WO2011044934A1 - Probiotic bacteria strains enabling of hydrolyzing prebiotic fibers and symbiotic compositions thereof - Google Patents

Probiotic bacteria strains enabling of hydrolyzing prebiotic fibers and symbiotic compositions thereof Download PDF

Info

Publication number
WO2011044934A1
WO2011044934A1 PCT/EP2009/063410 EP2009063410W WO2011044934A1 WO 2011044934 A1 WO2011044934 A1 WO 2011044934A1 EP 2009063410 W EP2009063410 W EP 2009063410W WO 2011044934 A1 WO2011044934 A1 WO 2011044934A1
Authority
WO
WIPO (PCT)
Prior art keywords
composition
composition according
bifidobacterium
dsm
enzyme
Prior art date
Application number
PCT/EP2009/063410
Other languages
French (fr)
Inventor
Giovanni Mogna
Gian Paolo Strozzi
Luca Mogna
Original Assignee
Probiotical S.P.A
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Probiotical S.P.A filed Critical Probiotical S.P.A
Priority to PCT/EP2009/063410 priority Critical patent/WO2011044934A1/en
Publication of WO2011044934A1 publication Critical patent/WO2011044934A1/en

Links

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
    • 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
    • A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
    • A23L33/21—Addition of substantially indigestible substances, e.g. dietary fibres
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01021—Beta-glucosidase (3.2.1.21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01025—Beta-mannosidase (3.2.1.25), i.e. mannanase
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/51—Bifidobacterium
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/51—Bifidobacterium
    • A23V2400/515—Animalis
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/51—Bifidobacterium
    • A23V2400/519—Breve
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/51—Bifidobacterium
    • A23V2400/521—Catenulatum
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/51—Bifidobacterium
    • A23V2400/531—Lactis

