Methods and Compositions for Improved Digestion of Milk Oligosaccharides

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Appl. No. 61/836,046, filed Jun. 17, 2013, the disclosure of which is incorporated by reference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under Grant Nos. ATT007079, HD061923, and HD065122, awarded by the National Institutes of Health. The Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

In addition to essential nutrients such as lactose lipids and proteins, human milk contains a large concentration of oligosaccharides. Human milk oligosaccharides (HMO) are complex and diverse molecules. These molecules are composed of glucose (Glc), galactose (Gal), N-acetylglucosamine (GlcNAc), and often contain fucose (Fuc) and/or N-acetylneuraminic acid (NeuAc), linked via several glycosidic bonds. The simplest oligosaccharides in human milk are trisaccharides where lactose can be sialylated to form sialyllactose, or fucosylated to form fucosyllactose. More complex HMO are also based on a lactose core that is conjugated with repeats of lacto-N-biose I (Galβ1-3GlcNAc; LNB; type-1 chain) or N-acetyllactosamine (Galβ1-4GlcNAc; type-2 chain), producing molecules with a degree of polymerization larger than 4 (Bode et al. (2012) Adv. Nutr. 3:383 S). These core structures can be modified by fucose and sialic acid residues via different linkages (De Leoz et al. (2012) J. Proteome Res. 11:4662). Although a large number of different HMO structures have been determined, a few isomers can represent up to 70% of the total molecules.

Remarkably, the energetic value of HMO for the infant is minimal. HMO are resistant to enzymatic hydrolysis from intestinal brush border membrane and pancreatic juices, and therefore the majority of these molecules transit the intestinal tract reaching the colon in intact form. During their transit HMO are believed to prevent pathoge colonization, by serving as decoy binding sites for epithelial glycans (Newburg et al. (2005) Annu Rev. Nutr. 25:37).

Human milk oligosaccharides (HMO) influence the composition of the intestinal microbiota in the first years of life. While the microbial community in breast-fed infants is largely dominated by the genus Bifidobacterium, formula-fed infants show increased bacterial diversity (Roger et al. (2010) Microbiol. 156:3329; Yatsunenko et al. (2012) Nature 486:222). This indicates that both pro- and antimicrobial elements in breast-milk account for these differences. A conceptual basis for co-evolution between bifidobacteria and milk glycans is supported by recent definition of the molecular mechanisms by which these microbes catabolize HMO. In Bifidobacterium longum subsp. infantis (B. infantis) ATCC 15697, these mechanisms include oligosaccharide transporters and intracellular glycosyl hydrolases (GH) such as fucosidases, hexosaminidases and sialidases (Gamido et al. (2012) Adv. Nutr. 3:415 S).

BRIEF SUMMARY OF THE INVENTION

Provided herein are methods for promoting growth of beneficial gut bacteria and/or increasing oligosaccharide consumption in an individual, comprising administering to the individual a composition comprising a bacterium that expresses heterologous alpha-fucosidase, thereby promoting growth of beneficial gut bacteria in the individual. In some embodiments, the heterologous alpha-fucosidase is GH-29. In some embodiments, the heterologous alpha-fucosidase is GH-95. In some embodiments, the bacterium is not Bifidobacterium longum subsp. infantis (B. infantis), or B. bifidum.

In some cases, the bacteria further expresses a second heterologous alpha-fucosidase. The second alpha-fucosidase can be GH-95 or GH-29. In some embodiments, the bacterium is selected from the group consisting of Lactobacillus and Bifidobacterium. In some embodiments, the Lactobacillus is selected from the group consisting of L. casei, L. paracasei, and L. rhamnosus. The Bifidobacterium can be selected from the group consisting of B. adolescentis, B. catenulatum, B. pseudocatenulatum, B. dentium, B. longum, and B. breve. In some embodiments, the bacterium is Bifidobacterium breve (B. breve).

In some embodiments, the method further comprises administering an oligosaccharide, e.g., an exogenous oligosaccharide. The oligosaccharide can be administered at the same time (e.g., in the same composition) or at a different time from the bacteria. The oligosaccharide can be a fucosylated oligosaccharide. In some embodiments, the composition comprises a milk oligosaccharide, a fucosylated milk oligosaccharide, or a human milk oligosaccharide. In some embodiments, the composition does not include an oligosaccharide containing an N-glycolylneuraminic acid residue.

In some embodiments, the oligosaccharide is selected from the group consisting of: an oligosaccharide consisting of 3 Hexose (Hex) moieties and 6 N-acetyl hexosamine (HexNAc) moieties; an oligosaccharide consisting of 4 Hex moieties and 3 HexNAc moieties; an oligosaccharide consisting of 3 Hex moieties and 4 HexNAc moieties; an oligosaccharide consisting of 6 Hex moieties and 2 HexNAc moieties; an oligosaccharide consisting of 3 Hex moieties, 4 HexNAc moieties and 1 fucose (Fuc) moiety; an oligosaccharide consisting of 4 Hex moieties and 4 HexNAc moieties; an oligosaccharide consisting of 3 Hex moieties and 5 HexNAc moieties; an oligosaccharide consisting of 4 Hex moieties, 4 HexNAc moieties, and 1 Fuc moiety; an oligosaccharide consisting of 5 Hex moieties and 4 HexNAc moieties; an oligosaccharide consisting of 3 Hex moieties, 5 HexNAc moieties, and 1 Fuc moiety; an oligosaccharide consisting of 4 Hex moieties and 5 HexNAc moieties; an oligosaccharide consisting of 3 Hex moieties and 6 HexNAc moieties; an oligosaccharide consisting of 5 Hex moieties, 4 HexNAc moieties, and 1 Fuc moiety; an oligosaccharide consisting of 4 Hex moieties, 5 HexNAc moieties, and 1 Fuc moiety; and an oligosaccharide consisting of 3 Hex moieties, 6 HexNAc moieties, and 1 Fuc moiety.

Further provided are compositions comprising a beneficial gut bacterial strain that expresses a heterologous alpha-fucosidase. In some embodiments, the alpha-fucosidase is GH-29 or GH-95. In some embodiments, the bacterial strain is not Bifidobacterium longum subsp. infantis (B. infantis) or B. bifidum. In some embodiments, the composition further comprising at least one oligosaccharide, such as a fucosylated oligosaccharide, a milk oligosaccharide, or a human milk oligosaccharide. In some cases the beneficial gut bacterial strain expresses at least two heterologous alpha-fucosidases. For example, the beneficial gut bacterial strain can express both GH-29 and GH-95.

In some embodiments, the composition includes a beneficial gut bacterial strain selected from the group consisting of Lactobacillus and Bifidobacterium. The Lactobacillus can be selected from the group consisting of L. casei, L. paracasei, and L. rhamnosus. The Bifidobacterium can be selected from the group consisting of B. adolescentis, B. catenulatum, B. pseudocatenulatum, B. dentium, B. longum, and B. breve. In some embodiments, the beneficial gut bacterial strain is Bifidobacterium breve (B. breve).

In some embodiments, the composition does not include an oligosaccharide containing an N-glycolylneuraminic acid residue. In some embodiments, the at least one oligosaccharide includes a milk oligosaccharide, a fucosylated oligosaccharide, or a human milk oligosaccharide.

In some embodiments, the at least one milk oligosaccharide is selected from the group consisting of: an oligosaccharide consisting of 3 Hexose (Hex) moieties and 6 N-acetyl hexosamine (HexNAc) moieties; an oligosaccharide consisting of 4 Hex moieties and 3 HexNAc moieties; an oligosaccharide consisting of 3 Hex moieties and 4 HexNAc moieties; an oligosaccharide consisting of 6 Hex moieties and 2 HexNAc moieties; an oligosaccharide consisting of 3 Hex moieties, 4 HexNAc moieties and 1 fucose (Fuc) moiety; an oligosaccharide consisting of 4 Hex moieties and 4 HexNAc moieties; an oligosaccharide consisting of 3 Hex moieties and 5 HexNAc moieties; an oligosaccharide consisting of 4 Hex moieties, 4 HexNAc moieties, and 1 Fuc moiety; an oligosaccharide consisting of 5 Hex moieties and 4 HexNAc moieties; an oligosaccharide consisting of 3 Hex moieties, 5 HexNAc moieties, and 1 Fuc moiety; an oligosaccharide consisting of 4 Hex moieties and 5 HexNAc moieties; an oligosaccharide consisting of 3 Hex moieties and 6 HexNAc moieties; an oligosaccharide consisting of 5 Hex moieties, 4 HexNAc moieties, and 1 Fuc moiety; an oligosaccharide consisting of 4 Hex moieties, 5 HexNAc moieties, and 1 Fuc moiety; and an oligosaccharide consisting of 3 Hex moieties, 6 HexNAc moieties, and 1 Fuc moiety.

Also provided are compositions comprising beneficial gut bacteria, wherein the bacteria express more than one heterologous alpha-fucosidase, the composition further comprising at least one oligosaccharide. In some embodiments, the bacteria are not Bifidobacterium longum subsp. infantis (B. infantis), or B. bifidum. In some embodiments, the more than one alpha-fucosidase includes GH-29. In some embodiments, the more than one alpha-fucosidase further includes GH-95. In some embodiments, the at least one oligosaccharide includes a fucosylated oligosaccharide. In some embodiments, the at least one oligosaccharide includes a milk oligosaccharide. In some embodiments, the at least one oligosaccharide includes a human milk oligosaccharide or a fucosylated human milk oligosaccharide.

In some embodiments, the beneficial gut bacteria are a strain selected from the group consisting of Lactobacillus and Bifidobacterium. The Lactobacillus can be selected from the group consisting of L. casei, L. paracasei, and L. rhamnosus. The Bifidobacterium can be selected from the group consisting of B. adolescentis, B. catenulatum, B. pseudocatenulatum, B. dentium, B. longum, and B. breve. In some cases, the beneficial gut bacterial strain is Bifidobacterium breve (B. breve).

Further provided are methods of administering any of the foregoing compositions. Fore example, in some embodiments, a method of promoting growth of beneficial gut bacteria and/or increasing oligosaccharide consumption in an individual, comprising administering any of the foregoing compositions to the individual. In some embodiments, administration is oral. In some embodiments, administration is rectal.

In addition, provided herein are methods for isolating a beneficial strain of Bifidobacterium. In some embodiments, the method comprises: screening a population of Bifidobacterium for presence of a nucleic acid sequence encoding GH-29 or GH-95 alpha-fucosidase; detecting the presence or absence of the nucleic acid encoding GH-29 or GH-95 alpha-fucosidase; and selecting a Bifidobacterium strain where the presence of the GH-29 or GH-95 nucleic acid is detected, thereby isolating a beneficial strain of Bifidobacterium. In some embodiments, the method comprises: screening a population of Bifidobacterium for presence of GH-29 or GH-95 alpha-fucosidase polypeptide; detecting the presence or absence of the GH-29 or GH-95 alpha-fucosidase polypeptide; and selecting a Bifidobacterium strain where the presence of the GH-29 or GH-95 polypeptide is detected, thereby isolating a beneficial strain of Bifidobacterium.

Also provided are methods of making a beneficial strain of Bifidobacterium comprising: transfecting a Bifidobacterium with an expression cassette comprising a heterologous polynucleotide encoding GH-29 or GH-95 operably linked to a promoter; and selecting for and isolating Bifidobacterium containing the expression cassette. In some cases, the Bifidobacterium is not a strain of Bifidobacterium longum subsp. infantis (B. infantis), or B. bifidum. In some cases, the Bifidobacterium is a strain of Bifidobacterium breve (B. breve).

Further provided are methods for promoting growth of beneficial gut bacteria in an individual, comprising administering to the individual a composition comprising a bacterium that expresses a first heterologous alpha-fucosidase and a second heterologous alpha-fucosidase, thereby promoting growth of beneficial gut bacteria in the individual. In some embodiments, the first or second alpha-fucosidase is GH-29. In other cases, the first or second alpha-fucosidase is GH-95. In some embodiments, the first alpha-fucosidase is GH-29 and the second alpha-fucosidase is GH-95.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Evolutionary relationship of B. breve strains used in the study. The tree is drawn to scale, with branch lengths in the same units (number of base substitutions per site) as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary history was inferred using the Minimum Evolution method, followed by 1000 bootstrap replicates.

FIG. 2. Phylogenetic relationship of homologous fucosidase Blon_—0248 in B. breve strains. The tree is drawn to scale, with branch lengths in the same units (number of amino acid substitutions per site) as those of the evolutionary distances used to infer the phylogenetic tree The evolutionary history was inferred using the Minimum Evolution method, followed by 1000 bootstrap replicates.

FIG. 3. Growth of B. breve on HMO. B. breve isolates were inoculated on semi-synthetic MRS medium supplemented with 2% w/v HMO (A) and 2FL (B). B. infantis ATCC 15697 and B. breve ATCC 15700 were included as high and low growth controls respectively. Fermentations were carried out in triplicate.

FIG. 4. Glycoprofiling of the HMO consumption by selected B. breve strains. (A) Total utilization of HMO. Consumption is calculated as the percent difference in HMO between the start and the end of exponential phase. (B) Glycoprofiles of the consumption of neutral non-fucosylated HMO by seven B. breve strains. B. infantis ATCC 15697 was included as positive control.

FIG. 5. Glycoprofiling of the consumption of fucosylated and acidic HMO by select B. breve strains. Consumption of eight fucosylated HMO (A), and eleven sialylated HMO (B) was calculated. B. infantis ATCC 15697 was included as positive control. HMO consumption is represented as the percent difference in HMO between the start and the end of exponential phase.

FIG. 6. Fold in change expression for genes encoding α-fucosidases from GH families 95 and 29 in B. breve SC95, during mid-exponential growth on HMO and 2FL. Growth on lactose was used as a control.

FIG. 7. Temporal glycoprofile of the consumption of select neutral and acidic HMO by Bifidobacterium breve SC95 at different stages in the exponential phase.

DETAILED DESCRIPTION

I. Introduction

Provided herein are strains of beneficial gut bacteria that express one or more glycohydrolases capable of hydrolyzing a human milk oligosaccharide, or structurally similar oligosaccharides. In some embodiments, the gut bacteria are genetically engineered, and express one or more heterologous polypeptides. In some embodiments, the gut bacteria express at least one heterologoud glycohydrolase as described herein.

