Characterizations and In Vitro Gut Microbiome Modulatory Effects of Gluco-Oligosaccharides Synthesized by the Acceptor Reactions of Glucansucrase 53

Abstract

The production of novel oligosaccharides with potential prebiotic effects is of interest to expand the current market and explore the effectiveness of new functional carbohydrate forms. The utilization of glucansucrases is a cost-effective and environmentally friendly biotechnological strategy for producing novel gluco-oligosaccharides through acceptor reactions. In this study, an active glucansucrase (GS53) was used to produce gluco-oligosaccharides via its acceptor reactions with glucose, maltose, and maltotriose, and these oligosaccharides were tested in terms of structure and their gut microbiome modulatory effects. The formations of oligosaccharides were monitored by TLC analysis, and GS53 was active for the three acceptors but not for the other sugars tested. The structural characterization of the gluco-oligosaccharides by ¹H NMR analysis revealed the glycosylation of each acceptor with α-(1 → 3) and α-(1 → 6) linkages, whereas LC-MS analysis demonstrated the formations of DP 8, DP 7, and DP 6 oligosaccharides with acceptors maltose, maltotriose, and glucose, respectively. In vitro fecal fermentation analysis, in which microbial short-chain fatty acids (SCFAs) and microbial compositional changes were assessed using gas chromatography and 16S rRNA sequencing, respectively, demonstrated that the gluco-oligosaccharides formed SCFAs—particularly propionate and butyrate—at levels comparable to those observed with inulin, a well-established prebiotic. Additionally, the gluco-oligosaccharides were found to promote the growth of Bifidobacterium adolescentis and Blautia OTUs, which are known to have important physiological functions beneficial to human health. Overall, these results demonstrate that gluco-oligosaccharides synthesized using GS53 through acceptor reactions exhibit prebiotic potentials and could be utilized in the future as dietary supplements as well as in the development of functional foods targeting colonic health.

1. Introduction

The functional food market is expanding rapidly in response to rising consumer demands, with prebiotics and candidate prebiotic compounds emerging as critical components in the development of novel functional food products [1]. Prebiotics are primarily valued for their ability to selectively modulate the composition and function of the gut microbiota by promoting beneficial bacterial groups, including, but not limited to, bifidobacteria and lactobacilli, which are associated with various health benefits [2]. The stimulation of beneficial microbes within the gut flora results in the modulation of the gut microbiome and suppresses pathogenic bacteria, helping shift host health towards homeostasis. During this process, immunomodulatory functions of prebiotics as well as their roles in reduction of the risks of certain metabolic diseases can occur [3,4,5]. Structurally, prebiotics are mainly carbohydrates comprising specific glycosidic linkages that cannot be broken down by digestive enzymes during their transit through the gastrointestinal system, and certain oligosaccharides are classified as effective prebiotic compounds [5]. The main examples of prebiotic oligosaccharides currently dominating the market with distinct applications are fructo-oligosaccharides and galacto-oligosaccharides [6]. In addition, there has been an increasing demand for the production of alternative novel oligosaccharides, which possess controlled and predictable structural and functional features and have the potential to act like prebiotics, in sustainable and cost- and scale-effective ways [7]. For this, biotechnological approaches have become promising methods, and different enzymes from distinct sources such as glucansucrases can be utilized to produce novel oligosaccharides with controlled and predictable structural and functional features [8].