Definitions

  • Probiotic bacteria strains enabling of hydrolyzxng prebiotic fibers and symbiotic compositions thereof.
  • the present invention relates to probiotic bacteria strains which are able to hydrolyze prebiotic fibers.
  • the present invention relates to symbiotic
  • compositions comprising at least one prebiotic fibers and at least one bacteria strain having at least an
  • alpha-galactosidase enzyme and/or a beta-mannosidase enzyme.
  • the present invention relates to the use of said symbiotic compositions in food and/or pharmaceutical field.
  • compositions comprising said symbiotic composition .
  • the bifidobacteria exert a saccharolytic action; that is, they obtain the energy required for their growth through the fermentation of the carbohydrates. Therefore, it would be desirable to provide suitable carbohydrates or saccharides as energy source for growing intestinal bacteria belonging to the intestinal microflora.
  • prebiotic fibers have a bifidogenic effect i.e. they promote the proliferation of the beneficial bifidobacteria and encourage their metabolic selectivity.
  • a healthy intestinal flora is not only important for our wellbeing, but also improves the absorbation of nutrients, accelerates transit and strengthens the immune system' s defences .
  • the bifidobacteria are able to use in the colon most of the non-digestible monosaccharides, di-saccharides and oligosaccharides taken with the diet, or formed at the colon level following to the hydrolytic action on more complex glucidic molecules from enzymes secreted by other microbial groups forming the intestinal microflora.
  • the bifidobacteria do not result able to directly ferment the polysaccharides, polymeric carbohydrates composed of long chains of monosaccharides (more than 10 units) .
  • the patent application WO2007/125558 Al discloses symbiotic compositions comprising non-digestible polysaccharides and Bifidibacteria which metabolize them and their uses.
  • the prebiotic fiber is selected from inulin and/or its analogues.
  • the bacteria strains belonging to the genus Bifidobacteria are capable of directly
  • the aim of the present invention is, therefore, to provide non-digestible di-saccharides and oligosaccharides directly in the colon by means of a food composition comprising suitable carbohydrates and specific bacteria strains which are able to hydrolysate such carbohydrates .
  • Table 1 shows the kinetic of p- ⁇ trophenyl-a-D- galactopyranoside hydrolysis by measuring the absorbance OD at 405 nm for the bacterial strains Bifidobacterium breve DSM 16604 (BR03), Bifidobacterium lactis IMG P-21384 (BS01) , respectively .
  • Table 2 shows a turbidity test where the use of
  • Table 3 shows the acidification curves for the bacterial strain Bifidobacterium breve DSM 16604 (BR03) stimulated (Stim.) and non-stimulated (N-stim.), and the bacterial strain Bifidobacterium lactis LMG P-21384 (BS01) stimulated (Stim.) and non-stimulated (N-stim.) have been measured as pH in function of time in hours (hrs) .
  • Table 4 shows the growth of the bacteria strain
  • Bifidobacterium breve DSM 16604 (BR03) in the presence of Partially Hydrolyzed Guar Gum (PHGG) , by measuring the absorbance OD at 560 nra.
  • prebiotic those substances or components of the diet ⁇ cell and reserve oligosaccharides and polysaccharides of the plants), neither digestible by the human digestive enzymes nor absorbable in the small
  • probiotic those living species- specific microorganisms which, when ingested or applied in a sufficient number, are able to induce in the consumer specific functional and beneficial effects on its state of health .
  • compositions/products generally defined by the term
  • hydrolyzate it is meant a material of lower molecular weight than the parent polysaccharide
  • oligosaccharides and sugars includes, but is not limited exclusively to, oligosaccharides and sugars .
  • the symbiotic composition of the present invention comprises at least one prebiotic component (or fiber) , and at least one probiotic component (or bacterial strain) .
  • the prebiotic component comprises at least one mannose and/or galactose containing carbohydrate, or hydrolysates thereof.
  • said carbohydrates are selected from mannans and galactooligosaccharides (GOS) , or
  • Galactooligosaccharides are non- digestible fibers derived from lactose containing chains of galactose monomers.
  • said mannans are selected from glucomannans and galactomannans , or hydrolysates thereof.
  • Glucomannans are neutral polysaccharides that comprise, in most cases, predominantly mannose residues with glucose as the second sugar.
  • the polysaccharides contain some acetylated residues and may contain some galactose side chains (Khanna, 2003).
  • Galactomannans are polysaccharides consisting of a mannose backbone with galactose side groups (more specifically, a (1-4) -linked beta-D-mannopyranose backbone with branchpoints from their 6-positions linked to alpha-D-galactose , i.e. l-6 ⁇ linked alpha D-galactopyranose) .
  • the glucomannans are selected from Konjac gums.
  • the Konjac gum ⁇ Amorphophallus konjac) has usually a
  • Mannose Glucose Ratio (MGR) of about 1,6:1, and a Degree of Polymerisation (DP) bigger than 6000 (Khanna, S. (2003) The Chemical , physical and Nutritional Properties of the Plant Polysaccharides Konjac Glucomannan. PhD Thesis, Glasgow
  • the sugars are arranged in blocks of mannose and glucose residues that are ⁇ -(1-4) with typically 1.6:1 mannose to glucose residues within the polysaccharides (Khanna, 2003) .
  • the galactomannans are selected from carrube, guar gums, and tara gums (the mannose to galactose ratio mannose : galactose is about 3:1).
  • the guar gum is a soluble fiber derived from the Indian cluster bean
  • the hydrolysates are selected from glucomannans partially hydrolysed and/or galactomannans partially
  • the probiotic component comprises at least one bacterial strain.
  • bacteria strains it is meant that the bacteria are present as live bacteria strains, for example in a freeze dried form or dried powders .
  • the bacterial strains are selected from the group consisting of bacteria strains having at least an
  • the bacteria strains of the present invention may contain more than two enzymes, for example three or four enzymes, rendering these strains enabling of hydrolyzing prebiotic fibers.
  • the bacteria strains of the present invention may contain an alpha-galactosidase enzyme, a beta-mannosidase enzyme, and a ⁇ -glucosidase enzyme; or an alpha-galactosidase enzyme, a beta-mannosidase enzyme, a ⁇ -glucosidase enzyme, and a beta-mannanase enzyme.
  • a-Galactosidases are a group of exotype carbohydrases which catalyze the cleavage of terminal a-1 , 6-liriked galactosyl residues from a wide range of substrates, including linear and branched
  • oligosaccharides oligosaccharides, polysaccharides and synthetic substrates such as p-nitrophenyl-a-D-galactopyranoside .
  • ⁇ -Mannosidase is a widely used exoglycosidase enzyme in glycobiology . The enzyme cleaves single terminal D-mannosyl residues, which are ⁇ 1-4) linked to the non-reducing end of oligosaccharides (glycans) or those present on the glycan moiety of glycoproteins with relative specificity.
  • the bacteria strains of the present invention are capable to directly ferment said prebiotic component as such, by using it as a carbon and energy source for its own growth and reproduction. Further, said bacteria are able to provide non-digestible monosaccharides, di-saccharides and oligosaccharides directly in the colon, thus substantially increasing the production in the colon of bifidobacteria already present in microflora and improving the intestinal microflora .
  • the preferred bacteria strains are selected from the group comprising :
  • Lactobacillus plantarum LMG P-21021 (LP01) it was deposited at BCCM LMG (Belgian Coordinated Collections of Microorganisms - Laboratorium voor Microbiologie,
  • the condition for cultivation are the following:
  • TPY broth trypticase lOg/1, phytone 5g/l, yeast extract 5g/l, glucose 10 g/1, tween 80 (1 ml/1), K2HPO4 2g/l, MgCl 2 *6 H 2 0 0,5 g/1, ZnS0 4 7 H20 0,25 g/1, CaCl 2 -2 H2O 0,15 g/1, L-cysteine-hydrochloride 0,5 g/1.
  • the symbiotic composition of the present invention includes, as a prebiotic, glucomannans , galactomannans and/or their hydrolysates and, as a probiotic capable of directly fermenting the above prebiotic fibers, the bacterial strain Bifidobacterium breve DSM 16604 (BR03), or Bifidobacterium breve DSM 16604 (BR03) and Bifidobacterium lactis LMG P-21384 (BS01); alternatively Bifidobacterium breve DSM 16604 (BR03) and Lactobacillus plantarum LMG
  • P-21021 LP01, preferably Bifidobacterium breve DSM 16604 (BR03), Bifidobacterium lactis LMG P-21384 (BS01), and
  • Lactobacillus plantarum LMG P-21021 (LP01) .
  • the weight ratio between Bifidobacterium breve DSM 16604 (BR03) and at least one further bacteria strain selected from Bifidobacterium lactis LMG P-21384 (BS01) , Bifidobacterium catenulatum/ pseudocatenulatum DSM 18350 (BA03), Bifidobacterium animalis subsp lactis DSM 18352 (BA05), Bifidobacterium catenulatum DSM 18353 ⁇ BCOl), and Lactobacillus plantarum LMG P-21021 ⁇ LP01 ⁇ is from 5:1 to 1:1, preferably 3:1.
  • the bacteria strain is selected from the group consisting of: Bifidobacterium breve DSM 16604 (BR03), Bifidobacterium lactis LMG P-21384 (BS01) , Bifidobacterium catenulatum/ pseudocatenulatum DSM 18350 (BA03), Bifidobacterium animalis subsp lactis DSM 18352 (BA05), Bifidobacterium catenulatum DSM 18353 (BCOl), and Lactobacillus plantarum LMG P-21021 ⁇ LPOD .
  • the bacteria strain is selected from the group consisting of: Bifidobacterium breve DSM 16604 (BR03), Bifidobacterium lactis LMG P-21384 (BS01), and Lactobacillus plantarum LMG P-21021 (LP01) .
  • the composition comprises a mixture of bacteria strains
  • composition comprises a mixture of bacteria strains
  • the symbiotic composition comprises the bacterial strain Bifidobacterium breve DSM 16604 (BR03) in combination with the bacterial strain Bifidobacterium lactis LMG P-21384 (BS01) and/or Lactobacillus plantarum LMG P-21021 (LP01) , and at least one prebiotic fiber selected from Konjac gums, carrube gums, guar gums, PHGG, or tara gums, and/or their hydrolysates.
  • the prebiotic fibers are Konjac gums and/or PHGG, and the weight ratio between
  • Bifidobacterium breve DSM 16604 (BR03) and Bifidobacterium lactis LMG P-21384 (BS01) or between Bifidobacterium breve DSM 16604 (BR03) and Lactobacillus plantarum LMG P-21021 fLPOl) in the symbiotic composition is from 5:1 to 1:1, preferably 3:1.
  • the symbiotic composition of the present invention includes, as a prebiotic, glucomannans , galactomannans and/or their hydrolysates and, as a probiotic capable of directly fermenting the above prebiotic fibers, the bacterial strain Bifidobacterium breve DSM 16604 (BR03) in combination with at least one further bacteria strain selected from the group consisting of: Bifidobacterium lactis LMG P-21384 (BS01), Bifidobacterium catenulatum/
  • pseudocatenulatum DSM 18350 BA03
  • Bifidobacterium animalis subsp lactis DSM 18352 BA05
  • Bifidobacterium catenulatum DSM 18353 BC01
  • Lactobacillus plantarum LMG P-21021 LP01
  • the prebiotic component is present in an amount comprised from 5 to 99 % by weight, based on the total weight of the symbiotic composition; preferably from 30 3 ⁇ 4 to 95 I, more preferably from 50 to 90 % by weight.
  • the probiotic component is present in an amount comprised from 1 to 15 % by weight, based on the total weight of the symbiotic composition; preferably, from 5 to 10 %.
  • composition if any, consists of the additional substances such as adjuvants and/ or excipients or proper
  • a pharmaceutical composition e.g. tablets
  • the symbiotic composition is comprised from 40 to 70% by weight, based on the total weight of the food composition, and the remaining part is made of pharmaceutically acceptable adjuvants and/or excipients .
  • a food composition e.g. yogurt or chocolate
  • the symbiotic composition is comprised from 1 to 15% by weight, based on the total weight of the food composition.
  • the prebiotic component includes from 50 % to 95 % by weight of PHGG based on the total quantity of said prebiotic component.
  • the symbiotic composition contains bacteria strains in an amount comprised from lxl0 6 to 1x10 11 CFU/g, with respect to the weight of the symbiotic composition, preferably from 1x10 s to lxlO 11 CFU/g.
  • the symbiotic composition contains bacteria strains in an amount comprised from lxlO 6 to lxlO 11 CFU/dose, respect to the weight of the composition,
  • compositions of the present invention are those for oral administration, for example, capsules, compressed beads, tablets, powders or granules in sachets (to be suspended or dissolved in water and soft drinks at the time of use) or analogous forms, effervescent formulations.
  • the prebiotic component is in dryed form and the probiotic component is in freeze-drying form.
  • the freeze-drying process of the probiotic component is carried out by using techniques and equipments generally employed in the freeze-drying processes of pharmaceutical and/or food compositions.
