WO2010099824A1 - Bacteria strains having a high anti-inflammatory activity - Google Patents

Bacteria strains having a high anti-inflammatory activity Download PDF

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Publication number
WO2010099824A1
WO2010099824A1 PCT/EP2009/052591 EP2009052591W WO2010099824A1 WO 2010099824 A1 WO2010099824 A1 WO 2010099824A1 EP 2009052591 W EP2009052591 W EP 2009052591W WO 2010099824 A1 WO2010099824 A1 WO 2010099824A1
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WIPO (PCT)
Prior art keywords
dsm
bacteria
probiotical
strains
bacterium
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PCT/EP2009/052591
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French (fr)
Inventor
Giovanni Mogna
Gian Paolo Strozzi
Luca Mogna
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Probiotical S.P.A.
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Publication date
Priority to CN2009801589932A priority Critical patent/CN102438637A/en
Priority to RU2011139211/10A priority patent/RU2011139211A/en
Priority to US13/254,730 priority patent/US20120064118A1/en
Priority to DK09779111.5T priority patent/DK2403510T3/en
Application filed by Probiotical S.P.A. filed Critical Probiotical S.P.A.
Priority to BRPI0924902-8A priority patent/BRPI0924902A2/en
Priority to EP09779111.5A priority patent/EP2403510B1/en
Priority to PL09779111T priority patent/PL2403510T3/en
Priority to PCT/EP2009/052591 priority patent/WO2010099824A1/en
Priority to AU2009341473A priority patent/AU2009341473B2/en
Priority to CA2754300A priority patent/CA2754300C/en
Priority to ES09779111T priority patent/ES2778836T3/en
Priority to PT97791115T priority patent/PT2403510T/en
Priority to KR1020117023442A priority patent/KR20110125667A/en
Publication of WO2010099824A1 publication Critical patent/WO2010099824A1/en
Priority to US14/535,068 priority patent/US20150283185A1/en

