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.     

Biochemical and Functional Properties of Wheat Gliadins: A Review

Gliadins account for 40–50% of the total storage proteins of wheat and are classified into four subcategories, α-, β-, γ-, and ω-gliadins. They have also been classified as ω5-, ω1, 2-, α/β-, and γ-gliadins on the basis of their primary structure and molecular weight. Cysteine residues of gliadins mainly form intramolecular disulfide bonds, although α-gliadins with odd numbers of cysteine residues have also been reported. Gliadins are generally regarded to possess globular protein structure, though recent studies report that the α/β-gliadins have compact globular structures and γ- and ω-gliadins have extended rod-like structures. Newer techniques such as Mass Spectrometry with the development of matrix-assisted laser desorption/ionization (MALDI) in combination with time-of-flight mass spectrometry (TOFMS) have been employed to determine the molecular weight of purified ω- gliadins and to carry out the direct analysis of bread and durum wheat gliadins. Few gliadin alleles and components, such as Gli-B1b, Gli-B2c and Gli-A2b in bread wheat cultivars, γ-45 in pasta, γ-gliadins in cookies, lower gliadin content for chapatti and alteration in Gli 2 loci in tortillas have been reported to improve the product quality, respectively. Further studies are needed in order to elucidate the precise role of gliadin subgroups in dough strength and product quality.     

Large scale isolation of gliadin fractions

The ω,- γ-, and α-gliadin groups were isolated by chromatography of 10 g of crude gliadin on Sulfopropyl Sephadex C50, followed by chromatographies of the SPS-C50 fractions on Sephadex G100. The alcohol-soluble glutenins separated from the SPS-C50 fractions containing the γ- and β-gliadins had lower contents of Asx, Ala and Tyr but higher contents of Thr, Ser and Met than the gliadins. On the basis of weights of the isolated fractions, the proportions of ω-, γ-, β- and α-gladin groups in Cappelle wheat were estimated to be about 15, 30, 30 and 25% of the total gliadin, respectively. This method of isolation of the four gliadin groups is suitable for all wheat varieties, especially for the isolation of α-gliadin.     

木瓜蛋白酶控制水解蚕豆水溶性蛋白及水解度对蚕豆蛋白功能性质的影响

针对蚕豆蛋白在pH较低的体系中溶解性差、乳化能力低,制约其在食品工业中应用等问题,应用808Titrando瑞士万通全自动滴定仪控制木瓜蛋白酶水解蚕豆蛋白,实现蚕豆蛋白的有限水解,探讨水解度对蚕豆蛋白水解物的溶解性、乳化能力及持水性等功能性质的影响。通过木瓜蛋白酶的控制水解获得水解度为2%～4%的蚕豆蛋白水解物;与蚕豆蛋白相比,在pH4.0～6.0体系、水解度为4%的蚕豆蛋白水解物的溶解度提高2～3倍;pH6.0、水解度为2%的蚕豆蛋白水解物的乳化能力是蚕豆蛋白的2.3倍,持水力是蚕豆蛋白的1.6倍。     

The influence of nitrogen fertilisation on quantities and proportions of different protein types in wheat flour

The flours of 13 wheat varieties grown at different levels of nitrogen fertilisation were characterised by the quantitative determination of flour protein groups and gluten protein types using a combined extraction/HPLC procedure. The results demonstrate that the quantities of albumins and globulins were scarcely influenced by different nitrogen fertilisation, whereas those of gluten proteins (gliadins, glutenins) were strongly influenced. The effect on gliadins was more pronounced than on glutenins, as well as the effect on major protein types (α-gliadins, γ-gliadins, LMW subunits of glutenin) in comparison with minor types (ω-gliadins, HMW subunits of glutenin). The proportions of hydrophilic proteins (ω-gliadins, HMW subunits of glutenin) were increased by high levels of nitrogen and those of hydrophobic proteins (γ-gliadin, LMW subunits of glutenin) were decreased. The degree of the effects on both quantities and proportions of flour protein groups and gluten protein types was strongly dependent on the variety. © 1998 SCI.     

