Activated bifidobacteria and methods of use thereof

Abstract

Some embodiments of the invention include a composition and method for treating dysbiosis in infants. The composition may include a mixture of activated bifidobacteria and a complex oligosaccharide wherein the complex oligosaccharide may be derived from a human or non-human source.

1 - 116 . (canceled) 
     
     
         117 . A method of preparing an activated  Bifidobacterium , the method comprising
 (a) incubating a purified strain of  Bifidobacterium  in a fermenter in the presence of a mammalian milk oligosaccharide,   (b) harvesting activated  Bifidobacterium  cells, and   (c) purifying the activated  Bifidobacterium  cells obtained in (b),   
       wherein the activated  bifidobacterium  contains a transport system capable of internalizing one or more oligosaccharides before said oligosaccharide is hydrolyzed and is further capable of hydrolyzing said internalized oligosaccharide, wherein said oligosaccharide has the structure of an oligosaccharide found in a mammalian milk. 
     
     
         118 . The method of  claim 117 , wherein the activated  Bifidobacterium  cells express a gene coding for a sialidase, a fucosidase, a sialic acid transporter, or a fucose transporter. 
     
     
         119 . The method of  claim 117 , wherein, in the activated  Bifidobacterium  cells, gene Blon_0042 is upregulated, gene Blon_2168 is downregulated, or a combination thereof. 
     
     
         120 . The method of  claim 117 , wherein any of the genes Blon_2334, Blon_2335, Blon_2336, Blon_2337, Blon_2338, Blon_2339, Blon_2344, Blon_2346, Blon_2347, and Blon_2331, is upregulated in the activated  Bifidobacterium  cells. 
     
     
         121 . The method of  claim 117 , wherein the activated  Bifidobacterium  cells comprise upregulated genes selected from the group consisting of Blon_0042, Blon_R0015, Blon_R0017, Blon_R0021, Blon_R0022, and combinations thereof, and/or downregulated genes selected from the group consisting of Blon_0518, Blon_0785, Blon_2167, Blon_2168 and combinations thereof. 
     
     
         122 . The method of  claim 117 , wherein the  Bifidobacterium  is  B. longum, B. breve, B. bifidum  or  B. pseudocatenulatum.    
     
     
         123 . The method of  claim 122 , wherein the  B. longum  is  B. longum  subsp.  infantis.    
     
     
         124 . The method of  claim 117 , wherein the mammalian milk oligosaccharide is isolated from a mammalian milk source. 
     
     
         125 . The method of  claim 124 , wherein the mammalian milk oligosaccharide is from a human or a bovine source, optionally wherein the bovine source is bovine colostrum. 
     
     
         126 . The method of  claim 117 , wherein the mammalian milk oligosaccharide is selected from the group consisting of Lacto-N-Tetraose, Lacto-N-Neotetraose, Lacto-N-Fucopentaose I, Lacto-N-Fucopentaose II, Lacto-N-Fucopentaose III, Lacto-N-Fucopentaose V, Lacto-N-Hexaose, Para-Lacto-N-Hexaose, Lacto-N-Neohexaose, Para-Lacto-N-Neohexaose, Monofucosyllacto-N-Hexaose II, Isomeric Fucosylated Lacto-N-Hexaose (1), Monofucosyllacto-N-Hexaose, Isomeric Fucosylated Lacto-N-Hexaose (3), Isomeric Fucosylated Lacto-N-Hexaose (2), Difucosyl-Para-Lacto-N-Neohexaose, Difucosyl-Para-Lacto-N-Hexaose, Difucosyllacto-N-Hexaose, Lacto-N-Neoocataose, Para-Lacto-N-Octanose, Iso-Lacto-N-Octaose, Lacto-N-Octaose, Monofucosyllacto-Nneoocataose, Monofucosyllacto-N-Ocataose, Difucosyllacto-N-Octaose I, Difucosyllacto-N-Octaose 11, Difucosyllacto-N-Neoocataose 11, Difucosyllacto-N-Neoocataose 1, Lacto-N-Decaose, Trifucosyllacto-N-Neooctaose, Trifucosyllacto-N-Octaose, Trifucosyl-Iso-Lacto-N-Octaose, 2′-fucosyllactose, 3-fucosyllactose, difucosyllactose, 3′-sialyllactose, 6′-sialyllactose, 3′-sialyl-3-fucosyllactose, sialyllacto-N-tetraose, and 6′-sialyllactosamine. 
     
