Compostions and method of use for h5 competent bifidobacterium longum subsp. infantis

Abstract

Bifidobacterium longum subsp. infantis comprising a functional H5 cluster, including the Bifidobacterium longum subsp. infantis EVC001 deposited under ATCC Accession No. PTA-125180 may be used in compositions for improving gut health in infants and adults.

1 . A composition comprising a  Bifidobacterium longum  subsp.  infantis  comprising a functional H5 gene cluster and at least one oligosaccharide having a Type I or Type II core. 
     
     
         2 . The composition of  claim 1 , wherein the  Bifidobacterium longum  subsp.  infantis  is  Bifidobacterium longum  subsp.  infantis  EVC001 deposited under ATCC Accession No. PTA-125180. 
     
     
         3 - 4 . (canceled) 
     
     
         5 . The composition of  claim 1 , wherein the  Bifidobacterium longum  subsp.  infantis  is activated; or
 wherein the  Bifidobacterium longum  subsp.  infantis  is activated by an activator, wherein the activator from Table 4; or   wherein the  Bifidobacterium longum  subsp.  infantis  contains a LNB transport system capable of internalizing one or more oligosaccharide having a Type I or Type II core before the oligosaccharide is hydrolyzed, and is capable of hydrolyzing the internalized oligosaccharide; or   wherein the  Bifidobacterium longum  subsp.  infantis  has been cultured in presence of at least one mammalian milk oligosaccharide; or   wherein the  Bifidobacterium longum  subsp.  infantis  has a higher binding affinity to mammalian mucosal cells than  Bifidobacteria  of the same species cultivated in the absence of complex oligosaccharides.   
     
     
         6 . (canceled) 
     
     
         7 . The composition of  claim 1 , wherein the composition further comprises an activator selected from Table 4; or
 wherein the composition further comprises galactooligosaccharide (GOS); or   wherein the composition further comprises a protein source rich in threonine, N-acetyl-threonine, gamma-glutamylthreonine, or a combination thereof; or   wherein the composition further comprises a secondary metabolite, and
 wherein the secondary metabolite comprises a short chain fatty acid, or 
 wherein the secondary metabolite comprises acetate, lactate, or combinations thereof. 
   
     
     
         8 - 11 . (canceled) 
     
     
         12 . The composition of  claim 1 , wherein the functional H5 cluster comprises Blon_2175, Blon_2176, and Blon_2177; or
 wherein the functional H5 cluster comprises Blon_2171, Blon_2173, Blon_2174, Blon_2175, Blon_2176, Blon_2177, and galT, or   wherein the  Bifidobacterium longum  subsp.  infantis  comprises upregulated genes selected from the group consisting of Blon_0042, Blon_R0015, Blon_R0017, Blon_R0021, Blon_R0022, and combinations thereof, or   wherein the  Bifidobacterium longum  subsp.  infantis  comprises downregulated genes selected from the group consisting of Blon_0518, Blon_0785, Blon_2167, Blon_2168 and combinations thereof; or   wherein the  Bifidobacterium longum  subsp.  infantis  comprises upregulated genes selected from the group consisting of Blon_0879, Blon_0880, Blon_0881, Blon_0882, Blon_2177, Blon_2334, Blon_2335, Blon_2336, Blon_2337, Blon_2338, Blon_2339, Blon_2343, Blon_2344, Blon_2346, Blon_2347, and Blon_2331; or   wherein the  Bifidobacterium longum  subsp.  infantis  expresses a gene coding for a sialidase or a fucosidase; or   wherein the  Bifidobacterium longum  subsp.  infantis  expresses a gene coding for a sialic acid or a fucose transporter.   
     
     
         13 - 18 . (canceled) 
     
     
         19 . The composition of  claim 1 , wherein the  Bifidobacterium longum  subsp.  infantis  is present at a concentration of from 1 Million cfu/g to 500 Billion cfu/g; or
 wherein the  Bifidobacterium longum  subsp.  infantis  is present at a concentration of from 5 Billion cfu/g to 100 Billion cfu/g; or   wherein the  Bifidobacterium longum  subsp.  infantis  is present at a concentration of from 10 Billion cfu/g to 50 Billion cfu/g or from 50 million cfu/g to 5 billion cfu/g.   
     