Thermal Properties of Yak α-Lactalbumin and β-Lactoglobulin: a DSC Study

A differential scanning calorimetry (DSC) method was used to investigate the denaturation temperature of yak α-lactalbumin (α-La), β-lactoglobulin (β-Lg), and a mixture of two proteins and the thermal properties of α-La and β-Lg in the presence of glucose, lactose, sucrose, NaCl, CaCl₂, and at various pH (4.0–10.0). The denaturation temperature (T _d) of α-La increased from 52.1 °C in the absence of β-Lg to 53.9 °C in the presence of β-Lg, while the T _d of β-Lg decreased from 81.4 °C in the absence of α-La to 79.9 °C in the presence of α-La. α-La was thermal stable in the range of pH 4.0–10.0, while β-Lg was more thermal stable in acidic pH than in alkaline pH. Sugars, Na⁺, and Ca²⁺ influenced the stabilization of the two proteins against thermal denaturation with greatly influenced for β-Lg. α-La kept reversibility in the presence of sugars, NaCl, CaCl₂, and over a wide pH range (4.0–10.0), with most of the reversibility values being greater than 90%. In contrast, β-Lg was completely irreversible whether in its native state or in the presence of the additives.     

Nitrogen extractability of sesame (Sesamum indicum L.) seed and the preparation of two protein isolates

Nitrogen extractability from sesame flour in water is greatest using a flour: solvent ratio of 1:40 and an extraction time of 15 min. At pH 4.0-6.5, <10% nitrogen is extracted, whilst >90% is extracted at pH 11.0. In the presence of NaCl (0.5-1.0M) nitrogen extractability is greatest at pH values > pH 4.0. The two sesame protein isolates produced were bland-tasting, light-coloured and contained approximately 95% protein. The alkali isolate (extracted in water at pH 10.0 and precipitated at pH 4.0) was readily solubilised and showed a good protein recovery (60%). The salt isolate (extracted in 1M NaCl at pH 6.0 and precipitated at pH 4.0) was less soluble and showed a lower protein recovery (50%). The sesame products were extracted sequentially using various solvents. Sesame flour proteins were mainly salt-soluble (67%), alkali isolate proteins mainly water-soluble (41%) and alkali-soluble (41%), and salt isolate proteins mainly alkali-soluble (35%). The amino acid composition of the sesame products is described. Oil-expelled cake showed poor nitrogen extractability (53% at pH 11.0 in water) and was, therefore, a poor source for protein isolate production.     

Preparation and Functional Properties of Enzymatically Deamidated Soy Proteins

Heat-denatured soy protein was hydrolyzed by Alcalase to 2.0% or 4.0% degree of hydrolysis (DH), heated again at 100°C and deamidated with B. circulans peptidoglutaminase. The extent of deamidation was 6.0% and 8.2% for 2.0 DH hydrolysates and 12.8% and 16.0% for 4.0 DH hydrolysates heated for 15 and 30 min, respectively. Deamidation increased protein solubility and substantially enhanced emulsifying activity under mildly acidic (pH 4–6) as well as alkaline conditions. Deamidation improved emulsion stability and foaming power of heat-denatured hydrolysed soy proteins. Enzymatically deamidated soy protein hydrolysates had improved functional properties compared to nondeamidated hydrolysates and the native soy protein.     

GLIADIN MODIFICATIONS CATALYZED BY GUINEA PIG LIVER TRANSGLUTAMINASE

Wheat gliadins, that are glutamine rich and lysine poor proteins, are good substrates for transglutaminases reactions. This study was conducted to determine the efficiency with which guinea pig liver transglutaminase catalyzes transfer and hydrolysis reactions of native and acylated gliadins. In all reactions, 35% of the total glutaminyl residues were modified. Neutral pH simultaneously enhanced glutaminyl residue hydrolysis and protein cross-linking, while acidic pH reduced the cross-linking reaction. Functional properties of two enzymatically modified gliadins and a chemically deamidated one were tested at neutral pH. A deamidation level of 25–27% appeared to be an optimum for the emulsification properties. Enzymatically modified gliadin showed better resistance to coalescence than the chemically deamidated one; a result that probably is related to the presence of high molecular weight polymers.     