     
         127 . The method of  claim 117 , further comprising drying the purified activated  Bifidobacterium  cells. 
     
     
         128 . The method of  claim 127 , wherein the drying is performed by spray drying or freeze drying, optionally wherein the freeze drying is performed in the presence of a suitable cryoprotectant, and optionally wherein the cryoprotectant is selected from the group consisting of glucose, lactose, raffinose, 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, and taurine. 
     
     
         129 . The method of  claim 127 , further comprising formulating the purified activated  Bifidobacterium  cells into a composition in the form of a dry powder, dry powder suspended in an oil, solution, packet, sachet, orally disintegrating tablet, foodstuff, capsule, lozenge, effervescent tablet, suppository, or enema, optionally wherein the capsule or tablet has an enteric coating, and optionally 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, hydroxy propyl methyl cellulose acetate succinate, polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymers, cellulose acetate trimellitate, sodium alginate, and Zein. 
     
     
         130 . The method of  claim 117 , further comprising formulating the purified activated  Bifidobacterium  cells into a composition such that the activated  Bifidobacterium  cells are at a concentration of from 1 billion cfu/g to 500 billion cfu/g, 1 billion cfu/g to 100 billion cfu/g, or 1 billion cfu/g to 50 billion cfu/g. 
     
     
         131 . The method of  claim 127 , further comprising formulating the purified activated  Bifidobacterium  cells into a composition to include a stabilizer, optionally wherein the stabilizer is a flow agent, and optionally wherein the stabilizer is a milk protein. 
     
     
         132 . The method of  claim 117 , further comprising formulating the activated  bifidobacterium  composition to include an isolated complex oligosaccharide. 
     
     
         133 . The method of  claim 132 , wherein the isolated complex oligosaccharide is isolated from a mammalian milk source selected from human, bovine, pigs, rabbits, goats, sheep, or camel, wherein the bovine source is bovine milk, bovine colostrum, bovine colostrum concentrate, bovine whey permeate, or mixtures thereof. 
     
     
         134 . The method of  claim 132 , wherein the complex oligosaccharide comprises at least one of (3Hex,4HexNac,1Fuc), (1Gal,1GlcNAc,1NeuAc), or (1Glu,1Gal,1NeuAc (3′ or 6′)), and/or wherein the complex oligosaccharide is from bovine colostrum and comprises any of Hex(4); Hex(4) HexNAc(2); and Hex(3) HexNAc(1) NeuAc(1) at levels greater than 1%, and/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, optionally wherein the complex oligosaccharide is at least 20%, at least 50%, or at least 80% of the weight of the composition. 
     
     
         135 . The method of  claim 132 , wherein the complex oligosaccharide comprises a fucosyllactose, sialyllactose, or derivatives thereof, optionally wherein the fucosyllactose, sialyllactose, or derivatives thereof are synthetically produced and purified oligosaccharides of 2′-fucosyllactose, 3′-fucosyllactose, difucosyllactose, lacto-N-fucosylpentose I, lacto-N-fucosylpentose II, lacto-N-fucosylpentose III, lacto-N-fucosylpentose V, 3′-sialyllactose, 6′-sialyllactose, 3′-sialyl-3-fucosyllactose, sialyllacto-N-tetraose, 6′-sialyllactosamine, or mixtures thereof, and/or wherein the fucosyllactose and/or sialyllactose comprises 1-5%, 5-20%, or 20-50% of the total oligosaccharides, and/or wherein the mass ratio of the complex oligosaccharide to the fucosyllactose and/or sialyllactose is from 20:1 to 1:10, and/or wherein the complex oligosaccharide is less than 50% fucosylated. 
     
     
         136 . The method of  claim 117 , further comprising formulating the purified activated  Bifidobacterium  cells into a composition which includes a secondary metabolite, optionally wherein the secondary metabolite is acetate, lactate, or a combinations thereof; or a short chain fatty acid.