Thermal aggregation,amino acid composition and in vitro digestibility of vicilin-rich protein isolates from three Phaseolus legumes: A comparative study

The heat-induced aggregation and the in vitro digestibility of vicilin-rich protein isolates from three Phaseolus legumes, including kidney bean, red bean and mung bean were investigated and compared, and their amino acid composition and free sulfhydryl (SH) group contents also evaluated. The results showed that the extent in the heat-induced aggregation varied with the type of the protein isolates, and the formation of new disulphide bonds (at the expense of free SH contents) was involved in the formation of the aggregates. The protein isolates with higher levels of hydrophobic and uncharged polar amino acids, and lower basic amino acid contents exhibited lower extent of their heat-induced aggregation. The in vitro pepsin plus trypsin digestibility was different for various native protein isolates. The digestibility was to a varying extent affected by the heat treatment. The influences of heating on the digestibility of these proteins were closely related to the extent of their heat-induced aggregation. The results suggest that the improvement of nutritional property of those vicilin-rich protein isolates by heat treatment is largely dependent upon their amino acid composition as well as the extent of heat-induced aggregation.     

Thermal properties of Phaseolus angularis (red bean) globulin

The thermal properties of Phaseolus angularis (red bean) globulin were studied by differential scanning calorimetry under various medium conditions. Red bean globulin (RGB) was fractionated by ion-exchange chromatography into a major fraction, with SDS–PAGE pattern corresponding to the 7S vicilin, and two minor fractions, probably representing residual vicilin and the 11S legumin, respectively. The thermogram of RBG showed a major endothermic peak at 86.4°C and a minor transition at 92.2°C. Vicilin exhibited two endothermic peaks (87.7 and 94.1°C), while legumin showed one transition at 89.5°C. The progressive increase in denaturation temperature (T_d) with increase in salt concentration, suggests a more compact conformation for RBG with higher thermal stability. Decreases in enthalpy and T_d were observed under the influence of highly acidic and alkaline pHs, chaotropic salts, and protein perturbants such as sodium dodecyl sulfate, urea and ethylene glycol, indicating partial denaturation and decrease in thermal stability. Dithiothreitol and N-ethylmaleimide have little effect on the thermal properties of RBG since the major protein component, vicilin, is devoid of disulfide bonds.     

Functional properties and in vitro trypsin digestibility of red kidney bean (Phaseolus vulgaris L.) protein isolate: Effect of high-pressure treatment

The effects of high-pressure (HP) treatment at 200–600 MPa, prior to freeze-drying, on some functional properties and in vitro trypsin digestibility of vicilin-rich red kidney bean (Phaseolus vulgaris L.) protein isolate (KPI) were investigated. Surface hydrophobicity and free sulfhydryl (SH) and disulfide bond (SS) contents were also evaluated. HP treatment resulted in gradual unfolding of protein structure, as evidenced by gradual increases in fluorescence strength and SS formation from SH groups, and decrease in denaturation enthalpy change. The protein solubility of KPI was significantly improved at pressures of 400 MPa or higher, possibly due to formation of soluble aggregate from insoluble precipitate. HP treatment at 200 and 400 MPa significantly increased emulsifying activity index (EAI) and emulsion stability index (ESI); however, EAI was significantly decreased at 600 MPa (relative to untreated KPI). The thermal stability of the vicilin component was not affected by HP treatment. Additionally, in vitro trypsin digestibility of KPI was decreased only at a pressure above 200 MPa and for long incubation time (e.g., 120 min). The data suggest that some physiochemical and functional properties of vicilin-rich kidney proteins can be improved by means of high-pressure treatment.     

Thermal analysis of flaxseed (Linum usitatissimum) proteins by differential scanning calorimetry

The thermal properties of flaxseed (whole or dehulled), dehulled and delipidated meal, as well as proteins extracted and isolated from the meal, were investigated by differential scanning calorimetry (DSC). A high denaturation temperature (T_d) of 114.7 °C was observed for the major fraction of flaxseed protein isolated by anion-exchange chromatography, representing the 11–12 S storage globulin. Marked decreases in T_d and enthalpy were observed at pH 3 compared with pH of 5 or higher, while the presence of high salt (1.0 M NaCl) resulted in higher thermal stability, enthalpy and greater cooperativity of the transition. Thermal analysis of the major fraction in the presence of chaotropic salts or protein structure perturbants (sodium dodecyl sulfate, urea, dithiothreitol, N-ethylmaleimide, ethylene glycol) suggested the contributions of hydrophobic and ionic interactions, hydrogen bonding as well as disulfide linkages or disulfide–sulfhydryl (SS–SH) interactions, to the thermal stability of flaxseed protein.     