     
         20 - 21 . (canceled) 
     
     
         22 . The composition of  claim 1 , wherein the oligosaccharide can be obtained from mammalian milk selected from human, bovine, pig, rabbit, goat, sheep, camel milk, or mixtures thereof; or
 wherein at least one of the oligosaccharide has a Type I core; or   wherein at least one of the oligosaccharide has a Type II core; or   wherein the composition further comprises an isolated complex oligosaccharide, and
 wherein the complex oligosaccharide is isolated from a mammalian milk source; and
 wherein the mammalian milk source is human, bovine, pigs, rabbits, goats, sheep, or camel; or 
 
 wherein the complex oligosaccharide is a mammalian milk oligosaccharide (MMO), and
 wherein the mammalian milk oligosaccharide (MMO) comprises lacto-N-biose, lacto-N-triose, N-acetyllactosamime, lacto-N-neotriose, lacto-N-tetraose, lacto-N-neotetraose, fucosyllactose, lacto-N-fucopentose, lactodifucotetraose, sialyllactose, disialyllactone-N-tetraose, 2′-fucosyllactose, 3′-sialyllactoseamin, 3′ fucosyllactose, 3′-sialyl-3-fucosyllactose, 3′-sialyllactose, 6′-sialyllactosamine, 6′-sialyllactose, difucosyllactose, lacto-N-fucosylpentose I, lacto-N-fucosylpentose II, lacto-N-fucosylpentose III, lacto-N-fucosylpentose V, sialyllacto-N-tetraose, their derivatives, or combinations thereof, or 
 wherein the mammalian milk oligosaccharide (MMO) comprises oligosaccharide molecules found in human milk oligosaccharides (HMO), bovine milk oligosaccharides (BMO), bovine colostrum oligosaccharides (BCO), goat milk oligosaccharides (GMO), or a combination thereof, and
 wherein the bovine source is from bovine milk, bovine colostrum, bovine colostrum concentrate, or mixtures thereof, or 
 
 
 wherein the bovine colostrum oligosaccharide comprises any of Hex(4); Hex(4) HexNAc(2); or Hex(3) HexNAc(1) NeuAc(1) at levels greater than 1%; or 
 wherein the complex oligosaccharide is from whey permeate; or 
 wherein the complex oligosaccharide comprises at least one of (3Hex,4HexNac,1Fuc), (1Gal,1GlcNAc,1NeuAc), or (1Glu,1Gal,1NeuAc (3′ or 6′)); or 
 wherein the complex oligosaccharide is less than 50% fucosylated; or 
 wherein the complex oligosaccharide comprises one of the following ratios of constituents: 1) a ratio of Hex(2) NeuAc(1):Hex(2) HexNAc(1) less than 5.0; 
 2) a ratio of Hex(2) HexNAc(1):Hex (3) HexNAc(1) of greater than 1.0; 
 3) a ratio of Hex(2) HexNAc(1):Hex (3) HexNAc(2) of greater than 2.0; 
 4) a ratio of Hex(3):Hex (3) HexNAc(1) NeuAc(1) of less than 100; or 
 5) a ratio of Hex(2) HexNAc(1):Hex (4) NeuAc(2) NeuGc(1) of greater than 10; or 
 wherein the complex oligosaccharide comprises a plurality of oligosaccharides with 3 to 10 residues (DP3-10 oligosaccharides); or 
 wherein the complex oligosaccharide comprises a plant-derived oligosaccharide, and 
 wherein the plant-derived oligosaccharide is from carrots, peas, broccoli, onions, tomatoes, peppers, rice, wheat, oats, bran, oranges, cocoa, olives, apples, grapes, sugar beets, cabbage, corn, or a mixture thereof, or
 wherein the plant-derived oligosaccharide is from orange peels, cocoa hulls, olive pomace, tomato skins, grape pomace, corn silage, or a mixture thereof, or 
 wherein the plant-derived oligosaccharides are between 2 and 10 sugar residues (DP2-DP10), between 3 and 10 sugar residues (DP3-DP10), between 5 and 10 sugar residues (DP5-DP10), or up to DP20. 
 
   
     
     
         23 - 33 . (canceled) 
     
     
         34 . The composition of  claim 1 , wherein the complex oligosaccharide is at least 20% of the weight of the composition; or
 wherein the complex oligosaccharide is at least 50% of the weight of the composition; or   wherein the complex oligosaccharide is at least 80% of the weight of the composition.   
     
