EP1102595A2 - Procedes mettant en oeuvre des bacteries probiotiques non productrices d'acide lactique pour augmenter la solubilite de substances nutritives - Google Patents

Procedes mettant en oeuvre des bacteries probiotiques non productrices d'acide lactique pour augmenter la solubilite de substances nutritives

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Publication number
EP1102595A2
EP1102595A2 EP99945016A EP99945016A EP1102595A2 EP 1102595 A2 EP1102595 A2 EP 1102595A2 EP 99945016 A EP99945016 A EP 99945016A EP 99945016 A EP99945016 A EP 99945016A EP 1102595 A2 EP1102595 A2 EP 1102595A2
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EP
European Patent Office
Prior art keywords
approximately
micrograms
ranges
milligrams
concentration
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP99945016A
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German (de)
English (en)
Inventor
Sean Farmer
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Ganeden Biotech Inc
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Ganeden Biotech Inc
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Priority to EP06075064A priority Critical patent/EP1719518A1/fr
Publication of EP1102595A2 publication Critical patent/EP1102595A2/fr
Ceased legal-status Critical Current

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Classifications

    • 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
    • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/02—Nutrients, e.g. vitamins, minerals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to the utilization of probiotic lactic acid-producing bacteria in a nutritional composition. More specifically, the present invention relates to the use of Bacillus coagulans for increasing the solubility of nutritional materials, preferably essential vitamins and minerals, within the gastrointestinal tract of mammals.
  • Probiotic microorganisms are those which confer a benefit when grow in a particular environment, often by inhibiting the growth of other biological organisms in the same environment.
  • Examples of probiotic organisms include bacteria and bacteriophages which possess the ability to grow within the gastrointestinal tract, at least temporarily, to displace or destroy pathogenic organisms, as well as providing other benefits to the host. See e.g., Salminen et al, 1996. Antonie Van Leeuwenhoek 70: 347-358; Elmer et al, 1996. JAMA 275: 870-876; Rafter, 1995. Scand. J. Gastroenterol. 30: 497-502; Perdigon et al, 1995. J. Dairy Sci.
  • probiotics are members of the normal colonic micro flora and are not viewed as being overtly pathogenic. However, these organisms have occasionally caused infections (e.g., bacteremia) in individuals who are, for example, immunocompromised. See e.g., Sussman, J. et al, 1986. Rev Infect. Dis. 8: 771-776; Hata, D. et al, 1988. Pediatr. Infect. Dis. 7: 669-671.
  • probiotic bacteria found in sour milk has been utilized since ancient times (i.e., long-before the discovery of bacteria) as a therapeutic treatment for dysentery and related gastrointestinal diseases. More recently, probiotic preparations were systematically evaluated for their effect on health and longevity in the early- 1900's (see e.g., Metchinikoff, ⁇ ., Prolongation of Life. Willaim Heinermann. London 1910), although their utilization has been markedly limited since the advent of antibiotics in the 1950's to treat pathological microbes. See e.g.. Winberg, et al, 1993. Pediatr. Nephrol. ⁇ : 509-514; Malin et al. Ann. Nutr. Metab.
  • lactic acid-producing bacteria e.g.. Bacillus. Lactobacillus and Streptococcus species
  • probiotics are the lactic acid-producing bacteria (i.e., Lactobacill ⁇ ) and Bifidobacteria, which are widely utilized in yogurts and other dairy products. These probiotic organisms are non-pathogenic and non-toxigenic. retain viability during storage, and possess the ability to survive passage through the stomach and small intestine. Since probiotics do not permanently colonize the host, they need to be ingested or applied regularly for any health- promoting properties to persist.
  • Commercial probiotic preparations are generally comprised of mixtures of Lactobacilli and Bifidobacteria, although yeast such as Saccharomyces have also been utilized.
  • gastrointestinal microflora has been shown to play a number of vital roles in maintaining gastrointestinal tract function and overall physiological health.
  • the growth and metabolism of the many individual bacterial species inhabiting the gastrointestinal tract depend primarily upon the substrates available to them, most of which are derived from the diet. See e.g., Gibson G.R. et al, 1995. Gastroenterology 106: 975-982; Christl, S.U. et al, 1992. Gut 33: 1234-1238.
  • Bifidobacteria are known to be involved in resisting the colonization of pathogens in the large intestine. See e.g., Yamazaki, S. et al, 1982. Bifidobacteria and Microflora 1: 55-60.
  • lactic acid producing bacteria also produce bacteriocins which are inhibitory metabolites which are responsible for the bacteria's anti-microbial effects. See e.g., Klaenhammer, 1993. FEMS Microbiol. Rev. 12: 39-85; Barefoot et al, 1993. J. Diary Sci. 76: 2366-2379.
  • selected Lactobacillus strains which produce antibiotics have been demonstrated as effective for the treatment of infections, sinusitis, hemorrhoids, dental inflammations, and various other inflammatory conditions. See e.g., U.S. Patent No. 5,439,995.
  • Lactobacillus reuteri has been shown to produce antibiotics which possess antimicrobial activity against Gram negative and Gram positive bacteria, yeast, and various protozoan. See e.g., U.S. Patent Nos. 5,413.960 and 5,439,678. Lactobacillus casei ssp, rhamnosus has been utilized to preserve animal feedstuffs, particularly S. rubidaea F 13299, alone or combined with an anti-fungal B. subtillis (swain FB260). See U.S. Patent No. 5,371,011.
  • Probiotics have also been shown to possess anti-mutagenic properties.
  • Gram positive and Gram negative bacteria have been demonstrated to bind mutagenic pyrolysates which are produced during cooking at a high temperature.
  • Studies performed with lactic acid-producing bacteria has shown that these bacteria may be either living or dead, due to the fact that the process occurs by adsorption of mutagenic pyrolysates to the carbohydrate polymers present in the bacterial cell wall. See e.g., Zang, X. Bacillus et al, 1990. J. Dairy Sci. 73: 2702-2710.