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    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • A—HUMAN NECESSITIES
    • 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
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/10—Laxatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/12—Antidiarrhoeals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Bacteria strains having a high anti-inflammatory activity having a high anti-inflammatory activity
  • the present invention relates to probiotic bacteria strains having a high anti-inflammatory activity.
  • the present invention relates to bacteria strains as strongly inducers of Interleukin-10 (IL-IO) production.
  • the present invention relates to the anti-inflammatory activity shown by said bacteria strains due to its enhancement of IL-IO production in peripheral blood mononuclear cells, with on the other hand a low capability to stimulate the production of the pro-inflammatory 11-12, thus leading to a high IL-10/IL- 12 ratio.
  • the present invention relates to the use of at least one bacterium strain for the preparation of a composition for the prevention or treatment of the inflammatory bowel diseases (IBD) and irritable bowel syndrome (IBS) .
  • the present invention relates to food products, such as probiotic dietary supplements containing at least one probiotic bacterium strain, as an active ingredient.
  • probiotics are live microorganisms which when administered in adequate amounts confer a health benefits on the host.
  • Probiotic lactobacilli and bifidobacteria are increasingly recognized as a way to prevent and/or treat intestinal disorders.
  • the intestinal immune system forms the largest part of the immune system. It interacts with a complex antigenic load in the form of food antigens, commensal bacteria, and occasional pathogens.
  • Dendritic cells are pivotal in earliest bacterial recognition and in shaping T cell responses . Dendritic cells sense antigen in tissues before migrating to draining lymphonodes, where they have the unique ability to activate and influence functional differentiation of naive Tcelis . Signals from DC can determine whether tolerance or an active immune response occurs to a particular antigen and furthermore influence whether a ThI or Th2 immune response predominates: DC upregulate the co-stimulatory molecules, CD80 and CD86, and produce IL-12 which contributes to a ThI response.
  • DC may produce IL-IO and IL-4 which promote the generation of a Th2 response or regulatory T cells .
  • Recognition of hazardous microbes, allergens and toxins as pathogenic agents activates the gastrointestinal immune system.
  • Antigen-specific Treg cells which mediate oral tolerance to commensal microbes, differentiate between harmless inhabitants of the gut and pathogens. A break in the development or maintenance of oral tolerance may result in an immense array of detrimental inflammatory disorders, including inflammatory bowel disease (IBD) and colitis.
  • IBD and colitis are conditions in which the immune system of patients reacts excessively to indigenous intestinal bacteria. Treg cell depletion in these disorders effectively breaches tolerance and allows for massive inflammation in the gut.
  • In vivo transfer of Treg cells suppresses the development of the above diseases, through IL-IO, TGF- ⁇ and CTLA ⁇ 4TMdependent mechanisms.
  • Probiotic strains can induce pro-inflammatory cytokines such as interleukin-1 (IL-I), IL-6, IL-12, tumor necrosis factor alpha (TNF- ⁇ ) , and gamma interferon (IFN- ⁇ ) as well as antiinflammatory cytokines such as IL-IO and transforming growth factor ⁇ .
  • IFN- Y and IL-12 potently augment the functions of macrophages and NK cells, which may be a possible mechanism of their anti-carcinogenic and anti-infectious activity.
  • induction of IL-IO and transforming growth factor ⁇ is assumed to participate in the down-regulation of inflammation, since these cytokines can inhibit the functions of macrophages and T cells and promote the development of regulatory T cells.
  • IL-IO is produced by many cells, including Th2 cells, DCs, monocytes, B cells, keratinocytes and regulatory T cells; it has an anti-inflammatory effect and primarily acts to inhibit the ThI response.
  • IL-IO drives the generation of a CD4+ T-cell subset, designated T regulatory cells 1 (TrI), suppressing antigen-specific immune responses and actively down-regulates a pathological immune response in vivo.
  • TrI T regulatory cells 1
  • IBD Inflammatory Bowel Disease
  • IL-10 is a cytokine of particular therapeutic interest since it has been shown in animal models that interleukin (IL) -10 ( -/-) mice spontaneously develop intestinal inflammation. It has been shown in animal models "hat probiotic strains displaying an in vitro potential to induce higher levels of the anti-inflammatory cytokine IL-IO and lower levels of the inflammatory cytokine IL-12, offer the best protection against in vivo colitis in the model.
  • Probiotic-mediated immunomodulation represents an interesting option in the management of IBD and it was shown that both the systemic and mucosal immune systems can be modulated by orally delivered bacteria.
  • candidate probiotics have been proven equally efficient due to the differences in survival and persistence of the strain in the gastro-intestinal tract, and/or to strain-specific interactions of the probiotic with the host immune system. The selection of a successful protective strain may therefore rely on the proper screening of a large number of candidate strains for their technological and immunomodulatory performance .
  • the Applicant has selected a group of bacteria strains which are able to solve the outstanding problems present in the prior art. According to a first aspect of the present invention, there is provided a group of bacteria strains or their cellular components having an immunoregulatory function through stimulation of Interleukin-10.
  • a food product containing at least one bacterium strain or its cellular components, as an active ingredient.
  • a composition containing at least one bacterium strain or its cellular components, for use as a medicament.
  • a use of at least one bacterium strain or its cellular components for the manufacture of a medicament for the prevention or treatment of inflammatory conditions of the large intestine and small intestine.
  • a use of at least one bacterium strain or its cellular components for the manufacture of a medicament for the prevention or treatment of functional bowel disorders is provided.
  • the Applicant has tested bacteria strains belonging to the following species: L, acidophilus , L. crispatus, L. gasseri , L. delbrueckii, L. salivarius , L. casei, L. paracasei n L. plantarum r L. rhamnosus , L. reuteri, L. brevis, L. buchneri , L. fermentum, B, adolescentis, B. angulatum, B. bifidum, B. breve, B. catenulatum, B. infantis, B. lactis r B. longum, B. pseudocatenulatum, and 5. thermophilus. Table 1 shows a group of bacteria strains which find a valid application in the contest of the present invention.
  • DSM 17104 plantarum LP 07 05 S. p. A.
  • DSM 18297 fermentum LFO8 06 S-P-A.
  • Lactobacillus DSM 19070 21.02.20 PROBIOTICAL plantarum LP 09 07 S. p. A.
  • Lactobacillus DSM 19071 21.02.20 PROBIOTICAL plantarum LP 10 07 S. p. A. La ctoba cill us DSM 19187 20 . 03 . 20
  • the bacteria strains or their cellular components contribute to the prevention or treatment of immune diseases including autoimmune diseases such as inflammatory bowel diseases, and contribute to maintenance of the immunological homeostasis (health maintenance) of mammals such as human beings, domestic animals, and pet animals.
  • immune diseases including autoimmune diseases such as inflammatory bowel diseases
  • immunological homeostasis health maintenance
  • the bacteria strains or their components according to the present invention are high in safety and can be orally administered.
  • the above microorganisms and the cellular components thereof are useful in that immunoregulatory cells can efficiently induced in the body by making use of the microorganism or the cellular components thereof as an active ingredient of pharmaceutical products, a food product, and the animal feeding stuff.
  • Figure 1 is a diagram showing an amount (pg/ml) of cytokine IL-IO production. Strain-specific patterns of IL-IO and IL-12 release for different microorganism strains.
  • Figure 2 is a diagram showing the IL-10/IL-12 ratio. Strain- specific IL-10/IL-12 ratio for different microorganism strains .
  • a bacterium strain is selected from the group consisting of L. paracasei LMG P-21380, L. plantarum LMG P-21021, Bifidobacterium lactis LMG P-21384, Bifidobacterium breve DSM 16604 or its cellular components, which induces the production of Interleukin-10.
  • said bacteria strains exhibit a IL-10/IL-12 ratio comprised from bigger than 1 and less than 150, preferably comprised from 10 and 100, more preferably comprised from 30 and 60.
  • the bacteria strain is Bifidobacterium breve DSM 16604 which induces the production of Interleukin-10 and exhibits a IL-10/I1-12 ratio which is comprised from 50 and 100, preferably from 70 and 80.
  • the bacteria strains may be in the form of live bacteria or dead bacteria or their cellular components.
  • a food product comprises at least one bacterium strain which is selected from the group consisting of L, pazacasei LMG F-21380, L. plantarum LMG P- 21C21, Bifidobacterium lactis LMG P-21384, and Bifidobacterium breve DSM 16604, as an active ingredient.
  • Said bacteria strains induce the production of Interleukin- 10. Further, said bacteria strains exhibit a IL-10/IL-12 ratio comprised from bigger than 1 and less than 150, preferably comprised from 10 and 100, more preferably comprised from 30 and 60.
  • the bacteria strain is Bifidobacterium breve DSM 16604 which induces the production of Interleukin-10 and exhibits a IL-10/I1-12 ratio which is comprised from 50 and 100, preferably from 70 and 80.
  • the bacteria strains may be in the form of live bacteria or dead bacteria or their cellular components.
