A versatile and scalable strategy for glycoprofiling bifidobacterial consumption of human milk oligosaccharides

Riccardo G Locascio, Milady R Niñonuevo, Scott R Kronewitter, Samara L Freeman, J Bruce German, Carlito B Lebrilla, David A Mills, Riccardo G Locascio, Milady R Niñonuevo, Scott R Kronewitter, Samara L Freeman, J Bruce German, Carlito B Lebrilla, David A Mills

Abstract

Human milk contains approximately 200 complex oligosaccharides believed to stimulate the growth and establishment of a protective microbiota in the infant gut. The lack of scalable analytical techniques has hindered the measurement of bacterial metabolism of these and other complex prebiotic oligosaccharides. An in vitro, multi-strain, assay capable of measuring kinetics of bacterial growth and detailed oligosaccharide consumption analysis by FTICR-MS was developed and tested simultaneously on 12 bifidobacterial strains. For quantitative consumption, deuterated and reduced human milk oligosaccharide (HMO) standards were used. A custom software suite developed in house called Glycolyzer was used to process the large amounts of oligosaccharide mass spectra automatically with (13)C corrections based on de-isotoping protocols. High growth on HMOs was characteristic of Bifidobacterium longum biovar infantis strains, which consumed nearly all available substrates, while other bifidobacterial strains tested, B. longum bv. longum, B. adolescentis, B. breve and B. bifidum, showed low or only moderate growth ability. Total oligosaccharide consumption ranged from a high of 87% for B. infantis JCM 7009 to only 12% for B. adolescentis ATCC 15703. A detailed analysis of consumption glycoprofiles indicated strain-specific capabilities towards differential metabolism of milk oligosaccharides. This method overcomes previous limitations in the quantitative, multi-strain analysis of bacterial metabolism of HMOs and represents a novel approach towards understanding bacterial consumption of complex prebiotic oligosaccharides.

© 2008 The Authors. Journal compilation © 2008 Society for Applied Microbiology and Blackwell Publishing Ltd.

Figures

Figure 1
Figure 1
Schematic of experimental procedure. Schematic illustration for the workflow utilized for the real‐time monitoring of bacterial growth, sample preparation, MS analysis, data collection and analysis. (a and b) Clonal expansion and real‐time monitoring of bacterial growth performed in anaerobic chamber. (c) Sample collection. (d) Sample inactivation, sterilization and clean‐up. (e) Internal standard addition. (f) TipTop PGC oligosaccharide isolation. (g) Sample MS analysis, data acquisition and processing. Time gains in sample processing as compared with a previously developed method (LoCascio et al., 2007).
Figure 2
Figure 2
Human milk oligosaccharide (HMO) consumption glycoprofiles. MALDI‐FTICR‐MS analysis of 12 bifidobacterial strains grown on a medium supplemented with 1.6% (w/v) HMO. HMO consumption is represented as the per cent difference in HMO abundance between the start and the end of fermentation, which was defined as the beginning of stationary phase (see Fig. S2 and Table S1 for detailed glycoprofiles and HMOs relative per cent abundance). Measurements are triplicates of individual biological and technical replicates. DP, degree of polymerization of the HMOs. *Fucosylated HMOs.
Figure 3
Figure 3
MALDI‐FTICR‐MS analysis of glycans remaining post fermentation by B. adolescentis (A) and B. infantis (B). Shown in the upper section are the full scan MS of the two samples along with the zoomed‐in mass spectra of four selected masses [m/z 1097 (1), 1243 (2), 1608 (3), 1754 (4)]. The lines point to the difference of the D/H ratio for each sample illustrating differential consumption of the two species (see text for details on the D/H methods).
Figure 4
Figure 4
HPLC‐Chip/TOF‐MS analysis of selectivity for specific glycan structures consumed by B. infantis ATCC 15697. Shown are nanoLC extracted ion chromatograms (XIC) of selected milk glycans recovered (A) after incubation with B. infantis ATCC 15697 and (B) before incubation (control). The complete disappearance (A) of all glycan peaks for specific oligosaccharide (m/z: 732, 878 and 1243) indicates that B. infantis consumes specific HMOs but does not discriminate for any specific isomers within each group of HMO consumed.