Glucansucrases (EC. 2.4.1.5), mainly encoded in the genomes of lactic acid bacteria (LAB), are effective examples of the enzymes that can be employed to produce novel indigestible oligosaccharides with prebiotic potential. Specifically, glucansucrases can catalyze acceptor reactions, with sucrose as the donor and a mono/di/oligosaccharide molecule as the acceptor; the glucose units are transferred to the acceptor sugar by forming (1,6)Glc/(1,3)Glc/(1,2)Glc/(1,4)Glc units in the growing oligosaccharide chain [9]. The formation of the linkage type, the length of the oligosaccharide formed, and the branched or unbranched nature of the oligosaccharides can be varied for different factors, such as donor-acceptor ratio, type of the acceptor molecule, and the structural features of the glucansucrases, the last of which plays the most critical role [9,10]. For instance, acceptor reactions of mellibiose with two different glucansucrases resulted in the formation of gluco-oligosaccharides with (1,6)Glc/(1,3)Glc units and (1,6)Glc/(1,4)Glc/(1,3)Glc units, respectively, confirming the crucial role of glucansucrases [11,12]. The structural features (i.e., linkage type, branching ratio, molecular weight) of indigestible carbohydrates like gluco-oliogosaccharides dictate their impacts on colonic microbiota composition and function, because microbial binding proteins, hydrolases, and transporters are quite structure-specific in their degradative activities [13,14]. Many different studies have highlighted the potential effectiveness of the glucansucrases forming distinct gluco-oligosaccharides with potential prebiotic and immuno-modulatory functions in a very cost-effective and sustainable approach [2,10,11,15,16,17]. In these cases only sucrose as a cheap substrate and an acceptor sugar that can be obtained from various by-products are required for the catalytic action of the glucansucrases, and unlike other oligosaccharide production strategies requiring harsh degradation conditions for depolymerization, oligosaccharides are formed in very mild conditions [15,16,17]. One example is a glucansucrase from the Leuconostoc citreum SK24.002 strain, which was demonstrated to produce gluco-oligosaccharides, utilizing maltose as the acceptor sugar. These maltose-derived GOSs, which possess varying degrees of polymerization (DP), have been observed to stimulate in vitro growth of Bifidobacterium and Lactobacillus strains in a similar way, with fructooligosaccharides acting as the carbon source. Among the tested oligosaccharides, the DP4 structure demonstrated the highest butyrate production potential [18]. In another example, the gluco-oligosaccharides produced by an acceptor reaction of glucansucrase E81 with melibiose and lactose were found to promote the growth of Bifidobacterium strains but did not have the same effect on Lactobacillus species [11,15]. Similarly, glucan-based oligosaccharides synthesized in the reaction medium of glucansucrase from L. mesenteroides 6055 with sucrose and maltose increased the number of Akkermansia, Bacteroides and Bifidobacterium species, while producing high levels of acetic acid, an important SCFA for regulating both microbiome and epithelial behavior [19]. The number of structurally variant gluco-oligosaccharides produced by glucansucrases is rapidly increasing, and it is critical to screen the prebiotic potential of these diverse oligosaccharides under fecal fermentation conditions to determine their potentially divergent impacts on gut microbiota modulation as well as for the formation of short-chain fatty acids (SCFA) as a function of their structural features.

Similarly, the numbers of whole-genome sequenced LAB strains have been increasing continuously and rapidly, and it is possible to identify more glucansucrases that might be responsible for the production of novel gluco-oligosaccharides with different structural and functional properties [20,21]. In this study, an active glucansucrase (GS53) obtained from Weissella cibaria PDER53A was tested for acceptor reactions with maltose, maltotriose, glucose, lactose, trehalose, raffinose, cellobiose, mannose, and melibiose as the acceptor sugars to produce potentially prebiotic gluco-oligosaccharides. At first, TLC analysis was used as an assay for formation of the gluco-oligosaccharides with different acceptor sugars to explore the differential activity of GS53. This was followed by the structural characterization of the GS53 gluco-oligosaccharides formed with the acceptor reactions of glucose, maltose, and maltotriose by NMR and LC-MS analysis. Finally, the gut microbiota modulation properties of the gluco-oligosaccharides produced with GS53 were determined through in vitro fecal fermentation analysis. Our results revealed that GS53 can be used for the production of novel gluco-oligosaccharides with prebiotic potentials, which can then be utilized as supplements or incorporated into food formulations for the production of functional food commodities targeting colonic health.

