Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
• • 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
  • 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
  • A23L33/30—Dietetic or nutritional methods, e.g. for losing weight
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
  • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
  • A61K35/745—Bifidobacteria
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
  • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
  • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • 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
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• • 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
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
• • 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
  • C12N1/205—Bacterial isolates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/225—Lactobacillus
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/225—Lactobacillus
  • C12R2001/23—Lactobacillus acidophilus
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/225—Lactobacillus
  • C12R2001/24—Lactobacillus brevis

Definitions

• the present invention relates to novel bacteria and metabolites thereof, their use in probiotic compositions and food products, methods for selection of probiotic bacteria and methods of personalising a probiotic composition or food product.
• the invention also relates to the use of said bacteria, metabolites and compositions for the prevention and/or treatment of gastrointestinal disorders.
• the human body is colonised with a myriad of microbes representing over 1000 bacterial species.
• the composition and density of the microbiota is specific for each body location.
• the majority of the bacterial biomass resides in the gastrointestinal tract (GIT), especially in the lumen of the large intestine, where two populations are present, the lumen and mucosa-associated populations that differ from each other.
• GIT gastrointestinal tract
• the microbiota has an important role in human health. It contributes to the maturation of the gut tissue, to host nutrition, pathogen resistance, epithelial cell proliferation, host energy metabolism and immune response.
• IBD inflammatory bowel disease
• IBS irritable bowel syndrome
• rheumatoid arthritis atopic eczema
• asthma atopic eczema
• type 1 diabetes atopic eczema
• microbiota of adults is fairly stable over time and unique within an individual.
• the similarity of the dominant microbial population is higher in monozygotic twins compared to unrelated subject suggesting the role of host genetic factors on the microbiota composition.
• Some animal studies suggest that the major histocompatibility complex is involved in the genetic regulation of gut microbiota. However, little is known about which genes or other factors determine or regulate the spectrum of microbial composition.
• the mucosal layer covering the gut epithelium has an important role as the first layer of host defences, but it also enables contacts between intestinal microbiota and the host.
• the mucus is mainly composed of mucins, large glycoproteins containing a protein core and attached oligosaccharides.
• the mucus layer prevents the direct contact of the bacteria with the epithelial cells in the colon, it provides adhesion sites for the GIT bacteria and has thus an important role in bacterial colonization. Besides adhesion sites, the secreted mucus provides endogenous substrate for bacteria.
• the mucus may be a major nutrient source in situations, where carbohydrates originating elsewhere are limited.
• Blood group antigens are attached to various components in the red blood cell membrane, and the antigens expressed on the red blood cell determine an individual's blood group.
• the main two blood groups are called ABO (with blood types A, B, AB, and O) and Rh (with Rh D-positive or Rh D-negative blood types).
• ABO blood group antigens are expressed in the mucus of secretor type individuals (roughly 80% of Western population).
• the expression of the ABO antigens is site-specific. For example, in the GIT the expression of fucosylated glycans including ABO blood group antigens decreases towards the distal parts of the intestine.
• Some microbes such as Helicobacter pylori and some other pathogenic bacteria and viruses, have been shown to use ABO blood group antigens as adhesion receptors (Boren et al. 1993, Imberty and Varrot 2008). ABO antigen binding ability has been reported also for Lactobacillus spp., which tend to adhere in a strain-specific manner (Uchida et al. 2006). Bifidobacteria and Bacteroides thetaiotaomicron are, for example, also able to specifically utilize blood group antigens, e.g. the glycan structures of ABO antigens (Martens et al. 2008, Xiao et al. 2010). The ABO blood group status of an individual also has an effect on the relative proportions of the host microbiota (Mäkivuokko et al. 2012).
• the present invention is based on the inventors' surprising discovery that certain bacterial strains showed enhanced in vitro adhesion to one or more of the ABO blood group antigens. This finding enables the selection of ABO blood group-specific bacteria for use in probiotic compositions and for the treatment or prevention of gastrointestinal disorders.
• probiotic bacterium may also demonstrate enhanced health effects by blocking the adhesion and invasion of certain pathogenic microbes which also bind to ABO antigens.
• a probiotic bacterium which adheres to an ABO antigen could also enhance the probiotic responses by providing tighter and longer-lasting contact between the host cells and probiotics. It could further provide the host easier access to any beneficial metabolites produced by the probiotics when probiotics are colonized tighter and more long-lasting.
