Comparative study of enzymatic hydrolysis of α/β- and γ-gliadins

Enzymatic hydrolysis of proteins and fractionation of hydrolysates is a route of diversifying their functional properties. Chymotryptic hydrolysis of different sulphur-rich gliadins (α/β- and γ-types), major wheat storage proteins, was studied. The peptides formed in the course of digestion were characterised by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS-PAGE) and reversed-phase high performance liquid chromatography (RP-HPLC). With reference to previous work, a general scheme of degradation was assessed for γ-gliadins. Limited hydrolysis released two types of polypeptides, comprising respectively the repetitive and the non-repetitive moieties of the protein. In spite of strong sequence homologies between the two groups of sulphur-rich gliadins, it was not possible to prepare similar peptide fractions from α/β-gliadins. They were more resistant to hydrolysis and the region where the two domains merge appeared inaccessible to chymotrypsin. Restricted accessibility of cleavage sites was attributed to the less expanded conformation of α/β-type than γ-type gliadins. A first step of scaling-up was performed. This offers opportunities to prepare functional peptides from wheat storage proteins.     

β-GLUCAN AND β-GLUCANASE IN BREWING

The development of endo β-glucanase during the micro-malting of barley, with and without the addition of gibberellic acid, was compared. Results indicated that the stimulative effect of gibberellic acid on the enzyme system was of only marginal practical importance. From the assessment of the enzyme activity in a number of commercial malts it would appear that the germination time used for some malts is too short to take full advantage of the critical phase of very rapid enzyme development. The viscosity-reducing power of β-glucanase was demonstrated in miniature brewery mashing experiments and details of the progressive degradation of β-glucan by the enzyme system were analysed by gel filtration methods. The β-glucan content of a number of varieties of barley was established.     

Synthesis of β‐Galactooligosaccharides from Lactose Using Microbial β‐Galactosidases

Abstract: Galactooligosaccharides (GOSs) are nondigestible oligosaccharides and are comprised of 2 to 20 molecules of galactose and 1 molecule of glucose. They are recognized as important prebiotics for their stimulation of the proliferation of intestinal lactic acid bacteria and bifidobacteria. Therefore, they beneficially affect the host by selectively stimulating the growth and/or activity of a limited number of gastrointestinal microbes (probiotics) that confer health benefits. Prebiotics and probiotics have only recently been recognized as contributors to human health. A GOS can be produced by a series of enzymatic reactions catalyzed by β‐galactosidase, where the glycosyl group of one or more D‐galactosyl units is transferred onto the D‐galactose moiety of lactose, in a process known as transgalactosylation. Microbes can be used as a source for the β‐galactosidase enzyme or as agents to produce GOS molecules. Commercial β‐galactosidase enzymes also do have a great potential for their use in GOS synthesis. These transgalactosyl reactions, which could find useful application in the dairy as well as the larger food industry, have not been fully exploited. A better understanding of the enzyme reaction as well as improved analytical techniques for GOS measurements are important in achieving this worthwhile objective.     

DETERMINATION OF α- AND β-AMYLASE IN MALT

The Analysis Committee of the European Brewery Convention carried out a collaborative trial on malts using the specific analysis methods for α- and β-amylase activities based on dyed substrates supplied by MegaZyme (Aust.) Pty. Ltd. The repeatability and reproducibility values for the methods were judged to be unsatisfactory and consequently the methods were not recommended for Analytica-EBC.     

Investigation of selected toxicological parameters of γ-pentachlorocyclohexene (γ-PCCH)

The toxic effects of the γ-hexachlorocyclohexane metabolite γ-pentachlorocyclohexene (γ-PCCH) were studied by acute and subacute (6 weeks) experiments. The investigations included cerebral convulsibility with chemoshock (Tetrazolium), reactivity with hot plate method, the learning ability with learning tests, and peripheral nervous activity (EMG). Nociceptive reaction time was not influenced, the learning process (6 weeks) was inhibited by γ-PCCH. The conduction velocity of the peripheral nerve was decreased. At the end of the 6th week liver enlargement was found.     

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.     

β-GLUCAN AND β-GLUCAN SOLUBILASE IN MALTING AND MASHING

During malting the water-insoluble β-glucan of barley is diminished whilst water-soluble gum is little decreased. The amount of β-glucan surviving into malt depends on variety but barleys rich in glucan give malts with high β-glucan levels. The β-glucan content of barley depends on variety and growth site. β-Glucan solubilase survives mashing and catalyses the release of hemicellulose into solution. There is no correlation between the β-glucan content of malt and the amount released into wort. However, barley adjuncts containing high levels of β-glucan give worts rich in β-glucan. β-Glucan dissolution in mashing is dependent on time, temperature, grist particle size and liquor: grist ratio. Use of adjuncts derived from barley contribute relatively more β-glucan in wort, coinciding with reduced rates of wort separation, but these can be increased by using a β-glucanase produced by growing the fungus Trichoderma viride on spent grains.     