2. Materials and Methods

2.1. Chemicals and Reagents

Glucansucrase 53 (GS53), encoded in the genome of Weissella cibaria PDER53A, was used in this study to produce the gluco-oligosaccharides, and the cloning, expression, purification, and activity tests were performed as described elsewhere (İspirli et al., in preparation). Sucrose, maltose, maltotriose, glucose, lactose, trehalose, raffinose, cellobiose, mannose, and melibiose were purchased from Sigma-Aldrich (Darmstadt, Germany) and TLC silica gel aluminum sheets were obtained from Merck (Darmstadt, Germany). All other chemicals used in this study were obtained from Sigma-Aldrich (Germany).

2.2. Production of Gluco-Oligosaccharides with the Acceptor Reaction of GS53

The formations of oligosaccharides by GS53 acceptor reactions were screened using TLC analysis. The donor-acceptor reactions were first performed in medium containing 1 mM CaCl₂ and 1 U GS53 enzyme, 0.4 g mL⁻¹ sucrose, 0.1 g mL⁻¹ acceptor sugars (maltose, maltotriose, glucose, lactose, trehalose, raffinose, cellobiose, mannose, and melibiose) and 20 mM sodium acetate at pH 5.4. These reactions identified maltose, maltotriose, and glucose as the most effective acceptor sugars. The reaction of maltose, maltotriose, and glucose as acceptor sugars was performed under the same reaction conditions at 37 °C until sucrose was depleted, followed by TLC analysis. Samples from each reaction were loaded onto TLC plates and run with acetonitrile–H2O (80:20) solvent. The products on the TLC plates were then developed using methanol/sulfuric acid/N-1-naphthyl dihydrochloride staining. After sucrose was consumed, residual sugars were removed using immobilized yeast, as previously described [22]. The reaction mixtures were then subjected to structural characterization.

2.3. Structural Characterization of Gluco-Oligosaccharides by NMR and LC-MS/QTOF

¹H NMR analysis was used for the structural characterization of the gluco-oligosaccharides formed by the acceptor reactions of GS53 with maltose, maltotriose, and glucose using previously described methodology [16]. Briefly, lyophilized oligosaccharide samples were dissolved in 500 μL D2O for analysis, and an Agilent Premium Compact 600 MHz NMR instrument was used for analysis. The ¹H NMR spectra of the gluco-oligosaccharides were analyzed using the MestReNova program (14.2.1). To confirm the formation of the oligosaccharides as well as to determine their final molecular masses, LC-MS/Q-TOF analysis was performed. The details of the analytical protocol are given elsewhere [23].

2.4. In Vitro Fecal Fermentation of Gluco-Oligosaccharides

In vitro fecal fermentation of gluco-oligosaccharides was performed in an anaerobic chamber (BACTRON300 Anaerobic Chamber; Shel Lab, Cornelius, OR, USA) under an atmosphere of 90% N₂, 5% CO₂, and 5% H₂, as previously described [24]. Fermentations were performed using gluco-oligosaccharides formed by the acceptor reactions of GS53 with maltose, maltotriose, and glucose, a commercially available inulin as a positive control (Sigma-Aldrich #P9135), and a carbon-free control group (blank). Substrates were weighed (50 mg) into 25 mL Balch tubes (Chemglass Life Sciences, Vineland, NJ, USA) for each time point (0, 6, 12, 24, and 48 h) and transferred to the anaerobic chamber. Carbonate-phosphate buffer was transferred to the anaerobic chamber to deoxygenate, and the next day 2 ml of autoclaved buffer was added to each sample tube and mixed.

Fecal material collected from three individuals who had not used antibiotics in the past 6 months, were between the ages of 25 and 45, and were not pregnant or lactating (for female donors) were placed in separate containers to account for individual diversity in the gut microbiota, tightly sealed, kept on ice, and transferred to the anaerobic chamber. Samples were then homogenized with carbonate-phosphate buffer [feces–buffer 1:3 (w/v)] and filtered through four layers of cheesecloth. Each tube containing the gluco-oligosaccharides and the control groups was inoculated with 0.5 mL of filtered fecal slurry. The tubes were immediately hermetically sealed with aluminum stoppers (Chemglass Life Sciences, Vineland, NJ, USA) and incubated at 37 °C at 150 rpm in a shaking incubator (Innova 42, New Brunswick Scientific, Edison, NJ, USA). Analysis was performed in triplicate for each sample. In addition, 2 mL aliquots were taken for DNA extraction and analysis at 0 and 24 h and stored at −20 °C until further use. Protocols involving human stool collection and use were approved by the Scientific Research Ethics Committee of Health Sciences of Necmettin Erbakan University (application number: 10684; approval number: 2022/259; date of approval: 6 July 2022).