  • a composition comprises at least one bacterium strain which is selected from the group consisting of L. paracasei LMG P-2138G, L. plantarum LMG P- 21021, Bifidobacterium lactis LMG P-21384, and Bifidobacterium breve DSM 16604 or its cellular components, as producer of Interleukin-10, for use as a medicament for the prevention or treatment of inflammatory conditions of the large intestine and small intestine or for the prevention or treatment of functional bowel disorders.
  • the inflammatory conditions are selected from the group comprising Crohn's disease and ulcerative colitis while the functional bowel disorders are selected from the group comprising diarrhea and constipation .
  • Said bacteria strains induce the production of Interleukin- 10. Further, said bacteria strains exhibit a IL-10/IL-12 ratio comprised from bigger than 1 and less than 150, preferably comprised from 10 and 100, more preferably comprised from 30 and 60.
  • the bacteria strain is Bifidobacterium breve DSM 16604 which induces the production of Interleukin-10 and exhibits an IL-10/I1-12 ratio which is comprised from 50 and 100, preferably from 70 and 80.
  • the bacteria strains may be in the form of live bacteria or dead bacteria or their cellular components .
  • the composition contains bacteria strains and/or their cellular components, as an active ingredients, in an amount comprised from IxIO ⁇ 1 to 1x10 ⁇ -1 CFU/g, respect to the weight of the composition, preferably from IxIO 8 to IxIO 11 CFU/g.
  • the composition contains bacteria strains and/or their cellular components, as an active ingredient, in an amount comprised IxIO ⁇ to IxK)H CFU/dose, preferably from IxIO 8 to IxIO 10 CFU/dose.
  • the dose may be of 1 g, 3 g, 5 g, and 10 g.
  • composition may further comprise additives and co- formulates pharmaceutically acceptable.
  • composition of the present invention may include vitamins (for example folic acid, riboflavin, vitamine E, ascorbic acid ⁇ , antioxidants compounds (for example polphenols, flavonoids and proanthocyanidines ) , aminoacid (for example glutamic, metionin) and also mineral (for example selenium and zinc) .
  • vitamins for example folic acid, riboflavin, vitamine E, ascorbic acid ⁇
  • antioxidants compounds for example polphenols, flavonoids and proanthocyanidines
  • aminoacid for example glutamic, metionin
  • mineral for example selenium and zinc
  • the composition of the present invention further includes at least a substance having preblotic properties in an amount comprised from 1 to 30% by weight, respect to the total weight composition, preferably from 5 to 20% by weight.
  • Said prebiotic substance preferably includes carbohydrates which are not digested and absorbed by the organism.
  • Said carbohydrates are preferably selected from: fructo- oligosaccharides (or FOS) , short-chain fructo- oligosaccharldes, inulin, isomalt-oligosaccharides , pectins, xylo-oligosaccharides (or XOS) , chitosan-o- ligosaccharides (or COS), beta-glucans, arable gum modified and re-sistant starches, polydextrose, D-tagatose, acacia fibers, bambu', carob, oats, and citrus fibers.
  • FOS fructo- oligosaccharides
  • short-chain fructo- oligosaccharldes inulin
  • isomalt-oligosaccharides pectins
  • xylo-oligosaccharides or XOS
  • COS chitosan-o- ligosaccharides
  • the bacteria strain Bifidobacterium breve DSM 16604 is in combination with at least one bacteria straxns selected from the group consisting of L. paracasei LMG P-21380, L. plantarum LMG P-21021, and Bifidobacterium lactis LMG P-21384.
  • the bacteria strains may be in the form of live bacteria or dead bacteria or their cellular components .
  • L. rhamnosus LR04
  • DSM 16605 I. paracasei
  • LMG P-21380 L. plantarum (LP Oi) LMG P-
  • Living (viable) and dead (killed) bacteria samples were prepared starting from frozen stocks collection as follows. Pure Lactobacillus strains were cultured in de Man, Rogosa and Sharpe broth (MRS, DeMar. et al. i960) while Bifidobacterium strains, were cultured in MRS or Tryptone Phytone Yeast broth (TPY f Scardovi 1966;, supplemented with 0.05% L-cysteme-hydrochlonde . The cultures were prepared at 37°C under anaerobic conditions for 16-22 hours. All bacteria were harvested by centrifugation (300Og for 15 min) during exponential and/ or stationary growth phase in order to collect cells.
  • Pelleted bacteria were then washed in phosphate buffered saline (PBS) and concentration was determined by means of colony-forming unit (CFU) counting.
  • CFU colony-forming unit
  • washed pelleted bacteria were diluted to a final working concentration of lxlO"CFU/mL in PBS containing 20% glycerol and stored at ⁇ 80°C until used for assay.
  • bacteria could be diluted in RPMX-1640 and the suspension aliquoted and stored at -2O 0 C.
  • the percentage of viability was not dependent on the time of storage. One fresh aliquot was thawed for every new experiment to avoid variability in the cultures between experiments .
  • one of rhe following procedures may be used. Heatkilled bacterial cultures were prepared by heating the above washed pelleted bacteria resuspended in distilled water at 10O 0 C for 30 min. Alternatively bacteria can be y- irradiated or sonicated. Apart from one of the above procedures used for having a dead bacteria sample, the above sample may be treated in a liquid form or in a freeze-dried one .
  • the bacteria strains of the present invention were co- cultured with PBMCs (Peripheral Blood Mononuclear Cells) in order to study the specific capability to induce cytokine production by immunopotent cells.
  • PBMCs Peripheral Blood Mononuclear Cells
  • PBMCs peripheral blood of healthy donor as described. Briefly, after Ficoll gradient centrifugation, mononuclear cells were collected, washed in PBS and adjusted to 2x10 ⁇ cells/mL in a complete medium consisting of RPMI 1640 supplemented with L-glutamin (300 mg/1) , penicillium
  • RPMI complete medium can also be obtained by RPMI-1640 supplemented with L-glutamin (300 mg/1), gentamicin
  • Monocytes can be purified from PBMCs by negative magnetic cell sorting.
  • the positively selected ceils can be used as source of peripheral blood lymphocytes (PBLs) .
  • Monocytes as well as PBLs . can be counted and resuspended at a concentration of 5xlQ 6 ceils/mL in complete RPMI medium.
  • PBMCs, Monocytes and PBLs mononuclear cells cryopreservation in liquid nitrogen, that cells, collected after Ficoll gradient centrifugation, were resuspen ⁇ ed at a concentration of 1x106 cells/mL in a complete medium consisting of RPMI 1640 supplemented with 10% DMSO (Dimethyl sulfoxide) .
  • DMSO Dimethyl sulfoxide
  • PBMCs cultures were set up in duplicate or triplicate in 96- well flat or round-bottom polystyrene microtitre plates . All cultures contained 0.1- 0.5xl0 6 PBMCs (or monocytes or PBLs) in complete medium. PBMCs were cultured in medium only or stimulated with phytoemoglutinine (PHA) at a final concentration of 50 ⁇ g/mL or lipopolisaccharides (LPS) at a final concentration of 0,5- i ⁇ g/mL . The co-cultures with the live bacteria samples were obtained by adding a thawed aliquot of live bacteria sample to the PBMCs cultures having a cell: bacteria ratio of 1:1, 1:10 or 1:200.
  • PHA phytoemoglutinine
  • LPS lipopolisaccharides
  • the above bacteria-cell optimal concentration can be determined after proliferation test with different relative concentration (for example varying concentrations of bacterial cell fractions from 10 6 to 10 9 CFU/ml) .
  • PBMCs were cultured with 5-20 ⁇ g/mL (preferably 10 ⁇ g/mL) of dead bacteria samples (heatkered, ⁇ -irradiated or sonicated) in freezed-dried form or with dead bacteria samples in the liquid form having a bacteria: cell ratio from
  • Control cultures contained unstimulated PBMCs, PHA-stimulated
  • PBMCs peripheral blood cells
  • monocytes peripheral blood cells
  • PBLs peripheral blood cells
  • Enzyme-Linked Immunosorbent ⁇ ssay using commercial kits (like Quantikine Kits, R&D Systems Minneapolis, MN), as instructed by the manufacturer, as well known at the skilled person in the art.
  • the minimum detectable dose of IL-IO and 11-12 was typically less " chan 3,9 pg/ml and 5,0 pg/ml f respectively.
  • Statistical analyses were performed with the Wilcoxon Mann- Whitney test to reveal significant differences between cytokine production in response to different strains of bacteria. Differences were considered to be significant at P ⁇ 0.05. Evaluation of IL-IO and IL-12 production
  • the Fig. 1 shows that strain-specific patterns of IL-10 and IL-12 release for different probiotic strains.
  • IL-10 concentrations were substantial with values ranging between 200 and 1700 pg/mL depending on the bacterial strain.
  • IL-12 production we also observed significant variations between strains, covering a range of cytokine levels of 10 to 1200 pg/mL.
  • Bifidobacterium breve BR 03 is able to module the immune responses by inducing the production of IL-10 by In vitro cultured mononuclear cells. Bifidobacterium breve BR 03 strongly induced IL-IO production (1688 pg/ml) . On the contrary, it has a low capability to stimulate the production of the pro-inflammatory IL-12 (22pg/ml) .
  • the pro-inflammatory cytokine IL-12 Is mainly produced by phagocytic and antigen-presenting cells (APCs) as a quick reaction against bacteria, intracellular parasites or other infectious agents .
  • APCs phagocytic and antigen-presenting cells
  • IL-12 will limit or inhibit differentiation of Th2 T cells, itself acting as an immunoregulatory molecule in the ThI response.
  • IL-12 will induce IFN- ⁇ and directly or indirectly activate natural killer cells, thus enhance further release of pro-inflammatory cytokines which promote an antigen-specific immune response.
  • IL-IO as a regulatory cytokine, is a potent inhibitor of IL-12 production by these phagocytic cells and may suppress the emergence of an unbalanced ThI response, such as the one seen in the gastrointestinal tract of IBD patients in a acute phase of inflammation; hence the importance In selecting probiotic strains with a favorable IL-10/IL-12 ratio.
  • Bifidobacterium breve BR 03 is the most potent "anti -inflammatory" strain eliciting the best IL-10/IL-12 ratio, as illustrated in Figure 2.
  • the figure 2 shows that strain-specific IL-10/IL-12 ratio for different probiotic strains.