The Impact of NaCl Substitution with KCl on Proteinase Activities Cell-Free Extract and Cell-Free Supernatant at Different pH Levels and Salt Concentrations: Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus

The effect of NaCl substitution with KCl at different pH levels (6.0, 5.5, and 5.0) and salt concentrations on proteinase activities of cell-free and supernatant of Lactobacillus delbrueckii ssp. bulgaricus 11824 (L. bulgaricus) and Streptococcus thermophilus MS (ST) was investigated. MRS broths were separately mixed with 4 salt treatments (NaCl only, 1NaCl:1KCl, 1NaCl:3KCl, and KCl only) at 2 different concentrations (5% and 10%) and incubated at 37 °C for 22 h. The cell pellets were used to prepare proteinase of cell-free extract and the cell-free supernatants were used as source of extracellular proteinases. The proteolytic activities and protein contents of both fractions were determined. The supernatants after incubation of both fractions with 3 milk caseins (α-, β-, κ-casein) were subjected to angiotensin-converting-enzyme inhibitory (ACE-inhibitory) activity and proteolytic activity by ortho-phthalaldehyde (OPA) method. Significant differences were observed in ACE-inhibitory activities and proteolytic (OPA) between salt treatments of cell-free extract and cell-free supernatant of L. bulgaricus and S. thermophilus at same salt concentration and same pH level. There was a significant effect of pH level and salt treatments interaction on ACE-inhibitory activity, OPA activity and azocasein activity. Practical Application: To reduce sodium concentration in cheese by substituting of NaCl with KCl, it was important to study the effect on starter culture proteinases which play a vital role in ripening and texture profile of cheese.     

Functionality of phosphorylated vicilin exposed to chemical and physical agents

The effects of phosphorylation with sodium trimetaphosphate (STMP) on functional and physicochemical properties of pea vicilin, as probed by high hydrostatic pressure and chemical denaturation were evaluated. The isoelectric point of unmodified and phosphorylated vicilin decreased in the presence of 0.5 M NaCl, resulting in a decrease of the solubility at pH 1.0. The gelation capacity of unmodified vicilin in the presence of NaCl decreased approximately 80% when compared with unmodified vicilin without NaCl. Increasing pressure from 0.1 MPa (atmospheric pressure) to 240 MPa significantly decreased the solubility of vicilin phosphorylated with 4% STMP at pH 1.0 and 4.0 by about 30%. Pressure had no effect on solubility of native vicilin. Pressure treatment at 240 MPa improved the gelation capacity of vicilin phosphorylated with 1% STMP. Glycerol decreased the gelation capacity of vicilin and its solubility in the acidic pH range.     

Sodium dodecyl sulphate electrophoresis of wheat gliadins

Polypeptide chain weights of α-, β- and γ-gliadin fractions have been estimated by polyacrylamide gel electrophoresis in the presence of mercaptoethanol and sodium dodecyl sulphate. Values lie in the range 32 000 to 44 000. When available data on the amino acid analyses were adjusted and compared, significant similarities emerged. It is suggested that these gliadins contain a minimum of 4 cystine residues/molecule. The results provide support for the hypothesis that glutenins and gliadins have evolved from a common precursor. The results of an amino acid analysis of a previously unreported β-gliadin component are included.     

Detection and quantification of adulteration of durum wheat flour by flour from common wheat using reverse phase HPLC

When common wheat (Triticum aestivum L) gliadins were separated by RP-HPLC, a major doublet peak eluted at 47.20 and 47.94 min. This peak was consistently found to be absent in Durum wheat (Triticum durum Desf) gliadins separated under identical conditions. In Durum wheat gliadins a characteristic small peak eluted at 48.30 min followed at 50.47, 510.37, 52.80 min by larger peaks. The peak area ratio of the peaks eluting at 50.47 and 51.37 min was found to be 2.18 (±0.14). This ratio was found to decrease proportionally on contamination of Durum wheat flour with flour from some common wheat varieties. This ratio alone was not enough to detect and quantify adulteration by all varieties of common wheat. An alternative method was found whereby the peak emerging between 47 and 49 min in the Durum wheat gliadin elution profile was expressed as a ratio of the total protein applied. This ratio was shown to increase when Durum wheat flour was adulterated with flour from common wheat thus enabling quantitative estimation of the level of adulteration. A third method of detecting adulteration of Durum wheat flour is also proposed in which the peak emerging between 47 and 49 min is collected and the protein separated by PAGE. The presence of more than one band of γ/β-gliadins is indicative of adulteration.     

