Effect of soaking,cooking and crude α-galactosidase treatment on the oligosaccharide content of cowpea flours

Abstract : The effects of soaking, cooking and crude α-galactosidase treatment on the level of stachyose and raffinose present in cowpea flours were investigated. Soaking for 16 h resulted in an average reduction of 26·2% for stachyose and 28·0% for raffinose, while cooking for 50 min resulted in a reduction of 28·6% for stachyose and 44·0% for raffinose. On the other hand, treatment of cowpea flours for 2 h at 50°C with crude fungal preparations having an α-galactosidase activity equivalent to 64 units μg^?1 protein, brought about a mean decrease of 82·3% for stachyose and 93·3% for raffinose. These results show that the enzyme treatment was more effective in removing the raffinose-family oligosaccharides and hence could be a useful technique for control of the flatulence-inducing activity of cowpea flours.     

Production of soya milk containing low flatulence‐causing oligosaccharides in a packed bed reactor using immobilised α‐galactosidase

Peanut α‐galactosidase was immobilised in calcium alginate beads and used to hydrolyse the flatulence‐causing oligosaccharides, raffinose and stachyose, in soya milk in batch and in packed bed reactor with recycle. The immobilised enzyme exhibited a slightly lower activity than the free enzyme. The activity yield of immobilised α‐galactosidase was 75.1% and the immobilisation yield was 82.6%. Batch hydrolysis using immobilised enzyme at 55 °C resulted in 96% reduction in the oligosaccharides after 12 h. For the continuous process, a packed bed reactor with recycle was used. More than 98% of the oligosaccharides were hydrolysed after 6 h of reaction at 55 °C. The immobilised enzyme also proved to be stable up to three repeated hydrolysis reactions.     

α-Amylase inhibitors in chick pea (cicer arietinum L)

The α-amylase inhibitory activity of 28 varieties of chick pea (Cicer arietinum L) was determined, and KGT/GBS-8 was found to have the greatest activity (81.4 units g^?1). The inhibitor was heat labile and the inhibitory activity decreased during germination.     

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

In 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.     

ENZYMIC QUANTIFICATION OF (1→3) (1→4)-β-D-GLUCAN IN BARLEY AND MALT

A simple and quantitative method for the determination of (1→3) (1→4)-β-D-glucan in barley flour and malt is described. The method allows direct analysis of β-glucan in flour and malt slurries. Mixed-linkage β-glucan is specifically depolymerized with a highly purified (1→3) (1→4)-β-D-glucanase (lichenase), from Bacillus subtilis, to tri-, tetra- and higher degree of polymerization (d.p.) oligosaccharides. These oligosaccharides are then specifically and quantitatively hydrolysed to glucose using purified β-D-glucosidase. The glucose is then specifically determined using glucose oxidase/peroxidase reagent. Since barley flours contain only low levels of glucose, and maltosaccharides do not interfere with the assay, removal of low d.p. sugars is not necessary. Blank values are determined for each sample allowing the direct measurement of β-glucan in maltsamples.α-Amylasedoes not interfere with the assay. The method issuitable for the routineanalysis of β-glucan in barley samples derived from breeding programs; 50 samples can be analysed by a single operator in a day. Evaluation of the technique on different days has indicated a mean standard error of 0–1 for barley flour samples containing 3–8 and 4–6% (w/w) β-glucan content.     

Evidence for the improvement of thermostability of the maltogenic α‐amylase of Aspergillus niger by negative pressure

Thermostability of the enzymes is influenced by the different parameters and pressure also influences the biological activity of the enzymes. Recently reported maltogenic α‐amylase from Aspergillus niger acts optimally on starch at 40°C and it was unstable above 40°C at atmospheric pressure. Calcium could stabilize the maltogenic α‐amylase activity up to 50°C at atmospheric pressure. But, at negative pressure (−200 mbar) enzyme was stable at temperatures higher than 50°C either in the presence or absence of the substrate, starch making it adoptable for starch processing. Enzyme showed higher affinity to the starch at negative pressure compared to the atmospheric pressure and change in the surface roughness of the enzyme is almost similar to the native state at 70°C and negative pressure. These results suggest that thermolabile enzymes can be used at negative pressures for industrial applications.     

Purification and characterisation of a novel α‐L‐rhamnosidase exhibiting transglycosylating activity from Aspergillus oryzae

A novel α‐L‐rhamnosidase was isolated and purified from Aspergillus oryzae NL‐1. The enzyme was purified 13.2‐fold by ultrafiltration, ion exchange and gel filtration chromatography with an overall recovery of 6.4% and specific activity of 224.4 U/mg, and the molecular mass of its subunit was approximately 75 kDa. Its optimal temperature and pH were 65 °C and 4.5, respectively. The enzyme was stable in the pH range 3.5–7.0, and it showed good thermostability at higher temperatures. The K_M, kcat and kcat/K_M values were 5.2 mm , 1624 s^?1 and 312 s^?1 mm ^?1 using pNPR as substrates, respectively. Moreover, the enzyme exhibited transglycosylating activity, which could synthesise rhamnosyl mannitol through the reactions of transglycosylation with inexpensive rhamnose as the glycosyl donor. Our findings indicate that the enzyme has potential value for glycoside synthesis in the food industry.     

