Hydrolysis Of Galactooligosaccharides Research Articles

Trans-\u03b2-galactosidase activity of pig enzymes embedded in the small intestinal brush border membrane vesicles

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.

Hydrolysis of galacto-oligosaccharides in soy molasses by α -galactosidases and invertase from Aspergillus terreus

Two α -galactosidase (P1 and P2) and one invertase present in the culture of Aspergillus terreus grown on wheat straw for 168 h at 28ºC were partially purified by gel filtration and hydrophobic interaction chromatographies. Optimum pH and temperatures for P1, P2 and invertase preparations were 4.5-5.0, 5.5 and 4.0 and 60, 55 and 65ºC, respectively. The K M app for Ï� -nitrophenyl-α -D-galactopyranoside were 1.32 mM and 0.72 mM for P1 and P2, respectively, while the K M app value for invertase, using sacarose as a substrate was 15.66 mM. Enzyme preparations P1 and P2 maintained their activities after pre-incubation for 3 h at 50ºC and invertase maintained about 90% after 6 h at 55 ºC. P1 and P2 presented different inhibition sensitivities by Ag+, D-galactose, and SDS. All enzyme preparations hydrolyzed galacto-ologosaccharides present in soymolasses.

Production of α-Galactosidase by Aspergillus oryzae through solid-state fermentation and its application in soymilk Galactooligosaccharide hydrolysis

α-Galactosidase was produced by Aspergillus oryzae on red gram plant waste-wheat bran based media in solid-state fermentation (SSF). Optimum temperature for α-galactosidase production was 35 0C and upto 4 cm of bed height of substrate had no inhibitory effect on enzyme production. Hydrolysis of galactooligosaccharides in soymilk was carried out by α-galactosidase. Optimum temperature and pH for the hydrolysis of raffinose and stachyose of soymilk were 55(0)C and 5.2-6.2, respectively. The enzymatic treatment for 3 h completely removed the raffinose oligosaccharides in soymilk. Crude extract also showed considerable amount of invertase activity.

Hydrolysis of Galacto-oligosaccharides in Soymilk by κ-carrageenan-entrapped α-galactosidase from Aspergillus Oryzae

α-Galactosidase was immobilized in κ-carrageenan. The optimum pH of the soluble enzyme and immobilized enzyme was 4.8. The optimum temperature of the soluble enzyme was 50 °C and that of the immobilized enzyme was increased to 53 °C. The immobilized enzyme was used in batch, repeated batch, and in the continuous mode to degrade the raffinose family sugars present in soymilk. Two hours incubation with free and immobilized α-galactosidase resulted in 88 and 75% reduction in raffinose family oligosaccharides in soymilk respectively. In the repeated batch, 61% reduction was obtained in the fourth cycle. A fluidized bed reactor was designed to treat soymilk continuously. The performance of immobilized α-galactosidase was also tested in a fluidized bed reactor at different flow rates and 92% reduction of raffinose family oligosaccharides in soymilk was obtained at 25 ml h−1 flow rate. The study revealed that immobilized α-galactosidase in continuous mode is efficient in reducing the oligosaccharides present in the soymilk.

Enzymatic properties of α-galactosidase from Trichoderma reesei in the hydrolysis of galactooligosaccharides

Enzymatic properties of the α-galactosidase (α-galactoside galactohydrolase, EC 3.2.1.22) from Trichoderma reesei in the hydrolysis of natural galactooligosaccharides and α -O-methyl D-galactopyranoside have been investigated in a wide range of substrate concentrations. The hydrolyses of α -O-methyl D-galactopyranoside and melibiose were inhibited by substrate at concentrations higher than 100 mM while in the hydrolysis of raffinose and stachyose such an effect was not observed. It was shown by 1H and 13C NMR spectroscopy and HPLC techniques that inhibition by the excess of α -O-methyl D-galactopyranoside or melibiose strongly correlated with formation of transglycosylation products. The product of autocondensation reaction with α -O-methyl D-galactopyranoside as substrate was found to be α -O-methyl galactopyranosyl-1,6-D-galactopyranoside. The stereochemical course of stachyose hydrolysis has been determined. The enzyme catalyses the hydrolysis with retention of anomeric configuration and is assumed to operate via a double displacement mechanism. Simultaneous hydrolysis of stachyose and raffinose effected by the α-D-galactosidase was studied by direct 1H NMR measurements. Cleavage of the terminal galactose residue of stachyose was found to be the rate-limiting step. Formation constants of enzyme-substrate complex for stachyose and raffinose were calculated. The suggested model can be used for simulating the two-substrate system and predicting the extent of stachyose hydrolysis.

Hydrolysis of galactooligosaccharides by commercial preparations of α‐galactosidase and β‐fruetofuranosidase: Potential for use as dietary additives

AbstractIn vitro and in vivo studies were conducted with six commercial enzyme preparations (SP249, Energex, Rohament CW, Novozyme 230 and crude α ‐galactosidase) to determine their effectiveness in hydrolysing galactooligosaccharides from soya bean and canola meal in the gastrointestinal tract of poultry. The use of the enzyme invertase to enhance galactoside hydrolysis was also studied. A wide range of α ‐galactosidase activity was observed in vitro, with crude α‐galactosidase from Mortirella vinacea and Novozyme 230 preparation showing the highest activity values of 4.3 and 1.5 nkat mg−1, respectively. All preparations with the exception of crude α‐galactosidase showed invertase activity which is known to convert raffinose and stachyose to the corresponding di‐and trisaccharide, melibiose and manninotriose. Although the activity of invertase was highest on sucrose, the Novozyme 230 preparation showed activity values of 4.2 and 2.3 nkat mg−1 toward raffinose and stachyose substrates, respectively. De novo synthesis of raffinose was observed when soya bean meal, canola meal or pure sucrose and galactose were incubated with certain enzyme preparations (ie Energex). In general, preparations possessing hydrolytic activity towards galactooligosaccharides showed very little synthesis of raffinose while preparations capable of generating raffinose were very weak in the hydrolysis of galactooligosaccharides. The best result in terms of galactooligosaccharide in vitro hydrolysis of canola and soya bean meal was obtained with a combination of α‐galactosidase and invertase. In the in vivo study with caecectomised hens, hydrolysis of galactooligosaccharides averaged 88% when crude α‐galactosidase (2 g kg−1) and invertase (1 g kg−1) were added to laying, hen diet containing 200 g soya bean meal per kilogram. A problem identified in the current study was that minerals such as calcium phosphate and calcium carbonate common in poultry diets inhibit the hydrolysis activity of α‐galactosidase, indicating that high levels of activity would be required to yield a response in practical poultry feeding.