Ultrathin-layer horizontal high-resolution two-dimensional electrophoresis of legume seed proteins with intermediate protein staining

Summary Legume seed proteins extracted with urea-containing buffer are fractionated by high-resolution 2D-electrophoresis (urea-IEF x pore gradient SDS-PAGE), both dimensions in horizontal ultrathin-layer polyacrylamide gel slabs. High reproducibility is obtained, because the first dimension is performed in a slab gel, where a large number of protein samples are separated under identical conditions. The gel of the first dimension (IEF) is fixed, stained with Coomassie Brilliant Blue G 250, and destained before application to the second-dimension gel (SDS-PAGE). Prestaining of the focused proteins does not alter the protein pattern obtained after SDS electrophoresis. Thus, bands are made visible before separation in the second dimension, and the amount of Ampholine in the dye front is reduced during electrophoresis. The first-dimension gels can easily be stored in the destaining solution until they are run in the second dimension. As the proteins are fixed in the gel, there is no loss of proteins and defocusing of bands due to diffusion during the SDS-equilibration procedure. Loading of the dimensionstable gel strip onto the second dimension gel is a very easy operation, since the ultrathin gel strip adheres to a plastic foil and is simply laid into a moulded gel through. The gel strip is not imbedded with polymerizing acrylamide or agarose solution like in the conventional gel-rod-techniques, because a very good surface contact is obtained between the two flat gels.

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Ultradiinnschicht horizontale, hochauflösende Zweidimensional-Elektrophorese von Leguminosensamen-Proteinen mit Protein-Zwischenfärbung

Zusammenfassung Mit harnstoffhaltigen Tris-Glycin-Puffer extrahierte Leguminosensamen-Proteine werden mit der hochauflösenden 2D-Elektrophorese (Harnstoff-IEF x Gradientengel-SDS-Elektrophorese) aufgetrennt, wobei beide Dimensionen in horizontalen ultradünnen, auf Folie polymerisierten Polyacrylamid-Flachgelen durchgeführt werden. Die Flachgel-Focussierung (1. Dimension) erlaubt die Trennung einer großen Anzahl von Proteinproben unter identischen Bedingungen, wodurch die Reproduzierbarkeit von 2D-Elektrophoresen erheblich verbessert wird. Das Gel der ersten Dimension (IEF) wird mit Coomassie BB G 250 gefdrbt, bevor die einzelnen Gelstreifen für die zweite Dimension (Gradientengel-SDS-Elektrophorese) verwendet werden. Dies hat den Vorteil, daß die focussierten Proteine bereits vor dem zweiten Trennungsschritt sichtbar gemacht und zudem die Trägerampholyte in der Farbstoff Front bei der SDS-Elektrophorese stark vermindert werden. Die vorgefärbten Focussierungsgele können problemlos in der Entfärbelösung für die SDS-Gradientengel-Elektrophorese aufbewahrt werden. Der Proteinverlust und die Banden-diffusion bei der SDS-Aquilibrierung werden durch die Fixierung im Focussierungsgel verhindert. Das 2-D-Proteinmuster wird durch das Vorfärben der focussierten Proteine nicht verdndert. Die dimensionsstabilen, auf Folie polymerisierten Focussierungsstreifen lassen sich einfach handhaben und müssen nicht wie Rundgele an die zweite Dimension aufpolymerisiert werden. Der Gel-Gel-Kontakt der beiden Flachgele ist ohne weitere Hilfsmaßnahmen ausgezeichnet.

     

Evaluation of aglianico grape skin and seed polyphenol astringency by SDS–PAGE electrophoresis of salivary proteins after the binding reaction

SDS–PAGE electrophoresis and densitometry analysis were carried out to evaluate the reactivity of Aglianico red grape skin and seed polyphenols with human salivary proteins in order to find a method able to assess their astringency. Analysis of the supernatant obtained after a tannin/human salivary protein binding assay and sensorial analysis showed that four proteins, lactoferrin, PRPbg1, PRPbg2 and α-amylase, were the proteins best able to distinguish tannin solutions characterised by different levels of astringency.     