     
         35 - 36 . (canceled) 
     
     
         37 . The composition of  claim 1 , wherein fucosyllactose and/or sialyllactose comprises 1-5% of the total oligosaccharides; or
 wherein fucosyllactose and/or sialyllactose comprises 5-20% of total oligosaccharides; or   wherein fucosyllactose and/or sialyllactose comprises 20-50% of total oligosaccharides; or   preferably wherein mass ratio of complex oligosaccharide to fucosyllactose and/or sialyllactose is from 20:1 to 1:10.   
     
     
         38 - 47 . (canceled) 
     
     
         48 . The composition of  claim 1 , wherein the composition provides a total dietary intake of oligosaccharide in an amount of 0.001-100 grams per day; or
 wherein the oligosaccharide is in an amount of 1-20 grams, 3-20 grams, or 5-10 grams; or   wherein the oligosaccharide is in an amount of 10, 15, 20, 25, 30, 35, 40, 45, or 50 grams.   
     
     
         49 - 57 . (canceled) 
     
     
         58 . The composition of  claim 1 , wherein the composition is in the form of a dry powder, a dry powder suspended in an oil, or as a solution, and
 wherein the dry powder is spray dried or freeze-dried; or   wherein the composition is freeze-dried in the presence of a suitable cryoprotectant; or   wherein the composition further comprises a cryoprotectant, and   wherein the cryoprotectant is glucose, lactose, raffmose, sucrose, trehalose, adonitol, glycerol, mannitol, methanol, polyethylene glycol, propylene glycol, ribitol, alginate, bovine serum albumin, carnitine, citrate, cysteine, dextran, dimethyl sulphoxide, sodium glutamate, glycine betaine, glycogen, hypotaurine, peptone, polyvinyl pyrrolidone, or taurine, mammalian milk oligosaccharides, chitin, chitosan, other polysaccharides, or a combination thereof; or   wherein the composition further comprising a stabilizer, and   wherein the stabilizer is a flow agent, or   wherein the stabilizer is a milk protein; or   wherein the composition is a powder with a water activity level of less than 0.35, less than 0.30, less than 0.25, less than 0.2, less than or less than 0.1; or   wherein the composition is an anhydrous composition; or   wherein the composition is suspended in an oil; or   wherein the composition is in the form of a dry powder suspending in an oil, and   wherein the oil is a medium chain triglyceride; or   wherein the composition is suspended in syrup having oligosaccharide at least 57%, wherein water activity is low enough to keep  Bifidobacterium longum  subsp.  infantis  dormant; or   wherein the composition is in form of a packet, sachet, orally disintegrating tablet, foodstuff, capsule, lozenge, effervescent tablet, suppository, enema, capsule, dry powder, dry powder suspended in an oil, chewable composition, syrup, or gel, and   wherein the capsule or tablet has an enteric coating, and   wherein the enteric coating comprises one or more of fatty acids, waxes, shellac, plastics, plant fibers, methyl acrylate-methacrylic acid copolymers, cellulose acetate succinate, hydroxy propyl methyl cellulose phthalate, hydroxyl propyl methyl cellulose acetate succinate, polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymers, cellulose acetate trimellitate, sodium alginate, and zein; or   wherein the composition is a pharmaceutical composition, dietary supplement, nutritional product, food product, probiotic, and/or prebiotic; or   wherein the composition is formulated as a unit dose medicament.   
     
     
         59 - 77 . (canceled) 
     
     
         78 . A method of increasing concentration of  Bifidobacterium  in gastrointestinal tract of a mammal by administering an effective amount of the composition of  claim 1  to a mammal in order to increase levels of  Bifidobacterium  in feces of the mammal to greater than 10% of the total microbiome found in the feces. 
     
     
         79 . A method of improving health of a mammalian gastrointestinal tract comprising administering to a mammal in need thereof a therapeutically effective amount of  Bifidobacterium longum  subsp.  infantis  having a functional H5 gene cluster and an oligosaccharide having a Type I or Type II core. 
     
     
         80 . The method of  claim 79 , further comprising administering complex oligosaccharide to the mammal, and
 wherein the complex oligosaccharide and the  Bifidobacterium longum  subsp.  infantis  are provided either together or separately, or   wherein the complex oligosaccharide is provided as a solution and the  Bifidobacterium longum  subsp.  infantis  is provided as an enteric-coated tablet or capsule, or   wherein the  Bifidobacterium longum  subsp.  infantis  and the complex oligosaccharide are provided in a liquid composition which comprises complex oligosaccharide at a level of from about 1 g/L to 50 g/L, or   wherein the complex oligosaccharide and  Bifidobacterium longum  subsp.  infantis  is provided in dry form and as an enteric-coated tablet or capsule; or   wherein the complex oligosaccharide is administered prior to the administration of  Bifidobacterium longum  subsp.  infantis ; or   wherein the complex oligosaccharide is administered contemporaneously with the administration of  Bifidobacterium longum  subsp.  infantis ; or   wherein the complex oligosaccharide is administered after the administration of  Bifidobacterium longum  subsp.  infantis.      
     
