Functional and conformational properties of phaseolin (Phaseolus vulgris L.) and kidney bean protein isolate: A comparative study

BACKGROUND: Kidney bean (Phaseolus vulgris L.) seed is an underutilised plant protein source with good potential to be applied in the food industry. Phaseolin (also named G1 globulin) represents about 50 g kg^?1 of total storage protein in the seed. The aim of the present study was to characterise physicochemical, functional and conformational properties of phaseolin, and to compare these properties with those of kidney bean protein isolate (KPI). RESULTS: Compared with kidney bean protein isolate (KPI), the acid‐extracted phaseolin‐rich protein product (PRP) had much lower protein recovery of 320 g kg^?1 (dry weight basis) but higher phaseolin purity (over 950 g kg^?1). PRP contained much lower sulfhydryl (SH) and disulfide bond contents than KPI. Differential scanning calorimetry analyses showed that the phaseolin in PRP was less denatured than in KPI. Thermal analyses in the presence or absence of dithiothreitol, in combination with SH and SS content analyses showed the contributions of SS to the thermal stability of KPI. The analyses of near‐UV circular dichroism and intrinsic fluorescence spectra indicated more compacted tertiary conformation of the proteins in PRP than in KPI. PRP exhibited much better protein solubility, emulsifying activity index, and gel‐forming ability than KPI. The relatively poor functional properties of KPI may be associated with protein denaturation/unfolding, with subsequent protein aggregation. CONCLUSION: The results presented here suggest the potential for acid‐extracted PRP to be applied in food formulations, in view of its functional properties. Copyright © 2009 Society of Chemical Industry     

Isolation and Characterization of Chickpea (Cicer arietinum L.) Seed Protein Fractions

Proteins of ground chickpea seeds were extracted with sodium hydroxide (NaOH) solution and precipitated with addition of acid (isoelectric precipitate (C-IP)) and by cryoprecipitation (cryoprecipitate (C-CP)). The protein isolates were characterized by Native PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), reversed-phase high performance liquid chromatography (RP-HPLC) and electrospray-ionization mass spectrometry (ESI/MS). Both the isoelectric precipitate and cryoprecipitate contained the globulin protein 11S legumins and 7S vicilins as the major protein fractions and 2S albumin proteins as a minor protein fraction. The major subunits of RP-HPLC protein fractions from both cryoprecipitate and isoelectric precipitate were found to contain subunits of both legumins and vicilins. SDS-PAGE identified legumin α-subunits with MW 40.6 and 39.5 kDa and legumin β- subunits with MW 23.5 and 22.5 kDa, and vicilin subunits with MW 70.2, 50.7, 35.0, 33.6, 18.9 and 15.5 kDa. ESI-MS molecular weights 35,366, 35,268 and 14,648 Da are likely vicilin subunits while the 24,894 Da is a legumin β-subunit.     

Resistance of the kidney bean reserve protein,phaseolin, to proteolysis in the porcine digestive tract

Abstract : Phaseolin, the major reserve protein of the seeds of the kidney bean, Phaseolus vulgaris L, was shown to be poorly degraded in the pig small intestine following an initial 7-day exposure to casein-based diets incorporating kidney bean meal. Substantial amounts of large polypeptide fragments, which were recognised by antibodies raised against native phaseolin, were identified in ileal digesta. Little of this immunoreactivity was detected in the faeces of these animals, suggesting that components of the hindgut microflora were able to degrade these fragments further. Prolonged exposure to dietary kidney beans resulted in an increase in the digestibility of phaseolin, suggesting that the small intestine and/or its microflora may possess significant adaptive capacity. In-vitro digestion of purified phaseolin by a sequence of pepsin, trypsin and chymotrypsin yielded products which closely resembled those isolated from the ileal digesla; analysis suggested that degradation of native phaseolin by the major gastrointestinal endopeptidases is restricted to a vulnerable central region of its constituent polypeptides.     

