Tailoring the natural rare sugars D-tagatose and L-sorbose to produce novel functional carbohydrates

Rare sugars, scarce in nature but with significant potential in food, pharmaceutical, and nutrition industries, are the focus of this research. These include ketohexoses (tagatose, sorbose, psicose), polyols (xylitol), and deoxygenated monosaccharides (L-ribose). While the Izumoring strategy has been a primary method for rare sugar synthesis, newer non-Izumoring enzymatic techniques have emerged, including enzymatic synthesis of novel disaccharides using rare sugars as acceptors. However, research on this latter approach, especially concerning the physiological functions of oligosaccharides containing rare sugars, remains limited. D-Tagatose, in particular, is commercially important due to its low-calorie sweetener properties and GRAS status (Generally Recognized as Safe) in the U.S. It's also authorized as a novel food in other countries. Although showing prebiotic potential and therapeutic properties, D-tagatose's absorption in the small intestine limits its colonic fermentation. This study aims to address this limitation by biosynthesizing non-digestible tagatose-based oligosaccharides via transfructosylation using levansucrase from *Bacillus subtilis* CECT 39 (SacB). The study also explores L-sorbose and D-psicose as potential acceptors, using NMR and molecular docking to characterize the products and elucidate the enzyme's mechanism and substrate preference.

Existing literature highlights various methods for rare sugar production, including the Izumoring strategy and several non-Izumoring enzymatic approaches. These approaches focus primarily on the synthesis of rare sugars themselves rather than their incorporation into larger oligosaccharide structures. Several studies have explored the use of bacterial enzymes for the synthesis of novel disaccharides, such as the xylosylation of D-psicose or the glucosylation of D-galactose. Studies on the transfructosylation of L-sorbose, yielding a β-(2→2)-linked disaccharide, have also been reported. However, a gap in the literature exists concerning the specific physiological functions of oligosaccharides containing rare sugars. The limited understanding of the interactions between these oligosaccharides and the gut microbiota emphasizes the need for further investigation, especially in the context of D-tagatose's known prebiotic properties and its limitations due to small intestinal absorption.

The study utilized levansucrase SacB from *Bacillus subtilis* CECT 39, overproduced and purified from *E. coli*. The enzyme's activity (total, hydrolytic, and transfructosylation) was determined. Transfructosylation reactions were conducted using sucrose as the fructosyl donor and D-tagatose, L-sorbose, or D-psicose as acceptors. Reaction conditions (pH, temperature, enzyme concentration, and substrate ratios) were optimized, focusing on D-tagatose. The resulting oligosaccharides were purified using preparative LC-RID and characterized structurally using 1D and 2D NMR (¹H, ¹³C, gCOSY, TOCSY, gHSQC, gHMBC, gHSQC-TOCSY). Molecular docking (AutoDock Vina) was used to investigate SacB's binding modes with the rare sugars. *In vitro* small intestinal digestibility was assessed using pig BBMV (brush border membrane vesicles), and the prebiotic properties of the main tagatose-based disaccharide were evaluated using *in vitro* fermentation with human fecal samples. 16S rRNA sequencing was used to analyze microbiome composition changes, and SCFA (short-chain fatty acid) levels were measured using GC-FID. Statistical analyses (ANCOM, LEfSe, metagenomeSeq, Pearson correlation) were used to interpret the results.

SacB efficiently transferred fructose from sucrose to D-tagatose (at C-1) and L-sorbose (at C-5), forming the main disaccharides β-D-fructofuranosyl-(2→1)-D-tagatopyranose and β-D-fructofuranosyl-(2→5)-α-L-sorbopyranose. Molecular docking supported these findings and explained the enzyme's preference. Further fructose transfer yielded oligosaccharides with higher polymerization degrees. Optimal conditions for D-tagatose fructosylation were a 300:300 g/L sucrose:D-tagatose ratio and a SacB concentration of 3.1 U/mL. The main tagatose-based disaccharide (β-D-Fru-(2→1)-D-Tag) showed high resistance (99.8% after 3 h) to digestion by pig BBMV. *In vitro* fecal fermentation showed that β-D-Fru-(2→1)-D-Tag modulated the gut microbiota similarly to FOS (fructooligosaccharides), promoting Bifidobacterium and Mitsuokella growth more than unmodified tagatose. β-D-Fru-(2→1)-D-Tag also selectively stimulated Eggerthella, Ruminococcus gnavus group, Romboutsia, Succinivibrio, and Sutterella. Positive correlations were found between certain genera (e.g., Bifidobacterium) and SCFA (acetic acid, lactic acid) levels, highlighting the prebiotic effect.

The results demonstrate the successful biosynthesis of novel, non-digestible tagatose-based oligosaccharides with prebiotic potential. The high resistance to small intestinal digestion, combined with the selective modulation of the gut microbiota, suggests that these oligosaccharides could be valuable as low-calorie sweeteners and prebiotics. The specific glycosidic linkages (β-(2→1) in the main disaccharide) likely play a crucial role in their prebiotic properties, offering a strategy to enhance the beneficial effects of tagatose. The findings highlight the potential of tailoring rare sugars to create functional food ingredients, expanding the application of these compounds in various industries. The similarities between β-D-Fru-(2→1)-D-Tag and commercial FOS suggest comparable modulatory effects on gut microbiota.

This study successfully synthesized novel non-digestible oligosaccharides from rare sugars using a levansucrase. The main tagatose-based disaccharide showed high resistance to intestinal digestion and exhibited a prebiotic effect, promoting beneficial bacterial growth. The approach demonstrated here offers a promising strategy for developing novel prebiotics and low-calorie sweeteners by tailoring rare sugars, potentially improving the bioavailability and gut health benefits.

The study used pig BBMV to simulate human small intestinal digestion. While this is a commonly used model, differences might exist between pig and human enzyme activities. Further studies using human BBMV would strengthen the findings. The *in vitro* fecal fermentation model, while informative, doesn't completely replicate the complex conditions of the human gut. Long-term *in vivo* studies are needed to confirm the prebiotic effects and overall health benefits of the synthesized oligosaccharides.