Three Novel Calcium Delivery Systems Based On Casein Phosphopeptides And Chitooligosaccharides: Fabrication,Characterization,in Vivo Bioactive Studies

Various existing dietary calcium supplements have some problems such as the limited solubility and bioavailability.The development of novel calcium delivery systems with higher calcium-loaded capacity and better absorption efficiency is of particular interest to solve above problems.Biopolymers including proteins and polysaccharides have been used to fabricate protective calcium delivery systems,with the aims to increase the calcium-loaded capacity of vehicles and control the release rate of calcium in body.In this study,three novel calcium delivery systems were fabricated using natural biopolymers including chitooligosaccharide（COS）and casein phosphopeptides（CPP）.Three calcium delivery systems were studied using Caco-2cell models,mice models,micro-CT scan technology,spectroscopy,and microscope observations.The research contents about the systems mainly consists of physical and chemical characteristics,morphological characteristics,and intermolecular forces,as well as the abilities to achieve the controlled calcium release,promote calcium absorption,and modulate gut microbiota in vivo.The main results are as follows:1 Fabrication of Amadori-type and TGase-type calcium delivery systemsGlycosylation reactions between CPP and COS,the Maillard reaction andreaction,were used to fabricate two calcium delivery systems.The optimal conditions for the Maillard reaction were as follows:the mass ratio of CPP:COS of 0.6:1,p H of 7.5,the heating time of 3 h,and the heating temperature of 95°C,as well as its calcium-loaded capacity was 43.2%higher than that of CPP.The optimal conditions forreaction were as follows:the mass ratio of CPP:COS of 1:1,the incubation time of 2.5 h,and the enzyme concentration of 12.5 U/g,as well as its calcium-loaded capacity was 59.3%higher than that of CPP.2 Characterization of Amadori-type and TGase-type calcium delivery systemsThe particle size and zeta potential determinations showed that two calcium delivery systems by two glycosylation reactions had a significantly larger microscopic size than CPP and COS,however,their dispersion stability was not decreased during two cross-linked reactions.The calcium phosphate crystallization and simulated gastrointestinal digestion experiments showed that two copolymers could prevent calcium crystallization to the best effect and prolong the degradation of CPP-Ca in the gastrointestinal tract.Fourier transform infrared spectroscopy indicated that the chemical grafting of Amadori-type copolymer generated in-NH₂ of CPP and C=O of COS,while TGase-type copolymer in glutamine residues of CPP（the acyl donor）and-NH₂ of glucosamine（Glc N）in COS（the acyl acceptor）.The images of scanning electron microscopy and atomic force microscopy exhibited an embedded structure in which CPP was wrapped by a scattered layer of COS.Additionally,Caco-2 cells model demonstrated that two delivery vehicles could enhance intestinal calcium absorption.3 In vivo bioactive studies of Amadori-type and TGase-type calcium delivery systemsAfter 8-week feeding period of Amadori-type and TGase-type calcium delivery systems,the maximum stress and bone weight index parameters of bone in mice were significantly higher than those in the model group（P<0.05）.It showed that two calcium delivery systems could effectively improve the ability of bones to resist external force deformation and improve the mineralization process of bones.In the micrographs of micro-CT in different groups,the trabecular separation（Tb.Sp）,Bone surface/bone volume ratio（BS/BV）,and the trabecular bone pattern factor（Tb.Pf）of two systems groups were significantly decreased compared to the model group（P<0.05）,as well as the bone surface density（BS/TV）was significantly increased compared to the model group（P<0.05）,indicating that two novel calcium delivery systems could enhance the calcium absorption and bone synthesis in vivo.Both two calcium delivery systems could maintain the structural integrity of the ileum villi.In addition,both Amadori-type and TGase-type calcium delivery systems could modulate gut microbiota in a prebiotic-like manner.Two systems could promote the relative abundances of Bifidobacterium and Lactobacillus in the cecum contents,and reduce the relative abundance of Escherichia Coli and C.perfringens.The inhibitory effect of TGase-type calcium delivery system on Escherichia Coli and C.perfringens was better than that of Amadori-type delivery system.4 Fabrication,characterization,and in vivo release studies of COS/TPP cross-linked calcium delivery systemsCPP-Ca-loaded core-shell microparticles were prepared by the ionic gelation between COS and TPP.The results of calcium-loaded analysis and texture profile analysis showed that the COS/TPP cross-linked core-shell microparticles possessed good resistance to deformation and suitable stability in an acidic environment,which could satisfy the requirements of slow disintegration and calcium release in the acid environment.The thermodynamic stability analysis and infrared spectroscopy analysis showed that the COS/TPP cross-linked calcium delivery systems be utilized as shell materials to encapsulate thermally sensitive CPP-Ca,and the crosslinking site between the COS and TPP molecules was the P-O bond.Morphology and structural properties showed that CPP-Ca was effectively wrapped by lots of network structures fabricated by COS/TPP coacervate.The simulated gastrointestinal digestion and the timed blood sampling experiments showed that core-shell microparticles could prolong the enzymatic hydrolysis of CPP-Ca in the gastric conditions,and promote the calcium absorption in small intestine.