Metabolism of biosynthetic oligosaccharides by human-derived Bifidobacterium breve UCC2003 and Bifidobacterium longum NCIMB 8809

https://doi.org/10.1016/j.ijfoodmicro.2019.108476Get rights and content

Highlights

  • Utilization of biosynthetic oligosaccharides by two bifidobacterial strains was tested.

  • Differences in utilization are linked to carbohydrate molecular weight and structure.

  • Transcriptome analysis was done for B. breve UCC2003 when grown on two trisaccharides.

  • Upregulated genes represented glycosyl hydrolases and sugar transport systems.

  • The role of β-galactosidases in hydrolysis of particular trisaccharides was demonstrated.

Abstract

This work aimed to investigate the ability of two human-derived bifidobacterial strains, i.e. Bifidobacterium breve UCC2003 and Bifidobacterium longum NCIMB 8809, to utilize various oligosaccharides (i.e., 4-galactosyl-kojibiose, lactulosucrose, lactosyl-oligofructosides, raffinosyl-oligofructosides and lactulose-derived galacto-oligosaccharides) synthesized by means of microbial glycoside hydrolases. With the exception of raffinosyl-oligofructosides, these biosynthetic oligosaccharides were shown to support growth acting as a sole carbon and energy source of at least one of the two studied strains. Production of short-chain fatty acids (SCFAs) as detected by HPLC analysis corroborated the suitability of most of the studied novel oligosaccharides as fermentable growth substrates for the two bifidobacterial strains, showing that acetic acid is the main metabolic end product followed by lactic and formic acids. Transcriptomic and functional genomic approaches carried out for B. breve UCC2003 allowed the identification of key genes encoding glycoside hydrolases and carbohydrate transport systems involved in the metabolism of 4-galactosyl-kojibiose and lactulosucrose. In particular, the role of β-galactosidases in the hydrolysis of these particular trisaccharides was demonstrated, highlighting their importance in oligosaccharide metabolism by human bifidobacterial strains.

Introduction

The gastrointestinal tract (GIT) of mammals is host to a complex and densely populated community of different microorganisms, known as the intestinal microbiota. Bacteria dominate the gut ecosystem, and Bifidobacterium is one of the main representative genera, being particularly abundant in healthy, breast-fed infants due to the ability of certain bifidobacteria to consume oligosaccharides found in human milk (Khonsari et al., 2016; Yatsunenko et al., 2012). In addition to human milk oligosaccharides, bifidobacteria can ferment a large variety of dietary oligosaccharides, although the ability to metabolize particular carbohydrates is species- and strain-dependent (de Vrese and Schrezenmeir, 2008).

Scientific evidence suggests that the composition and activity of the gut microbiota is responsive to diet. Thus, a substantial number of studies have demonstrated that dietary fibers, especially so-called non-digestible (i.e. not digested by the host) oligo-/polysaccharides, alter the number and/or activity of certain gut microbiota components, thereby leading to improvements in host health (Makki et al., 2018; Scott et al., 2008; So et al., 2018; Valdes et al., 2018). To date, several non-digestible carbohydrates, such as galacto-oligosaccharides (GOS), fructo-oligosaccharides (FOS) and inulin, have demonstrated such beneficial effects (Slavin, 2013), and for this reason are referred to as prebiotics. In this context, bifidobacteria are often targeted for prebiotic intervention as they have been reported to confer various health benefits, including, but not limited to, immune-modulation (Hidalgo-Cantabrana et al., 2016; Sanchis-Chordà et al., 2018; Turroni et al., 2014), restriction of pathogenic bacteria through competitive exclusion and production of short chain fatty acids (SCFAs; examples are butyric and propionic acids) (Ventura et al., 2012), as well as modulation of mucosal barrier function (Turroni et al., 2014).