     
         81 - 95 . (canceled) 
     
     
         96 . The method of  claim 79 , wherein the complex oligosaccharide is at least 20% of the weight of the composition administered daily; or
 wherein the complex oligosaccharide is at least 50% of the weight of the composition administered daily; or   wherein the complex oligosaccharide is at least 80% of the weight of the composition administered daily.   
     
     
         97 - 101 . (canceled) 
     
     
         102 . The method of  claim 79 , wherein the complex oligosaccharide is provided in a daily dose of from 1 to 20 grams; or
 wherein the complex oligosaccharide is provided in a daily dose of from 1 to 10 grams; or   wherein the  Bifidobacterium longum  subsp.  infantis  is provided in a daily dose of from 1 million to 100 billion colony forming units (CFUs); or   wherein the  Bifidobacterium longum  subsp.  infantis  is provided in a daily dose of from 5 to 50 billion CFUs.   
     
     
         103 - 105 . (canceled) 
     
     
         106 . The method of  claim 79 , wherein the mammal in need thereof is administered a dose once daily or in multiple, optionally two, three, four, five, six, sub-doses administered at appropriate intervals throughout the day; or
 wherein subsequent to said administration, level of administered  Bifidobacterium longum  subsp.  infantis  in feces of the mammal is greater than 20% of total microbiome found in the feces; or   wherein subsequent to said administration, level of administered  Bifidobacterium longum  subsp.  infantis  in feces of the mammal is greater than 50% of total microbiome found in the feces; or   wherein subsequent to said administration, level of administered  Bifidobacterium longum  subsp.  infantis  in feces of the mammal is greater than 70% of total microbiome found in the feces; or   wherein the method further comprises monitoring levels of  Bifidobacterium  in the feces of the mammal; or   wherein  Bifidobacterium longum  subsp.  infantis  is administered for at least 5 days; or   wherein the oligosaccharide is administered for a duration from 30 to 360 days; or   wherein administration of the composition to the mammal in need thereof continues for a duration, whereby population of  Bifidobacterium  is established in gut of the mammal; or   wherein administration of the composition to the mammal in need thereof continues so that population of  Bifidobacterium  is maintained in gut of the mammal; or   wherein a dose is administered to maintain levels of  Bifidobacterium  greater than at least 5% of total fecal microbiome of the mammal; or   wherein a dose is administered to maintain the levels of  Bifidobacterium  greater than at least 20% of total fecal microbiome of the mammal; or   wherein a dose is administered to maintain levels of  Bifidobacterium  greater than at least 50% of total fecal microbiome of the mammal.   
     
     
         107 - 117 . (canceled) 
     
     
         118 . The method of  claim 79 , wherein the mammal is a human, a cow, a pig, a rabbit, a goat, a sheep, a cat, a dog, a horse, or a camel; or
 wherein the mammal is a human infant, and
 wherein the infant was delivered via cesarean section, or 
 wherein the infant was delivered vaginally, or 
 wherein the infant is being fed with infant formula which contains no appreciable quantity of mammalian milk oligosaccharides, or 
 wherein the infant is from birth to about 36 months post-conception, or 
 wherein the infant is from birth to weaning age, or 
 wherein the infant is nursed by a mother who is FUC-2 deficient as measured by a genetic test or the absence of complex fucosylated oligosaccharides in her milk; or 
   wherein the human is a pregnant woman, and
 wherein the pregnant woman is in at least third trimester of pregnancy. 
   
     
     
         119 - 127 . (canceled) 
     
     
         128 . The method of  claim 79 , wherein the improvement of health is a reduction of colic in the mammal; or
 wherein the improvement of health is accelerating the development of the immune system in the mammal; or   wherein the improvement of health is a result of colonization of the mammalian gastrointestinal tract with  Bifidobacterium longum  subsp.  infantis  at levels that represent more than 20% of total gut microbiome as measured by fecal analysis.   
     
