Preparation Of Food-derived Peptide Iron Chelate And Establishment Of Its Absorption Evaluation Model

Food-derived peptides are the general term for small-molecule peptides that are formed by enzymatic hydrolysis or fermentation using animal and plant proteins as raw materials(including beans,grains,fish,poultry,etc.).In this essay,a series of studies were conducted using food-derived peptides BP253 and BP701 as raw materials.Firstly,the chelation process of food-derived peptide BP253 and iron salts was optimized and a preparation process of peptide iron chelate with higher yield was obtained which laid a theoretical foundation for the development of a new iron supplement.Secondly,the Caco-2 cell model is a classic model for studying the absorption of nutrients,and it takes 21 days to culture the Caco-2 cell model in normal media.Therefore,the effect of BP701 and differentiating agent BA on the Caco-2 cell model was considered to establish a new type of Caco-2 monolayer cell model with a short cycle,saving time and raw materials.The significant conclusions are summarized as follows:1.The single factor experiment and response surface analysis method were used to design the experiment to optimize the optimal process of preparing peptide iron chelates by chelating the food-derived peptide BP253 with iron salts.The response surface analysis adopted the Box-Behnken center combination experim-ental design principle.And it used three factors and three levels with chelate yield as response index,choosing peptide concentration,peptide iron mass ratio,and pH as experimental factors,which aimed to obtain an optimal peptide iron chelate prepara-tion process with a peptide concentration of 40 mg/mL,mass ratio of peptide iron of 4.5:1,the chelating temperature of 45?,the chelating time of 50 min,and the reaction pH of 7.0.The yield of peptide iron chelate was 11.48%.2.To get the best digestion conditions for Caco-2 cells,the experiment studied the effect of trypton concentration and digestion temperature on the digestion effect of Caco-2 cells.It was learned that trypton concentration and digestion temperature are very important factors for the effect of Caco-2 cells digestion(digested single cell ratio).Experiments showed that 0.25%trypsin-0.02%EDTA-Na2 with digesting three minutes at 37? was the best condition for digesting Caco-2 cells.3.The effect of different kinds of food-derived peptides on the proliferation rate of Caco-2 cells(action time was 24 hours)was studied by MTT assay,and the proliferation rate of BP701 on Caco-2 cells was 42.66%.Secondly,the effects of different concentrations of BP701(5,50,100,200,400 ?g/mL)on the proliferation rate of Caco-2 cells were studied(periods of 24 h,72 h,and 120 h,respectively),of which 200 ?g/mL of BP701 had the greatest value-added effect on Caco-2 cells.4.The effect of different concentration of BP701 on the growth curve of Caco-2 cells was studied by MTT assay.At the same time,the blank group was set to study the growth cycle of Caco-2 cells and had a basic understanding of the growth characteristics of Caco-2 cells,which ensured the accuracy and stability of the experiment.Studies showed that BP701 at a concentration of 5-200 ?g/mL promoted the proliferation of Caco-2 cells,and the proliferation of Caco-2 cells in the logarithmic growth phase was the most obvious,and the growth-promoting effect of the slow-growth phase was significantly lower.In the logarithmic phase,the value-added effect of the stationary phase gradually weakened,but it didn't change the natural growth of Caco-2 cells.5.The effect of BP701 on the establishment of Caco-2 cell model was studied.Measuring the transmissivity of phenol red and AKP activity could judge whether it was successful of Caco-2 cell model establishment.The results showed that the transmissivity of phenol red gradually decreased with the increase of culture time of Caco-2 monolayer cell model.After 9 days of culture in Caco-2 monolayer cell model,the transmissivity of phenol red gradually became stable(8.03%).The phenol red transmittance of the Caco-2 monolayer cell model cultured in normal medium was stable on the 15th day of model establishment(8.19%),which indicated that BP701 played a major role in the formation of monolayer Caco-2 cells.When the Caco-2 cell model was established for 21 days,AP/BL was equal to 1.124,which indicated that Caco-2 cells only grew without differentiation.This study showed that the addition of BP701 promoted the rapid formation of a dense membrane in Caco-2 cells but did not promote its differentiation.6.MTT assay was used to determine the toxic effects of differentiating agent BA on Caco-2 cells in serum with different concentrations,5 mmol/L and 2 mmol/L BA0(without serum),5 mmol/L and 2 mmol/L BAFBS(containing 16.5%of serum)had no obvious effect on the survival rate of Caco-2 cells,but when the concentra-tion of BA was 10 mmol/L,Caco-2 cells had obvious toxic effects.7.The effects of 2 mmol/L BA0 and BAFBS on the establishment of Caco-2 cell model were investigated.When the Caco-2 cell model was established for 17 days and 2 mmol/L of BA0 and BAFBS were added for 5 days.The AKP activity assay results showed that BA0 and BAFBS both significantly increased the AKP activity of the AP side than that of the BL side when BA0 and BAFBS had been added to the Caco-2 monolayer cell model for five days,which indicated that the Caco-2 monolayer cell model appeared to different polarization phenomenon.However,BA0(AP/BL=15.46)significantly promoted the differentiation of Caco-2 monolayer model compared to BAFBS(AP/BL=4.394).The differentiation of Caco-2 monolayer model cultured by BA0 was consistent with the differentiation of Caco-2 monolayer model cultured by normal medium for 21 days(AP/BL=14.50),which indicated that BA0 could achieve a good differentiation effect in a Caco-2 monolayer cell model only for 5 days.Therefore,2 mmol/L BA0 was selected for subsequent experiments to establish the Caco-2 monolayer cell model.8.The effect of the addition time point of BA0 on the establishment of Caco-2 cell model was studied.Time point of BA0 was added by observing the cell morphology under a microscope,phenol red transmittance,and AKP activity assay.At the fifth day of establishment of Caco-2 cell model,BA was added and kept five days.Some cells in the Caco-2 monolayer cell model were found to be lysed on the 9th day under the microscope,the transmissivity of phenol red increased with time,the activity of AKP on the AP side and BL side was small and the AP/BL was only 2.380,which indicated that BA0 was added on the fifth day to the Caco-2 cell model was not conducive to successful establishment of the model.It was observed that the Caco-2 cells had formed a dense and flat membrane under a microscope,and the phenol red was transparent when BA0 was added to the Caco-2 cell model on the 14th day of the establishment of the Caco-2 cell model.The rate of AKP activity was only 8.38%and AKP activity was determined to be AP/BL=14.94,which indicated that the Caco-2 cell model can be successfully established for only 18 days when BA was added on the 14th day of establishment of the Caco-2 cell model.This saves both raw materials and time compared to the establishment of the standard Caco-2 cell model for 21-22 days.