WO2000007571A2 - Formulations renfermant des micro-organismes a effet probiotique - Google Patents

Formulations renfermant des micro-organismes a effet probiotique Download PDF

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Publication number
WO2000007571A2
WO2000007571A2 PCT/AT1999/000192 AT9900192W WO0007571A2 WO 2000007571 A2 WO2000007571 A2 WO 2000007571A2 AT 9900192 W AT9900192 W AT 9900192W WO 0007571 A2 WO0007571 A2 WO 0007571A2
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WO
WIPO (PCT)
Prior art keywords
formulation according
microorganisms
lactobacillus
formulation
stabilized
Prior art date
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Ceased
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PCT/AT1999/000192
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German (de)
English (en)
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WO2000007571A3 (fr
Inventor
Helmut Viernstein
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Individual
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Priority to EP99938048A priority Critical patent/EP1102580A2/fr
Priority to AU52704/99A priority patent/AU5270499A/en
Publication of WO2000007571A2 publication Critical patent/WO2000007571A2/fr
Publication of WO2000007571A3 publication Critical patent/WO2000007571A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/065—Microorganisms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
    • A61K9/0007—Effervescent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to formulations with probiotic microorganisms.
  • probiotics A General View in: “The Lactic Acid Bacteria, Volume 1", BJB Wood ed. (1992), the probiotic microorganisms (common synonym “probiotics”) are , Elsevier Applied Science, 155/156) describes individual or mixed cultures of living microorganisms which are applied to humans and animals and which have a beneficial effect on the host by supporting the properties of the naturally present intestinal microflora. This means that only products that contain living or viable microorganisms, e.g. as freeze-dried cells, improve the health status of humans and animals and which can develop their effects in the mouth or the gastrointestinal tract.
  • MSB lactic acid bacteria
  • lactic acid bacteria of the genera Lactobacillus, Streptococcus, Lactococcus, Leuconostoc, Enterococcus, Bifidobacterium, Carnobacterium and Sporolactobacillus are in pharmaceutical preparations and food supplements according to the current state of knowledge and current taxonomy. increasingly found.
  • Many subspecies of the genera Lactobacillus, Bifidobacterium and Enterococcus are considered to be necessary natural intestinal inhabitants of humans and animals. They occur - depending on nutritional status, age, state of health and intestinal tract - in dynamically changing amounts.
  • Lactic acid bacteria are assessed according to their abilities in a similar way to other microorganisms. These are: 1. biochemical (eg the utilization of carbohydrate utilization)
  • physiological e.g. stimulation of intestinal peristalsis
  • the microflora of the intestine can be specifically supported by the application of certain MSB via medical indications or the way of supplementing food.
  • MSB medical indications or the way of supplementing food.
  • microorganisms are difficult to stabilize. In preparations, they are usually used dried as viable, lyophilized microorganisms. The display is the responsibility of the culture producers. The continuous production of the microorganisms in the required qualities, as well as their further processing into physiologically effective forms. Definitions and the subsequent storage period determine the biological activity of the material (Brennan et al., J. Food Prot. 46 (1983), 887-892).
  • the object of the present invention is to provide formulations containing probiotic microorganisms which, when properly administered in suspension form, guarantee that physiologically relevant germ contents enter the human or animal intestine and which can therefore be highly effective in the intestinal physiology. Linked to this is the task of maintaining the viability of the probiotics, especially the MSB, in the product itself.
  • formulations themselves can be produced on a large scale and reproducibly as medical formulations for therapeutic indications or as an additional or preventive food supplement for humans and animals.
  • a formulation comprising buffer substances and microorganisms stabilized by lyophilization and. optionally technologically necessary auxiliaries, wherein the microorganisms stabilized by lyophilization are dry incorporated into the matrix-forming buffer substances and these formulations can be dry, for example either in powder form or as tablets.
  • the preparations according to the invention develop their effect, which are predominantly in powder form or as a tablet, after appropriate dissolution and ingestion by the patient or consumer.
  • the phase of the disintegration of the formulation can be carried out, for example, in water without a substantial loss of the number of bacteria, even if acidic reactions occur when dissolved.