  • the probiotic component can also be formulated in a coated, encapsulated or microencapsulated form, so as to result gastroresistant .
  • the probiotic component can also be formulated in a controlled release form, so as to selectively release the active substances ⁇ the bacteria strains) in the gastrointestinal tract, in particular in the large intestine or the colon.
  • a controlled-release symbiotic composition can be prepared, by microencapsulating or micro-coating the probiotic component forming the
  • biocompatible polymers such as, for example, Eudragit of different type and structure
  • biocompatible polymers resistant to the gastric juices of the stomach and able to release said components after a proper residence time in the gastrointestinal tract, or at pH values typical of the colon.
  • the above microencapsulated symbiotic composition thus obtained will be used, for example, for the preparation of food and/or pharmaceutical compositions in form of tablets, capsules or beads.
  • the symbiotic compositions of the present invention are prepared in a traditional way by using, depending on the type of formulation that one wishes to prepare, preparative techniques known to the skilled person in the art .
  • a symbiotic composition can be prepared by intimately mixing the
  • probiotic and prebiotic components preferably with known coadjuvants and/or excipients, reducing them to the desired granulometry and moisture degree, before packing them for being storaged.
  • coadjuvants and/or excipients reducing them to the desired granulometry and moisture degree, before packing them for being storaged.
  • particularly preferred symbiotic compositions according to the present invention are as follows.
  • Symbiotic composition 3 :
  • Fructo-oligosaccharides 1000 mg
  • the symbiotic compositions of the present invention can further include other active substances in order to give a food and/or pharmaceutical composition which form another important aspect of the present invention. Therefore, the food and/or pharmaceutical compositions of the present invention can contain active substances selected form
  • antioxidants hypoglycemics, hypocholesterolemics ,
  • immunostimulants and immunomodulatings substances with an antiaterogenic , antimeteorism, antiulcer, laxative,
  • vitamins, amino acids, mineral salts, enzymes, or proper excipients and/or additives such as carriers, lubricants, dispersers, antiaggregating , flavourings, sweeteners, stabilizers', preservatives commonly used in the formulation pharmaceutical art.
  • maltodextrins starch, tween, fragrances, such as those of mandarin, grapefruit, strawberry, bilberry, all fruits, calcium carbonate, magnesium stearate, talc, saccharose, glucose, acesulfame, saccharin, aspartame, ascorbic acid, parabens , giutamine, arginine, superoxide dismutase, glutathione.
  • maltodextrins starch, tween, fragrances, such as those of mandarin, grapefruit, strawberry, bilberry, all fruits, calcium carbonate, magnesium stearate, talc, saccharose, glucose, acesulfame, saccharin, aspartame, ascorbic acid, parabens , giutamine, arginine, superoxide dismutase, glutathione.
  • BROS Bifidobacterium breve DSM 16604
  • Lactobacillus plantarum LMG P-21021 (LP01) (lOOXlO 9 CFU/g) : 50 mg;
  • Preferred food and/or pharmaceutical compositions are, for example, in form of capsules, compressed beads (in this case, to be administered together with the content of a prebiotic sachet, to be dissolved in water), tablets, solutions or suspensions ready to drink, powders or granules in sachets (to be suspended or dissolved in water and soft drinks at the time of use) or analogous forms, effervescent formulations.
  • the food and/or pharmaceutical compositions can contain bacteria strains previously coated or encapsulated, so as to result gastro resistant.
  • Said food and/or pharmaceutical compositions can also be formulated in a controlled release form, so as to selectively release the active substances in the gastrointestinal tract, in particular in the large intestine or the colon.
  • a controlled release form so as to selectively release the active substances in the gastrointestinal tract, in particular in the large intestine or the colon.
  • those food and/or pharmaceutical compositions arranged in dried forms . The drying of the compositions is carried out by using techniques and equipments generally employed and known to the skilled person.
  • the food and/or pharmaceutical compositions of the present invention are prepared in a traditional way by using,
  • a granular food and/or pharmaceutical composition to be suspended or dissolved in water at the time of use, will be prepared by intimately mixing the components of the composition (symbiotic composition,
  • a controlled-release food and/or pharmaceutical composition will be prepared, for example, by microencapsulating or micro-coating the microgranulated mixture of the substances forming the
  • composition with opportune mixtures of biocompatible polymers such as, for example, Eudragit of different type and
  • microencapsulated mixture thus obtained will be used, for example, for the preparation of tablets, capsules or beads.
  • the composition of the present invention can be administered in a variety of ways, depending oh the needs of the patient or the consumer.
  • the present invention relates to the use of a symbiotic composition, as above described, for the preparation of food (for example
  • gastrointestinal diseases inflammatory diseases of the intestines that may affect any part of the gastrointestinal tract from anus to mouth such as vomiting, diarrhea or weight loss, and for preventing and/or treating Crohn's disease, colitis (inflammatory bowel disease - IBD) , colitis ulcerosa, and irritable bowel syndrome (IBS) .
  • colitis inflammatory bowel disease - IBD
  • colitis ulcerosa colitis ulcerosa
  • IBS irritable bowel syndrome
  • the symbiotic composition of the present invention is used for the preparation of food products based on milk, such as yoghurt, fermented milks, fresh cheeses and other food products, such as fruit-j uices , functional drinks and integrators/ creams, desserts, puree, chocolate, stuffs and fillings used in the confectionery.
  • milk such as yoghurt, fermented milks, fresh cheeses and other food products, such as fruit-j uices , functional drinks and integrators/ creams, desserts, puree, chocolate, stuffs and fillings used in the confectionery.
  • the aforesaid populations of the probiotic strains relate to the bacterial population at the end of the manufacturing, while at the end of the shelf life, the bacterial populations are preferably between lxlO 8 CFU/dose and lxlO 11 CFU/dose, more preferably between lxlO 9 CFU/dose and 2xl0 10 CFU/dose
  • p-D-mannopyranoside have been used for evaluating the enzyme production of ⁇ -galactosidase and ⁇ -mannosidase . These latter enzymes hydrolyse the chains of galactose and the end chains of mannose respectively which form the galactomannans ⁇ Guy D. Duffaud, et al . ; Applied and Environment Microbiology, Jan. 1997, Vol. 63, No. 1, p. 169-177).
  • Live bacteria samples selected from Bifidobacterium breve DSM 16604 (BR03), Bifidobacterium lactis LMG P-21384 (BS01) , Bifidobacterium catenulatum/pseudocatenulatum DSM 18350
  • Lactis DSM 18352 BA05
  • Bifidobacterium catenulatum DSM 18353 BC01
  • Lactobacillus plantarum LMG P-21021 LP01
  • Lactobacillus strains were cultured in de Man, Rogosa and Sharpe broth (MRS, DeMan et al . 1960) while Bifidobacterium strain, were cultured in MRS or Tryptone Phytone Yeast broth (TPY, Scardovi 1986), supplemented with
  • the above cultured bacteria were harvested by centrifugation (3000 g for 15 min) during exponential and/ or stationary growth phase in order to collect cells. Concentrated cell pellets were resuspended in 200-500 ⁇ of Tris-HCl buffer (pH 7) and a small amount of quartz sand was added. The cell pellet resuspended in this way, was then disrupted in ice through a ultrasonic bath (4 cycles of
  • Protein concentration in cell extract was determined by the Bradford method with bovine serum albumin as a standard, as well known at the skilled person in the art.
  • ⁇ -Mannosidase and ⁇ -Galactosidase activities were determined by monitoring the release of p-ni trophenol from
  • the enzymatic assays for ⁇ -Mannosidase or a-Galactosidase were conducted as follows. For each assay, 1.1 ml aliquots of 10 mM of substrate (p-nitrophenyl-B-D-mannopyranoside or p-nitrophenyl-a-D-galactopyranoside) in 0.1 M sodium
  • phosphate buffer pH 7.4
  • the cuvettes were preincubated for at least 10 min to allow the substrate to reach the assay temperature (25°C but 37°C is also possible) .
  • 0.1 ml of sample at defined total protein concentration for example
  • Reagent C 100 mM Potassium Phosphate Buffer, pH 6.5 at 25°C; the Reagent C was prepared by adjusting 50 ml of Reagent A to pH 6.5 at 25°C by adding Reagent B,
  • sample defined concentration of the above crude enzyme preparation (for example 60 - 100 pg/ml) .
  • the reagent Fl has been used for the determination of unit concentration in a known ⁇ -galactosidase enzyme solution, as a control standard.
  • control standard a known ⁇ -galactosidase enzyme solution
  • the said bacteria were cultured in the same medium MRS, with the above polymers at 20 g/1 as the only carbon, source, instead of glucose.
  • the quantification of the a-galactosidase produced by stimulated bacteria has been performed by the aforementioned method after 3 sequential sub-cultures in a medium containing said polymers, as carbon source.
  • the stimulated cultures were also used to evaluate the different capability of the stimulated strain to growth in medium with galactomannan polymers as the only carbon source v/ith respect to the non-stimulated one. All the above cultures in normal MRS or in MRS with galactomannans (and without glucose) were prepared by an inoculum of 1% of a fresh, culture. The cultures of stimulated and non-stimulated ⁇ or unstimulated (N-stim. ) ) bacteria in MRS with
  • the bacteria strain Bifidobacterium breve DSM 16604 ⁇ BR03 is able to hydrolyse the synthetic substrate
  • the bacteria strain Bifidobacterium breve DSM 16604 hydrolyses the substrate better than other bifidobacteria strains which need about 3-6 hours more, as shown in Table 1.
  • Table 1 confirms that the bacteria strain Bifidobacterium breve DSM 16604 (BR03) has an hight and specific ability to metabolize galactomannans and their hydrolysates thereof such as PHGG . From a quantitative point of view, it has been calculated that the bacteria strain Bifidobacterium breve DSM 16604 (BR03) is able to synthesize and produce an amount concentration of os-Galactosidase in active form of about 350 mU/mg of proteins of the extract.
  • BS01 produces an amount of a-Galactosid.ase of about 250 mU/mg.
  • bacteria strain Bifidobacterium breve DSM 16604 ⁇ BR03 ⁇ is able to hydrolyse the synthetic substrate
  • Bifidobacterium breve DSM 16604 has a big and specific ability to metabolize galactomannans and their hydrolysates thereof such as PHGG.
  • the bacteria strain Bifidobacterium breve DSM 16604 (BR03) is able to synthesize and produce an amount of concentration of B-Mannosidase ⁇ indicated as Unit for mg of total proteins extracted ⁇ equal to or bigger than 1 U/mg, preferably bigger than 5 U/ ' mg.
  • the total amount of extra.ct.ed proteins from 10 9 cells is comprised from 1 to 1000 yg, preferably from 10 to 100 ug . Also for the remaining bacteria strains explicitly mentioned in the present application have been obtained, similar values. With respect to LGG, the ⁇ -Mannosidase produced by the bacteria strain Bifidobacterium breve DSM 16604 (BR03) is 50 times more concentrated.
  • bacteria is stimulated to produce more a-Galactosidase .
  • Bifidobacterium breve DSM 16604 (BR03) is stimulated the amount of concentration of a-Galactosidase is increased by more than 4 times. From experimental data has been
  • Bifidobacterium breve DSM 16604 increases from about 350 mU/mg (as amount of total extracted proteins) to about 1500 mU/mg after 3 sequential sub-cultures in a medium containing galactomannans, as the only carbon source.
  • galactomannans as for example PHGG
  • PHGG PHGG
  • the API ZYM system comprises enzymatic tests performed on dried substrates in cupules.
  • the enzyme activities tested are as follows: 1) Control, 2) Phosphatase alkaline, 3) Esterase (C4) , 4) Esterase Lipase (C8 ⁇ , 5 ⁇ Lipase (C14), 6) Leucine aminopeptidase , 7) Valine aminopeptidase , 8) Cystine
  • Bifidobacterium breve DSM 16604 (BR03) non-stimulated (or unstimulated (N-stim. ) ) : less than or equal to 5 nM;
  • Bifidobacterium breve DSM 16604 (BR03), stimulated (Stim. ) in PHGG: bigger than 40 nM.
  • the maximum value that can be count by ApyzymTM is 40 nM .
  • Bifidobacterium breve DSM 16604 (BR03) stimulated (Stim.) in PHGG contains more than 8 times the amount of ⁇ -glucosidase enzyme in comparison with the non-stimulated one (or unstimulated ⁇ M-stim. ) ) .
  • polysaccharides hydrolyzing enzyme like a-galactosidase but also ⁇ -glucosidase
  • suitable to metabolize that substrate like a-galactosidase but also ⁇ -glucosidase

