Preparation Of Gudas Macrocephalus Minced Peptide Calcium Complex And Its Effect On Calcium Absorption

In this paper,the codfish minced meat was hydrolyzed as the raw material by coupled enzymatic reaction in order to obtain the codfish minced peptide,and the peptide calcium complex?GMMP-Ca?was obtained by chelating.Caco-2 cells were used to verify the calcium absorption ability of GMMP-Ca.Conclusions of this study are as follows:1.A high-producing protease strain HSCD-3 was selected from the intestine of frozen sea cucumber through a casein plate screening and fermentation culture rescreening.Analyzed by 16S rDNA,the strain was identified as Bacillus.After 48 h of fermentation,the enzyme activity of the fermentation product was reached to 89.86U/mL.The crude enzyme,named BP-1,was successfully obtained by ultrafiltration and precipitation and freeze drying.The molecular weight of the protease was approximately30.8 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis?SDS-PAGE?.The optimum temperature of the protease was 50?,and it maintained 85%protease activity in the range of 4 to 50?;the optimum pH of this enzyme was 8.0,and it maintained more than 80%protease activity at pH 6¹0.Different metal ions with the concentration of 5 mM showed different effects on the activity of it.Na⁺and K⁺had no significant influence on protease activity,Ca²⁺,Fe²⁺and Cu²⁺had an inhibiting effect on protease activity.However,Mg²⁺,Mn²⁺and Ba²⁺had enhancing effect on protease activity.2.The basic components of codfish minced meat were 18.71%protein,0.80%ash,80.10%moisture and 0.46%crude fat,respectively.Taking the degree of hydrolysis?DH?and amount of soluble calcium binding as indexes,the hydrolysis conditions were optimized through single-factor tests?protease types,two enzyme ratios,hydrolysis time,temperature,pH,enzyme dosage,and liquid-to-liquid ratio?and response surface design.The results showed that the hydrolysis solution exhibited a better DH and amount of soluble calcium binding when trypsin and BP-1 were combined,.The optimal conditions for hydrolysis were as followed:p H 8.22,temperature 54.7?,hydrolysis time 5.1 h,and enzyme amount 300 U/g,the ratio of solid to liquid was 1:3,the ratio of two enzymes?trypsin:BP-1?was 1:2.The DH was 32.9%,and the amount of soluble calcium binding was reached to 23.30?g/mL under this optimal condition.The molecular weight distribution of the protein hydrolysate was determined by high performance liquid chromatography?HPLC?.The polypeptide component with molecular weight larger than 1 kDa accounted for 10.47%,while the polypeptide component with molecular weight less than 1 kDa accounted for 89.53%,and the component with molecular weight between 0.13-0.52 kDa accounted for 47%of the total.3.The peptide calcium chelation process was optimized by using Gadus macrocephalus minced peptides and calcium chloride as raw materials and the chelation rate as an indicator.Besides,the stability of chelates was also tested.The experimental data showed that the final concentration of 95%ethanol was the best choice to precipitat the peptide calcium complex.Through single factor experiments and response surface optimization design,the optimal chelation process was as followed:the ratio of peptide to calcium was 3:1and the reaction was conducted at 43.6?,pH 7.50 with the reaction time of 50 min.The chelation rate was?78.69±1.34?%.Fourier transform infrared spectroscopy?FTIR?showed that Gadus macrocephalus minced peptides bound with calcium ion at N-terminal and C-terminal.The stability analysis showed that the calcium peptide chelates had high anti-digestibility.After simulating digestion in the gastrointestinal tract,it still retained 85.15%calcium holding capacity;the chelates showed better acid-base stability and the calcium retention rate remained more than 80%at pH 5⁹.Phosphate was able to compete with the calcium chelate of codfish minced peptide for calcium ion.With the increase of phosphate concentration,the retention rate of calcium was significantly decreased?P<0.05?.When the concentration of phosphate was reached to 20 mmol/L,the retention of calcium ions dropped to 15.40%.Different concentrations of lactose and sodium chloride had no significant effects on the stability of chelates?P>0.05?.4.By measuring the transmembrane electrical resistance?TEER?and alkaline phosphatase?AKP?activity of single layer cells,the transporte rate of them in Caco-2cell monolayer model was determined.The results showed that when the cells were cultured for 15 days,the TEER of the monolayer reached to 500?×cm²,and the ratio of AKP on both sides of Caco-2 cells was more than 2.When the cells were cultured for 21days,the TEER of the monolayer reached 615±14.8?×cm²,and the ratio of AKP on both sides of Caco-2 cells was over than 5,which indicated that Caco-2 monolayer cells had formed compact,well-differentiated monolayers.The model was successfully established and could be used to simulate digestion and absorption in the small intestine.The results showed that the casein phosphor peptides calcium chelate?CPP-Ca?showed the highest retention rates,and the Gadus macrocephalus minced peptides calcium chelate?GMMP-Ca?was secondly,inorganic calcium was the lowest,but there was no significant difference?P>0.05?.On the transport rate,the inorganic calcium was the lowest,and the transfer rate of CPP-Ca was higher than that of GMMP-Ca,but there was no significant difference between the two groups?P>0.05?.On the absorption rate,the CPP-Ca was the highest,GMMP-Ca was secondly,and inorganic calcium was the lowest.Statistical analysis showed significant difference?P<0.05?.In summary,GMMP-Ca could promote cellular calcium absorption at the cellular level.