Textural properties of legume protein isolate and polysaccharide gels

The influence of legume proteins from lupin, pea and fababean on the formation of gels prepared by heat treatment in the absence or presence of xanthan gum, locust bean gum and NaCl was investigated. The resulting fracture and texture properties of gels not only are associated with the heating process used to form the gel but also depend on the conformational aspects of xanthan–locust bean gum in admixture with legume proteins, which after 10 days of aging reinforce the system. The fracture and textural properties are explained in terms of the effect of the protein–polysaccharide molecular structure and physicochemical conditions applied in the gel system during the gel preparation and measurements. Copyright © 2006 Society of Chemical Industry     

Molecular aspects of legume seed storage protein synthesis

The seed proteins of crop plants will provide an ever‐increasing proportion of protein in the human diet in the future. Research is currently under way with a variety of crop plants to try to identify the molecular mechanisms that regulate the production of seed proteins. This review, however, will be limited to studies of soybean (Glycine max) and common bean (Phaseolus vulgaris). The accumulation of storage proteins during seed maturation, the characteristics of the different proteins, and the results of studies of the messenger RNAs encoding the seed proteins have revealed a number of similarities as well as differences between common beans and soybeans. Ongoing research in the isolation and characterization of the genes encoding the seed proteins will provide the much needed background to further explore the molecular mechanisms that regulate the synthesis of these proteins.     

几种豆类蛋白在肉制品中的应用

我国有丰富的豆类资源,这些豆类是廉价、优质的天然植物蛋白质来源。豆类蛋白具有良好的营养价值和功能特性,越来越多地被应用于肉制品加工中,不仅能提高产品的出品率,改善产品的口感,而且能够大大地降低肉制品的生产成本。本文对大豆蛋白的特点、分类以及几种大豆蛋白在肉制品中的应用研究进行了综述、同时也简单介绍了豌豆蛋白、芸豆蛋白、鹰嘴豆分离蛋白、黑豆蛋白和绿豆蛋白等几种不同豆类蛋白在肉制品中的应用研究进展,为豆类加工和肉制品产业发展提供参考。     

Ultracentrifugation of salt-soluble proteins in ten legume species

The ultracentrifugation patterns of the total salt-soluble proteins in pea and northern bean showed four globulin fractions in flour proteins but only the 2S, 7S and 9S globulins were found in the isolates. Mung bean, lentil and lupin exhibited 2S, 7S and 11S globulins in flour and isolate proteins while chick-pea, field-pea, faba-bean and soya-bean proteins also contained the 15S fraction. Salt-soluble proteins from lima bean were unique in their composition of 5S, 7S and 15S fractions. Only pea bean and lentil failed to show high proportions of the 1-5S fraction in the flour proteins.     

Inhibitory effects of legume seed extracts on fish proteinases

Proteinase inhibitor extracts were prepared from various legume seeds. Black cowpea and soybean seeds showed the highest inhibitory activity against viscera proteinases (p < 0.05). Optimal extraction was attained by shaking the defatted legume seed powder in distilled water (3 ml g⁻¹) at ambient temperature for 1 h. Both extracts showed high thermal stability, but that from soybean was slightly less stable. The inhibitory activity of both extracts was retained over a broad pH range. The proteinase inhibitor extracts also reduced modori-inducing proteinase (MIP) activity. The sarcoplasmic MIP activities were more effectively inhibited than the myofibrillar-associated MIP activities. © 1999 Society of Chemical Industry     

Characterisation and fractionation studies on seed proteins of some of the leguminosae by starch-gel electrophoresis

This investigation supplements the previously reported characterisation of 32 cultivars of Phaseolus vulgaris and led to results for 5 cultivars of Vicia faba and 3 cultivars of Pisum sativum. Moreover, proteins of S1 fractions of commercial seeds of three Phaseolus vulgaris, two Vicia faba and three Pisum sativum cultivars were fractionated; electrophoretic patterns obtained for the albumin fractions (F1) are characteristic for the species used. By fractionation, three different globulin fractions (F2-F4) are obtained; after electrophoresis optimum cultivar differences appear in the legumin patterns (F2).     

