be produced by direct compression in a tablet press from a dried composition comprising probiotically active organisms and a stabilizing agent comprising both an antioxidant, a bulking agent and a gelatinizer.

The inventor of the present invention surprisingly discovered that a stabilizing agent comprising both an antioxidant, a bulking agent and a gelatinizer which was mixed with the probiotic cells while the cells still were suspended in an aqueous phase allowed the manufacture of a dried composition that can be formed into tablets by direct compression by use of a tablet press, while retaining cell viability. According to the invention, tablets containing 10⁵, 10⁶ , 10⁹ or even 10¹¹ CFU per g can be manufactured.

Various antioxidants have been reported to stabilize microorganisms during the drying process and also extend shelf life of the dried organism. However, as can be seen in example 15-17 in addition to antioxidants, both a gelatinizer and a bulking agent are required to obtain the optimal stabilization. According to the invention the antioxidant can be selected from the group consisting of gallates, L- ascorbic acid, lycopene, beta-carotene, lutein, resveratrol (trans-3,4',5-trihydroxystilbene) and isomers and derivatives thereof, tocopherols such as vitamin E [(+)-alpha-tocopherol], butylhydroxyanisol (BHA), butyl hydroxytoluen (BHT; 2,6-di-tert-butyI-p-cresoI), homocysteine, cysteine, cystathionine, methionine, gluthatione, salts thereof and combinations thereof. Also peptides containing any one of homocysteine, cysteine, cystathionine, methionine, gluthatione, pharmaceutically acceptable salts thereof, and combinations of any of the foregoing are contemplated to provide the protective effect. In a preferred embodiment the antioxidant is selected from the group consisting of propyl gallate (3,4,5- trihydroxybenzoic acid n-propyl ester) and sodium L-ascorbate and a mixture thereof and examples of the use of both propyl gallate and sodium L-ascorbate are provided in the experimental section below.

Although the invention is not limited to any specific mechanism or explanation, it is assumed that the bulking agent and the gelatinizer of the stabilizing agent during the drying process form a 3-dimensional matrix in which the probiotic microorganisms "hide" in spaces formed by the components of the stabilizing agent, and thus escape the mechanical damage during compression. By a "gelatinizer" is meant a compound - or mixture of compounds - which when dissolved in a suitable solvent, e.g. water, is able to form a gel i.e. a three dimensional matrix containing a major part of the solvent. Typically gelatinizers are macromolecules, such as gelatin, agar, or alginate. Preferred gelatinizers in the present invention are macromolecules having hydrocolloid properties. According to the invention the bulking agent may be selected from the group of bulking agents consisting of starches, dextrose, cellulose, saccharides, inositol, skimmed-milk powder, yeast extract, caseinate, polyoles and sugar alcohols and the gelatinizer may be selected from the group consisting of alginic acid, sodium alginate, potassium alginate, ammonium alginate, calcium alginate, agar, carragenan, locust bean gum, guar gum, guar flour, tragacanth, konjac glucomannan, propylene glycol alginate, PES (Processed Euchema seaweed), acacia gum, xanthan gum, karaya gum, tara gum, gellan gum, and combinations thereof.

In preferred embodiments the gelatinizer is an alginate or a mixture of alginates and the bulking agent is selected from the group of saccharides, inositol or skimmed milk powder. The saccharide may be any saccharides selected from the group consisting of sucrose, dextrose, fructose, arabinose, galactose, mannose, dextran, maltodextrin, rhamnose, ribose, xylose, tetrulose, ribulose, xylulose, psicose, sorbose, tagatose, lactose, maltose, isomaltose, maltotriose, raffinose, trehalose, cyclodextrins, maltodextrins, cellobiose, lactulose, maltulose, melibiose, neotrehalose, ® maltotriose, manninotriose, stachyose, Raftilose (registered trademark belonging to Raffinerie

Tirlemontoise S.A. (Tiense Suikerraffinaderij)), fructooligosaccharides and fructooligomers having prebiotic properties, pharmaceutically acceptable salts thereof, and combinations of any of the foregoing.

In particular preferred embodiments illustrated in the experiments the gelatinizer is an alginate or a mixture of alginates and the bulking agent is selected from the group of sucrose, inositol or skimmed milk.

It is further contemplated that the stabilizing agent in addition to an antioxidant, a bulking agent and a gelatinizer further may comprise one or more cryoprotective agent(s). We have surprisingly found that one or more of the compound(s) involved in the biosynthesis of nucleic acids or one or more derivative(s) of any such compounds may act as cryoprotective agent(s) of lactic acid bacteria. In particular, agents selected from the group of purine bases, pyrimidine bases, nucleosides and nucleotides have proven their value and is expected in combination with the antioxidant, the bulking agent and the gelatinizer further to protect the probiotically active organism. In particular inosine and IMP are preferred.

As previously mentioned it is assumed that the stabilizing agent of the present invention forms a 3- dimensionai matrix which protects the microorganisms from mechanical damage during the compression. It is contemplated that this in general provides protection of microorganisms to brief mechanical compression such as those occuring during the formation of tablets in a tablet press. Thus the invention can be extended to a wide range of cells selected from cultures comprising probiotically active microorganisms, including fungal species, yeast species and bacterial species and combinations thereof.

The range of probiotically active microorganisms, which are suitable for use according to the invention, includes probiotically active fungal species, yeast species and bacterial species. Examples of currently useful filamentous fungi include e.g. probiotically active Endothia parasitica and Trametes species, Aspergillus species, such as e.g. Aspergillus oryzae, Rhizopus species, Mucor species, Penicillium species such as e.g. Penicillium roqueforti and Penicillium candidum and Torulopsis species. Useful probiotically active organisms also include yeast species such as Pichia species, Torulopsis species, Kluyveromyces species such as e.g. Kluyveromyces maxianus and Kluyveromyces thermotolerans, Torulospora species such as e.g. Torulospora delbrueckii and furthermore the following Geotrichum candidum, Torula kefir, Candida valida, Debaryomyces species and Saccharomyces species e.g. Debaryomyces hansenii, Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces carl- bergensis and Saccharomyces kefir.

In presently preferred embodiments, the probiotically active bacterial species are selected from the group consisting of the genera Aerococcus, Bacillus, Bacteroides, Bifidobacterium, Clostridium, Enterococcus, Fusobactehum, Lactobacillus, Lactococcus, Leuconostoc, Melissococcus, Micrococcus, Pediococcus, Peptostrepococcus, Propionibacterium, Staphylococcus, Streptococcus and Weissella. Specific examples of important embodiments comprise probiotical active lactic acid bacterial species including Enterococcus faecalis, Enterococcus faecium, Lactobacillus acidophilus, Lactobacillus alimentarius, Lactobacillus casei Shirota, Lactobacillus casei subsp. paracasei, Lactobacillus casei subsp. casei, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbruckii subsp. lactis, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus farciminus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus paracasei subsp. paracasei, Lactobacillus rhamnosus, Lactobacillus plantarum,

Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sake, Lactococcus lactis, Lactocoocus lactis subsp. cremoris, Streptococcus faecalis, Streptococcus faecium, Streptococcus salivarius and Streptococcus thermophilus. Further examples of important embodiments comprise probiotically active Bifidobacterium species including Bifidobacterium infantis, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium lactis, Bifidobacterium animalis and Bifidobacterium breve.

Further bacterial species that are useful in the present invention can be selected from the group consisting of probiotically active Paenibacillus lautus, Bacillus coagulans, Bacillus licheniformis, Bacillus subtilis, Micrococcus varians, Pediococcus acidilactici, Pediococcus pentosaceus, Pediococcus acidi- lactici, Pediococcus halophilus, Staphylococcus carnosus and Staphylococcus xylosus.

In addition, the microorganism Lactobacillus casei ssp. rhamnosus strain LC-705, DSM 7061 described in EP publication No. 0576780, and described as Lactobacillus rhamnosus LC-705, DSM 7061 in US5908646, alone or in combination with a bacterium of the genus Propionibacterium or another strain of Lactobacillus casei is useful in the compressed tablets of the invention.

The invention is not, however, limited to these above mentioned particular microorganisms. The person skilled in the art would recognise those microorganisms, which may be useful in the method according to the invention. A very important embodiment of the present invention is to combine two or more of the above mentioned probiotically active organisms, such as e.g. a preparation comprising a probiotically active Lactobacillus species and a probiotically active Bifidobacterium species.

Generally, even closely related strains may express very different probiotic activities. Thus, particular important embodiments of the present invention comprise specific probiotical active bacterial strains selected from the group of strains consisting of: Bifidobacterium animalis strain DSM15954, Bifidobacterium longum subsp. infantis strain DSM15953, Bifidobacterium longum subsp. longum strain DSM15955, Enterococcus faecium strain DSM15958, , Lactobacillus acidophilus strain DSM13241 , Lactobacillus delbrueckii subsp. bulgaricus strain DSM15956, Lactobacillus helveticus strain DSM14998, Lactobacillus helveticus strain DSM14997, Lactococcus lactis strain DSM14797, Streptococcus thermophilus strain DSM15957, Lactobacillus fermentum strain ATCC55845 and Lactobacillus rhamnosus strain ATCC55826.

A sample of each of these strains have been deposited according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure as indicated in table 1 below: Table 1 : Strains deposited according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

Note: An accession number starting with "DSM" indicates that the strain was deposited with the DEUTSCHE SAMMLUNG VON MIKROORGANISMEN UND ZELLKULTUREN GmbH (DSMZ), Mascheroder Weg lb, D-38124 Braunschweig, GERMANY; a number starting with "ATCC" indicates that the strain was deposited with the American Type Culture Collection (ATCC), P.O. Box 1549, Manassas, VA 20108, USA.

It will be appreciated that useful probiotically active organisms can be of a genetically modified strain of one of the above organisms or any other organism useful in the method of the invention. It will be appreciated that the term "genetically modified" as used herein indicates any modification of DNA sequences coding for genes and modifications of sequences that regulate the expression of genes. Accordingly, genetic modification can be based on construction or selection of mutants of microorganism or it can be based on recombinant DNA technology.