2.5. Determination of SCFA Levels During Fecal Fermentation

SCFAs (acetic acid, butyric acid, and propionic acid), which account for 90–95% of microbial metabolites, are produced by fermentation by microorganisms in the colon. To quantify metabolic performance, 1 mL aliquots were taken from each tube at 0, 6, 12, 24, and 48 h time points for the determination of SCFAs. A total of 200 μL of an internal standard (157.5 mL of 4-methyl valeric acid, 1.47 mL of 85% phosphoric acid, and 39 mg of copper sulfate pentahydrate in a total volume of 25 mL) was added to each aliquot. Samples were stored at −20 °C until the time of analysis. For the analysis, samples were brought to room temperature and then centrifuged at 13,000 rpm for 10 min. The amount of SCFAs was determined using a gas chromatograph (GC-FID, GC-2030, Shimadzu, Kyoto, Japan) under the following conditions: column flow: 1.10 mL/min; oven starting temperature: 100 °C; FID temperature: 250 °C; 1 min at 100 °C and 5.5 min at 200 °C, as described previously [24].

2.6. Microbiota Composition Analysis

2.6.1. Extraction of DNA from Fecal Material

DNA from samples collected at 0 and 24 h of fermentation was isolated using the phenol-chloroform method described by [25]. Samples collected 24 h after inoculation were thawed at room temperature, then centrifuged at 13,000 rpm at 4 °C for 10 min, and the supernatant was removed. DNA extraction was performed using previously described and modified chemical-enzymatic lysis methods [26,27]. Details of the phenol-chloroform method used are presented in a previous study [28].

2.6.2. 16S rRNA Sequencing, Sequence Processing and Community Analysis

The conserved V4–V5 regions of the 16S rRNA gene were amplified using the universal microbial primers under the following PCR conditions: initial denaturation at 95 °C for 5 min; 22 cycles of denaturation at 98 °C for 20 s, annealing at 60 °C for 15 s, and extension at 72 °C for 30 s; and a final extension at 72 °C for 10 min, as described in detail in a previous study [24,28,29]. The PCR products obtained from the amplification process were barcoded with TruSeq dual indexing primers, and the KAPA HiFi HotStart DNA Polymerase Kit (KAPA Biosystems Inc., Wilmington, MA, USA) was used to amplify the DNA sequences for analysis. The amplified and barcoded 16S rRNA regions were sequenced using next-generation sequencing technology (Illumina Miseq with 2 × 250 cycles and V2 chemistry (Illumina, San Diego, CA, USA)) at the Purdue University Genomic Core Facility (West Lafayette, IN, USA) [29]. Sequencing data were processed using mothur version 1.48.0 (15 May 2024) according to the mothur MiSeq SOP (https://mothur.org/wiki/miseq_sop/, accessed on 15 May 2024), with previously described modifications [24]. Sequences were classified using the mothur-formatted Ribosomal Database Project version 16, to which species epithets had been added, allowing species-level classification of OTUs (operational taxonomic units, computational analogs of species).

2.7. Bioinformatics and Statistical Analysis

Diversity metrics were calculated using summary.single() and dist.shared() commands in mothur. Ecological α-diversity was measured using the Chao, Inverse Simpson, and Shannon indices, whereas Bray–Curtis was used to calculate β-diversity metrics. The analysis of molecular variance (AMOVA) test was used to determine whether centroids were significantly different between size fractions.