Capillary gel electrophoresis versus SDS PAGE of exudate from fresh pork

This investigation compared electrophoretic profiles of exudate from fresh normal pork in which the protein separation was undertaken by both sodium dodecyl sulphate polyacrylamide gel electrophoresis SDS PAGE and capillary gel electrophoresis. The profiles obtained using the two techniques were similar and in many investigations either method would be suitable. It is suggested that capillary gel electrophoresis offers a number of benefits over the traditional time consuming and labour intensive gel electrophoresis techniques which involves gel preparation, sample application, staining and eventually quantification of the zones by densitometry. For separation of proteins in meat-based systems the advantages of capillary gel electrophoresis include on-capillary detection, instrumental automation, rapid analysis time and low sample volumes while providing similar information to that obtained from SDS PAGE.     

塔里木盆地4种豆科植物种子成分比较

分析了塔里木盆地北部的疏叶骆驼刺、胀果甘草、铃铛刺和锦鸡儿种子中常规营养成分和油脂中脂肪酸组成及含量,结果表明:①锦鸡儿种子中粗脂肪和粗蛋白含量都很高,酸性洗涤纤维含量低,油脂中亚油酸含量高,可作为油脂植物资源开发利用;②疏叶骆驼刺和铃铛刺种子油中亚油酸和棕榈酸含量丰富,营养价值较高;③胀果甘草种子油中亚麻酸含量高达37.92%,亚油酸为32.22%,有很大的开发利用价值;④这4种植物种子油中的不饱和脂肪酸含量都较高,占所有鉴定脂肪酸的70%以上.     

Proteinase inhibition of fish muscle enzymes using legume seed extracts.

Seed extracts from indigenous and introduced legumes were prepared and used to search for inhibitors of fish muscle proteinases. Fish flesh extracts were prepared from samples of Merluccius productus (Pacific whiting or merluza) obtained off the Oregon coast and in the Gulf of California, respectively. The proteinase activity in the fish muscle for the Pacific whiting was the highest, followed by parasitized merluza. The lowest proteinase activity was for the nonparasitized merluza. Six out of 12 seed extracts reduced the proteinase activity from the fish flesh by more than 50%. The reduction of enzyme activity was higher for samples of fish flesh extracts from the Gulf of California than for the Oregon samples. Seed extracts also reduced the proteinase activity of commercial serine and cysteine proteinases such as trypsin, chymotrypsin, and papain. This inhibitory capacity was maintained even after heating the seed extracts to 90 degrees C for 15 min. Several seed extracts show promise for use as proteinase inhibitors during production of surimi, the intended commercial product of massive fisheries such as Pacific whiting or merluza.     

Application of Fourier transform infrared spectroscopy to legume seed flour analysis

The secondary structure of legume (Phaseolus vulgaris L. and Lens culinaris L.) proteins was investigated by studying the amide I infrared absorption band in whole seed flours, before and after dry heating and autoclaving thermal treatments. The analysis procedure, set up on 7S and different model proteins, shows that the content of β-sheet structures in lentil is higher than in common bean (47% vs. 32%). The dry heating does not appreciably affect secondary structures in lentil, while it causes a reduction of β-sheets (to 13%), an increase of aggregates, and the appearance of random coil structures in common bean. The autoclaving treatment produces high amounts of aggregates in both legumes. However, in lentil, random coil structures are lower than in common bean and some β-sheet structures are still detectable. These results indicate that multimeric heat-induced complexes of legume proteins have a high stability because of the high content in β-sheet structures, in particular in lentil, which may adversely affect protein utilization.     

The trypsin inhibitors present in seed of different grain legume species and cultivar

Trypsin inhibitors in a selection of grain legume seeds from different species and cultivars were studied. The results showed that trypsin inhibition content ranged from negligible in Lupinus spp. to very high in Glycine max. Although there is variation among cultivars, generally the highest TIU mg⁻¹ sample values occured in soybean (43–84) and common bean (21–25). Inhibitor content of different Lathyrus cultivars, ranged from 19–30 TIU mg⁻¹ sample. This was higher than the contents in chickpea (15–19 TIU mg⁻¹ sample) and pea (6–15 TIU mg⁻¹ sample). Lentil and faba bean had low values in most cvs (3–8, and 5–10 TIU mg⁻¹ sample, respectively). Trypsin inhibitor isoform analyses showed that the amount of TI detected, varied with legume species and variety.     