• probiotic bacteria are more resistant to acid and/or bile. These properties may be helpful to enable the bacterium or metabolite thereof to survive the conditions of the stomach and gastrointestinal tract, and are therefore advantageous properties for a probiotic bacterium. Acid tolerance may also be beneficial if the strain is fermented in a product such as yogurt.
• the bacterium of the invention may be a bacterial strain deposited as DSM 32111, DSM 32108, DSM 32107, DSM 32098, DSM 32104, DSM 32112, DSM 32109, DSM 32105, DSM 32110, DSM 32103, DSM 32099, DSM 32106, DSM 32097, DSM 32114, DSM 32115 or a mutant, a variant and/or a progeny thereof.
• a probiotic composition comprising a bacterium or metabolite thereof according to the invention and a suitable carrier.
• the probiotic composition of the invention may comprise a combination of 2, 3, 4, 5 or 6 bacterial strains or metabolites thereof according to the invention, optionally in combination with one or more further bacterial strains.
• the probiotic composition may further comprise a prebiotic component.
• the present invention also provides a method of producing a probiotic composition of the invention, the method comprising combining the selected bacterium or metabolite thereof with a suitable carrier.
• the invention further provides a food product comprising a probiotic composition according to the invention.
• a method of selecting a bacterium or metabolite thereof comprising:
• the admixtures are incubated in separate vials in step (b).
• the method is a fluorescence based method.
• bacteria are selected which have:
• the invention further provides a bacterium or metabolite thereof selected by the method of the invention.
• the invention provides the use of a bacterium or metabolite of the invention or selected by a method of the invention, or a probiotic composition of the invention, for the manufacture of a formulation for preventing and/or treating gastrointestinal disorders.
• the invention also provides a bacterium or metabolite of the invention or selected by a method of the invention, or a probiotic composition of the invention, for use in preventing and/or treating gastrointestinal disorders.
• the invention provides a method of preventing and/or treating gastrointestinal disorders comprising administering to a subject a bacterium or metabolite thereof according to the invention or selected by a method of the invention, or a probiotic composition of the invention, in a pharmaceutically effective amount.
• bacteria or “bacterial” is used herein to refer to any bacterial species, strains or combinations thereof, and is not limited to strains currently accepted as probiotics.
• bacterial strains used in the present invention are those that a suitable for human and/or animal consumption.
• a skilled person will be readily aware of specific species and or strains from within the genera described herein which are used in the food and/or agricultural industries and which are generally considered suitable for human and/or animal consumption.
• Such bacterial strains are typically non-pathogenic, and may be generally regarded as safe for human use (e.g. GRAS).
• bacteria is generally used to refer to whole bacteria, for example whole viable bacteria.
• Bacteria suitable for use in the present invention include, but are not limited to, Bifidobacterium, Brevibacterium, Propionibacterium, Lactococcus, Streptococcus, Lactobacillus, Enterococcus, Pediococcus, Leuconostoc and/or Oenococcus.
• the bacterium belongs to the genus Lactobacillus .
• suitable strains of Lactobacillus include L. acidophilus, L. amylovorus, L. brevis, L. casei, L. crispatus, L. fermentum, L. vaginalis, L. curvatis, L. de/brueckii bu/garicus, L. gasseri, L. helveticus, L. jensenii, L. mucosae, L. paracasei, L. plantarum, L. rhamnosus, L. silvarius and L. ruminis.
• the present invention provides the novel bacterial strains DGCC11884, DGCC11864, DGCC11860, DGCC11873, DGCC11852, DGCC11853, DGCC11865, DGCC1925, DGCC11866, DGCC5111, DGCC11854, DGCC11858, DGCC11887, DGCC11862 and DGCC11881.
• These strains have been deposited by DuPont Nutrition Biosciences ApS, Langebrogade 1, P.O.
• the invention further provides a mutant, a variant and/or a progeny of the deposited bacterial strains.
• mutant refers to any microorganism resulting from modification of the parent (i.e. deposited) strain.
• a mutant may be a microorganism resulting from genetically modifying a deposited strain.
• variant refers to a naturally occurring microorganism which is derived from the parent (i.e. deposited strain).
• a variant may be a microorganism resulting from adaption to particular cell culture conditions.
• progeny means any microorganism resulting from the reproduction or multiplication of any one of the deposited strains. Therefore, “progeny” means any direct descendant of any one of the deposited strains. As such, the progeny strain may itself be identified as the same strain as the parent (i.e. deposited) strain. It will be apparent to one skilled in the art that due to the process of asexual reproduction, a progeny strain will be genetically virtually identical to the parent strain. Accordingly, in one embodiment, the progeny may be genetically identical to the parent strain, and may be considered to be a “clone” of the parent strain. Alternatively, the progeny may be substantially genetically identical to the parent strain.
