Bifidobacterial β-Galactosidase-Mediated Production of Galacto-Oligosaccharides: Structural and Preliminary Functional Assessments.

Valentina Ambrogi,Valentina Ambrogi,Valentina Ambrogi,Valentina Ambrogi,Bas Kuipers,Francesca Bottacini,Francesca Bottacini,Elisa G Doyagüez,John Mac Sharry,Ellen O’Keeffe,Margriet Schoterman,Justin Van Breen,Cordula Lindner,Dan Walsh,F Javier Moreno,Ellen O’Keeffe,John Mac Sharry,Francesca Bottacini,Douwe Van Sinderen,Oswaldo Hernandez-Hernandez,F Javier Moreno,Justin Van Breen,Justin Van Breen,Barry Schoemaker,Maria Luisa Jimeno,Douwe Van Sinderen,Ellen O’Keeffe,Ellen O’Keeffe,Justin Van Breen,Linqiu Cao

doi:10.3389/fmicb.2021.750635

Abstract

In the current study the ability of four previously characterized bifidobacterial β-galactosidases (designated here as BgaA, BgaC, BgaD, and BgaE) to produce galacto-oligosaccharides (GOS) was optimized. Of these enzymes, BgaA and BgaE were found to be promising candidates for GOS production (and the corresponding GOS mixtures were called GOS-A and GOS-E, respectively) with a GOS concentration of 19.0 and 40.3% (of the initial lactose), respectively. GOS-A and GOS-E were partially purified and structurally characterized. NMR analysis revealed that the predominant (non-lactose) disaccharide was allo-lactose in both purified GOS preparations. The predominant trisaccharide in GOS-A and GOS-E was shown to be 3′-galactosyllactose, with lower levels of 6′-galactosyllactose and 4′-galactosyllactose. These three oligosaccharides have also been reported to occur in human milk. Purified GOS-A and GOS-E were shown to be able to support bifidobacterial growth similar to a commercially available GOS. In addition, GOS-E and the commercially available GOS were shown to be capable of reducing Escherichia coli adhesion to a C2BBe1 cell line. Both in vitro bifidogenic activity and reduced E. coli adhesion support the prebiotic potential of GOS-E and GOS-A.

Highlights

Bifidobacteria are common gut commensals that are abundant in the intestinal microbiota of naturally delivered, fullterm and breast-fed infants (Turroni et al, 2012; Moossavi et al, 2019)
GOS can be enzymatically synthesized by a transgalactosylation reaction employing β-galactosidases of different origin with solely lactose as substrate, where D-galactosyl moieties are transferred onto the D-galactose component of lactose, resulting in addition of a variable number galactosyl moieties and release of glucose as the by product (Torres et al, 2010; Vera et al, 2016). β-galactosidases belong to a class of hydrolytic enzymes (EC 3.2.1.23) which under standard physiological conditions are unable to carry out transgalactosylation activities
The enzymatic reaction was started with a high initialsubstrate concentration, which represents a saturated lactose solution in which not all available lactose is dissolved and the insoluble lactose can function as a substrate pool, which is able to maintain the relatively constant lactose concentration, prior to the clarification of the whole reaction mixture (Vera et al, 2012)

Summary

Introduction

Bifidobacteria are common gut commensals that are abundant in the intestinal microbiota of naturally delivered, fullterm and breast-fed infants (Turroni et al, 2012; Moossavi et al, 2019). HMOs are not digested by the human host, and will reach the intestinal tract, where they may be utilized as a primary carbon and energy source by specific species of the genera Bifidobacterium and Bacteroides (Brand-Miller et al, 1998; Jost et al, 2015) Based on these characteristics, HMOs are considered natural and highly effective prebiotics for infants. It has been demonstrated that galactosyl-lactose can physiologically act as a potent anti-inflammatory agent and can positively contribute to immune modulation during infancy Based on these properties it was suggested that GOS plays a role in protection against enteric inflammation in neonates (Newburg et al, 2016). Typhimurium strain SL1344nal it was shown that a particular GOS, produced through lactosebased transgalactosylation mediated by a cell free extract of Bifidobacterium bifidum NCIMB 41171, is able to inhibit adhesion of SL1344nal to HT-29 cells (Searle et al, 2009)

Results

Discussion

Conclusion