  • the (spatial) close contact between microorganisms and buffer substances is sufficient to survive even the acidic processes during the dissolution process if the formulation according to the invention is formulated as effervescent powder and substances such as organic acids (e.g. tartaric acid, citric acid), acidifying agents, various (acidic) ) Flavors, etc.
  • the buffer substances are also able to exert this protective effect when passing through the stomach.
  • the formulation according to the invention guarantees protection of the microorganisms stabilized by lyophilization from the natural acid barrier of the stomach, the microorganisms being protected by the action of the buffer substances of the formulation after their reconstitution.
  • the microorganisms therefore enter the intestine in a viable form, where they can develop their effectiveness.
  • formulations according to the present invention are outstandingly suitable for the production of preparations for a wide variety of medical indications and as an additional or preventive food supplement for humans and animals.
  • the microorganisms stabilized by lyophilization preferably contain auxiliaries for lyophilization, as well as residues from the nutrient medium. These lyophilisate compositions determine the hygroscopic properties of the lyophilisate.
  • Preferred lyophilisates have a w values in the range from 0.2 or below, in particular from 0.1 to 0.2, after the freeze-drying process. As a result, they are long-lasting, but they are also able to absorb large quantities of water quickly from the ambient air, which of course must be avoided during storage got to.
  • This problem can be technologically solved by further processing - tableting, filling, packaging - immediately after the introduction of lyophilized microorganisms into the effervescent matrix, so that contact with the ambient air is minimized.
  • the packaging must have an a w value of 0.1 to 0.2 in the long term so that free water is not available for active life processes in cells.
  • Packaging in composite films, sachets or for tablets in aluminum sleeves with desiccants are particularly suitable for this.
  • the storage temperature should be chosen so that 20 ° C is not exceeded.
  • the formulation according to the invention advantageously has an a w value of 0.1 to 0.2, in particular 0.1 to 0.15. Therefore, buffer matrices which are composed of completely anhydrous mineral constituents are preferably also used according to the invention. In principle, these are easy to prepare, but have so far not been used in the production of probiotic formulations.
  • Preferred buffer systems in the context of the present invention are phosphate buffers, citrate buffers, carbonate buffers, malate buffers and tartrate buffers with alkalis and alkaline earths (pH 4-7.5).
  • Strains from the families Lactobacillus, Enterococcus, Bifidobacterium and Enterobacteriaceae, preferably from the genera Enterobacter and Escherichia, are used in the formulations according to the invention as microorganisms stabilized or preserved by lyophilization, in particular strains from the genera Lactobacillus delbschreibii subs. bulgaricus, Lactobacillus acidophilus, Lactobacillus casei GG, Lactobacillus casei subsp. casei, Lactobacillus helveticus, Lactobacillus lactis, Lactobacillus salivarius, Lactobacillus plantarum, Streptococcus salivarius subsp.
  • thermophilus Enterococcus faecium, Bifidobacterium bifidum, Bifidobacterium infantis, Bififobacterium longum (as well as other Bifidobacterium species) and physiologically important subspecies of Escherichia coli and mixtures of two or more species of these microorganisms are particularly preferred.
  • yeast strains in particular Saccharomyces bulardii for the human field, or other probiotic yeasts, for example Kluyveromyces marxianus for animals, are suitable for the production of the formulations according to the invention.
  • microorganisms are those whose effects can be used for oral active immunization.
  • the formulations according to the invention advantageously comprise further formulation auxiliaries or technically necessary combinations of adjuvants.
  • antioxidants and amino acids may be necessary.
  • the substance classes mentioned are typically part of all natural systems of organic origin and are therefore also obtained on an industrial scale and used as additives. They are present in the concentrations used for the formulation according to the invention below the limit values permitted in medications and nutritional supplements and are completely harmless.
  • Auxiliaries used with preference are L-ascorbic acid and L-cysteine.
  • the decisive technological step for the formulations according to the invention lies in the use of buffer substances which, after being dissolved in a suitable solvent, such as water, fruit juices, mineral waters, etc., offer the microorganisms, which were also dry until then, optimal conditions for cell wall reconstitution and these then allow passage through the upper digestive tract, in particular the stomach, with the hydrochloric acid present in the stomach being buffered for a short time.
  • a suitable solvent such as water, fruit juices, mineral waters, etc.
  • composition When dispersing the pharmaceutical form, a composition should preferably be produced which corresponds to the currently valid WHO recipe for rehydration during or after diarrhea.