References

    1. Barrangou R., Altermann E., Hutkins R., Cano R., Klaenhammer T.R. Functional and comparative genomic analyses of an operon involved in fructooligosaccharide utilization by Lactobacillus acidophilus. Proc Natl Acad Sci USA. 2003;100:8957–8962.
    1. Bode L. Recent advances on structure, metabolism and function of human milk oligosaccharides. J Nutr. 2006;136:2127–2130.
    1. Bode L., Rudloff S., Kunz C., Strobel S., Klein N. Human milk oligosaccharides reduce platelet‐neutrophil complex formation leading to a decrease in neutrophil beta 2 integrin expression. J Leukoc Biol. 2004;76:820–826.
    1. Chaturvedi P., Warren C.D., Buescher C.R., Pickering L.K., Newburg D.S. Survival of human milk oligosaccharides in the intestine of infants. In: Newburg D.S., editor. Kluwer Academic; 2001. pp. 315–323.
    1. Coppa G.V., Gabrielli O., Pierani P., Catassi C., Carlucci A., Giorgi P.L. Changes in carbohydrate‐composition in human‐milk over 4 months of lactation. Pediatrics. 1993;91:637–641.
    1. Coppa G.V., Bruni S., Morelli L., Soldi S., Gabrielli O. The first prebiotics in humans – human milk oligosaccharides. J Clin Gastroenterol. 2004;38:S80–S83.
    1. Favier C.F., De Vos W.M., Akkermans A.D.L. Development of bacterial and bifidobacterial communities in feces of newborn babies. Anaerobe. 2003;9:219–229.
    1. Gibson G.R., Roberfroid M.B. Dietary modulation of the human colonic microbiota – introducing the concept of prebiotics. J Nutr. 1995;125:1401–1412.
    1. Hong P., Ninonuevo M.R., Lee B., Lebrilla C.B., Bode L. Human milk oligosaccharides reduce HIV‐1‐gp120 binding to dendritic cell‐specific ICAM3‐grabbing non‐integrin (DC‐SIGN) Br J Nutr. 2008
    1. Lievin V., Peiffer I., Hudault S., Rochat F., Brassart D., Neeser J.R., Servin A.L. Bifidobacterium strains from resident infant human gastrointestinal microflora exert antimicrobial activity. Gut. 2000;47:646–652.
    1. Lin H.C., Su B.H., Chen A.C., Lin T.W., Tsai C.H., Yeh T.F., Oh W. Oral probiotics reduce the incidence and severity of necrotizing enterocolitis in very low birth weight infants. Pediatrics. 2005;115:1–4.
    1. LoCascio R.G., Ninonuevo M.R., Freeman S.L., Sela D.A., Grimm R., Lebrilla C.B. Glycoprofiling of bifidobacterial consumption of human milk oligosaccharides demonstrates strain specific, preferential consumption of small chain glycans secreted in early human lactation. J Agric Food Chem. 2007;55:8914–8919. et al.
    1. Mussatto S.I., Mancilha I.M. Non‐digestible oligosaccharides: a review. Carbohydr Polym. 2007;68:587–597.
    1. Ninonuevo M.R., Park Y., Yin H., Zhang J., Ward R.E., Clowers B.H. A strategy for annotating the human milk glycome. J Agric Food Chem. 2006;54:7471–7480. et al.
    1. Ninonuevo M.R., Ward R.E., LoCascio R.G., German J.B., Freeman S.L., Barboza M. Methods for the quantitation of human milk oligosaccharides in bacterial fermentation by mass spectrometry. Anal Biochem. 2007;361:15–23. et al.
    1. Ouwehand A.C., Salminen S., Isolauri E. Probiotics: an overview of beneficial effects. Antonie Van Leeuwenhoek. 2002;82:279–289.
    1. Parche S., Beleut M., Rezzonico E., Jacobs D., Arigoni F., Titgemeyer F., Jankovic I. Lactose‐over‐glucose preference in Bifidobacterium longum NCC2705: glcP, encoding a glucose transporter, is subject to lactose repression. J Bacteriol. 2006;188:1260–1265.
    1. Roberfroid M. Prebiotics: the concept revisited. J Nutr. 2007;137:830s–837s.
    1. Ruiz‐Palacios G.M., Cervantes L.E., Ramos P., Chavez‐Munguia B., Newburg D.S. Campylobacter jejuni binds intestinal H(O) antigen (Fuc alpha 1, 2Gal beta 1, 4GlcNAc), and fucosyloligosaccharides of human milk inhibit its binding and infection. J Biol Chem. 2003;278:14112–14120.
    1. Sakata S., Kitahara M., Sakamoto M., Hayashi H., Fukuyama M., Benno Y. Unification of Bifidobacterium infantis and Bifidobacterium suis as Bifidobacterium longum. Int J Syst Evol Microbiol. 2002;52:1945–1951.
    1. Sela D.A., Chapman J., Adeuya A., Kim J.H., Chen F., Whitehead T.R. The complete genome sequence of Bifidobacterium longum subsp. infantis reveals adaptations for milk utilization within the infant microbiome. Proc Natl Acad Sci USA. 2008;105:18964–18969. et al.
    1. Servin A.L. Antagonistic activities of lactobacilli and bifidobacteria against microbial pathogens. FEMS Microbiol Rev. 2004;28:405–440.
    1. Ward R.E., Ninonuevo M., Mills D.A., Lebrilla C.B., German J.B. In vitro fermentation of breast milk oligosaccharides by Bifidobacterium infantis and Lactobacillus gasseri. Appl Environ Microbiol. 2006;72:4497–4499.

Source: PubMed

赞助者和合作者

医疗条件

药物干预

3
订阅