Bifidogenic effect and in vitro immunomodulatory roles of melibiose-derived oligosaccharides produced by the acceptor reaction of glucansucrase E81
Introduction
The probiotic and prebiotic market has continued to grow worldwide due to the increasing demand for the production of functional foods or supplements containing these unique components that are capable of positively affecting the consumer’s health [1]. The prebiotic market is dominated by fructo-oligosaccharides and galacto-oligosaccharides [2], and these and other oligosaccharides or polysaccharides have attracted researchers to study their functional characteristics [[3], [4], [5], [6]]. The production of potential prebiotic oligosaccharides can involve enzymatic transfer reactions using cheap substrates such as sucrose [[7], [8], [9], [10]] or be completed by the digestion of polysaccharides to their oligo-forms, with different techniques used to test their potential prebiotic roles [11,12]. In the context of the former production strategy, glucansucrases are very efficient enzymes responsible for the production of oligosaccharides using sucrose as the donor and different sugars as the acceptors [[13], [14], [15]]. Importantly, different oligosaccharides produced by glucansucrases have shown potential prebiotic activities [8,16,17], with more studies required to adequately elucidate the prebiotic activity of the different oligosaccharides produced in these reactions. Another important feature of nondigestible oligosaccharides is their immunomodulatory effect; however, the action mechanism of this characteristic is not clearly understood at this time [18,19]. Recently, malto-oligosaccharides produced by 4,6-α-glucanotransferase were shown to modulate anti-inflammatory, interleukin (IL)-4, and proinflammatory IL-12 cytokines levels under in vitro conditions [20], which revealed the immunomodulatory potential of the oligosaccharides produced by the glucansucrases. As these oligosaccharides can be produced using cheap substrates and processes under environmentally friendly conditions, more studies are warranted to comprehend their potential health-promoting functions such as their prebiotic roles and immunomodulatory abilities to possibly facilitate the realization of novel functional foods. Melibiose was previously shown to be a good acceptor for glucansucrases [13], but the oligosaccharides produced in the donor–acceptor reactions of melibiose have not been tested yet in detail with respect to their potential functional roles. In this study, we sought to unveil the potential functional roles of melibiose-derived oligosaccharides produced during donor–acceptor reactions.
Lactobacillus reuteri E81 is a sourdough isolate found to harbour glucansucrase and 4,6-α-glucanotransferase coding genes in its genome [21]. Recently the glucansucrase E81 was shown to be responsible for the production of an α-glucan with (α1→3) and (α1→6) glycosidic linkages using sucrose as the substrate and was also deemed capable of synthesizing DP = 9 malto-oligosaccharides using sucrose as a donor and maltose as the acceptor sugar [7,22]. In this study, we describe the production of melibiose-derived oligosaccharides using sucrose and melibiose as the donor and acceptor sugars, respectively. The melibiose-derived oligosaccharides produced in this study were monitored by TLC analysis and structurally characterised by NMR and liquid chromatography–mass spectrometry (LC-MS) analysis. Importantly, the induction of IL-4, IL-10, IL-12, and tumour necrosis factor alpha (TNF-α) cytokines in HT29 cells by melibiose-derived oligosaccharides was tested to show their immunomodulatory potentials. Finally, the bifidogenic potential of the melibiose-derived oligosaccharides was determined by testing the growth of the probiotic and pathogenic strains using this oligosaccharide mixture. This study suggested the production of melibiose-derived oligosaccharides by glucosyltransferase A gene (GtfA)-ΔN E81 with potential immunomodulatory and bifidogenic roles and is expected to help to unveil the potential of the oligosaccharides produced within the donor–acceptor studies to be used as novel food supplements.