Calculations and graphing were performed using GraphPad Prism^® (version 10, GraphPad Software Inc., La Jolla, CA, USA) statistical software. Statistical differences in the data were determined by one-way analysis of variance (ANOVA). Tukey’s comparison test was used to assess significant differences between groups, with an alpha value of 0.05.

3. Results and Discussion

3.1. Synthesis and Characterization of Gluco-Oligosaccharides Produced by GS53 Acceptor Reactions

In this study, an active glucansucrase GS53 from Weissella cibaria PDER53A was tested for its potential to form novel gluco-oligosaccharides. To determine the utilization of which acceptor sugars were used by GS53, reactions were performed with maltose, maltotriose, glucose, lactose, trehalose, raffinose, cellobiose, mannose, and melibiose. Among the tested sugars, GS53 was only active on maltose, maltotriose, and glucose as acceptors (herein referred to as GS53 maltose, GS53 maltotriose, and GS53 glucose, respectively) (Figure 1); no oligosaccharide formation was observed for the acceptor reaction of GS53 with lactose, trehalose, raffinose, cellobiose, mannose, and melibiose. The inability of GS53 to use these sugars as acceptors might be related to structural features of both GS53 and acceptor sugars and/or the donor–acceptor ratio [17,21,23]. Figure 1 demonstrates the formation of gluco-oligosaccharides with glucose, maltose, and maltotriose by TLC, with fructose removed from the reaction environment by immobilized yeast. Importantly, no glucose accumulation was observed, in contrast to the presence of the fructose in the reaction medium. This was due to the gluco-elongations to the acceptor sugars, resulting in the formation of gluco-oligosaccharides [30]. Overall, these findings revealed that, similar to other glucansucrases [22,31], GS53 efficiently utilized glucose, maltose, and maltotriose as acceptor sugars for the production of gluco-oligosaccharides.

Characterization of the gluco-oligosaccharides produced by GS53 was further per-formed by ¹H NMR and LC-MS/Q-TOF analysis. Figure 2 demonstrates the ¹H NMR spectrum of the gluco-oligosaccharides produced by the acceptor reactions of maltose (a), maltotriose (b), and glucose (c), respectively. The ¹H NMR spectrum for the acceptor reaction of maltose (Figure 2a) revealed the addition of the α-Glc → 3 and α-Glc → 6 units to acceptor maltose, detected with the signals at δ~5.20 and δ~4.83, respectively [21]. Additionally, the signals at δ 5.08 and δ 4.53 correspond to the reducing ends of the oligosaccharides Rα and Rβ, respectively. Signals at δ 5.36 (M) and δ 4.97 (L) originate from the α-Glc → 4 unit at the nonreducing end of the maltose acceptor and the α-D-Glcp-(1 → 5)-D-Fru (leucrose) unit [21]. Similar to the ¹H NMR spectrum of maltose, the ¹H NMR spectrum of the acceptor reaction of products of maltotriose (Figure 2b) showed signals indicating the addition of the α-Glc → 3 and α-Glc → 6 units to maltotriose, with peaks at δ~5.18 and δ~4.81, respectively, with a slight shift [21]. Apart from the new glycosylation units, similar peaks originating from maltotriose and leucrose were observed in the spectra of gluco-oligosaccharides formed by the acceptor reaction of GS53 with maltotriose (Figure 2b). Finally, the ¹H NMR spectrum of gluco-oligosaccharides formed by the acceptor reaction of glucose was observed, and both α-Glc → 6 units and α-Glc → 3 units were detected in the structure of oligosaccharides, where the former were the dominant units in the final structural confirmation (Figure 2c). Overall, these findings revealed that GS53 successfully transferred α-Glc → 6 units and α-Glc → 3 units directly to the acceptor sugars, whereas addition of the α-Glc → 6 units to the oligosaccharide chain was observed at high levels. Previously, utilizations of these three acceptors for the formation of distinct oligosaccharides were tested, and elongations of the oligosaccharide chain with glucose (Glc) units via (α1 → 4), (α1 → 3), (α1 → 2) and (α1 → 6) linkages depending on the glucansucrases tested were observed [10,21,30,32], suggesting the crucial role of the glucansucrase for the final structural characteristics of the oligosaccharides.