Abstract

The present invention relates to probiotic bacteria strains which are able to hydrolyze prebiotic fibers. Further, the present invention relates to symbiotic compositions comprising at least one prebiotic fibers and at least one bacteria strain having at least an alpha-galactosidase enzyme and/or a beta-mannosidase enzyme. Further, the present invention relates to the use of said symbiotic compositions in food and/or pharmaceutical field. Finally, the present invention relates to food or pharmaceutical compositions comprising said symbiotic composition.

Description

Probiotic bacteria strains enabling of hydrolyzxng prebiotic fibers and symbiotic compositions thereof.
DESCRIPTION
The present invention relates to probiotic bacteria strains which are able to hydrolyze prebiotic fibers.
Further, the present invention relates to symbiotic
compositions comprising at least one prebiotic fibers and at least one bacteria strain having at least an
alpha-galactosidase enzyme and/or a beta-mannosidase enzyme.
Further, the present invention relates to the use of said symbiotic compositions in food and/or pharmaceutical field.
Finally, the present invention relates to food or
pharmaceutical compositions comprising said symbiotic composition .
As most of the intestinal bacteria capable of playing a positive role in the ambit of the intestinal ecosystem, the bifidobacteria exert a saccharolytic action; that is, they obtain the energy required for their growth through the fermentation of the carbohydrates. Therefore, it would be desirable to provide suitable carbohydrates or saccharides as energy source for growing intestinal bacteria belonging to the intestinal microflora.
It is known that prebiotic fibers have a bifidogenic effect i.e. they promote the proliferation of the beneficial bifidobacteria and encourage their metabolic selectivity.
A healthy intestinal flora is not only important for our wellbeing, but also improves the absorbation of nutrients, accelerates transit and strengthens the immune system' s defences .
Generally, the bifidobacteria are able to use in the colon most of the non-digestible monosaccharides, di-saccharides and oligosaccharides taken with the diet, or formed at the colon level following to the hydrolytic action on more complex glucidic molecules from enzymes secreted by other microbial groups forming the intestinal microflora. On the contrary, the bifidobacteria, do not result able to directly ferment the polysaccharides, polymeric carbohydrates composed of long chains of monosaccharides (more than 10 units) .
The patent application WO2007/125558 Al discloses symbiotic compositions comprising non-digestible polysaccharides and Bifidibacteria which metabolize them and their uses. In this patent application the prebiotic fiber is selected from inulin and/or its analogues. The bacteria strains belonging to the genus Bifidobacteria are capable of directly
metabolizing said prebiotic fibers as such. However, there persists the need of being able to completely use also other long chain polysaccharides, not only inulin, during the transit of the same along all the extension of the colon, so as to ensure an improvement of their bifidogenic effect, and at the same time an improvement of the intestinal microflora.
The aim of the present invention is, therefore, to provide non-digestible di-saccharides and oligosaccharides directly in the colon by means of a food composition comprising suitable carbohydrates and specific bacteria strains which are able to hydrolysate such carbohydrates .
This and other aims, which will result apparent from the following detailed description, have been attained by the Applicant, which has completely unexpectedly found that, contrary to what is known in the art, a very limited number of bacterial strains belonging to the genes Bifidobacterium are able to hydrolyze at least one mannose and/or galactose containing carbohydrates, or hydrolysates thereof.
According to a first aspect of the present invention there are provided symbiotic compositions, as reported in the appended independent claim.
According to a second aspect of the present invention there is provided the use of said symbiotic compositions for preparing food and/or pharmaceutical products for improving the bifidogenic activity in the organism, as reported in the appended independent claim.
According to a third aspect of the present invention there are provided food and/or pharmaceutical products containing said symbiotic compositions, as reported in the appended independent claim.
Other aspects and features of the present invention will be more fully apparent from the following disclosure and appended dependent claims .
Table 1: shows the kinetic of p-ηίtrophenyl-a-D- galactopyranoside hydrolysis by measuring the absorbance OD at 405 nm for the bacterial strains Bifidobacterium breve DSM 16604 (BR03), Bifidobacterium lactis IMG P-21384 (BS01) , respectively .
Table 2 : shows a turbidity test where the use of
galactomannans by the bacterial strain Bifidobacterium breve DSM 16604 (BR03) stimulated (Stim.) and non-stimulated (N- stim. ) , and the bacterial strain Bifidobacterium lactis LMG P-21384 (BS01) stimulated (Stim.) and non-stimulated (N- stim. ) have been measured as absorbance OD at 560 nm in function of time measured in hours (hrs) . Table 3: shows the acidification curves for the bacterial strain Bifidobacterium breve DSM 16604 (BR03) stimulated (Stim.) and non-stimulated (N-stim.), and the bacterial strain Bifidobacterium lactis LMG P-21384 (BS01) stimulated (Stim.) and non-stimulated (N-stim.) have been measured as pH in function of time in hours (hrs) .
Table 4: shows the growth of the bacteria strain
Bifidobacterium breve DSM 16604 (BR03) in the presence of Partially Hydrolyzed Guar Gum (PHGG) , by measuring the absorbance OD at 560 nra.
By the term "prebiotic", it is meant those substances or components of the diet {cell and reserve oligosaccharides and polysaccharides of the plants), neither digestible by the human digestive enzymes nor absorbable in the small
intestine, which, once arrived in the colon, selectively stimulate the development and the activity of the microbial groups beneficial for the health of the individual.
By the term l!probiotic " , it is meant those living species- specific microorganisms which, when ingested or applied in a sufficient number, are able to induce in the consumer specific functional and beneficial effects on its state of health .
The association of probiotics with prebiotics gives rise to compositions/products generally defined by the term
11 symbiotic " .
By a term "hydrolyzate" , it is meant a material of lower molecular weight than the parent polysaccharide, and
includes, but is not limited exclusively to, oligosaccharides and sugars .
The symbiotic composition of the present invention comprises at least one prebiotic component (or fiber) , and at least one probiotic component (or bacterial strain) .
The prebiotic component comprises at least one mannose and/or galactose containing carbohydrate, or hydrolysates thereof.
In a preferred embodiment, said carbohydrates are selected from mannans and galactooligosaccharides (GOS) , or
hydrolysates thereof. Galactooligosaccharides (GOS) are non- digestible fibers derived from lactose containing chains of galactose monomers. Preferably, said mannans are selected from glucomannans and galactomannans , or hydrolysates thereof. Glucomannans are neutral polysaccharides that comprise, in most cases, predominantly mannose residues with glucose as the second sugar. The polysaccharides contain some acetylated residues and may contain some galactose side chains (Khanna, 2003). Galactomannans are polysaccharides consisting of a mannose backbone with galactose side groups (more specifically, a (1-4) -linked beta-D-mannopyranose backbone with branchpoints from their 6-positions linked to alpha-D-galactose , i.e. l-6~linked alpha D-galactopyranose) .
Preferably, the glucomannans are selected from Konjac gums. The Konjac gum {Amorphophallus konjac) has usually a
Mannose : Glucose Ratio (MGR) of about 1,6:1, and a Degree of Polymerisation (DP) bigger than 6000 (Khanna, S. (2003) The Chemical , physical and Nutritional Properties of the Plant Polysaccharides Konjac Glucomannan. PhD Thesis, Glasgow
Caledonian University, Glasgow, UK) .
The sugars are arranged in blocks of mannose and glucose residues that are β-(1-4) with typically 1.6:1 mannose to glucose residues within the polysaccharides (Khanna, 2003) . Preferably, the galactomannans are selected from carrube, guar gums, and tara gums (the mannose to galactose ratio mannose : galactose is about 3:1). The guar gum is a soluble fiber derived from the Indian cluster bean
{Cyamopsistetragonoloba{psox~alioides) ) , and it has (the mannose to galactose ratio mannose : galactose is about 2:1). Preferably, the hydrolysates are selected from glucomannans partially hydrolysed and/or galactomannans partially
hydrolysed, more preferably partially hydrolysed carrube, Partially Hydrolysed Guar Gum such as PHGG, or partially hydrolysed tara gum. The probiotic component comprises at least one bacterial strain.
In the context of the present invention, for "bacteria strains" it is meant that the bacteria are present as live bacteria strains, for example in a freeze dried form or dried powders . The bacterial strains are selected from the group consisting of bacteria strains having at least an
alpha-galactosidase enzyme and/or a beta-mannosidase enzyme. The bacteria strains of the present invention may contain more than two enzymes, for example three or four enzymes, rendering these strains enabling of hydrolyzing prebiotic fibers. For example, the bacteria strains of the present invention may contain an alpha-galactosidase enzyme, a beta-mannosidase enzyme, and a β-glucosidase enzyme; or an alpha-galactosidase enzyme, a beta-mannosidase enzyme, a β-glucosidase enzyme, and a beta-mannanase enzyme.
The a-Galactosidases ( a-D-galactoside galactohydrolase ) are a group of exotype carbohydrases which catalyze the cleavage of terminal a-1 , 6-liriked galactosyl residues from a wide range of substrates, including linear and branched
oligosaccharides, polysaccharides and synthetic substrates such as p-nitrophenyl-a-D-galactopyranoside . β-Mannosidase is a widely used exoglycosidase enzyme in glycobiology . The enzyme cleaves single terminal D-mannosyl residues, which are β{1-4) linked to the non-reducing end of oligosaccharides (glycans) or those present on the glycan moiety of glycoproteins with relative specificity.