• the mutant, variant or progeny may have at least 90, 95, 98, 99, 99.5 or 99.9% sequence identity over the entire length of the bacterial genome with their parent strain. Furthermore, the mutant, variant or progeny will retain the same phenotype as the deposited parent strain, for example the mutant or variant may demonstrate the same or equivalent level of in vitro adhesion to A, B and/or O blood type antigen as the parent strain.
• the term “metabolite” refers to all molecules produced or modified by the bacteria as a result of bacterial metabolism during growth, survival, persistence, transit or existence of bacteria during probiotic product manufacture and storage and during gastrointestinal transit in a mammal.
• examples include all organic acids, inorganic acids, bases, proteins and peptides, enzymes and co-enzymes, amino acids and nucleic acids, carbohydrates, lipids, glycoproteins, lipoproteins, glycolipids, vitamins, all bioactive compounds, metabolites containing an inorganic component, and all small molecules, for example nitrous molecules or molecules containing a sulphurous acid.
• a metabolite or metabolites are typically obtained from the supernatant of a cell culture from which the bacterial cells have been removed.
• the cells may be grown in MRS medium under anaerobic conditions for 6-24 hours at 37° C.
• the bacterial cell culture may be grown to a cell density of at least about OD 600 0.5, 1.0, 1.5, 2.0, 2.5, or 3.0, for example from 1.5 to 2.5 OD 600 .
• the cells may suitably be removed by centrifugation or by filtration. It will be apparent that the supernatant may be used directly in the formulations of the present invention, or that one or more of the metabolites may be isolated from the supernatant by any suitable means prior to use.
• Suitable metabolites for use in the present invention include, but are not limited to, metabolites from any of the above mentioned bacteria.
• the bacterium or metabolite thereof is characterised by its adhesion to a particular ABO blood type antigen, with reference to a control strain.
• the adhesion to the antigen may be measured by mixing the bacterium or metabolite thereof with the antigen, and measuring adhesion according to any suitable test or assay.
• the method is a fluorescence-based method.
• the blood group antigen may be biotinylated and combined with a sample of bacteria to be tested, then transferred to streptavidin coated plates and the attached bacteria detected with a suitable dye, such as a fluorescent dye.
• Antigen may be bound to any immobilized matrix for affinity binding assays and the binding of bacteria to it could be detected using suitable antibodies, fluorometric or colorimetric stains, or by labelling the bacteria with any labelling technique such as radioactive labelling. It could be done the other way around, by attaching bacterial components to immobilized matrix, and by investigating if the blood group antigens can bind.
• the antigen adhesion is compared to Lactobacillus crispatus LMG18199 (available from BCCM/LMG collection). According to another aspect of the invention, the antigen adhesion is compared to Lactobacillus reuteri RC-14 (available from Christian Hansen A/S, Denmark).
• the bacterium or metabolite thereof is selected on the basis of its resistance to bile and/or acid. These properties may be helpful to enable the bacterium or metabolite thereof to survive the conditions of the stomach and gastrointestinal tract, and are therefore advantageous properties for a probiotic bacterium.
• the bile resistance assay comprises incubating strain cultures in culture medium (e.g. MRS) containing a defined amount of dehydrated fresh bile (e.g. oxgall, available under the brand name DifcoTM from BD), such as 0.9% or 0.3% at 37° C. for 24 hours in anaerobic conditions. Growth is measured before and after incubation and the bile tolerance results are expressed as % growth (OD) with bile in comparison to growth without bile.
• MRS culture medium
• oxgall available under the brand name DifcoTM from BD
• the acid tolerance assay comprises incubating bacterial cells in suitable medium (e.g. PBS) at neutral pH (i.e. pH 7.2) levels and at a comparator acidic pH (such as pH 2.5 or pH 3.5) for at least 90 min at 37° C.
• suitable medium e.g. PBS
• neutral pH i.e. pH 7.2
• comparator acidic pH such as pH 2.5 or pH 3.5
• the ten-fold dilution series are grown on MRS agar, and colonies are counted after 48 h incubation in anaerobic conditions at 37° C.
• the results for acid tolerance are expressed as growth log reduction of CFU after exposure to acid conditions in comparison to neutral pH.
