Effect Of Enzymatic Dephosphorylation On The Structure And Digestibility Of Caprine Casein Micelles

Casein is one of the main protein components in infant formula milk powder,primarily derived from cow and caprine milk.The phosphorylation level of casein in caprine milk differs from that in human milk,with caprine casein having a higher degree of phosphorylation.This leads to differences in the microstructure of caprine casein micelles compared to human casein micelles,resulting in different digestibility in the infant stomach between formula-fed infants and breastfed infants.Currently,in the production of infant formula milk powder,there have been no reports on modifying casein at the molecular level to make it more similar to human milk.Previous research has focused on the dephosphorylation of cow casein and its effects on physicochemical properties,but there is a lack of relevant research on dephosphorylation of caprine casein micelles,particularly the effects of dephosphorylation on the structure and digestibility of caprine casein micelles.The composition and phosphorylation level of casein in cow milk and caprine milk are different,and the structure of cow casein micelles and caprine casein micelles are also different.Their optimal conditions for dephosphorylation may be different.Subsequently,a batch of caprine micellar casein concentrates powder was produced,and the impact of enzymatic dephosphorylation on the structural properties of caprine casein micelles and their digestibility in simulated infant gastrointestinal fluids was examined using this material.The primary research objectives and conclusions of this study are outlined below:1.The caprine casein micelles were dissociated at 4℃,and the changes in the levels of free proteins,calcium dissociation,phosphorylation levels of free caseins,and the structural changes of the dissociated casein micelles were studied.The results showed that after ultracentrifugation and resuspension of the ultrafiltration supernatant twice at 4℃,the dissociation of casein micelles reached equilibrium,with 36.5%total casein dissociating,among which the dissociation ofβ-,κ-,andα_s-casein reached 60.4%,31.4%,and 11.8%,respectively.At 25℃,freeβ-casein existed in the form of low-phosphorylated forms with 2 P,3 P,and 4 P,while at 4℃,the dissociation of calcium from the micelles resulted in the dissociation of high-phosphorylated forms ofβ-casein with 5 P and 6 P.After dissociation at4℃,the relative content of micellar calcium decreased from 72.0%to 50.2%,and the ratio of calcium to casein in micelles decreased from 21.8 to 19.4.The casein micelles still maintained an intact spherical morphology,but the mean square radius of gyration(R_g),hydrodynamic radius(R_h),and mass-average molar mass of the micelles decreased.The shape factor R_g/R_h,hydration level,and intrinsic fluorescence intensity of the micelles increased,and the heterogeneity of casein spatial distribution within the micelles decreased,indicating that the internal components of the micelles underwent rearrangement and the skeletal structure became looser after low-temperature induced dissociation.2.Caprine casein micelles were dephosphorylated using bovine intestinal alkaline phosphatase to investigate the optimal dephosphorylation conditions and the effects of dephosphorylation on the structure of caprine casein micelles.The results showed that optimal dephosphorylation can be achieved by mixing 5 mg/m L of heated caprine casein micelles solution with 0.8 U/m L of alkaline phosphatase solution in equal volumes,followed by incubating at p H 6.4.β-Casein showed marked dephosphorylation and formed multi-phosphorylated isoforms depending on dephosphorylation degree,and whereasα_s-andκ-caseins showed limited and fast dephosphorylation,respectively.With increasing dephosphorylation,both the colloidal calcium and the calcium sensitive micellar caseins,especiallyβ-casein,were gradually dissociated,and the calcium insensitive serumκ-casein was gradually associated with the micelles.The dissociatedβ-casein fraction was predominated by isoforms with lower number of phosphate groups.For micelles with increasing dephosphorylation,the molar mass decreased,the gyration and hydrodynamic radii decreased,the ratio of gyration to hydrodynamic radii and micellar hydration increased,the spherical morphology was generally retained,and the internal protein inhomogeneity disappeared progressively.3.Caprine casein micelle powders with dephosphorylation degrees of 0%(un-dephosphorylated),34%(partially dephosphorylated),and 63%(highly dephosphorylated)were prepared.The phosphorylation pattern ofβ-casein in the partially dephosphorylated caprine casein micelles was similar to that of human milkβ-casein.The impact of dephosphorylation on the digestibility of caprine casein micelles in vitro,simulating infant gastrointestinal fluids,was investigated.After dephosphorylation,the size of the curd particles of caprine casein micelles observed by the naked eye and using a confocal laser scanning microscope decreased.Highly dephosphorylated caprine casein micelles did not form curds,which might be related to the dissociation of calcium during dephosphorylation.The water-holding capacity of the curds increased,indicating that dephosphorylated caprine casein micelles became more porous.Dephosphorylated casein,particularlyβ-casein,exhibited a faster digestion rate in simulated infant gastric fluid,consistent with the significant dephosphorylation ofβ-casein and limited dephosphorylation ofα_s-casein.Additionally,dephosphorylation accelerated the release rate of free amino acids in caprine casein micelles.This suggests that dephosphorylated caprine casein micelles were easier to be digested and absorbed by infants.In conclusion,this study investigated the structural characteristics of caprine casein micelles and the role of phosphate groups for micelles through low-temperature dissociation.Subsequently,different dephosphorylation degrees of caprine casein micelles were prepared using enzymatic methods.The aim was to convert caprine casein micelles into a novel casein ingredient with similar phosphorylation patterns and micelle characteristics to human casein micelles.In vitro digestion experiments confirmed that dephosphorylation improved the digestibility of caprine casein micelles,indicating that the use of dephosphorylated caprine casein micelles in infant formula powders may have developmental value.