Abstract

The present invention relates to probiotic bacteria strains having a high anti-inflammatory activity. The present invention relates to bacteria strains as strongly inducers of Interleukin-10 (IL-10) production. In particular, the present invention relates to the anti-inflammatory activity shown by said bacteria strains due to its enhancement of IL-10 production in peripheral blood mononuclear cells, with on the other hand a low capability to stimulate the production of the pro-inflammatory 11-12, thus leading to a high IL-10/IL-12 ratio. Further, the present invention relates to the use of at least one bacterium strain for the preparation of a composition for the prevention or treatment of the inflammatory bowel diseases (IBD) and irritable bowel syndrome (IBS). Finally, the present invention relates to food products, such as probiotic dietary supplements containing at least one probiotic bacterium strain, as an active ingredient.

Description

DESCRIPTIOM
Bacteria strains having a high anti-inflammatory activity
The present invention relates to probiotic bacteria strains having a high anti-inflammatory activity. The present invention relates to bacteria strains as strongly inducers of Interleukin-10 (IL-IO) production. In particular, the present invention relates to the anti-inflammatory activity shown by said bacteria strains due to its enhancement of IL-IO production in peripheral blood mononuclear cells, with on the other hand a low capability to stimulate the production of the pro-inflammatory 11-12, thus leading to a high IL-10/IL- 12 ratio. Further, the present invention relates to the use of at least one bacterium strain for the preparation of a composition for the prevention or treatment of the inflammatory bowel diseases (IBD) and irritable bowel syndrome (IBS) . Finally, the present invention relates to food products, such as probiotic dietary supplements containing at least one probiotic bacterium strain, as an active ingredient.
It is known that probiotics are live microorganisms which when administered in adequate amounts confer a health benefits on the host. Probiotic lactobacilli and bifidobacteria are increasingly recognized as a way to prevent and/or treat intestinal disorders.
Most of our encounters with antigens or infectious agents occur at mucosal surfaces, which include the surface lining the gastrointestinal, respiratory and genitourinary tracts. Since probiotics are usually absorbed orally, they are thus ideally suited to influence the immune response at the "mucosal frontier" of the gastrointestinal tract, representing more than 300 m2.
The intestinal immune system forms the largest part of the immune system. It interacts with a complex antigenic load in the form of food antigens, commensal bacteria, and occasional pathogens. Dendritic cells (DC) are pivotal in earliest bacterial recognition and in shaping T cell responses . Dendritic cells sense antigen in tissues before migrating to draining lymphonodes, where they have the unique ability to activate and influence functional differentiation of naive Tcelis . Signals from DC can determine whether tolerance or an active immune response occurs to a particular antigen and furthermore influence whether a ThI or Th2 immune response predominates: DC upregulate the co-stimulatory molecules, CD80 and CD86, and produce IL-12 which contributes to a ThI response. Further, DC may produce IL-IO and IL-4 which promote the generation of a Th2 response or regulatory T cells . Recognition of hazardous microbes, allergens and toxins as pathogenic agents activates the gastrointestinal immune system. Antigen-specific Treg cells, which mediate oral tolerance to commensal microbes, differentiate between harmless inhabitants of the gut and pathogens. A break in the development or maintenance of oral tolerance may result in an astounding array of detrimental inflammatory disorders, including inflammatory bowel disease (IBD) and colitis. IBD and colitis are conditions in which the immune system of patients reacts excessively to indigenous intestinal bacteria. Treg cell depletion in these disorders effectively breaches tolerance and allows for massive inflammation in the gut. In vivo transfer of Treg cells suppresses the development of the above diseases, through IL-IO, TGF-β and CTLA~4™dependent mechanisms.
Probiotic strains can induce pro-inflammatory cytokines such as interleukin-1 (IL-I), IL-6, IL-12, tumor necrosis factor alpha (TNF-α) , and gamma interferon (IFN-γ) as well as antiinflammatory cytokines such as IL-IO and transforming growth factor β. IFN- Y and IL-12 potently augment the functions of macrophages and NK cells, which may be a possible mechanism of their anti-carcinogenic and anti-infectious activity. On the other hand, induction of IL-IO and transforming growth factor β is assumed to participate in the down-regulation of inflammation, since these cytokines can inhibit the functions of macrophages and T cells and promote the development of regulatory T cells. IL-IO is produced by many cells, including Th2 cells, DCs, monocytes, B cells, keratinocytes and regulatory T cells; it has an anti-inflammatory effect and primarily acts to inhibit the ThI response. IL-IO drives the generation of a CD4+ T-cell subset, designated T regulatory cells 1 (TrI), suppressing antigen-specific immune responses and actively down-regulates a pathological immune response in vivo.
Several intestinal conditions are under the umbrella of "Inflammatory Bowel Disease (IBD)", including Crohn's disease, ulcerative colitis and pouchitis.
In inflammatory bowel disease, IL-10 is a cytokine of particular therapeutic interest since it has been shown in animal models that interleukin ( IL) -10 ( -/-) mice spontaneously develop intestinal inflammation. It has been shown in animal models "hat probiotic strains displaying an in vitro potential to induce higher levels of the anti-inflammatory cytokine IL-IO and lower levels of the inflammatory cytokine IL-12, offer the best protection against in vivo colitis in the model.
Probiotic-mediated immunomodulation represents an interesting option in the management of IBD and it was shown that both the systemic and mucosal immune systems can be modulated by orally delivered bacteria. However, not all candidate probiotics have been proven equally efficient due to the differences in survival and persistence of the strain in the gastro-intestinal tract, and/or to strain-specific interactions of the probiotic with the host immune system. The selection of a successful protective strain may therefore rely on the proper screening of a large number of candidate strains for their technological and immunomodulatory performance .
Therefore, it remains the need to isolate and select bacteria strains having a marked anti-inflammatory activity. In particular, it remains the need to isolate and select specific bacteria strains as strongly inducers of IL-IO production. Further, it remains the need to isolate and select bacteria strains with a low capability to stimulate the production of the pro-inflammatory 11-12, thus leading to a IL-10/IL-12 ratio at least bigger than one. Finally it remains the need to find out and select bacteria strains which show high persistence in the gastro-intestinal tract due to their resistance to gastric juice, bile salts, pancreatic secretion and to adhesion to gut wall . Last but not least it is important to select bacteria strains without acquired antibiotic resistances .
The Applicant has selected a group of bacteria strains which are able to solve the outstanding problems present in the prior art. According to a first aspect of the present invention, there is provided a group of bacteria strains or their cellular components having an immunoregulatory function through stimulation of Interleukin-10.
According to a second aspect of the present invention, there is provided a food product containing at least one bacterium strain or its cellular components, as an active ingredient. According to a third aspect of the present invention, there is provided a composition containing at least one bacterium strain or its cellular components, for use as a medicament. According to a fourth aspect of the present invention, there is provided a use of at least one bacterium strain or its cellular components for the manufacture of a medicament for the prevention or treatment of inflammatory conditions of the large intestine and small intestine.
According to a fifth aspect of the present invention, there is provided a use of at least one bacterium strain or its cellular components for the manufacture of a medicament for the prevention or treatment of functional bowel disorders.