Advantageously, the bacteria strains of the present invention are capable to directly ferment said prebiotic component as such, by using it as a carbon and energy source for its own growth and reproduction. Further, said bacteria are able to provide non-digestible monosaccharides, di-saccharides and oligosaccharides directly in the colon, thus substantially increasing the production in the colon of bifidobacteria already present in microflora and improving the intestinal microflora .
The preferred bacteria strains are selected from the group comprising :
- Bifidobacterium breve DSM 16604 (BR03}, deposited on
20.07.2004 by Anidral Sri that has changed the name in
Probiotical SpA, Via Matter, 3 Novara 28100 (Italy);
- Bifidobacterium lactis LMG P-21384 (BS01) , deposited on 31.01.2002 by Anidral Sri that has changed the name in
Probiotical SpA, Via Mattel, 3 Novara 28100 (Italy);
- Bifidobacterium catenulatum/ pseudocatenulatum DSM 18350, deposited on 15.06.2006, which was previously classified as Bifidobacterium adolescentis (ΞΙ-3) with the same deposit number DSM 18350, by Anidral Sri that has changed the name in Probiotical SpA, Via Mattel, 3 Novara 28100 (Italy);
- Bifidobacterium animalis subsp lactis DSM 18352, deposited on 15.06.2006, which was previously classified as
Bifidobacterium adolescentis (EI-18) with the same deposit number DSM 18352, by Anidral Sri that has changed the name in Probiotical SpA, Via Matter, 3 Novara 28100 (Italy);
- Bifidobacterium catenulatum DSM 18353 (BCOl), deposited on 15.06.2006 by Anidral Sri that has changed the name in
Probiotical SpA, Via Mattel, 3 Novara 28100 (Italy); and
- Lactobacillus plantarum LMG P-21021 (LP01), deposited on 16.10.2001 by Laboratorio Microbiologic© Grana provolone S.r.l. that was assigned to Mofin S.r.l. on 31.01.2002, Via P. Custodi, 12 Novara 28100 (Italy).
The above bacteria strains have been deposited at the DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunsweig-Germany, or at Belgium Coordinated Collections of Microorganisms (BCCM) Laboratorium voor Microbiologie
Bacterlenverzameling {LMG) Univerisiteit Gent -.Belgium.
With reference to Lactobacillus plantarum LMG P-21021 (LP01) it was deposited at BCCM LMG (Belgian Coordinated Collections of Microorganisms - Laboratorium voor Microbiologie,
Bacterienverzameling Universiteit Gent) .
The condition for cultivation are the following:
- Medium: TPY broth: trypticase lOg/1, phytone 5g/l, yeast extract 5g/l, glucose 10 g/1, tween 80 (1 ml/1), K2HPO4 2g/l, MgCl2*6 H20 0,5 g/1, ZnS04 7 H20 0,25 g/1, CaCl2-2 H2O 0,15 g/1, L-cysteine-hydrochloride 0,5 g/1.
- pH before sterilization: 7,10 ± 0,1
- Sterilization at 121 °C for 15 minutes,
- pH after sterilization 6,6 ± 0,1
- Incubation temperature 37 °C,
- Incubation time 17± 1 hour,
- Short term storage at 5 °C,
- Interval of transfer 2-3 days.
- Long term storage (minus) -25 °C
- Condition for testing viability: growth in TPY broth at 37 °C.
In a preferred embodiment, the symbiotic composition of the present invention includes, as a prebiotic, glucomannans , galactomannans and/or their hydrolysates and, as a probiotic capable of directly fermenting the above prebiotic fibers, the bacterial strain Bifidobacterium breve DSM 16604 (BR03), or Bifidobacterium breve DSM 16604 (BR03) and Bifidobacterium lactis LMG P-21384 (BS01); alternatively Bifidobacterium breve DSM 16604 (BR03) and Lactobacillus plantarum LMG
P-21021 (LP01, preferably Bifidobacterium breve DSM 16604 (BR03), Bifidobacterium lactis LMG P-21384 (BS01), and
Lactobacillus plantarum LMG P-21021 (LP01) . Advantageously, in the symbiotic composition the weight ratio between Bifidobacterium breve DSM 16604 (BR03) and at least one further bacteria strain selected from Bifidobacterium lactis LMG P-21384 (BS01) , Bifidobacterium catenulatum/ pseudocatenulatum DSM 18350 (BA03), Bifidobacterium animalis subsp lactis DSM 18352 (BA05), Bifidobacterium catenulatum DSM 18353 {BCOl), and Lactobacillus plantarum LMG P-21021 {LP01} is from 5:1 to 1:1, preferably 3:1.
The bacteria strain is selected from the group consisting of: Bifidobacterium breve DSM 16604 (BR03), Bifidobacterium lactis LMG P-21384 (BS01) , Bifidobacterium catenulatum/ pseudocatenulatum DSM 18350 (BA03), Bifidobacterium animalis subsp lactis DSM 18352 (BA05), Bifidobacterium catenulatum DSM 18353 (BCOl), and Lactobacillus plantarum LMG P-21021 {LPOD .
Advantageously, the bacteria strain is selected from the group consisting of: Bifidobacterium breve DSM 16604 (BR03), Bifidobacterium lactis LMG P-21384 (BS01), and Lactobacillus plantarum LMG P-21021 (LP01) . In a preferred embodiment the composition comprises a mixture of bacteria strains
consisting of Bifidobacterium breve DSM 16604,
Bifidobacterium lactis LMG P-21384, and Lactobacillus
plantarum LMG P-21021, and a prebiotic component consisting of Konj ac gums. In another preferred embodiment the
composition comprises a mixture of bacteria strains
consisting of Bifidobacterium breve DSM 16604,
Bifidobacterium lactis LMG P-21384, and Lactobacillus
plantarum LMG P-21021, and a prebiotic component consisting PHGG .
Preferably, the symbiotic composition comprises the bacterial strain Bifidobacterium breve DSM 16604 (BR03) in combination with the bacterial strain Bifidobacterium lactis LMG P-21384 (BS01) and/or Lactobacillus plantarum LMG P-21021 (LP01) , and at least one prebiotic fiber selected from Konjac gums, carrube gums, guar gums, PHGG, or tara gums, and/or their hydrolysates. Advantageously, the prebiotic fibers are Konjac gums and/or PHGG, and the weight ratio between
Bifidobacterium breve DSM 16604 (BR03) and Bifidobacterium lactis LMG P-21384 (BS01) or between Bifidobacterium breve DSM 16604 (BR03) and Lactobacillus plantarum LMG P-21021 fLPOl) in the symbiotic composition is from 5:1 to 1:1, preferably 3:1.
In another preferred embodiment, the symbiotic composition of the present invention includes, as a prebiotic, glucomannans , galactomannans and/or their hydrolysates and, as a probiotic capable of directly fermenting the above prebiotic fibers, the bacterial strain Bifidobacterium breve DSM 16604 (BR03) in combination with at least one further bacteria strain selected from the group consisting of: Bifidobacterium lactis LMG P-21384 (BS01), Bifidobacterium catenulatum/
pseudocatenulatum DSM 18350 (BA03), Bifidobacterium animalis subsp lactis DSM 18352 (BA05), Bifidobacterium catenulatum DSM 18353 (BC01), and Lactobacillus plantarum LMG P-21021 (LP01) .
In the symbiotic compositions of the present invention, the prebiotic component is present in an amount comprised from 5 to 99 % by weight, based on the total weight of the symbiotic composition; preferably from 30 ¾ to 95 I, more preferably from 50 to 90 % by weight.
In turn, the probiotic component is present in an amount comprised from 1 to 15 % by weight, based on the total weight of the symbiotic composition; preferably, from 5 to 10 %.
The part lacking to 100% by weight of the symbiotic
composition, if any, consists of the additional substances such as adjuvants and/ or excipients or proper
additives /carriers . In a pharmaceutical composition (e.g. tablets) the symbiotic composition is comprised from 40 to 70% by weight, based on the total weight of the food composition, and the remaining part is made of pharmaceutically acceptable adjuvants and/or excipients .
In a food composition (e.g. yogurt or chocolate) the
symbiotic composition is comprised from 1 to 15% by weight, based on the total weight of the food composition. In a particularly preferred embodiment, the prebiotic component includes from 50 % to 95 % by weight of PHGG based on the total quantity of said prebiotic component. In a preferred embodiment, the symbiotic composition contains bacteria strains in an amount comprised from lxl06 to 1x1011 CFU/g, with respect to the weight of the symbiotic composition, preferably from 1x10s to lxlO11 CFU/g.
In a preferred embodiment, the symbiotic composition contains bacteria strains in an amount comprised from lxlO6 to lxlO11 CFU/dose, respect to the weight of the composition,
preferably from lxlO8 to lxlO11 CFU/dose. Particularly
preferred symbiotic compositions of the present invention are those for oral administration, for example, capsules, compressed beads, tablets, powders or granules in sachets (to be suspended or dissolved in water and soft drinks at the time of use) or analogous forms, effervescent formulations.
In a preferred embodiment, the prebiotic component is in dryed form and the probiotic component is in freeze-drying form. The freeze-drying process of the probiotic component is carried out by using techniques and equipments generally employed in the freeze-drying processes of pharmaceutical and/or food compositions. In another preferred embodiment, the probiotic component can also be formulated in a coated, encapsulated or microencapsulated form, so as to result gastroresistant . In another preferred embodiment, the probiotic component can also be formulated in a controlled release form, so as to selectively release the active substances {the bacteria strains) in the gastrointestinal tract, in particular in the large intestine or the colon. Thus, a controlled-release symbiotic composition can be prepared, by microencapsulating or micro-coating the probiotic component forming the
symbiotic composition with opportune mixtures of
biocompatible polymers (such as, for example, Eudragit of different type and structure) resistant to the gastric juices of the stomach and able to release said components after a proper residence time in the gastrointestinal tract, or at pH values typical of the colon.
The above microencapsulated symbiotic composition thus obtained will be used, for example, for the preparation of food and/or pharmaceutical compositions in form of tablets, capsules or beads. The symbiotic compositions of the present invention are prepared in a traditional way by using, depending on the type of formulation that one wishes to prepare, preparative techniques known to the skilled person in the art .
By way of absolutely non limiting example, a symbiotic composition can be prepared by intimately mixing the
probiotic and prebiotic components, preferably with known coadjuvants and/or excipients, reducing them to the desired granulometry and moisture degree, before packing them for being storaged. Representative, but not limiting examples of particularly preferred symbiotic compositions according to the present invention are as follows.
Symbiotic composition 1:
- Bifidobacterium breve DSM 16604 (BR03) (150X109 CFU/g) : 83 mg;