  • Lactobacilli have also been shown to possess the ability to degrade carcinogens (e.g., N-nitrosamines), which may serve an important role if the process is subsequently found to occur at the level of the mucosal surface. See e.g., Rowland, I.R. and Grasso, P., Appl Microbiol. 29: 7-12. Additionally, the co-administration of lactulose and Bifidobacteria longum to rats injected with the carcinogen azoxymethane was demonstrated to reduce intestinal aberrant crypt foci, which are generally considered to be pre-neoplastic markers. See e.g., Challa, A. et al, 1997. Carcinogenesis 18: 5175-21.
  • carcinogens e.g., N-nitrosamines
  • Purified cell walls of Bifidobacteria may also possess anti-tumorigenic activities in that the cell wall of Bifidobacteria infantis induces the activation of phagocytes to destroy growing tumor cells. See e.g., Sekine, K. et al, 1994. Bifidobacteria and Microflora 13: 65-77 ' . Bifidobacteria probiotics have also been shown to reduce colon carcinogenesis induced by 1.2-dimethylhydrazine in mice when concomitantly administered with fructo-oligosaccharides(FOS; see e.g.. Koo and Rao. 1991. Nutrit. Rev. 51: 137-146), as well as inhibiting liver and mammary tumors in rats (see e.g., Reddy and Rivenson. 1993. Cancer Res 55: 3914-3918).
  • FOS fructo-oligosaccharides
  • Antibiotics are widely used to control pathogenic microorganisms in both humans and animals.
  • the widespread use of anti-microbial agents, especially broad spectrum antibiotics has resulted in a number of serious clinical consequences.
  • the indiscriminate use of these chemicals has resulted in the generation of multiple antibiotic- resistant pathogens. See e.g., Mitchell, P. 1998. The Lancet 352: 462-463; Shannon, K., 1998. The Lancet 352: 490-491.
  • antibiotics often kill beneficial, non-pathogenic microorganisms (i.e., flora) within the gastrointestinal tract which contribute to digestive function and health.
  • relapse the return of infections and their associated symptoms
  • secondary opportunistic infections often result from the depletion of lactic acid-producing and other beneficial flora within the gastrointestinal tract.
  • lactic acid-producing or probiotic bacteria are extremely sensitive to common antibiotic compounds.
  • many individuals develop a number of deleterious physiological side-effects including: diarrhea, intestinal cramping, and sometimes constipation. These side-effects are primarily due to the non-selective action of antibiotics, as antibiotics do not possess the ability to discriminate between beneficial, non-pathogenic and pathogenic bacteria, both bacterial types are killed by these agents.
  • intestinal flora beneficial microorganisms
  • intestinal flora which normally colonize the gastrointestinal tract
  • the resulting change in the composition of the intestinal flora can result in vitamin deficiencies when the vitamin-producing intestinal bacteria are killed, diarrhea and dehydration and, more seriously, illness should a pathogenic organism overgrow and replace the remaining beneficial gastrointestinal bacteria.
  • beneficial and/or pathological microorganisms can also inhabit the oral cavity, the genital area and the vagina (see e.g., Thomason, et al, 1991. Am. J. Obstet Gynecol 165: 1210-1217; Marsh. 1993. Caries Res. 27: 72-76; Lehner, 1985. Vaccine 3: 65-68; Hill & Embil, 1986. Can. Med. Assoc. J. 134: 321-331).
  • anti-microbial drugs can similarly cause an imbalance in those microorganisms and the therapeutic use of probiotic bacteria, especially the Lactobacillus strains, which colonize those areas has been disclosed (see e.g., Winberg, et al, 1993. Pediatr. Nephrol 7: 509-514; Malm, et al, 1996. Ann. Mar. Metab. 40: 137-145. U.S. Patent No. 5.176.91 1 ).
  • Increasing numbers of pathogenic microorganisms have developed antibiotic resistance, requiring the development and use of second and third generation antibiotics.
  • Microorganisms that are resistant to multiple drugs have also developed, often with multiple drug resistance spreading between species, leading to serious infections that cannot be controlled by use of antibiotics.
  • Bacillus coagulans is a non-pathogenic gram positive spore-forming bacteria that produces L(+) lactic acid (dextrorotatory) in homo-fermentation. This microorganism has been isolated from natural sources, such as heat-treated soil samples inoculated into nutrient medium (see e.g., Bergey 's Manual of Systemic Bacteriology, Vol. 2, Sneath, P.H.A., et al, eds., (Williams & Wilkins. Baltimore, MD, 1986)).
  • Purified Bacillus coagulans strains have served as a source of various enzymes including, but not limited to: restriction endonucleases (see U.S. Patent No. 5,200,336); amylase (see U.S. Patent No. 4,980,180); lactase (see U.S. Patent No. 4,323,651); and cyclo-malto-dextrin glucano- transferase (see U.S. Patent No. 5,102,800).
  • Bacillus coagulans has been used to produce lactic acid (see U.S. Patent No. 5,079,164).
  • Bacillus coagulans designated Lactobacillus sporogenes, Sakaguti & Nakayama (ATCC 31284)
  • Bacillus natto to produce a fermented food product from steamed soybeans
  • Bacillus coagulans strains have also been used as animal feed additives for poultry and livestock to reduce disease and improve feed utilization and to, therefore, increase growth rate in the animals (see International Patent Application Nos. WO 9314187 and WO 9411492).
  • Bacillus coagulans strains and various other lactic acid-producing bacteria have proven to produce copious amounts of lactic acid and enzymes that have the ability to increase the solubility of nutritional materials, in particular vitamins and minerals.
  • the present invention discloses the discovery that, in digestion, bacterial enzymes and various other metabolic products of bacteria (e.g., lactic acid), play a vital role in the solubility and therefor the bioavailability, of vitamins and minerals, either as a component of food or as a result of nutritional supplementation.
  • bacteria e.g., lactic acid
  • the present invention discloses a probiotic composition
  • a probiotic composition comprising one or more Bacillus bacterial species or strains, combined with an effective amount of a bifidogenic oligosaccharide, preferably fructo-oligosaccharide(FOS), in a pharmaceutically- or nutritionally-acceptable carrier which is suitable for oral administration to a human or animal species, wherein said Bacillus: (i) is capable of growing at temperatures of about 20°C to about 65°C; ( / ' ) produces L(+) lactic acid (dextrorotatory); ( ) produces spores resistant to heat up to 98°C; and (iv) exhibits probiotic activity.