We further hypothesized that the α-Glc → 6 units in the GS53-synthesized gluco-oligosaccharides could demonstrate significant prebiotic potential, since these structures can reach the colon without degradation in the upper gastrointestinal system and can be fermented by beneficial colonic microorganisms. Further, it is possible that the ultimate ratio of glucose units and the final chain length of these oligosaccharides could influence their prebiotic effects.

Following the confirmation of the structure of the gluco-oligosaccharides formed with the successful acceptor reactions, the oligosaccharide mixtures were subjected to electrospray ionization tandem MS (ESI-MS/MS) analysis to determine the final degree of polymerization of the oligosaccharides. Figure 3 demonstrates the ESI-MS/MS profiles of the corresponding oligosaccharide mixtures, where the highest mass values of 1349 m/z, 1187 m/z, and 1025 m/z were observed for the gluco-oligosaccharides obtained by the acceptor reactions of maltose, maltotriose, and glucose, respectively (Figure 3a–c). These data revealed that GS53 produced DP 8, DP 7, and DP 6 gluco-oligosaccharides using maltose, maltotriose, and glucose, respectively. Previously, the formation of oligosaccharides with DP values of DP 9 [21], DP ≤ 6 [32,33], and DP~12 [30] were observed with the acceptor reactions of other glucansucrases with maltose and maltooligosaccharides, suggesting the glucansucrase-specific characteristics determine the final DP values of products. Overall, the structural characterization of the gluco-oligosaccharides produced by GS53 acceptor reaction demonstrated the addition of the high levels of α-Glc → 6 units together with low levels of α-Glc → 3 units to the oligosaccharide growing chain. The final DP values of these oligosaccharides were DP 8, DP 7, and DP 6 for maltose, maltotriose, and glucose acceptor reactions, respectively, suggesting the possibility that product gluco-oligosaccharides may differ in their final functional properties.

3.2. GS53 Gluco-Oligosaccharides Induced Short-Chain Fatty Acid (SCFA) Production Comparable to That of Inulin

To test the effects of GS53 oligosaccharides produced in this study on gut microbiota composition and metabolic outputs, a series of in vitro fecal fermentation assays were conducted, and microbial short-chain fatty acids (SCFAs—namely, acetate, propionate, and butyrate) generated throughout the fermentation were quantified (Figure 4 and Figure S1). Total SCFA amounts were calculated by summing acetate, propionate, and butyrate. Inulin was included in the fecal fermentation assays as a positive control. Our results revealed no significant differences (p > 0.05) between the samples and the control group regarding total SCFA, acetate, propionate, and butyrate levels (Figure S1). However, butyrate is an important SCFA for gut health. It was observed that after 24 h of fermentation, maltotriose resulted in the generation of significantly (p < 0.05) lower butyrate after 24 h of fermentation as compared to inulin and GS53 glucose (Figure S1). Therefore, the data obtained from the 24 h fermentation process were used for further analysis (Figure 4). These data clearly demonstrate that oligosaccharides synthesized in this study stimulate SCFA production to a similar extent as inulin. Inulin is well-established as a highly butyrogenic prebiotic compound [34,35]. Thus, these results further suggest that oligosaccharides synthesized herein may exhibit prebiotic potential.

3.3. GS53 Oligosaccharides Influenced the Structure (β-Diversity) and α-Diversity of Microbial Communities

To investigate the effects of gluco-oligosaccharides on colonic microbiota composition, the samples collected before the fermentation (initial) and after 24 h of fermentation were analyzed through 16S rRNA gene amplicon sequencing. The 24 h time points were chosen because, within the 24 h of fermentation, the SCFA generated by the fecal microbiota almost reached their plateau state (Figure 4), suggesting that the majority of the fermentation was completed within 24 h.