Studies on the protein composition and baking quality of einkorn lines

White flours from 23 einkorn breeding lines (assortment 1) and wholemeal flours from 24 einkorn lines (assortment 2) were investigated for their qualitative and quantitative protein compositions by means of a combined extraction/HPLC procedure. The HPLC patterns of the gliadin fractions enabled the differentiation of most einkorn samples. The absence of a group of γ-gliadins at the beginning of the γ-gliadin elution region was unique for einkorn compared to all other wheat species. Differences in the patterns of γ-gliadins allowed the classification of einkorns into four groups; a further subdivision of these groups was possible by the number of ω5-gliadins and the different patterns of α-gliadins and low-molecular-weight glutenin subunits. The total gluten protein (gliadins + glutenins) contents of einkorn flours were similar to or even higher than those of common wheat and spelt. Typical for einkorn flours was the extreme excess of gliadins over glutenins with ω5-gliadins being most abundant and high-molecular-weight glutenin subunits being extremely rare. Micro-tests on the mixing properties and baking performance of assortment 2 flours revealed remarkable differences. Dough development time was negatively correlated with the ratio of gliadins to glutenins and positively with the content of glutenins; bread volume was mainly dependent on the content of glutenins. In conclusion, the determination of the quantitative gluten protein compositions offers a reliable indication of the expected baking quality during the early stages of breeding.     

A third β-Gliadin from Cappelle-Desprez wheat

A third β-gliadin has been isolated from Cappelle-Desprez wheat. It is the middle member of a triplet of almost equally spaced bands that appears on starch-gel electro-phoresis. Surprisingly, it has no very close affinity with the fastest of these three bands. Although conforming to the amino acid composition expected in gliadins, the content of (His + Arg) is four less than in the fastest component, but the small molecular weight of only 27 000 compensates for this, so that the mobility at acid pH is only a little less than that of its faster companion. The protein appears to be single-chain and free from SH groups and carbohydrate. Of the (Glx+Asx) content, 96% is amidated. Published analyses of β-gliadins have been calculated to the same basis and compared.     

Comparative investigations of gluten proteins from different wheat species I. Qualitative and quantitative composition of gluten protein types

In contrast to the hexaploid common (bread) wheat, little information is available on the qualitative and quantitative compositions of gluten proteins from other cultivated wheat species. Therefore, representatives of hexaploid spelt, tetraploid durum wheat and emmer, and diploid einkorn were compared with three classes of common wheat (winter wheat, spring wheat, wheat rye hybrid). The flours were extracted to yield total endosperm proteins and the gluten protein fractions (gliadins and glutenin subunits). The extracts were characterised using sodium dodecyl sulfate polyacrylamide gel electrophoresis and reversed-phase HPLC; both methods revealed that gluten protein groups and types known from common wheat (ω-, α-, γ-gliadins, HMW and LMW subunits of glutenin) were present in all species. The HPLC platterns of gliadins and glutenin subunits from species with the same genome composition (common wheat/spelt or durum wheat/emmer) were related, and those of einkorn quite different. According to the quantities determined by reversed-phase HPLC, α-gliadins were predominant in most cases, followed by γ-gliadins and LMW subunits; ω-gliadins and HMW subunits were generally minor components. Common wheats were characterised by the highest proportions of total glutenins and HMW subunits, which are known to be important for breadmaking quality. Moreover, the lower ratio of gliadins to glutenins was typical. Emmer had the lowest proportions of total glutenins and of HMW and LMW subunits, together with einkorn the highest proportion of α-gliadins, and, by far, the highest ratio of gliadins to glutenins. The values for spelt and durum wheat were mostly in a medium range between common wheats, emmer, and einkorn, respectively. Amongst common wheats, spring wheat was characterised by more balanced quantities of α- and γ-gliadins, and wheat rye hybrid by the highest proportions of ω-gliadins. Received: 26 November 1999     

复合米糠蛋白-卵白蛋白的起泡特性及相关机理分析

考察复合米糠蛋白（rice bran protein,RBP）-卵白蛋白（ovalbumin,OVA）的起泡特性,并分析在特定pH值与NaCl浓度下溶液与泡沫中不同蛋白质的物化性质,以阐述两种蛋白之间相互作用对起泡特性的影响。结果表明,在pH 4.0条件下,两种蛋白质在起泡能力上可以产生协同作用,且当RBP-OVA质量比为3:1时,添加1% NaCl后RBP-OVA复合蛋白的起泡能力和泡沫稳定性均显著增加;而在pH 7.0、无NaCl的情况下两种蛋白在起泡特性上没有表现出特别明显的协同作用,当添加1% NaCl后二者在起泡能力方面反而表现出一定的拮抗作用。通过对pH 4.0、1% NaCl条件下溶液与泡沫中蛋白质的物化性质进行分析可知,因两种蛋白质在物化性质方面具有一定的互补性,可通过蛋白质之间的相互作用从不同的物化性质角度改善RBP-OVA复合蛋白的起泡能力与泡沫稳定性。     