  • a bifidogenic oligosaccharide preferably fructo-oligosaccharide(FOS)
  • a pharmaceutically- or nutritionally-acceptable carrier which is suitable for oral administration to a human or animal species
  • said Bacillus (i) is capable of growing at temperatures of about 20°C
  • the probiotic activity results from the vegetative growth of the isolated Bacillus, Lactobacillus, Bifidiobacterium, or Sporolactobacillus species that produces extracellular metabolites which function to increase the bioavailability of nutrients though increased solubility.
  • the present invention also discloses a therapeutic composition
  • a therapeutic composition comprising one or more Bacillus coagulans strain in combination with a therapeutically-effective amount of a short or long chain bifidogenic oligosaccharide, preferably fructo-oligosaccharide (FOS), in a pharmaceutically- or nutritionally-acceptable carrier suitable for oral administration to the digestive track of a mammal.
  • a short or long chain bifidogenic oligosaccharide preferably fructo-oligosaccharide (FOS)
  • FOS fructo-oligosaccharide
  • the Bacillus coagulans strain is included in the composition in the form of spores which are capable of germinating following ingestion; a dried cell mass; a stabilized gel or paste; or a stabilized liquid suspension.
  • the present invention also describes a method for decreasing the number of pathogenic organisms that inhibit the growth of vitamin-producing organisms within the gastrointestinal tract, wherein a composition is administered that would, preferably, include a complex of the RDA vitamins, minerals and trace minerals as well as those nutrients that have no established RDA. but have a beneficial role in healthy human or mammal physiology.
  • the preferred composition would include a minerals which are in either in the gluconate or citrate form, due to the established fact that these forms are more readily metabolized by lactic acid-producing bacteria.
  • FIG. 1 illustrates, in tabular form, the various metabolic characteristics of
  • FIG. 2 illustrates, in tabular form, the vitamins, their RDA, their ranges and respective preferred ranges, of the therapeutic compositions of the present invention.
  • FIG. 3 illustrates, in tabular form, the preferred major minerals and their respective RDA.
  • FIG. 4 illustrates, in tabular form, the preferred trace minerals and their respective ranges, as utilized in the therapeutic composition of the present invention.
  • probiotic ' ' refers to microorganisms (e.g., bacteria, yeast, viruses, and/or fungi) which form, at a minimum, a part of the transient or endogenous flora and. thus, possess a beneficial prophylactic and/or therapeutic effect upon the host organism.
  • Probiotics are generally known to be clinically-safe (i.e.. non-pathogenic) by those skilled within the art.
  • the Bacillus species is thought to result from competitive inhibition of growth of pathogens due to superior colonization, parasitism of undesirable microorganisms, lactic acid production and/or other extracellular products having anti-microbial activity, or combinations thereof.
  • the term "'nutritional supplement” refers to any type of orally ingestable material which can be digested and assimilated by the gastrointestinal tract, and from which a physiological, nutritional benefit may be derived. This material includes liquid and solid foodstuffs, vitamin and mineral supplements, and the like.
  • the present invention discloses the utilization of Bacillus, Lactobacillus, Bifiidiobacterium, or Sporolactobacillus bacterial species, preferably Bacillus coagulans, in nutritional compositions, in combination with a bifidogenic oligosaccharide, preferably fructo- oligosaccharides (FOS), as a probiotic for increasing the solubility of essential vitamin and mineral nutritional materials in mammals, particularly in humans.
  • the present invention therefore describes various nutritional compositions, methods for using the compositions and systems containing the nutritional compositions.
  • Non-pathogenic Bacillus species of the present invention include, but are not limited to: Bacillus coagulans; Bacillus coagulans Hammer; Bacillus brevis subspecies coagulans, Bacillus subtilis, Bacillus uniflagellatus, Bacillus lateropsorus, Bacillus laterosporus BOD, Bacillus megaterium, Bacillus polymyxa, Bacillus licheniformis, Bacillus pumilus, and Bacillus sterothermophilus, and any genetic variants thereof.
  • Exemplary lactic acid-producing Lactobacillus species include, but are not limited to: Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus DDS-1, Lactobacillus GG, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus gasserii. Lactobacillus jensenii, Lactobacillus delbruekii, Lactobacillus, bulgaricus, Lactobacillus salivarius and Lactobacillus sporogenes (also designated as Bacillus coagulans), and any genetic variants thereof.
  • Exemplary lactic acid-producing Sporolactobacillus species include all Sporolactobacillus species, for example. Sporolactobacillus P44, and any genetic variants thereof.
  • Exemplary lactic acid-producing Bifiidiobacterium species include, but are not limited to:
  • Bifidiobacterium adolescentis Bifiidiobacterium animalis. Bifiidiobacterium bifidum. Bifidiobacterium bifidus. Bifiidiobacterium breve. Bifiidiobacterium infantis. Bifidiobacterium infantis. Bifidiobacterium longum, and any genetic variants thereof.
  • the prophylactic and/or therapeutic effect of a lactic acid-producing bacteria of the present invention results, in part, from a competitive inhibition of the growth of pathogens due to: ( ) their superior colonization abilities; (ii) parasitism of undesirable microorganisms; (/ /) the production of lactic acid and/or other extracellular products possessing anti-microbial activity; or (iv) various combinations thereof.
  • the aforementioned products and activities of the lactic acid-producing Bacillus of the present invention act synergistically to produce the beneficial probiotic effect disclosed herein.
  • a probiotic bacteria which is suitable for use in the methods and compositions of the present invention ( ) possesses the ability to produce lactic acid; (ii) demonstrates beneficial function; and (iii) is non-pathogenic.
  • suitable bacteria have been identified and are described herein, although it should be noted that the present invention is not to be limited to currently-classified bacterial species insofar as the purposes and objectives as disclosed.
  • the physiochemical results from the in vivo production of lactic acid is key to the effectiveness of the probiotic lactic acid-producing bacteria of the present invention.