The overall structures of microbial communities were compared using the Bray–Curtis dissimilarity metric based on the relative abundance of OTUs at a 97% identity level (Figure 5a). The substrate was found to have a significant (p < 0.001, AMOVA) effect on microbial community composition, as the microbial communities of each sample were clustered accordingly, accounting for almost 76.5% of the total variation.

α-Diversity of the communities was calculated using the number of species observed and Chao indices, which account for richness, as well as inverse Simpson and Shannon indices, which account for both richness and evenness (Figure 5b–e). No significant (p > 0.05) differences in community richness values were observed among any of the samples. On the other hand, GS53 glucose had significantly (p < 0.05) lower Shannon and inverse Simpson indices compared to the blank and inulin controls. This suggests that GS53 glucose has significantly reduced evenness compared to inulin based on these indices. Furthermore, although no significant differences were observed (p > 0.05), GS53 glucose trended towards lower inverse Simpson and Shannon index values compared to GS53 maltose and GS53 maltotriose. This may be attributed to the presence of the additional α1 → 4 glycosidic linkages in GS53 maltose and maltotriose, which are absent in GS53 glucose; the degradation of a broader range of glycosidic linkages potentially requires the involvement of a more diverse microbial community.

3.4. GS53 Gluco-Oligosaccharides Promoted Butyrate-Producing Bacteria and OTUs Within Bifidobacterium

To investigate the effects of gluco-oligosaccharides on colonic microbiota composition, operational taxonomic unit (OTU, computational analogs of species) abundances were compared before and after (24 h post-inoculation) fermentation. The fold-changes (relative to time 0) in the relative abundances of the most abundant 50 microbial OTUs are illustrated in a heatmap (Figure 6, left panel), and the relative abundances of some of the most responsive taxa are given in bar graphs (Figure 6, right panel). Overall, our results revealed that the gluco-oligosaccharides synthesized in this study promote various microbial taxa that are known to have the ability to synthesize SCFAs and have important physiological functions; however, the degree of promotion was gluco-olgosaccharide type (structure)-dependent. For instance, 3.9-, 4.8-, 4.7-, and 8.0-fold increases (relative to time 0) were observed in the relative abundance of OTU10 Bifidobacterium adolescentis after 24 h of fermentation of inulin, GS53 glucose, GS53 maltoriose, and GS53 maltose, respectively. B. adolescentis exhibits health-promoting effects by improving intestinal barrier function, exerting anti-inflammatory and immune-regulatory effects, and producing neurotransmitters (i.e., gamma-aminobutyric acid (GABA)) and vitamins [36]. In addition, B. adolescentis is known to utilize dietary carbohydrates, and in turn, produce acetate and lactate [36]. Lactate produced by B. adolescentis could be possibly converted into butyrate by other butyrate-producing bacteria in the gut belonging to phylum Bacillota [37,38]. Thus, stimulation of B. adolescentis by the substrates may contribute to elevated butyrate formation during the fermentation observed in this study.

In addition, like inulin, a well-known prebiotic, fermentations of gluco-oligosaccharides by fecal microbiota significantly (p < 0.05) increased the relative abundances of OTUs classified within the genera Blautia (OTU9, OTU23, OTU29), Coprococcus (OTU26, OTU41), and Lachnospiraceae (OTU15, OTU44). These microorganisms are known to strongly generate through dietary carbohydrate fermentation [39,40]. Therefore, the elevated butyrate formation observed at the end of the fermentation assays may be attributed to significant consumption of gluco-oligosaccharides by these organisms. In addition, butyrate has garnered increasing attention due to its numerous health-promoting effects, both locally within the gut and systemically throughout the body. It serves as the principal energy source for colonocytes, exhibits potent anti-inflammatory properties, contributes to colorectal cancer prevention, and plays a crucial role in maintaining intestinal homeostasis [41].

