This work highlights the utility of brush border membrane vesicles (BBMV) from the pig small intestine as a reliable model for gathering information about the reaction mechanisms involved in the human digestion of dietary carbohydrates. Concretely, the elucidation of the transgalactosylation mechanism of pig BBMV to synthesize prebiotic galacto-oligosaccharides (GOS) is provided, unravelling the catalytic activity of mammalian small intestinal β-galactosidase towards the hydrolysis of GOS. This study reveals that pig BBMV preferably synthesizes GOS linked by β-(1 → 3) bonds, since major tri- and disaccharide were produced by the transfer of a galactose unit to the C-3 of the non-reducing moiety of lactose and to the C-3 of glucose, respectively. Therefore, these results point out that dietary GOS having β-(1 → 3) as predominant glycosidic linkages could be more prone to hydrolysis by mammalian intestinal digestive enzymes as compared to those linked by β-(1 → 2), β-(1 → 4), β-(1 ↔ 1) or β-(1 → 6). Given that these data are the first evidence on the transglycosylation activity of mammalian small intestinal glycosidases, findings contained in this work could be crucial for future studies investigating the structure-small intestinal digestibility relationship of a great variety of available prebiotics, as well as for designing tailored fully non-digestible GOS.

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Enzymatic transgalactosylation, in different concentrated carbohydrate solutions, was 2 investigated using brush border membrane vesicles (BBMV) from the pig small 3 intestine. When lactulose was incubated with BBMV, the hydrolytic activity of the 4 enzyme towards the disaccharide was observed to be very low compared to that towards 5 the lactose, but the linkage specificity 1→3), previously observed in lactose 6 solutions, was not significantly affected. As in the case of lactose, lactulose 7 transgalactosylation by BBMV synthesizes the corresponding 3´-galactosyl derivative 8 (β-Gal-(1→3)-β-Gal-(1→4)-β-Fru). Fructose released during lactulose hydrolysis was 9 found to be good acceptor for the transgalactosylation reaction, giving rise to the synthesis of the disaccharide β-Gal-(1→5)-Fru. When incubating an 80/20 mixture of lactulose/galactose, the presence of galactose did not affect the qualitative composition of the transglycosylated substrate but enhanced the synthesis of β-Gal-(1→5)-Fru and decreased the synthesis of β-(1→3) glycosidic bonds. The marked tendency for synthesizing this linkage indicates that under hydrolytic conditions, -Gal-(1→3)-Galand β-Gal-(1→5)-Fru glycosidic bonds would be preferentially digested.

A simple process for purifying lactulose in commercial lactulose syrups was developed.The objective was to establish a selective enzymatic hydrolysis of lactose and epilactose and further removal of monosaccharides by activated charcoal and water/ethanol solutions. Four commercial-galactosidases from different microorganisms were tested obtaining significant differences in their activities towards the three disaccharides present in the lactulose syrup. galactosidases from Bacillus circulans (Biolactasa ® NTL*2) and Bifidobacterium bifidum (Saphera ® 2600 L) showed a reduced enzymatic activity towards lactulose and a high enzymatic activity towards lactose. These two enzymes were chosen to optimize the lactose hydrolysis methodology using Response Surface Methodology (RSM). Once chosen the optimum enzymatic conditions to selectively hydrolyse lactose present in the lactulose syrup, the hydrolysed sample was treated with activated charcoal and water/ethanol solutions to eliminate all monosaccharides. The proposed method offers a product with high purity (> 94%) and high recovery (>80%) of lactulose.

Extruded spaghetti-type pasta systems were obtained separately either from native or oxidized starch prepared via wet chemistry with the aim of evaluating the effect of oxidation modification of starch. In addition to this, the butyrylation reaction (butyrate (Bu) esterification—short-chain fatty acid) using native or oxidized starch was analyzed under reactive extrusion (REx) conditions with and without the addition of a green food-grade organocatalyst (l(+)-tartaric acid) with the purpose of developing potentially health-promoting spaghetti-type pasta systems in terms of increasing its resistant starch (RS) values. These would be due to obtaining organocatalytic butyrylated starch or not, or the manufacture of a doubly modified starch (oxidized-butyrylated—starch oxidation followed by organocatalytic butyrylation) or not. To this end, six pasta systems were developed and characterized by solid-state 13C cross-polarization magic angle spinning nuclear magnetic resonance (CP MAS NMR) spectroscopy, degree of substitution (DS), attenuated total reflectance Fourier transform infrared (ATR/FTIR) spectroscopy, X-ray diffraction (XRD), thermogravimetric analysis (TGA), pancreatic digestion, free Bu content analysis and in vitro starch digestibility. The results obtained here suggest that starch oxidation hydrolytically degrades starch chains, making them more susceptible to enzymatic degradation by α-amylase. However, the oxidized starch-based pasta systems, once esterified by Bu mainly on the amylose molecules (doubly modified pasta systems) increased their RS values, and this was more pronounced with the addition of the organocatalyst (maximum RS value = ~8%). Interestingly, despite the checked chemical changes that took place on the molecular structure of starch upon butyrylation or oxidation reactions in corn starch-based spaghetti-type pasta systems, and their incidence on starch digestibility, the orthorhombic crystalline structure (A-type starch) of starch remained unchanged.

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