The Applicant has tested bacteria strains belonging to the following species: L, acidophilus , L. crispatus, L. gasseri , L. delbrueckii, L. salivarius , L. casei, L. paracasein L. plantarumr L. rhamnosus , L. reuteri, L. brevis, L. buchneri , L. fermentum, B, adolescentis, B. angulatum, B. bifidum, B. breve, B. catenulatum, B. infantis, B. lactis r B. longum, B. pseudocatenulatum, and 5. thermophilus. Table 1 shows a group of bacteria strains which find a valid application in the contest of the present invention.
TABLE 1
Deposit Deposit
N0 Bacterium strain Depositor number date
1 Streptococcus LMG p_ 5.05.199 ANIDRAL thermophilus B39 18383 8 S. R. L.
2 Streptococcus LMG P- 5.05.199 ANIDRAL thermophilus T003 18384 8 S. R. L.
3 Lactobacillus LMG 16.10.20
MOFIN S. R. L. pentosus 9/1 ei 21019 01
4 Lactobacillus LMG P- 16.10.20
MOFIN S. R. L. plantarum 776/1 bi 21020 01
5 Lactobacillus
LMG P- 16.10.20 plantarum 476LL 20 MOFIN S. R. L. 21021 OI bi
6 Lactobacillus LMG P- 16. 10 .20
MOFIK S .R. L. plantarum PR ci 21022 01
7 Lactobacillus LMG P- 16. 10 .20
MOFIN S .R. L. plantarum 776/2 hi 21023 01
Lactobacillus easel
LMG P- 31.01.20 ANIDRAL ssp. paracasei 21380 02 S. R. L. 181A/3 aiai
Lactobacillus belonging to the LMG P- 31.01.20 ANIDRAL acidophilus group 21381 02 S. R. L. 192A/1 aiai
10 Bifidobacterium LMG P- 31 .01.20 ANIDRAL longum 175A/1 aiai 21382 02 S. R. L.
11 Bifidobacterium LMG P- 31 .01.20 ANIDRAL breve 195A/1 aid 21383 02 S. R. L.
12 Bifidobacterium LMG P- 31 .01.20 ANIDRAL lactis 32A/3 aiai 21384 02 S. R. L.
13 Lactobacillus LMG P- 31 .01.20
MOFIN S. R. L. plantarum 501/2 gi 21385 02 Lactococcus lactis
LMG P- 15.03.20 ssp. lactis 501/4 MOFIN S. R. L. 21387 02 hi Lactococcus lactis
LMG P- 31.01.20 ssp. lactis 501/4 MOFIN S. R. L, 21388 02 ci
Lactobacillus LMG P- 15.03.20
MOFIN S. R. L. plantarum 501/4 Ii 21389 02
Streptococcus 18.06.20 PROBIOTICAL
DSM 16506 thermophilus GBl 04 S .p. A.
Streptococcus 18.06.20 PROBIOTICAL
DSM 16507 thermophilus GB5 04 S. p. A.
Bifidobacterium 20.07.20 PROBIOTICAL
DSM 16603 longum BL 03 04 S. p. A.
Bifidobacterium 20.07.20 PROBIOTICAL
DSM 16604 breve BR 03 04 S. p. A. Lactobacillus casei 20.07.20 PROBIOTICAL
DSM 16605 ssp. rhamnosus LR 04 04 S. p. A. Lactobacillus
20.07.20 PROBIOTICAL delbrueckii ssp, DSM 16606 04 S. p. A. bulgaricus LDB 01 Lactobacillus
20.07.20 PROBIOTICAL delbrueckii ssp. DSM 16607 04 S. p. A. bulgaricus LDB 02
Streptococcus 20.07.20 PROBIOTICAL
DSM 16590 thermophilus YO2 04 S. p. A.
Streptococcus 20.07.20 PROBIOTICAL
DSM 16591 thermophilus YO3 04 S. p. A.
Streptococcus 20.07.20 PROBIOTICAL
DSM 16592 thermophilus YO4 04 S. p. A.
Streptococcus 20.07.20 PROBIOTICAL
DSM 16593 thermophilus Y05 04 S. p. A.
Bifidobacterium 21.07.20 PROBIOTICAL
DSM 16594 adolescentis BA 03 04 S. p. A.
Bifidobacterium 21.07.20 PROBIOTICAL
DSM 16595 adolescentis BA 04 04 S. p. A. Bifidobacterium 21.07.20 PROBIOTICAL
DSM 16596 breve BR 04 04 S .p. A. Bifidobacterium
21.07.20 PROBIOTICAL pseudocatenulatum DSM 16597 04 S. p. A. BP 01 Bifidobacterium
21.07.20 PROBIOTICAL pseudocatenulatum DSM 16598 04 S. p.A. BP 02
Staphylococcus 01.02.20 PROBIOTICAL
DSM 17102 xylosus SX 01 05 S. p. A.
Bifidobacterium 01.02.20 PROBIOTICAL
DSM 17103 adolescentis BA 02 05 S. p. A.
Lactobacillus 01.02.20 PROBIOTICAL
DSM 17104 plantarum LP 07 05 S. p. A.
Streptococcus 21.12.20 PROBIOTICAL
DSM 17843 thermophilus YO8 05 S. p. A.
Streptococcus 21.12.20 PROBIOTICAL
DSM 17844 thermophilus YO9 05 S. p. A.
Streptococcus 21.12.20 PROBIOTICAL
DSM 17845 thermophilus YOlOO 05 S. p. A.
Lactobacillus 24.05.20 PROBIOTICAL
DSM 18295 fermentum LFO 6 06 S .p. A.
Lactobacillus 24.05.20 PROBIOTICAL
DSM 18296 fermentum LFO 7 06 S. p. A.
Lactobacillus 24.05.20 PROBIOTICAL
DSM 18297 fermentum LFO8 06 S-P-A.
Lactobacillus 24.05.20 PROBIOTICAL
DSM 18298 fermentum LFO9 06 S. p. A.
Lactobacillus 24.05.20 PROBIOTICAL
DSM 18299 gasseri LGSOl 06 S. p. A.
Lactobacillus 24.05.20 PROBIOTICAL
DSM 18300 gasseri LGS02 06 S. p. A.
Lactobacillus 24.05.20 PROBIOTICAL
DSM 18301 gasseri LGS03 06 S. p. A.
Lactobacillus 24.05.20 PROBIOTICAL
DSM 18302 gasseri LGS04 06 S. p. A. Bifidobacterium 15.06.20 PROBIOTICAL
DSM 18350 adolescentis EI-3 06 S. p. A.
Bifidobacterium 15.06.20 PROBIOTICAL
DSM 18351 adolescentis EI-15 06 S. p. A.
Bifidobacterium 15.06.20 PROBIOTICAL
DSM 18352 adolescentis EI-18 06 S-P-A.
Bifidobacterium 15.06.20 PROBIOTICAL
DSM 18353 catenulatum EI-20 06 S. p. A.
Streptococcus 13.09.20
DSM 18613 MOFIN S. R. L. thermophilus FRa i 06
Streptococcus DSM 18614 13.09.20 MOFIN S. R. L. thermophilus LB2bi 06
Streptococcus DSM 18615 13.09.20 MOFIN S. R. L. thermophilus LRci 06
Streptococcus DSM 18616 13.09.20 MOFIN S. R. L. thermophilus FP4 06
Streptococcus DSM 18617 13.09.20 MOFIN S. R. L. thermophilus ZZ5F8 06
Streptococcus DSM 18618 13.09.20 MOFIN S. R. L. thermophilus TEO4 06
Streptococcus DSM 18619 13.09.20 MOFIN S. R. L. thermophilus Sl ci 06
Streptococcus DSM 18620 13.09.20 MOFIN S. R. L. thermophilus 641bi 06
Streptococcus DSM 18621 13.09.20 MOFIN S. R. L. thermophilus 06 277A/lai Streptococcus DSM 18622 13.09.20 MOFIN S. R. L. thermophilus 06 277A/2ai
Streptococcus DSM 18 623 13. 09. 20 MOFIN S .R .L. thermophilus IDCIl 06
Streptococcus DSM 18 624 13. 09. 20 MOFIN S .R .L. thermophilus ML3di 06
Streptococcus DSM 18 625 13. 09. 20 MOFIN S .R .L. thermophilus TEO3 06 Streptococcus DSM 19057 21.02.20 MOFIN S. R. L. thermophilics G62 07
Streptococcus DSM 19058 21.02.20 MOFIN S. R. L. thermophilus Gl 192 07
Streptococcus DSM 19059 21.02.20 MOFIN S. R. L. thermophilus GBl 8 07
Streptococcus DSM 19060 21.02.20 MOFIN S. R. L. thermophilus CCR21 07
Streptococcus DSM 19061 21.02.20 MOFIN S. R. L. thermophilus G92 07
Streptococcus DSM 19062 21.02.20 MOFIN S. R. L. thermophilus G69 07
Streptococcus DSM 19063 21.02.20 PROBIOTICAL thermophilus YO 10 07 S. p. A.
Streptococcus DSM 19064 21.02.20 PROBIOTICAL thermophilus YO 11 07 S. p. A.
Streptococcus DSM 19065 21.02.20 PROBIOTICAL thermophilus YO 12 07 S. p. A.
Streptococcus DSM 19066 21.02.20 PROBIOTICAL thermophilus YO 13 07 S. p. A.
Weissella ssp. DSM 19067 21.02.20 PROBIOTICAL WSP 01 07 S. p. A.
Weissella ssp. DSM 19068 21.02.20 PROBIOTICAL WSP 02 07 S. p. A.
Weissella ssp. DSM 19069 21.02.20 PROBIOTICAL WSP 03 07 S. p. A.
Lactobacillus DSM 19070 21.02.20 PROBIOTICAL plantarum LP 09 07 S. p. A.
Lactococcus lactis DSM 19072 21.02.20 PROBIOTICAL NS 01 07 S. p. A.
Lactobacillus DSM 19071 21.02.20 PROBIOTICAL plantarum LP 10 07 S. p. A. La ctoba cill us DSM 19187 20 . 03 . 20
PROBIOTICAL fermen tum 07
S . p . A . LF 10 Lactobacillus DSM 19188 20.03.20 PROBIOTICAL fermentum 07 S. p. A. LF 11
82 Lactobacillus easel DSM 19739 27.09.20 PR03I0TICAL ssp, rhamnosus LR 05 07 S. p. A.
Bifidobacterium DSM 19818 30.10.20
PROBIOTICAL bifidum 07 S. p. A.
BBOl
84 Lactobacillus DSM 19948 28.11.20 PROBIOTICAL delbrveckii LD 01 07 S. p. A.
85 Lactobacillus DSM 19949 28.11.20 PROBIOTICAL delbrueckii LD 02 07 S. p. A.
86 Lactobacillus DSM 19950 28.11.20 PROBIOTICAL delbrueckii LD 03 07 S. p. A.
87 Lactobacillus DSM 19951 28.11.20 PROBIOTICAL delbrueckii LD 04 07 S. p. A.
88 Lactobacillus DSM 19952 28.11.20 PROBIOTICAL delbrueckii LD 05 07 S. p. A.
89 Lactobacillus DSM 21717 06.08.20 PROBIOTICAL acidophilus LA 02 08 S. P. A.
90 Lactobacillus DSM 21718 06.08.20 PROBIOTICAL paracasei LPC 08 08 S. P. A.
91 Lactobacillus DSM 21980 14.11.20 PROBIOTICAL pentosυs LPS 01 08 S. P. A.
92 Lactobacillus DSM 21981 14 .11 .20 PROBIOTICAL rhamnosus LR 06 08 S. P. A.
The bacteria strains or their cellular components, according to the present invention, contribute to the prevention or treatment of immune diseases including autoimmune diseases such as inflammatory bowel diseases, and contribute to maintenance of the immunological homeostasis (health maintenance) of mammals such as human beings, domestic animals, and pet animals.
In other words, the bacteria strains or their components according to the present invention are high in safety and can be orally administered. Thus, the above microorganisms and the cellular components thereof are useful in that immunoregulatory cells can efficiently induced in the body by making use of the microorganism or the cellular components thereof as an active ingredient of pharmaceutical products, a food product, and the animal feeding stuff.
Other aspects and features of the invention will be more fully apparent from the following disclosure and appended claims .
Figure 1 is a diagram showing an amount (pg/ml) of cytokine IL-IO production. Strain-specific patterns of IL-IO and IL-12 release for different microorganism strains.
