Influence of other whey proteins on the heat-induced aggregation of α-lactalbumin

The thermally induced aggregation of α-lactalbumin in solution and in the presence of serum protein-free casein micelles has been studied using gel permeation chromatography. No aggregation of α-lactalbumin was detected after heating at 90°C for 24 min. However, addition of β-lactoglobulin or serum albumin to the serum protein-free casein micelles + α-lactalbumin system caused aggregation of the α-lactalbumin, the rate and extent of this aggregation being dependent upon the concentration of free sulphydryl groups present in the other whey protein. It is therefore the sulphydryl group which is important and which appears to function by inducing cleavage of intramolecular disulphide bonds in the α-lactalbumin. This leads to the formation of intermolecular disulphide bridges and hence to aggregation of the α-lactalbumin.     

Susceptibility of an industrial α-lactalbumin concentrate to cross-linking by microbial transglutaminase

The susceptibility of an industrial α-lactalbumin concentrate to cross-linking with a microbial transglutaminase from Streptoverticillium mobaraense was investigated. At a protein concentration of 0.5% w v⁻¹, the maximum cross-linking was observed at 50°C, pH 5 and at 5 h of incubation time. Results from sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis showed that most of the monomeric form of α-lactalbumin was converted to polymers too large to move into the gel matrix. Addition of ethylenediamine tetraacetic acid or SDS prior to the incubation of protein–enzyme mixture, further enhanced the transglutaminase reaction with the industrial α-lactalbumin. Results from reverse phase chromatography indicated that cross-linking caused a broadening of the α-lactalbumin peak with little change in the average hydrophobicity of the protein. In contrast to the reported results on pure α-lactalbumin, the industrial α-lactalbumin concentrate showed considerable cross-linking with transglutaminase even without the reduction of the disulphide bonds. This difference was attributed to the partially unfolded secondary structures in the industrial α-lactalbumin concentrate.     

Evidence for a molten globule state in an oligomeric plant protein

Three different mustard globulin (isoforms), purified and isolated, using three extraction solutions (differing in ionic strength) were studied for structural integrity as a function of pH. Evidence of a molten globule state (a reversible intermediary state between the native and fully denatured forms) was obtained. This phenomenon may ultimately prove to be important in the translocation of these proteins across biological membranes at the time of their biosynthesis. Circular dichroism, hydrophobic probe, fluorescence spectral scans and differential scanning calorimetry were used to study this phenomenon. Secondary and tertiary structures (circular dichroism (CD) data) were found to be similar for globulins from higher ionic strength extractions, but different from the globulin from distilled water extraction; however, for all three isoforms, little change in secondary structure fractions as a function of pH was observed. Changes in tertiary structure (near-UV CD and intrinsic fluorescence data) as a function of pH were observed for all three globulin isoforms with greatest changes in tertiary structure being seen in the acidic pH range, i.e. 3–5. In contrast, all globulins were shown to undergo the least conformational change in the pH range of 6–9.     

α 1,4 — α 1,6 Glucopolysaccharides Contained in Developing Maize Kernels

Corn grains with different filling times were fractionated in order to obtain their α 1,4 — α 1,6 glucopolysaccharide composition and structure. Sugary, waxy, flint and several hybrids were the varieties analyzed. There is an increase in the total content of polysaccharides, as the grain is completed. But, no differences in composition and structure were detected between the first sample analyzed (15th day after pollination) and the last one, which corresponds to the complete grain filling period. The methodology developed by us, allowed us to observe that the introduction of su character on the hybrids (F x S and S x F) has influence on the final polysaccharide structure. Also, in the F x W hybridα 1,4 — α 1,6 glucans, their structural characteristics were different from those of F or W pure.     

Interactions between aroma compounds and β-lactoglobulin in the heat-induced molten globule state

The present study aims to elucidate the binding of small hydrophobic ligands onto the molten globule state of β-lactoglobulin (BLG). The conversion of the native BLG into a molten globule state was induced by heat treatment at acidic pH. The molten globule state was evidenced by far and near-UV circular dichroism spectra. β-Ionone and guaiacol exhibited a higher binding ability to BLG in the heat-induced molten globule state compared to unheated BLG, as assessed by protein surface hydrophobicity measurements, using 6-propionyl-2-(dimethylamino)naphthalene (PRODAN) fluorescent probe. The binding sites of the two aroma compounds were determined by 2D nuclear magnetic resonance (NMR) spectroscopy. The less tightly packed structure of the molten globule favoured ligand binding, in particular within the central cavity. The greater flexibility of the calyx entrance, and the conformational change of loop EF induced an easier access of the central cavity after the thermal treatment.     