Improving health and/or reducing the risk of (chronic) disease are some of the forces driving the development of functional foods for humans. There is both scientific and commercial interest in the concept of prebiotics which aim to beneficially modulate gut microbiota composition and associated bacterial metabolic activities. Significant scientific efforts are ongoing to identify novel and possibly improved compounds that possess such beneficial or prebiotic potential. In this sense, the (bio)synthesis of various oligosaccharides whose structural features make them promising candidates as new prebiotic ingredients has been described in recent years. Briefly, these oligosaccharides were produced by enzymatic synthesis using microbial glycoside hydrolases (GHs) acting on, in most cases, sucrose as donor and using different di- or trisaccharides as acceptors to produce 4-galactosyl-kojibiose (Díez-Municio et al., 2012a), lactulosucrose (Díez-Municio et al., 2012b), lactosyl-oligofructosides (Díez-Municio et al., 2015), raffinosyl-oligofructosides (Díez-Municio et al., 2016a) and lactulose-derived galacto-oligosaccharides (GOS-Lu) (Cardelle-Cobas et al., 2008; Martínez-Villaluenga et al., 2008). Following their synthesis, the ability of these particular novel carbohydrates to promote growth of specific probiotic strains has been investigated (Cardelle-Cobas et al., 2011; García-Cayuela et al., 2014), as well as their prebiotic potential using in vitro batch-culture fermentation systems inoculated with human faecal slurries (Cardelle-Cobas et al., 2009; Cardelle-Cobas et al., 2012; Díez-Municio et al., 2016b). Moreover, in the case of GOS-Lu, this prebiotic effect has been further corroborated by in vivo studies using different rat models (Fernández et al., 2018; Hernández-Hernández et al., 2012; Marín-Manzano et al., 2013). Remarkable findings were obtained regarding their strong bifidogenic effect by selectively promoting similar Bifidobacterium levels as compared to those produced by well-established prebiotics, such as lactulose and FOS. Nevertheless, further and precise information is needed regarding the affected bifidobacterial species, the associated enzymatic machinery required for the metabolism of these novel oligosaccharides and the corresponding metabolic end products.

Therefore, the main objective of this study was to gather knowledge on the utilization of a series of novel dietary oligosaccharides by specific beneficial human gut commensals represented by pure cultures of strains belonging to Bifidobacterium breve and Bifidobacterium longum (i.e. B. breve UCC2003 and B. longum NCIMB 8809), as well as to describe specific metabolic pathways required for their growth. Further insights were inferred from the relationship between structural features (i.e., monomer and glycosidic linkage type) and bifidogenic properties.

Section snippets

Commercial carbohydrates

Glucose (Glc), galactose (Gal), lactose (β-D-Gal-(1 → 4)-D-Glc), lactulose (β-D-Gal-(1 → 4)-D-Fru), maltose (α-D-Glc-(1 → 4)-D-Glc) and raffinose (α-D-Gal-(1 → 6)-α-D-Glc-(1 → 2)-β-D-Fru) were purchased from Sigma-Aldrich (St. Louis, MO, USA), and kojibiose (α-D-Glc-(1 → 2)-D-Glc) was purchased from Carbosynth (Berkshire, UK).

Production of oligosaccharides

The oligosaccharides employed in this study were synthesized by transglycosylation acceptor reactions catalyzed by microbial glycoside hydrolases and further structurally

Chemical structure and degree of purity of the novel oligosaccharides

Synthesized oligosaccharides with a variety of structural features, such as monomer composition and type of glycosidic linkage, were employed in this study in order to determine their potential bifidogenic properties. The enzymes and starting substrates used for their production are specified in Section 2.1.2. Table 1 shows the chemical structures and degree of purity of the carbohydrates assessed in the present study. With the exception of GOS-Lu, the degree of purity of the novel

Conclusions

The data assembled in this study provide information on the abilities of B. breve UCC2003 and B. longum NCIMB 8809 to grow on a number of novel oligosaccharides. Overall, the tested biosynthetic oligosaccharides supported reasonable to good growth of, at least, one of the two studied strains with the exception of RFOS which appears to represent a poor substrate for either of the strains. The identification of key genes encoding for carbohydrate transport systems and glycoside hydrolases,

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was financed by project AGL2017-84614-C2-1-R (Spanish Ministry of Economy and Competitiveness) and by The APC Microbiome Institute (under Science Foundation Ireland (SFI) grant number: SFI/12/RC/2273-P1 and SFI/12/RC/2273-P2). M. Esteban-Torres is supported by IRC Grant (GOIPD/2017/1302). L.R-A. thanks the Spanish Research Council (CSIC) and the Spanish Ministry of Economy and Competitiveness for a Juan de la Cierva contract. She is also supported by a postdoctoral scholarship from

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