α-Glucosidase (Bacillus stearothermophilus)

The procedure for obtaining anthocyanin-enriched extracts from black rice was optimized by response surface methodology, and the effects of the optimized extract on in vitro starch digestibility were investigated in a wheat flour gel model. The experimental results were well-described by a polynomial multiple regression model (R² = 0.8812, p = 0.0546) with regard to anthocyanin content in anthocyanin-enriched extracts from black rice. The optimal conditions for obtaining anthocyanin-enriched extracts from black rice were 50.78% ethanol and 1 N HCl (0.60 mL), yielding a predicted anthocyanin content of 624.27 mg cyanidin 3 glucoside extract. The optimized anthocyanin-enriched extract was a stronger inhibitor of α-glucosidase than acarbose. Furthermore, the predicted glycemic index values of gels prepared with the optimized extract were significantly lower than that of wheat flour gel. These results indicate that the optimized extract suppressed starch hydrolysis by inhibiting digestive enzymes.

Food-derived Weissella spp. have gained attention during recent years as efficient dextran producers. Weissella confusa Cab3 dextransucrase (WcCab3-DSR) was isolated applying PEG fractionation and used for in vitro synthesis of dextran and glucooligosaccharides. WcCab3-DSR had a molar mass of 178 kDa and was activated by Co²⁺ and Ca²⁺ ions. Glycerol and Tween 80 enhanced enzyme stability, and its half-life at 30°C increased from 10 h to 74 h and 59 h, respectively. The ¹H and ¹³C NMR spectral analysis of the produced dextran confirmed the presence of main chain α-(1→6) linkages with only 3.0% of α-(1→3) branching, of which some were elongated. An HPSEC analysis in DMSO revealed a high molecular weight of 1.8 × 10⁷ g/mol. Glucooligosaccarides produced through the acceptor reaction with maltose, were analyzed with HPAEC-PAD and ESI-MS/MS. They were a homologous series of isomaltooligosaccharides with reducing end maltose units. To the best of our knowledge, this is a first report on native W. confusa dextransucrase.

In this paper, anion exchange chromatography coupled with pulsed amperometric detection has been successfully applied for the fine analysis of isomaltooligosaccharides (IMO) syrups where previous reported methods suffered from a lack of homologue oligosaccharides resolution. These syrups are made of a very complex mixture of glucose oligosaccharides characterized at the same time by their DP value (from 2 to ~15) and linkage types [α-(1–2, 3 or 6) and non-IMO α-(1–4)] and position. A mix of available commercial standards (17 species) was completely separated on a CarboPac PA-100 column at a flow rate of 1 mL min⁻¹ and with a gradient of sodium acetate in 100 mM sodium hydroxide. The method was validated according to calibration curve, precision, recovery tests, limits of detection and quantitation. Calibration curves presented correlation coefficients greater than 0.98. The analytical method has been applied on real syrups, keeping a high performance separation of structurally close molecules and giving, for six determinations, very low relative SD for the available standard molecules (0.3–5.8%). The accuracy of the proposed method was tested by recovery measurements: first by spiking maltose on three different syrups and then by spiking six different sugar standards (20, 50 and 75% of the initial content) on a single syrup. Good recovery results (respectively, 96.5–99.7 and 97.1–102.7%) were found. The method was found sensible with limits of detection (signal-to-noise ratio of 3) between 0.048 and 0.124 µg mL^-1 and limits of quantification (signal-to-noise ratio of 10) between 0.159 and 0.412 µg mL^-1.

The impact of high hydrostatic pressure and temperature on the stability and catalytic activity of α-amylase from barley malt has been investigated. Inactivation experiments with α-amylase in the presence and absence of calcium ions have been carried out under combined pressure–temperature treatments in the range of 0.1-800 MPa and 30-75°C. A stabilizing effect of Ca²⁺ ions on the enzyme was found at all pressure–temperature combinations investigated. Kinetic analysis showed deviations of simple first-order reactions which were attributed to the presence of isoenzyme fractions. Polynomial models were used to describe the pressure-temperature dependence of the inactivation rate constants. Derived from that, pressure-temperature isokinetic diagrams were constructed, indicating synergistic and antagonistic effects of pressure and temperature on the inactivation of α-amylase. Pressure up to 200 MPa significantly stabilized the enzyme against temperature-induced inactivation. On the other hand, pressure also hampers the catalytic activity of α-amylase and a progressive deceleration of the conversion rate was detected at all temperatures investigated. However, for the overall reaction of blocked p-nitrophenyl maltoheptaoside cleavage and simultaneous occurring enzyme inactivation in ACES buffer (0.1 M, pH 5.6, 3.8 mM CaCl₂), a maximum of substrate cleavage was identified at 152 MPa and 64°C, yielding approximately 25% higher substrate conversion after 30 min, as compared to the maximum at ambient pressure and 59°C.

Wheat bran is a major dietary fibre source, comprising mainly hemicelluloses of the arabinoxylan type, and cellulose. Environmental scanning electron microscopy was used to associate wheat bran solubilisation by enzymatic treatments (cellulase, cellobiohydrolase, α-glucosidase, xylanase, arabinofuranosidase, α-xylosidase, -amylase, α-amylase, amyloglucosidase, -glucosidase) with tissue structure modifications. When cellulase or xylanase were used alone or in association with other enzymes, separation of outer layers (epicarp and mesocarp) from the endocarp and aleurone layers was observed. Starch granule removal was observed only with a cocktail of enzymes.