Section snippets
Production of GtfA-ΔN and purification
The glucansucrase gene (GtfA) from Lactobacillus reuteri E81 (accession number: MH051191) was previously cloned in pET15b (Novagen, Madison, WI) expression vector as its N-terminally truncated variant [7]. This construct was used to produce GtfA-ΔN in E. coli BL21 Star (DE3) (Invitrogen) and the activity of the GtfA-ΔN was determined as described previously [7]. For the production of GtfA-ΔN at high levels, pET15b-GtfA-ΔN was induced with isopropyl-β-d-1-thiogalactopyranoside (IPTG, final
Biosynthesis of melibiose-derived oligosaccharides with GtfA-ΔN
Lactobacillus reuteri E81 harbours the active GtfA gene and, recently, we expressed the GtfA gene in GtfA-ΔN form, which was shown to be an efficient glucansucrase under in vitro conditions responsible for the production of an α-glucan with (α 1→3) and (α 1→6) glycosydic linkages and malto-oligosaccharides with the acceptor reaction of maltose [7]. In this study, the production of melibiose-derived oligosaccharides using sucrose as the donor and melibiose as the acceptor sugar by GtfA-ΔN was
Conclusion
In this study, GtfA-ΔN E81 was reported to produce melibiose-derived oligosaccharides by using sucrose and melibiose as the donor and acceptor sugars, respectively. Structural characterisation of the melibiose-derived oligosaccharides revealed the formation of DP 3 and DP 4 melibiose-derived oligosaccharides containing (1,6)Glc and/or (1,3)Glc units, respectively. Melibiose-derived oligosaccharides produced in this study showed immunomodulatory functions by triggering anti-inflammatory cytokine
Declaration of Competing Interest
The authors declare that there is no conflict of interest.
Acknowledgments
This study was financially supported by TUBİTAK (The Scientific and Technological research council of Turkey) with the grant number 116O523.
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2021, Food BioscienceCitation Excerpt :aureus and Y. enterocolitica demonstrating that they were not able to utilize DP 3 cellobiose-derived oligosaccharides as carbon source during the 48 h of incubation period (Table 2). Previous studies also demonstrated the inability of pathogenic strains to use glucansucrase-based oligosaccharides as carbon sources supporting their potential as prebiotics (Holt, Miller-Fosmore, & Côté, 2005; İspirli, Colquhoun, et al., 2019; İspirli & Dertli, 2020a, 2020b; İspirli, Kaya, & Dertli, 2019). The glycosidic linkages and structural characteristics of the glucansucrase-based oligosaccharides can be the key issue for their role to certain pathogenic strains as melibiose and maltose-derived oligosaccharides were demonstrated to be utilized as carbon sources by pathogenic strains (Gregory L Côté et al., 2003).
Synthesis and characterization of Bifidogenic raffinose-derived oligosaccharides via acceptor reactions of glucansucrase E81
2021, LWTCitation Excerpt :aureus and Y. enterocolitica, did not grow with DP 4 raffinose-derived oligosaccharides as C source. Previously, similar findings of no-growth was observed for the pathogenic strains when they were cultivated with glucansucrase-derived oligosaccharides including raffinose-derived oligosaccharides (Holt, Miller-Fosmore, & Côté, 2005; İspirli, Colquhoun, et al., 2019; İspirli & Dertli, 2020a, 2020b; İspirli, Kaya et al., 2019). It should be noted that in another study, a low level of utilization of maltose and melibiose-derived oligosaccharides by the pathogenic strains was reported suggesting the importance of the structural features of the glucansucrase-derived oligosaccharides (Côté et al., 2003).
Production of lactose derivative hetero-oligosaccharides from whey by glucansucrase E81 and determination of prebiotic functions
2021, LWTCitation Excerpt :No growth was observed for L. reuteri and L. plantarum whereas L. rhamnosus demonstrated high growth rate with the lactose derivative hetero-oligosaccharides at 0.5% concentration (Table 1). This was in accordance with previous findings as strain specific properties for Lactobacilli determined their utilisation for both glucansucrase-based oligosaccharides and lactose derivative hetero-oligosaccharides produced by different enzymes (Bivolarski et al., 2018; Gopal, Sullivan, & Smart, 2001; İspirli, Colquhoun, et al., 2019; İspirli & Dertli, 2020; İspirli, Kaya, & Dertli, 2019). Medium level of growth was observed for B. longum and B. animalis strains with the tested oligosaccharides suggesting a moderate level of bifidogenic effect for lactose derivative hetero-oligosaccharides produced by the acceptor reactions of glucansucrase with whey and lactose (Table 1).