OTU2 Bacteroides ovatus, OTU3 B. uniformis, and their OTU4 Bacteroides sp. were among the most responsive microbial taxa during the fermentation of given substrates. At the end of the fermentation, inulin, GS53 glucose, GS53 maltoriose, and GS53 maltose resulted in 11.6-, 19.7-, 16.5-, and 11.7-fold increases (relative to time 0) in the relative abundance of OTU2 B. ovatus, respectively. Similarly, at the end of the fermentation, inulin, GS53 glucose, GS53 maltoriose, and GS53 maltose resulted in 9.8-, 17.4-, 17.1-, and 15.4-fold increases (relative to time 0), respectively, in the relative abundance of OTU3 B. uniformis. Furthermore, at the end of the fermentation, inulin did not cause significant (p > 0.05) change in the relative abundance of OTU4 Bacteroides, but GS53 glucose, GS53 maltoriose, and GS53 maltose resulted in 39.1-, 27.6-, and 23.3-fold increases (relative to time 0) in the relative abundance of this OTU, respectively. These increases could be attributed to the superior ability of Bacteroides members to utilize dietary glucans [42]. Moreover, in addition to generating acetate, Bacteroides species (including B. ovatus) are known to produce large amounts of propionate as a result of carbohydrate fermentation through the succinate pathway [43,44]. It is likely that the high concentrations of propionate observed at the end of substrate fermentation in this study may be attributed largely to the increased activity of Bacteroides OTUs.

Another taxon stimulated by gluco-oligosaccharides was OTU7 Parabacteroides distasonis, with the degree of promotion varying in a structure-dependent manner. For example, at the end of the fermentation, the relative abundance of OTU7 P. distasonis in GS53 matotriose was significantly higher than that in GS53 glucose and GS53 maltose. Parabacteroides spp., including P. distasonis, have been previously observed to respond differentially in their growth rates on divergent resistant glucan structures [45]. Increased growth of this species on gluco-oligosaccharides could be attributed to the fact that, like Bacteroides, Parabacteroides species have broad carbohydrate utilization capacity [46]. Parabacteroides spp. generally ferment to acetate [47] and succinate [48], which could be either absorbed into the bloodstream [46] or further converted to other metabolites like propionate or butyrate by other neighboring organisms in the gut [49]. Increased abundance of OTU7 P. distasonis could be a contributing factor to elevated acetate and, indirectly, the propionate and butyrate production observed in this study. Moreover, P. distasonis has previously been shown to provide beneficial health effects, such as alleviating inflammatory arthritis [50], pancreatitis [47], insulin resistance [51], obesity, and metabolic dysfunction [48]. Because of these health-promoting effects, some recent papers have recognized P. distasonis as a next-generation probiotic [52,53,54]. Another next-generation probiotic, Bacteroides uniformis, as noted above, is believed to positively influence the production of health-promoting short-chain fatty acids (SCFAs) as a result of the significant stimulation of its growth by GS53 oligosaccharides.

4. Conclusions

In this study, glucansucrase GS53 was effectively employed to utilize gluco-oligosaccharides with different structural features using sucrose as donor and maltose, maltotriose, and glucose as acceptors. Structural analysis revealed that oligosaccharides derived from maltose and maltotriose contained α-(1 → 3), α-(1 → 4), and α-(1 → 6) linkages, whereas those synthesized on glucose lacked α-(1 → 4) linkages. The degree of polymerization was found to be 8, 7, and 6 for maltose-, maltotriose-, and glucose-based oligosaccharides, respectively. In vitro fecal fermentation analysis revealed that the gluco-oligosaccharides resulted in the generation of propionate and butyrate at levels comparable to those observed with inulin, a well-established prebiotic. Furthermore, the gluco-oligosaccharides were found to promote the growth of Bifidobacterium adolescentis, Blautia, and Lachnospiraceae OTUs, which are known to have important physiological functions beneficial to human health. Collectively, these findings underscore the prebiotic potentials of gluco-oligosaccharides synthesized using GS53 through acceptor reactions and further suggest that these oligosaccharides may be beneficial as dietary supplements and in the development of functional foods aimed at enhancing colonic health. Future studies should focus on investigating the properties of these oligosaccharides for food applications and validate their prebiotic potentials through studies in animal models and clinical trials.