Purification and physicochemical characterization of ovine β‐lactoglobulin and α‐lactalbumin

Ovine whey proteins were fractionated and studied by using different analytical techniques. Anion‐exchange chromatography and reversed‐phase high‐performance liquid chromatography (HPLC) showed the presence of two fractions of β‐lactoglobulin but only one of α‐lactalbumin. Gel permeation and sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis allowed the calculation of the apparent molecular mass of each component, while HPLC coupled to electrospray ionisation‐mass spectrometry (ESI‐MS) technique, giving the exact molecular masses, demonstrated the presence of two variants A and B of ovine β‐lactoglobulin. Amino acid compositions of the two variants of β‐lactoglobulin differed only in their His and Tyr contents. Circular dichroism spectroscopy profiles showed pH conformation changes of each component. The thermograms of the different whey protein components showed a higher heat resistance of β‐lactoglobulin A compared to β‐lactoglobulin B at pH 2, and indicated high instability of ovine α‐lactalbumin at this pH.     

ANALYSIS OF HOPS AND HOP PRODUCTS FOR α-ACIDS OR ISO-α-ACIDS

Two methods based on the resolution of mixtures of hop compounds by chromatography on Sephadex columns have been adopted by E.B.C. and A.S.B.C. as ‘International Methods’.     

In vitro inhibitory effects of cranberry bean (Phaseolus vulgaris L.) extracts on aldose reductase, α‐glucosidase and α‐amylase

The in vitro inhibitory activities of different seed extracts prepared from cranberry bean mutant SA‐05 and its wild‐type variety Hwachia against aldose reductase, α‐glucosidase and α‐amylase were examined. The results indicated that the polyphenolics‐rich extracts obtained using 800 g kg^?1 methanol and 500 g kg^?1 ethanol demonstrated inhibitory activities against aldose reductase (IC₅₀ of 0.36–0.46 mg mL^?1) and α‐glucosidase (IC₅₀ of 1.32–1.94 mg mL^?1). The 500 g kg^?1 ethanol extracts also showed α‐amylase inhibitory activities (IC₅₀ of 70.11–80.22 μg mL^?1). Subsequent extracts, prepared further with NaCl and H₂O from precipitates of 800 g kg^?1 methanol or 500 g kg^?1 ethanol extracts, exhibited potent α‐amylase inhibitory activities (IC₅₀ of 17.68–38.68 μg mL^?1). A combination of 500 g kg^?1 ethanol extraction plus a subsequent H₂O extraction produced highest polyphenolics and α‐amylase inhibitors. The SA‐05 α‐amylase inhibitor extracts showed greater inhibitory activities than that of Hwachia. Thus, cranberry bean mutant SA‐05 is an advantageous choice for producing anti‐hyperglycaemic compounds.     

Production of α‐amylase by Aspergillus oryzae As 3951 in solid state fermentation using spent brewing grains as substrate

BACKGROUND: The optimisation of nutrient levels for the production of α‐amylase by Aspergillus oryzae As 3951 in solid state fermentation (SSF) with spent brewing grains (SBG), an inexpensive substrate and solid support, was carried out using response surface methodology (RSM) based on Plackett–Burman design (PBD) and Box–Behnken design (BBD). RESULTS: In the first optimisation step a PBD was used to evaluate the influences of related factors. Corn steep liquor, CaCl₂ and MgSO₄ were found to be the most compatible supplements to the substrate of SBG and influenced α‐amylase activity positively. In the second step the concentrations of these three nutrients were optimised using a BBD. The final concentrations (g/g dry substrate basis) in the medium optimised with RSM were 1.8% corn steep liquor, 0.22% CaCl₂ and 0.2% MgSO₄ · 7H₂O using SBG as the solid substrate. The average α‐amylase activity reached 6186 U g^?1 dry substrate under the optimised conditions at 30 °C after 96 h. Under the optimised conditions of SSF an approximately 17.5% increase in enzyme yield was observed. CONCLUSION: SBG was found to be a good substrate for the production of α‐amylase by A. oryzae As 3951 under SSF. Copyright © 2007 Society of Chemical Industry     

Inhibitory activities of kaempferol,galangin, carnosic acid and polydatin against glycation and α‐amylase and α‐glucosidase enzymes

The activities of four natural phenolics, kaempferol, galangin, carnosic acid and polydatin in scavenging free radicals, inhibiting advanced glycation end‐product (AGE) formation, α‐amylase and α‐glucosidase and trapping methylglyoxal (MGO), were evaluated in this study. Carnosic acid and galangin had the highest activity in scavenging free radicals. Kaempferol and galangin had the greatest activity in preventing bovine serum albumin (BSA) against glycation and reducing glycated proteins. Polydatin had the greatest performance in trapping MGO to reduce glycation reaction. However, there was no significant difference for kaempferol, galangin and carnosic acid in inhibiting AGE formation by BSA‐MGO reaction. Kaempferol, galangin and carnosic acid were the competitive inhibitors for α‐amylase, while kaempferol and carnosic acid were noncompetitive inhibitors for α‐glucosidase. However, polydatin showed as a mixed noncompetitive inhibitor for both α‐amylase and α‐glucosidase. The results indicated that the four natural phenolics have potential in inhibiting AGE production and the digestive enzymatic activity with different mechanisms.     