The present results show that certain glycohdrolases increase growth of beneficial gut bacteria on a human milk oligosaccharide (HMO) substrate. In some embodiments, alpha-fucosidases of the GH-29 family are associated with growth on HMO. In some embodiments, alpha-fucosidases of the GH-95 family are associated with growth on HMO. In some embodiments, bacteria that express multiple alpha-fucosidases (e.g., a GH-29 alpha-fucosidase and a GH-95 alpha-fucosidase) are capable of growing on human milk oligosaccharide, or structurally similar oligosaccharides.

The present results show that bacteria that express GH-29, express GH-95, or express multiple alpha-fucosidases (e.g., a GH-29 alpha-fucosidase and a GH-95 alpha-fucosidase), either endogenously or heterologously, can establish a beneficial microbiome in the gut of an individual to which HMO have been administered (e.g., a breastmilk-fed infant, or a human ingesting HMO). Alternatively, or in addition, administering HMO to a subject can be used to select for the establishment of a beneficial microbiome in the gut by selecting for beneficial bacteria that express GH-29, express GH-95, or express multiple alpha-fucosidases (e.g., a GH-29 alpha-fucosidase and a GH-95 alpha-fucosidase) in comparison to other microorganisms. Moreover, administering compositions of beneficial bacteria that express (e.g., heterologously) one or more of the glycohydrolases described herein, the composition further including a human milk oligosaccharide, can provide a therapeutic for, e.g. establishing a beneficial gut microbiome in a subject and selecting against for the growth of the beneficial gut bacteria in comparison to other microorganisms.

Disclosed herein is isolation of a representative number of strains of Bifidobacterium, and characterization of the molecular mechanisms for their consumption of milk oligosaccharides. Bifidobacterium breve, B. infantis, B. longum subsp. longum (B. longum), and B. bifidum are the species most frequently detected in breast-fed infant feces (Avershina et al. (2013) Appl. And Env. Microbiol. 79:497; Roger et al. (2010) Microbiol. 156:3329). In general, B. breve and B. infantis are more exclusively found in infants, and B. longum and B. bifidum are found in both infants and adults. While several strains of B. bifidum and B. infantis grow vigorously on HMO in vitro, this phenotype has been largely unexplored for larger numbers of B. breve and B. longum subsp. longum isolates. Only one strain of B. breve, ATCC 15700, was shown to utilize lacto-N-tetraose (LNT) primarily, contrasting with the versatility in HMO species consumption observed by B. infantis (Asakuma et al. (2011) J. Biol. Chem. 286:34583; LoCasio et al. (2007) J. Agric. Food Chem. 55:8914).

II. Definitions

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. See, e.g., Lackie, DICTIONARY OF CELL AND MOLECULAR BIOLOGY, Elsevier (4th ed. 2007); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, N.Y. 1989); Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of this invention.

The term “glycohydrolase” as used herein refers to an enzyme that catalyzes the hydrolysis of glycosides. Similarly, the term “alpha-fucosidase” as used herein refers to a glycohydrolase that is specific for, or substantially specific for, alpha-fucosides. Alpha-fucosidases include those enzymes found in glycoside hydrolase family 29 (GH-29) and glycoside hydrolase family 95 (GH-95). Exemplary glycohydrolases include SEQ ID NOs: 1-6, polypeptides encoded by SEQ ID NOs: 7-12, or polypeptides or nucleic acids substantially identical, or substantially similar, thereto.

As used herein, the term “oligosaccharide” refers to polymeric carbohydrates that contain 3 to 20 monosaccharides covalently linked through glycosidic bonds. In some embodiments, the oligosaccharides are purified from human milk, bovine milk, or the milk of any other suitable mammal. In some cases, the oligosaccharides are purified from whey, cheese, or other dairy products, e.g., purified away from oligosaccharide-degrading enzymes in milk, whey, cheese, or other dairy products. Purified oligosaccharides can be further modified as described herein. Alternatively, the oligosaccharides can be synthesized or partially synthesized (e.g., synthesized from a purified oligosaccharide starting material) as described herein. Compositions described herein can include a mixture of oligosaccharides that have been purified, partially synthesized, or synthesized.

The term human milk oligosaccharide (HMO) can refer to a number of complex oligosaccharides found in human milk, or oligosaccharides that are structurally similar to, or structurally identical to oligosaccharides found in human milk. Consequently, HMO need not be derived from human milk or human milk products and can be partially synthesized, synthesized de novo, or derived from the milk of another organism. HMO compositions can include mixtures of oligosaccharides that have been purified, partially synthesized, or synthesized. HMO compositions further include chemically modified oligosaccharides found in human milk, or oligosaccharides that are structurally similar to, or structurally identical to oligosaccharides found in human milk as described herein. Human milk oligosaccharides can, in some embodiments, include fucosyl oligosaccharides.

Among the monomers of milk oligosaccharides are D-glucose (Glc), D-galactose (Gal), N-acetylglucosamine (GlcNAC), L-fucose (Fuc), and sialic acid [N-acetylneuraminic acid (NeuAc)]. Elongation may be achieved by attachment of GlcNAc residues linked in β1-3 or β1-4 linkage to a Gal residue followed by further addition of Gal in a β-1-3 or β-1-4 bond. Most HMOs carry lactose at their reducing end. From these monomers, a large number of core structures may be formed. Further variations may occur due to the attachment of lactosamine, Fuc, and/or NeuAc. See, e.g., Kunz, C. et al., Annual. Rev. Nutri., 20:699-722 (2000) for a further description of HMOs. Human milk oligosaccharides can also be found in, or purified from, the milk of other mammals, provided that they are identical or substantially identical to the human milk oligosaccharides.

Hexose (Hex) represents a residue of glucose or galactose or mannose.

Fucose (Fuc) represents a residue of Deoxyhexose.

HexNAc represents a residue of N-acetylglucosamine or N-acetylgalactosamine.

NeuAc represents a residue of N-acetyl neuraminic acid (sialic acid).

The term “Bifidobacterium” and its synonyms refer to a genus of anaerobic bacteria having beneficial properties for humans. Bifidobacteria is one of the major genera of bacteria that make up the gut flora, the bacteria that reside in the gastrointestinal tract and have health benefits for their hosts. See, e.g., Guarner F and Malagelada J R. Lancet (2003) 361, 512-519, for a further description of Bifidobacterium in the normal gut flora.

The term “beneficial gut bacteria” or the like refers to live microorganisms that reside in the gut or can be introduced into the gut of an individual and confer a health benefit on the host. In some cases, the beneficial gut bacteria can aid in the digestion of carbohydrates, proteins, or fatty acids that are not efficiently digested, or not digested at all, by the host. In some cases, the beneficial gut bacteria generate metabolites that are beneficial to the host such as fatty acids, vitamins, or modulators of the immune system. In some cases, the beneficial gut bacteria inhibit the growth of pathogenic bacteria in the gut.

Exemplary embodiments of beneficial gut bacteria include lactobacilli (e.g., L. casei, L. paracasei, and L. rhamnosus) and bifidobacteria (e.g., B. adolescentis, B. catenulatum, B. pseudocatenulatum, B. dentium, B. bifidum, B. longum, B. infantis, and B. breve). Exemplary embodiments of beneficial gut bacteria further include, but are not limited to, the foregoing lactobacilli and bifidobacteria that express an alpha-fucosidase such as GH-29, or an alpha-fucosidase such as GH-95. In some cases, the beneficial gut bacteria further include, but are not limited to, the foregoing bifidobacteria and bifidobacteria that express at least two alpha-fucosidases. For example, beneficial gut bacteria further include, but are not limited to, Lactobacilli and Bifidobacteria that express GH-29 and GH-95.

A “prebiotic” or “prebiotic nutrient” is generally a non-digestible food ingredient that beneficially affects a host when ingested by selectively stimulating the growth and/or the activity of one or a limited number of bacteria in the gastrointestinal tract, e.g., beneficial gut bacteria. As used herein, the term “prebiotic” refers to the above described non-digestible food ingredients in their non-naturally occurring states, e.g., after purification, chemical or enzymatic synthesis as opposed to, for instance, in whole human milk. Pre-biotics can be administered separately from beneficial gut bacteria, or in conjunction with beneficial gut bacteria. As used herein, “in conjunction with” refers to at the same time as, substantially the same time as, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 45, or 60 minutes before or after, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 hours before or after, or about 1 or 2 days before or after the administration of gut bacteria.

A “probiotic” refers to beneficial gut bacteria that when administered in adequate amounts confer a health benefit on the host.

An “expression cassette” refers to a nucleic acid construct, which when introduced into a host cell (e.g., a microorganism, such as a Bifidobacterium or a Lactobacillus), results in transcription and/or translation of a RNA or polypeptide, respectively. An expression cassette typically includes a sequence to be expressed, and sequences necessary for expression of the sequence to be expressed. The sequence to be expressed can be a coding sequence or a non-coding sequence (e.g., an inhibitory sequence). The sequence to be expressed is generally operably linked to a promoter. The promoter can be a heterologous promoter or a promoter that is derived from the host plant. Generally, an expression cassette is inserted into an expression vector to be introduced into a host cell. The expression vector can be viral or non-viral.

“Recombinant” refers to a human manipulated polynucleotide or a copy or complement of a human manipulated polynucleotide. For instance, a recombinant expression cassette comprising a promoter operably linked to a second polynucleotide may include a promoter that is heterologous to the second polynucleotide as the result of human manipulation (e.g., by methods described in Sambrook et al., Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)). A recombinant expression cassette may comprise polynucleotides combined in such a way that the polynucleotides are extremely unlikely to be found in nature. For instance, human manipulated restriction sites or plasmid vector sequences may flank or separate the promoter from the second polynucleotide. One of skill will recognize that polynucleotides can be manipulated in many ways and are not limited to the examples above. A recombinant protein is one that is expressed from a recombinant polynucleotide, and recombinant cells, tissues, and organisms are those that comprise recombinant sequences (polynucleotide and/or polypeptide).

A polynucleotide sequence is “heterologous to” an organism or a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified from its original form. For example, a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is different from naturally-occurring variants.

The term “exogenous,” in reference to a polypeptide or polynucleotide, refers to polypeptide or polynucleotide which is introduced into a cell or organism (e.g., a microorganism, such as a Bifidobacterium or a Lactobacillus) by any means other than by mating.

The term “transgenic,” e.g., a transgenic microorganism, such as a transgenic Bifidobacterium or Lactobacillus, refers to a recombinantly modified organism with at least one introduced genetic element. The term is typically used in a positive sense, so that the specified gene is expressed in the transgenic organism. However, a transgenic organism can be transgenic for an inhibitory nucleic acid, i.e., a sequence encoding an inhibitory nucleic acid is introduced. The introduced polynucleotide can be from the same species or a different species, can be endogenous or exogenous to the organism, can include a non-native or mutant sequence, or can include a non-coding sequence.

In the case of both expression of transgenes and inhibition of endogenous genes (e.g., by antisense, or sense suppression) one of skill will recognize that a polynucleotide sequence need not be identical and can be “substantially identical” to a sequence of the gene from which it was derived.

The term “promoter” refers to regions or sequence located upstream and/or downstream from the start of transcription and which are involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A “bacterial promoter” is a promoter capable of initiating transcription in bacterial cells (e.g., Bifidobacterium or Lactobacillus). In some cases, a bacterial promoter can originally derive from the same species of microorganism into which it is introduced. In other cases, a bacterial promoter may derived from another species of bacteria or from another organism (e.g., a viral, fungal, plant, animal, or mammalian promoter) that is capable of initiating transcription in bacterial cells.

A “constitutive promoter” refers to a promoter that is capable of initiating transcription under nearly all conditions, whereas an “inducible promoter” initiates transcription under specific conditions such as the presence of an inducer (e.g., allolactose, arabinose, tryptophan, IPTG) or a signal (e.g., heat, cold, low phosphate,). In some embodiments, a promoter is inducible if the transcription levels initiated by the promoter under a specific cellular condition are at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1000-fold higher or more as compared to the transcription levels initiated by the promoter in the absence of that condition.

The term “express,” “expresses,” “expressing,” or the like, as in “a bacterium that expresses” refers to a bacterium that has polynucleotide encoding a specific gene (e.g., a glycohydrolase such as an alpha-fucosidase, including GH-29 or GH-95) that is capable of being expressed. In some cases, the gene can express constitutively. In other cases, the gene can be expressed only under certain conditions (e.g., it is inducible).

The term “modulate” as in to “modulate a gene” or “modulate expression” of a gene refers to increasing or decreasing the expression, activity, or stability of a gene. For example, a gene may be modulated by increasing or decreasing the amount of RNA that is transcribed from the gene or altering the rate of such transcription. Decreased expression may include expression that is reduced by 5%, 10%, 15%, 20%, 25%, 30%, 50%, 75%, 80%, 90%, 95%, 99% or more. Increased expression, or over expression, includes expression that is increased by 1%, 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more. In some cases expression may be increased by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1000-fold higher. Expression may be modulated in a constitutive or inducible manner as provided herein.

Two nucleic acid sequences or polypeptides are said to be “identical” if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below. The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. When percentage of sequence identity is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acids residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated according to, e.g., the algorithm of Meyers & Miller, Computer Applic. Biol. Sci. 4:11-17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA).

The term “substantial identity” of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 25% sequence identity. Alternatively, percent identity can be any integer from at least 25% to 100% (e.g., at least 25%, 26%, 27%, 28%, . . . , 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%), preferably calculated with BLAST using standard parameters, as described below. One of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 40%. Percent identity of polypeptides can be any integer from at least 40% to 100% (e.g., at least 40%, 41%, 42%, 43%, . . . , 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%). In some embodiments, substantially identical polypeptide share at least 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Unless otherwise indicated, the comparison window extends the entire length of a reference sequence. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection.

One example of a useful algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

“Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.

The following six groups each contain amino acids that are conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

(see, e.g., Creighton, Proteins (1984)).

An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.

III. Glycohydrolases

Alpha-Fucosidases

Described herein are alpha-fucosidases (e.g., GH-29 or GH-95 family glycohydrolases) that are capable of hydrolyzing components of human milk oligosaccharides, and other saccharides of similar structure. The GH-95 and GH-29 alpha-fucosidases play a role in degrading α-1,2- and α-1,3/4-fucosylated milk oligosaccharides, respectively, and also glycoconjugates, in the gut of host organisms. The glycohydrolases can be expressed in bacteria (e.g., beneficial gut bacteria) and confer the ability of the bacteria to hydrolyze components of oligosaccharides (e.g., milk oligosaccharides, or human milk oligosaccharides). In some cases, the glycohydrolases can thus confer the ability of the bacteria to utilize oligosaccharides (e.g., milk oligosaccharides, or human milk oligosaccharides) as a carbon and/or nitrogen source. In some cases, this can provide a selective advantage as compared to other microorganisms present.

