Physicochemical Modification Of Kidney Bean(Phaseolus Vulgaris L) Proteins And Its Related Structure-Function Mechanism

The aim of this study was to explore utilization means for proteins from red kidney bean (Phaseolus vulgaris L). Physiochemical and functional properties of phaseolin (the main storage globulin) and red kidney bean protein isolate (KPI) was studied and compared, the related mechanism was elucidated. Physiochemical modifications were used to modify KPI for improving its functional properties. The possible relationship between structure and functional properties of KPI was also discussed. Main results are as follows:Phaseolin showed excellent functional properties, its protein solubility (PS), emulsifying activity index (EAI), and gel-forming ability were much higher than those of KPI (P < 0.05). Differential scanning calorimetry analyses suggested that phaseolin was less denatured than KPI, the enthalpy change (ΔH) of phaseolin was about 20 J g?1, while enthalpy change (ΔH) of KPI was only 11.2 J g?1. The exposed SH (SHE), total SH (SHT) and SS content of phaseolin was significantly lower than those of KPI. Near-UV CD spectra and intrinsic fluorescence spectrum analyses confirmed much loss of tertiary conformation of KPI, relative to phaseolin, which may be attributed to acid and alkaline treatment during KPI preparation resulted in protein denaturation, exposure of hydrophobic groups.The effects of microfluidization on functional properties as well as conformational properties of KPI were investigated by solubility and turbidimetric measurements, DSC, fluorescence spectrum and Far-UV CD. The microfluidization led to dissociation of insoluble aggregates, thus improved PS and EAI of KPI, in a pressure dependent manner. Fluorescence emission spectra and far- UV CD spectrum analyses showed that both the tertiary conformation and the secondary structure of the proteins in KPI were nearly unaffected by the microfluidization treatment. DSC analysis indicated the microfluidization-treated KPI samples presented similar Td andΔH, relative to that of untreated KPI.The effects of high-pressure (HP) treatment at 200–600 MPa on functional properties and in vitro trypsin digestibility of vicilin-rich red kidney bean (Phaseolus vulgaris L.) protein isolate (KPI) were investigated. HP-induced conformation changes were also evaluated by Try fluorescence spectrum, surface hydrophobicity (H0) and free sulfhydryl (SH) contents analyses. HP treatment at 200 MPa led to dissociation of soluble aggregates (at void volume), while HP treatment at 400 and 600 MPa led to dissociation of insoluble aggregates in KPI, thus improve PS. HP treatment at 200 and 400 MPa significantly increased emulsifying activity index (EAI) and emulsion stability index (ESI); however, EAI and ESI were significantly decreased at 600 MPa (relative to untreated KPI). The in vitro trypsin digestibility of KPI was decreased only at a pressure above 200 MPa and for long incubation time (e.g., 120 min). HP treatment resulted in gradual unfolding of protein structure, as evidenced by gradual increases in SHE and H0 as well as decrease inΔH. However, only 600 MPa HP treatment resulted in loss of tertiary conformation of KPI, as evidenced by Try fluorescence spectrum analyses. Interestingly, theΔH of HP treated KPI (at 600 MPa) was 11.2 J g-1, account for 60% of that of untreated KPI.The degree of N-acylation sharply increased to about 93-94 % with the anhydride levels increasing from 0 to 0.1 (acetylation) or 0.2 g g-1 (succinylation). Further increase in the ratio just resulted in a slight increase (about 2% to 3%) in extent of acylation. The O-acylation (Thr, Ser) distinctly occurred only when degree of N-acylation was higher than 93-94 %.The acylation treatment (at a ratio of anhydride to protein of 0.05 g/g) resulted in a shift of the minimal of PS profile of KPI toward a more acidic pH. In the succinylation case, the PS progressively increased with the increase in ratio of anhydride to protein. Whereas in the acetylation case, the PS gradually increased to a maximum (from 70% to about 85%) at an anhydrideto-protein ratio of 0.2 g/g, and then on the contrary decreased upon further increase in ratio of anhydride to protein. Acylation, especially succinylation remarkably improved EAI at neutral pH. Succinylation resulted in a marked decrease in mechanical moduli of heat-induced gels of KPI, while the mechanical moduli were, on the contrary, increased by acetylation. Additionally, in vitro trypsin digestibility was improved by the acylation in an anhydride-type and level-dependentmanner.The succinylation led to progressive and significant decrease in H0, from 879 (control) to 118-119 (at degree of N-acylation of 97 %). Whereas in the acetylation case, the H0 decreased first, and reached a minimum (at degree of N-acylation of about 93 %) and then increased to a value (957) even higher than control. There was a close and negative relationship between Ip and degree of N-acylation, the Ip of acetylated KPI was similar or slightly higher than that of succinylated KPI. On the other hand, zeta potential at neutral pH of acylated KPI samples also linearly decreased with the increase in degree of N-acylation.In short, microfluidization, high-pressure, acetylation and succinylation resulted in increase in PS, EAI and ESI of KPI. Succinylation is the most effective for improving functional properties of KPI, the PS, EAI and ESI of succinylated KPI are close to those of phaseolin. These modification technologies led to dissociation of insoluble aggregate. Microfluidization treatment had little effect on the conformation of KPI. HP led to protein unfolding of KPI, however, only HP treatment at 600 MPa resulted in change in tertary conformation of KPI. Upon acylation marked structural unfolding (or change in tertiary conformation) occurred when the degree of O-acylation began to increase.