Research Of Kidney Bean(phaseolus Vulgaris L)proteins Fibrils And Gelation Mechanism

Kidney bean is one of important leguminous plant, which yield is below in soybean. Kidney bean embody abundant nutrition, so the bean has great research significance and value. The bean exploitation was not enough for the second place of legumes. The aim of the thesis was researched and utilized the properties of fibril aggregation and gelation from kidney bean protein. Physicochemical and functional properties of phaseolin(the main storage globulin) and kidney bean protein isolate(KPI) were studied from micro to macro aspects. The possible mechanisms of fibrils aggregation were discussed in the thesis. Main results are as follows:Phaseolin could form amyloid fibrils by heating at low pH and low ionic strength. The study compared with the physicochemical properties of phaseolin fibrils. The thesis research the protein conformational variation under the different influencing factors conditions of heat induced. The results showed that the purity of phaseolin(92-95%)was higher than kidney bean protein isolate(84-90%). The viscosity of phaseolin solution increased with increasing heating time. SDS-PAGE showed that the conformation of phaseolin was affected by heating time. DLS and Congo red test proved the phaseolin fibril increased with heating time, and phaseolin fibrils much better thanβ-lactoglobulin at the same condition. Th T fluorescence intensity increased with increasing heating time. CD detected the secondary conformation of phaseolin fibril, which proved -helix structure transformed toβstructure. The growth of phaseolin fibrils were observed by AFM and TEM. AFM 3D images displayed the specific information of phaseolin fibrils.DLS showed that the characters of different influencing factors phaseolin fibrils, stirring and seed are the best methods for phaseolin fibrils. Th T fluorescence and CD detected the secondary conformation of phaseolin fibril, and proved stirring and seed have best effect for phaseolin. When adjust protein solution pH from 2 to 7,βstructure transformed to -helix. AFM showed morphology of phaseolin fibrils under different conditions. Compared withβ-lactoglobulin fibril, phaseolin fibrils have more bigger, and more curved.The theory of percolation model and fractal model applied to KPI gelation at ionic strength 0-300 mM. Percolation theory model could detect the minimum critical concentration of KPI gelation. Fractal model could suggest that KPI gelation network structure formed by short and thick fibrils. SEM images showed that the network of KPI gelation. Th T fluorescence kinetics test reflected the growth of phaseolin fibrils andβstructure of phaseolin. AFM 3D images suggested that the globulin may be form spiral structure, and arrange in spiral shaope, and the length could reach several micrometers. Phaseolin fibril formation has at least two stages: a reversible formation of linear aggregates, followed by slow strengthening of the fibrils.The extraction of phaseolin(60%) is higher than KPI(32%), so research KPI has more practial significance. DSC shows KPI denatured temperature(Td)and enthalpy(ΔH)increased with increasing ionic strength. The turbidity and mechanical modulus of KPI gelation increased with increasing ionic strength. The KPI gelation critical concentration decreased with increasing ionic strength. The isoelectric point of KPI solution is 4.62 at pH 2 and different ionic strength (0-300 mM). Zeta potential decreased with ionic strength, and hydrodynamic radius increased with ionic strength. AFM images showed that the length of KPI fibrils increased with increasing ionic strength in 100 mM. Chaotic aggregation images at 200 and 300 mM ionic strength.Samples showed typical amyloid fibrils features, when Congo red staining freeze-dried phaseolin fibrils samples. The results of Th T fluorescence intensity became decreased than before freeze-dried. Zeta potential and hydrodynamic radius changed a few than before freeze-dried. The difference of freeze-dried samples could be observed by AFM. The solution viscosity and gelation mechanical modulus could improve by putting phaseolin fibrils into protein solution.In general, heat-induced Kidney bean protein has typical properties of amyloid fibrils at low pH and low ionic strength. Physicochemical and functional properties changed a lot. Formation of linear aggregation has the width 10 nm and length 2-3μm in 720 min heating time. Stirring, ionic strength, heating time, temperature, seed and pH are all have important influence on properties of Kidney bean protein fibrils. The gelation theory of Percolation model and fractal model was fit to KPI gelation. The freeze-dried phaseolin fibrils have the properties before freeze, but the phaseolin aggregation degree changed a few.