As used herein the term "mutant" is comprised by the conventional meaning of that term i.e. it refers to strains obtained by subjecting a microbial strain to any conventionally used mutagen ization treatment including treatment with a chemical mutagen such as ethanemethane sulphonate (EMS) or N-methyl- N'-nitro-N-nitroguanidine (NTG), UV light or to spontaneously occurring mutants which are selected on the basis of a desired characteristic such as e.g. antibiotic and/or gastric acid-resistance. It is also possible to select useful genetically modified organisms according to the invention by random mutagenesis or by selection of spontaneously occurring mutants, i.e. without the use of recombinant DNA technology. It is envisaged that mutants of the above-mentioned organisms also can be provided by recombinant DNA technology including site-directed mutagenesis, PCR techniques and other in vitro or in vivo modifications and insertion of DNA sequences.

As seen from the examples, the advantage of adding a complete stabilizing agent comprising both antioxidant, bulking agent and gelatinizer is a considerable improved survival rate compared to compositions where only antioxidant and bulking agent is added. In general, formulations of the complete stabilizing agent according to the present invention are easily found which results in that more than 25% of the initial content of viable cells (i.e. the total number of viable cells present in, the mixture prior to compression) are viable in the freshly prepared compressed tablet. Thus, an interesting embodiment of the present invention is a probiotic composition in the form of a compressed tablet comprising a complete stabilizing agent, said agent resulting in that the viability of the cells during compression in a tablet press does not decrease by more than 75 %, preferably by no more than 60 %, and in particular by no more than 55%. The decrease in viability of the cells is calculated as: ([the total number of viable cells present in 1 g of the mixture prior to compression] - [the total number of cells present in 1 g of the freshly prepared tablet composition]) divided by [the total number of viable cells present in 1 g of the mixture prior to compression], multiplied by [100%].

Although compression force is a critical parameter the effect of the stabilizing agent can be extended to a wide range of different compression forces, while the viability of the cells is retained (see Example 11 - 13).

Whereas the stabilizing effect of the stabilizing agent can be observed in very different compositions, a preferred embodiment is a probiotic composition in the form of a compressed tablet wherein the directly compressible formulation on a solids basis comprises: (i) from about 0.1 to 50 % w/w, preferably from about 0.1 to 10 % w/w, and most preferably from about 1 to 6% w/w of probiotically active organisms; (ii) from about 0.001 to 20 % w/w, preferably from about 0.002 to 5 % w/w, and most preferably from about 0.02 to 1.25 % w/w of an antioxidant; and (iii) from about 0.01 to 10 % w/w, preferably from about 0.02 to 3 % w/w, and most preferably from about 0.1 to 2 % w/w of a gelatinizer and (IV) a bulking agent. A probiotic composition in the form of a compressed tablet, wherein said antioxidant is selected from the group consisting of propylgallate and sodium L-ascorbate, and wherein said gelatinizer is an alginate or a mixture of alginates, and wherein said bulking agent is selected from the group of saccharides, inositol, skimmed-milk powder and a mixture thereof is preferred. In a particular preferred composition, the probiotic composition in the form of a compressed tablet in addition comprise glyceryl behenate. Low amounts of glyceryl behenat are generally used as a lubricant for tablet manufacturing. As illustrated in example 18, the present inventors have surprisingly discovered that when rather large amounts of glyceryl behenat are added to the formulation of the freeze-dried granulates the compressed tablets show an unexpected long disintegration time measured as disintegration in water acording to Ph. Eur. 4^th Ed. Section 2.9.1.: "Disintegration of tablets and capsules". Inexample 18 the glyceryl behenat concentration is 25% w/w in the final product, however the effect are contemplated to cover a range of concentrations such as from about 10 to 50 %, preferably from about 15 to 30 % w/w, and most preferably from about 20 to 40% w/w of the final product without imposing any adverse effects on viability.

The individual steps in the method provided by the invention for the preparation of a probiotic composition in the form of a compressed tablet can be described in details. The first step in the process is the culture of one or more cultures of probiotically active organisms to obtain a concentrated aqueous suspension of the cells. Cultures may be propagated by fermentation in any of several ways. In the Materials Section one method of fermentation is provided. The procedure is well suited to the propagated strains. However, this method only serve to illustrate the invention and should not be construed so as to limit the invention, the person skilled in the art would recognize that other media and conditions of fermentation would apply for other probiotic microorganisms and will know to select these.

After fermentation the organisms typically need to be concentrated to obtain a suspension containing 10⁵ or more colony forming units (CFU) per g. The concentration can conveniently be performed by centrifugation, microfiltration, ultrafiltration, reverse osmosis, sedimentation, precipitation and partitioning techniques etc. In many situations it will be advantageous that the concentration suspension contains very much more than 10⁵ CFU per g. It may be advantageous that the concentration suspension contains more than 10⁷ , or more than 10⁹ , or even more than 10¹¹ CFU per g.

Following the concentration step the concentrated suspension is mixed with the stabilizing agent which comprises both an antioxidant, a bulking agent and a gelatinizer. The stabilizing agent may be added to the concentrated suspension in the form of a mixture of dry ingredients or in the form of an aqueous solution comprising an antioxidant, a bulking agent and a gelatinizer. To obtain a composition that is suitable for further formulation the water content of the mixture of the concentrated suspension and the stabilizing agent is reduced to obtain a dry composition. The drying can be obtained by a number of procedures such as e.g. freeze-drying, fluidized bed-drying, spray-drying. In an useful embodiment, the probiotically active organism is freeze dried by (i) freezing the mixture of the concentrated culture and the stabilizing agent to get a frozen composition, followed by (ii) the sublimation of water from the frozen composition. In one preferred embodiment the mixture of the concentrated culture and the stabilizing agent is frozen by adding the mixture dropwise into liquid N₂ forming frozen granula of the mixture. The frozen culture granula are collected and then freeze-dried. In an preferred embodiment the sublimation of water from the frozen material is performed while retaining the material at a temperature below 40°C, preferably below 10°C, more preferably below 0°C, even more preferably below -10°C and most preferably below -18°C. Typically, but not necessarily the process is performed on trays in a conventional industrial freeze-drying plant. A feasible method for the process is described in DE2805676 and FR2393251. US4655047 describes an alternative device and method that can be used for the cryogenic pelletization of liquids.

According to an embodiment of the invention, the sublimation of water from the frozen material is performed to a relative humidity of about 1 % to 25%, preferably from about 2% to 20% w/w and most preferably from about 3% to 15%.

In a preferred embodiment of the invention the composition of the dried material resulting from the drying step of the process on a solids basis comprises: (i) from about 1 to 80 % w/w, preferably from about 10 to 75 % w/w, and most preferably from about 12 to 65% w/w of dried probiotic organisms; (ii) from about 0.03 to 25 % w/w, preferably from about 0.1 to 20 % w/w, and most preferably from about 0.35 to 12 % w/w of an antioxidant; (iii) from about 1 to 40 % w/w, preferably from about 1 to 15 % w/w, and most preferably from about 1.5 to 10 % w/w of a gelatinizer; and (iv) from about 20 to 80 % w/w, preferably from about 30 to 72 % w/w of a bulking agent.

If this preferred procedure is followed, the resulting granulate should be reduced to a convenient size. The reduction of size can be done by any of a number of methods e.g. by crushing, grinding or as described in the "Materials" section, by milling to obtain a granulate. Typically, the milling is performed at a temperature below 25°C. In one embodiment the size of freeze-dried material is reduced to obtain a granulate of about 60 μ (10^"6 m) to about 600 μ. In an preferred embodiment the freeze-dried material is reduced to obtain a granulate having an average particle size less than 0,1 mm.

In most situations, as will be apparent to the skilled person, the granulate should be blended with one or more additional excipients to form a directly compressible formulation i.e. a formulation that forms a tablet when it is compressed for a short period of time in a tablet press. Representative examples of such additional excipients can be sought in the examples section. In one particular preferred embodiment the granulate is mixed with one or more pharmaceutically acceptable excipients which comprise glyceryl behenate.

The granulate is finally compressed into tablets. An illustrative example of the tabletting procedure is given in the materials section. However, the tabletting procedure should be optimised with respect to the specific combination of probiotic organism, stabilizing agent and additional excipients. Based on his general knowledge, the skilled person is capable of identifying optimal process, and will know that the applied compression force, the compression speed, the dwell-time and also the shape of the punches are critical factors to consider.

As can be seen from the Examples [11 ff] the compression force is a critical parameter. According to a preferred embodiment the compressible formulation is subjected to a pressure of about 1 to 50 kN/cm², preferably from about 1 to 30 kN/cm², more preferably from about 1 to 20 kN/cm², even more preferably from about 1 to 10 kN/cm² and most preferably from about 1 to 3 kN/cm . In a particular preferred embodiment (example 10) the compression force is 2 kN/cm². As seen from the experiments the general trend is that the lower the compression force the higher the survival rate. Nomally, a low compression force results in less coherent tablets, however the use of excipients which comprise glyceryl behenate results in a tablet with good coherency even when small compression forces are used and constitutes a preferred embodiment of the invention.

Also the dwell-time, i.e. the time during which the compression is maximal, needs to be considered. According to a preferred embodiment the compression time, i.e. the short period of time wherein the compressible formulation is compressed in the tablet press, is less than 1 s, preferably the time is less than 0.5 s, more preferably less than 0.1 s and most preferably less than 0.03 s. However, the dwell- time is even shorter. Thus the conditions that probiotic cells are exposed to during the compression in a tablet press is completely different from the conditions probiotic cells are exposed to during extrusion where compression typically last for several minutes.