• the bacterium or metabolite thereof is characterised by both its adhesion to a particular ABO blood type antigen, with reference to a control strain, and by its acid and/or bile tolerance. Accordingly, the invention provides a bacterium or metabolite thereof characterised by:
• the invention further provides a probiotic composition comprising one or more bacterial strains that have been selected according to methods of the invention.
• the composition may comprise a single strain, or a combination of 2, 3, 4, 5, 6, 7, 8, 9 or 10 strains of the invention.
• the probiotic composition may further comprise additional strains, for example commercially available probiotic bacterial strains.
• the probiotic composition of the invention further comprises one or more further bacterial strains.
• the further micro-organism may be a bacterium from one or more of the following genera: Lactococcus, Streptococcus, Pediococcus, Enterococcus, Leuconostoc, Carnobacterium, Propionibacterium, Bifidobacterium, Lactobacillus, Brevibacterium , and Vagococcus .
• the at least one further probiotic microorganism is selected from the genera Lactobacillus, Streptococcus, Enterococcus, Bifidobacterium and Saccharomyces.
• the further probiotic microorganism is a bacterium preferably a probiotic lactic acid bacterium and/or a probiotic Bifidobacterium .
• the further microorganism is from the genus Lactobacillus or the genus Bifidobacterium or is a mixture thereof.
• the microorganism may be a strain from the species L. acidophilus, L. curvatus, L. rhamnosus, L. casei, L. paracasei, L. salivarius, B. lactis. B animalis, B. longum and/or B. bifidum .
• the microorganism may be a strain from the species L. acidophilus, L. curvatus, L. salivarius and/or B. lactis.
• the bifidobacterium may be any bifidobacterium having a probiotic effect, typically strains belonging to the species Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium bifidum and Bifidobacterium adolescentis are used.
• the Lactobacillus bacterium may be any of the following: Lactobacillus acidophilus, Lactobacillis amylovorus. Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus lactis, Lactobacillus paracasei, Lactobacillus pentosaceus, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus , and Lactobacillus salivarius,
• the probiotic composition of the invention further comprises one or more, such as a combination of 2, 3, 4 or 5 of the following commercially available strains: Lactobacillus acidophilus NCFM; Bifidobacterium lactis BL-04; Lactobacillus paracasei LPC37; Bifidobacterium lactis HNO19, and/or Bifidobacterium lactis Bi-07.
• probiotic refers to a live microorganism which, when administered in adequate amounts, confers a health benefit on the recipient. They are suitable for human consumption and therefore are non-pathogenic and non-toxic. These probiotic strains generally have the ability to survive the passage through the upper part of the digestive tract. Probiotic bacteria typically exercise their beneficial effect on health on the one hand via ecological interactions with the resident flora in the digestive tract, and on the other hand via their ability to influence the immune system in a positive manner via the “GALT” (gut-associated lymphoid tissue). Probiotic bacteria, when given in a sufficient number, have the ability to progress live through the intestine.
• GALT gut-associated lymphoid tissue
• the probiotic bacteria used in accordance with the present invention may comprise from 10 6 to 10 12 CFU of bacteria/g of support, and more particularly from 10 8 to 10 12 CFU of bacteria/g of support, typically 10 9 to 10 12 CFU/g for the lyophilized form.
• the bacterium may be administered at a dosage of from about 10 6 to about 10 12 CFU of microorganism/dose, typically about 10 8 to about 10 12 CFU of microorganism/dose.
• per dose it is meant that this amount of microorganism is provided to a subject either per day or per intake, typically per day.
• the microorganism is to be administered in a food product (for example in a yoghurt)—then the yoghurt will typically contain from about 10 8 to 10 12 CFU of the microorganism.
• this amount of microorganism may be split into multiple administrations each consisting of a smaller amount of microbial loading—so long as the overall amount of microorganism received by the subject in any specific time (for instance each 24 hour period) is from about 10 6 to about 10 12 CFU of microorganism, such as about 10 8 to about 10 12 CFU of microorganism.
• an effective amount of at least one strain of a microorganism may be at least 10 6 CFU of microorganism/dose, for example from about 10 6 to about 10 12 CFU of microorganism/dose, such as about 10 8 to about 10 12 CFU of microorganism/dose.
• CFU stands for “colony-forming units”.
• support is meant the food product, dietary supplement or the pharmaceutically acceptable support or carrier.
• bacterium or metabolite alone according to the present invention (i.e. without any support, diluent or excipient)
• they are typically administered on or in a carrier or support as part of a product, in particular as a component of a food product, a dietary supplement or a pharmaceutical formulation.