- PHGG: 6400 mg; - Insoluble fibers: 117 mg.
Symbiotic composition 2:
- Bifidobacterium breve DSM 16604 (BR03 ) (150X109 CFU/g} : 83 mg;
- Lactobacillus plantarum LMG P-21021 {LP01} (150X109 CFU/g) : 83 mg ;
- PHGG : 5500 mg;
- Insoluble fibers: 116 mg;
- Apple flavor: 100 mg;
- Malic acid: 20 mg .
Symbiotic composition 3 :
- Bifidobacterium breve DSM 16604 (BR03 ) (150X109 CFU/g ) : 167 mg;
- Bifidobacterium iactis LMG P-21384 (BS01) (150X109 CFU/g} : 83 mg ;
- PHGG: 2500 mg;
- Fructo-oligosaccharides (FOS) : 1000 mg;
- Blueberry natural flavor: 300 mg;
- Malic acid: 22 mg;
- Sucralose: 8 mg .
The symbiotic compositions of the present invention can further include other active substances in order to give a food and/or pharmaceutical composition which form another important aspect of the present invention. Therefore, the food and/or pharmaceutical compositions of the present invention can contain active substances selected form
antioxidants, hypoglycemics, hypocholesterolemics ,
immunostimulants and immunomodulatings , substances with an antiaterogenic , antimeteorism, antiulcer, laxative,
antidiarrheal activity, vitamins, amino acids, mineral salts, enzymes, or proper excipients and/or additives, such as carriers, lubricants, dispersers, antiaggregating , flavourings, sweeteners, stabilizers', preservatives commonly used in the formulation pharmaceutical art.
By way of absolutely non limiting example, among the
particularly preferred excipients and additives that can be present in the food and/or pharmaceutical compositions, there may be mentioned maltodextrins , starch, tween, fragrances, such as those of mandarin, grapefruit, strawberry, bilberry, all fruits, calcium carbonate, magnesium stearate, talc, saccharose, glucose, acesulfame, saccharin, aspartame, ascorbic acid, parabens , giutamine, arginine, superoxide dismutase, glutathione. Representative, but not limiting examples of particularly preferred food and/or pharmaceutical compositions according to the present invention are as follows .
1 Pharmaceutical composition (tablets) :
- Bifidobacterium breve DSM 16604 (BROS) (100X109 CFU/g) : 100 mg;
- PHGG: 600 mg;
- Macrocrystalline Cellulose: 300 mg ;
- Calcium hydrogen phosphate: 110 mg (anhydrous) ;
- Sodium carboxymethylcellulose : 65 mg;
- Talc: 35 mg;
- Stearic acid: 40 mg;
- Magnesium stearate: 25 mg.
2. Food composition (yogurt 125 grams or fermented milk 125 grams ) :
- Bifidobacterium breve DSM 16604 (BR03) (100X109 CFU/g): 100 mg;
- Lactobacillus plantarum LMG P-21021 (LP01) (lOOXlO9 CFU/g) : 50 mg;
- PHGG: 3 grams; - Frutto-oligosaccharides (FOS) : 1 grams.
Preferred food and/or pharmaceutical compositions are, for example, in form of capsules, compressed beads (in this case, to be administered together with the content of a prebiotic sachet, to be dissolved in water), tablets, solutions or suspensions ready to drink, powders or granules in sachets (to be suspended or dissolved in water and soft drinks at the time of use) or analogous forms, effervescent formulations. In a preferred embodiment the food and/or pharmaceutical compositions can contain bacteria strains previously coated or encapsulated, so as to result gastro resistant.
Said food and/or pharmaceutical compositions can also be formulated in a controlled release form, so as to selectively release the active substances in the gastrointestinal tract, in particular in the large intestine or the colon. Among the preferred embodiments of the present invention, there may be mentioned those food and/or pharmaceutical compositions arranged in dried forms . The drying of the compositions is carried out by using techniques and equipments generally employed and known to the skilled person.
The food and/or pharmaceutical compositions of the present invention are prepared in a traditional way by using,
depending on the type of formulation that one wishes to prepare, preparative techniques known to the skilled in the food and' pharmaceutical art. By way of absolutely non
limiting example, a granular food and/or pharmaceutical composition, to be suspended or dissolved in water at the time of use, will be prepared by intimately mixing the components of the composition (symbiotic composition,
coadjuvants, excipients), reducing them to the desired granulometry and moisture degree, before packing them in single-dose sealed sachets. In turn, a controlled-release food and/or pharmaceutical composition will be prepared, for example, by microencapsulating or micro-coating the microgranulated mixture of the substances forming the
composition with opportune mixtures of biocompatible polymers (such as, for example, Eudragit of different type and
structure) resistant to the gastric juices of the stomach and able to release said components after a proper residence time in the gastrointestinal tract, or at pH values typical of the colon. The microencapsulated mixture thus obtained will be used, for example, for the preparation of tablets, capsules or beads. The composition of the present invention can be administered in a variety of ways, depending oh the needs of the patient or the consumer.
In one of its preferred aspects, the present invention relates to the use of a symbiotic composition, as above described, for the preparation of food (for example
supplements) and/or pharmaceutical compositions, for carrying out the improvement and/or the restoration of the bifidogenic activity in the organism, for ameliorating of the intestinal microflora and the gastrointestinal microflora, for
preventing and/or treating intestinal diseases,
gastrointestinal diseases, inflammatory diseases of the intestines that may affect any part of the gastrointestinal tract from anus to mouth such as vomiting, diarrhea or weight loss, and for preventing and/or treating Crohn's disease, colitis (inflammatory bowel disease - IBD) , colitis ulcerosa, and irritable bowel syndrome (IBS) .
In a particularly preferred aspect, the symbiotic composition of the present invention is used for the preparation of food products based on milk, such as yoghurt, fermented milks, fresh cheeses and other food products, such as fruit-j uices , functional drinks and integrators/ creams, desserts, puree, chocolate, stuffs and fillings used in the confectionery.
In the contest of the present invention, the aforesaid populations of the probiotic strains relate to the bacterial population at the end of the manufacturing, while at the end of the shelf life, the bacterial populations are preferably between lxlO8 CFU/dose and lxlO11 CFU/dose, more preferably between lxlO9 CFU/dose and 2xl010 CFU/dose
Selection of the bacteria strains
Two synthetic substrates such as
p-nitrophenyl-a-D-galactopyranoside and p-nitrophenyl
p-D-mannopyranoside , have been used for evaluating the enzyme production of α-galactosidase and β-mannosidase . These latter enzymes hydrolyse the chains of galactose and the end chains of mannose respectively which form the galactomannans {Guy D. Duffaud, et al . ; Applied and Environment Microbiology, Jan. 1997, Vol. 63, No. 1, p. 169-177).
All the bacteria strains explicitly mentioned in the present application have duly tested as shown below.
Live bacteria samples selected from Bifidobacterium breve DSM 16604 (BR03), Bifidobacterium lactis LMG P-21384 (BS01) , Bifidobacterium catenulatum/pseudocatenulatum DSM 18350
(BA03), Bifidobacterium animalis subsp. Lactis DSM 18352 (BA05) , Bifidobacterium catenulatum DSM 18353 (BC01),and Lactobacillus plantarum LMG P-21021 (LP01) were prepared starting from frozen stocks collection as follows. Pure
Lactobacillus strains were cultured in de Man, Rogosa and Sharpe broth (MRS, DeMan et al . 1960) while Bifidobacterium strain, were cultured in MRS or Tryptone Phytone Yeast broth (TPY, Scardovi 1986), supplemented with
0.05% L-cysteine-hydrochloride . The cultures were prepared at 37°C under anaerobic conditions for 16-22 h.
For β-mannosidase and cx-galactosidase determination in ceil extracts, the above cultured bacteria were harvested by centrifugation (3000 g for 15 min) during exponential and/ or stationary growth phase in order to collect cells. Concentrated cell pellets were resuspended in 200-500 μΐ of Tris-HCl buffer (pH 7) and a small amount of quartz sand was added. The cell pellet resuspended in this way, was then disrupted in ice through a ultrasonic bath (4 cycles of
10 min, vortexing every 2 cycles). Ceil debris was removed by centrifugation at 10, 000 g for 10 min, and, the soluble fraction (cell extract) was used as the crude enzyme
preparation, that can contain the two enzymes of interest { β-Mannosidase and a-Galactosidase).
Protein concentration in cell extract was determined by the Bradford method with bovine serum albumin as a standard, as well known at the skilled person in the art. β-Mannosidase and α-Galactosidase activities were determined by monitoring the release of p-ni trophenol from
p-nitrophenyl- β-D-mannopyranoside or
p-nitrophenyl-ci-D-galactopyranoside , respectively .
The enzymatic assays for β-Mannosidase or a-Galactosidase were conducted as follows. For each assay, 1.1 ml aliquots of 10 mM of substrate (p-nitrophenyl-B-D-mannopyranoside or p-nitrophenyl-a-D-galactopyranoside) in 0.1 M sodium
phosphate buffer (pH 7.4) were pipetted into capped cuvettes. The cuvettes were preincubated for at least 10 min to allow the substrate to reach the assay temperature (25°C but 37°C is also possible) . After the preincubation, 0.1 ml of sample at defined total protein concentration (for example
60 - 100 pg/ml of the above crude enzyme preparation) was added to the cuvettes and mixed promptly. The release of the p-nitrophenol (PNP) was measured by monxtoring the change in absorbance at 405 nm (Guy D. Duffaud, et al . ; Applied and Environment Microbiology, Jan. 1997, Vol. 63, No. 1,
p. 169-177) . For- a quantitative determination of β- mannosidase and a-galactosidase concentration the same above assay was conducted adding 0,1 ml of dilution of standard enzymes instead of the sample (crude enzyme preparation) , A blank containing the same amount of sample in 0.1 M sodium phosphate buffer (pH 7.4) was used as a control. At
temperatures below 100°C and pH below 9.5, non enzymatic release of PNP was found to be negligible. One unit of β- mannosidase or a-galactosidase activity was defined as the amount of enzyme releasing 1 umol of p-Ni trophenol (PNP) per minute under the above specified assay conditions, such as pH 6.5 at 25°C, With reference to the quantification of a- galactosidase concentration in cell extracts it was also determined as follow, according to the below reaction (I) :