Reflections about the functional potential of legume proteins A Review

The term “functional potential” was introduced to better approach to the understanding of the relationships between the structure and the functional properties of food proteins. Although in practice the complex of structural features of a protein contributes to its functionality, it is very useful to regard the functional potential of the protein surface on the one side and to look on special effects of protein conformation (role of compact or unfolded state) on the functionality on the other side. This point of view may help to design the best strategy of modification of the protein structure and functionality. The special situation of the oligomeric legume storage protein, i. e. legumin‐like 11 S globulins and vicilin‐like 7 S globulin, and the structural and functional modification of 11 S globulin by limited tryptic hydrolysis and by acylation are discussed in this paper.     

Determination of soya protein in meat products by standard curves obtained from SDS gel electrophoresis

Summary A method based on densitometry of sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoretograms has been developed for the assessment of soya protein in fresh and heated meat products that also contain other additives. The protein is extracted with 3% SDS and 3 % 2-mercaptoethanol in a tris/borate buffer, pH 8.2. The electrophoretic separation is carried out on gels containing 12% polyacrylamide and SDS, pH 9.18. The method shows reproducable results. Qualitative identification is possible in products heated to less than 100 °C in the centre with standard deviation ±0,6% when the addition of soya protein is 1–5%,and ±1,5% when the addition is higher. The electrophoretic method can presumably also be used for obtaining standard graphs for determination of casein.

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Identifizierung von Sojaeiweiß in Fleischerzeugnissen mit Hilfe einer aus der SDS Gel Elektrophorese entwickelten Standardkurve

Zusammenfassung Diese Methode basiert auf der densitometrischen Auswertung eines Dodecylsulfat-Polyacrylamid-Elektrophoresegels und dient der Identifizierung von Sojaeiweiß in rohen und erhitzten Fleischwaren, die noch andere Zusätze erhalten. Die Proteine werden in Tris/Borsäure-Puffer (pH 8,2) mit 3% SDS and 3% 2-Mercaptoethanol aufgelöst. Die Elektrophorese wurde in 12% Polyacrylamid mit SDS, pH 9,18, durchgeführt. Die qualitative Bestimmung ist sowohl in rohen als in hocherhitzten Fleischwaren durchführbar. Die quantitative Bestimmung ist nur möglich in Produkten, die nicht über 100 °C erhitzt sind. Bei Anwendung der Standardkurve muß mit einer Standardabweichung von ±0,6% gerechnet werden, wenn die Zusätze zwischen 1–5% Sojaeiweiß und ± 1,5%, wenn die Zusatze größer sind. Die elektrophoretische Methode ist wahrscheinlich auch für eine Bestimmung von anderem Fremdeiweiß anwendbar.

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This work was supported by funds from the Danish Agricultural and Veterinary Research Council     

Complex gels of proteins and acid polysaccharides]

The authors studied the thermomechanical properties of complex gels of gelatin and sodium alginate which had been produced on the basis of their insoluble (type I) and soluble (type II) complexes. Compared to gelatin gels of the same concentration, the complex gels I have higher melting points. The latter depend on the pH value at which the insoluble complexes of gelatin and sodium alginate were separated. Up to temperatures from 70 degrees--80 degrees C., no melting of the complex gels II was observed. The aging time and the composition of the soluble complexes of gelatin and sodium alginate exert no marked effects on the properties of the complex gels II. The soluble complexes of gelatin and sodium alginate can form gels in 7 M urea solutions.     

Analysis of kimchi microflora using denaturing gradient gel electrophoresis

A polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) technique was used to determine the microfloral composition during the fermentation of kimchi, a traditional Korean fermented vegetable food. The kimchi was fermented at 10 degrees C or 20 degrees C for 30 or 20 days, respectively. DGGE of the partially amplified 16S rDNA was performed and the most intense bands sequenced. The application of this culture-independent molecular technique determined that the lactic acid bacteria Weissella confusa, Leuconostoc citreum, Lactobacillus sakei, and Lactobacillus curvatus were the main microorganisms responsible for kimchi fermentation.