• a carrier or support as part of a product, in particular as a component of a food product, a dietary supplement or a pharmaceutical formulation.
• these products typically contain additional components well known to those skilled in the art.
• compositions which can benefit from the composition may be used in the present invention.
• These include but are not limited to foods, particularly fruit conserves and dairy foods and dairy food-derived products, and pharmaceutical products.
• the bacterial composition of the present invention may be formulated as a nutritional supplement.
• the bacterial composition may be in the form of, for example, a capsule, tablet, powder or emulsion.
• a typical probiotic ingredient is a freeze-dried powder containing, for example, 10 10 -10 12 viable probiotic bacterial cells per gram.
• the powder may further comprise a suitable carrier, such as skim milk or sugars, typically oligosaccharides such as sucrose or trehalose.
• the bacterial composition may be encapsulated using a carrier such as alginate, starch or xanthan.
• a carrier such as alginate, starch or xanthan.
• a typical capsule preparation may contain approximately 10°-10 11 viable probiotic bacterial cells per capsule.
• the probiotic composition of the present invention may additionally contain one or more prebiotics.
• prebiotic component refers to any compound, nutrient or additional microorganism used to support or enhance a desired probiotic health effect or to assist the growth and/or activity of probiotic bacteria.
• prebiotics are carbohydrates (such as oligosaccharides), but the definition does not preclude non-carbohydrates.
• the most prevalent forms of prebiotics are nutritionally classed as soluble fibre. To some extent, many forms of dietary fibre exhibit some level of prebiotic effect.
• a prebiotic is a selectively fermented ingredient that allows specific changes, both in the composition and/or activity in the gastrointestinal microflora that confers benefits upon host well-being and health.
• the prebiotic may be used according to the present invention in an amount of 0.01 to 100 g/day, such as 0.1 to 50 g/day, or 0.5 to 20 g/day. In one embodiment, the prebiotic may be used according to the present invention in an amount of 1 to 10 g/day, such as 2 to 9 g/day, or 3 to 8 g/day. In another embodiment, the prebiotic may be used according to the present invention in an amount of 5 to 50 g/day, such as 10 to 25 g/day.
• dietary sources of prebiotics include soybeans, inulin sources (such as Jerusalem artichoke, jicama, and chicory root), raw oats, unrefined wheat, unrefined barley and yacon.
• prebiotics examples include alginate, xanthan, pectin, locust bean gum (LBG), inulin, guar gum, galacto-oligosaccharide (GOS), fructo-oligosaccharide (FOS), polydextrose (i.e.
• lactitol lactosucrose
• soybean oligosaccharides isomaltulose (PalatinoseTM), isomalto-oligosaccharides, gluco-oligosaccharides, xylo-oligosaccharides, manno-oligosaccharides, beta-glucans, cellobiose, raffinose, gentiobiose, melibiose, xylobiose, cyclodextrins, isomaltose, trehalose, stachyose, panose, pullulan, verbascose, galactomannans, and all forms of resistant starches.
• a particularly preferred example of a prebiotic is polydextrose.
• the probiotic composition of the present invention may be used as, or in the preparation of, a food product.
• a food product is used in a broad sense and covers food for humans as well as food for animals (i.e. a feed). In one aspect, the food is for human consumption.
• the food may be in the form of a solution or as a solid—depending on the use and/or the mode of application and/or the mode of administration.
• a typical probiotic food product may contain approximately 10 9 -10 11 viable probiotic bacterial cells per daily dose.
• the probiotic bacteria may be incorporated in the food product as a probiotic ingredient, such as a freeze-dried powder, or may be cultured in the product.
• composition of the present invention may be used in conjunction with one or more of: a nutritionally acceptable carrier, a nutritionally acceptable diluent, a nutritionally acceptable excipient, a nutritionally acceptable adjuvant, and a nutritionally active ingredient.
• the probiotic composition of the present invention can be used as an ingredient in soft drinks, a fruit juice or a beverage comprising whey protein, health teas, cocoa drinks, milk drinks and lactic acid bacteria drinks, yoghurt and drinking yoghurt, cheese, ice cream, water ices and desserts, confectionery, biscuits cakes and cake mixes, snack foods, balanced foods and drinks, fruit fillings, care glaze, chocolate bakery filling, cheese cake flavoured filling, fruit flavoured cake filling, cake and doughnut icing, instant bakery filling creams, fillings for cookies, ready-to-use bakery filling, reduced calorie filling, adult nutritional beverage, acidified soy/juice beverage, aseptic/retorted chocolate drink, bar mixes, beverage powders, calcium fortified soy/plain and chocolate milk, calcium fortified coffee beverage.