Alpha-fucosidases hydrolize fucosides to fucose. See, e.g., Levvy, G. A. and McAllan, A. Mammalian fucosidases. 2. alpha-L-Fucosidase. Biochem. J. 80 (1961) 435-439. Glycohydrolases of the GH-29 family are exo-acting alpha-fucosidases found in archaea, bacteria, and eukarya. In some cases, the GH-29 alpha-fucosidases herein are of bacterial origin. However, in other cases, they can be of from an organism of any phylogenetic kingdom as long as they can be expressed in a beneficial gut bacteria.

In some embodiments, GH-29 alpha-fucosidases (E.C. number 3.2.1.51) described herein can specifically release α-1,3- and α-1,4-linked fucosyl residues from 3-fucosyllactose, various Lewis blood group substances (a, b, x, and y types), and lacto-N-fucopentaose II and III. In some cases, GH-29 alpha-fucosidases described herein can cleave fucose from LNFPIII (α1-3). In some cases, GH-29 alpha-fucosidases described herein do not show activity on small oligosaccharides (2FL and 3FL), glycoconjugates containing α-1,2-fucosyl residue, or on synthetic α-fucoside (p-nitrophenyl-α-1-fucoside). In some cases, the GH-29 alpha-fucosidases described herein exhibit a greater activity against longer-chain fucosylated oligosaccharides. GH-29 alpha-fucosidases described herein can confer the ability of gut bacteria to utilize, e.g. oligosaccharides, milk oligosaccharides, human milk oligosaccharides, fucosyl oligosaccharides, 3-fucosyllactose, or lacto-N-fucopentaose II as a carbon source (e.g., as a sole carbon source).

Exemplary GH-29 alpha-fucosidases include a fucosidase domain. In some cases, the GH-29 alpha-fucosidases include additional domains such as a carbohydrate binding domain. In some cases, exemplary fucosidases can also include a FIVAR domain, and/or a transmembrane domain. Consequently, in some cases, the GH-29 alpha-fucosidases, can be expressed (e.g., heterologously expressed) as fragment polypeptides such that the catalytic activity and growth on HMO phenotype are preserved, but non-essential domains or fragments are removed or replaced. The essential features of GH-29 alpha-fucosidases are known in the art and are described in, e.g. Ashida et al., Glycobiology, 19(9), 1010-17 (2009); and Sela et al., Applied and Enviromental Microbiology, 78, 795-803 (2012).

Similarly, GH-95 glycohydrolases are 1,2-alpha-L-fucosidases which hydrolyze Fuca1-2Gal linkages at the non-reducing end of an oligosaccharide. In some cases, a GH-95 glycohydrolase as used herein cannot hydrolyze the fucoysl linkage when the Gal residue is further modified. In some cases, the GH-95 glycohydrolases provided herein are predicted to cleave α1-2, α1-3, 2FL, 3FL, and Fuca1-2Gal substrates. GH-95 alpha-fucosidases described herein can confer the ability of gut bacteria to utilize, e.g. oligosaccharides, milk oligosaccharides, human milk oligosaccharides, or fucosyl oligosaccharides as a carbon source (e.g., as a sole carbon source).

Exemplary GH-95 alpha-fucosidases can include an N-terminal domain, a catalytic domain and/or an Ig-like domain. Consequently, in some cases, the GH-95 alpha-fucosidases, are expressed (e.g., heterologously expressed) as fragment polypeptides such that the catalytic activity and growth on HMO phenotype are preserved, but non-essential domains or fragments are removed or replaced. The essential features of GH-95 alpha-fucosidases are known in the art and are described in, e.g., Katayama, et al., Journal of Bioscience and Bioengineering, 99(5), 457-65 (2005); and Sela et al., Applied and Enviromental Microbiology, 78, 795-803 (2012).

Provided herein are bacterial alpha-fucosidase polypeptides (e.g., any of GH-29: SEQ ID NOs: 1-4 or GH-95: SEQ ID NOs: 5 or 6) and polynucleotides encoding such polypeptides (e.g., any of SEQ ID NOs: 7-10, and 11 or 12 respectively).

Also described herein are polypeptides substantially identical to the sequences exemplified herein, polynucleotides and expression cassettes encoding such alpha-fucosidase polypeptides or a mutation or fragment thereof, and vectors or other constructs for alpha-fucosidase polypeptide expression in a microorganism (e.g., a Bifidobacterium or a Lactobacillus). Also described herein are polypeptides which are substantially similar to the exemplified sequences (e.g., SEQ ID NOs: 1-6). Polypeptides which are “substantially similar” share sequences as noted above except that residue positions which are not identical may differ by conservative amino acid changes.

Polynucleotides that selectively hybridize to, and/or are substantially identical to, one of SEQ ID NOs: 7-12 are also provided herein. The phrase “selectively (or specifically) hybridizes to” refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA). Polynucleotides that selectively hybridize to any one of SEQ ID NOs: 7-12 can be of any length, e.g., at least 10, 15, 20, 25, 30, 50, 100, 200 500 or more nucleotides or having fewer than 500, 200, 100, or 50 nucleotides, etc.

Other Glycohydrolases

Provided herein are other glycohydrolases that are capable of hydrolyzing components of human milk oligosaccharides, and other saccharides of similar structure. The glycohydrolases can be expressed in bacteria (e.g., beneficial gut bacteria) and confer the ability of the bacteria to hydrolyze components of human milk oligosaccharides and other saccharides of similar structure. In some cases, the glycohydrolases can thus confer the ability of the bacteria to utilize the human milk oligosaccharides as a carbon and/or nitrogen source. In some cases, this can provide a selective advantage as compared to other microorganisms present.

For example, bacteria expressing glycohydrolases capable of hydrolyzing components of human milk oligosaccharides can grow more quickly, or become a larger portion of the microbiome in the gut of a subject that is consuming human milk oligosaccharides, as compared to bacteria that do not express such glycohydrolases. In some embodiments, this selective advantage can be utilized by providing glycohydrolases capable of hydrolyzing components of human milk oligosaccharides to bacteria known or suspected of being beneficial. In other cases, bacteria known or suspected of being beneficial can be assayed to determine their glycohydrolases and thus an pre-biotic composition or formulation can be applied to select for the beneficial bacteria.

Glycohydrolases described herein include alpha-sialidases, beta-galactosidases, beta-hexosaminidases, and alpha-fucosidases. Alpha-sialidases (EC:3.2.1.18 COG4409) are enzymes which catalyze the hydrolysis of alpha-(2->3)-, alpha-(2->6)-, alpha-(2->8)-glycosidic linkages of terminal sialic acid residues in oligosaccharides, glycoproteins, glycolipids, colominic acid, and synthetic substrates. Members of this family contain multiple BNR (bacterial neuraminidase repeat) repeats or Asp-boxes. The repeats are short, however the repeats are never found closer than 40 residues together suggesting that the repeat is structurally longer. These repeats are found in a variety of non-homologous proteins, including bacterial ribonucleases, sulphite oxidases, reelin, netrins, sialidases, neuraminidases, some lipoprotein receptors, and a variety of glycosyl hydrolases. See, e.g., Schauer, R. Sialic acids. Adv. Carbohydr. Chem. Biochem. 40 (1982) 131-234.

Beta-galactosidase (EC: 3.2.1.23 COG1874) catalyzes hydrolysis of terminal non-reducing beta-D-galactose residues in beta-D-galactosides. This class comprises a widespread group of enzymes that hydrolyze the glycosidic bond between two or more carbohydrates, or between a carbohydrate and a non-carbohydrate moiety. A classification system for glycosyl hydrolases, based on sequence similarity, has led to the definition of 85 different families. See, e.g., Kuby, S. A. and Lardy, H. A. Purification and kinetics of beta-D-galactosidase from Escherichia coli, strain K-12. J. Am. Chem. Soc. 75 (1953) 890-896.

N-acetyl-beta-hexosaminidase (EC:3.2.1.52 COG3525) catalyzes the hydrolysis of terminal non-reducing N-acetyl-D-hexosamine residues in N-acetyl-beta-D-hexosaminides. This class comprises a widespread group of enzymes that hydrolyze the glycosidic bond between two or more carbohydrates, or between a carbohydrate and a non-carbohydrate moiety. See, e.g., Isolation of beta-N-acetylhexosaminidase, beta-N-acetylglucosaminidase, and beta-N-acetylgalactosaminidase from calf brain. Biochemistry. 6 (1967) 2775-82.

IV. Beneficial Gut Bacteria

As described herein, beneficial gut bacteria include those that reside in the gut of an individual or can be introduced into the gut of an individual (e.g., are capable of growth in the gut without pathogenesis) and confer a health benefit. In some embodiments, the beneficial gut bacteria express an alpha-fucosidase, such as GH-29. In other embodiments, the beneficial gut bacteria express a GH-95 alpha-fucosidase. In some cases, the beneficial bacteria express at least two alpha-fucosidases, such as a GH-29 and a GH-95 alpha-fucosidase. The alpha-fucosidases can be endogenous glycohydrolases, i.e., the glycohydrolases occur naturally in the strain. In other cases, at least one (e.g., 1, 2, 3, or 4) of the alpha-fucosidases are heterologous. In some cases, the heterologous gene is introduced as a recombinant expression cassette, and the beneficial gut bacteria is transgenic. In other cases, the heterologous gene is introduced by a natural process, such as bacterial mating, and the beneficial gut bacteria expresses a heterologous gene and yet the bacteria is not transgenic.

In general, beneficial gut bacteria are selected from species that are normally found in the gut of a human infant, a breast-fed human infant, a formula-fed human infant (e.g., a milk, soy, or corn based formula), an adolescent, an adult, or a cow or other animal. Beneficial gut bacteria are selected from species of bacteria that do not cause pathogenesis in the host organism. In some embodiments, the beneficial gut bacteria are selected from species of bacteria that are only opportunistically pathogenic in cases of immune-deficiency or autoimmune disease. Beneficial gut bacteria include lactobacilli and bifidobacteria.

In some embodiments, the beneficial gut bacteria can metabolize carbohydrates that cannot be digested by the host, such as one or more oligosaccharides (e.g., milk oligosaccharides, or human milk oligosaccharides). For example, the beneficial gut bacteria can express a GH-29 alpha-fucosidase, a GH-95 alpha-fucosidase, or multiple alpha-fucosidases (e.g., a GH-29 and a GH-95 alpha-fucosidase) and thus be capable of digesting one or more oligosaccharides (e.g., milk oligosaccharides, or human milk oligosaccharides). In some embodiments, the beneficial gut bacteria can generate metabolites that serve as nutrients for the host, serve an immunomodulatory function (e.g., reduce inflammation or stimulate mucosal epithelium), or signal the enteric nervous system. In still other embodiments, the beneficial gut bacteria regulate epithelial cell turnover, promote epithelial restitution, and/or reorganize tight junctions in the gut epithelium.

In some cases, the beneficial bacteria produce a conjugated linoleic acid or convert a conjugated linoleic acid. Conjugated linoleic acids are a family of linoleic acid isomers. Conjugated linoleic acids can be converted to linoleic acid or alpha-L-linoleic acid by bacterial strains in the gut. Inability of the gut microbiome to convert conjugated linoleic acids has been associated with digestive diseases, gluten sensitivity and/or dysbiosis. Dysbiosis is associated with inflammatory bowel disease and chronic fatigue syndrome. Described herein are methods of providing a gut microbiome (or a component thereof) to a subject in need thereof that is capable of producing or converting a conjugated linoliec acid.

V. Oligosaccharides

In some embodiments, GH-29 and/or GH-95 expressing bacteria as described herein are formulated with or administrated in conjunction with an oligosaccharide. Oligosaccharides described herein include human milk oligosaccharides (HMO) and oligosaccharides of a similar structure. In some embodiments, the oligosaccharides include those that are not digestible, or not substantially digestible, in a human gut without the aid of beneficial gut bacteria. Oligosaccharides herein include galacto-oligosaccharides (GOS) and oligosaccharides derived from a mammal such as a cow, a goat, a sheep, a horse, a buffalo, or a yak. In some embodiments, oligosaccharide containing compositions are adminstered to a subject in order to select for the growth and/or colonization of beneficial bacteria in the gut.

Human milk oligosaccharides (HMO) include, e.g., those described in U.S. Pat. No. 8,197,872. Human milk oligosaccharide compositions include compositions containing one or more of the following: 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 II, Difucosyllacto-N-Neoocataose II, Difucosyllacto-N-Neoocataose I, Lacto-N-Decaose, Trifucosyllacto-N-Neooctaose, Trifucosyllacto-N-Octaose, and Trifucosyl-Iso-Lacto-N-Octaose. In some cases, HMO compositions can contain at least two or more of the foregoing oligosaccharides (e.g., 3, 4, 5, 6, 7, 8, 9, or more).

The HMOs described herein can be derived using any of a number of sources and methods known to those of skill in the art. For example, HMOs can be purified from human milk using methods known in the art. One such method for extraction of oligosaccharides from pooled milk entails the centrifugation of milk at 5,000×g for 30 minutes at 4° C. and fat removal. Ethanol can then be added to precipitate proteins. After centrifugation to sediment precipitated protein, the resulting solvent can be collected and dried by rotary evaporation. The resulting material can be adjusted to the appropriate pH (e.g., 6.8) with, for example, a phosphate buffer, and β-galactosidase can be added. After incubation, the solution can be extracted with chloroform-methanol, and the aqueous layer collected. Monosaccharides and disaccharides can removed by selective adsorption of HMOs using solid phase extraction with graphitized nonporous carbon cartridges. The retained oligosaccharides can be eluted with, e.g., water-acetonitrile (60:40) with 0.01% trifluoroacetic acid. (See, e.g., Ward et al., Appl. Environ. Microbiol. (2006), 72: 4497-4499; Gnoth et al., J. Biol. Chem. (2001), 276:34363-34370; Redmond and Packer, Carbohydr. Res., (1999), 319:74-79.) Individual HMOs can be further separated using methods known in the art such as capillary electrophoresis, HPLC (e.g., high-performance anion-exchange chromatography with pulsed amperometric detection; HPAEC-PAD), and thin layer chromatography. See, e.g., Splechtna et al., J. Agricultural and Food Chemistry (2006), 54: 4999-5006.

