Effect of Transglutaminase on Protein Electrophoretic Pattern of Rice,Soybean, and Rice‐Soybean Blends

The interactions taking place in composite dough containing rice flour and soybean proteins (5% w/w) in the presence of transglutaminase, an enzyme with cross‐linking activity, were studied using different electrophoretic analyses. The interaction between rice proteins and soybean proteins was intensified by the formation of new intermolecular covalent bonds catalyzed by transglutaminase and the indirect formation of disulfide bonds among proteins. The main protein fractions involved in those interactions were both β‐conglycinin and glycinin of soybean and the glutelins of the rice flour, although albumins and globulins were also cross‐linked. The addition of soybean proteins to rice flour improves the amino acid balance and they also might play an important role on the rice dough properties because soybean proteins interact with rice proteins, yielding protein aggregates of high molecular weight.     

Conjugation of sodium caseinate and gum arabic catalyzed by transglutaminase

Conjugation of the milk protein sodium caseinate and a protein-containing polysaccharide, gum arabic, was achieved through the use of the cross-linking enzyme transglutaminase. The extent of conjugation was monitored by size exclusion separation coupled with a multiangle laser light scattering detector. The elution times of gum arabic solutions incubated with transglutaminase were unchanged over time, whereas incubation of sodium caseinate with transglutaminase resulted in shorter elution times as reaction time increased, indicating the formation of cross-linked caseinate polymers. However, when mixtures of caseinate and gum arabic were incubated with transglutaminase, the elution times were decreased markedly, indicating conjugation between the protein and polysaccharide. The molecular masses of the conjugates increased from approximately 950 to 1600 kDa. This method of protein-polysaccharide conjugation offers noticeable advantages over previously used methods, and the conjugates produced may exhibit unique functional properties.     

Biochemical Analysis and Rheological Properties of Gluten Modified by Transglutaminase

A transglutaminase from Streptoverticillium sp. was used to create new covalent intermolecular cross‐links between proteins in gluten. This modification induced drastic changes in its physicochemical properties as well as in its rheological behavior. To understand these changes, we characterized the gluten extractability in acetic acid and identified the proteins of supernatant and pellet by immunoblotting using antibodies specific for each prolamin class. The proportion of soluble proteins decreased drastically after transglutaminase treatment due to the formation of large insoluble polymers as shown by SDS‐PAGE. Among the constitutive proteins of gluten, the high molecular weight glutenin subunits were the most affected in the transglutaminase reaction. The rheological behavior of gluten after 18 hr of incubation with transglutaminase was studied in shear by dynamic measurements over 10^‐3 – 10¹ Hz frequency range and by creep and recovery tests. The behavior of treated glutens remained that of a transient network, but the viscoelastic response was shifted toward shorter times and the steady‐state viscosity was greatly increased. The enzymatic treatment caused a considerable reinforcement of the network. The modified glutens were also less sensitive to thermal processing than unmodified glutens, as shown by a lower amplitude of variation of storage modulus G′ with temperature after enzymatic treatment.     

Purification and substrate specificity of transglutaminases from blood and Streptoverticillium mobaraense.

A procedure for a fast and simple purification of bovine plasma transglutaminase was developed, which resulted in a homogeneous enzyme preparation. Two different procedures were developed for the purification of pig erythrocyte transglutaminase, both of which resulted in partial purification. Both enzymes were used in cross-linking reactions of alpha-lactalbumin, beta-lactoglobulin, bovine serum albumin, casein, hemoglobin, glycinin, and myosin. The substrate specificity was compared to that of bacterial transglutaminase isolated from Streptoverticillium mobaraense. The bacterial transglutaminase caused cross-linking of a wider range of proteins and, thus, exhibited a lower substrate specificity than the blood transglutaminases. In addition, differences exist in the necessity of the addition of reducing agents. These differences allow specific applications of blood and bacterial transglutaminases at protein cross-linking in single or complex protein systems.     