• the probiotic composition can further be used as an ingredient in food products such as American cheese sauce, anti-caking agent for grated & shredded cheese, chip dip, cream cheese, dry blended whip topping fat free sour cream, freeze/thaw dairy whipping cream, freeze/thaw stable whipped tipping, low fat and light natural cheddar cheese, low fat Swiss style yoghurt, aerated frozen desserts, hard pack ice cream, label friendly, improved economics & indulgence of hard pack ice cream, low fat ice cream: soft serve, barbecue sauce, cheese dip sauce, cottage cheese dressing, dry mix Alfredo sauce, mix cheese sauce, dry mix tomato sauce and others.
• food products such as American cheese sauce, anti-caking agent for grated & shredded cheese, chip dip, cream cheese, dry blended whip topping fat free sour cream, freeze/thaw dairy whipping cream, freeze/thaw stable whipped tipping, low fat and light natural cheddar cheese, low fat Swiss style yoghurt, aerated frozen desserts, hard pack ice cream, label friendly, improved
• dairy product as used herein is meant to include a medium comprising milk of animal and/or vegetable origin.
• milk of animal origin there can be mentioned cow's, sheep's, goat's or buffalo's milk.
• milk of vegetable origin there can be mentioned any fermentable substance of vegetable origin which can be used according to the invention, in particular originating from soybeans, rice or cereals.
• the food product employed according to the invention is a fermented milk or humanized milk.
• the present invention may be used in connection with yoghurt production, such as fermented yoghurt drink, yoghurt, drinking yoghurt, cheese, fermented cream, milk based desserts and others.
• yoghurt production such as fermented yoghurt drink, yoghurt, drinking yoghurt, cheese, fermented cream, milk based desserts and others.
• composition can be further used as an ingredient in one or more of cheese applications, meat applications, or applications comprising protective cultures.
• the present invention also provides a method of preparing a food or a food ingredient, the method comprising admixing the composition according to the present invention with another food ingredient.
• the present invention relates to products that have been contacted with the probiotic composition of the present invention (and optionally with other components/ingredients), wherein the composition is used in an amount to be capable of improving the nutrition and/or health benefits of the product.
• the term “contacted” refers to the indirect or direct application of the composition of the present invention to the product.
• the application methods include, but are not limited to, treating the product in a material comprising the composition, direct application by mixing the composition with the product, spraying the composition onto the product surface or dipping the product into a preparation of the composition.
• the composition of the present invention is typically admixed with the product.
• the composition may be included in the emulsion or raw ingredients of a foodstuff.
• the composition may be applied as a seasoning, glaze, colorant mixture, and the like.
• composition is made available on or to the surface of a product to be affected/treated. This allows the composition to impart one or more of the following favourable characteristics: nutrition and/or health benefits.
• compositions of the present invention may be applied to intersperse, coat and/or impregnate a product with a controlled amount of a microorganism.
• the composition is used to ferment milk or sucrose fortified milk or lactic media with sucrose and/or maltose where the resulting media containing all components of the composition—i.e. said microorganism according to the present invention—can be added as an ingredient to yoghurt milk in suitable concentrations—such as for example in concentrations in the final product which offer a daily dose of 10 6 -10 10 cfu.
• the microorganism according to the present invention may be used before or after fermentation of the yoghurt.
• the probiotic bacteria should remain effective through the normal “sell-by” or “expiration” date during which the food product is offered for sale by the retailer.
• the effective time may usefully extend past such dates until the end of the normal freshness period when food spoilage becomes apparent.
• the desired lengths of time and normal shelf life will vary from foodstuff to foodstuff and those of ordinary skill in the art will recognise that shelf-life times will vary upon the type of foodstuff, the size of the foodstuff, storage temperatures, processing conditions, packaging material and packaging equipment.
• the bacterial composition of the present invention may be used as, or may be added to, a functional food.
• a functional food means food which is capable of providing not only a nutritional effect and/or a taste, but is also capable of delivering a further beneficial effect to consumer, such as an additional health benefit.
• functional foods are ordinary foods that have components or ingredients (such as those described herein) incorporated into them that impart to the food a specific functional benefit, such as a medical or physiological benefit, other than a purely nutritional effect.
• nutraceuticals Some functional foods are nutraceuticals.