The microflora of the gastrointestinal (Gl) tract is under normal circumstances a stable ecosystem where the composition of the microbial flora remain relatively constant in the respective segments of the Gl. This ecological system is created by the indigenous microorganisms and the host providing a number of favourable habitats for microbial growth. The stomach, which is a part of the gastrointestinal tract, is acidic (pH 1-1.5) where only a few acid tolerant organisms, such as Lactobacillus, are able to survive. Thus, due to its acidic condition the stomach acts as a barrier of the passage of undesired pathogenic organisms. The pH of the intestinal tract is neutral to alkaline and is consequently a site for major bacterial growth. Due to a neutral pH in the large intestines, bacteria are present in vast numbers in this Gl segment. The characteristic microflora of the large intestine consists mainly of anaerobic bacteria such as Bifidobacterium, Streptococcus and Lactobacillus spp. or the obligate anaerobes, Clostridium and Bacterioides spp., but also facultative aerobes such as e.g. Escherichia coli are present in smaller numbers.

One of the most important properties of this ecosystem is to prevent the colonization of potentially pathogenic microorganisms which may lead to undesirable conditions such as flatulence, diarrhoea, constipation, liver damage, irritable bowel syndrome, inflammatory bowel disease and bowel cancer (Cummings (1991); Bingham (1999)). Probiotic organisms may prevent the colonization of potentially pathogenic microorganisms by what has been referred to as "colonization resistance" or "barrier effect" (van der Waaij et al. (1971); Vollaard and Clasener (1994)). By "colonization resistance" or "barrier effect" is referred to mechanisms used by bacteria already present in the gut to maintain their presence in this environment and to avoid colonization of the same intestinal sites by freshly ingested microorganisms, for instance pathogens. Thus it is a still further aspect of the present invention to provide a method for reducing the occurrence of infections with pathogens in the gastrointestinal tract, the method comprising administering a tableted composition according to the invention comprising at least one probiotically active organism to a human or an animal. Besides, the composition of the invention is useful for the prevention of infections with acid labile pathogenic organisms in patients using antacida and other drugs having the effect of increasing the pH of the gastric juice.

In the present context, the expression "reducing the occurrence of infections" indicates that the above- mentioned infections or symptoms caused by the presence of pathogens in the gastrointestinal tract occurs at a reduced frequency or seriousness as compared to a human or animal subject who or which is not being treated according to the method of the invention. In the present context treatment is also to be construed as encompassing prevention or prophylaxis in addition to cure.

A number of reports indicate that intake of probiotic microorganisms may not only reduce the occurrence of infections, but indeed contribute to the treatment of disease. Thus, according to the present invention a method is also provided for treatment of a condition in an animal or a human characterised by undesired effects such as e.g. diarrhoea by ingestion of tabletted probiotics according to the invention. In addition to diarrhoea, probiotic organisms have been implicated in the prevention or treatment of a wide range of diseases such as antibiotic-associated disorders, gastroenteritis, diarrhoea (including traveler's diarrhoea, acute infantile diarrhoea), lactose intolerance, intestinal infections and colonization by pathogenic bacteria (including Helicobacter pylori and Clostridium difficile), irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), colonic cancer, urogenital infections and tumours, vaginal infections, allergy (especially atopic eczema), vaccination, cholesterolaemia and even hypertension (Fuller (1989); Elmer (2001); Sanders (1999); Salminen (2001); Andersson et al. (2001)).

It is furthermore documented that probiotic microorganisms produce essential vitamins and nutrients required by the intestinal cells and furthermore assist with degradation of certain nutrients and even activate cell-mediated immune effector functions. Thus probiotic microorganisms can improve the general health status of a mammal. (Conway (1996); Wold (2001)).

Consequently, it is a useful embodiment of the present invention to provide a composition according to the invention to use for the preparation of a medicament for treating or preventing of diseases or improving the general health status of a mammal. In particular it is contemplated that the use for the preparation of a medicament for treating or preventing any of the above mentioned diseases, syndromes or conditions, including the preparation of a medicament that is suitable for reducing the occurrence of infections with pathogens, are useful embodiments of the invention.

In general the composition will be used to prepare a medicament that is suitable for oral administration, however it is also within the scope of the invention to prepare a medicament which is suitable for being dissolved. Even a medicament with prolongated dissolving time is contemplated as illustrated by example 18.

The following Examples serve to illustrate the invention and should by no means be construed so as to limit the scope of the invention.

Materials and methods

Cultures:

The following 2 cultures have been used: Lactobacillus acidophilus DSM13241 (this microorganism was deposited with the DSMZ-DEUTSCHE SAMMLUNG VON MIKROORGANISMEN UND

ZELLKULTUREN GmbH (DSMZ), Mascheroder Weg lb, D-38124 Braunschweig, GERMANY on the 20 January 2000 and converted to a deposit in accordance with the Budapest treaty on 30 September 2003 and given the DSMZ -accession number DSM13241) and Bifidobacterium animalis DSM15954 (this microorganism was deposited with the DSMZ in accordance with the Budapest treaty on 30 September 2003 and given the DSMZ -accession number DSM15954).

Fermentation media and fermentation conditions:

The culture was grown in MRS broth (Merck, Damstadt, Germany) in a 100 I fermentation tank at 37°C, stirred at 20 rpm. 1 % of the culture mentioned above was used as inoculum. The anaerobic fermentation was run with nitrogen in the headspace and a pressure of about 2 bar. using standard condition to The cultures were allowed to acidify to about pH 5.5. The pH was subsequently maintained at 5.5 by controlled addition of NH₄OH. When no further base consumption was detected, the respective culture was cooled down to about 10°C.

Postfermentation treatment of cultures:

Following cooling, the bacteria in fermentation broths were concentrated 10-20 times by centrifugation. Then a stabilizing agent comprising different combinations of an antioxidant, a bulking agent and a gelatinizer was added and subsequently the culture was frozen as pellets in liquid nitrogen at one atmosphere of pressure. The frozen culture granulate was then collected and freeze-dried in a conventional freeze drying plant.

Tabletting:

Milling:

The freeze-dried granulate / culture was milled in a Quadro Comil 197S, Screen 2A045R (circular holes, 0 1,143 mm), at rotation speed: 2800 rpm. Mixing:

A standard granulate (1000 gram) was mixed with the following composition (w/w): 5,0 % freeze-dried and milled concentrate of culture. 2,0 % Silicium Dioxide (Syloid AL-1 FP, GRACE Davidson). 92,5 % Microcrystalline cellulose (Avicel PH 112, FMC) or Lactitol (Lacty-Tab, Purac) respectively as indicated in individual examples. 0,5 % Magnesium Stearate (MF-2-V, Ackros).

The milled freeze-dried concentrate of culture was mixed with Silicium dioxide and Microcrystalline Cellulose / Lactitol for 60 seconds at Position 70 Level II in Quintech Micromixer, "big bowl". Magnesium Stearate was added and the Granulate was mixed for 20 seconds.

Tabletting:

The granulate was compressed into tablets with the following parameters:

Tablet Press: Korsch 106, Instumented, 6 punches, rotary press

Punches: 010 mm, normale concave.

Target tablet weight: 550 mg.

Target speed: 10.000 tablets/hour

Compression force: Tablets were collected at target Compression force: 3, 6, 10, and 15 kN or as indicated in the specific examples.

Survival rate:

Total cell counts are performed according to Chr. Hansen, Analytical Method QAm-017 on the freeze- dried milled culture concentrates, and on tablets collected at the different compression forces. Before analysis the tablets were crushed gently using mortar and pestle.

Survival rate is calculated as Total viable cell count in the tablet /Total viable cell count in the freeze- dried milled culture concentrate * Dilution factor (20) * 100 = Survival rate in percent.

In addition, the invention relates to a dried composition obtainable through the process of claim 18 step (c) optionally in the form of a granulate having a reduced particle size obtained by crushing, grinding or milling. Said dried composition may further comprise a component having a buffering capacity at a pH in the range of from about 2 to about 3.9. Said component having a buffering capacity may be present in an amount of from about 1 % to about 15% or 20 % of the dry weight of said composition, and said component having a buffering capacity preferably comprises an organic acid selected from the group consisting of citric acid, tartaric acid, malonic acid, phosphoric acid, malic acid, maleic acid, acetic acid, fumaric acid and lactic acid, optionally in combination with its corresponding salt, and dried fruit juice and fruit pulp from acidic fruits. The invention also relates to a probiotic composition in the form of a compressed tablet as defined in the claims herein and further comprising a component having a buffering capacity at a pH in the range of from about 2 to about 3.9, and wherein said component having a buffering capacity preferably is present in an amount of from about 1 % to about 10% or 15% of the dry weight of said compressed tablet. Said component having a buffering capacity comprises an organic acid selected from the group consisting of citric acid, tartaric acid, malonic acid, phosphoric acid, malic acid, maleic acid, fumaric acid, acetic acid and lactic acid, optionally in combination with its corresponding salt, and dried fruit juice and fruit pulp from acidic fruits.

EXAMPLE 1

A concentrated suspension of L. acidophilus (LA-5™) (DSM13241 ) was prepared as described and mixed (blended) with an incomplete "stabilizing agent" consisting of an antioxidant (Na-ascorbate) and an bulking agent (inositol) but no gelatinizer to obtain a final composition in the freeze-dried product as listed below. The suspension and the "stabilizing agents" was mixed at about 10°C, for 30 min. minutes. After mixing, the concentrated culture was frozen as pellets and freeze-dried as described.

100g of L. acidophilus (LA-5™) freeze-dried granulate have the following composition:

Cells 32g Protein/peptides 20g

Inositol and carbohydrates 8g

Salts of organic acids 29g

Na-ascorbate (Antioxidant) 9g

Water 2g

The freeze-dried granulate is then milled and mixed into the tablet granulate as described, the tablet granulate was composed by:

Freeze-dried, milled LA-5™ (FD LA-5™) 5 % w/w Silicium dioxide (Syloid AL-1 FP, GRACE Davidson), 2 % w/w

Microcrystalline cellulose (Avicel PH 112, FMC) 92,5 % w/w

Magnesium stearate (MF-2-V, Akcros) 0,5 % w/w

Finally, the tablet granulate was compressed into tablets (0 12 mm) subjecting the granulate to a compression force of 5 kN/cm²:

Survival rate after tablet compression was found to be: 36 % of the initial cell count in the milled freeze- dried material. EXAMPLE 2

A concentrated suspension of L. acidophilus (LA-5™) (DSM13241) was prepared as described and mixed (blended) with a complete stabilizing agent comprising an antioxidant (propyl gallate), a bulking agent (sucrose) and a gelatinizer (sodium alginate) to obtain a final composition in the freeze-dried product as listed below. The suspension and the stabilizing agent was mixed at about 10°C, for 30 minutes. After mixing, the concentrated culture was frozen as pellets and freeze-dried as described.