Phaseolin in vitro pepsin digestibility: role of acids and phenolic compounds

Great Northern bean (Phaseolus vulgaris L.) phaseolin proteolysis at 37 degrees C, varying HCl concentrations (10 mM to 1 M), phaseolin:pepsin ratios ranging from 5:1 to 100:1 (w/w), and incubation times up to 24 h was investigated. The results suggest that phaseolin is not resistant to in vitro pepsin hydrolysis. At a phaseolin-to-pepsin ratio of 100:1 (w/w), native phaseolin was completely digested in 24 h when incubated in 50 mM HCl, while heat-denatured phaseolin (30 min at 100 degrees C, boiling water bath) was digested in 1 h under similar conditions. When incubated at 37 degrees C for 24 h, acid alone, even at as low a concentration as 10 mM, caused a partial breakdown of native phaseolin. The degree of phaseolin hydrolysis by HCl was dependent on the acid concentration used. The rate of native phaseolin hydrolysis increased with increasing HCl concentration rather than pepsin concentration. Common food acids were able to partially hydrolyze phaseolin. Among the food acids tested, oxalic acid was the most effective in hydrolyzing phaseolin. Spectroscopic studies revealed a significant change in secondary and tertiary structures when native phaseolin was incubated in dilute HCl. None of the tested phenolic compounds adversely affected phaseolin hydrolysis by pepsin.     

Extraction and characterization of Foeniculum vulgare pectins and their use for preparing biopolymer films in the presence of phaseolin protein

Pectins from Foeniculum vulgare were extracted under acidic conditions. The obtained pectins were mainly composed of uronic acid but also contained traces of rhamnose, galactose, and arabinose. Extracted pectins were used as a carbohydrate source to prepare biopolymer films in the absence and in the presence of phaseolin protein. The swelling characteristics of the films were examined as a function of ionic strength, pH, and the applied osmotic stress. The swelling behavior was dominated by a Donnan-type effect, which decreases with increasing ionic strength and counterion valency. In all cases the swelling of films containing phaseolin was reduced, suggesting a network formation between protein and pectins. Mechanical property studies have also estimated the validity of the obtained novel biopolymer films in terms of mechanical resistance.     

Susceptibility of phaseolin to in vitro proteolysis is highly variable across common bean varieties (Phaseolus vulgaris)

A study was conducted to investigate the amino acid (AA) composition and the susceptibility to in vitro proteolysis (pepsin, 120 min and pancreatin, 240 min) of a collection of purified phaseolins ( n = 43) in unheated or heat-treated form. The AA composition of phaseolin varied little across bean varieties. At 360 min of in vitro proteolysis, the degree of hydrolysis varied from 11 to 27% for unheated and from 57 to 96% for heated phaseolins ( P < 0.001). Heat treatment markedly increased the susceptibility of phaseolin to proteolysis ( P < 0.001). The AA scores (AAS) and the protein digestibility corrected for AAS indicated S-containing AA as the limiting AA (39 +/- 3 and 30 +/- 5%, respectively). In conclusion, susceptibility to proteolysis of heat-treated phaseolin rather than its AA composition affects the nutritional value of phaseolin estimated in vitro. Therefore, it should be the criterion of choice in breeding programs aimed at improving the nutritional value of common beans for humans.     

Wild-weed-crop complexes of common bean (Phaseolus vulgaris L., Fabaceae) in the Andes of Peru and Colombia, and their implications for conservation and breeding

During germplasm explorations carried out in Peru and Colombia, interbreeding complexes of wild and cultivated common bean were observed in both countries, eight in Apurimac and Cusco departments of Peru and eight in Cundinamarca and Boyaca´ departments of Colombia. The existence of complexes was evidenced both by segregation of wild and cultivated morphological traits in certain populations, and by the presence of genetically stabilized weedy types which were assumed to have arisen from past hybridization. Observations on phaseolin seed protein confirmed that genetic exchange was occurring. Phaseolin types introduced from other regions were in incipient stages of introgression into local populations. On the other hand, local phaseolin types were observed in all phases of the complexes from totally wild to fully cultivated beans, suggesting that the complexes had undergone a long evolution. Complexes could be an effective means to generate genetic variability, introgressing genes from wild populations into cultivated types and complementing modern plant breeding programs. The conservation of such complexes depends on the continued existence of the wild, weedy and cultivated beans in close proximity; on the maintenance of a semi-domesticated environment; and on the willingness of farmers to leave weedy types in the field.     

A note on the variation of phaseolin among common bean landraces collected in Eastern Asia

Summary Phaseolin seed protein was used as a marker to reveal the origin, Mesoamerican or Andean, of common bean (Phaseolus vulgaris) accessions cultivated in some countries (Korea, China, and Japan) of eastern Asia. Andean cultivars (T and C phaseolin patterns) are predominant in this geographical area. Introductions from Middle America, represented by cultivars with S phaseolin type, also occurred at lower rate. In all cases, genotypes with larger seeds have been favoured.     

