Effect Of PH Shifting-based De-calcification On The Structure Of Casein Micelle And The Functionality Of Milk Protein Concentrate

Milk protein concentrate?MPC?is a functional dairy protein ingredient that is produced from skim milk by ultrafiltration,diafiltration and spray-drying.MPC is unique in its high-protein content as well as low lipid and low lactose content,while the defect in solubility,heat stability,interfacial activity and other functionality limit its application in food.The functionality of MPC is mainly affected by the structure of casein micelle?i.e.,the aggregation state of casein?,while the structure of casein micelle is mainly maintained by both calcium-bridging and hydrophobic interaction.The removal of calcium within casein micelle will change the balance between electrostatic repulsive force and hydrophobic interaction,which might lead to the targeted modification of the structure of casein micelle and hence the improvement of the functionality of MPC.Among the various de-calcification methods,the acidification of skim milk has the advantage of being simple and efficient.However,the structure of casein micelle is complicated and also is sensitive to the changes in environment factors.Therefore,both the selection of proper acidification condition and the investigaton of how the structure of casein micelle is gradually changed by skim milk acidification during the manufacturing process of MPC,are the key to achieve both the modification of casein micelle structure and the improvement of MPC functionality.Based on the manufacturing process of MPC,this study progressively investigated the effect of skim milk acidification on the structure of casein micelle in skim milk,retentate and MPC,using TEM,SAXS,SEM,RP-HPLC,CLSM and other technique.Besides,a series of MPC with different casein micelle structure were obtained and thus the effect of casein micelle structure on the solubility,heat stability,emulsifiability and foamability of MPC was also investigated.The effect of acidification mode?GDL,HCl?and extent?pH 6.7-4.8?on the distribution of calcium and the structure of casein micelle in skim milk was investigated.Colloid calcium is progressively dissociated as the pH decreases,and a sharp increase in the dissociation of colloid calcium appears between pH 5.8-5.4.The dissociation of micellar casein also reaches a maximum between pH 5.8 and 5.4,with the corresponding amount of free casein accounting for 23%of total casein in skim milk,and the preferential dissociation of each casein follows the order of?>?>?_s1>?_s2,which is inversely correlated to their levels of phosphorylation.Between pH 6.7-6.0,the structure of casein micelle is generally maintained;between p H5.8-5.4,the casein micelle is partially disintegrated into non-micellar casein aggregate;between p H 5.2-4.8,the isoelectric aggregaton of casein commences.With diminishing pH,the?-casein hairy layer gradually collpase and the internal structure of casein micelle becomes more homogenous in spatial distribution of casein.The comparison of different acidification mode shows that GDL induce higher extent of dissociation for both colloidal calcium and micellar casein.GDL results in homogeneous acidification,while HCl leads to the formation of local casein aggregate.Therefore,GDL and pH 6.7-5.4 are chosen for following study.The effect of skim milk acidification?pH 6.7-5.4?on the distribution of calcium and the structure of casein micelle in retentate was investigated.Membrane filtration comprises four processing stages each with a concentration factor of 3,and the retentate from each stage corresponds to MPC56,MPC70,MPC80 and MPC85,respectively.The pH of retentate decreases with decreasing pH of skim milk and increases with increasing stage,and the maximum increase is 0.3-0.4.The dissociation of colloidal calcium increases with decreasing pH and increasing stage.The dissociation of micellar casein also increases with decreasing pH and increasing stage,and a sharp increase in dissociation appears between pH 5.8-5.4,with the corresponding highest amount of free casein accounting for 82%of total casein in skim milk.Between pH 6.7-6.0,the structure of casein micelle is generally maintained with increasing stage;between pH 5.8-5.6,the structure of casein micelle is partially disintegrated with increasing stage;at pH 5.4,the structure of casein micelle is completely disrupted with increasing stage.During ultrafiltration and diafiltration of acidified skim milk,the calcium-bridging within micelle further decreases,the pH of retentates increases and the ionic strength of retentate decreases,thus leading to the increase in electrostatic repulsive force within micelle and hence the disintegration of micellar skeleton.The