Selective Coacervation Behavior Of Pea Whey Proteins With Polysaccharides And Its Application On Stabilizing Acid Emulsion

Pea whey proteins,as a by-product of pea protein isolate,mainly exist in pea whey with large amount and physiological activity,which is a good protein resource.Selective coacervation is a potential protein purification technology,which can achieve rapid separation and purification of target proteins by selectively binding proteins with polysaccharides.In this paper,PA1a and PA2 were taken as the main research object to study the selective complex coacervation behavior between polysaccharides with different charge properties and proteins,the effect of p H,protein/polysaccharide mass ratio and charge density on selective coacervation was investigated to determine the selective coacevation conditions for the separation or purification of PA1a and PA2.Finally,pea whey protein and polysaccharide coacervates were used to prepare acid-stable emulsion to expand its application range.Main research results are as follows:The selective coacervation of pea whey protein with the cationic polysaccharide-chitosan was studied.The main proteins of pea whey were identified as PA2,PA1a and lectin using MALDI-TOF/TOF-MS.When the dialyzed pea whey proteins(removal of salt ions and small molecules)coacervated with chitosan,the turbidimetric titration and zeta potential results showed that at maximum turbidity(p H_max),charge neutrality of complexes was fulfilled;the maximal protein recovery was obtained at the mass ratio of 1:1(38.16%).At p H_max,lectin hardly participated in the formation of coacervates;the proportions of PA1a and PA2 in coacervates were related to the protein to polysaccharide mass ratios.At high protein/polysaccharide mass ratios(?40:1),only PA2 transferred into in coacervates.After removal of chitosan by centrifugation,high-puritied PA2(93.26%)could be obtained.Meanwhile,the selective coacervation behavior of original pea whey proteins with chitosan was studied.It was found that the presence of salt ions and other small molecules could affect the strength of complex coacervation,the critical p H(p H_c,p H_?1)shifted to lower p H and the protein recovery decreased.The conditions for the selective purification of PA2 shifted from the protein/polysaccharide mass ratio of 40:1 to 20:1.In order to purify PA1a,the selective coacervation behavior of anionic polysaccharides(sulfated(SPS)and carboxylated polysaccharides(CPS))with PA2 and PA1amixtures(PPM)were studied.It was found that PPM-SPS system had higher critical p H_cand protein recovery than that of PPM-CPS;both PA2 and PA1a participated in the formation of coacervates,but PA2 preferentially transferred into coacervates than PA1a,which made enrichment of single PA1a in the supernatant when mass ratio decreased to a certain extent.With the increase in polysaccharide charge density,the protein recovery rate increased gradually,and less amount of polysaccharide were required to purify the PA1a.Among the four given systems,PPM-CMC system showed the highest PA1a recovery(59.40%)and the purity of PA1a was 92.65%determined by SEC-HPLC.Zeta potential analysis showed that PA2 had higher isoelectric point and carried more positive charge than PA1a;ITC results indicated that the binding affinity of PA2 with DS(1.098±0.114×10⁶M^-1)was higher than that of PA1a(0.439±0.061×10⁶M^-1),might a reason why PA2 preferentially participated in coacervation.Finally,the pea whey protein-polysaccharide coacervates was used to prepare emulsions,and the emulsifying property and physical stability of emulsions at acidic conditions were investigated.As a control,emulsions stabilized by pea whey protein separated into two layers immediately at p H 5 and p H 6.During 14 days'storage,the creaming behavior improved progressively at p H 3?p H 7.On the contrary,emulsions stabilized by pea whey proteins-dextran sulfate coacervates showed good stability in the p H 3?p H 7 at first two days,and the creaming index increased gradually in the following days;however,after heat treatment(95°C for 30min),the creaming index of emulsions was signifiacantly lower than that of unheated emulsions.Emulsions formed by heated pea proteins(95°C,30 min)showed higher protein adsorption percentage and interface protein content than those formed by unheated proteins,facilitating proteins adsorbed at the oil-water interface contained higher percentages of lectin and PA2 in emulsions.