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.     

Reduction efficiency of the neurotoxin β‐ODAP in low‐toxin varieties of Lathyrus sativus seeds by solid state fermentation with Aspergillus oryzae and Rhizopus microsporus var chinensis

Solid state fermentation of several low‐toxin varieties of grass pea (Lathyrus sativus L) seeds with Aspergillus oryzae and Rhizopus microsporus var chinensis removed the neurotoxin β‐ODAP (3‐N‐oxalyl‐L ‐2,3‐diaminopropanoic acid) to a considerable degree from the seed meal. The detoxification efficiency was statistically significant and ranged from 52.4% (p < 0.01) to 82.2% (p < 0.001), which was lower than for a high‐toxin variety processed by the same fermentation procedure (94.8%, p < 0.001). While the content of β‐ODAP decreased, those of other free protein amino acids, especially glutamic acid, histidine, threonine, tyrosine, valine and lysine, increased dramatically in the fermented seeds. Efforts to remove the neurotoxin from Lathyrus sativus either by breeding or by food processing to obtain toxin‐free grass pea seeds have been made worldwide for several decades. The efficiencies of various reported processing methods are summarised and compared. © 2000 Society of Chemical 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.     

Structural characterisation of β‐cyclodextrin crosslinked by adipic acid

This study was conducted to investigate the structural characterisation of β‐cyclodextrin (β‐CD) crosslinked by adipic acid. β‐CD was treated with different concentrations (0%, 5%, 10% and 15%, w/v) of adipic acid. Different instruments, such as scanning electron microsope (SEM), Fourier‐transform infrared (FT‐IR) spectroscopy and ¹H and ¹³C nuclear magnetic resonance (NMR) spectra were used to find out chemical structure in the crosslinked β‐CD. SEM analysis suggested that crosslinking β‐CD with 15% adipic acid changed the original morphology and considerably increased the particle size of the raw material. FT‐IR spectroscopy data showed that an intensive absorption band at 1706 cm^?1 was present in the β‐CD samples treated with 10% and 15% adipic acid, indicating a crosslinking between hydroxyl groups of β‐CD and carboxyl groups of adipic acid. NMR spectra revealed that the ester linkages between hydroxyl groups of β‐CD and carboxyl groups of adipic acid were formed after crosslinking of β‐CD with adipic acid.     

Enzymatic Properties of β‐1,3‐Glucanase from Streptomyces sp Mo.

Streptomyces sp Mo endo‐β‐1,3‐glucanase was found to have hydrolyzing activity toward curdlan and released laminarioligosaccharides selectively. The molecular weight was estimated to be 36000 Da and its N‐terminal amino acid sequence was VTPPDISVTN. The optimal pH was 6 and the enzyme was found to be stable from pH 5 to 8. The optimal temperature was 60 °C and the activity was stable below 50 °C. The enzyme hydrolyzed selectively curdlan containing only β‐1,3 linkages. The enzyme had 89% relative activity toward Laminaria digitata laminarin, which contains a small amount of β‐1,6 linkages compared with curdlan, while Eisenia bicyclis laminarin with a higher amount of β‐1,6‐linkages, was not hydrolyzed. Mo enzyme adsorbed completely on curdlan powder. The enzymatic hydrolysis of curdlan powder resulted in the accumulation of laminaribiose (yield 81.7%). Trisaccharide was inevitably released from the hydrolysis of laminarioligosaccharides with 5 to 7 degrees of polymerization (DP). Although the enzyme cleaved off disaccharide (DP 2) from tetrasaccharide (DP 4), the reaction rate was lower than those of DP 5 to 7. The results indicated that the active site of Mo endo‐β‐1,3‐glucanase can efficiently recognize glucosyl residue chain of greater than DP 5 and hydrolyzes the β‐1,3 linkage between the 3rd and 4th glucosyl residue.     

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.     