Figure 2 is a diagram showing the IL-10/IL-12 ratio. Strain- specific IL-10/IL-12 ratio for different microorganism strains .
The invention will be fully described by means of the following description without any limiting effects . In a preferred embodiment a bacterium strain is selected from the group consisting of L. paracasei LMG P-21380, L. plantarum LMG P-21021, Bifidobacterium lactis LMG P-21384, Bifidobacterium breve DSM 16604 or its cellular components, which induces the production of Interleukin-10. Further, said bacteria strains exhibit a IL-10/IL-12 ratio comprised from bigger than 1 and less than 150, preferably comprised from 10 and 100, more preferably comprised from 30 and 60. Advantageously, the bacteria strain is Bifidobacterium breve DSM 16604 which induces the production of Interleukin-10 and exhibits a IL-10/I1-12 ratio which is comprised from 50 and 100, preferably from 70 and 80.
The bacteria strains may be in the form of live bacteria or dead bacteria or their cellular components.
In another preferred embodiment a food product comprises at least one bacterium strain which is selected from the group consisting of L, pazacasei LMG F-21380, L. plantarum LMG P- 21C21, Bifidobacterium lactis LMG P-21384, and Bifidobacterium breve DSM 16604, as an active ingredient. Said bacteria strains induce the production of Interleukin- 10. Further, said bacteria strains exhibit a IL-10/IL-12 ratio comprised from bigger than 1 and less than 150, preferably comprised from 10 and 100, more preferably comprised from 30 and 60. Advantageously, the bacteria strain is Bifidobacterium breve DSM 16604 which induces the production of Interleukin-10 and exhibits a IL-10/I1-12 ratio which is comprised from 50 and 100, preferably from 70 and 80. The bacteria strains may be in the form of live bacteria or dead bacteria or their cellular components.
In a further preferred embodiment a composition comprises at least one bacterium strain which is selected from the group consisting of L. paracasei LMG P-2138G, L. plantarum LMG P- 21021, Bifidobacterium lactis LMG P-21384, and Bifidobacterium breve DSM 16604 or its cellular components, as producer of Interleukin-10, for use as a medicament for the prevention or treatment of inflammatory conditions of the large intestine and small intestine or for the prevention or treatment of functional bowel disorders. The inflammatory conditions are selected from the group comprising Crohn's disease and ulcerative colitis while the functional bowel disorders are selected from the group comprising diarrhea and constipation .
Said bacteria strains induce the production of Interleukin- 10. Further, said bacteria strains exhibit a IL-10/IL-12 ratio comprised from bigger than 1 and less than 150, preferably comprised from 10 and 100, more preferably comprised from 30 and 60. Advantageously, the bacteria strain is Bifidobacterium breve DSM 16604 which induces the production of Interleukin-10 and exhibits an IL-10/I1-12 ratio which is comprised from 50 and 100, preferably from 70 and 80. The bacteria strains may be in the form of live bacteria or dead bacteria or their cellular components .
In a preferred embodiment, the composition contains bacteria strains and/or their cellular components, as an active ingredients, in an amount comprised from IxIO^1 to 1x10^-1 CFU/g, respect to the weight of the composition, preferably from IxIO8 to IxIO11 CFU/g.
In a preferred embodiment, the composition contains bacteria strains and/or their cellular components, as an active ingredient, in an amount comprised IxIO^ to IxK)H CFU/dose, preferably from IxIO8 to IxIO10 CFU/dose. The dose may be of 1 g, 3 g, 5 g, and 10 g.
The composition may further comprise additives and co- formulates pharmaceutically acceptable.
The composition of the present invention may include vitamins (for example folic acid, riboflavin, vitamine E, ascorbic acid}, antioxidants compounds (for example polphenols, flavonoids and proanthocyanidines ) , aminoacid (for example glutamic, metionin) and also mineral (for example selenium and zinc) .
In another particularly preferred embodiment, the composition of the present invention further includes at least a substance having preblotic properties in an amount comprised from 1 to 30% by weight, respect to the total weight composition, preferably from 5 to 20% by weight. Said prebiotic substance preferably includes carbohydrates which are not digested and absorbed by the organism. Said carbohydrates are preferably selected from: fructo- oligosaccharides (or FOS) , short-chain fructo- oligosaccharldes, inulin, isomalt-oligosaccharides , pectins, xylo-oligosaccharides (or XOS) , chitosan-o- ligosaccharides (or COS), beta-glucans, arable gum modified and re-sistant starches, polydextrose, D-tagatose, acacia fibers, bambu', carob, oats, and citrus fibers. Particularly preferred prebiotics are the short-chain fructo-oligosaccharides (for simplicity shown herein- below as FOSs-c.c); said FOSs-c.c. are not digestable glucides, generally obtained by the conversion of the beet sugar and including a saccharose molecule to which three glucose molecules are bonded. In a preferred embodiment the bacteria strain Bifidobacterium breve DSM 16604 is in combination with at least one bacteria straxns selected from the group consisting of L. paracasei LMG P-21380, L. plantarum LMG P-21021, and Bifidobacterium lactis LMG P-21384. The bacteria strains may be in the form of live bacteria or dead bacteria or their cellular components .
The following bacteria strains have been tested. Three Lactobacillus strains: L. rhamnosus (LR04) DSM 16605, I. paracasei (LPC 00) LMG P-21380, L. plantarum (LP Oi) LMG P-
21021, and two Bifidobacterium strains: B. lactis (BS 01) LMG P-21384, and B. breve (BR 03) DSM 16604 belonging to the most representative species of probiotic bacteria, were selected based on their resistance to acid, digestive enzyme, and bile and other characteristics such as antibiotic resistance and safety of use.
Living (viable) and dead (killed) bacteria samples were prepared starting from frozen stocks collection as follows. Pure Lactobacillus strains were cultured in de Man, Rogosa and Sharpe broth (MRS, DeMar. et al. i960) while Bifidobacterium strains, were cultured in MRS or Tryptone Phytone Yeast broth (TPYf Scardovi 1966;, supplemented with 0.05% L-cysteme-hydrochlonde . The cultures were prepared at 37°C under anaerobic conditions for 16-22 hours. All bacteria were harvested by centrifugation (300Og for 15 min) during exponential and/ or stationary growth phase in order to collect cells. Pelleted bacteria were then washed in phosphate buffered saline (PBS) and concentration was determined by means of colony-forming unit (CFU) counting. With reference to the preparation of living (viable) bacteria samples, washed pelleted bacteria were diluted to a final working concentration of lxlO"CFU/mL in PBS containing 20% glycerol and stored at ~80°C until used for assay. Alternatively bacteria could be diluted in RPMX-1640 and the suspension aliquoted and stored at -2O0C.
Survival of bacteria upon freezing and thawing was determined by amount of live bacteria by means of colony- forming unit
(CFU) counting and/ or with staining for cFDA (live) and PI
(dead) . For all strains tested, >80% was alive upon thawing.
The percentage of viability was not dependent on the time of storage. One fresh aliquot was thawed for every new experiment to avoid variability in the cultures between experiments .
With reference to the preparation of the dead bacteria samples, one of rhe following procedures may be used. Heatkilled bacterial cultures were prepared by heating the above washed pelleted bacteria resuspended in distilled water at 10O0C for 30 min. Alternatively bacteria can be y- irradiated or sonicated. Apart from one of the above procedures used for having a dead bacteria sample, the above sample may be treated in a liquid form or in a freeze-dried one .
The bacteria strains of the present invention were co- cultured with PBMCs (Peripheral Blood Mononuclear Cells) in order to study the specific capability to induce cytokine production by immunopotent cells.
PBMCs were isolated from peripheral blood of healthy donor as described. Briefly, after Ficoll gradient centrifugation, mononuclear cells were collected, washed in PBS and adjusted to 2x10^ cells/mL in a complete medium consisting of RPMI 1640 supplemented with L-glutamin (300 mg/1) , penicillium
{100 U/ml), streptomycin (64 ϋ/ml and 10% heat inactivated FCS (Fetal Calf Serum) .