Characterization of protein isolates from different Indian chickpea (Cicer arietinum L.) cultivars

Protein isolates prepared by alkaline solubilization followed by isoelectric precipitation and freeze drying from desi (PBG-1, PDG-4, PDG-3, GL-769, and GPF-2) and kabuli (L-550) chickpea cultivars were evaluated for functional (water and oil absorption capacities, least gelation concentration, foaming capacity and stability) and thermal properties. Significant difference (P ? 0.05) in properties of kabuli and desi chickpea protein isolates was observed. Kabuli chickpea protein isolate showed significantly (P ? 0.05) higher ash (1.14%), protein (94.4%), L^∗, ΔE value, oil absorption capacity (OAC) and lower water absorption capacity (WAC) than their corresponding desi chickpea protein isolates. The solubility-pH profile of different protein isolates showed minimum solubility in the pH between 4.0 and 5.0 and two regions of maximum solubility at pH 2.5 and 7.0. Foaming capacity of all protein isolates increased with the increase in concentration. Kabuli chickpea protein isolate showed the highest foam stability (94.7%) after 120 min of storage. The thermal properties of protein isolates from different chickpea cultivars were studied by differential scanning calorimetry (DSC). Protein isolates from both the chickpea types differed significantly (P ? 0.05) in peak denaturation temperature (T_d) and heat of transition (ΔH). Kabuli type protein isolate exhibited lower T_d and ΔH value as compared to those from desi types. The interrelationships between characteristics of protein isolates showed a significant (P ? 0.05) negative correlation of T_d with protein content and OAC. It was also observed that cultivars with high fat content had high ΔH and lower WAC.     

Study of Thermal Properties of Oat Globulin by Differential Scanning Calorimetry

The thermal behavior of oat globulin was studied by differential scanning calorimetry (DSC). The effects of pH, salts, and of various structure perturbants upon thermal characteristics were determined. Raising or lowering pH from near neutrality reduced denaturation temperature. (T_d), enthalpy (ΔH) and cooperativity indicated by increase in width at half height (ΔT_1/2). The effect of salts on thermal stability was related to their position in the lyotrophic series and suggests involvement of hydrophobic interaction in the thermal stability of oat globulin. Increasing concentrations of urea progressively lowered T_d and ΔH and increased ΔT_1/2; sodium dodecyl sulfate (SDS) lowered ΔH without affecting T_d; ethylene glycol (EC) lowered T_d without changing ΔH. Dithiothreitol did not affect DSC characteristics suggesting that disulfide bonds do not contribute to the thermal response of oat globulin.     

Effects of transglutaminase treatment on the thermal properties of soy protein isolates

The effects of covalent cross-linking of microbial transglutaminase (MTGase) on the thermal properties of soy protein isolates (SPI), including the thermal denaturation and glass transition were investigated by conventional and modulated differential scanning calorimetry (DSC). The MTGase treatment significantly increased the thermal denaturation temperatures (including the on-set temperature of denaturation, T_m and the peak temperature of denaturation, T_d) of glycinin and β-conglycinin components of SPI (P ⩽ 0.05), and the thermal pretreatment of SPI further increased the extent of this improvement. The MTGase treatment also improved the ability of SPI to resist the urea-induced denaturation. Modulated DSC analysis showed that there were two glass transition temperatures (T_g) in the reversible heat flow signals of native SPI (about 5% moisture content), approximately corresponding to 45 and 180 °C, respectively. These T_g values of SPI were significantly decreased by the MTGase treatment (at 37 °C for more than 2 h) (P ⩽ 0.05). The improvement in the hydration ability of protein and the formation of high molecular biopolymers may account for the changes of thermal properties of soy proteins caused by the MTGase cross-linking.     

Comparative study of the composition and thermal properties of protein isolates prepared from nine Phaseolus vulgaris legume varieties

Beans contain high amounts of protein and, as demonstrated with other legumes, may contain peptides with bioactive properties. Dozens of dry bean (Phaseolus vulgaris) varieties are grown in Canada, and many vary not only in terms of their protein content but also in their composition. In this study, the composition, molecular structure, and thermal properties of nine varieties of P. vulgaris were studied using electrophoresis, reversed-phase high-performance liquid chromatography (RP-HPLC), differential scanning calorimetry (DSC), and Fourier-transform infrared spectroscopy (FTIR). The predominant protein observed in each variety was vicilin. RP-HPLC chromatogram showed protein isolates (PIs) of white, great northern and black beans to have different profiles of low-molecular-weight proteins. Additionally, black bean PIs were missing proteins in the molecular mass (MM) range of 60 to 97 kDa. DSC analysis showed cranberry and light red kidney bean PIs to have significantly lower denaturation temperatures compared to the other varieties. RP-HPLC chromatograms of cranberry and light red kidney bean PIs were also very different. FTIR spectroscopy showed the predominant secondary structures in all varieties to be ??-sheets and random coils. Increasing the temperature from 25 °C to 95 °C resulted in a reduction in ??-sheet structures and an increase in the content of random coils and antiparallel ??-sheet structures. Overall there were no major dissimilarities observed in the FTIR profiles of the nine P. vulgaris varieties.     