  • Lactic acid production markedly decreases the pH (i.e., increases acidity) within the local micro-floral environment and does not contribute to the growth of many undesirable, physiologically-deleterious bacteria, fungi, and viruses.
  • the bacterial enzymes and various other metabolic products of the probiotic bacteria serve to inhibit the growth of competing pathogenic bacteria, as well as concomitantly increasing the solubility and therefore the bioavailability, of vitamins and minerals within the gastrointestinal tract.
  • Typical lactic acid-producing bacteria useful as a probiotic of this invention are efficient lactic acid producers which include non-pathogenic members of the Bacillus genus which produce bacteriocins or other compounds which inhibit the growth of pathogenic organisms.
  • Exemplary lactic acid-producing, non-pathogenic Bacillus species include, but are not limited to: Bacillus coagulans; Bacillus coagulans Hammer; and Bacillus brevis subspecies coagulans.
  • Bacillus species which are preferred in the practice of the present invention include, but are not limited to the lactic acid-producing Bacillus coagulans and Bacillus laevolacticus.
  • Various other non-lactic acid-producing Bacillus species may be utilized in the present invention so long as they produce compounds which possess the ability to inhibit pathogenic bacterial or mycotic growth. Examples of such suitable non-lactic acid-producing Bacillus include, but are not limited to: Bacillus subtilis. Bacillus uniflagellatus. Bacillus lateropsorus, Bacillus laterosporus BOD. Bacillus megaterium. Bacillus polymyxa. Bacillus licheniformis. Bacillus pumilus. and Bacillus sterothermophilus.
  • Bacillus species particularly those species having the ability to form spores (e.g., Bacillus coagulans), are a preferred embodiment of the present invention.
  • the Bacillus species utilized in the practice of the present invention may selected from the group comprising: Bacillus coagulans, Bacillus subtilis, Bacillus laterosporus and Bacillus laevolacticus, all of which have the ability to form spores, and can colonize tissue aerobically.
  • Bacillus coagulans including, but not limited to many different strains available through commercial and public sources, such as the American Tissue Culture Collection (ATCC).
  • ATCC American Tissue Culture Collection
  • Bacillus coagulans strains are available as ATCC Accession Numbers 15949, 8038.
  • Bacillus subtilis strains are available as ATCC Accession Numbers 10783, 15818, 15819, 27505, 13542, 15575, 33234, 9943, 6051a, 25369, 1 1838, 15811, 27370, 7003, 15563, 4944, 27689, 43223, 55033, 49822, 15561, 15562, 49760, 13933, 29056, 6537, 21359, 21360, 7067, 21394, 15244, 7060, 14593, 9799, 31002, 31003, 31004, 7480, 9858, 13407, 21554.
  • Bacillus laterosporus strains are available as ATCC Accession Numbers 6456, 6457, 30 29653, 9141, 533694, 31932 and 64, including Bacillus laterosporus BOD.
  • Bacillus laevolacticus strains are available as ATCC Accession Numbers 23495. 23493, 23494, 23549 and 23492. It should be noted, however, that although many of the examples herein refer to the Bacillus coagulans species in particular, it is intended that any of the Bacillus species can be used in the compositions, articles of manufacture, systems and method of the present invention.
  • a Bacillus species is particularly suited for the present invention due to the properties in common between species of the Bacillus genus, including, but not limited to, the ability to form spores which are relatively resistant to heat and other conditions, making them ideal for storage (shelf-life) in product formulations, and ideal for survival and colonization of tissues under conditions of pH, salinity, and the like on tissues subjected to microbial infection.
  • probiotic Bacillus coagulans is non-pathogenic and is generally regarded as safe (i.e., GRAS classification) by the U.S. Federal Drug Administration (FDA) and the U.S. Department of Agriculture (USDA), and by those individuals skilled within the art.
  • Additional useful characteristics of the Bacillus species include, but are not limited to: non-pathogenicity. being aerobic, facultative and heterotrophic. thus rendering these species safe, and able to colonize skin, mucous membrane tissues, and various other tissues of interest.
  • Bacillus species possesses the ability to produce heat-resistant spores, it is particularly useful for making pharmaceutical compositions which require heat and pressure in their manufacture. Accordingly, formulations that include the utilization viable Bacillus spores in a pharmaceutically-acceptable carrier are particularly preferred for making and using compositions disclosed in the present invention.
  • the growth of these various Bacillus species to form cell cultures, cell pastes, and spore preparations is generally well-known within the art.
  • the exemplary culture and preparative methods which are described herein for Bacillus coagulans may be readily utilized and/or modified for growth and preparation of the other Bacillus strains, as well as the lactic acid-producing bacteria disclosed in the present invention.
  • the exemplary methods and compositions which are described herein using Bacillus coagulans as a probiotic for controlling, treating, or reducing microbial infections may also be readily utilized with other Bacillus species.
  • Bacillus coagulans is exemplary and preferred as a probiotic for biological control of various microbial pathogens. Because Bacillus coagulans forms heat-resistant spores, it is particularly useful for making pharmaceutical compositions for treating microbial infections.
  • Probiotic Bacillus coagulans has been categorized as "safe" by the U.S. Federal Drug Administration (FDA), the U.S. Department of Agriculture (USDA), and by those skilled within the art.
  • beneficial properties include its ability to reduce the numbers of pathogenic organisms that can kill vitamin producing organisms in the gut, its production of lactase which is a beneficial attribute for those individuals suffering from lactose intolerance, its production of bacteriocins that are inhibitory to those intestinal organisms that produce carcinogens in the colon, and its well known ability to produce L(+) lactic acid, which functions to both metabolize and increase the solubility, absorption, and therefore the bioavailability of vitamins and minerals with the gastrointestinal tract.
  • the Gram positive rods of Bacillus coagulans have a cell diameter of greater than 1.0 ⁇ m with variable swelling of the sporangium, without parasporal crystal production.