Physicochemical characterisation of mung bean (Phaseolus aureus) protein isolates

Protein isolates were prepared from mung bean (Phaseolus aureus) flour by two different methods, ie by traditional alkaline water extraction/isoelectric precipation and by micellisation, and studied with regard to chemical composition and protein fractional distribution. Micellisation decreased the phytic acid content, whereas the ‘isoelectrical’ isolate preparation resulted in an increase in phytic acid. Using size exclusion liquid chromatography (SE‐HPLC) and analytical ultracentrifugation, it has been shown that the major protein component in both isolates is a 7S globulin, which was most enriched in the micelle isolate. The results of one‐ and two‐dimensional SDS gel electrophoresis give evidence for the absence of interchain disulphide bonds in the polypeptide chains, which are typical for the 7S globulin class. The presence of minor protein zones corresponding to disulphide‐linked α and β chains of 11S globulin‐like storage proteins in the protein isolates has also been shown. © 2000 Society of Chemical Industry     

Structure-Digestibility Relationship of Legume 7S Proteins

Structure-digestibility relationship was investigated in in vitro model systems for phaseolin and vicilin, the major 7S storage proteins of dry bean and green peas, respectively. Native phaseolin was more resistant to trypsinolysis than vicilin, while heating caused a reversal of proteolysis rates. Conformational studies using far-uv and near-uv CD spcctroscopy suggested the native conformatin of vicilin to be far more flexible to thermal treatment and SDS-induced environmental changes; however, neither the thermal treatment nor the anionic detergent caused a complete randomization of structure in either proteins studied. Ironically, the flexible native conformation of vicilin seemed to induce greater undesirable changes upon heating so as to confer resistance to proteolysis. The possible role of secondary and quarternary structures of these two proteins is descussed in relation to their digestibility.     

Functional properties of 8S globulin fractions from 15 mung bean (Vigna radiata (L.) Wilczek) cultivars

The functional properties including solubility, water absorption capacity, oil absorption capacity, foaming properties and emulsifying properties of 8S globulin fractions from 15 mung bean cultivars were investigated in this study. In addition, the effects of pH on solubility, foaming properties and emulsifying properties were studied. The functional properties of the 8S globulin fractions varied significantly among the different mung bean varieties and exhibited better performance in solubility and emulsion stability compared with soya bean 7S protein. A negative correlation was found between water absorption capacity and oil absorption capacity. Remarkable differences in polypeptides constituents were observed in 8S globulin fractions, and the ratio of 11S/8S globulins has a positive effect on water absorption capacity while a negative effect on oil absorption capacity. As a function of pH, the emulsifying activity indexes of the 8S globulin fractions were found to be distinctly dependent on the solubility, while no significant correlation was found between the emulsifying stability and solubility, nor between the foaming properties and solubility. The foaming capacity showed a strong correlation with foam stability.     

EFFECT OF BEAN (Phaseolus vulgaris) ALBUMINS ON PHASEOLIN IN VITRO DIGESTIBILITY, ROLE OF TRYPSIN INHIBITORS

The in vitro digestibility of albumin proteins from Phaseolus vulgaris was low (26–32%) and when heated (99C/30 min) it was further reduced to 13–18%, independent of the variety. Phaseolin (the main bean globulin) had, after heating, a digestibility similar to casein, but when heated in mixtures containing as low as 10% of the albumin fraction, it suffered a drastic decrease in its susceptibility to proteolysis. The chemical interactions that occur among the components of the albumin fraction that reduce its digestibility do not occur between phaseolin and albumins. Interactions between phaseolin and albumins depend on pH of heating and are responsible for differences in molecular weight distribution of peptides formed by the action of pepsin, but did not alter overall digestibility of the mixture. The low digestibility indeed was due to heat-stable trypsin inhibitors found in the acid-soluble fraction of the albumins. Lectins and α-amylase inhibitors, glycoproteins of the albumin fraction, showed, in similar experiments, no effect on phaseolin digestibility, in spite of being very poorly digested even after heating.     

Immunodetection of legume proteins resistant to small intestinal digestion in weaned piglets

Experiments were conducted to investigate the biochemistry of digestion of the major storage proteins from soya bean, pea, faba bean, blue lupin, and chickpea seeds in the ileum of piglets. Hyperimmune plasmas against the crude protein extracts and the purified 11S and 7S globulin fractions of each legume seed and an anti‐pea albumin PA2 and lectin antibody were used. They served to probe immunoblots of feed protein extracts and ileal digesta samples. Globally, the recognition by plasmas of intact or partially digested proteins in ileal digesta was rather faint, in agreement with the fairly high in vivo digestibility data obtained with these legume seed proteins. Nevertheless, immunoreactive polypeptides found in digesta of piglets fed pea, faba bean and chickpea belonged mainly to proteins of the 7S family, and to other proteins including low‐molecular weight components such as PA2 albumin and lectin in the case of pea. In piglets fed lupin, nearly intact polypeptides from the 11S family were detected. To conclude, the present immunochemical study conducted on ileal digesta of piglets revealed a few dietary legume proteins of the vicilin and albumin families. Legumin proteins were demonstrated unequivocally in the case of lupin and white chickpea. Copyright © 2003 Society of Chemical Industry     