Molecular forces involved in heat-induced pea protein gelation: Effects of various reagents on the rheological properties of salt-extracted pea protein gels

The molecular forces involved in the gelation of heat-induced pea protein gel were studied by monitoring changes in gelation properties in the presence of different chemicals. At 0.3 M concentration, sodium thiocyanate (NaSCN) and sodium chloride (NaCl) showed more chaotropic characteristic and enhanced the gel stiffness, whereas sodium sulfate (Na₂SO₄) and sodium acetate (CH₃COONa) stabilized protein structure as noted by increasing denaturation temperatures (T_d) resulting in reduced storage moduli (G′). To determine the involvement of non-covalent bonds in pea protein gelation, guanidine hydrochloride (GuHCl), propylene glycol (PG), and urea were employed. The significant decrease in G′ of pea protein gels with the addition of 3 M GuHCl and 5 M urea indicated that hydrophobic interactions and hydrogen bonds are probably involved in pea protein gel formation. The increase in G′ with increasing PG concentration (5–20%), demonstrated hydrogen bonds and electrostatic interaction involvement. No significant influence was observed on G′ with addition of different concentrations of β-mercaptoethanol (2-ME), low levels of dithiothreitol (DTT), and up to 25 mM N-ethylmaleimide (NEM), which indicated that disulfide bonds are not required for gel formation, but data at higher DTT and NEM concentrations and slow cooling rates showed a minor contribution by disulfide bonds. Reheating and recooling demonstrated that gel strengthening during the cooling phase was thermally reversible but not all the hydrogen bonds disrupted in the reheating stage were recovered when recooled.     

Thermal properties of globulin from rice (Oryza sativa) seeds

The thermal properties of rice (Oryza sativa) seed globulin were studied by differential scanning calorimetry (DSC) under the influence of various medium conditions. The denaturation temperatures (T_d) of crude and purified rice globulin were 97.6 and 98.5 °C, respectively. Increasing salt concentration enhanced thermal stability of rice globulin. The effect of chaotropic anions on DSC characteristics of rice globulin followed the order of the lyotropic series indicating progressive decreases in both T_d and enthalpy (ΔH). Extreme pHs and protein perturbants (sodium dodecyl sulfate at above 20 mM, dithiothreitol, urea and ethylene glycol) caused marked protein denaturation and loss of thermal stability. In contrast, N-ethylmaleimide and SDS at below 20 mM exerted a stabilizing effect on rice globulin. Pre-heat treatments led to partial unfolding of rice globulin, and the level of denaturation increased progressively with increasing heating time.     

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.     

A comparison between the properties of seed,starch, flour and protein separated from chemically hardened and normal kidney beans

The physicochemical, functional and thermal properties of starch, flour and protein isolates obtained from chemically hardened kidney beans were evaluated. A rapid chemical hardening procedure (soaking in acetate buffer, pH = 4.0, 37 °C, 6 h) was used to produce hardened kidney beans. Chemical hardening altered physicochemical, cooking, hydrating and textural properties of beans to a significant level (P < 0.05). Soaked and cooked chemically hardened beans had a higher value for different textural parameters than their normal counterparts. Chemical hardening increased cooking time (from 49 to 123 min) of beans and decreased swelling power and solubility of starch. The turbidity value of gelatinized starch suspensions from chemically hardened beans was significantly lower than that from normal beans. Chemical hardening of beans caused significant increase in transition temperatures (T_o, T_p, T_c) and enthalpy of gelatinization (ΔH_gel) of both starch and flour. Chemically hardened bean starch showed significantly higher pasting temperature (94.9 °C) as compared to normal bean starch (83.2 °C). Flour and protein isolate from chemically hardened beans showed significantly lower water absorption, oil absorption, foaming capacity and gelling ability than those from normal beans. The onset temperature (T_m), peak denaturation temperature (T_d) and heat of transition or enthalpy (ΔH) of protein isolate from hardened and normal beans did not differ significantly. Copyright © 2007 Society of Chemical Industry     