Alternatively, enzymatic methods can be used to synthesize the HMOs described herein. In general, any oligosaccharide biosynthetic enzyme or catabolic enzyme (with the reaction running in reverse) that converts a substrate into any of the HMO oligosaccharides (or their intermediates) may be used. For example, prebiotic galacto-oligosaccharides have been synthesized from lactose using the β-galactosidase from L. reuteri (See, Splechtna et al., J. Agricultural and Food Chemistry (2006), 54: 4999-5006). The reaction employed is known as transgalactosylation, whereby the enzyme β-galactosidase hydrolyzes lactose, and, instead of transferring the galactose unit to the hydroxyl group of water, the enzyme transfers galactose to another carbohydrate to result in oligosaccharides with a higher degree of polymerization (Vandamme and Soetaert, FEMS Microbiol. Rev. (1995), 16:163-186). The transgalactosylation reaction can proceed intermolecularly or intramolecularly. Intramolecular or direct galactosyl transfer to D-glucose yields regioisomers of lactose. Through intermolecular transgalactosylation di-, tri-, and tetra saccharides and eventually higher oligosaccharides specific to Bifidobacteria are produced. A related method utilizes the β-galactosidase of Bifidobacterium bifidum NCIMB 41171 to synthesize prebiotic galacto-oligosaccharides (See, Tzortzis et al., Appl. Micro. and Biotech. (2005), 68:412-416).

Another approach to the synthesis of the carbohydrates of the invention that combines elements of the methods outlined above entails the chemical or enzymatic synthesis of or isolation of oligosaccharide backbones containing Lacto-N-biose, or Lacto-N-tretrose from non-human mammalian milk sources (e.g., cows, sheep, buffalo, goat, horse, yak, etc.) and enzymatically adding Lacto-N-biose, Fucose and Sialic Acid units as necessary to arrive at the HMO. For this purpose, a variety of bifidobacterial carbohydrate modifying enzymes, such as those disclosed in PCT Publication WO 2008/033520 can be utilized. Examples of such oligosaccharide modifying enzymes include sialidases, silate O-Acetylesterases, N-Acetylneuraminate lyases, N-acetyl-beta-hexosaminidase, beta-galactosidases, N-acetylmannosamine-6-phosphate 2-epimerases, alpha-L-fucosidases, and fucose dissimilation pathway proteins, among others, which may be used to catalyze a biosynthetic reaction under the appropriate conditions.

Alternatively, conventional chemical methods may be used for the de novo organic synthesis of or conversion of pre-existing oligosaccharides into the HMO oligosaccharides described herein. See, e.g., March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th Edition.

Galacto-oligosaccharide (GOS) compositions include, e.g., those described in U.S. Pat. No. 8,425,930. GOS are naturally occurring in human milk, however, commercial GOS preparations are often produced by enzymatic treatment of lactose with β-galactosidases from different sources such as fungi, yeast and/or bacteria, yielding a mixture of oligomers with varied chain lengths (Angus, F., Smart, S, and Shortt, C. 2005. In Probiotic Dairy Products ed. Tamine, A. pp. 120-137. Oxford: Blackwell Publishing). Thus, the basic structure of GOS includes a lactose core at the reducing end which is elongated typically with up to six galactose residues. GOS structural diversity dependents on the enzyme used in the trans-galactosylation reaction, and the experimental conditions such as pH and temperature (Dumortier, V., et al. 1990. Carbohydr Res 201:115-23.).

In some embodiments, GOS compositions herein include those with a relatively high degree of polymerization (DP). The “DP” of a GOS refers to the total number of sugar monomer units that are part of a particular oligosaccharide. For example, a tetra GOS has a DP of 4, having 3 galactose moieties and one glucose moiety. In some cases, the GOS compositions include a GOS that has been enriched for DP 4-5 galacto-oligosaccharides, a GOS that has been enriched for DP 6-8 galacto-oligosaccharides, and a GOS that has been enriched for DP 3 galacto-oligosaccharides. Exemplary levels of enrichment can include GOS that contains at least 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the particular DP galacto-oligosaccharides by weight. Other exemplary levels of enrichment can include GOS that contains at least 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the particular galacto-oligosaccharides of a particular DP or higher by weight. In some cases, the enriched GOS compositions have less than 10% or less than 5% of sugar monomers (e.g., galactose) and optionally less than 10% or less than 5% of dimeric galacto-oligosaccharides. In some embodiments, the enriched compositions of the invention also have less than 10% or less than 5% of trimeric (DP3) galacto-oligosaccharides. In some cases, the GOS compositions contain a mixed population of galacto-oligosaccharides, for example a composition containing a mix of galacto-oligosaccharides of DP 3, 4, 5, 6, 7, 8, 9, or 10, or other combinations thereof. Methods of purifying or preparing GOS compositions are known in the art (see, e.g., U.S. Pat. No. 8,425,930).

VI. Formulations

In general, any food or beverage that can be consumed by human infants or adults or animals may be used to make formulations containing the probiotic compositions described herein (e.g., compositions containing a bacteria expressing a GH-29 alpha-fucosidase, a GH-95 alpha-fucosidase, or expressing multiple alpha-fucosidases such as GH-29 and GH-95 fucosidases). Preferable foods include those with a semi-liquid consistency to allow easy and uniform dispersal of the prebiotic or probiotic compositions described herein. Accordingly, such food items include, without limitation, dairy-based products such as cheese, cottage cheese, yogurt, and ice cream. Fruits and vegetables targeted for infants/toddlers, such as apple sauce or strained peas and carrots (e.g., those from Gerber Products Company; Fremont, Mich.) are also suitable for use in the present invention. Both infant cereals such as rice- or oat-based cereals (e.g., Gerber) and adult cereals such as Musilix may also be suitable for use in this invention. In addition to foods targeted for human consumption, animal feeds may also be supplemented with the prebiotic and probiotic compositions of the invention.

Alternatively, the prebiotic and probiotic compositions of the invention may be used to supplement a beverage. Examples of such beverages include, without limitation, infant formula, follow-on formula, toddler's beverage, milk, fermented milk, fruit juice, fruit-based drinks, and sports drinks Many infant and toddler formulas are known in the art and are commercially available, including, for example, Carnation Good Start (Nestle Nutrition Division; Glendale, Calif.) and Nutrish A/B produced by Mayfield Dairy Farms (Athens, Term.). Other examples of infant or baby formula include those disclosed in U.S. Pat. No. 5,902,617. Other beneficial formulations of the compositions of the present invention include the supplementation of animal milks, such as cow's milk, which are normally lacking in HMOs.

Alternatively, the prebiotic and probiotic compositions of the present invention can be formulated into pills or tablets or encapsulated in capsules, such as gelatin capsules. Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers. Lozenge or candy forms can comprise the compositions in a flavor, e.g., sucrose, as well as pastilles comprising the compositions in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art. The inventive prebiotic or probiotic formulations can also contain conventional food supplement fillers and extenders such as, for example, rice flour.

In some embodiments, the prebiotic or probiotic composition can further comprise a non-human protein, non-human lipid, non-human carbohydrate, or other non-human component. For example, in some embodiments, the compositions of the invention comprise a bovine (or other non-human) milk protein, a soy protein, betalactoglobulin, whey, soybean oil or starch.

Alternatively, the prebiotic and probiotic compositions of the present invention can be administered to the subject in a manner that administers the composition to the gut, but bypass the oral cavity (e.g., the mouth or esophagus) or the stomach. For example, the compositions can be administered rectally, directly to the colon, or directly to the small intestine. In some cases, the method may include techniques to deliver the composition to the colon without delivering the composition to the small intestine.

The dosages of the prebiotic and probiotic compositions of the present invention can be varied depending upon the requirements of the individual and will take into account factors such as age (infant versus adult), weight, and reasons for the need for administration of or selection for beneficial gut bacteria (e.g., antibiotic therapy, chemotherapy, disease, or age). The amount administered to an individual, in the context of the present invention should be sufficient to establish colonization of the gut with beneficial bacteria over time. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that may accompany the administration of a prebiotic or probiotic composition of the present invention. The dosage range, effective as a food supplement and for reestablishing beneficial bacteria in the intestinal tract, ranges from about 1 micrograms/L to about 25 grams/L. A further advantageous range is about 100 micrograms/L to about 15 grams/L. Another useful range is 1 gram/L to 10 grams/L. In one embodiment, a concentration of 8 grams/L is preferred. (See, e.g., Knol et al., J. Pediatric Gastro. and Nutr. (2005) 40:36-42.) When used, Bifidobacteria may be included in the formulations of the invention in an amount of 10⁶ to 10¹² colony forming units (CFU). A further advantageous range is 10⁸ to 10¹² CFU. In one advantageous embodiment, 10¹⁰ CFU of Bifidobacteria may be included in the formulations of the invention.

It will be appreciated that it may be advantageous for some applications to include other pre-biotic factors in the formulations of the present invention. Such additional components may include, but are not limited to, fructooligosaccharides such as Raftilose (Rhone-Poulenc, Cranbury, N.J.), inulin (Imperial Holly Corp., Sugar Land, Tex.), and Nutraflora (Golden Technologies, Westminister, Colo.), as well as xylooligosaccharides, galactooligosaccharides, soyoligosaccharides, lactulose/lactitol, among others.

The present invention includes methods of making any of the above-described compositions. For example, the invention provides for methods of combining at least one or more oligosaccharides described herein with a non-human protein, non-human lipid, non-human carbohydrate, or other non-human component to produce a synthetic prebiotic or probiotic food. For example, in some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the oligosaccharides described herein are combined with a non-human protein, non-human lipid, non-human carbohydrate, or other non-human component. In some embodiments, at least one or more oligosaccharide of the present invention are combined with a bovine (or other non-human) milk protein, a soy protein, beta-lactoglobulin, whey, soybean oil or starch.

VII. Examples

Introduction

Human milk contains a high concentration of complex oligosaccharides that influence the composition of the intestinal microbiota in breast-fed infants. Select species such as Bifidobacterium longum subsp. infantis and B. bifidum can utilize human milk oligosaccharides (HMO) in vitro as the sole carbon source, while B. longum subsp. longum and B. breve are less adapted to these substrates. We sought to examine the adaptations of a more representative number of B. breve strains to human milk oligosaccharides. For this purpose, a number of B. breve isolates from breast-fed infant feces were characterized for the presence of different glycosyl hydrolases that participate in HMO utilization, as well as by their ability to grow on HMO or specific HMO species such as lacto-N-tetraose (LNT) and fucosyllactose. All B. breve strains showed a vigorous growth on lacto-N-tetraose and lacto-N-neotetraose (LNnT), and in general growth on total HMO was moderate for most of the strains, with several strain differences. Growth and consumption of fucosylated HMO was strain-dependent, primarily in isolates possessing a Glycosyl Hydrolase family 29 α-fucosidase. Glycoprofiling of the spent supernatant after HMO fermentation by select strains revealed that all B. breve can utilize sialylated HMO to a certain extent, especially sialyl-lacto-N-tetraose. Interestingly, this oligosaccharide was depleted before neutral lacto-N-tetraose by strain SC95. The present results indicate that the HMO consumption phenotype in B. breve is variable. Specific strains, however, display adaptations to substrates including fucosylated and sialylated HMO. The present results provide a rationale for the predominance of this species in breast-fed infant feces, and a more accurate picture of the ecology of the developing infant intestinal microbiota.

Example 1

Isolation and Identification of Bifidobacterium from Breast-Fed Infant Feces

Materials and Methods

Subjects.

Fecal samples were collected from 40 exclusively breast-fed term infants at 3 and 4 months of age. None of the infants enrolled in this study had received antibiotic treatment, infant-formula or solid food. Parents transferred their infant fecal samples into sterile plastic tubes and were instructed to immediately store the samples in −20° C. until transported by study personnel. Fecal samples were transported on dry ice and stored at −80° C. before processing.

Microbial Isolations.

For isolation of Bifidobacterium, 100 mg of each fecal sample was taken aseptically, transferred to a sterile tube, diluted tenfold with 1% peptone water (Becton Dickinson, Sparks, Md.), and homogenized by vortexing. Ten-fold dilutions were prepared with 1% peptone water and inoculated on modified BSM agar (mBSM). Modified BSM agar was prepared by supplementing de Man Rogosa Sharpe (MRS) media (Becton Dickinson, Sparks, Md.) with 15 g/L agar, 500 mg/L L-cysteine-HCl, 100 mg/L mupirocin, 25 mg/L kanamycin, 4.28 mg/L polymixin B, 25 mg/L iodoacetate, 20 mg/L nalidixic acid and 25 mg/mL of 2,3,5-triphenyltetrazoliumclhoride (Sigma). The plates were incubated for 48 h at 37° C. in an anaerobic chamber (Coy Laboratory Products, Grass Lake, Mich.), in an atmosphere containing 5% carbon dioxide, 5% hydrogen, and 90% nitrogen. Resulting colonies were streaked onto mBSM agar, and after two passages they were grown in MRS broth supplemented with 0.05% L-cysteine-HCl and stored at −80° C. in 25% glycerol. Prior to each assay all bacteria strains were subcultured twice in MRS broth supplemented with 0.05% L-cysteine-HCl and incubated at 37° C. for 18 h in an anaerobic chamber.

Additional B. breve strains were obtained from the Japanese Collection of Microorganism (RIKEN BioResource Center, Japan), the American Type Culture Collection (Manassas, Va.), and the University of California-Davis Viticulture and Enology Culture Collection (Table 1).

TABLE 1

List of Bifidobacterium strains used in this study.

Additional strain

Code

Species

information^a

Source

UCC2003

B. breve

O'Connell et al., 2011

Infant

nursing

stool

ATCC15700

B. breve

JCM1192; DSM20213

Infant feces

ATCC15698

B. breve

JCM1273; DSM20091

Infant feces

ATCC15701

B. breve

JCM7016

Infant feces

JCM7017

B. breve

Human feces

JCM7019

B. breve

Infant feces

JCM7020

B. breve

Infant feces

S-17c

B. breve

Roy et al. 1996

Infant feces

S-46

B. breve

Roy et al. 1996

Infant feces

SC81

B. breve

This study

Infant feces

SC95

B. breve

This study

Infant feces

SC139

B. breve

This study

Infant feces

SC154

B. breve

This study

Infant feces

SC500

B. breve

This study

Infant feces

SC506

B. breve

This study

Infant feces

SC508

B. breve

This study

Infant feces

SC522

B. breve

This study

Infant feces

SC559

B. breve

This study

Infant feces

SC567

B. breve

This study

Infant feces

SC568

B. breve

This study

Infant feces

SC573

B. breve

This study

Infant feces

SC580

B. breve

This study

Infant feces

SC670

B. breve

This study

Infant feces

KA179

B. breve

This study

Infant feces

ATCC15697

B. longum subsp.

JCM1222; DSM20088

Infant feces

infantis

JCM 10602

B. animalis subsp.

DSMZ 10140

Dairy

lactis

product

^aThe original strain numbers are also noted, if known. JCM, Japan Collection of Microorganisms, ATCC, American Type Culture Collection; DSMZ, German Collection of Microorganisms and Cell Culture.