Inhibitory potential of phenylpropanoid glycosides from Ligustrum purpurascens Kudingcha against α‐glucosidase and α‐amylase in vitro

The leaves of Ligustrum purpurascens are used in a Chinese traditional tea called small‐leaved kudingcha, which is rich in phenylpropanoid glycosides (PPGs) and has many beneficial properties. Two critical exoacting glycoside hydrolase enzymes (glucosidases) involved in carbohydrate digestion are α‐glucosidase and α‐amylase. We investigated the properties of PPGs from L. purpurascens for inhibiting α‐amylase and α‐glucosidase activity in vitro and found IC₅₀ values of 1.02 and 0.73 mg mL^?1, respectively. The patterns of inhibiting both α‐amylase and α‐glucosidase were mixed‐inhibition type. Multispectroscopy and molecular docking studies indicated that the interaction between PPGs and α‐amylase and α‐glucosidase altered the conformation of enzymes, with binding at the site close to the active site of enzymes resulting in changed enzyme activity. Our studies may help in the further health use of small‐leaved kudingcha.     

α‐Glucosidase and α‐amylase inhibitory activities of phloroglucinal derivatives from edible marine brown alga,Ecklonia cava

BACKGROUND: Diabetes mellitus (DM) is a chronic metabolic disorder characterized by defects in insulin secretion and action, which can lead to damaged blood vessels and nerves. With respect to effective therapeutic approaches to treatment of DM, much effort has being made to investigate potential inhibitors against α‐glucosidase and α‐amylase from natural products. The edible marine brown alga Ecklonia cava has been reported to possess various interesting bioactivities, which are studied here. RESULTS: In this study, five phloroglucinal derivatives were isolated from Ecklonia cava: fucodiphloroethol G ( 1 ), dieckol ( 2 ), 6,6′‐bieckol ( 3 ), 7‐phloroeckol ( 4 ) and phlorofucofuroeckol A ( 5 ); compounds 1, 3 and 4 were obtained from this genus for the first time and with higher yield. The structural elucidation of these derivatives was completely assigned by comprehensive analysis of nuclear magnetic spectral data. The anti‐diabetic activities of these derivatives were also assessed using an enzymatic inhibitory assay against rat intestinal α‐glucosidase and porcine pancreatic α‐amylase. Most of these phlorotannins showed significant inhibitory activities in a dose‐dependent manner, responding to both enzymes, especially compound 2 , with the lowest IC₅₀ values at 10.8 µmol L^?1 (α‐glucosidase) and 124.9 µmol L^?1 (α‐amylase), respectively. Further study of compound 2 revealed a non‐competitive inhibitory activity against α‐glucosidase using Lineweaver‐Burk plots. CONCLUSION: These results suggested that Ecklonia cava can be used for nutritious, nutraceutical and functional foods in diabetes as well as for related symptoms. Copyright © 2009 Society of Chemical Industry     

Effect of α‐tocopherol and α‐tocotrienol on the performance of Chilean hazelnut oil (Gevuina avellana Mol) at high temperature

The high temperature antioxidant efficiency of α‐tocopherol, α‐tocotrienol and a mixture of both on hazelnut oil were evaluated. Crude hazelnut oil (HZO), crude hazelnut oil treated with alumina (THZO), as well as three samples of THZO to which 150 mg kg^?1 of α‐tocopherol, 140 mg kg^?1 of α‐tocotrienol or a mixture containing 70 mg kg^?1 of α‐tocopherol and 70 mg kg^?1 of α‐tocotrienol, were added and submitted to thermal treatment at 180°C for 18 h. The addition of tocols to THZO decreased the formation of polar compounds and increased its oxidative stability in all the systems studied. However, α‐tocopherol showed a higher antioxidant capacity than α‐tocotrienol at high temperature. In addition, α‐tocotrienol showed a more rapid degradation rate than α‐tocopherol under the conditions studied. Copyright © 2004 Society of Chemical Industry     

THE DETERMINATION OF α- AND β-ACIDS IN HOPS AND HOP PRODUCTS USING HPLC

A rapid reversed phase HPLC method for the analysis for α- and β-acids in hops and hop products is described and has been evaluated. The method uses citric acid in the eluent as a complexing agent to overcome the irreversible adsorption effects shown by some columns, thus allowing optimum eluent pH to be selected. The precision of the method for analysis of hop extract has been determined giving relative standard deviations of 1·0% and 2·1% for α- and β-acids respectively. General agreement with results obtained using a polarimetric α-acids analysis method for hop extracts and hops has been demonstrated.