  • the formulation according to the invention is made available as a shower, the buffer substances being part of the dry shower bases.
  • the formulation according to the invention disintegrates, CO 2 is formed , which displaces the oxygen present in the solution, and thus leads to more favorable survival conditions for anaerobic and microaerophilic germs.
  • the effervescent basics are preferably divided as effervescent granules (cf., for example, European Pharmacopoeia, 3rd edition (1997), 1847/1848 and Hagers Handbook of Pharmaceutical Practice, 5th edition, page 723).
  • the buffer granules are manufactured using the technologically customary granulation methods, e.g. by thermal granulation in heatable mixers, by granulation with reactive or non-reactive liquids in suitable commercial devices, whereby granulation can also be carried out in a vacuum (e.g. evacuable mixer with chopper with subsequent drying and screening, the TOPO® process or fluidized bed granulation) . Spiked with the intended microorganisms, these granules can then be tableted or filled into sachets.
  • a vacuum e.g. evacuable mixer with chopper with subsequent drying and screening, the TOPO® process or fluidized bed granulation
  • the formulation according to the invention preferably contains one or more active pharmaceutical ingredients, in particular an antidarrheal active ingredient, as a result of which a superadditive, anti-diarrhetic effect results from the combination of these substances while at the same time restoring a healthy intestinal flora.
  • active ingredients for the formulation according to the invention can be, for example, loperamide, domperidone and ofloxacin, which themselves have no germ-damaging effect on lactic acid bacteria.
  • the active formulation according to the invention preferably preferably comprises
  • 0.1 - 30% by weight of microorganisms stabilized by lyophilization in particular 0.1 to 5% by weight, and - 10 - 99.9% by weight, in particular 70 to 99% by weight of buffer substances (shower base) and, if necessary, 0 - 80% by weight, in particular 0 to 2% by weight, of auxiliaries.
  • the buffer substances, but also auxiliary substances and lyophilisates, are preferably in granular form or in powder form.
  • a particularly preferred formulation contains microorganisms of the genus Lactobacillus casei GG stabilized by lyophilization.
  • the present invention relates to the use of the formulations of the intestinal physiologically active according to the invention for the preparation of preparations for medical indications, in particular for gastrointestinal disorders, or as a food supplement and animal food supplement.
  • formulations according to the invention are particularly suitable in all cases in which the natural intestinal flora has been impaired or destroyed, for example in the effective accompanying and aftertreatment during or after administration of antibiotics.
  • formulations according to the invention can also include glucose and / or oligosaccharides and / or other saccharides which are favorable for the growth of germs in the intestinal lumen, as well as polysaccharides and related substances (inulins, dietary fibers).
  • Example 1 Stable formulations of effervescent granules (tablets) with lactic acid bacteria as an effective principle
  • Lactobacillus casei GG (LGG, Valio, Finland) approx. 10 8 CfU (colony forming units) / dose
  • Enterococcus faecium (Ef) (strain M74, Medipharm, Sweden) approx.
  • Bifidobacterium lactis (strain Bb 12, Hansen 's Biosystems / Denmark) approx. 10 10 CfU / dose
  • Example 2 Incubation of the Formulations According to the Invention in Artificial Gastric Juice (aMS)
  • the buffer substances are mixed with bacterial lyophilisates in a weight ratio of 249: 1.
  • One part of lyophilisate is sufficient to achieve a germ density of approximately 10 8 -10 9 CfU / g of formulation mass.
  • germ densities in ranges above 10 12 CfU / g are achieved, which are of course preferred for commercial products according to the invention.
  • the incubation is carried out at a body temperature of 37 ° C to simulate as physiological conditions as possible.
  • SWB shaking water bath
  • REF reference sample
  • the course of the reduction in the number of bacteria is shown in Table 1.
  • Column 2 of Table 1 shows the course of the bacterial count as it results from the use of the buffer substance and LGG according to the invention in the case of incubation in aMS.
  • Column 3 contains the REF data.
  • Column 4 shows how aMS affects unprotected lactic acid bacteria in the form of a lyophilisate (Lyo).
  • composition of the effervescent mixture (according to Bauer et al. "Pharmaceutical Technology", 5th edition, page 316):
  • Table 4 below lists the survival germ counts of LGG after incubation in artificial gastric juice and in parallel after incubation in water (REF).
  • Example 4 Effect of domperidone on the survivability of lactic acid bacteria
  • Table 6 shows that the active ingredient domperidone has very little influence on the survivability of the lactic acid bacteria during the incubation. -The combination of domperidone and artificial gastric juice causes a slightly greater decrease in the number of bacteria than water. E.g. Effect of ofloxacin on the survivability of lactic acid bacteria