  • Bacillus coagulans is a non-pathogenic. Gram positive, spore-forming bacteria that produces L(+) lactic acid (dextrorotatory) under homo-fermentation conditions. It has been isolated from natural sources, such as heat-treated soil samples inoculated into nutrient medium (see e.g., Bergey's Manual of Systemic Bacteriology, Vol. 2, Sneath, P.H.A. et al, eds.. Williams & Wilkins, Baltimore, MD, 1986).
  • Bacillus coagulans strains have served as a source of enzymes including endonucleases (e.g., U.S. Patent No. 5,200,336); amylase (U.S. Patent No. 4,980,180); lactase (U.S. Patent No. 4,323,651) and cyclo-malto-dextrin glucano-transferase (U.S. Patent No. 5,102,800).
  • Bacillus coagulans has also been utilized to produce lactic acid (U.S. Patent No. 5,079,164).
  • a strain of Bacillus coagulans also referred to as Lactobacillus sporogenes; Sakaguti & Nakayama, ATCC No.
  • Bacillus coagulans strains have been used as animal feeds additives for poultry and livestock to reduce disease and improve feed utilization and, therefore, to increase growth rate in the animals (International PCT Patent Applications No. WO 9314187 and No. WO 9411492). In particular, Bacillus coagulans strains have been used as general nutritional materials and agents to control constipation and diarrhea in humans and animals.
  • Bacillus coagulans bacteria utilized in the present invention are available from the American Type Culture Collection (ATCC, Rockville, MD) using the following accession numbers: Bacillus coagulans Hammer NRS 727 (ATCC No. 1 1014); Bacillus coagulans
  • Bacillus coagulans bacteria are also available from the Deutsche Sarumlung von Mikroorganismen und Zellkuturen GmbH (Braunschweig, Germany) using the following accession numbers: Bacillus coagulans Hammer 1915 (DSM No. 2356); Bacillus coagulans Hammer 1915 (DSM No. 2383, corresponds to ATCC No. 11014); Bacillus coagulans Hammer (DSM No. 2384, corresponds to ATCC No. 1 1369); and Bacillus coagulans Hammer (DSM No. 2385, corresponds to ATCC No. 15949). Bacillus coagulans bacteria can also be obtained from commercial suppliers such as Sabinsa Corporation (Piscataway, NJ) or K.K. Fermentation (Kyoto, Japan). Bacillus coagulans strains and their growth requirements have been described previously
  • Bacillus coagulans can also be isolated from natural sources (e.g., heat-treated soil samples) using well-known procedures (see e.g., Bergey ' s Manual of Systemic Bacteriology, Vol. 2, p. 1 1 17. Sneath. P.H.A. et al, eds.. Williams & Wilkins. Baltimore. MD. 1986).
  • Bacillus coagulans had previously been mis-characterized as a Lactobacillus in view of the fact that, as originally described, this bacterium was labeled as Lactobacillus sporogenes (See Nakamura et al. 1988. Int. J. Syst. Bacteriol 38: 63-73).
  • Bacillus coagulans is aerobic and facultative, grown typically in nutrient broth, pH 5.7 to 6.8, containing up to 2% (wt/vol) NaCl, although neither NaCl nor KCI are an absolute requirement for growth.
  • a pH value ranging from approximately pH 4 to pH 6 is optimum for initiation of sporulation. It is optimally grown at about 30°C to about 55°C, and the spores can withstand pasteurization. It exhibits facultative and heterotrophic growth by utilizing a nitrate or sulfate source.
  • Bacillus coagulans can be grown in a variety of media, although it has been found that certain growth conditions produce a culture which yields a high level of sporulation. For example, sporulation is enhanced if the culture medium includes 10 mg/liter of manganese sulfate, yielding a ratio of spores to vegetative cells of about 80:20. In addition, certain growth conditions produce a bacterial spore which contains a spectrum of metabolic enzymes particularly suited for the present invention (i.e., the control of microbial infections). Although spores produced by these particular growth conditions are preferred, spores produced by any compatible growth conditions are suitable for producing a Bacillus coagulans useful in the present invention. (i) Culture of Vegetative Bacillus coagulans
  • Bacillus coagulans is aerobic and facultative, and is typically cultured at pH 5.7 to 6.8, in a nutrient broth containing up to 2% (by wt) NaCl, although neither NaCl, nor KC1 are required for growth.
  • a pH of about 4.0 to about 7.5 is optimum for initiation of sporulation (i.e., the formation of spores).
  • the bacteria are optimally grown at 30°C to 45°C, and the spores can withstand pasteurization. Additionally, the bacteria exhibit facultative and heterotrophic growth by utilizing a nitrate or sulfate source.
  • the various metabolic characteristics of Bacillus coagulans is illustrated in FIG. 1.
  • Bacillus coagulans can be cultured in a variety of media, although it has been demonstrated that certain growth conditions are more efficacious at producing a culture which yields a high level of sporulation. For example, sporulation is demonstrated to be enhanced if the culture medium includes 10 mg/1 of MgS0 4 sulfate, yielding a ratio of spores to vegetative cells of approximately 80:20.
  • certain culture conditions produce a bacterial spore which contains a spectrum of metabolic enzymes particularly suited for the present invention (i.e., production of lactic acid and enzymes for the enhanced probiotic activity and biodegradation).
  • the spores produced by these aforementioned culture conditions are preferred, various other compatible culture conditions which produce viable Bacillus coagulans spores may be utilized in the practice of the present invention.
  • Suitable media for the culture of Bacillus coagulans include: PDB (potato dextrose broth); TSB (tryptic soy broth); and NB (nutrient broth), which are all well-known within the field and available from a variety of sources.
  • media supplements which contain enzymatic digests of poultry and/or fish tissue, and containing food yeast are particularly preferred.
  • a preferred supplement produces a media containing at least 60% protein, and about 20% complex carbohydrates and 6% lipids.
  • Media can be obtained from a variety of commercial sources, notably DIFCO (Newark, NJ); BBL (Cockeyesville, MD); Advanced Microbial Systems (Shakopee. MN); and Troy Biologicals (Troy, MD.
