Preparation,Isolation And Structural Analysis Of Chicken Cartilage Peptides And Their Effects On The Growth Mechanism Of Osteoblast

Chicken cartilage is rich in collagen and chondroitin sulfate,which has excellent bone health protection effect.In this study,chicken cartilage,a by-product of chicken meat processing,was used as raw material to prepare chicken cartilage hydrolysates with different enzymatic parameters.The optimal chicken cartilage hydrolysate was selected based on the characteristic analysis and functional evaluation.And the 9h-hydrolysate was subjected for further isolation,purification and structuaral analysis to obtain oligopeptides with clear amino acid sequences.The proliferation,differentiation and apoptosis effects of chicken cartilage peptides on MC3T3-E1 cells were evaluated.Besides,the protection mechanisms of chicken sternal peptides on cadmium-induced apoptosis model were also investigated.In this experiment,the hydrolysis effects of different proteases,enzyme to substrate ratio,pH,temperature and time on chicken cartilage were analyzed using single-factor experiments.Results showed that the optimal chicken cartilage hydrolysate?Alcalase,[E/S]0.50%,pH 7.0,55°C and 9 h?was obtained with the protein recovery rate of 57.00%,degree of hydrolysis?DH?of 26.53%,percentage of small molecular weight?<1 kDa?of 61.62%,DPPH·scavenging IC50 value of 5.14 mg/mL and ORAC value of 0.57?mol TE/mg peptides.Pearson correlation analysis showed that the DH and molecular weight distribution?<1 kDa?of enzymatic hydrolysates were significantly correlated with their ORAC activities,and the correlation coefficients r were 0.737 and 0.534,respectively.The optimal enzymatic hydrolysis of chicken cartilage was further purified using DEAE-52 anion exchange chromatography and gel exclusion chromatography,then obtained F1-c fraction with the higest ORAC value of 3.23?mol TE/mg peptides.Finally,the components of F1-c fraction were analyzed using UPLC-MS/MS.Five chicken cartilage polypeptides were first identified including GGAP,QIGPA,QLGPA,MPKYA and QGPAN.The effects of each peptide on the profileration effect of MC3T3-E1 cells were detected using MTT and EdU assay.The results showed that the cell viability of the cells which incubated with 0.1 mM of QIGPA,QLGPA,MPKYA and QGPAN were increased by 39.34%,21.44%,21.23%and 37.06%,respectively.Among them,QIGPA could promote the synthesis of cellular DNA,and the percentage of positive cells was as high as 16.89%.The degree of differentiation of MC3T3-E1 cells was increased as th extended of culture time.Chicken cartilage?0.1 mM?did not significantly affect the alkaline phosphate?ALP?activity of MC3T3-E1 cells for 3 days.On the contrary,additional chicken cartilage peptides could significantly increase the ALP of the cells.When the differentiation time was extended to 14days,the average optical density?AOD?of NBT-formazan in the cells treated with QIGPA,MPKYA and QGPAN was significantly higher than the differentiation control group?p<0.05?with the AOD value above 0.61.The ALP activity of QIGPA and QGPAN was up to 200.03and 276.56 nmol/min/mg protein.These results indicated that chicken cartilage peptides QIGPA,MPKYA and QGPAN are more favourable to promote the differentiation of MC3T3-E1 cells.MC3T3-E1 cells were incubated with 3.2?M Cd?Ac?₂ for 48 h to establish a cadmium-induced osteoblast apoptosis model.The protection effect of chicken cartilage peptides?0.5 mM?was evaluated based on this cell model.The results of CCK-8 assay showed that the cell viability of MC3T3-E1 cells which treated with GGAP,QIGP,QLGPA and QGPAN was increased by 21.03%,38.54%,42.71%and 13.69%,respectively.The results of Annexin V-FITC/PI assay showed that the proportion of early apoptotic cells were13.96%,5.48%,13.04%and 8.20%,respectively.In addition,the percentage of late apoptotic and necrotic cells were accounted for 14.01%,8.40%,3.16%and 12.04%,respectively.These results revealed that four chicken cartilage peptides could significantly reverse the cadmium-induced apoptosis?p<0.05?.Among then,the ratio of JC-1 red/green fluorescent intensity was increased by 15.71%and 25.95%compared to the cadmium-induced model group after the treatment of QIGPA and QLGPA.These results revealed that they could significantly reverse the decrease of mitochondrial membrane potential induced by cadmium?p<0.05?.In addition to MPKYA,the other four chicken cartilage peptides could protect against the cadmium-induced apoptotic damage by inhibiting the phosphorylation of p-38 and ERK 1/2.In conclusion,QIGPA,MPKYA and QGPAN which identified from chicken cartilage hydrolysates could promote the proliferation and differentiation of MC3T3-E1 cells;while GGAP,QIGPA,QLGPA and QGPAN could protect the osteoblasts against cadmium-induced apoptosis.These results suggested that chicken cartilage peptides could be selected as potential nutritional active ingredients to protect bone health by promoting osteoblasts proliferation and differentiation,as well as slowing down the cadmium-induced apoptosis.In addition,they could also be used as raw materials for the development of bone-related functional foods and food for special medical purpose,which would have broad application prospects in the protection of bone health.