Comparative investigations of gluten proteins from different wheat species

The only commercially available immunoassay for gliadin determination in gluten-free food which has been ring-tested and in use for many years, is a test kit based on monoclonal antibodies against -gliadins. Various studies of the literature have shown that different gliadin standards resulted in different calibration curves, and it has been proposed that the affinity of -gliadins to the monoclonal antibodies varied among wheat varieties. To clarify this fundamental problem, total gliadins and the -gliadins from a winter wheat ("Rektor"), a spring wheat ("CWRS"), a wheat rye hybrid ("Herzog") and varieties of spelt, durum wheat, emmer and einkorn, were isolated and analyzed by means of an enzyme-linked immunoabsorbent assay (ELISA) kit based on antibodies against -gliadins. Additionally, single - and -gliadins of Rektor wheat were studied. The results demonstrated that the calibration curves derived for total gliadins differed, in parts, strongly from that of the kit gliadin standard; only the curves for the durum wheat and spelt gliadins were in congruence with the kit gliadin. Single -gliadins revealed strong differences between and within wheat species and ELISA equivalents had a range from 10 to 220% according to kit gliadin. The affinity of -gliadins was not correlated with the calibration curves of total gliadins. Some of the -gliadins of Rektor wheat showed ELISA equivalents similar to those of -gliadins. Because the proportions of -gliadins in total gliadins were significantly higher than those of -gliadins, the unspecific binding of -gliadins contributed much more to the total affinity of gliadins than the specific binding of -gliadins. -Gliadins, however, did not show any detectable affinity.     

Functionality of native and denatured cashew nut kernel protein isolates at isoelectric pH as a function of salt concentration

The reduced solubility of proteins near the isoelectric pH limits their use in food formulations whose pH lies in the range 5.0–6.0 because of poor functionality. In the present study, the effect of salt on the functionality of native and denatured cashew nut kernel protein isolates at the isoelectric pH was investigated. Both isolates showed improvement in their functional properties, but the improvement was greater for the denatured protein isolate. The solubilities of denatured and native protein isolates at the isoelectric pH increased from 26.4 g l^?1 and 64 g l^?1, respectively, without salt to maxima of 363 and 308 g l^?1, respectively, at 0.75 M salt concentration. The water binding capacity of the isolates increased with increase in NaCl concentration from 1.70 ml g^?1 to 1.77, 1.82, 1.92 and 2.2 ml g^?1 for denatured protein isolate and from 1.45 ml g^?1 to 1.65, 1.69, 1.82 and 1.97 ml g^?1 for native protein isolate at 0.25, 0.50, 0.75 and 1.0 M salt concentrations, respectively. When the properties of the isolates in 0.75 M NaCl solutions were compared with those in salt‐free water there were 15% and 116% increases in emulsifying capacity, 40‐fold and 45‐fold increases in emulsifying activity and 4.6‐fold and 40‐fold increases in emulsion stability for native and denatured protein isolates, respectively, whilst the corresponding foaming capacities increased from 4 to 5.5 and 0 to 8.9 ml g^?1 protein. Statistically, no difference in the foaming capacity of either of the isolates was observed above 0.5 M NaCl. The foam stability also exhibited similar behaviour. Copyright © 2004 Society of Chemical Industry     

Synergistic enhancement in the co-gelation of salt-soluble pea proteins and whey proteins

This paper investigated the enhancement of thermal gelation properties when salt-soluble pea proteins were co-gelated with whey proteins in NaCl solutions, using different blend ratios, total protein concentrations, pH, and salt concentrations. Results showed that the thermal co-gelation of pea/whey proteins blended in ratio of 2:8 in NaCl solutions showed synergistic enhancement in storage modulus, gel hardness, paste viscosity and minimum gelation concentrations. The highest synergistic enhancement was observed at pH 6.0 as compared with pH 4.0 and 8.0, and at the lower total protein concentration of 10% as compared with 16% and 22% (w/v), as well as in lower NaCl concentrations of 0.5% and 1.0% as compared with 1.5%, 2.0%, 2.5%, and 3.0% (w/v). The least gelation concentrations were also lower in the different pea/whey protein blend ratios than in pure pea or whey proteins, when dissolved in 1.0% or 2.5% (w/v) NaCl aqueous solutions.