100g of L. acidophilus (LA-5™) freeze-dried granulate contains the following composition:

Cells 32g

Protein/peptides 11 g

Carbohydrates (bulking agent) 28g

Salts of organic acids 24g Na-alginate (gelatinizer) 4g

Propyl gallate (Antioxidant) 0,3g

Water 2g

Freeze-dried, milled LA-5™ (FD LAF) 5 % w/w

Silicium dioxide (Syloid AL-1 FP, GRACE Davidson), 2 % w/w

Microcrystalline cellulose (Avicel PH 112, FMC) 92,5 % w/w Magnesium stearate (MF-2-V, Akcros) 0,5 % w/w

Finally, the tablet granulate was compressed into tablets (0 12 mm) subjecting the granulate to a compression force of 5 kN/cm². Survival rate after tablet compression was found to be: 73 % of the initial cell count in the milled freeze-dried material.

EXAMPLE 3

A concentrated suspension of L. acidophilus (LA-5™) (DSM13241) was prepared as described and mixed (blended) with a complete stabilizing agent comprising an antioxidant (Na-ascorbate), a bulking agent (inositol) and a gelatinizer (sodium alginate) to obtain a final composition in the freeze-dried product as listed below. The suspension and the stabilizing agent was mixed at about 10°C, for 30 minutes. After mixing, the concentrated culture was frozen as pellets and freeze-dried as described. 100g of L. acidophilus (LA-5™) freeze-dried granulate contains the following composition :

Cells 16g

Protein/peptides 31 g

Inositol and carbohydrates (Bulking agent) 20g Salts of organic acids 17g

Na-alginate (gelatinizer) 5g

Na-ascorbate (Antioxidant) 10g

Water 2g

The freeze-dried granulate is milled before it is mixed into the tablet granulate.

A tablet granulate is mixed:

Freeze-dried, milled LA-5™ (FD LaK) 5 % w/w Silicium dioxide (Syloid AL-1 FP, GRACE Davidson) 2 % w/w

Microcrystalline cellulose (Avicel PH 112, FMC) 92,5 % w/w

Magnesium stearate (MF-2-V, Akcros) 0,5 % w/w

and tablets (0 12 mm) made at a compression force of 5 kN/cm². Survival rate after tablet compression was found to be: 73 % of of the initial cell count in the milled freeze-dried material.

EXAMPLE 4

A concentrated suspension of B. animalis DSM15954 was prepared as described and mixed (blended) with an incomplete "stabilizing agent" comprising only an antioxidant (Na-ascorbate) and a bulking agent (inositol) but no gelatinizer to obtain a final composition in the freeze-dried product as listed below. The suspension and the stabilizing agent was mixed at about 10°C, for 30 minutes. After mixing, the concentrated culture was frozen as pellets and freeze-dried as described.

100g of B. animalis (BB-12) freeze-dried granulate contains the following composition:

Cells 38g

Protein/peptides 18g

Inositol and carbohydrates (Bulking agent) 15g

Salts of organic acids 17g Na-ascorbate (Antioxidant) 10g

Water 2g

The freeze-dried granulate is milled before it is mixed into the tablet granulate. A tablet granulate is mixed:

Freeze-dried, milled BB-12 (FD BB-12) 5 % w/w

Silicium dioxide (Syloid AL-1 FP, GRACE Davidson) 2 % w/w

Lactitol (Lacty-Tab, Purac) 92,5 % w/w Magnesium stearate (MF-2-V, Akcros) 0,5 % w/w

, and tablets (0 12 mm) made at a compression force of 9 kN/cm². Survival rate after tablet compression was found to be: 50 % of the initial cell count in the milled freeze-dried material.

EXAMPLE 5 A concentrated suspension of B. animalis DSM15954 was prepared as described and mixed (blended) with a complete stabilizing agent comprising an antioxidant (Na-ascorbate), a bulking agent (inositol) and a gelatinizer (sodium alginate) to obtain a final composition in the freeze-dried product as listed below. The suspension and the stabilizing agent was mixed at about 10°C, for 30 minutes. After mixing, the concentrated culture was frozen as pellets and freeze-dried as described.

Cells 17g

Protein/peptides 8g Inositol and carbohydrates (Bulking agent) 16g

Salts of organic acids 20g

Na-alginate (gelatinizer) 11 g

Na-ascorbate (Antioxidant) 25g

Water 2g

A tablet granulate is mixed:

Freeze-dried, milled BB-12 (FD BbK) 5 % w/w Silicium dioxide (Syloid AL-1 FP, GRACE Davidson) 2 % w/w

Lactitol (Lacty-Tab, Purac) 92,5 % w/w

Magnesium stearate (MF-2-V, Akcros) 0,5 % w/w and tablets (0 12 mm) made at a compression force of 9 kN/cm². Survival rate after tablet compression was found to be: 76 % of the initial cell count in the milled freeze- dried material.

EXAMPLE 6

A concentrated suspension of B. animalis DSM15954 was prepared as described and mixed (blended) with a complete stabilizing agent comprising an antioxidant (propyl gallate), a bulking agent (sucrose) and a gelatinizer (sodium alginate) to obtain a final composition in the freeze-dried product as listed below. The suspension and the stabilizing agent was mixed at about 10°C, for 30 minutes. After mixing, the concentrated culture was frozen as pellets and freeze-dried as described.

100g of B. animalis (BB-12) freeze-dried granulate contains the following composition :

Cells 37g

Protein/peptides 11 g

Carbohydrates (Bulking agent) 33g Salts of organic acids 13g

Na-alginate (gelatinizer) 4g

Propylgallate (Antioxidant) 0,4g

Water 2g

A tablet granulate is mixed:

Freeze-dried, milled BB-12 (FD BAF) 5 % w/w

Silicium dioxide (Syloid AL-1 FP, GRACE Davidson) 2 % w/w Lactitol (Lacty-Tab, Purac) 92,5 % w/w

Magnesium stearate (MF-2-V, Akcros) 0,5 % w/w

, and tablets (0 12 mm) made at a compression force of 9 kN/cm². Survival rate after tablet compression was found to be: 54 % of the initial cell count in the milled freeze-dried material.

EXAMPLE 7

A concentrated suspension of B. animalis DSM15954 was prepared as described and mixed (blended) with a complete stabilizing agent comprising an antioxidant (propyl gallate), a bulking agent (sucrose) and a gelatinizer (sodium alginate) to obtain a final composition in the freeze-dried product as listed below. The suspension and the stabilizing agent was mixed at about 10°C, for 30 minutes. After mixing, the concentrated culture was frozen as pellets and freeze-dried as described. 100g of B. animalis (BB-12) freeze-dried granulate contains the following composition:

Cells 37g

Protein/peptides 11g

Carbohydrates (Bulking agent) 33g

Salts of organic acids 13g

Na-alginate (gelatinizer) 4g

Propylgallate (Antioxidant) 0,4g

Water 2g

A tablet granulate is mixed:

Freeze-dried, milled BB-12 (FD BAF) 5 % w/w Silicium dioxide (Syloid AL-1 FP, GRACE Davidson) 2 % w/w

Microcrystalline cellulose (Avicel PH 112, FMC) 92,5 % w/w

Magnesium stearate (MF-2-V, Akcros) 0,5 % w/w

, and tablets (0 12 mm) made at a compression force of 5 kN/cm². Survival rate after tablet compression was found to be: 74 % of the initial cell count in the milled freeze-dried material.

EXAMPLE 8

A concentrated suspension of B. animalis DSM15954 was prepared as described and mixed (blended) with a complete stabilizing agent comprising an antioxidant (Na-ascorbate), a bulking agent (inositol) and a gelatinizer (sodium alginate) to obtain a final composition in the freeze-dried product as listed below. The suspension and the stabilizing agent was mixed at about 10°C, for 30 minutes. After mixing, the concentrated culture was frozen as pellets and freeze-dried as described.

Cells 17g

Protein/peptides 8g

Inositol and carbohydrates ( Bulking agent) 16g

Salts of organic acids 20g Na-alginate (gelatinizer) 11g

Na-ascorbate (Antioxidant) 25g

Water 2g The freeze-dried granulate is milled before it is mixed into the tablet granulate.

A tablet granulate is mixed: Freeze-dried, milled BB-12 (FD BbK) 5 % w/w 5 Silicium dioxide (Syloid AL-1 FP, GRACE Davidson) 2 % w/w Microcrystalline cellulose (Avicel PH 112, FMC) 92,5 % w/w Magnesium stearate (MF-2-V, Akcros) 0,5 % w/w

, and tablets (0 12 mm) made at a compression force of 5 kN/cm². Survival rate after tablet 10 compression was found to be: 103 % of the initial cell count in the milled freeze-dried material.

EXAMPLE 9 A concentrated suspension of B. animalis DSM15954 was prepared as described and mixed (blended with the stabilizing agent Na-ascorbate (antioxidant) and inositol (bulking agent) but no gelatinizer to a 15 final composition in the freeze-dried product as listed below. The suspension and the "stabilizing agents" was mixed at about 10°C, for 30 min. minutes. After mixing the concentrated culture was frozen as pellets, and freeze-dried as described.

A concentrated suspension of B. animalis DSM15954 was prepared as described and mixed (blended) 20 with an incomplete "stabilizing agent" comprising only an antioxidant (Na-ascorbate) and a bulking agent (inositol) to obtain a final composition in the freeze-dried product as listed below. The suspension and the "stabilizing agent" was mixed at about 10°C, for 30 minutes. After mixing, the concentrated culture was frozen as pellets and freeze-dried as described.