Physicochemical and structural properties of oat globulin polymers formed by a microbial transglutaminase

Oat globulin was polymerized by a microbial transglutaminase (TG), and some physicochemical and functional properties of polymers were studied. Reversed-phase HPLC revealed that the number of epsilon-(gamma-glutamyl) lysine isopeptide bonds formed after 4 h of enzyme incubation was 2.21 micromol/g of protein. SDS-PAGE showed that the oat globulin acidic polypeptides (AP) were more susceptible to polymerization than the basic polypeptides (BP), and the reactivities of both AP and BP were enhanced by the addition of other substrate proteins. Differential scanning calorimetry showed that both the denaturation temperature and denaturation enthalpy were decreased after TG treatment. Fourier transform infrared spectroscopy revealed marked increases in the intensity of two intermolecular beta-sheet bands associated with aggregate formation but little conformational changes in the polymerized protein. TG incubation led to progressive changes in flow properties of oat globulin dispersions, indicating enhanced pseudoplasticity and increased viscosity and yield stress.     

Charge modifications to improve the emulsifying properties of whey protein isolate

Whey protein isolate was modified by ethylene diamine in order to shift its isoelectric point to an alkaline pH. The extent of the modification was studied using SDS-PAGE and MALDI-TOF mass spectrometry. The modified whey proteins were used as an emulsifier to stabilize oil-in-water emulsions at acidic and neutral pH ranges, and their emulsifying properties were compared with that of the unmodified whey proteins and with the previously studied ethylene diamine modified sodium caseinate. The emulsifying activity of the modified whey proteins was similar to that of the unmodified ones, but the stability of an emulsion at pH 5 was significantly improved after the modification. Charge and coverage of droplet surface and the displacement of the interfacial proteins by surfactant Tween 20 were further studied as a function of pH. As compared with the unmodified whey proteins, the modified ones were proven to cover the interface more efficiently with extensive surface charge at pH 5, although the interfacial layer was less resistant to the surfactant displacement.     

Enzyme-aided modification of chicken-breast myofibril proteins: effect of laccase and transglutaminase on gelation and thermal stability

The effect of laccase and transglutaminase (TG) on cross-linking, gelation, and thermal stability of salt-soluble chicken-breast myofibril proteins was investigated at pH 6. Both enzymes modified the protein pattern detected by SDS-PAGE. Identification of proteins by peptide mass mapping showed that myosin heavy chain (MHC) and troponin T were the most affected proteins. These proteins faded or disappeared as a function of the incubation time with both enzymes on SDS-PAGE. The molecular weight of actin was not, however, affected by either enzyme. The effects that the enzymes had on the gel formation of chicken-breast myofibrils were studied in 0.35 and 0.60 M NaCl solutions at 3% protein content and a constant temperature of 40 degrees C by using a small deformation viscoelastic measurement. TG substantially increased the storage modulus (G') of 3% protein in 0.35 M NaCl. Without the enzymes, gelation was insignificant in 0.35 M NaCl. The increased solubility of the proteins at 0.60 M NaCl intensified gelation with TG. G' increased 32 and 64% at dosages of 10 and 100 nkat of TG, respectively. Also, laccase increased G' of the gel in 0.60 M salt concentration. However, a high laccase dosage decreased the magnitude of G' below the control level. Differential scanning calorimetric (DSC) measurements indicated slightly reduced myosin heat stability after TG pretreatment and increased actin heat stability with both enzymes. Maximum transition temperatures did not alter with either enzyme.     

Analysis of the contribution of Mesoamerican and Andean gene pools to European common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) germplasm and strategies to establish a core collection

