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.     

Effects of malting on β‐glucanase and phytase activity in barley grain

The effects of malting on β‐glucan and phytate were investigated in one naked and one covered barley by a full factorial experiment with three factors (steeping temperature, moisture content and germination temperature) each with two levels. Analysis of total content of β‐glucan in the malted samples showed small changes after steeping at the high temperature (48 °C), while steeping at the lower temperature (15 °C) gave a significantly lower content. This trend was even stronger for β‐glucan unextractable at 38 °C. Analysis of the activity of β‐glucanase for the samples steeped at 15 °C showed a strong increase over the time of germination, while those steeped at 48 °C had a much slower development. The other two factors influenced the outcome to a small extent, mainly because the steeping temperature was the most important factor overall where any changes in β‐glucan and β‐glucanase were observed. When β‐glucan was extracted at 100 °C, a larger yield was obtained, and this was influenced by the steeping temperature in a much stronger way than for β‐glucan extracted at 38 °C. Determination of average molecular weight for β‐glucan extracted at 100 °C gave a lower value for samples steeped at 15 than at 48 °C. The design did not have any large effects on phytate degradation and phytase activity. However, it indicated that selective control of the enzymes might be possible, since phytase activity was barely affected by the parameters studied, while β‐glucanase was heavily affected. © 2002 Society of Chemical Industry     

The (1–3, 1–4)‐β‐Glucanases in Malting Barley: Enzyme Survival and Genetic and Environmental Effects

Eighteen barley genotypes used in Brazilian malting barley breeding programs were characterized in relation to (1–3, 1–4)‐β‐glucanase activity in green and kilned malt. They were tested to determine the loss of enzyme activity during kilning in the malting process and the environmental effects on enzyme activity were measured. The genotypes analyzed showed great variation regarding the enzyme activity in both kinds of malt, in a range from 531.94 to 934.31 U/kg in green malt, and from 187.02 to 518.40 U/kg in dry malt. The mean enzyme activity loss during kilning was close to 60%, very similar to the results obtained in other studies. The loss among genotypes varied from 8.04% to 71.54%. The enzyme activity varied significantly under the different environments tested, showing existence of environmental effects on the genotypes analyzed. Embrapa 127 was the genotype that exhibited the highest enzyme activity in finished malt although it had shown a low activity in green malt, reflecting a negligible loss of activity during kilning. The data indicate promising results to malting barley breeding due to the wide variability exhibited by genotypes as to enzyme activity and levels of isoenzyme with high thermostability.     

Screening of β‐Glucosidase and β‐Xylosidase Activities in Four Non‐Saccharomyces Yeast Isolates

The finding of new isolates of non‐Saccharomyces yeasts, showing beneficial enzymes (such as β‐glucosidase and β‐xylosidase), can contribute to the production of quality wines. In a selection and characterization program, we have studied 114 isolates of non‐Saccharomyces yeasts. Four isolates were selected because of their both high β‐glucosidase and β‐xylosidase activities. The ribosomal D1/D2 regions were sequenced to identify them as Pichia membranifaciens Pm7, Hanseniaspora vineae Hv3, H. uvarum Hu8, and Wickerhamomyces anomalus Wa1. The induction process was optimized to be carried on YNB‐medium supplemented with 4% xylan, inoculated with 106 cfu/mL and incubated 48 h at 28 °C without agitation. Most of the strains had a pH optimum of 5.0 to 6.0 for both the β‐glucosidase and β‐xylosidase activities. The effect of sugars was different for each isolate and activity. Each isolate showed a characteristic set of inhibition, enhancement or null effect for β‐glucosidase and β‐xylosidase. The volatile compounds liberated from wine incubated with each of the 4 yeasts were also studied, showing an overall terpene increase (1.1 to 1.3‐folds) when wines were treated with non‐Saccharomyces isolates. In detail, terpineol, 4‐vinyl‐phenol and 2‐methoxy‐4‐vinylphenol increased after the addition of Hanseniaspora isolates. Wines treated with Hanseniaspora, Wickerhamomyces, or Pichia produced more 2‐phenyl ethanol than those inoculated with other yeasts.     

Characterization of the Supermolecular Structure of Polydatin/6‐O‐α‐Maltosyl‐β‐cyclodextrin Inclusion Complex