• the term “nutraceutical” means a food which is capable of providing not only a nutritional effect and/or a taste satisfaction, but is also capable of delivering a therapeutic (or other beneficial) effect to the consumer. Nutraceuticals cross the traditional dividing lines between foods and medicine.
• the present invention relates to the use of a bacterium or metabolite thereof which is demonstrates binding to at least one ABO blood type antigen, or a probiotic composition comprising one or more such bacteria, for the prevention and/or treatment of gastrointestinal disorders.
• gastrointestinal disorder includes any disease or disorder relating to the gastrointestinal tract.
• the disorder may, for example, be one that is known to be associated with an altered composition and diversity of the GIT microbiota.
• the gastrointestinal disorder may be inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), Crohn's disease, ulcerative colitis, constipation or diarrhoea.
• IBD inflammatory bowel disease
• IBS irritable bowel syndrome
• Crohn's disease Crohn's disease
• ulcerative colitis constipation or diarrhoea.
• composition of the present invention may be used in conjunction with one or more of: a pharmaceutically acceptable carrier, a pharmaceutically acceptable diluent, a pharmaceutically acceptable excipient, a pharmaceutically acceptable adjuvant, a pharmaceutically active ingredient.
• a pharmaceutically acceptable support may be for example a support in the form of compressed tablets, tablets, capsules, ointments, suppositories or drinkable solutions. Other suitable forms are provided below.
• the pharmaceutical may be in the form of a solution or as a solid—depending on the use and/or the mode of application and/or the mode of administration.
• the bacterium or metabolite thereof or probiotic composition may be used according to the present invention in any suitable form—whether when alone or when present in a combination with other components or ingredients.
• the bacteria used in the present invention may be referred to herein as “the composition”.
• combinations comprising the composition of the present invention and other components and/or ingredients i.e. ingredients—such as food ingredients, functional food ingredients or pharmaceutical ingredients) may be used in any suitable form.
• the bacterium or metabolite thereof or probiotic composition may be used according to the present invention in the form of solid or liquid preparations or alternatives thereof.
• solid preparations include, but are not limited to tablets, capsules, dusts, granules and powders which may be wettable, spray-dried or freeze-dried.
• liquid preparations include, but are not limited to, aqueous, organic or aqueous-organic solutions, suspensions and emulsions.
• Suitable examples of forms include one or more of: tablets, pills, capsules, ovules, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed-, modified-, sustained-, pulsed- or controlled-release applications.
• the tablets may also contain one or more of: excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine; disintegrants such as starch (such as corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates; granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia; lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.
• excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine
• disintegrants such as starch (such as corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicate
• Examples of nutritionally acceptable carriers for use in preparing the forms include, for example, water, salt solutions, alcohol, silicone, waxes, petroleum jelly, vegetable oils, polyethylene glycols, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.
• Suitable excipients for the forms include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols.
• composition of the present invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, propylene glycol and glycerin, and combinations thereof.
• the forms may also include gelatin capsules, fibre capsules, fibre tablets or fibre beverages.
• the formulation should ideally be able to remain stable during transit though the gastrointestinal tract; for example, it may be resistant to proteolytic degradation, stable at acid pH and resistant to the detergent effects of bile.
• Acid tolerance of the strains was tested in pH 2.5 and pH 3.5 for 1.5 h and bile tolerance in 0.9% and 0.3% Oxgall (Difco) bile concentrations for 24 h (Saarela et al. 2009). Briefly, the strains were cultivated in duplicate in MRS broth in anaerobic conditions at 37° C. for 18 h. The pelleted cells were washed twice with 10 ml PBS pH 7.2 and resuspended in 0.01 mol/L PBS pH 7.2 so that the optical density (OD 600 ) of the sample was 1 (equals to 1 ⁇ 10 8 CFU/ml). Acid tolerance was tested by incubating cells in PBS pH 7.2.
• bile tolerance results were expressed as % growth (OD) in MRS with 0.9% or 0.3% Oxgall in comparison to growth (OD) in MRS without bile. Additionally, acid and bile tolerance of the strains was compared with that of Lactobacillus rhamnosus LGG (VTT E-96666) strain. All the measurements were performed in duplicates and repeated twice for most of the strains.
• the ABH adhesion capability was tested for all strains (H antigen corresponding to blood group 0 phenotype).
• the tested blood group antigens A, B and H were suspended in PBS pH7.2 and biotinylated.
• the stains were cultivated in anaerobic conditions in MRS plates at 37° C. for 48-72 h.
