Study On Mechanism And Inhibition Of Protein Aggregation Induced By The Gas-liquid Interface In Foam Fractionation

Foam fractionation,due to its high efficiency at low concentrations,low costs and free pollution,has a great potential in effectively reducing the costs of protein separation in the downstream processing of biotechnology.However,the large number of gas-liquid interfaces in foam fractionation often induce protein aggregation and result in a loss of protein functionality.As a result,the industrial applications of foam fractionation in protein separation are limited.For inhibiting the interface-induced protein aggregation in foam fractionation,its molecular mechanism and kinetics and its correlations with the separation performances were studied using bovine serum albumin(BSA)as a model protein.Then,the method for effectively inhibiting the gas-liquid interface-induced protein aggregation was proposed and its mechanism was studied at a molecular level.Finally,the application of the method in the foam fractionation of bromelain from the crude extract of pineapple peel was studied.Firstly,the relationship between the structual changes of BSA and the aggregation of BSA in foam fractionation was studied with BSA concentration and pH as the structual inducing factors for BSA.The results show that the phenylalanine residues-containing hydrophobic zones were the sites for the aggregation of BSA molecules;with decreasing BSA concentration or pH,the BSA molecules suffered a higher-level structural unfolding and the phenylalanine residues-containing hydrophobic zones were more exposed to the solution;resultantly,the aggregation ability of BSA molecules was improved and the molecular weights of the BSA aggregates increased;in addition,intermolecular ?-sheet was formed between the aggregated BSA molecules;its content increased as the BSA structure became more unfolded.Secondly,the whole foam fractionation process was divided into three stages: adsorption in liquid phase,foam rising and defoaming.In each stage,the relationship between the mass flux of BSA adsorbed at the gas-liquid interface and the production rate of BSA aggregates was studied.On this basis,the effects of enhanced interfacial adsorption and enhanced foam drainage on the aggregation kinetics of BSA in foam fractionation were discussed.The results show that the production rate of BSA aggregates had a linear correction with the change in the mass flux of BSA adsorbed at the gas-liquid interface in the stages of adsorption in liquid phase and foam rising while in the defoaming stage,it was determined by the mass flux and the relative content of BSA adsorbed at the gas-liquid interface in the rising foam;adsorption in liquid phase was the limiting stage for protein aggregation induced by the gas-liquid interface;enhancing the adsorption of BSA at the gas-liquid interface and enhancing foam drainage both increased the production rate of BSA aggregates in the whole foam fractionation process while enhancing foam drainage resulted in the formation of BSA aggregates of higher molecular weight and even insoluble aggregates;furthermore,enhancing foam drainage allowed BSA aggregates to be more readily distributed in the residual solution.Thirdly,the relationship between the gas-liquid interface-induced aggregation of BSA and its separation performances was studied with sodium citrate as an aggregation enhancer.The results show that enhancing BSA aggregation induced by the gas-liquid interface improved the stability of the rising foam and thus weakened bubble coalescence;resultantly,the enhanced aggregation effectively improved the recovery percentage of BSA,but reduced the enrichment ratio.Fourthly,?-cyclodextrin was used to inhibit the aggregation of BSA in foam fractionation and the inhibition meachnism was also studied.The results show the hydrophobic cavity of ?-cyclodextrin held the side chains of tyrosine and phenylalanine residues at the moelcular surface of BSA and screened the hydrophobic groups near the acting sites;as a result,?-cyclodextrin could effectively inhibit the aggregation of BSA induced by the gas-liquid interface in foam fractionation;however,?-cyclodextrin reduced the separation performances of BSA by reducing the protein interficial adsorption.Finally,?-cyclodextrin was used in foam fractionation of bromelain from the crude extract of pineapple peel and its effects on the recovery of bromelain activity were studied.The results show that compared to the case without ?-cyclodextrin,the addition of ?-cyclodextrin(0.40 g/L)and enhancing the adsorption of bromelain in liquid phase increased the activity enrichment ratio,specific activity and the activity recovery percentage by50.5%,71.0% and 40.7%,respectively.In conclusion,the studies on the molecular mechanism and kinetics of protein aggregation induced by the gas-liquid interface and its correlations with the separation performances provided important theoretical supports for the effective inhibition of protein aggregation in foam fractionation.On this basis,the studies on the mechanism of ?-cyclodextrin inhibiting the interface-induced protein aggregation and the development of the technology of ?-cyclodextrin coupled with protein adsorption enhancement had practical implications to reducing the loss of protein functionality in foam fractionation and the industrial applications of foam fractionation of proteins.