     
         129 - 130 . (canceled) 
     
     
         131 . A method of preparing activated  Bifidobacterium longum  subsp.  infantis , comprising
 preparing activated  Bifidobacterium longum  subsp.  infantis  by cultivating  Bifidobacterium  by incubating the  Bifidobacterium longum  subsp.  infantis  comprising a functional H5 cluster, including the  Bifidobacterium longum  subsp.  infantis  EVC001 deposited under ATCC Accession No. PTA-125180, bacteria under conditions whereby gene Blon_0042 is upregulated, gene Blon_2168 is downregulated, or combinations thereof; or   preparing activated  Bifidobacterium longum  subsp.  infantis  by cultivating  Bifidobacterium  by incubating the  Bifidobacterium longum  subsp.  infantis  comprising a functional H5 cluster, including the  Bifidobacterium longum  subsp.  infantis  EVC001 deposited under ATCC Accession No. PTA-125180, bacteria under conditions whereby one or more of the genes Blon_0042, Blon_0881, Blon_2175, Blon_2176, Blon_2177, Blon_2331, Blon_2334, Blon_2335, Blon_2336, Blon_2337, Blon_2338, Blon_2339, Blon_2343, Blon_2344, Blon_2346, and Blon_2347 is upregulated, and/or Blon_2168 is downregulated; or   preparing activated commensal  Bifidobacterium longum  subsp.  infantis  EVC001 deposited under ATCC Accession No. PTA-125180 by culturing said bacterial sp. in presence of a mammalian milk oligosaccharide (MMO) or an activator selected from compounds listed in Table 4, whereby bacterial cells in the culture medium are activated, and
 wherein the MMO or the activator from Table 4 is added in an amount sufficient to induce expression of a gene and/or a protein encoding for a sialidase, a fucosidase, or an alpha-N-acetylgalactosaminidase, or genes listed in Table 1 or Table 2 in bacterial cells, or 
 wherein a starting media composition comprises one or more activators from Table 4 in an amount from 0.1 to 3% by weight/vol of the starting media composition, or wherein the MMO and/or the activator constitutes a carbon source and consumption of the carbon source by  Bifidobacterium longum  subsp.  infantis  cells both increases cellular biomass and activates a transport system capable of internalizing one or more oligosaccharides before the oligosaccharide is hydrolyzed and consequently the  Bifidobacterium longum  subsp.  infantis  cells are further capable of hydrolyzing the oligosaccharide, wherein the oligosaccharide has the structure of an oligosaccharide found in a mammalian milk, and
 wherein the mammalian milk is human, bovine, pig, rabbit, goat, sheep, camel, buffalo milk, or mixtures thereof, or 
 
 wherein the activated bacterial cells have a higher binding affinity to mammalian mucosal cells than bacterial cells of the same species cultivated on non-activating monomers or dimers, or 
 wherein activation of the bacterial cells comprises upregulating Blon_0881 and Blon_2343 in  Bifidobacterium longum  subsp.  infantis  or functional homologs in other bacterial species, the homologs being expressed during activation of other bacterial species, or 
 wherein activation comprises upregulating expression of glucosamine-6-phosphate isomerase and carbohydrate ABC transporter membrane protein from  Bifidobacterium longum  subsp.  infantis , or 
 wherein activation of  Bifidobacterium  cells comprises upregulating genes selected from the group consisting of Blon_0042, Blon_R0015, Blon_R0017, Blon_R0021, Blon_R0022, Blon_2177, and combinations thereof, and/or downregulating genes selected from the group consisting of Blon_0518, Blon_0785, Blon_2167, Blon_2168 from  Bifidobacterium longum  subsp.  infantis , or 
 wherein  Bifidobacterium  cells comprise an upregulated Blon_0042 gene from  Bifidobacterium longum  subsp.  infantis , or 
 wherein  Bifidobacterium  cells comprise a downregulated Blon_2168 and/or Blon_2177 gene from  Bifidobacterium longum  subsp.  infantis , or 
 wherein activation of  Bifidobacterium  cells comprise upregulating genes selected from the group consisting of Blon_0882, Blon_0881, Blon_0880, Blon_0879, Blon_2334, Blon_2335, Blon_2336, Blon_2337, Blon_2338, Blon_2339, Blon_2344, Blon_2346, Blon_2347, Blon_2331, and combinations thereof. 
   
     
     
         132 - 144 . (canceled)