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Abstract

L'invention concerne une formulation renfermant des micro-organismes stabilisés et des substances tampon. Cette formulation facilite le passage des micro-organismes, après leur dispersion et leur absorption dans le tube digestif, à travers l'estomac, tout en maintenant leur vitalité.
PCT/AT1999/000192 1998-08-06 1999-07-29 Formulations renfermant des micro-organismes a effet probiotique Ceased WO2000007571A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP99938048A EP1102580A2 (fr) 1998-08-06 1999-07-29 Formulations renfermant des micro-organismes a effet probiotique
AU52704/99A AU5270499A (en) 1998-08-06 1999-07-29 Formulations having probiotically active microorganisms

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AT0136098A AT407008B (de) 1998-08-06 1998-08-06 Formulierungen mit probiotisch wirksamen mikroorganismen
ATA1360/98 1998-08-06

Publications (2)

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WO2000007571A2 true WO2000007571A2 (fr) 2000-02-17
WO2000007571A3 WO2000007571A3 (fr) 2000-05-11

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EP (1) EP1102580A2 (fr)
AT (1) AT407008B (fr)
AU (1) AU5270499A (fr)
WO (1) WO2000007571A2 (fr)

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WO2002007741A1 (fr) * 2000-07-25 2002-01-31 Borody Thomas J Therapie par recolonisation avec des probiotiques
WO2002005829A3 (fr) * 2000-07-17 2002-05-02 Hansens Lab Procede permettant de reduire les changements dans la flore gastro-intestinale nuisibles du point de vue ecologique intervenus chez des patients suivant un traitement medicamenteux
EP1287745A1 (fr) 2001-08-24 2003-03-05 The Procter & Gamble Company Composition masicatoire avec des substances probiotiques
WO2007098924A3 (fr) * 2006-02-28 2007-12-13 Lohmann Animal Health Gmbh Composition stabilisant l'eau, procede de production et d'utilisation associes
WO2010094727A1 (fr) * 2009-02-23 2010-08-26 Chr. Hansen A/S Procédé pour produire une composition de bactéries d'acide lactique
US20110223251A1 (en) * 2008-08-28 2011-09-15 Chr-Hansen A/S Bacterial composition
FR2963239A1 (fr) * 2010-08-02 2012-02-03 Vetalis Formes galeniques effervescentes solides pour animaux
WO2012035454A1 (fr) * 2010-09-16 2012-03-22 Yeditepe Universitesi Granule effervescent biopesticide lyophilisé et procédé de production de celui-ci
CN103550401A (zh) * 2013-11-19 2014-02-05 浙江美保龙生物技术有限公司 一种复合微生态制剂泡腾片的制备方法
WO2015154140A1 (fr) * 2014-04-09 2015-10-15 Kambouris Shares Pty Ltd Procédés et compositions d'administration d'un probiotique
US9649343B2 (en) 2011-03-09 2017-05-16 National Institutes of Health (NIH); U.S. Department of Health and Human Services (DHHS); The United States of America, NIH Division of Extramural Inventions and Tehnology Resources (DEITR) Compositions and methods for transplantation of colon microbiota
US9901603B2 (en) 2015-05-14 2018-02-27 Crestovo Holdings Llc Compositions for fecal floral transplantation and methods for making and using them and device for delivering them
US9962413B2 (en) 2010-08-04 2018-05-08 Crestovo Holdings Llc Compositions for fecal floral transplantation and methods for making and using them and devices for delivering them
US10092601B2 (en) 2016-10-11 2018-10-09 Crestovo Holdings Llc Compositions and methods for treating multiple sclerosis and related disorders