25 100g of B. animalis (BB-12) freeze-dried granulate contains the following composition:

Cells 38g Protein/peptides 18g Inositol and carbohydrates (Bulking agent) 15g

30 Salts of organic acids 17g Na-ascorbate (Antioxidant) 10g Water 2g

35 The freeze-dried granulate is milled before it is mixed into the tablet granulate.

A tablet granulate is mixed:

Freeze-dried, milled BB-12 (FD BB-12) 5 % w/w Silicium dioxide (Syloid AL-1 FP, GRACE Davidson) 2 % w/w

Microcrystalline cellulose (Avicel PH 112, FMC) 92,5 % w/w

Magnesium stearate (MF-2-V, Akcros) 0,5 % w/w

, and tablets (0 12 mm) made at a compression force of 5 kN/cm². Survival rate after tablet compression was found to be: 57 % of the initial cell count in the milled freeze-dried material.

EXAMPLE 10

A concentrated suspension of L. acidophilus (LA-5™) (DSM13241 ) was prepared as described and mixed (blended) with a complete stabilizing agent comprising an antioxidant (propyl gallate), a bulking agent (sucrose) and a gelatinizer (sodium alginate) to obtain a final composition in the freeze-dried product as listed below. The suspension and the stabilizing agent was mixed at about 10°C, for 30 minutes. After mixing, the concentrated culture was frozen as pellets and freeze-dried as described.

Cells 32g

Protein/peptides 11 g

Carbohydrates (bulking agent) 28g

Salts of organic acids 24g

Na-alginate (gelatinizer) 4g Propylgallate (Antioxidant) 0,3g

Water 2g

Cells 37g

Protein/peptides 11 g

Carbohydrates (Bulking agent) 33g Salts of organic acids 13g

Na-alginate (gelatinizer) 4g

Propylgallate (Antioxidant) 0,4g

Water 2g

A mixture of both BB-12 granulate and LA-5™ granulate were used for the tablet granulate. It is mixed:

Freeze-dried, milled BB-12 (FD BAF) 1,2 % w/w Freeze-dried, milled LA-5™ (FD LAF) 6 % w/w

Silicium dioxide (Syloid AL-1 FP, GRACE Davidson) 2 % w/w

Glyceryl behenate (Compritol 888 ATO, Gattefosse) 25 % w/w

Microcrystalline cellulose (Vivapur 14, J.Rettenmaier & Sόhne) 15 % w/w

Calciumhydrogenphosphat (Anhydrous Emcompress, Penwest) 50,8 % w/w

, and tablets (0 10 mm) made at a compression force of 2 kN/cm². Survival rate after tablet compression was found to be: 82 % of the initial cell count in the milled freeze-dried material.

Example 11 In this experiment the compositions described in Example 2 (a composition with a complete stabilizing agent comprising Propyl gallate, Sucrose and Sodium alginate), in Example 3 (a composition with a complete stabilizing agent comprising Na-ascorbate, Inositol and Sodium alginate) and in Example 1 (a composition with a incomplete "stabilizing agent" consisting of Na-ascorbate and Inositol but no Sodium alginate) were prepared and the tablet granulates were compressed into tablets (0 12 mm) subjecting the granulates to a range of compression forces of approximately 3, 6, 10, and 15 kN/1.13 cm² as indicated in Figure 1. In all cases the granulate comprised Lactobacillus acidophilus strain DSM13241.

As seen from Figure 1 a complete stabilizing agent, which comprises alginate is superior to a incomplete "stabilizing agent" without alginate. Furthermore it is seen that the compression force is inversely related to survival rate, and furthermore that the effect of the stabilizing agent can be extended to a wide range of different compression forces.

Example 12:

In this experiment the compositions described in Example 7 (a composition with a complete stabilizing agent comprising Propyl gallate, Sucrose and Sodium alginate), Example 8 (a composition with a complete stabilizing agent comprising Na-ascorbate, Inositol and Sodium alginate) and Example 9 (a composition with a incomplete "stabilizing agent" consisting of Na-ascorbate and Inositol but no Sodium alginate) were prepared and the tablet granulates were compressed into tablets (0 12 mm) subjecting the granulates to a range of compression forces of approximately 3, 6, 10, and 15 kN/1.13 cm² as indicated in Figure 2. The granulate in all cases comprised Bifidobacterium animalis strain DSM 15954.

As seen from Figure 2 a complete stabilizing agent that comprises alginate is superior to a partial "stabilizing agent" without alginate. Thus the effect is not species specific to Lactobacillus acidophilus, but can be extended to the unrelated species Bifidobacterium animalis. Furthermore, it is seen that the compression force is inverse related to survival rate, and furthermore that the effect of the stabilizing agent can be extended to a wide range of different compression forces.

Example 13:

In this experiment the compositions described in Example 6 (a composition with a complete stabilizing agent comprising Propyl gallate, Sucrose and Sodium alginate), Example 5 (a composition with a complete stabilizing agent comprising Na-ascorbate, Inositol and Sodium alginate) and Example 4 (a composition with a incomplete "stabilizing agent" consisting of Na-ascorbate and Inositol but no Sodium alginate) were prepared and the tablet granulates were compressed into tablets (0 12 mm) subjecting the granulates to a range of compression forces of approximately 3, 6, 10, and 15 kN/1.13 cm² as indicated in Figure 3. The granulate in these formulations comprised Bifidobacterium animalis strain DSM15954.

Furthermore, formulations comprising 1 (a complete stabilizing agent comprising Alginate and Propyl gallate), 2 (a complete stabilizing agent comprising Alginate and Sodium ascorbate) and 3 (an incomplete "stabilizing agent" comprising Sodium ascorbate, but no Alginate) and formulated with Lactobacillus acidophilus strain DSM13241 were formed into tablets (0 12 mm) subjecting the granulates to a range of compression forces of approximately 3, 6, 10, and 15 kN/1.13 cm² as indicated in Figure 4.

The general trend from this experiment is similar to the trends in example 11 and 12. However the result of example 13 indicates that the antioxidant component of the stabilizing agent should be selected with regard to the additional excipients applied.

Example 14:

Analytical Procedure Q-AM-017, "Enumeration of microorganisms" Chr-Hansen A/S (Denmark).

AREA OF APPLICATION

This method is used for enumeration of lactic acid bacteria in various starter cultures and for counting of cross contaminants. The method is applicable only together with the concerned culture's analytical programme according to current quality control (Qc) procedures, why reference must be given to the analytical parameters herein. PRINCIPLE

The method is a quantitative method where the result is reported as CFU/g. A known amount of sample is homogenized with diluent and decimal dilutions are prepared. Appropriate dilutions are mixed with MRS medium (Oxoid) or spread on the surface. After incubation all colonies are counted.

SAMPLING

Take samples according to established microbiological principles so that the sample is as representative as possible of the product to be examined.

APPARATUS AND GLASSWARE

Bottles a 250 ml

Tubes a 20 ml with caps

Autoclave, operating at ± 1 °C pH-meter sensitive to ± 0.2

Balance, operating at ± 0.01 g

Whirlmixer

Stomacher

Sterile Stomacher bags, 400 ml Incubator, operating ± 1 °C

Water bath, operating ± 1 °C

Sterile pipettes

Petri dishes, 9 cm

Sterile Drigalski spatulas

MEDIA

Table 2. Diluent, Contents

Preparation Suspend the ingredients in 1000 ml of distilled water. Heat to boiling point under frequent agitation. Dispense the diluent into bottles or tubes and autoclave at 121°C for 15 minutes.

pH after autoclaving: 7.0 ± 0.2. Contents in bottles after autoclaving: 99.0 ± 1.0 ml. Contents in tubes after autoclaving: 9.00 ± 0.05 ml. If the diluent (Table 2) is to be used immediately then cool to 20^CC or lower.

Storage

The prepared diluent (Table 2) may be stored for 6 months at 5°C in a dark place.

Preparation

Suspend the ingredients in 1000 ml of distilled water. Heat to boiling point under frequent agitation till complete solution. Distribute the medium into bottles and autoclave at 121 °C for 15 min. pH after autoclaving: 6.8 ± 0.2.

If the medium is to be used immediately, cool it to approx. 47°C in a water bath. Before use 2 ml 50% glucose solution has to bee added to 200 ml of MRS Agar (Oxoid) for all CR-cultures.

Is the medium used for spread plating pour 12-15 ml of melted medium into Petri dishes and let the medium set and dry for 30 min in a Clean Bench.

Glucose solution

2.0 g glucose is thawed in 100 ml. distilled water. The solution is then sterile filtered by use of a 0.20 nM filter.

MRS-Glucose Agar

Immediately before use 2 ml. of 50% glucose solution is added to a 200 ml. MRS Agar (Oxoid).

Storage

The prepared MRS Agar (Oxoid) may be stored dark for 6 months at 5°C. Poured plates packed in plastic bags may be stored dark for 10 days at 5°C.

PROCEDURE NB - The analytical period from weighing out the sample until the sample is pour plated or spread plated must not exceed 30 min.

Before beginning the microbiological examination, melt the medium in a boiling water bath or by boiling in an autoclave, and then cool it to 47 ± 1 °C in a water bath.

Note - if prepoured plates are to be used the surface of the medium must be dry before plating.

In the Analytical Programme or Qc of the concerned product the following are given: a) The amount of grammes (X) to be used for the first dilution (D1) b) Minutes in Stomacher (M) c) The appropriate dilutions (D2) d) Amount to be seeded (A ml) e) The incubation parameters f) Plating method

Preparation of dilutions

Weigh X gram of product into a sterile Stomacher bag and add by weighing the sufficient amount of sterile diluent to make the first dilution (D1). Place the bag in the Stomacher and treat for (M) minutes. If convenient, pour the contents of the bag into an empty, sterile bottle. By use of a sterile pipette transfer 0.1 or 1.0 ml from the lowest dilution into a bottle or tube with sterile diluent to make the next dilution (which now is the lowest!).