Reaction (I)
(p-Nitrophenyl-a-D~Galactopyranoside + HsO) ot~Galactosidase
Figure imgf000020_0001
{p-Ni trophenol (PNP) + D-Galactose)
For evaluating the above enzymes the following reagents were used :
A. 100 mM Potassium Phosphate Monobasic Solution,
B. 100 mM Potassium Phosphate Dibasic Solution,
C. 100 mM Potassium Phosphate Buffer, pH 6.5 at 25°C; the Reagent C was prepared by adjusting 50 ml of Reagent A to pH 6.5 at 25°C by adding Reagent B,
D. 9.9 mM p-Nitrophenyl-a-D-Galactopyranoside Solution (PNP- Ga 1.)
E. 200 mM Borate buffer, pH 9.8 with 1 M NaOH at 25 °C,
F. 1. a-Gaiactosidase Enzyme Solution (SIGMA G8507); a fresh solution containing 0.5 units /ml of
a-Galactosidase in cold Reagent C is prepared.
2. sample: defined concentration of the above crude enzyme preparation ( for example 60 - 100 pg/ml) .
We performed the test as follows (SIGMA Quality Control Test Procedure_ Enzymatic Assay of a-galactosidase„ EC 3.2.1.22) Reagents added to Test Blank
Reagent C 0,70 ml 0,70 ml
Reagent D 0,20 ml 0,20 ml
Mix by swirling and equilibrate to 25°C- Then add to:
Reagent Fl (standard) or F2 (sample) 0,10 ml —
Immediately mix by swirling and incubate at 25°C for 5 minutes. Then add to:
Reagent E 2 ml 2 ml
Reagent Fl (standard) or F2 (sample) — 0 / 10
Mix by swirling and record the A 405 nm for both the Test and Blank, using a spectrophotometer.
The reagent Fl has been used for the determination of unit concentration in a known α-galactosidase enzyme solution, as a control standard. In order to calculate the a-galactosidase concentration for the preparation of the standard curve concerning the above known α-galactosidase enzyme solution (referred to as control standard) and unknown sample
(referred to as cell extracts or crude enzyme preparations) the herewith expressions have been used:
(A405 nm Test - A405 nm Blank} (3.0) (df) Units /ml enzyme ~ =
(18.5) (5) (0.1)
Where
3.0 = Total volume of assay
df = Dilution factor
5 = Conversion, factor for 5 minutes to 1 minute
18.5 = Millimoiar extinction coefficient of p-Hitrophenol at
405 nm
0.1 = Volume (in milliliter) of enzyme used units/ml enzyme
Unit/mg solid =
mg soiid/mi enzyme units/ml enzyme
Unit/mg protein =
mg protein/ml enzyme
With reference to the stimulation of selected bacteria to use at least one mannose and/or galactose containing
carbohydrates such as for example galactomannan polymers, the said bacteria were cultured in the same medium MRS, with the above polymers at 20 g/1 as the only carbon, source, instead of glucose. The quantification of the a-galactosidase produced by stimulated bacteria has been performed by the aforementioned method after 3 sequential sub-cultures in a medium containing said polymers, as carbon source.
The stimulated cultures were also used to evaluate the different capability of the stimulated strain to growth in medium with galactomannan polymers as the only carbon source v/ith respect to the non-stimulated one. All the above cultures in normal MRS or in MRS with galactomannans (and without glucose) were prepared by an inoculum of 1% of a fresh, culture. The cultures of stimulated and non-stimulated {or unstimulated (N-stim. ) ) bacteria in MRS with
galactomannans were incubated in the just mentioned
conditions: 37°C till 24 h, measuring the pH values and torbidxty every 2 hours, in order to evaluate the bacterial differential growth.
Results
The bacteria strain Bifidobacterium breve DSM 16604 {BR03) is able to hydrolyse the synthetic substrate
p-nitrophenyl-a-D-galactopyranoside in a very efficient way in the first 30-60 minutes of the incubation step.
The bacteria strain Bifidobacterium breve DSM 16604 (BR03) hydrolyses the substrate better than other bifidobacteria strains which need about 3-6 hours more, as shown in Table 1. Table 1 confirms that the bacteria strain Bifidobacterium breve DSM 16604 (BR03) has an hight and specific ability to metabolize galactomannans and their hydrolysates thereof such as PHGG . From a quantitative point of view, it has been calculated that the bacteria strain Bifidobacterium breve DSM 16604 (BR03) is able to synthesize and produce an amount concentration of os-Galactosidase in active form of about 350 mU/mg of proteins of the extract. The above amount is 20-30% bigger than that produced by BS01 which is in turn quite efficient to hydrolyse galactomannans. Indeed, BS01 produces an amount of a-Galactosid.ase of about 250 mU/mg. Further, the bacteria strain Bifidobacterium breve DSM 16604 {BR03} is able to hydrolyse the synthetic substrate
p-ni trophenyl-^¾-D-mannopyranoside in a very efficient way. Thus, it is confirmed that the bacteria strain
Bifidobacterium breve DSM 16604 (BR03) has a big and specific ability to metabolize galactomannans and their hydrolysates thereof such as PHGG. The bacteria strain Bifidobacterium breve DSM 16604 (BR03) is able to synthesize and produce an amount of concentration of B-Mannosidase {indicated as Unit for mg of total proteins extracted} equal to or bigger than 1 U/mg, preferably bigger than 5 U/'mg.
The total amount of extra.ct.ed proteins from 109 cells is comprised from 1 to 1000 yg, preferably from 10 to 100 ug . Also for the remaining bacteria strains explicitly mentioned in the present application have been obtained, similar values. With respect to LGG, the β-Mannosidase produced by the bacteria strain Bifidobacterium breve DSM 16604 (BR03) is 50 times more concentrated.
Further, the Applicant has demonstrated that the enzyme activity of cx-Galactosidase in a Bifidobacteria can be stimulated. When a Bifidobacteria is cultured with only galactomannans, as carbon source (as for example PHGG), which require a specific enzyme for being metabolized, said
bacteria is stimulated to produce more a-Galactosidase .
It has been estimated that when the bacteria strain
Bifidobacterium breve DSM 16604 (BR03) is stimulated the amount of concentration of a-Galactosidase is increased by more than 4 times. From experimental data has been
demonstrated that the amount of concentration of
α-Galactosidase, produced by the bacteria strain
Bifidobacterium breve DSM 16604 (BR03), increases from about 350 mU/mg (as amount of total extracted proteins) to about 1500 mU/mg after 3 sequential sub-cultures in a medium containing galactomannans, as the only carbon source.
When the bacteria strain Bifidobacterium breve DSM 16604 (BR03) is stimulated it results consequently more effective in using the galactomannans as the only carbon source, see Tables 2 and 3 where the growth of the strain is monitoring on the bases of turbidity (Optical Density} and pH
respectively. Based on the differences in turbidity between the growth of BR03 stimulated and not stimulated it has been calculated that when BR03 is cultured in the presence of galactomannans an increase of 12% in the bacterial biomass is obtained in the next culture in the same condition {with galactomannans as the only carbon source), see Table 2.
The above experiment of growth in a medium with
galactomannans (as for example PHGG) as only carbon source, has been repeated for the bacteria strain Bifidobacterium breve DSM 16604 (BR03) after stimulation {sequential subcultures in the presence of partially hydrolysed
galactomannans such that PHGG) . The only difference was that the inoculation step was done using 1/16 of inoculum respect to the standard one (1%) which was used in the above
experiment. This inoculums size allow to more appreciate the difference between the growth of BR03 stimulated and the growth of not stimulated (calculated around 12% in the condition of above experiment), see Table 4. A test performed on Bifidobacterium breve DSM 16604. (BR03) which was stimulated and growth in PHGG, at same conditions as those explained above, with galactomannans as only carbon source, has shown that the bacteria strain Bifidobacterium breve DSM 16604 (BR03)is able to enhance the production of a further enzyme which is different from β-mannosidase or oi-galactosidase . The test has been performed by the use of Apyzym™ (N°25200 from BioMerieux) according to manufacturer protocol. As well known at the skilled person in the art, the API ZYM system, is a rapid semi quantitative procedure which allows the detection of 19 enzymatic reactions.
The API ZYM system comprises enzymatic tests performed on dried substrates in cupules. The enzyme activities tested are as follows: 1) Control, 2) Phosphatase alkaline, 3) Esterase (C4) , 4) Esterase Lipase (C8}, 5} Lipase (C14), 6) Leucine aminopeptidase , 7) Valine aminopeptidase , 8) Cystine
aminopeptidase , 9) Trypsin, 10) Chymotrypsin, 11) Phosphatase acid, 12} Phosphoamidase , 13) Alpha galactosidase , 14) Beta galactosidase , 15) Beta glucuronidase, 16) Alpha glucosidase, 17} Beta glucosidase, 18} N-acetyl-beta-glucosaminidase , 19) Alpha mannosidase, 20) Alpha fucosidase. After the inoculums of the strain in the API ZYM system and the incubation at 37°C for four hours in the dark, 0.02 mL each of reagent A and B (ready to use solutions contained in the commercial kit) was added to each cupule following the manufacturers directions. The reactions were allowed to develop for five minutes . The reactions were blindly graded 0-5 according to the color intensity compared to the
manufacturers interpretation scheme. Tests given grade 0 and 1 were regarded as negative and 2, 3, 4 and 5 were considered moderate to strong reactions and were regarded as positive, corresponding to an enzyme concentration of respectively 10, 20, 30 e > 40 nanomoles . The results obtain with Bifidobacterium breve DSM 16604 (BR03) on the substrate tested
6~Br-2 -naphtyl- βθ-galactopyranoside for the tested Enzyme, β-glucosidase (cellulose) are reported:
Bifidobacterium breve DSM 16604 (BR03) non-stimulated (or unstimulated (N-stim. ) ) : less than or equal to 5 nM;
Bifidobacterium breve DSM 16604 (BR03), stimulated (Stim. ) in PHGG: bigger than 40 nM. The maximum value that can be count by Apyzym™ is 40 nM .
From the above test, it comes out that Bifidobacterium breve DSM 16604 (BR03) stimulated (Stim.) in PHGG contains more than 8 times the amount of β-glucosidase enzyme in comparison with the non-stimulated one (or unstimulated {M-stim. ) ) .
This suggests that the growth of the strain with the specific substrate like galactomannans as only carbon source in the medium is able to stimulate the strain Bifidobacterium breve DSM 16604 (BR03)to produce an increase amount of
polysaccharides hydrolyzing enzyme (like a-galactosidase but also β-glucosidase) , suitable to metabolize that substrate.