  • a culture of Bacillus coagulans Hammer bacteria (ATCC No. 31284) was inoculated and grown to a cell density of approximately 1x10 s to lxlO 9 cells/ml in nutrient broth containing: 5.0 g Peptone; 3.0 g Meat Extract; 10-30 mg MnSO 4 and 1 ,000 ml distilled water, the broth was then adjusted to pH 7.0.
  • the bacteria were cultured by utilization of a standard airlift fermentation vessel at 30°C.
  • the range of MnS0 4 acceptable for sporulation was found to be 1.0 mg/1 to 1.0 g/1.
  • the vegetative bacterial cells can actively reproduce up to 65°C. and the spores are stable up to 90°C.
  • Bacillus coagulans Hammer bacterial cells or spores were collected using standard methods (e.g.. filtration, centrifugation) and the collected cells and spores may subsequently be lyophilized, spray dried, air dried or frozen.
  • the supernatant from the cell culture can be collected and used as an extracellular agent secreted by Bacillus coagulans which possesses anti-microbial activity useful in a formulation of this invention.
  • a typical yield obtained from the aforementioned culture methodology is in the range of approximately lxl0 9 to lxlO 13 viable spores and, more typically, approximately lxl0"to 1.5x10" cells/spores per gram prior to being dried. It should also be noted that the Bacillus coagulans spores, following a drying step, maintain at least 90%> viability for up to 7 years when stored at room temperature. Hence, the effective shelf-life of a composition containing Bacillus coagulans Hammer spores at room temperature is approximately 10 years.
  • a culture of dried Bacillus coagulans Hammer bacteria (ATCC No. 31284) spores may be prepared as follows. Approximately lxlO 7 spores were inoculated into one liter of culture medium containing: 24 g (wt./vol.) potato dextrose broth; 10 g of an enzymatic-digest of poultry and fish tissue; 5 g of fructo-oligosaccharides (FOS); and 10 g MnSO 4 . The culture was maintained for 72 hours under a high oxygen environment at 37°C so as to produce a culture having approximately 15x10'° cells/gram of culture. The culture was then filtered to remove the liquid culture medium and the resulting bacterial pellet was resuspended in water and lyophilized. The lyophilized bacteria were ground to a fine "powder" by use of standard good manufacturing practice (GMP) methodologies. The powder is then combined into Formulation 1 or Formulation 4 as described in Specific Example B to form dry powder compositions.
  • GMP standard good manufacturing
  • a one liter culture of Bacillus coagulans was prepared as described in Section 2( ), except that the fructo-oligosaccharide (FOS) was omitted.
  • the culture was maintained for 5 days as described, at which time FOS was added at a concentration of 5 g/liter. and the culture was continued. Subsequently, 20 ml of carrot pulp was then added at day 7, and the culture was harvested when the culture became saturated (i.e., no substantial cell division).
  • the culture was first autoclaved for 30 minutes at 250°F. and then centrifuged at 4000 r.p.m. for 15 mm. The resulting supernatant was collected and filtered in a Buchner funnel through a 0.8 ⁇ m filter. The filtrate was collected and further filtered through a 0.2 ⁇ m Nalge vacuum filter. The resulting final filtrate was then collected (an approximate volume of 900 ml) to form a liquid containing an extracellular product.
  • FOS fructo-oligosaccharide
  • Bifidogenic oligosaccharides are a class of carbohydrates particularly useful for preferentially promoting the growth of a lactic acid-producing bacteria of the present invention.
  • These oligosaccharides include, but are not limited to: fructo- oligosaccharides (FOS); gluco-oligosaccharides (GOS); other long-chain oligosaccharide polymers of fructose and/or glucose; and the trisaccharide - raffinose. All of these aforementioned carbohydrates are not readily digested by pathogenic bacteria.
  • the preferential growth of lactic acid-producing bacteria is promoted by the utilization of these bifidogenic oligosaccharides due to the nutrient requirements of this class of bacterium, as compared to pathogenic bacteria.
  • Bifidogenic oligosaccharides are long chain polymers that are utilized almost exclusively by the indigenous Bifidobacteria and Lactobacillus in the intestinal tract and can be similarly utilized by Bacillus.
  • physiologically deleterious bacteria such as Clostridium, Staphylococcus, Salmonella and Escherichia coli cannot metabolize FOS, or other bifidogenic oligosaccharides. and therefor use of these bifidogenic oligosaccharides in combination with a lactic acid-producing bacteria of the present .
  • Bacillus coagulans allows these beneficial, probiotic bacteria to grow and effectively compete with, and eventually replace any undesirable, pathogenic microorganisms within the gastrointestinal tract.
  • compositions of the present invention provides a synergistic effect thereby increasing the effectiveness of the probiotic-containing compositions disclosed herein.
  • This synergy is manifested by selectively increasing the ability of the probiotic bacterium to grow by, for example, increasing the level of nutrient supplementation which preferentially selects for growth of the probiotic bacteria over many other bacterial species within the infected tissue.
  • Bifidobacteria and Lactobacillus are also producers of lactic acid.
  • Bifidogenic oligosaccharides enable these aforementioned probiotic organisms to proliferate preferentially over the undesirable bacteria within the gastrointestinal tract, thereby augmenting the probiotic state of the body by further enhancing the solubility of these nutrients (whether of food origin or as a result of nutritional supplement augmentation).
  • the presence of the bifidogenic oligosaccharides in the compositions of the present invention allows for more effective microbial inhibition by increasing the ability of all varieties of probiotic bacteria to grow, and therefore provide said benefit.
  • the bifidogenic oligosaccharide of the present invention may be used either alone, or in combination with a lactic acid-producing microorganisms in a therapeutic composition. More specifically, due to the growth promoting activity of bifidogenic oligosaccharides, the present invention contemplates a composition comprising a bifidogenic oligosaccharide present in a concentration sufficient to promote the growth of lactic acid-producing microorganisms. As shown herein, these concentrations amounts can vary widely, as the probiotic microorganisms will respond to any metabolic amount of nutrient oligosaccharide, and therefore the present invention need not be so limited.
  • a preferred and exemplary bifidogenic oligosaccharide is fructo-oligosaccharide (FOS), although other carbohydrates may also be utilized, either alone or in combination.
