Construction And Calcium Absorption Promotion Mechanism Of Calcium Delivery Systems Based On Duck Egg White Peptides And Chitosan Oligosaccharide

The calcium loading amount,stability and intestinal absorption function of calcium supplements were the key factors affecting the bioavailability of calcium.All the existing calcium supplements had the limitation of low bioavailability.The development of new calcium supplements with high calcium-loading capacity,strong stability and excellent calcium absorption effect is an important way to solve the above problems.It is an effective strategy to construct the delivery system with high calcium carrying capacity to achieve slow release of calcium in vivo and improve the bioavailability of calcium.Three different assembly modes of DPs/COS delivery calcium system were constructed based on the egg white peptide（DPs）and chitosan oligosaccharide（COS）in this study,which aiming at improving calcium loading amount and regulating the stability of Ca-chelates.Additionally,active peptide and oligosaccharide（prebiotics）could cooperate to improve the bioavailability of calcium while calcium was released slowly in intestinal.Caco-2 monolayer cell model,the low Ca feeding induced calcium deficiency mice model,the phytic acid induced calcium deficiency mice model,in vitro caecum fermentation model and the Gallus-gallus model were used to investigate the calcium release regularity,the effect of calcium absorption and bone synthesis in vivo and the ability of calcium release of three calcium delivery systems.Moreover,this study aimed to investigate whether COS could play the synergistic role in promoting calcium absorption while regulating intestinal health as prebiotic.The main research results were as follows:1.Study on the construction,characterization and in vitro Ca release of COS-TPP/DPs-Ca systemCOS-TPP/DPs-Ca delivery system was based on the ion cross-linking reaction between COS and sodium tripolyphosphate（TPP）,then loaded with DPs-Ca.The optimal reaction conditions were as follows:DPs-Ca（DPs molecular weight<1 k Da）,COS concentration was 4%,the p H of TPP solution was 9,and the mass ratio of COS:TPP was 2.27:1.The result of thermal stability experiment showed that the ion cross-linking COS-TPP/DPs-Ca delivery system significantly improved the thermal stability of DPs-Ca.Moreover,it was observed by scanning electron microscopy（SEM）that DPs-Ca was embedded in the network structure of COS-TPP shell-nuclear particles.Finally,the result of in vitro simulating gastrointestinal digestion experiments showed that COS-TPP/DPs-Ca had resistance ability to gastric juice digestion and significantly delayed the release time of Ca²⁺in the intestine.2.Study on the construction,characterization and in vitro Ca release of heat-induced DPs-COS calcium delivery systemHeat-induced DPs-COS calcium delivery system was based on the Maillard reaction between DPs and COS.The optimal reaction conditions were as follows:p H=6.5,mass ratio of DPs（<1 k Da）:COS（1 k Da）=0.8:1（w/w）,temperature 85°C,reaction time 3 h.Under the optimal reaction conditions,the soluble calcium binding amount of delivery system increased by 248.5%compared with DPs.Firstly,the result of thermal stability analysis showed that the DPs-COS system had better thermal stability compared with DPs and COS.Secondly,the result of FTIR analysis showed that the covalent condensation of ammonia was formed by the-NH₂ of DPs and C=O of COS in the Heat-induced DPs-COS system.Finally,the result of in vitro simulated gastrointestinal digestion experiments and Caco-2 monolayer cell model showed that heat induced DPs-COS-Ca significantly prolonged the release time of Ca²⁺in the intestine.Heat-DPs-COS system could significantly increase the transport amount of calcium by 109.21%compared with Ca Cl₂（p<0.05）.3.Study on the construction,characterization and in vitro Ca release of TGase-induced DPs-COS calcium delivery systemTGase-induced DPs-COS calcium delivery system was based on the acyl transfer reaction catalyzed by TGase between DPs and COS to form a stable DPs-COS system,and then chelated Ca²⁺to construct the calcium delivery system.The optimal reaction conditions were:mass ratio of DPs（<1 k Da）:COS（1 k Da）=0.5:1,TGase dosage 15U/g,reaction time 3 h.Under the optimal reaction conditions,the soluble calcium binding amount of delivery system increased by 204.6%compared with DPs and showed good thermodynamic stability.The results of FTIR showed that the TGase-induced DPS-COS system consisted of glutamine residue of DPs as acyl donor and primary amine of Glc N in COS as acyl acceptor.Moreover,the results of in vitro experiments,such as the Caco-2 monolayer cell model and in vitro simulation of gastrointestinal digestion,indicated that TGase calcium delivery system could effectively prolong the degradation time of DPs-Ca in the gastrointestinal environment,and promote the intermembrane transport and uptake of Ca²⁺in Caco-2 cell,in which TGase-DPs-COS system could significantly increase the transport amount of calcium by 76.50%and 28.96%compared with Ca Cl₂ and DPs+Ca²⁺,respectively（p<0.05）.4.Comparative study of calcium absorption promoting activity in calcium delivery system based on low calcium diet modelThree kinds of calcium delivery system supplements were used by daily and alternate day intragastric administration to investigate the effects of calcium absorption and bone synthesis in mice induced by low calcium diet.The results showed that after8 weeks of continuous intragastric administration of three calcium delivery systems,the bone