membrane filtration process of MPC85 and p H gradient of 6.7,6.0,5.7 and 5.4 are chosen for following study.The effect of skim milk acidification?pH 6.7,6.0,5.7,5.4?and retentate pH restoration on the distribution of calcium and the structure of casein micelle in MPC was investigated.For MPC prepared from unrestored retentate,the recovery increases with decreasing pH of skim milk,while retentate pH restoration sharply increases the recovery.The dissociation of colloidal calcium increases with decreasing pH of skim milk,while retentate pH restoration slightly decreases the content of free calcium and slightly increases the content of colloidal calcium.The dissociation of micellar casein also increases with decreasing pH of skim milk and a sharp increase in dissociation appears between p H 5.7-5.4,while retentate p H restoration further increases the dissociation of micellar casein,with the corresponding highest amount of free casein accounting for 94%of total casein in skim milk.Retentate p H restoration will further increase the net negative charge of micellar casein,leading to the further increase in electrostatic repulsive force within micelle and hence the further disintegration of micellar skeleton.The MPC prepared in this section is used for following study.The effect of skim milk acidification?pH 6.7,6.0,5.7,5.4?and retentate pH restoration on the main functionality of MPC was investigated.The initial solubility of each MPC is above 88%and slightly increases with decreasing pH of skim milk,while retentate pH restoration further increases the initial solubility.During storage,the rate of solubility loss decreases with decreasing p H of skim milk,while retentate pH restoration further decreases the rate of solubility loss and the solubility remains almost unaltered at pH 5.4.The solubility loss is mainly caused by casein aggregation and the preferential solubilization of each casein follows the order of?>?>?_s1>?_s2.For MPC prepared from unrestored retentate,the heat stability decreases with decreasing pH of skim milk,while retentate pH restoration sharply increases the heat stability.The emulsifiability and emulsion stability as well as the foamability and foam stability increases with decreasing pH of skim milk,and a sharp increase appears between pH 5.7-5.4,while retentate pH restoration further increases the emulsifiability and emulsion stability as well as the foamability and foam stability.The effect of blending retentate derived from highly acidified?pH 5.4?and unacidified?pH 6.7?skim milk on the main functionality of MPC was investigated.When the blending ratio of retentate derived from highly acidified and unacidified skim milk is at and above20:80,the solublity of MPC is significantly improved.At the same de-calcification ratio,MPC obtained by blending retentate with high-acidification and without acidification and by acidification at a certain pH have no significant difference in casein micelle structure.Besides,the two MPC have the same extent of improvement in solubility and have no significant difference in heat stability,emulsifiability and emulsion stability as well as foamability and foam stability.When the retentate derived from highly acidified and unacidified skim milk are blended,the casein and calcium will re-distribute between serum and casein micelle until the blended retentate system reach an equilibrium state,resulting in no significant difference in casein micelle structure and main functionality between MPC obtainded by two different manufacture processes at the same de-calcification ratio.Taken together,both the modification of casein micelle structure and the improvement of MPC85 functionality were achieved by skim milk acidification.When skim milk is acidified to about pH 6.0,the resulting MPC85 retains the general structure of casein micelle and also has a relatively high solubility,and hence can be used for the development and production of cheese,yogurt,turbid dairy protein beverage and other similar product.When skim milk is acidified to pH 5.7-5.4,the resulting MPC85 has a high solubility and the corresponding dispersion has high emulsifiability,foamability,heat stability,viscosity and clarity,and hence can be used for the development and production of ice cream,whipped cream,milk foam,semi-transparent and transparent dairy protein beverage and other similar product.Moreover,blending retentate derived from highly acidified and unacidified skim milk at different ratio can result in a standardized control of the de-calcification ratio of MPC and also a series of MPC with different de-calcification ratio by the same batch of processing,which will further lead to an improvement in the overall performance of membrane filtration process.