Identification of Bifidobacteria by 16S rDNA Sequencing.

Genomic DNA was obtained from 1 ml of each culture, and centrifuged for 5 min at 2000×g. The bacterial pellet was resuspended and incubated for 30 min at 37° C. with enzymatic lysis buffer 20 mM Tris-Cl pH 8.0, 2 mM sodium EDTA, 1.2% Triton X-100, and 40 mg/ml lysozyme (Sigma, Mo.). After enzymatic lysis, bacterial DNA was isolated from the samples using the DNeasy tissue kit (Qiagen, Valencia, Calif.) according to the manufacturer instructions. DNA quality and yield was checked using a Nanodrop spectrophotometer (Wilmington, Del.); the DNA was then stored at −20° C. until further use. To identify the isolates at species level, the 16S rDNA gene was amplified by PCR using the universal primers 27F 5′-AGAGTTTGATCCTGGCTCAG and 1492R 5′-TACGGTTACCTTGTTACGA on an Applied Biosystems 2720m Thermal Cycler (Applied Biosystems, Mountain View, Calif.). One μl of extracted DNA was added to 50 μl reaction mixture containing 50 pmol of primers, 500 mM of each dNTP, 0.1 vol of 10×PCR buffer, 2.5 mM MgCl2, and 1 U AmpliTaq gold polymerase (Applied Biosystems). Amplification mixtures were subjected to 4 min of denaturation at 94° C., 30 cycles of 94° C. for 30 s, 55° C. for 40 s, and 72° C. for 1 min 30 s, followed by a final extension period of 7 min at 72° C. The resulting amplicons were separated on a 1% agarose gel, followed by GelRed staining (Phenix Research Products, Candler, N.C.), and purification using a QIAquick PCR Purification Kit (Qiagen, Valencia, Calif.). Sequencing was performed on an ABI 3730 Capillary Electrophoresis Genetic Analyzer using BigDye Terminator chemistries at the University of California Davis DNA Sequencing Facility. The sequences were analyzed using BioEdit 7.0 (available at the website at mbio.ncsu.edu/BioEdit/BioEdit.html), and checked by nucleotide-nucleotide BLAST comparison at the NCBI database for species identification.

Multilocus Sequence Typing (MLST) of Strains.

MLST analysis of B. breve isolates targeted intragenic regions of seven housekeeping genes clpC, purF, gyrB, fusA, Iles, rplB, rpoB. The PCR reaction was prepared as above and cycling conditions were optimized for every primer set (Table 2). The reaction included an initial denaturation at 95° C. for 4 min, followed by 35 cycles of 95° C. for 30 s, annealing at 60-67° C. for 30 s, elongation at 72° C. for 60 s, final extension at 72° C. for 7 min, and holding at 4° C. The PCR products were separated and sequenced as above.

TABLE 2

MLST genes and primers.

Expected

Amplicon

Anneling

size

Temp.

PCR primer (5′-3′)*^,a

(bp)

(° C.)

GAG TAC CGC AAG TAC ATC GAG

 748

63

CAT CCT CAT CGT CGA ACA GGA AC

CAT TCG AAC TCC GAC ACC GA

 977

62

GTG GGG TAG TCG CCG TTG

AGC TGC ACG CBG GCG GCA AGT TCG

 811

66

GTT GCC GAG CTT GGT CTT GGT CTG

ATC GGC ATC ATG GCY CAC ATY GAT

 784

66

CCA GCA TCG GCT GMA CRC CCT T

ATC CCG CGY TAC CAG ACS ATG

 789

66

CGG TGT CGA CGT AGT CGG CG

GGA CAA GGA CGG CRT SCC SGC CAA

 498

67

ACG ACC RCC GTG CGG GTG RTC GAC

GGC GAG CTG ATC CAG AAC CA

1057

62

GCA TCC TCG TAG TTG TAS CC

*Upper sequence, forward primer; Lower sequence, reverse primer.

^aIn the primer sequence R indicates (A/G), S (C/G), Y (C/T).

Sequencing data for all loci was edited using BioEdit 7.0 and aligned using CLUSTAL W. Phylogenetic analysis and concatenations of the sequenced loci were performed using the Molecular Evolutionary Genetic Analysis (MEGA) software version 5 (megasoftware.net). Descriptive evolutionary analysis including mol % G+C content, number of polymorphic sites, nucleotide diversity it/site, average number of nucleotide differences k were calculated using DnaSP version 5.10 (Table 3). Allelic sequences were assigned (see Cai et al. (2007) Microbiol. 153:2655) (Table 4). A minimum evolution tree of the concatenated loci was calculated using MEGA 5.0 (FIG. 1).

TABLE 3

Descriptive evolutionary analysis of MLST data.

Fragment

polymor-

Alle

analyzed

G + C

phic

frequen-

Gene

(bp)*

(mol %)

sites

cies

π

k

clpC

678 (0.25)

61.97

14

8

0.00404

2.739

purF

855 (0.56)

62.33

65

11

0.01308

11.18116

gyrB

688 (0.33)

60.17

14

12

0.00335

2.30435

fusA

753 (0.35)

60.35

14

8

0.00319

2.4058

Iles

743 (0.22)

61.51

41

12

0.0148

10.78986

rplB

428 (0.51)

64.75

8

5

0.00273

1.16667

rpoB

965 (0.27)

62.89

16

11

0.00353

3.4058

*Percentage of the gene is given in parenthesis.

π = mean pairwise nucleotide difference per site.

k = mean pairwise nucleotide difference per sequence.

TABLE 4

Allelic profiles of 24 B. breve strains analyzed by MLST.

Allele

Strains

ST^a

clpC

purF

gyrB

fusA

Iles

rplB

rpoB

UCC2003

1

1

1

1

1

1

1

1

ATCC15700

2

1

2

2

1

2

2

2

ATCC15698

3

1

3

3

1

3

3

3

ATCC15701

4

2

4

4

1

4

3

4

JCM7017

4

2

4

4

1

4

3

4

JCM7019

5

3

5

5

7

5

3

5

JCM7020

6

4

6

5

8

6

3

6

S-17c

7

1

7

6

1

4

3

2

S-46

8

2

4

4

1

4

3

7

SC81

9

5

8

7

2

7

3

8

SC95

10

6

9

8

1

3

3

3

SC139

11

7

3

8

1

8

3

4

SC154

12

2

1

8

1

3

3

1

SC500

13

1

10

3

2

3

4

9

SC506

14

6

9

8

3

3

1

3

SC508

15

1

1

9

1

9

5

2

SC522

9

5

8

7

2

7

3

8

SC559

16

1

1

10

1

10

3

10

SC567

17

1

11

8

6

11

3

1

SC568

10

6

9

8

1

3

3

3

SC573

18

2

4

11

1

3

3

3

SC580

9

5

8

7

2

7

3

8

SC670

19

1

1

12

5

11

3

11

KA179

20

8

3

8

4

12

3

9

^aST Indicates specific sequence type.

Results

Around 500 isolates were identified by 16S rDNA sequencing, and a total of 461 isolates were identified as Bifidobacterium. Seven species of bifidobacteria were detected, and the species longum, which includes subspecies longum and infantis, was found to be more represented, followed by B. breve with 77 strains (Table 5).

TABLE 5

Distribution of isolates of bifidobacteria from

breast-fed infants identified by 16S DNA.

Number of isolates

Species

identified

B. longum/B. infantis

297

B. breve

77

B. pseudocatenulatum

45

B. bifidum

22

B. dentium

8

B. adolescentis

7

B. animalis

5

We further investigated the identity of the B. breve isolates at the strain level by MLST (Deletoile et al. (2010) Res Microbiol. 161:82). The analysis also included nine strains from culture collections (Table 1). A total of 172 single nucleotide polymorphisms (SNPs) were found in seven loci, and they generated between 8 rplB and 65 purF polymorphic sites (Table 3). Twenty different allelic profiles were identified in the 86 B. breve isolates analyzed (Table 4). Some strains isolated from the unrelated infants in the study shared similar profiles, and we conservatively considered them as different strains. This resulted in a library of 24 strains of B. breve (Table 1), for which a consensus phylogenetic tree of the concatenated MLST data is shown in FIG. 1.

Example 2

Characterization of Glycosyl Hydrolases from Isolated Strains

Materials and Methods

In order to study the potential adaptations of the B. breve isolates for growth on HMO, we first determined the presence of three key GH classes required to cleave HMO into its constituent monosaccharides. β-galactosidase activity was not observed because it is widespread in the Bifidobacterium genus. α-fucosidases (Blon_—2336, Blon_—2335, Blon_—0248/0426, Blon_—0346), α-sialidases (Blon_—2348, Blon_—0646), and β-hexosaminidase Blon_—0459 protein sequences identified in the genome of B infantis ATCC 15697 were aligned with homolgous sequences from the GeneBank database (Accession numbers showed in Table 6) using Bioedit 7.0 and degenerated primers were designed to amplify conserved regions (Table 7). To differentiate between Blon_—0248 and Blon_—0426, strains positive for fucosidase Blon_—0248/0426 were also amplified with the primers designed to amplify the complete gene in B. infantis ATCC 15697 (Table 7). PCR reactions were prepared as above with 200 pmol of primers. Cycling conditions were optimized for every primer set (Table 7), and consisted of an initial denaturation at 95° C. for 4 min, followed by 35 cycles of 95° C. for 30 s, annealing at 45-55° C. for 30 s, elongation at 72° C. for 60 s, final extension at 72° C. for 7 min; and holding at 4° C. The resulting amplicons were separated and sequenced as above. B. infantis ATCC 15697 and B. animalis JCM 10602 were used as positive and negative control strains, respectively.

TABLE 6

Genebank accession numbers for glycosyl hydrolase.

Glycosyl

hydrolase

name

Protein sequences accession numbers

Blon_2335

YP_002323771.1; ZP_06596922.1; ZP_03742645.1;

ZP_03167824.1; NP_241708.1; ZP_03474775.1;

YP_003010680.1; ZP_04552485.1; ZP_07812017.1

Blon_2336

YP_002323772.1; WP_003795385.1;

WP_007588699.1; YP_001297867.1;

WP_006775425.1; YP_003822597.1;

WP_008706707.1; WP_009776262.1

Blon_0248/0426

YP_002321754.1; ZP_08285605.1; ZP_08026776.1;

ZP_06607921.1; ZP_06184004.1; YP_002533924.1;

ZP_05989289.1; ZP_02477566.1; YP_001851141.1;

ZP_03212758.1; ZP_05280631.1

Blon_0346

YP_002321848.1; ZP_02040503.1; ZP_02079496.1;

ZP_08131039.1; ZP_05718978.1; YP_004456548.1;

P_003242853.1;YP_002547035.1

Blon_2348

YP_002323784.1; WP_003818390.1; ACH92844.1;

WP_003796112.1; ACH92824.1; BAD66680.2

Blon_0646

YP_002322131.1; YP_007554019.1

Blon_0459

YP_002321953.1., YP_007555353.1

TABLE 7

Glycosyl hydrolase gene and qPCR primers.

Expected

amplicon

Anneling

size 

Temp.

Primer name

Primer sequence (5′-3′)^a

(bp)

(° C.)

Blon_2335F

GARATGAAYTAYTGGATG

960

56° C.

Blon_2335R

TTNCCRTCDATYTGRAANGGNGG

Blon_2336F

AARCAYCAYGAYGGNTTYTG

600

55° C.

Blon_2336R

ACYTCNGCNGGRTACCA

Blon_0248/0426F 

TAYGCNGARTGGTAY

210

45° C.

Blon_0248/0426R 

TCRTGRTGYTTNGTNGT

Blon_0346F

YTNGAYTTYCAYACNWS

740

48° C.

Blon_0346R 

TCRTGRTGYTTNGTNGT

Blon_2348F 

ATHACNGCNGAYATHAC

250

45° C.

Blon_2348R 

TCNACNACYTTRTTYTCRTC

Blon_0646F 

CCACCAGACATGGAACAGTG

220

60° C.

Blon_0646R 

AAATCGCCGAAGGTGATATG

Blon_0459F 

CCCCACCCTCGACTGGCTCA

510

62° C.

Blon_459R

CTTCGAGGTGGCACAGG

0248WF

ACCAACAACCAGCAACCAAT

135

56° C.

0248WR

ATCGAATACGGCACCTTCAG

0426WF

ACCAACAACCAGCAACCAAT

135

56° C.

0426WR

GACCGCCTTCATGGATAAGA

RNP-F

AACCTGATGATCGGACGACG

182

60° C.

RNP-R

GGCAAACTGCTCATCCAACG

60° C.

GH29-F

GGACTGAAGTTCGGCGTGTA

160

60° C.

GH29-R

TCGTTGTCCTCCTCCGAGAT

60° C.

GH95-F

CGCGGACTACCGCAGATATT

163

60° C.

GH95-R

ATCGAACATTGCCTCTGCCA

60° C.

^aIn the primer sequence R indicates (A/G), W (A/T), S (C/G), Y (C/T), H (A/C/T), D (A/G/T), N (A/C/G/T).

Results

The genome of B. breve UCC2003 (O'Connell et al. (2011) PNAS 108:11217) contains an α-fucosidase, an α-sialidase and a β-hexosaminidase with significant homology to cognate enzymes in B. infantis ATCC 15697. No homology was found to the same glycosyl hydrolases in B. bifidum genomes. Based on this, we used degenerate primers to look for genes encoding these GH in the assembled B. breve strains (Table 8). All of the B. breve strains possessed a gene homologous to β-hexosaminidase Blon_—0459 in B. infantis (Gamido et al. (2012) Mol Cell Proteomics 11:775), an α-fucosidase similar to Blon_—2335 in B. infantis ATCC 15697 (Sela et al. (2012) Appl. Env. Microbiol. 78:795) and all strains excepting JCM 7020 possessed an α-sialidase, related to Blon_—0646 in B. infantis (Sela et al. (2011) J. Biol. Chem. 286:11909). Moreover, five strains possessed a second α-fucosidase, homolog to locus tag Blon_—0248 in B. infantis ATCC 15697 (Sela et al. (2012) Appl. Env. Microbiol. 78:795) that belongs to GH family 29 (Table 8 and FIG. 2).

