Optimization Of Chelating Process Parameters Of Casein Zinc,Analysis Of The Binding Sites And Exploration Of The Absorption

Casein,as a full-price protein,contains eight essential amino acids for the human body.It can be used as a source of high-quality amino acids with rich source and low price.Zinc ions have a natural tendency to form stable complexes with peptides and proteins.Casein zinc chelate is an ideal zinc supplements that are easily absorbed by the human and can effectively increase the absorption rate of zinc.Providing a reference for the development of new micronutrient zinc supplements by studying the process parameters of casein zinc chelate,analyzing its binding sites and exploring the absorption and transport preliminary in the body.The specific tests are as follows:（1）The effect of zinc addition,reaction temperature and reaction time on the process of casein zinc chelate was investigated by using the mass of zinc in casein-zinc chelate as an evaluation index.According to the single factor experiment,the process was designed by Design Expert 8.06b software and analyzed by Response Surface Methodology.The results showed that the optimum conditions for chelating casein with Zn（CH₃COO）₂ were 205 mg of zinc addition,15.6℃of reaction temperature and 19 min of reaction time.The weight of zinc in casein zinc chelate can reach 13.025 mg,that is 65.125 mg zinc ions can be combined with 1 g casein.（2）The changes of the elements in the casein before and after the chelating reaction were observed by scanning electron microscopy.The binding sites of casein-zinc chelate were analyzed by infrared spectroscopy,Solid-State ¹H and ³¹P MAS NMR.The results showed that there are chelated zinc ions on the surface of casein after the chelation reaction.The hydroxyl group of the serine phosphate residue in the casein component is the main site of the chelating reaction between casein and zinc ion,meanwhile,the coordination reaction took place between zinc ion and casein in the free amino group or its surroundings.（3）In order to clarify the theoretical basis and technical support of casein zinc chelate transport in vivo,an in vitro digestion/Caco-2 cell model was established to simulate the absorption and transport of zinc ions.Compared the absorption and transport of zinc sulfate,zinc gluconate and casein zinc chelate in vitro digestion/Caco-2 cell model.Compared the effects of zinc sulfate,zinc gluconate and casein zinc chelate digestive juice on the expression of hZIP4,ZIP5,MT1,ZnT1,and ZnT2 mRNA in Caco-2 cells.The results showed that the Papp value of casein zinc chelate was significantly higher than that of zinc sulfate and zinc gluconate in an in vitro digestion/Caco-2 cell model,when the low and medium dose zinc ions were added to the matrix.At the same zinc concentration,the cumulative transport of casein zinc chelate in an in vitro digestion/Caco-2 cell model was significantly higher than that of zinc gluconate and zinc sulfate.Compared with zinc sulfate and zinc gluconate,casein zinc chelate digestive juice significantly promoted the expression of ZIP5,MT1,ZnT1,ZnT2 mRNA in Caco-2 cells,and significantly inhibited the expression of ZIP4 mRNA.Combined the results of transport assays and genetic tests showed that the form of zinc casein chelate is faster than added zinc in the form of zinc sulfate and zinc gluconate in an in vitro digestion/Caco-2 cell model.