The contents in the bottle are mixed by shaking the bottle for 7 sec 20-25 times in an angle of 30°. The contents in the tube are mixed on a whirlmixer at maximum speed for 3x1 sec.

Allow the foam to settle and repeat point 4 and 5 until the appropriate dilution/s (D2) is/are reached.

Pour plate

By use of a sterile pipette transfer A ml of the appropriate dilution/s (D2) into Petri dishes. Pour 10-12 ml of melted medium at not more than 47 ± 1 °C, into each Petri dish and mix well with the sample. Pour 10-12 ml of melted medium into an empty Petri dish as a control of sterility. Leave the dishes on a clean horizontal surface until the medium has set. Invert the dishes and incubate according to the concerned products Qc.

Spread plate

By use of a sterile pipette transfer A ml of the appropriate dilution/s (D2) on to the surface of the medium. Spread the sample all over the medium by use of a sterile Drigalski spatula. Use an uninoculated Petri dish with medium as a control of sterility. Let the sample be absorbed by the medium before the dishes are inverted and incubated according to the concerned products Qc.

COUNTING OF COLONIES

For total viable cell counts Petri dishes containing between 30-300 colonies are chosen. All colonies are counted.

For counting of cross contaminants Petri dishes not containing more than 300 colonies are chosen. All colonies are counted. Note - By counting of cross contaminants the product to be analyzed may produce pin point colonies which will make a cloud in the back ground. Therefore only colonies bigger than the pinpoint colonies in the cloud are counted.

CALCULATION

After counting a χ² -test must be carried out on the plate counts according to standard statistical procedures.

Note - The χ² -test is not carried out when the method is used for cross contaminants.

If the χ² -test is not accepted the results must be rejected and the analysis repeated.

If the χ² -test is accepted the mean number (N) of CFU/g is calculated according to below:

N = (∑c )/((n1+0.1 n2)d)

where:

∑c is the sum of colonies counted on all Petri dishes; n1 is the number of Petri dishes in the first dilution; n2 is the number of Petri dishes in the second dilution; d is the dilution factor corresponding to the first dilution.

REPORTING OF RESULTS

The calculated count may be reported as in the example above or as a rounded number with two significant digits.

Results which are reported externally should always be rounded.

Example: 19 184 is rounded to 19 000 and is reported as 1.9 x 10^.

294 x 10⁸ is rounded to 290 x 10⁸ and is reported as 2.9 x 10¹⁰.

For a three-digit number, round the third digit to the nearest zero: - If the third digit is 5 and the preceding digit is an even number, round the number down. If the preceding digit is an odd number round the number up.

Example: 28 500 is rounded to 28 000 and 11 500 rounded to 12 000. Example 15:

A concentrated suspension of L. acidophilus (LA-5™) (DSM13241 ) was prepared as described in "Materials and Methods" and mixed (blended) with different amounts of additives. Please see table 3 below. The additives were various combinations of an antioxidant, a bulking agent and a gelatinizer. Five different formulations of the freeze-dried product, see table 3, was prepared. The concentrated suspension and the various components were mixed at about 10°C, for 30 min. minutes. After mixing, the concentrated culture was frozen as pellets and freeze-dried as described previously.

Table 3: Composition of various formulations

The concentrated suspension of L. acidophilus (LA-5™) (DSM13241) was formulated with various combinations of an antioxidant, a bulking agent and a gelatinizer. The amounts given are based on the content in the final freeze-dried granulate. The % is % w/w.

The stability of the different final compositions was measured by determining viable counts in the final freeze-dried granulate after storage at different conditions. Viable cell counts are expressed as Colony Forming Units (CFU) pr. g of final freeze-dried granulate as described in example 14.

Table 4: Viable cell counts in the freeze dried granulate after freezing and after storage at 30°C.

Viable cell counts (CFU/g final product see example 14) after freeze drying and after storage of the granulate at 30°C for 3 weeks in closed alu-bags as well as we as in an open container in a climate chamber at 30°C, 15% Relative Humidity for 3 weeks.

From this experiment it can be concluded that the storage stability of formulation 5 that is formulated with the complete stabilizing agent comprising both an anti-oxidant, a bulking agent and a gelatinizer seems to be much better than the incomplete "stabilizing agent" formulations such as formulation 1 , 2, 3 or 4.

Example 16: A concentrated suspension of L. acidophilus (LA-5™) (DSM13241 ) was prepared as described and mixed (blended) with different combinations of additives as described in example 1.

The different freeze-dried granulates were then milled and mixed into the tablet granulate as described, the tablet granulate was composed by:

Tablets: Formulation 1-5

Freeze-dried, milled LA-5™ formulation 1-5 6 % w/w

Silicon dioxide (Syloid AL-1 FP, GRACE Davidson) 2 % w/w

Microcrystalline cellulose (Vivapur 14, JRS Pharma) 15 % w/w Calcium hydrogen phosphate (Anhydrous Emcompress, Penwest) 77 % w/w

Tablets: Formulation 6

Freeze-dried, milled LA-5™ formulation 5 6 % w/w Silicon dioxide (Syloid AL-1 FP, GRACE Davidson) 2 % w/w Glyceryl behenate (Compritol 888 ATO, Gattefosse) 25 % w/w Microcrystalline cellulose (Vivapur 14, JRS Pharma) 15 % w/w Calcium hydrogen phosphate (Anhydrous Emcompress, Penwest) 52 % w/w

Milling and mixing:

The freeze-dried granulate / culture was milled in a Quadro Comil 197S, Screen 2A045R (circular holes, 0 1 ,143 mm), at rotation speed: 2800 rpm.

The milled freeze-dried concentrate of culture was mixed with Silicon dioxide, Microcrystalline cellulose and Calcium hydrogen phosphate and for formulation 6 Glyceryl behenate for 60 seconds at Position 70 Level II in Quintech Micromixer, "big bowl".

Compression:

The granulate was compressed into tablets with the following parameters:

Tablet Press: Korsch 106, Instrumented, 6 punches, rotary press Punches: 010 mm, normal concave.

Target tablet weight: 400 mg. Target speed: 10.000 tablets/hour

Target tablet hardness: 45N

The tablets as well as the freeze dried granulates were stored under similar conditions at -20°C until enumeration of microorganisms according to the procedure in example 14.

Table 5: Viable cell counts in the freeze dried granulate and in tablets.

As can be seen in the table the best survival to compression was found in formulation 3, 5 and 6 which appeared comparable.

Example 17:

A concentrated suspension of L. acidophilus (LA-5™) (DSM13241 ) was prepared as described in "Materials and Methods" and mixed (blended) with different amounts of additives. As in example 15, five different formulations of the freeze-dried product, see table 3, was prepared. The concentrated suspension and the various components were mixed at about 10°C, for 30 min. minutes. After mixing, the concentrated culture was frozen as pellets and freeze-dried as described previously.

Freeze-dried, milled LA-5™ formulation 1-5 6 % w/w Silicon dioxide (Syloid AL-1 FP, GRACE Davidson) 2 % w/w Glyceryl behenate (Compritol 888 ATO, Gattefosse) 25 % w/w Microcrystalline cellulose (Vivapur 14, JRS Pharma) 15 % w/w Calcium hydrogen phosphate (Anhydrous Emcompress, Penwest) 52 % w/w Milling and mixing:

The milled freeze-dried concentrate of culture was mixed with Silicon dioxide, Glyceryl behenate, Microcrystalline cellulose and Calcium hydrogen phosphate for 60 seconds at Position 70 Level II in Quintech Micromixer, "big bowl".

Compression:

The granulate was compressed into tablets with the following parameters: Tablet Press: Korsch 106, Instrumented, 6 punches, rotary press

Punches: 010 mm, normal concave.

Target tablet weight: 400 mg.

Target speed: 10.000 tablets/hour

Target tablet hardness: 45N

Formulation 4 could not be milled and therefore no tablets were made of formulation 4.

The concentration of viable cells (CFU/g) after manufacturing of tablets and the concentration of cells after storage of the tablets at 30°C for 3 weeks in alu-bags and storage of the tablets in an open container in a climate chamber at 30°C/15% RH for 3 weeks is as follows:

Table 6: Viable cell counts in tablets stored dry at -20°C and tablets stored at 30°C.

From this example it can be concluded that that the formulation containing a complete stabilizing agent comprising both an oxidant, a bulking agent and a gelatinizer result the best survival of the probiotic organism to the combined effect of compression and storage. Example 18:

A concentrated suspension of L. acidophilus (LA-5™) (DSM13241 ) was prepared as described and mixed (blended) with different combinations of additives as described in example 1.

The freeze-dried granulates are then milled and mixed into the tablet granulate as described, the tablet granulate was composed by:

Formulation A Freeze-dried, milled LA-5™ formulation 5 6 % w/w

Silicon dioxide (Syloid AL-1 FP, GRACE Davidson) 2 % w/w

Microcrystalline cellulose (Vivapur 14, JRS Pharma) 15 % w/w

Calcium hydrogen phosphate (Anhydrous Emcompress, Penwest) 77 % w/w

Formulation B

Milling and mixing:

Compression: The granulate was compressed into tablets with the following parameters:

Tablet Press: Korsch 106, Instrumented, 6 punches, rotary press

Punches: 010 mm, normal concave.