Dynamic surface tension and adsorption properties of β-casein and β-lactoglobulin

The adsorption kinetic behaviour of β-lactoglobulin and β-casein solutions studied by dynamic surface tension measurements was interpreted by diffusion-controlled models. Approximate solutions of the model led to diffusion coefficients for short and long adsorption times. The coefficients associated with the short time region were found to be unexpectedly high. From the long time approximation, the coefficients reflect a slower process in the adsorption layer which is possibly superimposed by rearrangement processes.     

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.     

α-D-Polyglucane-Iodine Complexes

The temperature-dependent dyeability of oligoglucanes and polyglucanes with I₂/KI solution is based on the interaction between iodine and the C-atoms of glucosidic bonds (Vetter and Thorn [1, 2]). Only when the helix is compressed, the expansion of the lumen that is necessary for the attachment of iodine bands is achieved as suggested by Freudenberg et al. in 1939 [3]. The development of colour starts with the maltododecaosis (just over two spirals) within a temperature range of 20 to 25°C adsorbing one pentaiodide and bringing about a pale shade of pink. The rich blue tone is achieved with pure amyloses forming a maximum of about 610 to 620 nm. Unramified polysaccharides which had been synthesised by our team from 30 glucoside residues, still showed a purple colour with a maximum of 510 nm at temperatures from 20 to 25°C. Defined polysaccharides from 40 residues are bluish-purple (just 7 spirals of a tension-free helix) with a maximum absorption of about 550 nm. According to our knowledge, the rich blue tone requires at least 50 glucoside residues linked in an unramified chain [1, 2]. Amylopectin from potatoes has a maximum of 575 to 580 nm which shifts to 555 to 560 nm after partial break-down into β-dextrin by β-amylase (EC 3.2.1.2) and does a long-wave re-shift to 570 nm after splitting off of the side-chain stumps in the basic structure by pullulanase (EC 3.2.1.41). In the case of maize, the maximum is 535 nm for amylopectin, 520 nm for β-dextrin, and 555 nm for the basic structure. This means that in amylopectin from potatoes the helical sections available for the adsorption of iodine bands are longer than those in amylopectin from maize [4]. In glycogen iodine accumulates in a diffuse manner without forming any long bands. For this reason, an absorption shoulder of merely 400 to 500 nm is found by photometry [4]. A treatment with β-amylase makes the situation even worse; only when the side-chain stumps are separated by pullulanase does the formation of iodine bands in the basic structure of the glycogen improve to a maximum of 500 nm. This maximum corresponds to unramified sections of less than 30 glucoside residues [4, 2, 6].     

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.     

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.     

Enzymatic digestion and antioxidant activity of the native and molten globule states of camel α-lactalbumin: Possible significance for use in infant formula

Native- and molten globule states of α-lactalbumin (α-La) from camel and bovine milk were used for comparative assessment of digestibility and antioxidant activity. The proteolysis assessments were performed in the presence of gastrointestinal enzymes, using the o-phthaldialdehyde assay, and the antioxidant activity was carried out using a 2,20-azinobis(3-ethylenebenzothiazoline-6-sulfonic acid based method. Camel and bovine α-La revealed similar sensitivity to proteolysis by pepsin. The degree of hydrolysis (DH) of camel α-La by either trypsin or chymotrypsin was noticeably higher than that of the bovine protein counterpart. This can be explained by the different conformational and structural features of these proteins, as shown by studies of intrinsic- and 8-anilinonaphthalene-1-sulfonic acid fluorescence. The greater antioxidant activity of camel α-La could be explained by the higher content of antioxidant amino acid residues and different conformational features between bovine and camel α-La. The results may suggest that α-La produced from camel milk may be used for infant formulae as an alternative to that produced from bovine milk.     

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.     

Purification of α-D-Glucose-1-phosphate

A method is described for the purification of α-D-glucose-1-phosphate. The compound can be isolated from reaction mixtures containing carbohydrate, inorganic phosphate and glucose-1-phosphate by a one step chromatographic proces using an anion-exchange resin with potassiumacetate as eluting buffer. Glucose-1-phosphate can than be precipitated directly from the potassiumacetate solution by the addition of ethanol, because potassiumacetate is very soluble in ethanol and ethanol-water mixtures.     

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.