  • FOS can be obtained from a variety of natural sources, including commercial suppliers. As a product isolated from natural sources, the components can vary widely and still provide the beneficial agent, namely FOS. Typically, FOS possesses a polymer chain length of from approximately 4 to 200 sugar units, with the longer lengths being preferred. For example, the degree of purity can vary widely so long as biologically-functional FOS is present in the final formulation.
  • Preferred FOS formulations contain at least 50% by weight of fructo-oligosaccharides compared to simple(mono or disaccharide) sugars such as glucose, fructose or sucrose, preferably at least 80% fructo-oligosaccharides (FOS), more preferably at least 90% and most preferably at least 95% FOS.
  • Sugar content and composition can be determined by any of a variety of complex carbohydrate analytical detection methods as is well known.
  • Preferred sources of FOS include, but are not limited to: inulin; Frutafit IQ TM (Imperial Suiker Unie; Sugar Land, Texas); NutraFlora TM (Americal Ingredients, Inc.; Anaheim, CA); and Fruittrimfat Replacers and Sweeteners (Emeryville. CA).
  • Bifidogenic oligosaccharides such as GOS, and other long chain oligosaccharides are also available from commercial vendors.
  • the various active agents comprising the therapeutic composition of the present invention are combined with a carrier that is physiologically compatible with the gastrointestinal tract tissue of a human or animal to which it is administered. More specifically, the carrier is. preferably, substantially inactive except for surfactant properties which are used in making a suspension of the active ingredients.
  • the therapeutic compositions may also include other physiologically-active constituents which do not interfere with the efficacy of, for example, the Bacillus coagulans and FOS as active agents in said composition.
  • a typical therapeutic composition of the present invention will contain (within a one gram dosage formulation) from approximately 2xl0 7 to lxl 0 10 colony forming units (CFU) of viable bacteria or bacterial spores and approximately 10 milligrams (mg) to one gram of fructo- oligosaccharides (FOS).
  • CFU colony forming units
  • FOS fructo- oligosaccharides
  • the therapeutic compositions of the present invention may also include known antioxidants, buffering agents, and other agents such as coloring agents, flavorings, vitamins or minerals.
  • a preferred therapeutic composition may also contain one or more of the following minerals: calcium citrate (15-350 mg); potassium gluconate (5-150 mg); magnesium citrate (5-15 mg); and chromium picollinate (5-200 ⁇ g).
  • calcium citrate (15-350 mg
  • potassium gluconate (5-150 mg
  • magnesium citrate (5-15 mg)
  • chromium picollinate 5-200 ⁇ g
  • a variety of salts may be utilized, including calcium citrate, potassium gluconate, magnesium citrate and chromium picollinate.
  • Thickening agents may be added to the compositions such as polyvinylpyrrolidone, polyethylene glycol or carboxymethylcellulose.
  • Preferred additional components of a therapeutic composition of this invention can include assorted colorings or flavorings, vitamins, fiber, enzymes and other nutrients.
  • Preferred sources of fiber include any of a variety of sources of fiber including, but not limited to: psyllium. rice bran, oat bran, corn bran, wheat bran, fruit fiber and the like. Dietary or supplementary enzymes such as lactase, amylase, glucanase, catalase, and the like enzymes can also be included. Chemicals used in the present compositions can be obtained from a variety of commercial sources, including Spectrum Quality Products, Inc (Gardena, CA), Sigma Chemicals (St. Louis, MI), Seltzer Chemicals, Inc., (Carlsbad, CA) and Jarchem Industries, Inc., (Newark. NJ).
  • the various active agents are combined with a carrier which is physiologically compatible with the gastrointestinal tissue of the species to which it is administered.
  • Carriers can be comprised of solid-based, dry materials for formulation into tablet, capsule or powdered form; or the carrier can be comprised of liquid or gel-based materials for formulations into liquid or gel forms.
  • the specific type of carrier, as well as the final formulation depends, in part, upon the selected route(s) of administration.
  • the therapeutic composition of the present invention may also include a variety of carriers and/or binders.
  • a preferred carrier is micro-crystalline cellulose (MCC), added in an amount sufficient to complete the one gram dosage total weight.
  • Carriers can be solid-based dry materials for formulations in tablet, capsule or powdered form, and can be liquid or gel-based materials for formulations in liquid or gel forms, which forms depend, in part, upon the routes of administration.
  • Typical carriers for dry formulations include, but are not limited to: trehalose. malto- dextrin, rice flour, micro-crystalline cellulose (MCC) magnesium sterate, inositol, FOS, GOS, dextrose, sucrose, and like carriers.
  • MCC micro-crystalline cellulose
  • Exemplary anti-caking agents include MCC, talc, diatomaceous earth, amorphous silica and the like, and are typically added in an amount of from approximately 1% to 95% by weight.
  • dry formulations which are subsequently rehydrated are preferred to initially hydrated formulations.
  • Dry formulations e.g., powders
  • may be added to supplement commercially available foods e.g., liquid formulas, strained foods, or drinking water supplies.
  • Suitable liquid or gel-based carriers include but are not limited to: water and physiological salt solutions; urea; alcohols and derivatives (e.g., methanol, ethanol, propanol, butanol); glycols (e.g., ethylene glycol, propylene glycol, and the like).
  • water-based carriers possess a neutral pH value (i.e., pH 7.0).
  • the compositions may also include natural or synthetic flavorings and food-quality coloring agents, all of which must be compatible with maintaining viability of the lactic acid-producing microorganism.
  • Well-known thickening agents may also be added to the compositions such as corn starch, guar gum, xanthan gum, and the like.
  • a liquid-based composition containing spores is provided, it is desirable to include a spore germination inhibitor to promote long term storage. Any spore germination inhibitor may be used.
  • preferred inhibitors include: hyper-saline carriers, methylparaben, guargum, polysorbates, preservatives, and the like.