Electrospray MS‐based characterization of β‐carbolines – mutagenic constituents of thermally processed meat

The β‐carbolines 1‐methyl‐9H‐pyrido [3,4‐b]indole and 9H‐pyrido[3,4b]indole have been implicated as having causative roles in a number of human diseases, such as Parkinson's disease and cancer. As they can be formed during the heating of protein‐rich food, a number of analytical methodologies have been proposed for their detection and quantification in foodstuff. For this purpose, LC‐MS and LC‐MS/MS have emerged as the most specific analytical methods, and the quantification is based on the occurrence of unusual ions, such as [M+H‐(H^?)]⁺ and [M+H‐2H]⁺. In this study, we have investigated the formation of these ions by accurate‐mass electrospray MS/MS and demonstrated that these ions are formed from gas‐phase ion‐molecule reactions between water vapor present in the collision cell and the protonated molecule of 1‐methyl‐9H‐pyrido [3,4‐b]indole and 9H‐pyrido[3,4b]indole. Although this reaction has been previously described for heterocyclic amine ions, it has been overlooked in the most of recent LC‐MS and LC‐MS/MS studies, and no complete data of the fragmentation are reported. Our results demonstrate that additional attention should be given with respect to eliminating water vapor residues in the mass spectrometer when analysis of β‐carbolines is performed, as this residue may affect the reliability in the results of quantification.     

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.     

The role of pulsed electric fields treatment in enhancing the stability of amino acid – sugar complexes:‐ interactions between L‐Phenylalanine and β‐Cyclodextrin

In this work, the effect of pulsed electric fields (PEF) treatment on the interactions between amino acids (using L‐Phenylalanine: L‐Phe) and sugar (using β‐Cyclodextrin: β‐CD) complex was analysed by fluorescence spectroscopy, Raman spectroscopy and simultaneous thermal analyzer. Moreover, the molecular dynamics of β‐CD–L‐Phe inclusion complex treated by PEF was calculated by molecular modelling. The results indicated that β‐CD–L‐Phe complexes are formed by a molar ratio of 1:1, and the stability constant of such complexes increased from 147 to 614 M^?1 by PEF treatment. Thermal characterisations of β‐CD–L‐Phe complexes indicated that the PEF treatment could increase the yield of complexes. The PEF treatment resulted in an increase in the reaction enthalpy of β‐CD–L‐Phe inclusion complexes by DSC curve. These results show that PEF treatment has the potential to promote the chemical processing, especially the small organic molecules participate in inclusion or cross‐linking reaction.     

Formation of γ‐gluconolactone in a wine‐like model system

By experiments performed in wine‐like and must‐like model solutions, we have shown that intramolecular gluconic acid esterification leads to the formation of not only the well‐known δ‐gluconolactone (glucono‐5‐lactone) but also γ‐gluconolactone (glucono‐4‐lactone). To our knowledge, the presence of the latter is reported for the first time under conditions similar to those in grape musts and wines. Equilibrium between these lactones and gluconic acid was reached within 24 h of preparing a solution of gluconic acid, and in both wine‐like and must‐like model solutions the concentrations of γ‐ and δ‐gluconolactone represented the same proportions of the acid, ie about 60 and 40 mg g^?1 gluconic acid respectively. Owing to their chemical structures, not only δ‐gluconolactone but also γ‐gluconolactone could certainly contribute to sulphur dioxide binding in musts and wines containing gluconic acid. © 2002 Society of Chemical Industry     

Extraction and Isolation of β‐Glucan from Grain Sources—A Review

A collective report on the extraction and isolation of β‐glucan from grain sources, namely, oat, barley, and wheat is presented. An analysis on the effect of medium, pH, and temperature on the purity and yield of the β‐glucan derived under acidic/alkaline/aqueous/enzymatic conditions is also made. Water extraction and alkali extraction processes are preferred as the yield and recovery of extracted β‐glucan were good. Cost‐effective development of the process for deriving high molecular weight β‐glucan is the current requirement for its wide applications in food and pharmaceutical industries.     

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.