Target tablet weight: 400 mg. Target speed: 10.000 tablets/hour Target tablet hardness: 45N Table 7: Disintegration in water was conducted according to Ph.Eur 2.9.1 :

Formulation B that contains a high level of Glyceryl behenate has a much slower disintegration than formulation A that does not contain Glyceryl behenate. REFERENCES Andersson et al. (2001) Scandinavian Journal of Nutrition/Naringsforskning Vol 45:58-75. Bingham SA (1999): High-meat diets and cancer risk. Proc Nutr Soc 58:243-8. Conway, PL (1996): Microbial ecology of the human large intestine. In: Macfarlane & Gibson, eds. Human Colonic Microflora, CRC Press; 1-24; Cummings et al. (1991 ) The control and consequences of bacterial fermentation in the human colon. J Appl Bacteriol 1991 ;70:443-59. Elmer GW (2001). Probiotics: "living drugs". Am J Health Syst Pharm. 58(12):1101-9) Fuller R. (1989) Probiotics in man and animals. J Appl Bacteriol 66: 365-78. Jouany JP et al. (1998) Reprod Nutr Dev. 38(4):401-16). Laulund S (1994) Commercial Aspects of Formulation, Production and Marketing of Probiotic Products. Pp. 159-173 In: Gibson, Human Health: The Contribution of Microorganisms, Springer Verlag, Berlin, 1994 Salminen (2001 ), Scandinavian Journal of Nutrition/Naringsforskning Vol 45:8-12, 2001 ; Sanders (1999) Foodtechnology 53(11 ), 67-77; Shah NP (2000) Symposium on probiotic bacteria, J Dairy Sci 83, 894-907 Van der Waaij et al. (1971 ) Colonization resistance of the digestive tract in conventional and antibiotic-treated mice. Hyg (Lond), 69: 405-11. Vollaard and Clasener (1994) Colonization resistance. Antimicrob Agents Chemother, 38: 409-14.) Wold (2001) Scandinavian Journal of Nutrition/Naringsforskning Vol 45:76-85. DE2805676, (Niels J. T. Jespersen), 18 August 1978. FR2393251 , (Niels J. T. Jespersen), 29 December 1978. US4655047, (George A. Temple), 7 April 1987. EP1213347, (Gulden J-Yilmaz), 12 June 2002. WO 00/07571 , (Helmut Viernstein), 17 February 2000.

INDICATIONS RELATING TO DEPOSITED MICROORGANISM OR OTHER BIOLOGICAL MATERIAL (PCT Rule 13 bis)

A. The indications made below relate to the deposited microorganism or other biological material referred to in the description on page , line 3 B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet [)? Name of depositary institution Deutsche Sammlung von Mikroorganismen und Zellkulturen Address of depositary institution (including postal code and country) Mascheroder Weg 1 b D-38124 Braunschweig Germany

Date of deposit Accession Number 30 September 2003 DSMZ 15954 C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet ~\ As regards the respective Patent Offices of the respective designated offices, the applicants request that a sample of the deposited microorganisms only be made available to an expert nominated by the requester until the date on which the patent is granted or the date on which the application has been refused or withdrawn or is deemed to be withdrawn.

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated States)

E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable) The indications listed below will be submitted to the International Bureau later (specify the general nature of the indications e.g., "Accession Number of Deposit")

For receiving Office use only For International Bureau use only This sheet was received with the international application | | This sheet was received by the International Bureau on:

Authorized officer Authorized officer

Form PCT O/134 (Julyl998; reprint January 2004) INDICATIONS RELATING TO DEPOSITED MICROORGANISMS (PCT Rule I2bis) Additional sheet

In addition to the microorgansms indicated on page xx of the description, the following microorganism has been deposited with DSM-Deutsche Sammlung von Mikroorganismen und Zellkulturen Mascheroder Weg IB, D-38124 Braunsweig, Germany and American Type Culture Collection 10801 University Blvd Manassas, Virginia 20110-2209 USA

respectively, on the date and under the accession number as stated below:

Accession no. Date of deposit Description page no Line no.

DSMZ 14797 05 February 2002 6 27

DSMZ 14997 15 May 2002 6 27

DSMZ 14998 15 May 2002 6 27

DSMZ 13241 20 January 2000 6 25

DSMZ 15953 30 September 2003 6 24

DSMZ 15954 30 September 2003 6 23

DSMZ 15955 30 September 2003 6 25

DSMZ 15956 30 September 2003 6 26

DSMZ 15957 . 30 September 2003 6 28

DSMZ 15958 30 September 2003 6 25

ATCC 55845 25 October 1996 6 28

ATCC 55826 03 October 1996 6 29

C. ADDITIONAL INDICATIONS ,

As regards the respective Patent Offices of the respective designated offices, the applicants request that a sample of the deposited microorganisms only be made available to an expert nominated by the requester until the date on which the patent is granted or the date on which the application has been refused or withdrawn or is deemed to be withdrawn.

Claims

1. A probiotic composition in the form of a compressed tablet comprising dried, viable, probiotically active organisms and a stabilizing agent comprising a combination of 1) an antioxidant, 2) a bulking agent and 3) a gelatinizer.

2. The probiotic composition according to claim 1 , which in addition comprises glyceryl behenate.

3. The probiotic composition according any of the preceeding claims, wherein the antioxidant is selected from the group consisting of gallates, L-ascorbic acid, lycopene, beta-carotene, lutein, tocopherols, butylhydroxyanisol (BHA), butylhydroxytoluen, homocysteine, cysteine, cystathionine, methionine, gluthatione, salts thereof and combinations thereof and peptides containing any one of homocysteine, cysteine, cystathionine, methionine, gluthatione, pharmaceutically acceptable salts thereof and combinations of any of the foregoing.

4. The probiotic composition according to any of the preceeding claims, wherein said antioxidant is selected from the group consisting of propylgallate and sodium L-ascorbate and a mixture thereof.

5. The probiotic composition according to any one of the preceding claims, wherein the bulking agent is selected from the group consisting of saccharides, inositol, skimmed-milk powder, yeast extract, caseinate, polyoles and sugar alcohols and combinations thereof.

6. The probiotic composition according to any one of the preceding claims, wherein the gelatinizer is selected from the group consisting of alginic acid, sodium alginate, potassium alginate, ammonium alginate, calcium alginate, agar, carrageenan, locust bean gum, guar gum, guar flour, tragacanth, konjac glucomannan, propylene glycol alginate, PES, acacia gum, xanthan gum, karaya gum, tara gum, gellan gum and combinations thereof.

7. The probiotic composition according to any one of the preceding claims, wherein said gelatinizer is an alginate or a mixture of alginates.

8. The probiotic composition according to any one of the preceding claims, wherein the bulking agent is selected from the group consisting of inositol, sucrose, dextrose, fructose, arabinose, galactose, mannose, rhamnose, ribose, xylose, tetrulose, ribulose, xylulose, psicose, sorbose, tagatose, lactose, maltose, isomaltose, maltotriose, dextran, maltodextrin, raffinose, trehalose, cyclodextrins, cellobiose, ® lactulose, maltulose, melibiose, neotrehalose, maltotriose, manninotriose, stachyose, Raftilose , fructooligosaccharides and fructooligomers having prebiotic properties, pharmaceutically acceptable salts thereof and combinations of any of the foregoing.

9. The probiotic composition according to any one of the preceding claims, wherein said bulking agent is selected from the group of saccharides, inositol, skimmed-milk powder and a mixture thereof.

10. The probiotic composition according to any one of the preceding claims, wherein the stabilizing agent further comprises one or more cryoprotective agent(s) selected from the group consisting one or more compound(s) involved in the biosynthesis of nucleic acids or one or more derivative(s) of any such compounds.

11. The probiotic composition according to claim 10, wherein the one or more cryoprotective agent(s) is/are selected from the group of purine bases, pyrimidine bases, nucleosides and nucleotides.

12. The probiotic composition according to any one of the preceding claims, wherein the probiotically active organisms is a dried culture of cells selected from the group of cultures consisting of probiotically active fungal species, yeast species and bacterial species and combinations thereof.

13. The probiotic composition according to any one of the preceding claims, wherein the probiotically active organism is a bacterial species selected from the group consisting a bacterial species belonging to the genera of Aerococcus, Bacillus, Bacteroides, Bifidobacterium, Clostridium, Enterococcus, Fusobacterium, Lactobacillus, Lactococcus, Leuconostoc, Melissococcus, Micrococcus, Pediococcus, Peptostreptococcus, Propionibacterium, Staphylococcus, Streptococcus, Weissella and combinations thereof.

14. The probiotic composition according to any one of the preceding claims, wherein the probiotic cells is a dried culture of probiotical active bacterial strain selected from the group of strains consisting of: Bifidobacterium animalis strain DSM15954, Bifidobacterium longum subsp. infantis strain DSM15953, Bifidobacterium longum subsp. longum strain DSM15955, Enterococcus faecium strain DSM15958, Lactobacillus acidophilus strain DSM13241 , Lactobacillus delbrueckii subsp. bulgaricus strain DSM15956, Lactobacillus helveticus strain DSM14998, Lactobacillus helveticus strain DSM14997, Lactococcus lactis strain DSM14797, Streptococcus thermophilus strain DSM15957, Lactobacillus fermentum strain ATCC55845, and Lactobacillus rhamnosus strain ATCC55826, and combinations thereof.

15. The probiotic composition according to any one of the preceding claims, wherein the probiotic composition comprises a complete stabilizing agent said agent resulting in that the viability of the cells does not decrease by more than 60 % when compressed to form a compressed tablet.

16. The probiotic composition according to any one of the preceding claims, wherein the directly compressible formulation comprises on a solids basis: (i) from about 0.1 to 50 % w/w, preferably from about 0.1 to 10 % w/w, and most preferably from about 1 to 10% w/w of probiotically active organisms;

(ii) from about 0.001 to 20 % w/w, preferably from about 0.002 to 5 % w/w, and most preferably from 5 about 0.02 to 1.25 % w/w of an antioxidant;

(iii) from about 0.01 to 10 % w/w, preferably from about 0.02 to 3 % w/w, and most preferably from about 0.1 to 2 % w/w of a gelatinizer; and a bulking agent

10 17. The probiotic composition according to claim 16, wherein said antioxidant is selected from the group consisting of propylgallate and sodium L-ascorbate, and wherein said gelatinizer is an alginate or a mixture of alginates, and wherein said bulking agent is selected from the group of saccharides, inositol, skimmed-milk powder and a mixture thereof.

15 18. The probiotic composition according to claims 2 to 17, which have a disintegration time in water conducted according to Ph. Eur 4^,h Ed 2.9.1 of 40 minuttes or more.