Thanh法提取7S、11S球蛋白功能特性的研究

以中豆36为原料,分别提取大豆分离蛋白(SPI)、7S、11S球蛋白,系统地比较了它们的功能性质的差异。结果表明:SPI、7S及11S的功能特性之间存在较大差异,11S球蛋白具有较强的凝胶性、吸油性和溶解度;7S球蛋白具有较高的持水性和乳化性。     

Characterization of protein fractions from chickpea (Cicer arietinum L.) and oat (Avena sativa L.) seeds using proteomic techniques

Albumin, globulin and glutelin fractions were prepared from chickpea and oat seeds using sequential extractions. Molecular characteristics of individual protein fractions were investigated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in combination with proteomic techniques. SDS-PAGE results revealed that chickpea albumin and globulin fractions (C-Ab and C-Gb) showed protein bands with molecular weights (MWs) related to subunits of legumin (11S globulin) and pea vicilin (7S globulin); oat protein fractions (O-Ab, O-Gb and O-Gt) showed most protein bands with MWs related to subunits of oat 12S globulin (avenalin). With proteomic analysis, eighteen tryptic peptides from chickpea globulin fraction showed sequence homology that corresponded to chickpea legumin α- and β-subunit (NCBI accession number: gi|6273402; theoretical mass 56,216 Da) while sixteen tryptic peptides from chickpea albumin fraction (C-Ab) were identified as chickpea provicilin precursor (NCBI accession number: gi|82173888; theoretical mass 51,390 Da); fifteen tryptic peptides from oat protein fractions were identified with origin from oat 12S seed storage globulin 1 (NCBI accession number: gi|134918; theoretical mass 58,508 Da). The identified tryptic peptide, ALIVPQNFAIAAK, was commonly found in chickpea glutelin fraction (C-Gt), rice glutelin fraction (R-Gt), and oat albumin, globulin and glutelin fractions (O-Ab, O-Gb and O-Gt).     

Physicochemical, functional and angiotensin converting enzyme inhibitory properties of amaranth (Amaranthus hypochondriacus) 7S globulin

BACKGROUND: Amaranth 7S globulin is a minor globulin component and its impact on the properties of an amaranth protein ingredient depends on its proportion in the variety of amaranth being considered. Some physicochemical, functional and angiotesin I‐converting enzyme (ACE) inhibitory properties of amaranth vicilin were studied in this work and compared with the 11S globulin. RESULTS: Fluorescence spectroscopy results indicated that 7S globulin tryptophans were more exposed to the solvent and, by calorimetry, the 7S globulin denaturation temperature (T_d) was found lower than the 11S globulin T_d, suggesting a more flexible structure. The 7S globulin surface hydrophobicity was higher than that of the 11S globulin, which is in agreement with the better emulsifying properties of the 7S globulin. The solubility in neutral buffer of the 7S globulin (851 ± 25 g kg^?1) was also higher than that of the 11S globulin (195 ± 6 g kg^?1). Bioinformatic analyses showed the presence of ACE inhibitory peptides encrypted in 7S tryptic sequences and peptides released after in vitro gastrointestinal digestion showed a high ACE‐inhibitory capacity (IC₅₀ = 0.17 g L^?1), similar to that of 11S globulin peptides. CONCLUSION: Compared with the 11S globulin, the 7S globulin presents similar ACE inhibitory activity and some functional advantages, better solubility and emulsifying activity, which suits some food requirements. The functional behavior has been related with the structural properties. Copyright © 2011 Society of Chemical Industry     

In vitro and in vivo protein hydrolysis of beans (Phaseolus vulgaris) genetically modified to express different phaseolin types

Experiments were conducted to study whether phaseolin type could influence proteolysis susceptibility and nutritional value of total bean protein. The DOR-390 bean cultivar was genetically modified to express different phaseolin types (S, T or I). Beans were soaked and autoclaved. A sequential hydrolysis was carried out in vitro with pepsin and pancreatin. Differences in the degree of protein hydrolysis among bean lines started at 30 min and remained until 240 min, with the S bean proteins presenting lower values (P < 0.05). Subsequently, rats were fed with diets containing beans expressing different phaseolin types as the only source of protein for N digestibility and nutritional value determination. No differences (P > 0.05) in ileal protein digestibility and rat growth were observed. In conclusion, the differences in in vitro hydrolysis between bean lines expressing different phaseolin types had no consequences on growth and N retention in rats.     