US10195235B2 (en) 2016-08-03 2019-02-05 Crestovo Holdings Llc Methods for treating ulcerative colitis
US11026978B2 (en) 2016-10-11 2021-06-08 Finch Therapeutics Holdings Llc Compositions and methods for treating multiple sclerosis and related disorders
US11040073B2 (en) 2017-04-05 2021-06-22 Finch Therapeutics Holdings Llc Compositions and methods for treating diverticulitis and related disorders
US11166990B2 (en) 2018-07-13 2021-11-09 Finch Therapeutics Holdings Llc Methods and compositions for treating ulcerative colitis
US11202808B2 (en) 2015-05-22 2021-12-21 Arizona Board Of Regents On Behalf Of Arizona State University Methods for treating autism spectrum disorder and associated symptoms
US11213549B2 (en) 2016-10-11 2022-01-04 Finch Therapeutics Holdings Llc Compositions and method for treating primary sclerosing cholangitis and related disorders
US11357801B2 (en) 2016-06-15 2022-06-14 Arizona Board Of Regents On Behalf Of Arizona State University Methods for treating autism spectrum disorder and associated symptoms
US11433102B2 (en) 2017-04-05 2022-09-06 Finch Therapeutics Holdings Llc Compositions and methods for treating Parkinson's disease (PD) and related disorders
US11439672B2 (en) * 2003-12-05 2022-09-13 Ganeden Biotech, Inc. Methods for the dietary management of irritable bowel syndrome and carbohydrate malabsorption
US11542560B2 (en) 2012-05-25 2023-01-03 Board of Regents on Behalf of Arizona State University Microbiome markers and therapies for autism spectrum disorders
US11819523B2 (en) 2016-07-01 2023-11-21 Regents Of The University Of Minnesota Compositions and methods for C. difficile treatment
US11865145B2 (en) 2017-08-07 2024-01-09 Finch Therapeutics Holdings Llc Compositions and methods for maintaining and restoring a healthy gut barrier
US11890306B2 (en) 2017-05-26 2024-02-06 Finch Therapeutics Holdings Llc Lyophilized compositions comprising fecal microbe-based therapeutic agents and methods for making and using same
US11911419B2 (en) 2018-09-27 2024-02-27 Finch Therapeutics Holdings Llc Compositions and methods for treating epilepsy and related disorders
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WO2002005829A3 (fr) * 2000-07-17 2002-05-02 Hansens Lab Procede permettant de reduire les changements dans la flore gastro-intestinale nuisibles du point de vue ecologique intervenus chez des patients suivant un traitement medicamenteux
US9623056B2 (en) 2000-07-20 2017-04-18 Crestovo Llc Probiotic recolonisation therapy
US9610308B2 (en) 2000-07-25 2017-04-04 Crestovo Llc Probiotic recolonisation therapy
US9867858B2 (en) 2000-07-25 2018-01-16 Crestovo Holdings Llc Probiotic recolonisation therapy
US9789140B2 (en) 2000-07-25 2017-10-17 Crestovo Holdings Llc Probiotic recolonisation therapy
US9737574B2 (en) 2000-07-25 2017-08-22 Crestovo Llc Probiotic recolonisation therapy
US9962414B2 (en) 2000-07-25 2018-05-08 Crestovo Holdings Llc Probiotic recolonisation therapy
US9901604B2 (en) 2000-07-25 2018-02-27 Crestovo Holdings Llc Probiotic recolonisation therapy
WO2002007741A1 (fr) * 2000-07-25 2002-01-31 Borody Thomas J Therapie par recolonisation avec des probiotiques
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AU5270499A (en) 2000-02-28
AT407008B (de) 2000-11-27

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