Alternatively a RPMI complete medium can also be obtained by RPMI-1640 supplemented with L-glutamin (300 mg/1), gentamicin
(500 μg/mL) , penicillin (100 U/mL) , streptomycin (64 U/ml) and 20% heat-inactivated human AB serum or 10% FCS.
Monocytes can be purified from PBMCs by negative magnetic cell sorting. The positively selected ceils can be used as source of peripheral blood lymphocytes (PBLs) . Monocytes as well as PBLs . can be counted and resuspended at a concentration of 5xlQ6 ceils/mL in complete RPMI medium. For mononuclear cells (PBMCs, Monocytes and PBLs) cryopreservation in liquid nitrogen, that cells, collected after Ficoll gradient centrifugation, were resuspenόed at a concentration of 1x106 cells/mL in a complete medium consisting of RPMI 1640 supplemented with 10% DMSO (Dimethyl sulfoxide) .
PBMCs cultures were set up in duplicate or triplicate in 96- well flat or round-bottom polystyrene microtitre plates . All cultures contained 0.1- 0.5xl06 PBMCs (or monocytes or PBLs) in complete medium. PBMCs were cultured in medium only or stimulated with phytoemoglutinine (PHA) at a final concentration of 50μg/mL or lipopolisaccharides (LPS) at a final concentration of 0,5- iμg/mL . The co-cultures with the live bacteria samples were obtained by adding a thawed aliquot of live bacteria sample to the PBMCs cultures having a cell: bacteria ratio of 1:1, 1:10 or 1:200.
The above bacteria-cell optimal concentration can be determined after proliferation test with different relative concentration (for example varying concentrations of bacterial cell fractions from 106 to 109 CFU/ml) .
With reference to the co-cultures test with dead bacteria samples, PBMCs were cultured with 5-20 μg/mL (preferably 10 μg/mL) of dead bacteria samples (heatkiiled, γ-irradiated or sonicated) in freezed-dried form or with dead bacteria samples in the liquid form having a bacteria: cell ratio from
50:1 to 250:1 {preferably 200:1) .
Control cultures contained unstimulated PBMCs, PHA-stimulated
PBMCs, monocytes, PBLs all without bacteria strains or live bacteria sample only.
The plates were incubated at 370C in 5% CO2 - The supernatants of cultures were collected at 24, 48, 72 hours and 5 days, clarified by centrifugation and stored at -2O0C until cytokine analysis. Neither medium acidification nor bacterial proliferation was observed. Cytokines IL-IO and IL-12 levels were measured by standard
Enzyme-Linked Immunosorbent Λssay (ELISA) using commercial kits (like Quantikine Kits, R&D Systems Minneapolis, MN), as instructed by the manufacturer, as well known at the skilled person in the art.
Briefly, standards and samples ( supernatants from the above co-cultured) were added into the plates and incubated for 2h at room temperature. The specific horseradish peroxidase- conjugated antibody was added to ail wells after they were washed 4 times, and the plates were incubated for 1 hour at room temperature. The plates were then washed and incubated for 30 minutes with 3-3f , 5 , 5 ' -tetramethylbenzidine substrate reagent solution. The reaction was stopped by the addition of 1.8 M H2SO4. The absorbency of ail ELISAs was read at 450 nm with a microtiter plate reader. Standard curves for the cytokines were constructed.
The minimum detectable dose of IL-IO and 11-12 was typically less "chan 3,9 pg/ml and 5,0 pg/mlf respectively. Statistical analyses were performed with the Wilcoxon Mann- Whitney test to reveal significant differences between cytokine production in response to different strains of bacteria. Differences were considered to be significant at P<0.05. Evaluation of IL-IO and IL-12 production
The in vitro immune- stimulation by 5 live bacterial strains of PBMCs collected from healthy donors, revealed distinct capability of the strains to induce IL-IO and IL-12, so that IL-IO and IL-12 levels displayed a strain-specific pattern, as shown in Fig. 1.
The Fig. 1 shows that strain-specific patterns of IL-10 and IL-12 release for different probiotic strains. One experiment representative of 5.
Variations of IL-10 concentrations were substantial with values ranging between 200 and 1700 pg/mL depending on the bacterial strain. For the IL-12 production, we also observed significant variations between strains, covering a range of cytokine levels of 10 to 1200 pg/mL.
Bifidobacterium breve BR 03 is able to module the immune responses by inducing the production of IL-10 by In vitro cultured mononuclear cells. Bifidobacterium breve BR 03 strongly induced IL-IO production (1688 pg/ml) . On the contrary, it has a low capability to stimulate the production of the pro-inflammatory IL-12 (22pg/ml) .
The capacity of the probiotic strain B. breve BR 03 to boost the production of IL-10 differed considerably between other strains studied, among which can be considered the most potent inducers, see Fig. 1.
In addition to a high IL-IO induction potential, it is Important to minimize the IL-12 induction by the probiotic bacteria, when considering selecting a strain for an antiinflammatory application. The pro-inflammatory cytokine IL-12, Is mainly produced by phagocytic and antigen-presenting cells (APCs) as a quick reaction against bacteria, intracellular parasites or other infectious agents . In addition to an important role in the first line of defence against infection, IL-12 will limit or inhibit differentiation of Th2 T cells, itself acting as an immunoregulatory molecule in the ThI response. IL-12 will induce IFN-γ and directly or indirectly activate natural killer cells, thus enhance further release of pro-inflammatory cytokines which promote an antigen-specific immune response. This IL-12 production enhancing feedback mechanism, mediated by IFN-Y, is potentially leading to uncontrolled cytokine production. Fortunately, IL-IO, as a regulatory cytokine, is a potent inhibitor of IL-12 production by these phagocytic cells and may suppress the emergence of an unbalanced ThI response, such as the one seen in the gastrointestinal tract of IBD patients in a acute phase of inflammation; hence the importance In selecting probiotic strains with a favorable IL-10/IL-12 ratio.
Evaluation of IL-IO/ IL -12 r&t±o
It _s possible to use the IL-10/IL-12 ratio to distinguish between strains exhibiting a"pro-" versus "anti-inflammatory" profile (low versus high IL-10/IL-12 ratio, respectively) . This approach was found to be useful to identify strains with marked opposite profiles and can be used as a standardized in vitro test, allowing preliminary classification of candidate probiotic strains according to their immune modulation capacity that would be predictive of their in vivo effect. The importance of the ratio between these two cytokines was also recently demonstrated by Peran et al .. In the study, administration of a specific strain of Lactobacillus salivarius ssp. salivarius facilitates the recovery of the inflamed tissue in the TNBS model of rat colitis. This beneficial effect was partly associated to the ability of the strain to modify the cytokine profile in macrophages, reducing the amount of inflammatory cytokine IL- 12, while increasing the amount of the anti- inflammatory cytokine IL-IO.
The use of PBMC from a diversity of healthy human donors to screen the immunomodulatory activity of candidate probiotic strains by direct stimulation appears to be a good predictive indicator of in vivo anti-inflammatory strains. Despite the fact that this assay does not clarify the physiological mechanism (s) involved, it seems to mimic how the immune system may sense the bacterial strain and consequently polarise the immune response. Strains leading to a high IL-10/IL-12 ratio would more easily slow down an early ThI response .
In this context, assessing effects of 5 different probiotic bacteria, we found that Bifidobacterium breve BR 03 is the most potent "anti -inflammatory" strain eliciting the best IL-10/IL-12 ratio, as illustrated in Figure 2.
The figure 2 shows that strain-specific IL-10/IL-12 ratio for different probiotic strains. One experiment representative of 5.
Taking into account the above, all the bacteria strains identified in the present invention show:
- a strong capability to induce the anti- inflammatory IL-IO production,
low capability to stimulate the production of the proinflammatory IL- 12,
potent "anti - inflammatory" activity eliciting a high IL- 10/ IL- 12 ratio ,
high persistence in the gastro- intestinal tract due to their resistance to gastric juice, bile salts, pancreatic secretion and to adhesion to gut wall, and
- safe to use having none acquired antibiotic resistances,