Differential scanning calorimetric study of meals and constituents of some food grain Legumes

Two thermal transitions were observed in soya bean meal by differential scanning calorimetry (DSC) and on the basis of previous data were ascribed to the denaturation of the 11S globulin, glycinin, and the 7S globulin, β-conglycinin. Three DSC transitions were apparent in Vicia faba meal; one was associated with starch gelatinisation, and was absent from thermograms of soya bean because the latter stores oil rather than starch. The remaining two transitions were identified with the 11S globulin, legumin, and a 7S globulin, vicilin. The denaturation parameters of legumin and glycinin were very similar, in contrast to those of vicilin and β-conglycinin. From this it was concluded that legumin and glycinin probably represent homologous proteins, whereas the two 7S proteins possess intrinsically different structures to one another. Cowpeas (Vigna unguiculata) and dry beans (Phaseolus vulgaris) afforded two major transitions which were assigned to 7S globulins and starch. From its DSC profile, the 7S globulins of cowpea appeared heterogeneous, but nevertheless possessed denaturation characteristics similar to those of vicilin of V. faba. In contrast, the thermal behaviour of the 7S globulin of dry beans (glyco-protein II) was distinct from that of any of the other 7S globulins investigated. Three protein transitions were observed in the three lupin species examined. As in the case of soya bean, no starch transition was present. On the basis of the correspondence in transition temperature, one of the protein transitions was assigned to the 11S globulin component. Overall, this study indicates the potential of DSC as a means for obtaining data on seed protein homology from whole meals rather than the extracted proteins.     

In Vitro Enzymatic Hydrolysis of Phaseolin, the Major Storage Protein of Phaseolus Vulgaris L.

Susceptibility of the major storage protein, phaseolin, to different commercially available proteinases was studied in vitro for 17 varieties of Phaseolus vulgaris L. using electrophoretic techniques. Among serine proteinases, trypsin, subtilisin and pronase E readily hydrolyzed the native phaseolin. while chymotrypsin was less effective. Native phaseolin was markedly resistant to a carboxyl proteinase (pepsin) and somewhat resistant to a thio proteinase (papain). Major breakdown products of native phaseolin subunits were of a similar size irrespective of the enzyme used. and had an approximate MW of 22–25 kilodaltons. SDS-PAGE indicated that heated phaseolin from all the varieties investigated was effectively hydrolyzed by subtilisin, pronase E. pepsin and papain within 30 min, while chymotrypsin and trypsin exhibiting restricted specificity were slightly less effective.     

Forces Involved in Soy Protein Gelation: Effects of Various Reagents on the Formation, Hardness and Solubility of Heat-Induced Gels Made from 7S, 11S, and Soy Isolate

The effects of various reagents on the formation, hardness and solubility of heat-induced gels of soybean 7S, 11S globulins and isolate were studied. Gels were formed in 30 mM Tris HCl buffer (pH 8.0) with or without reagents by heating at 80°C for 30 min. The results indicated that electrostatic interactions and disulfide bonds are involved in the formation of 11S globulin gels; mostly hydrogen bonding in 7S globulin gels and hydrogen bonding and hydrophobic interactions in soy isolate gels. Analyses of the proteins solubilized from the gels indicated that the basic subunits of 11S globulin interact with 7S globulin in soy isolate gels. The contribution of certain acidic subunits to network formation in US soy isolate gels is limited     

大豆7S和11S球蛋白的结构和功能性质

主要介绍大豆７Ｓ和１１Ｓ球蛋白的结构和功能性质，大豆蛋白质各个成分的分子量有所不同，按超速离心分离系数可分为２Ｓ，７Ｓ１１Ｓ和１５Ｓ４个组份。７Ｓ组份占总蛋白质的３０．９％，它是由４种不同大豆蛋白民组成，１１Ｓ组份占总大豆蛋白质的４１％，而且都是单一的１１Ｓ球蛋白，１１Ｓ球蛋白的等电点为ｐＨ４．６４。     