Thermal properties of buckwheat proteins as related to their lipid contents

The thermal properties of buckwheat proteins (BP) as related to their lipid contents were studied using differential scanning calorimetry (DSC). BP samples with three levels of lipid content (2.5%, 6.5% and 17.8%, w/v) were obtained by selecting different extraction and de-fatting techniques. In the DSC thermograms of all BPs, there were two observable endothermic peaks with similar denaturation temperatures of about 80 and 102 °C, corresponding to the 8S and 13S globulins, respectively. The enthalpy changes (ΔH) of buckwheat globulins are also unaffected by the presence of lipids (2.5–17.8%), however, the width at half peak height (ΔT_1/2) presenting the cooperativity of the transition from native to denatured state was related to the lipid content. DSC analyses in the presence of some protein perturbants (e.g., urea, SDS and DTT) showed that the structure of buckwheat globulins (especially 13S globulins) are mainly maintained by hydrogen bonding and hydrophobic interactions, and the presence of lipids may disturb the hydrophobic interactions of these globulins. The disulfide bonds only pay an important role in those globulins with high (17.8%) or low (2.5%) lipid contents. These results suggest that the presence of lipid affect the thermal properties of buckwheat globulins, especially the ability to resist the denaturant-induced denaturation, and a suitable amount of lipids be favorable for the maintenance of native protein conformation of 13S globulins.     

Physicochemical, functional and structural properties of vicilin-rich protein isolates from three Phaseolus legumes: Effect of heat treatment

The physicochemical, functional and structural properties of vicilin-rich protein isolates from kidney, red and mung beans (KPI, RPI and MPI) were investigated, and the influences of heating (at 95 °C for 30 min) were also evaluated and compared. In the untreated samples, the thermal stability, free SH contents and surface hydrophobicity were different. The differences seemed to be closely related to the differences in extent of aggregation and/or tertiary and secondary conformational structures among proteins. The heating resulted in extensive denaturation of the protein, significant decreases in free SH groups and increases in surface hydrophobicity, but to a varying extent, depending on the type of protein isolates. The protein solubility and emulsifying activities were significantly improved by the heating. The tertiary and secondary structures of these proteins were also to a various extent affected. The conformational structures of proteins in RPI were most flexible, and susceptible to the heating, while the proteins in KPI were most heat-stable in structures. The results clearly indicated close relationships between functional properties of these vicilin-rich protein isolates and their conformational structures.     

Thermal and surface behavior of yeast protein fractions from Saccharomyces cerevisiae

An easy and inexpensive method of fractionation of a yeast homogenate was proposed and it is based on differential centrifugation steps of insoluble components and subsequent precipitations of soluble fractions. In this fractionation, the effect of addition of protease inhibitor was studied. The procedure, which was performed in mild conditions in order to minimize protein denaturation, produced four fractions that proceed from distinct parts of the yeast cell and with a different chemical composition: Fr I, Fr II, Fr III and Fr IV. Thermal and surface behavior of these samples was also analyzed. Fr I and Fr II, mainly composed by cell wall debris and membrane cell components, respectively, exhibited an adsorption rate (Δγ/Δt^1/2) ten-fold higher than Fr III and Fr IV, composed by nucleoproteins and cytoplasmic proteins. All fractions exhibited a unique DSC endotherm with different peak temperature (T_p) and enthalpy values (ΔH). Fr IV exhibited the highest T_p value (74 °C) and less affected by inhibitor absence. Fr I and Fr II showed the highest ΔH values (27-47 J/g protein) but they were markedly affected reducing their enthalpy values and increasing their surface properties in absence of protease inhibitor.     

Conformational and thermal properties of phaseolin,the major storage protein of red kidney bean (Phaseolus vulgaris L.)

BACKGROUND: A previous study of various functional and physicochemical properties of phaseolin indicated good potential of phaseolin for application in food formulations in view of its excellent functional properties. The aim of the present study was to explore the conformational and thermal properties of phaseolin in the presence of protein structural perturbants by intrinsic fluorescence emission spectroscopy and differential scanning calorimetry. Raman spectroscopy was also used to characterise the secondary structures of phaseolin. RESULTS: The Raman spectrum of phaseolin indicated that β‐sheets and random coils were the major secondary structures. Intrinsic fluorescence emission spectroscopy confirmed the structural peculiarity and compactness of phaseolin, as evidenced by the absence of any shift in emission maximum (λ_max) in the presence of structural perturbants such as sodium dodecyl sulfate (SDS), guanidine hydrochloride, urea and dithiothreitol (DTT). Increasing NaCl concentration enhanced the thermal stability of phaseolin. Addition of chaotropic salts (1 mol L^?1) caused progressive decreases in thermal stability following the lyotropic series of anions. Decreases in thermal denaturation temperature (T_d) and enthalpy change (ΔH) were observed in the presence of protein perturbants such as SDS, urea and ethylene glycol, indicating partial denaturation and a decrease in thermal stability. DTT and N‐ethylmaleimide had little effect on the thermal properties of phaseolin, confirming that phaseolin, a 7S globulin, is devoid of inter‐polypeptide disulfide bonds. CONCLUSION: The data presented here demonstrate the contributions of hydrophobic and electrostatic interactions and hydrogen bonding to the conformational stability of phaseolin. Copyright © 2010 Society of Chemical Industry     

