| Casein phosphopeptide is a bioactive peptide that can effectively promote the calcium absorption and utilization of body.At present,it has been successfully added as a food additive into products such as calcium,milk powder and so on.And it has good effect of calcium absorption and with a very broad market prospect.In this paper,based on the previous study,high-performance liquid chromatography was successfully used to enrich activity monomer P5 of casein phosphopeptide,and a new method for detecting content of casein phosphopeptide was developed.Meanwhile activity of casein phosphopeptide promoting calcium absorption in vitro and in vivo was evaluated and the calcium absorption mechanism of CPP was preliminarily explored by Caco-2 cell model.The main research contents and results are listed as follows:A novel method for the detection of casein phosphopeptide has been developed by TiO2 solid-phase microextraction.The solid-phase microextraction column was used to absorb phosphopeptides accurately in casein hydrolysate,which is casein phosphopeptide.The recovery rate and accuracy of experiments were designed.The recovery rate was 97%and the relative standard deviation of accuracy was 11.58%.It has good reproducibility,high sensitivity,and can absorb the phosphopeptides of samples specifically,which can be used to detect the content of casein phosphopeptide qualitatively and quantitatively.CPP with calcium activity evaluation in vitro we also conducted.In vitro,the effects of calcium and peptide ratio,pH,temperature and water bath time on the calcium in vitro of casein phosphopeptide were investigated.The calcium and peptide ratio,pH,temperature,and bath time were selected as independent variables,and each factor had three levels.In vitro combined calcium content was used as an indicator to design a response surface test to optimize the optimal conditions for influencing casein phosphopeptide in vitro.The results showed that the optimal theoretical conditions were as follows:calcium-peptide ratio 1:2,pH 8.2,temperature 40℃,water bath time 10min.Under these conditions,three parallel experiments were conducted.The average calcium content of CPP in vitro was 9.32 mg.Compared with the theoretical predictive value,the relative error is 3.58%.The good repeatability and reliable results indicate that the response surface analysis method can be used to optimize the in vitro binding calcium content of CPP.CPP activity evaluation and metabolism in vivo.Rat model was used to evaluate the calcium-absorbing activity of casein phosphopeptide in vivo,and to explore the regulation of calcium metabolism and histochemical localization by combining with the stable radionuclides.Animals were given gavage isotope 42Ca and casein phosphopeptide samples,and samples were taken at different times to collect urine and feces,and organs and blood samples were collected on alternate days.After the sample was digested by wet method,it was found that the sample contained a large amount of potassium,sodium,and other ions,causing spectral interference and inaccurate detection.To end this,the use of ME-1 chelated cartridge solid-phase extraction 42Ca was developed to effectively remove spectral interference ions such as potassium and sodium,and high-resolution ICP-MS was used to determine the isotope 42Ca content in the sample.At the same time,high-performance liquid chromatography was used to track the whereabouts of casein phosphopeptide,paving the way for subsequent cell experiments.The results showed that after several optimization experiments,the retention rate of calcium ions in solution reached（95.00±5.00）%,the removal rate of potassium ions reached（93.00±4.25）%,and the removal rate of sodium ions reached（87.65±11.35）%.The results of animal experiments have shown that non-radioactive calcium isotopes can be used to characterize the absorption and metabolism of calcium in animals,and to control their whereabouts.Isotope calcium is first absorbed into the bloodstream by the small intestine,and part of it enters the tissue fluid and is transported to various organs.The other part of the isotope calcium is not absorbed and excreted in the urine.Meanwhile,by comparing the total urinary calcium excretion of CPP1,CPP2,P5 and labeled calcium group,the results showed that the CPP sample had significant calcium absorption effect,and the urinary calcium excretion in the CPP sample group was significantly lower than that in the blank control,especially the CPP active monomer component P5.The difference was significant（P<0.05）.Preliminary exploration of the mechanism of calcium absorption by CPP.From the results of cell experiments,the fluorescence intensity of the CPP sample group was higher than that of the normal calcium intake group and the blank group,and there was a significant difference among the sample groups（P<0.05）.The experimental results are consistent with the results of animal experiments.The active monomer P5 has the strongest calcium absorption capacity.At 30 min,the intracellular calcium ion fluorescence intensity reached the highest point,and then remained stable,indicating that all the calcium ions in the solution entered the cell within 30 min to reach saturation.At the same time,through this experiment,it can be shown that active transport mechanism occurs in the process of calcium absorption of CPP,which is that calcium ions penetrate the cell membrane into the cell.In addition,through the Caco-2 cell model,it was also found that CPP was not digested by cell-producing enzymes during transmembrane transport.It was speculated that CPP exists in solution in intact peptides or enters cells but does not cross membranes to the bottom.From the results of Western Blot experiments,it is known that CPP-H and CPP-P5 groups promote the expression of TRPV5 and TRPV6 on the cell membrane significantly higher than the blank group.It was shown that active transport occurs during the process of promoting calcium absorption,and the expression of channel proteins on the cell membrane increases,and calcium ions are more rapidly taken into cells. |
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