Enzymatic Synthesis and Structural Characterization of Novel Trehalose-Based Oligosaccharides.

Trehalose, α-d-glucopyranosyl-(1↔1)-α-d-glucopyranoside, is a disaccharide with multiple effects on the human body. Synthesis of new trehalose derivatives was investigated through transgalactosylation reactions using ß-galactosidase from four different species. ß-galactosidases from Bacillus circulans (B. circulans) and Aspergillus oryzae (A. oryzae) were observed to be the best biocatalysts, using lactose as the donor and trehalose as the acceptor. Galactosyl derivatives of trehalose were characterized using nuclear magnetic resonance spectroscopy. Trisaccharides were the most abundant oligosaccharides obtained followed by the tetrasaccharide fraction (19.5% vs 8.2% carbohydrates). Interestingly, the pentasaccharide [ß-Galp-(1→4)]3-trehalose was characterized for the first time. Greater oligosaccharide production was observed using ß-galactosidase from B. circulans than that obtained from A. oryzae, where the main structures were based on galactose monomers linked by ß-(1→6) and ß-(1→4) bonds with trehalose in the ending. These results indicate the feasibility of commercially available ß-galactosidases for the synthesis of trehalose-derived oligosaccharides, which might have functional properties, excluding the adverse effects of the single trehalose.

In vitro digestion of polysaccharides: InfoGest protocol and use of small intestinal extract from rat.

Starch, dextran, pectin and modified citrus pectin were subjected to intestinal digestion following InfoGest protocol and a rat small intestine extract (RSIE) treatment. Gastric stage did not show any modification in the structure of the carbohydrates, except for modified pectin. Regarding intestinal phases, starch was hydrolyzed by different ways, resulting in a complementary behavior between InfoGest and RSIE. Contrarily, digestion of dextran was only observed using RSIE. Similar situation occurred in the case of pectins with RSIE, obtaining a partial hydrolysis, especially in the modified citrus pectin. However, citrus pectin was the less prone to hydrolysis by enzymes. The results demonstrated that InfoGest method underestimates the significance of the carbohydrates hydrolysis at the small intestine, thus indicating that RSIE is a very reliable and useful method for a more realistic study of polysaccharides digestion.

Behaviour of citrus pectin and modified citrus pectin in an azoxymethane/dextran sodium sulfate (AOM/DSS)-induced rat colorectal carcinogenesis model.

Large intestine cancer is one of the most relevant chronic diseases taking place at present. Despite therapies have evolved very positively, this pathology is still under deep investigation. One of the recent approaches is the prevention by natural compounds such as pectin. In this paper, we have assessed the impact of citrus pectin and modified citrus pectin on colorectal cancer in rats (Rattus norvegicus F344) to which azoxymethane and DSS were supplied. The lowest intake of food and body weight were detected in animals fed with citrus pectin, together with an increase in the caecum weight, probably due to the viscosity, water retention capacity and bulking properties of pectin. The most striking feature was that, neither citrus pectin nor modified citrus pectin gave rise to a tumorigenesis prevention. Moreover, in both, more than 50% of rats with cancer died, probably ascribed to a severe dysbiosis state in the gut, as shown by the metabolism and metagenomics studies carried out. This was related to a decrease of pH in caecum lumen and increase in acetate and lactic acid levels together with the absence of propionic and butyric acids. A relevant increase in Proteobacteria (Enterobacteriaceae) were thought to be one of the reasons for enteric infection that could have provoked the death of rats and the lack of cancer prevention. However, a reduction of blood glucose and triacylglycerides level and an increase of Bifidobacterium and Lactobacillaceae were found in animals that intake pectin, as compared to universal and modified citrus pectin feeding.

Evaluation of the impact of a rat small intestinal extract on the digestion of four different functional fibers.

The degree of digestion, modulated by rat small intestinal extract on different functional fibers was investigated. In general, inulin-type fructans and fructooligosaccharides were the most resistant to the enzymatic digestion. Results evidenced the high-resistance of fructosyl-fructose bonds. This fits well with the concept of prebiotic carbohydrates. However, the mixture of melibiose, manninotriose and verbascotetraose (α-GOS) from peas, with a considerably lower molecular weight (0.6 kDa) than the fructans studied, were highly digested (61.2%). Interestingly, the Gal-(1 → 6)-Gal bonds present into the manninotriose and verbascotetraose were more prone to be hydrolyzed than Gal-(1 → 6)-Glc (melibiose). However, when melibiose was the only disaccharide present in the reaction mixture, the hydrolysis was also high (67.7%). The use of small intestinal enzymatic preparations is a realistic approximation to evaluate the digestion of different carbohydrates, thus, showing that recognized non-digestible carbohydrates can also be partially digested.

Kinetic study on the digestibility of lactose and lactulose using small intestinal glycosidases.

Lactose is mostly hydrolysed in the small intestine, whereas lactulose, recognised prebiotic carbohydrate, reaches the colon to be fermented by the intestinal microbiota. Digestibility of these substrates was investigated by an in vitro digestion model using a Rat Small Intestine Extract (RSIE). A kinetic study of lactose digestion showed levels of hydrolysis (82.8%) at 0.2 mg*mL-1 and the highest hydrolysis rate constant (kobt). Considering these conditions, lactulose showed high resistance to intestinal digestion by RSIE, resulting in low hydrolysis degrees (20.4%) after 5 h reaction. These results underline the suitability of these intestinal extracts under the studied conditions, as a reliable tool to evaluate carbohydrate digestion and support the evidences towards the higher resistance of galactosyl-fructose linkages during its intestinal degradation.

In Vitro Digestibility of Galactooligosaccharides: Effect of the Structural Features on Their Intestinal Degradation.

Small intestinal brush border membrane vesicles from pig were used to digest galactooligosaccharides from lactose (GOS) and from lactulose (OsLu). Dissimilar hydrolysis rates were detected after digestion. Predominant glycosidic linkages and monomeric composition affected the resistance to intestinal digestive enzymes. The ß(1→3) GOS mixture was the most susceptible to hydrolysis (50.2%), followed by ß(1→4) (34.9%), whereas ß(1→6) linkages were highly resistant to digestion (27.1%). Monomeric composition provided a better resistance in ß(1→6) OsLu (22.8%) compared to ß(1→6) GOS (27.1%). This was also observed for ß-galactosyl fructoses and ß-galactosyl glucoses, where the presence of fructose provided higher resistance to digestion. Thus, the resistance to small intestinal digestive enzymes highly depends upon the structure and composition of prebiotics. Increasing knowledge in this regard could contribute to the future synthesis of new mixtures of carbohydrates, highly resistant to digestion and with potential to be tailored prebiotics with specific properties, targeting, for instance, specific probiotic species.