Cystathionine γ-lyase of Saccharomyces cerevisiae: Structural gene and cystathionine γ-synthase activity

Purification of Saccharomyces cerevisiae cystathionine γ-lyase (γ-CTLase) was hampered by the presence of a protein migrating very close to it in various types of column chromatography. The enzyme and the contaminant were nevertheless separated by polyacrylamide gel electrophoresis. N-terminal amino acid sequence analysis indicated that they are coded for by CYS3(CYI1) and MET17(MET25), respectively, leading to the conclusion that CYS3 is the structural gene for γ-CTLase and that the contaminant is O-acetylserine/O-acetylhomoserine sulfhydrylase (OAS/OAH SHLase). Based on these findings, we purified γ-CTLase by the following strategy: (1) extraction of OAS/OAH SHLase from a CYS3-disrupted strain; (2) preparation of antiserum against it; (3) identification of a strain devoid of the OAS/OAH SHLase protein using this antiserum; and (4) extraction of γ-CTLase from this strain. Purified γ-CTLase had cystathionine γ-synthase (γ-CTSase) activity if O-succinylhomoserine, but not O-acetylhomoserine, was used as substrate. From this notion we discuss the evolutional relationship between S. cerevisiae γ-CTLase and Escherichia coli γ-CTSase.     

SIMULTANEOUS DETERMINATION OF α, β AND ISO-α ACIDS IN HOPS AND HOP PRODUCTS

A method for the simultaneous analysis of α, β and iso-α acid in hops, hop extracts and isomerised hop extracts is described. It is based on the use of reversed phase high performance liquid chromatography and quantitative evaluation of the hop compounds is carried out with a computing integrator. The isomerisation reaction can be examined in detail, particularly in connection with the production of hop derived haze forming compounds in isomerised hop extracts used for post fermentation bittering.     

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.     

Sweet taste and solution properties of α,α-trehalose

α,α-Trehalose, a naturally occurring non-reducing disaccharide (α, D-glucopyranosyl 1–1 α-D-glucopyranoside) is investigated by solution parameters and sensorial effects. Interactions between α, α-trehalose and water give rise to hydration effects which are measurable by intrinsic viscosity [n], apparent specific volume V°₂, and NMR relaxation times. Water-solute interactions are prime determinants of taste qualities. An interpretation of sweetness intensity and persistence of α,α-trehalose is therefore offered, based on its solution properties and its effect on water structure. From the results, it appears that α,α-trehalose undergoes a better packing among water molecules than other sugars. α,α-Trehalose is found to be less sweet but more persistent than sucrose, fructose, glucose or maltose.     

KINETICS OF β-GLUCAN DEGRADATION IN WORT BY EXOGENOUS β-GLUCANASES TREATMENT

Three commercial β-glucanases, one bacterial (Cereflo 200L from Novo) and two fungal (Biobeta from Gist-Brocades and Filtrase from Biocon) have been studied with regard to the hydrolysis of β-glucan in sweet and hopped wort. At temperatures below 70°C these processes follow first order kinetics with rate constants being directly proportional to the enzyme concentrations. The rate constant for bacterial β-glucanase Cereflo 200L shows a negative dependence on temperature but positive with wort pH, whereas the reverse is the case for the two fungal β-glucanases. Within the ranges of pH and temperature tested the bacterial β-glucanase has 2–5 times the activity of the fungal ones. No evidence for synergic or competitive effects between bacterial and fungal β-glucanases have been found.     

ESTIMATION OF α- AND β-ACIDS IN HOP EXTRACTS BY HPLC

A procedure relying on high performance liquid chromatography for the estimation of α-acids and β-acids in hop extracts has been collaboratively tested by members of a Sub-Committee of the Institute of Brewing Analysis Committee and is recommended for use. No significant differences were found between precision values obtained using peak height and peak area measurements. For α-acids, the mean repeatability (r₉₅) and reproducibility (R₉₅) values were 1-3 and 2-4% m/m respectively over the range 41–62% m/m. For β-acids they were 0-9 and 2-0% m/m respectively over the range 11 to 35% m/m. The precision values were judged to be independent of concentration.