Common bean (Phaseolus vulgaris L.) was introduced in Europe from both Mesoamerican and Andean centres of origin. In this study, a collection including 544 accessions from all European regions showed that the Andean phaseolin types ‘T’ (45.6%) and ‘C’ (30.7%) prevailed over the Mesoamerican ones ‘S’ (23.7%), and accessions with cuboid seed shape (34.9%), maroon coat darker colour seed (44.3%), uniform seed colour (69.6%) were the most frequent. European accessions with phaseolin ‘S’ showed a significantly larger average seed size compared to those from America in the same phaseolin class while those presenting ‘T’ and ‘C’ phaseolin did not. This suggests that, during crop expansion in Europe, sampling or selection favoured the large-seeded races within the Mesoamerican ‘S’ gene pool or, possibly, introgression from Andean germplasm did occur. A core collection was developed using sampling approaches based on the information available in the genebank databases and on phaseolin patterns. Four sampling strategies were used: simple random sampling, and three random-stratified samplings, by logarithm of frequency of accessions by country, by European region, and by phaseolin pattern, respectively. Two sampling strategies resulted in core collections significantly different for phaseolin electrophoretic patterns from the whole collection. Stratification by phaseolin patterns increased the frequency of ‘S’ types (‘C’ type = 33%, ‘T’ type = 5.7% and ‘S’ type = 31.3%). The core collections were validated using seven seed characters, and no significant difference was observed in all strategies. This first developed European bean core collection will help to assess the contribution of the two American gene pools to the European germplasm and their relative importance for breeding purposes.     

Cross-linking and rheological changes of whey proteins treated with microbial transglutaminase

Modification of the functionality of whey proteins using microbial transglutaminase (TGase) has been the subject of recent studies. However, changes in rheological properties of whey proteins as affected by extensive cross-linking with TGase are not well studied. The factors affecting cross-linking of whey protein isolate (WPI) using both soluble and immobilized TGase were examined, and the rheological properties of the modified proteins were characterized. The enzyme was immobilized on aminopropyl glass beads (CPG-3000) by selective adsorption of the biotinylated enzyme on avidin that had been previously immobilized. WPI (4 and 8% w/w) in deionized water, pH 7.5, containing 10 mM dithiothreitol was cross-linked using enzyme/substrate ratios of 0.12-10 units of activity/g WPI. The reaction was carried out in a jacketed bioreactor for 8 h at 40 degrees C with continuous circulation. The gel point temperature of WPI solutions treated with 0.12 unit of immobilized TGase/g was slightly decreased, but the gel strength was unaffected. However, increasing the enzyme/substrate ratio resulted in extensive cross-linking of WPI that was manifested by increases in apparent viscosity and changes in the gelation properties. For example, using 10 units of soluble TGase/g resulted in extensive cross-linking of alpha-lactalbumin and beta-lactoglobulin in WPI, as evidenced by SDS-PAGE and Western blotting results. Interestingly, the gelling point of WPI solutions increased from 68 to 94 degrees C after a 4-h reaction, and the gel strength was drastically decreased (lower storage modulus, G'). Thus, extensive intra- and interchain cross-linking probably caused formation of polymers that were too large for effective network development. These results suggest that a process could be developed to produce heat-stable whey proteins for various food applications.     

Production and characterization of rabbit polyclonal antibodies to almond (Prunus dulcis L.) major storage protein.

Rabbits were immunized with purified almond major protein (AMP), the primary storage protein in almonds. Rabbit anti-AMP polyclonal antibodies (PA) could detect AMP when as little as 1-10 ng/mL were used to coat microtiter plates in a noncompetitive enzyme linked-immunosorbent assay (ELISA). Competitive inhibition ELISA assays detected the AMP down to 300 ng/mL. PA recognized the AMP in protein extracts from all U.S. major marketing cultivars of almonds (Mission, Neplus, Peerless, Carmel, and Nonpareil) with specific reactivity of 52.6-75% as compared to that of the AMP alone. Immunoreactivity of protein extracts prepared from commercial samples of blanched almonds, roasted almonds, and almond paste was respectively reduced by 50.0%, 56.6%, and 68.4% (noncompetitive ELISA) when compared to the immunoreactivity of the AMP. Moist heat (121 degrees C, 15 min) pretreatment of the AMP reduced the PA reactivity by 87% (noncompetitive ELISA). Exposing AMP to pH extremes (12.5 and 1.5-2.5) caused a 53% and 57% reduction in PA reactivity, respectively (noncompetitive ELISA). PA showed some cross-reactivity with the cashew major globulin, and to a lesser extent, the Tepary and Great Northern bean major storage protein (7S or phaseolin). The presence of almonds in a commercial food was detected using PA in a competitive ELISA.     