Claims

1. A composition comprising:
- at least a mannose and/or galactose containing
carbohydrate, or hydrolysates thereof, and
- at least one bacteria strain selected from the group consisting of bacteria strains having at least an
alfa-galactosidase enzyme and/or a beta-mannosidase enzyme.
2. The composition according to claim 1, wherein said at least one mannose and/or galactose containing carbohydrate is selected from mannans , or hydrolysates thereof.
3. The composition according to claim 2 , wherein said mannans are selected from the group consisting of
glucomannans and galactomannans , or hydrolysates thereof.
4. The composition according to any one claim 3, wherein said glucomannans are selected from Konjac gums.
5. The composition according to any one claim 3, wherein said galactomannans are selected from carrube, guar gums and tara gums .
6. The composition according to any one claims 1-5, wherein said hydrolysates are selected from glucomannns partially hydrolysed and/or galactomannas partially hydrolysed, more preferably carrube partially hydrolysed, guar gums partially hydrolysed such as PHGG, or tara gums partially hydrolysed.
7. The composition according to any one claim 1, wherein said mannose and/or galactose containing carbohydrate is selected from galactooligosaccharides (GOS) , or hydrolysates thereof .
8. The composition according to any one claims 1-7, wherein said at least one bacteria strain is selected from the group comprising :
- Bifidobacterium breve DSM 16604,
- Bifidobacterium lactis LMG P-21384,
- Bifidobacterium catenulatum/pseudocatenulatum DSM 18350,
- Bifidobacterium animalis subsp. Lactis DSM 18352,
- Bifidobacterium catenulatum DSM 18353, and
- Lactobacillus plantarum LMG P-21021.
9. The composition according to claim 8, wherein the bacteria strains are selected from the group comprising
Bifidobacterium breve DSM 16604, Bifidobacterium lactis LMG P-21384, and Lactobacillus plantarum LMG P-21021.
10. The composition according to any one claims 1-9, wherein said composition comprises the bacterial strain
Bifidobacterium breve DSM 16604 and/or Bifidobacterium lactis LMG P-21384 and/or Lactobacillus plantarum LMG P-21021, and Konjac gums and/or PHGG .
11. The composition according to any one claims 1-10, wherein said composition is a symbiotic composition
comprising at least one mannose and/or galactose containing carbohydrate, preferably in an amount comprised from 5 to 95 % by weight, based on the total weight of the
composition; and at least one bacteria strain, preferably in an amount comprised from 1 to 15 % by weight, based on the total weight of the composition.
12. The composition according to any one claims 1-11, wherein said at least one bacteria strain has an alpha-galactosidase enzyme, a beta-mannosidase enzyme, and a β-glucosidase enzyme .
13. A pharmaceutical composition comprising a composition according to any one claims 1-12, for use as a medicament.
14. A food composition comprising a composition according to any one claims 1-12.
15. Use of a composition according to any one claims 1-12 for the preparation of a food or pharmaceutical composition for the improvement and/or the restoration of the bifidogenic activity in the organism; for ameliorating of the intestinal microflora and the gastrointestinal microflora; for
preventing and/or treating intestinal diseases,
gastrointestinal diseases, inflammatory diseases of the intestines that may affect any part of the gastrointestinal tract from anus to mouth such as vomiting, diarrhea or weight loss; and for preventing and/or treating Crohn's disease, colitis (inflammatory bowel disease - IBD) , colitis ulcerosa, and irritable bowel syndrome {IBS}.
PCT/EP2009/063410 2009-10-14 2009-10-14 Probiotic bacteria strains enabling of hydrolyzing prebiotic fibers and symbiotic compositions thereof WO2011044934A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/EP2009/063410 WO2011044934A1 (en) 2009-10-14 2009-10-14 Probiotic bacteria strains enabling of hydrolyzing prebiotic fibers and symbiotic compositions thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/EP2009/063410 WO2011044934A1 (en) 2009-10-14 2009-10-14 Probiotic bacteria strains enabling of hydrolyzing prebiotic fibers and symbiotic compositions thereof

Publications (1)

Publication Number Publication Date
WO2011044934A1 true WO2011044934A1 (en) 2011-04-21

Family

ID=42244337

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2009/063410 WO2011044934A1 (en) 2009-10-14 2009-10-14 Probiotic bacteria strains enabling of hydrolyzing prebiotic fibers and symbiotic compositions thereof

Country Status (1)

Country Link
WO (1) WO2011044934A1 (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016518441A (en) * 2013-05-14 2016-06-23 プロバイオティカル・ソシエタ・ペル・アチオニＰｒｏｂｉｏｔｉｃａｌ Ｓ．Ｐ．Ａ． Lactic acid bacteria-containing composition for use in preventive and / or therapeutic treatment of recurrent cystitis
US10982184B2 (en) 2011-05-09 2021-04-20 Probiotical S.P.A. Bacterial strains capable of metabolizing oxalates
US11110135B2 (en) 2011-05-09 2021-09-07 Probiotical S.P.A. Bacterial strains belonging to the genus Bifidobacterium for use in the treatment of hypercholesterolaemia
US11446340B2 (en) 2011-05-09 2022-09-20 Probiotical S.P.A. Probiotic bacterial strains and symbiotic composition containing the same intended for infant food

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006013588A1 (en) * 2004-08-05 2006-02-09 Anidral S.R.L. Folic acid producing bifidobacterium bacterial strains, formulations and use thereof
WO2006054135A1 (en) * 2004-11-16 2006-05-26 Anidral S.R.L. Probiotic bacteria based composition and use thereof in the prevention and/or treatment of respiratory pathologies and/or infections and in the improvement of the intestinal functionality
WO2007125558A1 (en) * 2006-05-03 2007-11-08 Anidral S.R.L. Symbiotic composition comprising non-digestible polysaccharides and bifidobacteria which metabolize them and its uses
WO2009071578A1 (en) * 2007-12-03 2009-06-11 Probiotical S.P.A. Composition based on probiotic bacteria in association with a prebiotic and use thereof in the prevention and/or treatment of respiratory pathologies and/or infections and in the improvement of intestinal functionality
WO2009138300A1 (en) * 2008-05-16 2009-11-19 Probiotical S.P.A. Use of probiotic bacteria for the treatment of hyperhomocysteinaemia

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006013588A1 (en) * 2004-08-05 2006-02-09 Anidral S.R.L. Folic acid producing bifidobacterium bacterial strains, formulations and use thereof
WO2006054135A1 (en) * 2004-11-16 2006-05-26 Anidral S.R.L. Probiotic bacteria based composition and use thereof in the prevention and/or treatment of respiratory pathologies and/or infections and in the improvement of the intestinal functionality
WO2007125558A1 (en) * 2006-05-03 2007-11-08 Anidral S.R.L. Symbiotic composition comprising non-digestible polysaccharides and bifidobacteria which metabolize them and its uses
WO2009071578A1 (en) * 2007-12-03 2009-06-11 Probiotical S.P.A. Composition based on probiotic bacteria in association with a prebiotic and use thereof in the prevention and/or treatment of respiratory pathologies and/or infections and in the improvement of intestinal functionality
WO2009138300A1 (en) * 2008-05-16 2009-11-19 Probiotical S.P.A. Use of probiotic bacteria for the treatment of hyperhomocysteinaemia

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10982184B2 (en) 2011-05-09 2021-04-20 Probiotical S.P.A. Bacterial strains capable of metabolizing oxalates
US11110135B2 (en) 2011-05-09 2021-09-07 Probiotical S.P.A. Bacterial strains belonging to the genus Bifidobacterium for use in the treatment of hypercholesterolaemia
US11446340B2 (en) 2011-05-09 2022-09-20 Probiotical S.P.A. Probiotic bacterial strains and symbiotic composition containing the same intended for infant food
JP2016518441A (en) * 2013-05-14 2016-06-23 プロバイオティカル・ソシエタ・ペル・アチオニＰｒｏｂｉｏｔｉｃａｌ Ｓ．Ｐ．Ａ． Lactic acid bacteria-containing composition for use in preventive and / or therapeutic treatment of recurrent cystitis
US11110136B2 (en) 2013-05-14 2021-09-07 Probiotical S.P.A. Composition comprising lactic acid bacteria for use in the preventive and/or curative treatment of recurrent cystitis

Similar Documents

Publication Publication Date Title
Hernández-Hernández et al. Effect of prebiotic carbohydrates on the growth and tolerance of Lactobacillus
KR101108428B1 (en) Lactic acid bacteria isolated from mother's milk with probiotic activity and inhibitory activity against body weight augmentation
Saulnier et al. Microbiology of the human intestinal tract and approaches for its dietary modulation
RU2551315C2 (en) Recovering, identifying and describing strains with probiotic activity recovered from faeces of exclusively breast-fed children
US6783780B1 (en) Preparation that contains oligosaccharides and probiotics
CN100469869C (en) Embedding protection method for beneficial bacteria of intestinal tract
US10597740B2 (en) Bifidobacterium longum CBT BG7 strain for promotion of growth and nutraceutical composition for promotion of growth containing the same
KR100607319B1 (en) Galactomannan oligosaccharides and methods for the production and use thereof
EP2012596A1 (en) Symbiotic composition comprising non-digestible polysaccharides and bifidobacteria which metabolize them and its uses
US20150306121A1 (en) Processing of natural polysaccharides by selected non-pathogenic microorganisms and methods of making and using the same
EP1513541B1 (en) Method for preventing or alleviating symptoms of malabsorption from the gastrointestinal tract
US11026983B2 (en) Bifidobacterium breve CBT BR3 strain for promotion of growth and nutraceutical composition for promotion of growth containing the same
KR20040022386A (en) Bifidobacteria and preparations containing them
Maske et al. A review on enzyme-producing lactobacilli associated with the human digestive process: From metabolism to application
CN104717890A (en) Fermented infant formula with non digestible oligosaccharides
CN102946892A (en) A composition for use in the therapy of lactose intolerance or conditions arising from lactase deficiency
Sriphannam et al. A selected probiotic strain of Lactobacillus fermentum CM33 isolated from breast-fed infants as a potential source of β-galactosidase for prebiotic oligosaccharide synthesis
WO2011044934A1 (en) Probiotic bacteria strains enabling of hydrolyzing prebiotic fibers and symbiotic compositions thereof
CA3223260A1 (en) Probiotic composition for the treatment of increased intestinal permeability
US8568712B2 (en) Enzyme and prebiotic combinations for enhancing probiotic efficacy
US20090098087A1 (en) Method of treating lactose intolerance using genetically engineered bacteria
Chaia et al. Dairy propionibacteria from milk or cheese diets remain viable and enhance propionic acid production in the mouse cecum
KR100868777B1 (en) Food composition with Bifidobacterium adolescentis to utilize RS-3 type resistant starch
WO2020204910A1 (en) Stable probiotic composition for the management of lactose intolerance
US8889119B2 (en) Enzyme and prebiotic combinations for enhancing probiotic growth and efficacy

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 09736919

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 09736919

Country of ref document: EP

Kind code of ref document: A1