Preparation Of Phosvitin Phosphopeptide Calcium Complex And Its Mechanism Of Promoting Calcium Absorption

Phosvitin(PV)is a phosphorylated glycoprotein that exists in eggs yolk.It contains a large number of phosphorylated amino acids,which endows PV with strong emulsification,anti-oxidation and the ability of binding to metal ions.This topic used PV as the research object to prepares phosvitin phosphopeptides calcium complex(PPP-Ca)and studied the interaction between Ca²⁺and PV enzymatic hydrolysate,and explored the mechanism of PPP-Ca in promoting and regulating the intestinal Ca²⁺absorption and transport in the body.And,further research was carried out at the co-culture cellular level.The main research contents and experimental results are as follows:(1)The pretreatment phosvitin(HTMP-PV)was prepared by high temperature and medium pressure(HTMP),then combined with trypsin and thermolysin to prepare phosvitin phosphopeptides(PPP),and finally prepared the optimal conditions for preparing phosvitin phosphopeptides calcium complex(PPP-Ca).The HTMP pretreatment method was determined as follows:10 mg/m L PV,p H 6.5,120?,0.1 MPa for 30 min.The SDS-PAGE showed the molecular weight of HTMP-PV have a significant decrease.The degree of hydrolysis(DH%)and calcium binding rate were used as indicators to screen the enzyme amountand and the enzymatic hydrolysis time of thermolysin.The results showed that the optimal enzymatic conditions for thermolysin were 10 mg/m L HTMP-PV,2000 U/g protein,p H 6.8,68?for 24 h.After successive enzymolysis under the optimal conditions of trypsin and thermolysin,the calcium binding rate of the enzymatic hydrolysate was 43.01%,and the DH%was 45.55%.The reaction temperature and reaction time of the preparation of PPP-Ca have no significant effect on the peptide calcium chelation process.The peptide calcium mass ratio was 7:1,p H 9.5,and its calcium binding rate was the highest,which could reached more than 97%.(2)The structural changes of before and after the combination of PPP and calcium were analyzed through fourier infrared spectroscopy(FTIR),Zeta potential,XRD and other methods.The results showed that calcium was mainly bound to the phosphate group of the PPP,and other amino and carboxyl groups also provided some calcium binding site.Zeta potential showed that the Zeta value of PV dropped from-42.47 m V to-57.13 m V after treatment by HTMP+complex enzymolysis,indicating that the negative charge increased.The Zeta value of PPP-Ca increased to-24.2 m V,and the electronegativity was reduced.At the same time,the XRD pattern confirmed that the characteristic peaks of Ca²⁺was to be masked when PPP combined with Ca²⁺.Further determine the acid-base stability,thermal stability and simulated digestion stability of PPP-Ca.It was found that PPP-Ca was more sensitive to acidic environment than medium-alkaline environment,and have good thermal stability and in vitro simulated digestion stability.(3)The low-calcium mice model was built to explore the mechanism of PPP-Ca in the intestinal calcium absorption and transport in vivo.The AKP of low-calcium control group was effectively increased(18.93 Gold units/100 m L)caused by low-calcium diet.The results showed that PPP-Ca could effectively alleviate the abnormal increase in serum AKP,which is reduced to 11.09 Gold units/100 m L,reaching the level of normal control group(10.89 Gold units/100 m L).Femoral calcium content indicators found that the femoral calcium content of mice in the low-calcium control group was 80.47 mg/g,and the femoral calcium content of the PPP-Ca group reached 94.33 mg/g,indicating that PPP-Ca could effectively improve bone calcium.The expression of TRPV6 calcium channel-related protein in the duodenum was detected by Western Blotting,and it was found that PPP-Ca can affect the expression of TRPV6 channel-related protein.(4)The Caco-2 cell monolayer model was successfully constructed,then detected the amount of calcium transport and the relative expression of m RNA of TRPV6 calcium ion pathway-related protein genes,which explore calcium promotion absorption mechanism of peptide calcium complexes.In evaluating the integrity of the cell model,the results showed that the Caco-2 cell monolayer membrane polarization was obvious after 21 days,and the TEER value reached 860?·cm^-2or more,and the permeability of fluorescent yellow was 7.79×10^-6cm/s,could been used for transport experiments.In the measurement of calcium transport and TRPV6 calcium ion pathway-related protein gene m RNA expression indicators,the results was found that the calcium transport of Caco-2cells showed a certain concentration-dependent after treatment with different concentrations of Ca Cl₂solution.The calcium transport volume of PPP-Ca was 5.66?g after treatment for 3 h,which was significantly higher than the calcium transport volume of the Ca Cl₂group(3.07?g),indicating that PPP-Ca can promote the Ca²⁺transport volume of the Caco-2 cell monolayer.PPP-Ca can affect the m RNA expression of genes related to the TRPV6 calcium pathway in Caco-2 cells.(5)The co-culture system of osteoblasts(MC3T3-E1 cells)and osteoclast precursor cells(RAW264.7 cells)was set up to explore the effects of PPP and PPP-Ca on the differentiation of osteoblasts and osteoclasts.The results showed that PPP and PPP-Ca can effectively increase the AKP secretion of MC3T3-E1 cells in the co-culture system.For example,the AKP values of PPP and PPP-Ca groups were 2.39 Kings unit/100 m L and 2.77 Kings unit/100 m L in the 1:1 seeding density group of MC3T3-E1 cells and RAW264.7 cells,which were significantly higher than the 2.05 Kings unit/100 m L of the control group.At the same time,the amount of TRAP secretion of RAW264.7 cells of PPP and PPP-Ca reduced in the co-culture system.The TRAP values of control group was 4.26 U/L,which significantly higher than the TRAP values of PPP group(2.69 U/L)and PPP-Ca groups(2.461 U/L).These results indicated that PPP and PPP-Ca could down regulate the activity of TRAP in RAW264.7 cells,thus affecting cell differentiation.The expression levels of OPG,RANKL,NFATC-1 and RANK m RNA in the MC3T3-E1cell and RAW264.7 cell co-culture system were measured by RT-PCR.The results showed that PPP and PPP-Ca can significantly up-regulate the expression of OPG,RANKL and RANK m RNA in the co-culture system and down-regulates the expression of NFATC-1 m RNA.