A DSC study on the effects of various maltodextrins and sucrose on protein changes in frozen‐stored minced blue whiting muscle

A DSC heat denaturation study on the effects of various maltodextrins and sucrose on protein changes in minced blue whiting muscle during frozen storage at −10 and −20 °C was carried out. All maltodextrins slowed down the decreases in the denaturation enthalpies (ΔH_d) ascribed to myosin and actin, making evident a noticeable effectiveness against protein denaturation, especially at −20 °C. Sucrose was as effective as maltodextrins at −20 °C, but was the least effective treatment at −10 °C. Significant correlations between both ΔH_ds and either protein solubility or formaldehyde production were found at each storage temperature. A low protein sensitivity to the small amounts of formaldehyde produced during the first weeks of storage and errors associated with the determination of enthalpies led to poorer correlations at −20 °C. Maximum denaturation temperatures (T_max) correlated with protein solubility only at −20 °C. No clear relationship between either T_max and the effectiveness of cryostabilisation was found, as T_max also depends on the effectiveness of the treatments against the thermal denaturation of proteins. © 2001 Society of Chemical Industry     

Studies on the thermal behaviour of pea (Pisum sativum)vicilin

The effect of heat on pea (Pisum sativum L) vicilin has been studied using differential scanning calorimetry (DSC), gel filtrtion chromatography and turbidimetry. By adjusting the variables of pII, ionic strength and protein concentration, two processes could be identified from the DSC thermograms: protein denaturation and protein aggregation. The results are discussed in terms of electrostatic and hydrophobic interactions. A mechanism for the thermal aggregation of pea vicilin is proposed.     

Studies of Thermal Denaturation of Oat Globulin by Differential Scanning Calorimetry

Thermal denaturation of oat globulin was studied by differential scanning calorimetry (DSC). Prior heat treatments at 100°C and 110°C resulted in a progressive decrease in enthalpy (ΔH) indicating partial denaturation. Marked increase in denaturation temperature (T_d) and onset temperature (T_m) and decrease in width at half peak height (ΔT_1/2) suggest that the preheated protein assumed a more compact conformation or associated to a complex structure with higher thermal stability and cooperativity. The heated globulin was segregated into soluble and insoluble fractions containing native and denatured protein respectively. The denaturation kinetics of oat globulin was studied and results show a reaction order of 2.5 and an activation energy of 505 KJ/mol. Heat treatments caused a pronounced increase in activation energy and pre-exponential factor.     

Thermal characteristics of flaxseed (Linum usitatissimum L.) proteins

Thermal characteristics of ion-exchange protein fractions from dehulled delipidated meal of four flaxseed cultivars grown at three locations were evaluated by differential scanning calorimetry (DSC) to examine genotypic and environmental effects. DSC was performed in a “dry” state on four, 0.28, 0.35, 0.45 and 0.50 M NaCl lyophilized protein fractions. Flaxseed proteins exhibited two thermal events between 83 and 115 °C with distinct shoulders present in the 0.45 and 0.50 M fractions of all cultivars. Cultivar and location effects were significant for the first thermal transition (_Td1$T_{{\mathrm{d}}_1}$) of the 0.28 and 0.35 M fractions, and the second transition (_Td2$T_{{\mathrm{d}}_2}$) of the 0.35, 0.45 and 0.50 M fractions. The enthalpy of the first transition ΔH₁ of the 0.35 and 0.50 M fractions was cultivar dependent, while that of the second transition ΔH₂ was cultivar and location specific for the 0.35, 0.45 and 0.50 M fractions. The 0.28 and 0.45 M fractions displayed the lowest and highest thermal transition temperatures, respectively. All protein fractions consisted of reversing and nonreversing thermal events as assessed by modulated DSC. Flaxseed cultivars, when grouped by thermal characteristics of protein fractions, can be differentiated by principal component analysis.