• a single colony was re-inoculated to 10 ml MRS broth and cultivated overnight in anaerobic conditions at 37° C.
• the cells were washed twice with 10 ml PBS pH 7.2 and resuspended in PBS pH 7.2, so that the OD (600 nm) of the sample was set to 1 (equals to 1 ⁇ 10 8 cfu/ml).
• the 1 ml sample and 1 ml 10 ⁇ g/ml biotinylated antigen solution were mixed together and incubated in slow shaking for 30 min at room temperature, and 100 ⁇ l of the mixture transferred in to the Delfia Streptavidin coated 96-well plates (Wallac), which were washed twice with 200 ⁇ l PBS pH7.2, followed by wash with SuperBlock (Pierce) three times (2 ⁇ 200 ⁇ l+1 ⁇ 100 ⁇ l) and once with 200 ⁇ l sterile water.
• streptavidin—biotin interaction the plates were incubated for 30 min at room temperature on slow agitation before washing of the well three times with 200 ⁇ l sterile water. Between the washes the plate was incubated for 5 min.
• Syto9 dye diluted 1:6) (Invitrogen) was added to the well and incubated for 15 min in dark.
• the intensity of Syto-9 stain in well was measured with Wallac Viktor 2 1420 multilabel counter (Perkin Elmer). All the measurements were done in four replicates and the results were repeated at least twice.
• the adhesion of the strains was compared to Lactobacillus reuteri RC-14 (available from Christian Hansen A/S, Denmark).
• Acid Acid tolerance tolerance Other strain log log Average acid DGCC code/ change in change in tolerance, log Deposit strain commercial pH 2.5 for pH 2.5 for change in pH number(s) number ID Identification 1.5 h 1.5 h 2.5 for 1.5 h DSM 32107 11860 LX11860 L. brevis ⁇ 5.2 ⁇ 5.2 DSM 32363 4022 LA0893 L. acidophilus ⁇ 5.7 ⁇ 4.3 ⁇ 5.0 PTA-4800 9868 LS-33 L. salivarius ⁇ 4.6 ⁇ 4.8 ⁇ 4.7 DSM 32109 11865 LX11865 L. fermentum ⁇ 4.3 ⁇ 5.1 ⁇ 4.7 DSM 32112 11853 LX11853 L.
• strains were selected according to their suitability for use in a probiotic composition. Table 11 below shows those strains that performed better than control strains based on A, B and O antigen adhesion, and which strains showed enhanced bile and/or acid tolerance. A, B and/or O adhesion of five strains (DSM 32104, DSM 32112, DSM 32109, DSM 32110 and DSM 32106) was equal or better than the adhesion of control strain L. crispatus LMG18199, which adheres well to the A antigen (Uchida et al. 2006).
• Rats Male rats weighing approximately 200-220 g are surgically prepared for electromyography of the abdominal muscles in order to measure the pain response to colonic distension later on. Rats are then gavaged with each probiotic (daily dose of 10 7 , 10 8 and/or 10 9 CFU) or saline for 7-21 days before a colorectal distension test. Rats are accustomed to polypropylene tunnels 3-5 days before the distension test. The balloon used for distention is taken from an embolectomy probe and is inserted into the rectum at 1 cm from the anus and fixed at the basis of the tail. Increasing levels of distensions are then induced with the probe connected to a barostat. A functional ingredient desensitizes the colonic wall and leads to an increased pain threshold or decreased contraction of the abdominal muscles. Contractions corresponding to each pressure level are measured and compared between groups.
• TNBS trinitrobenzene sulfonic acid
• Fresh tissue specimens are visually scored by an experienced scientist to determine the level of inflammation.
• RNA is extracted and converted to cDNA with a commercially available kit.
• inflammation markers such as IL-6, TNF-alpha, IL-1beta, matrix metalloproteinases (MMPs) and their tissue inhibitors, in colonic tissue is analyzed with RT-qPCR.
• Histological samples are cut from tissues embedded in paraffin, and are visualized under a microscope to enumerate infiltrated immune cells.
• Inflammation markers such as IL-6, TNF-alpha, IL-1beta, hsCRP are then determined with commercially available ELISA kits.
• Lipopolysaccharides are analyzed from serum samples using a standard kit based on a Limulus amebocyte extract (the LAL kit), which is commercially available.
• Gut microbiota is analyzed from feces or caecum by extracting DNA. Samples then undergo gut microbial sequencing. Results are analyzed by a trained bioinformatician.

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