TABLE 8

Presence of glycosyl hydrolases and growth in different HMO by B. breve strains

Glycosyl hydrolases^a

α-fucosidase

α-sialidase

β-hexosaminidase

Bacterial growth

Strain

GH95

GH29

GH33

GH20

HMO^b

LNT

LNnT

2FL

3FL

3SL

6SL

UCC2003

+

−

+

+

+

+++

+++

−

+

−

−

ATCC15700

+

−

+

+

+

+++

+++

−

−

−

−

ATCC15698

+

−

+

+

++

+++

+++

−

−

−

−

ATCC15701

+

−

+

+

+++

+++

+++

−

−

−

−

JCM7017

+

−

+

+

++

+++

+++

−

−

−

−

JCM7019

+

−

+

+

++

+++

+++

+

+

−

−

JCM7020

+

+

−

+

++

+++

+++

−

−

−

−

S-17c

+

−

+

+

+

+++

+++

−

+

−

−

S-46

+

−

+

+

++

+++

+++

−

+

−

−

SC81

+

−

+

+

++

+++

+++

−

−

−

−

SC95

+

+

+

+

+++

+++

+++

+++

+

−

−

SC139

+

−

+

+

++

+++

+++

−

−

−

−

SC154

+

+

+

+

+++

+++

+++

−

−

−

−

SC500

+

−

+

+

++

+++

+++

−

−

−

−

SC506

+

+

+

+

++

+++

+++

−

−

−

−

SC508

+

−

+

+

+

+++

+++

−

−

−

−

SC522

+

−

+

+

++

+++

+++

−

+

−

−

SC559

+

−

+

+

++

+++

+++

−

−

−

−

SC567

+

−

+

+

++

+++

+++

−

−

−

−

SC568

+

+

+

+

++

+++

+++

+++

+

−

−

SC573

+

−

+

+

+

+++

+++

−

+

−

−

SC580

+

−

+

+

++

+++

+++

−

+

−

−

SC670

+

−

+

+

+

+++

+++

−

−

−

−

KA179

+

−

+

+

++

+++

+++

+

−

+

+

ATCC15697

+

+

+

+

+++

+++

+++

+++

+++

+++

+++

JCM10602

−

−

−

−

−

−

−

−

−

−

−

^aPositive amplification + indicates that the sequence of the PCR product is >55% homologous at the aminoacid level to the respective GH gene in B. infantis ATCC15697.

^bLevel of growth was classified as Negative −: OD <0.200; Low +: OD 0.200-0.550; Moderate ++: OD 0.550-0.750; High +++: OD >0.750

Example 3

Characterization of the Growth of Isolated Strains on Human Milk Oligosaccharides

Materials and Methods

The 24 B. breve strains in Table 1 were tested for growth in the presence of seven different substrates: HMO (Ward et al. (2006) Appl. Env. Microbiol. 72:4497), LNT, lacto-N-neotetraose (LNnT), 2′-fucosyllactose (2FL), 3′-fucosyllactose (3FL) (Glycom, Denmark), 3′-sialyllactose (3SL), and 6′-sialyllactose (6SL) (GenChem. Inc. Korean). B. infantis ATCC 15697, and B. animalis JCM 10602 were included as positive and negative control for growth in HMO, respectively. Two μl of each resulting overnight culture was used to inoculate 200 μl of modified MRS (mMRS) medium supplemented with 2% (w/v) of each sterile-filtered substrate as the sole carbohydrate source, and another 2 μl inoculated into mMRS without added sugar. The media was supplemented with 0.05% (w/v) L-cysteine, and in all the cases the cultures in the wells of the microliter plates were covered with 30 μl of sterile mineral oil to avoid evaporation. The incubations were carried out at 37° C. in an anaerobic chamber (Coy Laboratory Products, Grass Lake, Mich.). Cell growth was monitored in real time by assessing optical density (OD) at 600 nm using a BioTek PowerWave 340 plate reader (BioTek, Winoosky, Vt.) every 30 min preceded by 15 seconds shaking at variable speed. Two biological replicates and three technical replicates each were performed for every studied strain. Maximum ODs and growth rates were calculated and expressed as the mean of all replicates with the respective standard deviation. These calculations were performed as described in Breidt et al. (1994) J. Rapid Meth. Autom. Microbiol. 3:59) The OD obtained for each strain grown on the different substrates, was compared with the OD obtained in the absence of sugar source. This difference in OD (ΔOD) was used as a parameter to evaluate the strain's ability for growing on the different substrates.

Results

Growth behavior on HMO and maximum OD values obtained were parameters to classify this panel in three groups (Table 8).

In general, a moderate growth on HMO was witnessed for all the strains (Table 8 and Table 9), with some strain level differences (Table 8). Interestingly, three strains (SC95, SC154 and ATCC 15701) exhibited remarkable growth on HMO compared to the type strain B. breve ATCC 15700, but still lower overall growth and growth rate relative to B. infantis ATCC 15697 (FIG. 3A and Table 9).

TABLE 9

Kinetic analysis of bacterial growth in 2% HMO.

Kinetic parameters in 2% HMO

Growth

rate

Lag time

Generation

Max. OD

Strain

(1/h)

SD

(h)

SD

time (h)

SD

(600 nm)

SD

UCC2003

6.70E−02

±7.55E−03

3.070

±0.132

4.531

±0.543

0.524

±0.055

ATCC15700

6.16E−02

±1.12E−02

5.420

±0.042

5.007

±1.001

0.538

±0.025

ATCC15698

7.98E−02

±2.14E−03

4.260

±0.118

3.772

±0.102

0.656

±0.065

ATCC15701

5.77E−02

±2.22E−03

2.445

±0.881

5.225

±0.206

0.779

±0.040

JCM7017

7.19E−02

±4.50E−03

5.549

±0.096

4.199

±0.257

0.656

±0.021

JCM7019

9.94E−02

±4.55E−03

7.653

±0.310

3.033

±0.135

0.655

±0.014

JCM7020

8.51E−02

±2.08E−03

4.016

±0.083

3.538

±0.087

0.661

±0.015

S-17c

7.26E−02

±7.21E−03

6.161

±0.237

4.170

±0.398

0.540

±0.013

S-46

8.36E−02

±9.27E−03

4.413

±0.073

3.627

±0.378

0.71

±0.033

SC81

1.07E−01

±4.81E−03

6.331

±0.108

2.825

±0.129

0.715

±0.033

SC95

1.20E−01

±6.43E−03

4.655

±0.047

2.523

±0.131

0.859

±0.029

SC139

9.41E−02

±9.16E−03

5.390

±0.204

3.219

±0.297

0.667

±0.015

SC154

7.54E−02

±5.22E−03

6.295

±0.166

4.007

±0.281

0.768

±0.031

SC500

5.24E−02

±3.65E−03

13.512

±0.362

5.759

±0.404

0.558

±0.026

SC506

5.92E−02

±2.66E−03

3.806

±0.050

5.088

±0.222

0.731

±0.0007

SC508

4.26E−02

±2.37E−03

5.157

±0.070

7.086

±0.390

0.277

±0.054

SC522

4.88E−02

±1.14E−02

1.050

±0.223

6.439

±1.715

0.698

±0.047

SC559

6.26E−02

±1.27E−03

6.311

±0.137

4.807

±0.098

0.612

±0.0015

SC567

5.76E−02

±5.49E−03

9.953

±0.765

5.256

±0.529

0.567

±0.042

SC568

6.26E−02

±2.91E−03

6.216

±0.524

4.815

±0.220

0.680

±0.034

SC573

3.31E−02

±3.45E−03

3.419

±0.123

9.168

±0.933

0.306

±0.014

SC580

6.34E−02

±3.82E−03

2.045

±0.204

4.762

±0.284

0.727

±0.028

SC670

3.38E−02

±5.98E−03

9.886

±0.234

9.083

±1.505

0.332

±0.054

KA179

1.13E−01

±2.83E−03

6.990

±1.144

2.673

±0.066

0.606

±0.038

ATCC15697

2.07E−01

±3.29E−03

3.930

±0.051

1.452

±0.022

1.295

±0.015

JCM10602

1.10E−02

±1.45E−03

14.919

±2.389

27.578

±3.368

0.180

±0.025

All B. breve strains grew on LNT and LNnT to high cell densities and at levels comparable to B. infantis ATCC 15697 (Table 8). Interestingly, a few strains were able to grow on fucosylated HMO (FIG. 3B and Table 8). The isolates SC95 and SC568 grew well on 2FL, to a similar extent than B. infantis ATCC 15697. Using 3FL as the sole carbon source, only the strains SC95 and JCM 7019 showed growth (Table 8). Finally, growth on 3SL and 6SL was only observed for one strain (Table 8).

Example 4

Glycoprofiling and Gene Expression Analysis of Isolated Strains

Materials and Methods

Glycoprofiling.

Bacterial cultures in mMRS with 2% HMO were collected at the end of the exponential phase and centrifuged at 12000×g for 30 min. In the case of B. breve SC95, the samples were collected at three different points in the growth curve, approximately OD_600nm=0.2, 0.5 and 0.8. At least two biological replicates were performed in triplicate. Supernatants were filtered using a multiscreen 96-well filtration plate 0.22 μm (Millipore, Billerica, Mass.) prior to storage at −80° C. Remaining oligosaccharides were recovered from the supernatants (25 μl) and reduced to their alditol forms with 1M NaBH₄ at 65° C. for 1.5 h. Each replicate was desalted by solid-phase extraction on graphitized carbon cartridges. Salts were removed with 6 mL of deionized water and oligosaccharides were eluted with 20% acetonitrile in water (v/v) and with 40% acetonitrile in 0.01% trifluoroacetic acid (v/v). SPE fractions were combined and dried under vacuum. Samples were reconstituted in 100 μl of deionized water and diluted 50-fold for LC-MS analysis.

An Agilent high performance liquid chromatography chip time of flight (HPLC-Chip/TOF) mass spectrometer equipped with a capillary pump for sample loading and a nano pump for chromatographic separation was used for HMO analysis. Separation was performed on a microfluidic chip equipped with an enrichment and nano-LC analytical column, both packed with porous graphitized carbon. Briefly, HMO were separated by a 65 min gradient using a binary solvent system consisting of 3% acetonitrile/water in 0.1% formic acid (v/v) solvent A and 90% acetonitrile/water in 0.1% formic acid (v/v) solvent B. HMO were analyzed in positive ion mode, with a mass range between 300-2000 m/z. Agilent's Masshunter software version B.03.01 was used for data acquisition and data analysis.

HMO monosaccharide composition was determined using accurate mass within ±20 ppm mass error of theoretically calculated masses. Specific structures were assigned to HMO peaks by matching the reproducible retention time to that reported in annotated HMO libraries. Percent consumption was calculated using a label-free method, employing the un-inoculated HMO pool as an external standard. Total HMO consumption was calculated with respect to the un-inoculated control by normalizing the summed abundance of all identified oligosaccharide spectra in ion counts in the bacterial supernatant to that of the control using the following equation:

[

1

-

(

∑

i

=

1

n





API





bacteria





sample

∑

i

=

1

n



API





un-incoluated





control

)

]

×

100

where API is absolute peak intensity and n is the number of identified HMO. The consumption of individual HMO species was quantitated in the same manner, in which the absolute peak intensity of a specific HMO structure was normalized to the peak intensity of the corresponding structure in the un-inoculated control.

Gene Expression Analysis.

The full nucleotide sequences of the genes encoding a GH95 and a GH29 α-fucosidase in the strain B. breve SC95 generated were used to design qPCR primers using the primer-BLAST tool at NCBI (Table 7). For relative quantification, the rnpA gene protein component of ribonuclease P complex was used. B. breve SC95 was grown as described above in mMRS supplemented with either 2% lactose, 2% HMO or 2% 2FL in a microplate reader, and cultures were taken at mid-exponential phase OD 0.6-1.0. Samples were immediately pelleted at 12000×g for 1 min and stored in RNA later Ambion. RNA extraction, cDNA conversion and qPCR were performed (Gamido et al. (2012) Anaerobe 18:430).

Results

Based on their growth kinetic parameters and ability to utilize certain glycans, six strains of B. breve were selected to examine the consumption of 22 different oligosaccharides during growth on total HMO. This included strains SC95, SC154, SC568, SC580, ATCC15701, and JCM7019, as well as B. infantis ATCC 15697 and B. breve ATCC 15700 as positive and negative controls respectively. The supernatant was collected at the end of the exponential phase during growth on HMO, and remaining oligosaccharides were purified and reduced, and later detected and quantified by nano HPLC/CHIP TOF MS. Specific oligosaccharide and isomers were identified using two oligosaccharide structures libraries (Wu et al. (2011) J. Proteome Res. 10:856; Wu et al. (2010) J. Proteome Res. 9:4138). The oligosaccharides quantified include the most abundant neutral and sialylated HMO, and Table 10 shows their names, masses, chemical structure, and degree of polymerization (DP).

Among the six strains selected, total consumption of HMO ranged between 23 and 42%. These values are lower compared to B. infantis ATCC 15697 (64% consumption) but clearly higher than B. breve ATCC 15700 (FIG. 4A). FIG. 4B shows the consumption of neutral non-fucosylated HMO in more detail. We observed that for the B. breve strains the consumption patterns were similar. All strains were able to deplete LNT/LNnT from the culture media to a high extent. Among three major hexaoses found in HMO, a preference for lacto-N-neohexaose (LNnH) was observed, over lacto-N-hexaose (LNH) and para-lacto-N-hexaose (p-LNH).

In general, the ability of B. breve to metabolize fucosylated HMO was lower compared to B. infantis, which showed high consumption levels for all the HMO tested (FIG. 5A). However, strains SC95, SC154 and SC568 showed a significant consumption of monofucosylated 2FL and LNFPII and difucosylated DFpLNHII and DFLNH-A HMO (FIG. 5A). Interestingly, even though 2FL cannot support the growth in vitro of strain SC154 (Table 8), this strain utilizes larger fucosylated HMO.

We observed that growth on fucosylated HMO was more prominent in strains which possessed an additional GH29 α-fucosidase (FIG. 5A and Table 8). We evaluated the relative gene expression of this gene in strain SC95. Growth on 2FL as the sole carbon source up-regulated 40-fold the expression of the GH29 fucosidase gene (FIG. 6). The expression of a GH95 fucosidase was not altered by growth on 2FL, suggesting that the presence of the GH29 fucosidase gene endows these strains with the ability to consume fucosylated oligosaccharides. In contrast, growth on total HMO did not affect the expression of these genes.