Calcium ions make phytohemagglutinin resistant to trypsin proteolysis

To investigate the mechanism of phytohemagglutinin (PHA) susceptibility or resistance to the action of proteolytic enzymes, its in vitro proteolysis by trypsin was studied. It was found that Ca (2+) gives resistance to the native PHA molecule to trypsin proteolysis. In the absence of Ca (2+) trypsin performs a thorough hydrolysis of PHA. At the first stage of trypsin hydrolysis of PHA the formation of a relatively stable high molecular mass product occurs (PHA-T) as a result of non-co-operative proteolysis. At the second stage, the degradation of PHA-T occurs, and this degradation is performed by parallel co-operative proteolysis. This type of proteolysis differs from the action of trypsin on phaseolin, the main storage protein from common bean ( Phaseolus vulgaris L.). The implications of Ca (2+)influence of PHA hydrolysis by trypsin are discussed.     

The common bean populations from Basilicata (Southern Italy). An evaluation of their variation

The variation within a collection constituted by 36 populations of common bean (Phaseolus vulgarisL.) recently collected in Basilicata region (Southern Italy) was studied. These populations are cultivated in marginal areas of the region mainly for farms' self-consumption. An appreciable variation of seed shape, colour and type of pattern was observed; 34 populations are of climbing growth habit. The frequency of the phaseolin types within the collection was investigated. C, T and S phaseolin types were observed, the type C being predominant (18 of 36 landraces); intra-population variation was detected only for two landraces. Local populations were compared with 28 commercial cultivars widely grown in the Basilicata region and accounting for 80–90% of common bean production. Significant divergences in growth habit and frequency of phaseolin types were observed: 22 cultivars had bush growth habit and 20 showed T phaseolin type. These results stress the urgency of actions devoted to the safeguard of this local germplasm. In fact, the diffusion of alloctonous and not typical germplasm is the first step towards the erosion of useful or valuable genes present in those established local populations.     

Can Phaseolin Patterns Help Resolve the Phaseolus–Vigna Complex?

Although common bean (Phaseolus vulgaris L.) is cultivated throughout India, the Himalayas hold largest diversity of bean germplasm. No studies on characterization of phaseolin types on this germplasm have been conducted earlier. In order to determine whether the common bean cultivars collected from various areas in the Northern Himalayas represent introductions from the Central American and Andean domestication centers or are local domesticates, we have analyzed the electrophoretic variation (SDS-PAGE) of phaseolin types in several bean accessions. A few species of Vigna were also included in this study to determine whether phaseolin (vignin in Vigna) patterns can be used to resolve the Phaseolus–Vigna complex. The present investigation on phaseolin (globulin) patterns of Phaseolus vulgaris and Vigna spp. clearly shows much variability in globulin patterns. Three new types of phaseolin patterns were recorded. An attempt to resolve phylogenetic problems in this complex was made using the phaseolin data.     

Transglutaminase catalyzed cross-linking of sodium caseinate improves oxidative stability of flaxseed oil emulsion

Sodium caseinate was modified by transglutaminase catalyzed cross-linking reaction prior to the emulsification process in order to study the effect of cross-linking on the oxidative stability of protein stabilized emulsions. The extent of the cross-linking catalyzed by different dosages of transglutaminase was investigated by following the ammonia production during the reaction and using SDS-PAGE gel. O/W emulsions prepared with the cross-linked and non-cross-linked sodium caseinates were stored for 30 days under the same conditions. Peroxide value measurement, oxygen consumption measurement, and headspace gas chromatography analysis were used to study the oxidative stability of the emulsions. The emulsion made of the cross-linked sodium caseinate showed an improved oxidative stability with reduced formation of fatty acid hydroperoxides and volatiles and a longer period of low rate oxygen consumption. The improving effect of transglutaminase catalyzed cross-linking could be most likely attributed to the enhanced physical stability of the interfacial protein layer against competitive adsorption by oil oxidation products.     

Substrate specificities of microbial transglutaminase for primary amines

Transglutaminase (epsilon-glutaminyl-peptide:amine gamma-glutaminyl-transferase, EC 2.3.2.13) (TGase) is an enzyme that catalyzes acyl transfer reactions between primary amines and Gln residues in proteins and peptides. The substrate specificity of TGase for primary amines was investigated to incorporate various functional groups into proteins and peptides. In this study, microbial transglutaminase and guinea pig liver transglutaminase were used. For the primary amines to be incorporated into benzyloxycarbonyl-L-Gln-Gly (Z-Gln-Gly), they were required to have more than four carbon chains without side chains between the functional groups. These results suggest that with appropriate primary amines as spacers, various functional groups, carboxyl groups, phosphate groups, saccharides, and so on, can be incorporated into proteins by using TGase.