Claims

1. A bacterium strain selected from the group consisting of L. paracasei LMG P-2138G, L, plantarum LMG P-21021, Bifidobacterium lactis LMG P-21384, and Bifidobacterium breve DSM 166C4 or its cellular components, as inducer of Interleukin-10.
2. The bacterium strain according to claim 1, wherein said bacterium exhibits a IL-10/IL-12 ratio comprised from bigger than 1 and less than 150, preferably comprised from 10 and 100, more preferably comprised from 30 and 60.
3. The bacterium strain Bifidobacterium breve DSM 16604 according to claim 1 or 2, as inducer of Interleukin-10 and exhibiting an IL-lO/Il-12 ratio which is comprised from 50 and 100, preferably from 70 and 80.
4. The bacterium strain according to any one claims 1 to 3, wherein said bacterium is in the form of live bacterium or dead bacterium or its cellular components.
5. A food product comprising at least one bacterium strain according to any one claims 1 to 4, as an active ingredient .
6. A composition comprising at least one bacterium strain according to any one claims 1 to 4, for use as a medicament .
7. The composition according to claim 6, for use as a medicament for the prevention or treatment of inflammatory conditions of the large intestine and small intestine .
8. The composition according to claim 7, wherein the inflammatory conditions are selected from the group comprising Crohn's disease and ulcerative colitis.
9. The composition according to claim 6, for use as a medicament for the prevention or treatment of functional bowel disorders.
10. The composition according to claim 9, wherein the functional bowel disorders are selected from the group comprising diarrhea and constipation.
11. Use of at least one bacterium strain according to any one claims 1 to 4 for the manufacture of a medicament for the prevention or treatment of inflammatory conditions of the large intestine and small intestine .
12. The use according to claim 11, wherein the inflammatory conditions are selected from the group comprising Crohn's disease and ulcerative colitis.
13. Use of at least one bacterium strain according to any one claims 1 to 4 for the manufacture of a medicament for the prevention or treatment of functional bowel disorders.
14. The use according to claim 13, wherein the functional bowel disorders are selected from the group comprising diarrhea and constipation.
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CN116083323A (en) * 2023-03-16 2023-05-09 威凯海思(山东)生物工程有限公司 Bifidobacterium lactis HC2786 capable of relieving anaphylactic reaction, and product and application thereof

Citations (4)

* 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
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
WO2008038075A2 (en) * 2006-09-27 2008-04-03 Mofin S.R.L. Microbial liquid cultures having high stability and fermentative activity
EP2002842A2 (en) * 2006-03-31 2008-12-17 Morinaga Milk Industry Co., Ltd. Interleukin production regulator, pharmaceutical composition or food comprising the interleukin production regulator, and method for production of the interleukin production regulator

Patent Citations (4)

* 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
EP2002842A2 (en) * 2006-03-31 2008-12-17 Morinaga Milk Industry Co., Ltd. Interleukin production regulator, pharmaceutical composition or food comprising the interleukin production regulator, and method for production of the interleukin production regulator
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
WO2008038075A2 (en) * 2006-09-27 2008-04-03 Mofin S.R.L. Microbial liquid cultures having high stability and fermentative activity

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
IMAOKA AKEMI ET AL: "Anti-inflammatory activity of probiotic Bifidobacterium: enhancement of IL-10 production in peripheral blood mononuclear cells from ulcerative colitis patients and inhibition of IL-8 secretion in HT-29 cells.", WORLD JOURNAL OF GASTROENTEROLOGY : WJG 28 APR 2008, vol. 14, no. 16, 28 April 2008 (2008-04-28), pages 2511 - 2516, XP009117210, ISSN: 1007-9327 *
LEBLANC J G ET AL: "Anti-inflammatory properties of lactic acid bacteria: Current knowledge, applications and prospects", ANTI-INFECTIVE AGENTS IN MEDICINAL CHEMISTRY, BENTHAM SCIENCE PUBLISHERS, HILVERSUM, NL, vol. 7, no. 3, 1 July 2008 (2008-07-01), pages 148 - 154, XP009105259, ISSN: 1871-5214 *
MADSEN K L ET AL: "LACTOBACILLUS SPECIES PREVENTS COLITIS IN INTERLEUKIN 10 GENE-DEFICIENT MICE", GASTROENTEROLOGY, ELSEVIER, PHILADELPHIA, PA, vol. 116, no. 5, 1 May 1999 (1999-05-01), pages 1107 - 1114, XP008023587, ISSN: 0016-5085 *
PATHMAKANTHAN SHRI ET AL: "Lactobacillus plantarum 299: Beneficial in vitro immunomodulation in cells extracted from inflamed human colon.", February 2004, JOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, VOL. 19, NR. 2, PAGE(S) 166-173, ISSN: 0815-9319, XP009117202 *
PENA J A ET AL: "Probiotic Lactobacillus spp. Diminish Helicobacter hepaticus-Induced Inflammatory Bowel Disease in Interleukin-10-Deficient Mice", INFECTION AND IMMUNITY, AMERICAN SOCIETY FOR MICROBIOLOGY. WASHINGTON, vol. 73, no. 2, 1 February 2005 (2005-02-01), pages 912 - 920, XP003001378, ISSN: 0019-9567 *

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WO2018183941A2 (en) 2017-03-30 2018-10-04 Progenity Inc. Treatment of a disease of the gastrointestinal tract with live biotherapeutics
CN108707557A (en) * 2018-03-23 2018-10-26 景岳生物科技股份有限公司 Prevent, improve or slow down probiotic composition and its application of cancer of pancreas
CN108707557B (en) * 2018-03-23 2021-04-16 景岳生物科技股份有限公司 Probiotic composition for preventing, improving or slowing pancreatic cancer and application thereof
IT201900016805A1 (en) * 2019-09-20 2021-03-20 Sofar Spa Bacterial strains, their compositions and their use for the treatment of gastrointestinal disorders
CN114727640A (en) * 2019-09-20 2022-07-08 索法尔公司 Bacterial strains, compositions thereof and use thereof for the treatment of gastrointestinal disorders
WO2021053639A1 (en) * 2019-09-20 2021-03-25 Sofar S.P.A. Bacterial strains, their compositions and their use for the treatment of gastrointestinal disorders
CN110893195A (en) * 2019-09-30 2020-03-20 内蒙古伊利实业集团股份有限公司 Lactobacillus paracasei ET-22 with function of relieving intestinal inflammation
CN110893195B (en) * 2019-09-30 2023-03-14 内蒙古伊利实业集团股份有限公司 Lactobacillus paracasei ET-22 with function of relieving intestinal inflammation
US11751597B2 (en) 2019-11-05 2023-09-12 Alfasigma S.P.A. Compositions comprising bacterial strains for use in increasing the bioavailability of amino acids derived from proteins, and related food product methods and systems
CN112786113A (en) * 2019-11-07 2021-05-11 特安康股份有限公司 Method for establishing individualized probiotic database and application of method in screening probiotics
EP4070805A4 (en) * 2019-11-20 2024-02-21 Inner Mongolia Yili Ind Group New application of bifidobacterium lactis bl-99 in inhibiting intestinal inflammation
WO2022011902A1 (en) * 2020-07-17 2022-01-20 佛山市朗芯生物科技有限公司 Lactobacillus paracasei and application thereof in preparing medicine for treating ulcerative colitis
US11767503B2 (en) 2020-08-24 2023-09-26 BYHEALTH Co., Ltd. Bifidobacterium breve 207-1 and use thereof
KR102624867B1 (en) * 2020-08-24 2024-01-15 바이헬스 컴퍼니 리미티드 Bifidobacterium breve 207-1 and uses thereof
US20220333062A1 (en) * 2020-08-24 2022-10-20 BYHEALTH Co., Ltd. Bifidobacterium breve 207-1 and use thereof
KR20220047875A (en) * 2020-08-24 2022-04-19 바이헬스 컴퍼니 리미티드 Bifidobacterium brib 207-1 and uses thereof
EP4012019A4 (en) * 2020-08-24 2023-01-25 Byhealth Co., Ltd. Bifidobacterium breve 207-1 and use thereof

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