Acidic HMO represents approximately 15% of total HMO. We thus screened the consumption of eleven sialylated HMO in the spent supernatants of the listed strains during growth on total HMO (FIG. 5B). The levels of consumption were very similar among the strains tested, and comparable to B. infantis. In particular LSTb (sialyl-LNT b), sialyl-lacto-N-hexaose and mass 4121a/4121b were preferentially utilized by B. breve (FIG. 5B).

Finally, to elucidate possible substrate preferences in a B. breve strain with high HMO consumption, we monitored the consumption of nine representative oligosaccharides at different points during the fermentation of HMO by strain SC95 (FIG. 7). These HMO correspond to neutral and acid HMO that were consumed at levels higher than 40%. Remarkably, glycoprofiling of early exponential growth revealed that acidic HMO disappeared from the media first, together with LNnH. In contrast, LNT was metabolized first during the mid-exponential phase, and is majorly depleted at the end of the growth on HMO. We also observed that, while monofucosylated HMOs 2FL and LNFP II are depleted from the media at the mid-exponential phase and not later, difucosylated HMO appear to be steadily consumed during the three growth points (FIG. 7).

SUMMARY AND CONCLUSION

B. breve is one of the most representative species of bifidobacteria found in the infant intestinal microbiota. In order to determine whether free HMO contribute to the persistence of B. breve in the infant intestinal microbiota, we evaluated in detail the adaptations of a significant number of strains of B. breve to HMO.

The dominance of B. breve in this community has been supported by several studies, especially in breast-fed infants, where this species together with B. longum and B. infantis can largely outnumber other microorganisms. Breast milk itself is another habitat for this species, which, in addition to the vaginal and fecal microbiota of the mother, contribute to intestinal colonization of the infant. Some strains of this species are currently studied by their probiotic properties, for example in the production of conjugated linoleic acid or important immunomodulatory activities.

Since the predominance of bifidobacteria in breast-fed infants can be attributed in part to bioactive agents in milk such as HMO, the utilization of these substrates in vitro and in vivo is an important reflection of the adaptations of intestinal microorganisms to the environmental conditions prevalent in the infant gut. HMO utilization has only been shown for the type strain of B. breve ATCC 15700 (JCM 1192), and results indicate that this microorganism has a limited ability to consume HMO, almost exclusively LNT. Here we have expanded these observations and concluded that several infant-associated strains of B. breve can readily utilize HMO, consuming significantly larger amounts of total HMO compared to the type strain ATCC 15700. The HMO consumption in B. breve is however moderate by comparison to B. infantis ATCC 15697.

Mass spectrometry-based analysis of the HMO remaining after growth provides a detailed representation of the preferences of these strains for different oligosaccharide subsets present in the HMO pool. For example, all strains showed a vigorous growth on LNT and LNnT as a sole carbon source, and the molecular mass representing both oligosaccharide species (709) was the most consumed in pooled HMO. The utilization of LNnT is interesting since this oligosaccharide is not readily fermented by all species of Bifidobacterium found in the infant gut. Moreover growth on LNnT was shown to enable B. infantis to outcompete Bacteroides fragilis in a mouse model.

HMO with mass 1074 Da represent approximately 10% of the total HMO, and includes three neutral isomers, LNH, LNnH and p-LNH (Table 10). Interestingly, LNnH is the most abundant of the three isomers and it was selectively cleared from the growth media compared to the other two isomers. This indicates structure-based preferences in HMO consumption in B. breve (FIG. 4B and Table 10).

Strain-dependent differences were more evident in growth of B. breve on fucosylated HMO. Fucosidase activity has not been described previously in B. breve, and while all the strains studied possessed a gene encoding a GH95 α-fucosidase, we observed that the presence of a second α-fucosidase from GH29 in isolates SC95, SC568 and SC 154 correlated with their consistent growth and consumption of fucosylated HMO (FIG. 5A and Table 8). Some strains with this additional GH29 α-fucosidase did not, however, grow on 2FL and 3FL. These smaller HMO are imported by different transport mechanisms. For example, in B. infantis, 2FL and larger fucosylated HMO are likely imported by different solute binding proteins.

Remarkably, all B. breve strains consume pooled acidic HMO to a significant extent, and an α-sialidase was identified. All strains glycoprofiled showed a preferential consumption of select acidic HMO such as LSTb and S-LNH, but not smaller HMO, which might additionally explain why growth on 3SL and 6SL was negligible (Table 8).

The present results indicate that the mechanisms of HMO consumption in B. breve are very similar to B. infantis, with a preference to import intact oligosaccharides followed by intracellular degradation, rather than the extracellular degradation observed by B. bifidum. For example, B. breve strain ATCC 15700 can quickly deplete LNT from the spent media and the absence of intermediate monosaccharides indicates that this strain imports this substrate. Moreover, the GH genes identified in this study lacked signal peptides, indicating intracellular localization. Finally, the sequences obtained are homologous to previously characterized enzymes in B. infantis, including β-hexosaminidases Blon_—0459, two α-fucosidases Blon_—2335 and Blon_—0248 and an α-sialidase Blon_—0646, indicating a common origin.

The present results provide a rationale for the predominance of B. breve in the infant intestinal microbiota, improving our understanding about the ecology of this unique environment. The genetic variation of these strains and the strain-dependent character of the HMO utilization are factors to consider in probiotic and prebiotic studies. Better characterization of the diversity and physiology of beneficial strains of bifidobacteria, and more selective substrates that allow their implantation in the intestine, can be used to design selective synbiotic preparations.

VIII. Informal sequence listing

GH-29, Bifidobacterium longum subsp. infantis (B. infantis), Blon_0248,

Genbank Accession YP_002321754 (AfcB); SEQ ID NO: 1:

   1

mvlfmanpqr pkmyekfvhd tpewfkgagl gifahwgsys vpawaepiga lgtfddpvyw

  61

nthcpyaewy wntmsikgsp aaehqkevyg dmpyedfidm wkaeafdpad madlfaraga

 121

ryfvpttkhh egitlwkapd ndgwntvdrg phrdlvkefa damrdkglkf gvyyssgldw

 181

hkepnmpilg dgeygpqsed yarymyshvm dlideyqpsi lwgdidvpki seedndfsva

 241

rlfehyydvv pdgvvndrwg lthwdfrtve yeqgkelmgk gmwemtrgig ysfgynqmed

 301

adsymtgpea vklladvvsm ggn111digp daagripelq rqclegmadw mdvnspsihd

 361

vepvpeasps gegdgepwvr wtgdgksvya vvdaagrvpl riaadavdad savtlggsav

 421

avdadgdvlt advpasevag pqvvhfvrr

GH-29, Bifidobacterium longum subsp. infantis (B. infantis), Blon_0426,

Genbank Accession YP_002321924 (AfcB); SEQ ID NO: 2:

   1

mvlfmanpqr pkmyekfvhd tpewfkgagl gifahwgsys vpawaepiga lgtfddpvyw

  61

nthcpyaewy wntmsikgsp aaehqkevyg dmpyedfidm wkaeafdpad madlfaraga

 121

ryfvpttkhh egitlwkapd ndgwntvdrg phrdlvkefa damrdkglkf gvyyssgldw

 181

hkepnmpilg dgeygpqsed yarymyshvm dlideyqpsi lwgdidvpki seedndfsva

 241

rlfehyydvv pdgvvndrwg lthwdfrtve yeqgkelmgk gmwemtrgig ysfgynqmed

 301

adsymtgpea vklladvvsm ggn111digp daagripelq rqclegmadw mdvnspsihd

 361

vepvpeasps gegdgepwvr wtgdgksvya vvdaagrvpl ridagavdvd satilgggnv

 421

vveadgdmlt veipatdvag pqvvrfarh

GH-29, Bifidobacterium breve (B. breve) SC95, (AfcB); SEQ ID NO: 3:

   1

mvlfmanpqr pkmyekfvhd tpewfkgagl gifahwgsys vpawaepiga lgtfddpvyw

  61

nthcpyaewy wntmsikgsp aaehqkevyg dmpyedfidm wkaeafdpad madlfaraga

 121

ryfvpttkhh egitlwkapd ndgwntvdrg phrdlvkefa damrdkglkf gvyyssgldw

 181

hkepnmpilg dgeygpqsed yarymyshvm dlidkyqpsi lwgdidvpki seedndfsva

 241

rlfehyydvv pdgvvndrwg lthwdfrtve yeqgkelmgk gmwemtrgig ysfgynqmed

 301

adsymtgpea vklladvvsm ggn111digp daagripelq rqclegmadw myvnspsihd

 361

vepvpeasps gegdgepwvr wtgdgksvya vvdaagrvpl riaadavdad savtlggsav

 421

avdadgdvlt advpasevag pqvvhfvrr*

GH-29, Bifidobacterium bifidum, Genbank Accession BAH80310.1 (AfcB); 

SEQ ID NO: 4:

   1

mlhtasrgcs rswlrrltal iaysalafva 1pnvavaadp meyldvsfgg tfaadtyttg

  61

gdevakgpvt khgsiptkld gggitlaggt ngvtftstas fsesgkvnkg fraemeyrtt

 121

qtpsnlatlf samgnifvra ngsnleygfs tnpsgstwnd ytksvtlpsn nvkhiiqlty

 181

1pgadgaast lqlsvdgvag etatsaagel aaysdsvgnk fgigyevnpa sgaasrglag

 241

dvfrarvads dapweildas qllhvnfngt fsgtsytaas geqmlgslvs rsanpsisns

 301

avtlgggtag fdftptdftl gdneaitrpl vaelrftptq tgdnqtlfga ggnlflryes

 361

nklvfgastk sgdnwtdhki esaaatgaeh vvsvayvpnk agtgaklvmr vdggdaqtkd

 421

itglaylnss ikgkvgfgnd vhtdalsrgf vgslseirla etsanfttne fklvysqvsc

 481

dtsgikeant fdvepaecea alktklsklr ptegqadyid wgqigflhyg intyynqewg

 541

hgnedpsrin ptgldtdqwa ksfadggfkm imvtvkhhdg felydsrynt ehdwantava

 601

krtgekdlfr kivasakkyg lkvgiyyspa dsymerkgvw gnnsarvert iptivenddr

 661

agkvasgklp tfkykatdyg aymlnqlyel lteygdisev wfdgaqgnta gtehydygvf

 721

yemirrlqpq aiqanaayda rwvgnedgwa rqtewspqaa yndgvdkvsl kpgqmapdgk

 781

lgsmssvlse irsgaanqlh wypaevdakn rpgwfyrasq spasvaevvk yyeqstgrns

 841

qyllnvppsd tgkladadaa glkglgeela rrygtdlalg ksatvaasan dtavaapklt

 901

dgsklssdka vgntptytid lgstvavdav kisedvrnag qqiesatlqg rvngtwtnla

 961

tmttvgqqrd lrftsqnida irlvvnssrg pvrlsrlevf hteseiqtga rayyidptaq

1021

tagdgftkdk pmtsieqlhd vtvapgsvif vkagteltgd favfgygtkd epitvttyge

1081

sdkattasfd gmtagltlkq alkalgkdda gwvvadsata pasrvyvpqd eisvhaqssq

1141

nsgaeaaral dgdsstswhs qyspttasap hwvtldlgks renvayfdyl aridgnnnga

1201

akdyevyvsd dpndfgapva sgtlknvayt qrikltpkng ryvkfviktd ysgsnfgsaa

1261

emnvellpta veedkvatpq kptvdddadt ytipdiegvv ykvdgkvlaa gsvvnvgded

1321

vtvtvtaepa dgyrfpdgvt spvtyeltft kkggekppte vnkdklhati tkaqaidrsa

1381

ytdeslkvld dklaaalkvy dddkvsqddv daaeaalsaa idalktkptt pggegekpge

1441

gekpgdgnkp gdgkkpgdvi aktgastmgv vfaalamvag avvtleakrk snr

GH-95, Bifidobacterium longum subsp. infantis (B. infantis), Blon_2335,

Genbank Accession YP_002323771.1 (AfcA); SEQ ID NO: 5:

   1

mkltfdgiss hweegipfgn grmgavlcse pdadvlylnd dtlwsgypha etspltpeiv

  61

akarqassrg dyvsatriiq datqrekdeq iyepfgtaci rysseagerk hvkrsldlar

 121

alagesfrlg aadvhvdawc sapddllvye msssapvdas vsvtgtflkq trissgsdsd

 181

arqativvmg qmpglnvgsl ahvtdnpwed erdgigmaya gafsltvtgg eitviddvlq

 241

csgvtglslr frslsgfkgs aeqperdmtv ladrlgetia awpsdsraml drhvadyrrf

 301

fdrvgvrlgp andddeevpf aeilrskedt phrletlsea mfdfgrylli sssrphtqps

 361

nlqgiwnhkd fpnwysaytt niniemnywm tgpcalkeli eplvamnrel lepghdaaga

 421

ilgcggsavf hnvdiwrral pangeptwaf wpfgqawmcr nlfdeylfnq desylasiwp

 481

imrdsarfcm dflsdtehgl apapatspen yfvvdgetia vahtsentta ivrnllddli

 541

haaqtmpdld dgdkalvrea estraklaav rvgsdgrile wndelveadp hhrhlshlye

 601

lhpgagitan tprleeaark slevrgddgs gwsivwrmim warlrdaeha eriigmflrp

 661

veadaetdll gggvyasgmc ahppfqidgn lgfpaalaem lvqshdgmvr ilpalpedwh

 721

egsfhglrar gglsvdaswt ddaieytlrc tkpatitliv dgtdatqvrl spdepfkglv

 781

rr

GH-95, Bifidobacterium bifidum, Genbank Accession AAQ72464.1 (AfcA); 

SEQ ID NO: 6:

   1

mkhramssrl mplvascatv gmllaglpvs avavgttraa asdassstta titpsadttl

  61

qtwtseknss maskpyigtl qgpsqgvfge kfestdaadt tdlktglltf dlsaydhapd

 121

satfemtylg yrgnptatdt dtikvtpvdt tvctnnatdc ganvatgatk pkfsindssf

 181

vaeskpfeyg ttvytgdait vvpantkkvt vdvteivrqq faegkkvitl avgetkktev

 241

rfassegtts lngatadmap kltvsystkd dlkpsadttl qawaseknek kntaayvgal

 301

qpegdygdfg ekfkstdvhd vtdakmglmt fdlsdytaap ehsiltltyl gyagadktat

 361

atdkvkvvav dtsrctgtap cdtnnatwan rpdfevtdtt ktatshafay gskkysdgmt

 421

vesgnakkvl ldvsdvikae fakfsagate kkitlalgel nksdmrfgsk evtsltgate

 481

amqptlsvtk kpkaytlsie gptkvkyqkg eafdkaglvv katstadgtv ktltegnged

 541

nytidtsafd sasigvypvt vkynkdpeia asfnayvias vedggdgdts kddwlwykqp

 601

asqtdatata ggnygnpdnn rwqqttlpfg ngkiggtvwg evsrervtfn eetlwtggpg

 661

sstsynggnn etkgqngatl ralnkqlang aetvnpgnit ggenaaeqgn ylnwgdiyld

 721

ygfndttvte yrrdlnlskg kadvtfkhdg vtytreyfas npdnvmvarl taskagklnf

 781

nvsmptntny sktgetttvk gdtltvkgal gnngllynsq ikvvldngeg tlsegsdgas

 841

lkvsdakavt lyiaaatdyk qkypsyrtge taaevntrva kvvqdaankg ytavkkahid

 901

dhsaiydrvk idlgqsghss dgavatdall kayqrgsatt aqkreletiv ykygryltig

 961

ssrensqlps nlqgiwsvta gdnahgntpw gsdfhmnvnl qmnywptysa nmgelaepli

1021

eyveglvkpg rvtakvyaga ettnpettpi gegegymaht entaygwtap gqsfswgwsp

1081

aavpwilqnv yeayeysgdp alldrvyall keeshfyvny mlhkagsssg drlttgvays

1141

peqgplgtdg ntyesslvwq mlndaieaak akgdpdglvg nttdcsadnw akndsgnftd

1201

ananrswsca ksllkpievg dsgqikewyf egalgkkkdg stisgyqadn qhrhmshllg

1261

lfpgdlitid nseymdaakt slryrcfkgn vlqsntgwai gqrinswart gdgnttyqlv

1321

elqlknamya nlfdyhapfq idgnfgntsg vdemllqsns tftdtagkky vnytnilpal

1381

pdawaggsys glvargnftv gttwkngkat evrltsnkgk qaavkitagg aqnyevkngd

1441

tavnakvvtn adgasllvfd ttagttytit kkasanvpvt gvtvtganta tagdtvtlta

1501

tvapanatdk svtwstsdaa vatvnangvv ttkkagkvti tatsngdktk fgsieitvsa

1561

atvpvtsvtv agdaamtvdg eqtltatvap atatdktvtw kssdatvatv dangkvvakk

1621

agevtitata ggvsgtlkit vsdkaptvip vqsvtvtgkq elvegasttl tatvapadat

1681

dktvtwkssd esvatvdkdg vvtakkagtv titataggvs gtlhitvtak pvetvpvtsv

1741

evtveagttv svgktlqata tvkpgnatnk kvtwkssdes iatvdangvi takkagkvvi

1801

tatstdgtdk sgsveitvvd etkptpdhks vkadtgdvta gktgtvtepk dvagwksrsi

1861

ikqgklgkae iadgtivyaa gdktgddsfv vqytmadgtv idvtysvtvk aaetgkndgd

1921

gkgdgvaktg aavgalaglg lmllavgvsv vmirrkhsa

GH-29, Bifidobacterium longum subsp. infantis (B. infantis), Blon_0248,

derived from Genbank Accession NC_011593, (AfcB); SEQ ID NO: 7:

   1

atggtgttgt tcatggccaa tccacagcgt cccaagatgt atgagaagtt cgtgcacgat

  61

acacccgaat ggttcaaggg cgccggtctc ggcatcttcg cccactgggg ttcgtattcg

 121

gtgccggcat gggcggagcc gatcggtgcg cttggcacct ttgacgatcc ggtgtactgg

 181

aacacccact gcccgtatgc ggaatggtat tggaacacga tgagcatcaa gggctcgccg

 241

gcggccgagc atcagaagga agtctacggt gacatgccgt atgaggactt catcgacatg

 301

tggaaggccg aggcgttcga ccccgcggac atggccgacc tgttcgcacg cgccggtgcc

 361

cggtacttcg tgccgaccac gaagcatcac gaaggcatca cgctgtggaa ggcccccgac

 421

aacgatgggt ggaataccgt ggaccgtggt ccgcatcgcg atctggtcaa ggaattcgcc

 481

gacgccatgc gcgacaaggg actgaagttc ggcgtgtact actcctcggg cctcgactgg

 541

cacaaggagc ccaacatgcc gattctcggc gacggggaat acgggccgca gagcgaggac

 601

tacgcccgct atatgtactc gcatgtgatg gacctcatcg acgaatacca gccgtccatc

 661

ctgtggggag atatcgacgt gccgaagatc tcggaggagg acaacgattt cagcgtggcc

 721

cgactgttcg agcattacta cgacgtggtg ccggatggtg tggtcaacga ccgctggggc

 781

ctgacccatt gggacttccg caccgtcgaa tacgaacagg gcaaggagct catgggcaag

 841

ggcatgtggg agatgacccg aggcatcggc tactccttcg gctacaacca gatggaggac

 901

gccgactcct acatgaccgg tccggaggcg gtgaagttgc tcgccgacgt ggtctccatg

 961

ggcggcaacc tgctgctcga catcggcccc gacgccgccg gacgcatccc cgaactgcag

1021

cgtcagtgcc tcgagggcat ggccgactgg atggacgtga actcgccgag tatccatgat

1081

gtcgaaccgg tgccggaagc ctcgccttcc ggagaggggg acggcgagcc atgggtccgt

1141

tggaccggag acggcaagag cgtctatgcc gtcgtcgatg ctgcgggcag ggttccgctg

1201

cgcatcgccg ccgatgctgt ggacgcggat tccgccgtga cgcttggcgg atccgcagtc

1261

gccgtggacg ccgacggcga cgtgctgacc gccgatgttc cggcctcgga agtggcgggg

1321

ccgcaggtcg tgcacttcgt ccgtcgctga

GH-29, Bifidobacterium longum subsp. infantis (B. infantis), Blon_0426,

derived from Genbank Accession NC_011593, (AfcB); SEQ ID NO: 8:

   1

atggtgttgt tcatggccaa tccacagcgt cccaagatgt atgagaagtt cgtgcacgat

  61

acacccgaat ggttcaaggg cgccggtctc ggcatcttcg cccactgggg ttcgtattcg

 121

gtgccggcat gggcggagcc gatcggtgcg cttggcacct ttgacgatcc ggtgtactgg

 181

aacacccact gcccgtatgc ggaatggtat tggaacacga tgagcatcaa gggctcgccg

 241

gcggccgagc atcagaagga agtctacggt gacatgccgt atgaggactt catcgacatg

 301

tggaaggccg aggcgttcga ccccgcggac atggccgacc tgttcgcacg cgccggtgcc

 361

cggtacttcg tgccgaccac gaagcatcac gaaggcatca cgctgtggaa ggcccccgac

 421

aacgatgggt ggaataccgt ggaccgtggt ccgcatcgcg atctggtcaa ggaattcgcc

 481

gacgccatgc gcgacaaggg actgaagttc ggcgtgtact actcctcggg cctcgactgg

 541

cacaaggagc ccaacatgcc gattctcggc gacggggaat acgggccgca gagcgaggac

 601

tacgcccgct atatgtactc gcatgtgatg gacctcatcg acgaatacca gccgtccatc

 661

ctgtggggag atatcgacgt gccgaagatc tcggaggagg acaacgattt cagcgtggcc

 721

cgactgttcg agcattacta cgacgtggtg ccggatggtg tggtcaacga ccgctggggc

 781

ctgacccatt gggacttccg caccgtcgaa tacgaacagg gcaaggagct catgggcaag

 841

ggcatgtggg agatgacccg aggcatcggc tactccttcg gctacaacca gatggaggac

 901

gccgactcct acatgaccgg tccggaggcg gtgaagttgc tcgccgacgt ggtctccatg

 961

ggcggcaacc tgctgctcga catcggcccc gacgccgccg gacgcatccc cgaactgcag

1021

cgtcagtgcc tcgagggcat ggccgactgg atggacgtga actcgccgag tatccatgat

1081

gtcgaaccgg tgccggaagc ctcgccttcc ggagaggggg acggcgagcc atgggttcgt

1141

tggaccggag acggcaagag cgtctatgcc gtcgtcgatg ctgcgggcag ggttccgctg

1201

cgcatagatg cgggtgcggt cgatgtggat tccgcaacca ttcttggcgg tggcaacgtt

1261

gtcgtggagg cggacggcga tatgctgacc gtggagattc ccgcgacaga cgtcgccggc

1321

cctcaggtcg tgcgttttgc tcgacactaa

GH-29, Bifidobacterium breve SC95, (AfcB), SEQ ID NO: 9:

   1

atggtgctgt tcatggccaa tccgcagcgt cccaagatgt atgagaagtt cgtgcacgat

  61

acacccgaat ggttcaaggg cgccggtctc ggcatcttcg cccactgggg ttcgtattcg

 121

gtgccggcat gggcggagcc gatcggtgcg cttggcacct ttgacgatcc ggtgtactgg

 181

aacacccact gcccgtatgc ggaatggtat tggaacacga tgagcatcaa gggctcgccg

 241

gcggccgagc atcagaagga agtctacggt gacatgccgt atgaggactt catcgacatg

 301

tggaaggccg aggcgttcga ccccgcggac atggccgacc tgttcgcacg cgccggtgcc

 361

cggtacttcg tgccgaccac gaagcatcac gaaggcatca cgctgtggaa ggcccccgac

 421

aacgatgggt ggaataccgt ggaccgtggt ccgcatcgcg atctggtcaa ggaattcgcc

 481

gacgccatgc gcgacaaggg actgaagttc ggcgtgtact actcctcggg cctcgactgg

 541

cacaaggagc ccaacatgcc gattctcggc gacggggaat acgggccgca gagcgaggac

 601

tacgcccgct atatgtactc gcatgtgatg gacctcatcg acaaatacca gccgtccatc

 661

ctgtggggag atatcgacgt gccgaagatc tcggaggagg acaacgattt cagtgtggcc

 721

cgactgttcg agcattacta tgacgtggtg ccggatggtg tggtcaacga ccgctggggc

 781

ctgacccatt gggacttccg caccgtcgaa tacgaacagg gcaaggagct catgggcaag

 841

ggcatgtggg agatgacccg aggcatcggc tactccttcg gctacaacca gatggaggac

 901

gccgactcct acatgaccgg tccggaggcg gtgaagttgc tcgccgacgt ggtctccatg

 961

ggcggcaacc tgctgctcga catcggcccc gacgccgccg gacgcatccc cgaactgcag

1021

cgtcagtgcc tcgagggcat ggccgactgg atgtacgtga actcgccgag tatccatgat

1081

gtcgaaccgg tgccggaagc ctcgccttcc ggagaggggg acggcgagcc atgggtccgt

1141

tggaccggag acggcaagag cgtctatgcc gtcgtcgatg ctgcgggcag ggttccgctg

1201

cgcatcgccg ccgatgctgt ggacgcggat tccgccgtga cgcttggcgg atccgcagtc

1261

gccgtggacg ccgacggcga cgtgctgacc gccgatgttc cggcctcgga agtggcgggg

1321

ccgcaggtcg tgcacttcgt ccgtcgctga

GH-29, Bifidobacterium bifidum, Genbank Accession, AB474964.1 (AfcB);

SEQ ID NO: 10:

   1

atgctacaca cagcatcaag aggatgctcg cgttcgtggc tgcgcagact caccgcattg

  61

atagcggtct cggcgctcgc gttcgtggca ttgccgaacg tcgcggtggc ggcggatccg

 121

atggaatacc tcgatgtgtc gttcggcggc acgttcgctg cagacaccta caccacaggt

 181

ggcgacgagg tggcgaaggg ccccgtgacc aagcacggca gcataccgac caagcttgac

 241

ggcggcggca tcaccctcgc tggcggcacc aacggcgtga cattcacctc gaccgcgagc

 301

ttcagcgaga gtgggaaggt gaacaaggga ttccgcgccg aaatggagta ccgtacgacg

 361

cagacgccca gcaacctcgc cacattgttc tccgccatgg gcaacatctt cgtgcgggcg

 421

aacggcagca acctcgaata cggcttctcc acgaaccctt ccggcagtac atggaacgac

 481

tacacaaagt ccgtgacgct gccttccaac aatgtgaagc acatcatcca gctgacatat

 541

ctgccgggag ccgacggcgc tgcctcgacg ttgcagttgt cggtggatgg cgtggccggc

 601

gagaccgcca cctccgcggc cggcgagctc gcggccgtca gcgattccgt cgggaacaag

 661

ttcgggatcg gctacgaggt gaaccccgct tccggcgcgg cgagccgcgg tcttgccggt

 721

gacgtgttcc gcgcgcgtgt cgccgattcg gacgccccgt gggagattct tgacgcatcc

 781

cagctgctgc atgtcaattt caacggcacg ttcagcggca cctcatatac cgcggcgagc

 841

ggcgagcaga tgctgggctc gctggtgtcg cgctcggcca atccgtccat ctcgaactcc

 901

gccgtcacgc tgggcggcgg cacggccgga ttcgatttca cgcccacgga cttcaccctc

 961

ggtgacaacg aggccatcac ccgcccgctg gtcgcggagc tgcgcttcac cccgacgcag

1021

accggcgaca accagaccct gttcggcgcg ggcggcaacc tgttcctgcg ctacgagtcg

1081

aacaagctcg tgttcggcgc ctccaccaag tccggcgata attggaccga ccacaagatc

1141

gagtccgcgg ccgccacggg tgcggagcac gtcgtgtcgg tggcgtacgt gcccaataag

1201

gccggcaccg gcgcgaagct tgtcatgcgc gtggatggcg gcgacgccca gaccaaggac

1261

atcactggtc tggcttacct gaattcgagc atcaagggca aggtcggctt cggcaacgac

1321

gtgcataccg acgcgctcag ccgcggcttc gtcggctcgc tgagcgagat ccgcctggcc

1381

gaaacctccg cgaacttcac caccaacgaa ttcaagctgg tctactctca ggtcagctgc

1441

gacacgtcgg gcatcaagga ggcgaatacc ttcgacgtgg agcccgccga gtgcgaggcc

1501

gcgcttaaga ccaagctgtc caagctgcgt ccgaccgaag ggcaggccga ctacatcgac