Legume seed vicilins (7S storage proteins) interfere with the development of the cowpea weevil (Callosobruchus maculatus (F))

Vicilins (7S storage proteins) isolated from the seeds of the legumes Vigna unguiculata (cowpea), Vigna angularis (adzuki bean), Canavalia ensiformis (jack bean), Glycine max (soybean), Phaseolus vulgaris (common bean) and Phaseolus lunatus (lima bean) were shown to be immunologically related and to bind to a chitin matrix. The effect of the isolated vicilins on the development of the cowpea weevil Callosobruchus maculatus was examined. Vicilins from all non-host seeds, including those of the C maculatus-resistant cowpea line IT81D-1045, strongly inhibited larval development (ED₅₀ of 1·07±0·32% to 2·22±0·64%). Vicilins from the C maculatus-susceptible cowpea CE-31 and adzuki bean seeds were the exception with ED₅₀ of 6·25±0·75% and 5·40±1·54%, respectively. These results correlate well with the host range of C maculatus and are possibly a reflection of the low digestibility of vicilins by insect midgut proteinases in addition to the ability they show in binding to chitin-containing structures like the ones found in the bruchid midguts. © 1998 SCI.     

蚕豆蛋白的提取分离及相对分子质量的测定

分析了蚕豆清蛋白和球蛋白的组成及其亚基的相对分子质量.测得蚕豆清蛋白经过SepharoseCL 6B柱层析可洗脱出2个组分,球蛋白可洗脱出3个组分.SDS PAGE分析表明,蚕豆球蛋白7种亚基的相对分子质量分别为:54000,41400,38000,33600,18300,17500,16700.蚕豆清蛋白5种亚基的相对分子质量分别为:59000,55700,42000,38000,18600.     

Biopolymers Produced by Cross-linking Soybean 11S Globulin with Whey Proteins using Transglutaminase

Heterogeneity of biopolymers was determined by cross-linking acetylated-11S acidic subunits (Ac-11S) of soy protein with α-lactalbumin and β-lactoglobulin. The extent of polymerization was determined by electrophoresis and HPLC. Differential scanning calorimetry (DSC) was used to determine thermal properties of starting proteins and biopolymers. HPLC data demonstrated the absence of biopolymers from Ac-11S, acetylated α-lactalbumin and acetylated β-lactoglobulin when each was incubated separately with transglutaminase (TG). However, Ac-11S formed biopolymers with α-lactalbumin and β-lactoglobulin when TG was added. TG catalyzed the formation of heterologous and homologous biopolymers from whey protein isolate (WPI) and soybean 11S globulin (11S). Cross-linking WPI and 11S provided biopolymers with improved heat stability which may be useful to provide functionality to food products.     

In vitro digestibility of processed and fermented soya bean,cowpea and maize

Tropical legumes, ie soya bean and cowpea, were pre‐treated and subsequently fermented using pure cultures of Rhizopus spp. Impact of soaking, cooking and fermentation of the legumes on their digestibility was determined using an in vitro digestion method. Processing of white maize included, amongst others, natural lactic acid fermentation, cooking and saccharification using barley malt. An in vitro method was standardised to carry out comparative determinations of the dry matter digestibility of cereal and legume food samples as a function of processing conditions, without attempting to exactly mimic gastrointestinal digestion. Using this method based on upper digestive tract digestion, it was observed that digestibility of the legumes increased during cooking and fermentation. Cooking improved the total digestibility of both soya bean and cowpea from 36.5 to 44.8% and from 15.4 to 40.9% respectively. Subsequent fungal fermentation increased total digestibility only by about 3% for both soya bean and cowpea. Digestibility was also influenced by fungal strain and fermentation time. Cooking and subsequent saccharification using malt almost tripled total digestibility of white maize from 25.5 to 63.6%, whereas lactic fermentation of maize had no effect on in vitro dry matter digestibility. Although total digestibility of cooked legumes was only slightly improved by mould fermentation (3% for both soya bean and cowpea), the level of water‐soluble dry matter of food samples increased during fermentation with Rhizopus oryzae from 7.0 up to 27.3% for soya bean and from 4.3 up to 24.1% for cowpea. These fermented products could therefore play a role as sources of easily available nutrients for individuals suffering from digestive disorders. © 2000 Society of Chemical Industry