calcium content,shaft weight index,maximum bone load and maximum stress in mice were significantly increased compared with the model group（p<0.05）,all the three calcium delivery systems could effectively improve the degree of bone calcification and the ability to resist external deformation.Then,micro-CT was used to analyze the microstructure of mouse femur bone,the results showed that the structural model index（SMI）,bone surface area to bone volume ratio（BS/BV）,bone trabecular separation（Tb.Sp）,bone trabecular pattern factor（Tb.Pf）of the three calcium delivery system groups were significantly lower than those of the model group（p<0.05）;bone trabecular thickness（Tb.Th）,bone trabecular surface area density（BS/TV）,number of bone trabecular（Tb.N）,bone volume fraction（BV/TV）were significantly increased compared with model group（p<0.05）,indicating that the three calcium delivery systems could significantly improve the bone deformation resistance and reduced the risk of osteoporosis.After 8 weeks of three calcium delivery systems,the bone indexes of Heat-induced and TGase-induced calcium delivery system groups were significantly better than those of model group（p<0.05）,and recovered to the level of normal group,while there was no significant difference in maximum load on tibia between COS-TPP/DPs-Ca group and model group（p>0.05）.The Heat-type and TGase-type calcium delivery systems of alternate-day gavage had the same or better effect of promoting calcium absorption in vivo than DPs-Ca with the same calcium dose,and were better than Ca CO₃ with the same calcium dose.Interestingly,the calcium supplementation effect of COS-TPP/DPs-Ca was not as good as that of DPs-Ca,but the effect was still better than that of Ca CO₃ group.According to the intestinal histological observation,the relative abundance of intestinal flora and the analysis of metabolites,the three kinds of calcium delivery system supplements could maintain the integrity of intestinal villi structure,promote the proliferation and differentiation of small intestinal goblet cells,and increase the length and area of intestinal villi,improve the abundance of beneficial bacteria,such as Lactobacillus and Bifidobacterium,to optimize the intestinal flora structure;facilitating the synthesis of short-chain fatty acids（SCFAs）such as acetic acid,propionic acid and butyric acid;improve the expression of intestinal brush margin genes such as SI and SGLT1,regulate intestinal health,and promote the absorption of nutrients in small intestine.5.Study on calcium absorption promotion activity and mechanism of calcium delivery system in phytic acid inhibiting calcium absorption modelHeat-type and TGase-type DPs-COS binary system could effectively prevent Ca²⁺from forming insoluble calcium phosphate and delay the formation time of phosphate crystallization.In the Caco-2 monolayer cell model,Ca²⁺transport was inhibited by35.3%in the presence of 5 mmol/L phytic acid（PA）,and the Ca²⁺transport capacity was close to that of the Ca group without PA when PA,Ca,and Heat-type or TGase-type DPs-COS were present together.In the phytic acid inhibited calcium absorption mice model,the length,diameter and maximum load of femur in Heat type and TGase type DPs-COS system group significantly increased compared with model group（p<0.05）.SEM analysis of mice femur showed that both DPs-COS systems could improve the absence of large amounts of matrix on the femur surface caused by PA,and the fan-shaped surface pits were significantly improved.According to intestinal histological observation,intestinal flora relative abundance and metabolites analysis,both DPs-COS systems could improve the small intestinal structure disorders caused by PA,which are shown as follows:The villi length,villi area,the number of goblet cells on villi,and the contents of acetic acid and propionic acid in intestinal tract in the two DPs-COS groups were significantly higher than those in PA group（p<0.05）;The abundance of Lactobacillus in two DPs-COS groups was significantly higher than that in PA group（p<0.05）,the relative abundance of Escherichia coli was significantly lower than that of PA group（p<0.05）.6.Study on the synergistic effect and mechanism of calcium delivery system to promote calcium absorption through probioticsThe function and mechanism of regulating intestinal health in vivo and in vitro by Heat-type and TGase-type DPs-COS system and DPs-COS-Ca calcium delivery system were studied by using in vitro fermentation of rat cecal contents and Gallus-gallus model.The results showed that both Heat-DPs-COS and TGase-DPs-COS could increase the concentration of short-chain fatty acids and lactic acid,and had dose-effect relationship and time-effect relationship.By adding Heat-DPs-COS and TGase-DPs-COS,the inhibition effect of PA was reversed,and the abundance of beneficial bacteria（Lactobacillus and Bifidobacterium）was significantly increased,while the abundance of harmful bacteria（Escherichia coli）was significantly decreased,and the concentrations of acetic acid and lactic acid were restored to normal levels.The two DPs-COS-Ca ternary calcium delivery systems also had good effects on promoting the production of acetic acid and lactic acid and regulating intestinal flora.The Gallus-gallus model showed that the intestinal damage caused by dietary anti-nutrition factors could be reversed by the simultaneous injection of PA and DPs-COS system.DPs-COS did not lose its intestinal regulation function after chelating calcium,suggesting that DPs,COS and Ca could play a synergistic role in regulating intestinal health.