Polydatin is the main bioactive ingredient in many medicinal plants, such as Hu‐zhang (Polygonum cuspidatum), with many bioactivities. However, its poor aqueous solubility restricts its application in functional food. In this work, 6‐O‐α‐Maltosyl‐β‐cyclodextrin (Malt‐β‐CD), a new kind of β‐CD derivative was used to enhance the aqueous solubility and stability of polydatin by forming the inclusion complex. The phase solubility study showed that polydatin and Malt‐β‐CD could form the complex with the stoichiometric ratio of 1:1. The supermolecular structure of the polydatin/Malt‐β‐CD complex was characterized by ultraviolet–visible spectroscopy (UV), Fourier transform infrared spectroscopy (FT‐IR), X‐ray diffractometry (XRD), thermogravimetric/differential scanning calorimetry (TG/DSC), and proton nuclear magnetic resonance (¹H‐NMR) spectroscopy. The changes of the characteristic spectral and thermal properties of polydatin suggested that polydatin could entrap inside the cavity of Malt‐β‐CD. Furthermore, to reasonably understand the complexation mode, the supermolecular structure of polydatin/Malt‐β‐CD inclusion complex was postulated by a molecular docking method based on Autodock 4.2.3. It was clearly observed that the ring B of polydatin oriented toward the narrow rim of Malt‐β‐CD with ring A and glucosyl group practically exposed to the wide rim by hydrogen bonding, which was in a good agreement with the spectral data.     

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.     

Chemical Constituents of Gold‐red Apple and Their α‐Glucosidase Inhibitory Activities

Ten compounds were isolated and purified from the peels of gold‐red apple (Malus domestica) for the 1st time. The identified compounds are 3β, 20β‐dihydroxyursan‐28‐oic acid (1), 2α‐hydroxyoleanolic acid (2), euscaphic acid (3), 3‐O‐p‐coumaroyl tormentic acid (4), ursolic acid (5), 2α‐hydroxyursolic acid (6), oleanolic acid (7), betulinic acid (8), linolic acid (9), and α‐linolenic acid (10). Their structures were determined by interpreting their nuclear magnetic resonance and mass spectrometry (MS) spectra, and by comparison with literature data. Compound 1 is new, and compound 2 is herein reported for the 1st time for the genus Malus. α‐Glucosidase inhibition assay revealed 6 of the triterpenoid isolates as remarkable α‐glucosidase inhibitors, with betulinic acid showing the strongest inhibition (IC₅₀ = 15.19 μM). Ultra‐performance liquid chromatography‐electrospray ionization MS analysis of the fruit peels, pomace, flesh, and juice revealed that the peels and pomace contained high levels of triterpenes, suggesting that wastes from the fruit juice industry could serve as rich sources of bioactive triterpenes.     

Effect of High Hydrostatic Pressure‐Temperature Combinations on the Activity of β‐Glucanase from Barley Malt

β‐Glucanase from barley malt is known to be thermolabile but important in the mashing process. Therefore, the potential of increasing the thermostability of β‐glucanase in ACES buffer (0.1 M, pH 5.6) by high hydrostatic pressure has been investtigated. Inactivation of the enzyme as well as changes of the conversion rate in response to combined pressure‐temperature treatments in the range of 0.1–900 MPa and 30–75°C were assessed by analyzing the kinetic rate constants. A significant stabilization of β‐glucanase against temperature‐induced inactivation was detected at 400 MPa. With increasing pressure up to 600 MPa the catalytic activity of β‐glucanase was progressively decelerated. However, for the overall depolymerization reaction of β‐glucans in ACES buffer (0.1 M, pH 5.6) a maximum was identified at 215 MPa and 55°C yielding approximately 2/3 higher degradation of β‐glucan after 20 min as compared to the maximum at ambient pressure (45°C).     

Thermal modifications of structure and codenaturation of α‐lactalbumin and β‐lactoglobulin induce changes of solubility and susceptibility to proteases

Study of heat denaturation of major whey proteins (β‐lactoglobulin or α‐lactalbumin) either in separated purified forms, or in forms present in fresh industrial whey or in recomposed mixture respecting whey proportions, indicated significant differences in their denaturation depending on pH, temperature of heating, presence or absence of other co‐denaturation partner, and of existence of a previous thermal pretreatment (industrial whey). α‐Lactalbumin, usually resistant to tryptic hydrolysis, aggregated after heating at ⪈85°C. After its denaturation, α‐lactalbumin was susceptible to tryptic hydrolysis probably because of exposure of its previously hidden tryptic cleavage sites (Lys‐X and Arg‐X bonds). Heating over 85°C of β‐lactoglobulin increased its aggregation and exposure of its peptic cleavage sites. The co‐denaturation of α‐lactalbumin with β‐lactoglobulin increased their aggregation and resulted in complete exposure of β‐lactoglobulin peptic cleavage sites and partial unveiling of α‐lactalbumin tryptic cleavage sites. The exposure of α‐lactalbumin tryptic cleavage sites was slightly enhanced when the α‐lactalbumin/β‐lactoglobulin mixture was heated at pH 7.5. Co‐denaturation of fresh whey by heating at 95°C and pH 4.5 and above produced aggregates stabilized mostly by covalent disulfide bonds easily reduced by β‐mercaptoethanol. The aggregates stabilized by covalent bonds other than disulfide arose from a same thermal treatment but performed at pH 3.5. Thermal treatment of whey at pH 7.5 considerably enhanced tryptic and peptic hydrolysis of both major proteins.