19. A process for the preparation of a probiotic composition in the form of a compressed tablet comprising viable probiotically active organisms comprising the following steps: 20 (a) culturing one or more cultures of probiotically active organisms to obtain a concentrated aqueous suspension of the cells, said suspension containing at least 10⁵ CFU per g;

(b) mixing into the concentrated aqueous suspension of the cells a stabilizing agent comprising both an 25 antioxidant, a bulking agent and a gelatinizer to obtain a mixture;

(c) reducing the water content of the mixture to obtain a dried composition;

(d) optionally reducing the particle size of said dried composition of step (c) by crushing, grinding or 30 milling to obtain a granulate;

(e) optionally blending the granulate of step (d) with one or more pharmaceutically acceptable binders, lubricants, diluents, disintegrating agents, colouring agents, flavouring agents, flow-inducing agents, melting agents or other excipients and optionally further dry blending the granulate of step (d) with a

35 component having a buffering capacity at a pH in the range of from 2 to about 3.9 to form a directly compressible formulation; and

(f) compressing the granulate of step (d) or (e) for a short period of time in a tablet press to form a compressed tablet of the desired shape.

20. The process according to claim 18, wherein the granulate of step (d) have an average particle size less than 0,1 mm.

5 21. The process according to claim 19 to 20, wherein during step (e), the granulate of step (d) is mixed with one or more pharmaceutically acceptable excipients which comprise glyceryl behenate.

22. The process according to claims 19 to 21, wherein said dried composition is obtained by (i) freezing the mixture of step (b) to obtain a frozen composition and (ii) sublimation of water from the frozen

10 composition.

23. The process according to claims 19 to 22, wherein the probiotically active organisms is a dried culture of cells selected from the group of cultures consisting of probiotically active fungal species, yeast species and bacterial species and combinations thereof.

15 24. The process according to any of claims 19 to 23, wherein the probiotically active organism is a bacterial species selected from the group consisting of the genera Aerococcus, Bacillus, Bacteroides, Bifidobacterium, Clostridium, Enterococcus, Fusobacterium, Lactobacillus, Lactococcus, Leuconostoc, Melissococcus, Micrococcus, Pediococcus, Peptostreptococcus, Propionibacterium, Staphylococcus,

20 Streptococcus and Weissella and combinations thereof.

25. The process according to any of claims 19 to 24, wherein the probiotically active organism is a bacterial strain selected from the group consisting of Bifidobacterium animalis strain DSM15954, Bifidobacterium longum subsp. infantis strain DSM15953, Bifidobacterium longum subsp. longum strain

25 DSM15955, Enterococcus faecium strain DSM15958, Lactobacillus acidophilus strain DSM13241 , Lactobacillus delbrueckii subsp. bulgaricus strain DSM15956, Lactobacillus helveticus strain DSM14998, Lactobacillus helveticus strain DSM14997, , Lactococcus lactis strain DSM14797, Streptococcus thermophilus strain DSM15957, Lactobacillus fermentum strain ATCC55845, and Lactobacillus rhamnosus strain ATCC55826, and combinations thereof.

30 26. The process according to any of claims 19 to 25, wherein the short period of time of step (f) is less than 1 s, preferably the time is less than 0.5 s, more preferably less than 0.1 s and most preferably less than 0.03 s

35 27. The process according to any of claims 19 to 26, wherein the antioxidant is selected from the group consisting of gallates, L-ascorbic acid, lycopene, beta-carotene, lutein, tocopherols, butylhydroxyanisol (BHA), butylhydroxytoluen, homocysteine, cysteine, cystathionine, methionine, gluthatione, salts thereof and combinations thereof and peptides containing any one of homocysteine, cysteine, cystathionine, methionine, gluthatione, pharmaceutically acceptable salts thereof, and combinations of any of the foregoing.

28. The process according to any of claims 19 to 27, wherein the bulking agent is selected from the group consisting of saccharides, inositol, skimmed-milk powder, yeast extract, caseinate, polyoles and sugar alcohols and combinations thereof.

29. The process according to any of claims 19 to 28, wherein the gelatinizer is selected from the group consisting of alginic acid, sodium alginate, potassium alginate, ammonium alginate, calcium alginate, agar, carragenan, locust bean gum, guar gum, guar flour, tragacanth, konjac glucomannan, propylene glycol alginate, PES, acacia gum, xanthan gum, karaya gum, tara gum, gellan gum, pectin and combinations thereof.

30. The process according to any of claims 19 to 29, wherein the bulking agent is selected from the group consisting of inositol, sucrose, dextrose, fructose, arabinose, galactose, mannose, rhamnose, ribose, xylose, tetrulose, ribulose, xylulose, psicose, sorbose, tagatose, lactose, maltose, isomaltose, maltotriose, dextran, maltodextrin, raffinose, trehalose, cyclodextrins, cellobiose, lactulose, maltulose, ® melibiose, neotrehalose, maltotriose, manninotriose, stachyose, Raftilose , fructooligosaccharides and fructooligomers having prebiotic properties, pharmaceutically acceptable salts thereof and combinations of any of the foregoing.

31. The process according to any of claims 19 to 30, wherein the stabilizing agent further comprises one or more cryoprotective agent(s) selected from the group consisting of one or more compound(s) involved in the biosynthesis of nucleic acids or one or more derivative(s) of any such compounds.

32. The process according to claim 31 , wherein the one or more cryoprotective agent(s) is/are selected from the group consisting of purine bases, pyrimidine bases, nucleosides and nucleotides.

33. The process according to any of claims 22 to 32, wherein step (c) comprises a freezing of the mixture performed by adding the mixture dropwise into liquid N₂ to obtain said dried composition in the form of frozen granulae of the mixture.

34. The process according to any of claims 22 to 33, wherein step (c) comprises a further sublimation of water from the frozen composition said sublimation being performed while retaining the material at a temperature below 40°C, preferably below 10°C, more preferably below 0°C, even more preferably below - 0°C and most preferably below -18°C.

35. The process according to claim 34, wherein said sublimation of water from the frozen composition is performed to a relative humidity of about 1 % to 25%, preferably from about 2% to 20% w/w and most preferably from about 3% to 15%.

5 36. The process according to any one of claims 19 to 35, wherein during step (f) the compressible formulation is subjected to a pressure of about 1 to 50 kN/cm², preferably from about 1 to 30 kN/cm², more preferably from about 1 to 20 kN/cm², even more preferably from about 1 to 10 kN/cm², most preferably from about 1 to 3 kN/cm².

10 37. The process according to any of claim 19 to 36, wherein the dried material resulting from step (c) comprises on a solids basis:

(i) from about 1 to 80 % w/w, preferably from about 10 to 75 % w/w, and most preferably from about 12 to 65% w/w of dried probiotic organisms. 15 (ii) from about 0.03 to 25 % w/w, preferably from about 0.1 to 20 % w/w and most preferably from about 0.35 to 12 % w/w of an antioxidant; and

(iii) from about 1 to 40 % w/w, preferably from about 1 to 15 % w/w, and most preferably from about 1.5 20 to 10 % w/w of a gelatinizer; and

(iv) from about 20 to 80 % w/w, preferably from about 30 to 72 % w/w of a bulking agent.

38. The process according to any one of claims 19 to 37, wherein said antioxidant is selected from the 25 group consisting of propylgallate and sodium L-ascorbate, and wherein said gelatinizer is an alginate or a mixture of alginates, and wherein said bulking agent is selected from the group of saccharides, inositol, skimmed-milk powder, maltodextrins and a mixture thereof.

39. A method for reducing the occurrence of infections with pathogens, the method comprising

30 administering a tabletted composition according to claims 1 to 18 comprising at least one probiotically active organism.

40. Use of the composition according to any of claims 1 to 18 for the preparation of a medicament for the treatment or prevention of a disease, syndrome or condition, or for improving digestion of nutrients,

35 or for improving the general health status of a human being or a vertebrate animal.

41. The use according to claim 40, wherein said disease, syndrome or condition is selected from the group consisting of antibiotic-associated disorders, gastroenteritis, diarrhoea including traveller's diarrhoea and acute infantile diarrhoea, lactose intolerance, gastrointestinal infections and colonization of the gastrointestinal tract by pathogenic bacteria including Helicobacter pylori and Clostridium difficile, irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), colonic cancer, urogenital infections and tumours, vaginal infections, allergy (especially atopic eczema), vaccination, chόlesterolaemia and hypertension. 5

42. The use according to any one of claims 40 or 41 , wherein the medicament is suitable for preventing or treating infections with pathogens.

43. The use according to any one of claims 40 to 42, wherein the medicament is suitable for oral 10 administration.

44. A dried composition obtainable through the process of claim 19 step (c) optionally in the form of a granulate having a reduced particle size obtained by crushing, grinding or milling.

15 45. The dried composition according to claim 44 further comprising a component having a buffering capacity at a pH in the range of from about 2 to about 3.9.

46. The dried composition according to any one of claims 44 and 45, wherein said component having a buffering capacity is present in an amount of from about 1 % to about 15% of the dry weight of said

20 composition.

47. The dried composition according to any one of claims 44 through 46, wherein the component having a buffering capacity comprises an organic acid selected from the group consisting of citric acid, tartaric acid, malonic acid, phosphoric acid, malic acid, maleic acid, fumaric acid, acetic acid and lactic

25 acid, optionally in combination with its corresponding salt, and dried fruit juice and fruit pulp from acidic fruits.

48. The probiotic composition according to any one of claims 1 through 18 further comprising a component having a buffering capacity at a pH in the range of from about 2 to about 3.9.

30 49. The probiotic composition according to claim 48, wherein said component having a buffering capacity is present in an amount of from about 1 % to about 10% of the dry weight of said compressed tablet.

35 50. The probiotic composition in the form of a compressed tablet according to any one of claims 48 and 49, wherein the component having a buffering capacity comprises an organic acid selected from the group consisting of citric acid, tartaric acid, malonic acid, phosphoric acid, malic acid, maleic acid, acetic acid, fumaric acid and lactic acid, optionally in combination with its corresponding salt, and dried fruit juice and fruit pulp from acidic fruits.

40