  • aqueous and oleaginous carriers e.g., white petrolatum, isopropyl myristate, lanolin or lanolin alcohols, mineral oil. sorbitan mono-oleate. propylene glycol. cetylstearyl alcohol, together or in various combinations
  • detergents e.g., polyoxyl stearate or sodium lauryl
  • Suitable carriers comprise water-in-oil or oil-in-water emulsions and mixtures of emulsifiers and emollients with solvents such as sucrose stearate. sucrose cocoate. sucrose distearate, mineral oil, propylene glycol. 2-ethyl-l,3 polyoxypropylene-15-stearyl ether and water.
  • Emulsions containing water, glycerol stearate, glycerin, mineral oil, synthetic spermaceti, cetyl alcohol, butylparaben. propylparaben and methylparaben are all commercially available.
  • Preservatives may also be included in the carrier and include, methylparaben, propylparaben, benzyl alcohol and ethylene diamine tetraacetate (EDTA) salts.
  • Well-known flavorings and/or colorants may also be included in the carrier.
  • Preservatives may also be included within the carrier including methylparaben, propylparaben, benzyl alcohol and ethylene diamine tetraacetate salts.
  • Well-known flavorings and/or colorants may also be included within the carrier.
  • the composition of the carrier can be varied so long as it does not interfere significantly with the pharmacological activity of the active ingredients or the viability of the Bacillus coagulans spores.
  • a therapeutic composition of the present invention can be formulated to be suitable for oral administration in a variety of ways, for example in a liquid, a powdered food supplement, a paste, a gel, a solid food, a packaged food, a wafer, and the like as described in more detail in the Examples. Other formulations will be readily apparent to one skilled within the art.
  • a nutrient supplement component of a composition of this invention can include any of a variety of nutritional agents, as are well known, including vitamins, minerals, essential and non- essential amino acids, carbohydrates, lipids, foodstuffs, dietary supplements, and the like.
  • Preferred compositions comprise vitamins and/or minerals in any combination.
  • Vitamins for use in a composition of this invention can include vitamins B, C. D. E. folic acid, K, niacin, and like vitamins.
  • the composition can contain any or a variety of vitamins as may be deemed useful for a particularly application, and therefore, the vitamin content is not to be construed as limiting. Typical vitamins are those, for example, recommended for daily consumption and in the recommended daily amount (RDA). although precise amounts can vary.
  • FIG. 2 illustrates, in tabular form, the vitamins, their RDA. their ranges and respective preferred ranges, of the therapeutic compositions of the present invention.
  • the composition would preferably include a complex of the RDA vitamins, minerals and trace minerals as well as those nutrients that have no established RDA. but have a beneficial role in healthy human or mammal physiology.
  • the preferred major minerals and their respective RDA are shown in FIG. 3.
  • the preferred mineral format would include those that are in either the gluconate or citrate form because these forms are more readily metabolized by lactic acid bacteria.
  • FIG. 4 illustrates, in tabular form, the preferred trace minerals and their respective ranges, as utilized in the therapeutic composition of the present invention.
  • the invention contemplates a composition comprising a viable lactic acid-producing bacteria in combination with any material to be adsorbed, including but not limited to nutrient supplements, foodstuffs, vitamins, minerals, medicines, therapeutic compositions, antibiotics, hormones, steroids, and the like compounds where it is desirable to insure efficient and healthy abso ⁇ tion of materials from the gastrointestinal track into the blood.
  • the amount of material included in the composition can vary widely depending upon the material and the intended pu ⁇ ose for its abso ⁇ tion, such that the invention is not to be considered as limiting.
  • the present invention contemplates a method for increasing the bioavailability of vitamins and minerals using lactic acid-producing bacteria, preferably Bacillus coagulans spores. It should be noted however that the scope of the invention is not to be limited to any particular member of the Bacillus genera, but can include any member of the Bacillus, Lactobacillus, Bifidobacterium, Sporolactobacillus, Streptococcus, and/or Enterococcus genus.
  • the aforementioned method comprises administration of a therapeutic composition of the present invention containing the active ingredients, to a human or animal, in various dosage regimens as described herein to achieve the desired nutritional result.
  • Administration of a nutritional composition is, preferably, to the gastrointestinal tract by use of a gel, suspension, aerosol spray, capsule, tablet, wafer, powder or semi-solid formulation (e.g., a suppository) containing a therapeutic composition of the present invention, all formulated using methods well-known within the art.
  • Administration of the compositions containing the active ingredients effective in producing lactic acid and/or the appropriate enzymes for solublizing vitamins or minerals generally consist of from one to ten unit dosages of 10 milligrams to 10 grams per dosage of said composition for a total time period of from one day to one month. Unit dosages are generally given once every twelve hours, and up to once every four hours. Preferably, one to four dosages of the therapeutic composition is administered per day.
  • Exemplar formulations are provided in the Specific Examples section, infra.
  • Bacillus coagulans 5x10 8 viable spores (approximately 35 mg)
  • Fructo-oligosaccharides 250 mg
  • MCC microcrystaline cellulose
  • Bacillus coagulans 2.5x10 8 viable spores (approximately 17.5 mg)
  • Fructo-oligosaccharides 100 mg
  • Bacillus coagulans 2.5x10 8 viable spores (approximately 17.5 mg)
  • Vitamin A 10,000 IU
  • MCC microcrystaline cellulose

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Abstract

La présente invention concerne des compositions et des procédés thérapeutiques mettant en oeuvre des bactéries probiotiques non productrices d'acide lactique pour augmenter la solubilité et la biodisponibilité de substances nutritives, de préférence des vitamines et des minéraux, à l'intérieur du tractus gastro-intestinal d'un animal ou d'un humain. Les compositions thérapeutiques de l'invention mettent en oeuvre des espèces de Bacillus ou, de préférence, de Bacillus coagulans, en tant qu'espèces bactériennes probiotiques non productrices d'acide lactique. Ces compositions thérapeutiques peuvent également contenir une ou plusieurs vitamines ou minéraux pour augmenter, de manière exogène, l'ingestion de ces substances.
EP99945016A 1998-08-07 1999-08-06 Procedes mettant en oeuvre des bacteries probiotiques non productrices d'acide lactique pour augmenter la solubilite de substances nutritives Ceased EP1102595A2 (fr)

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