Evaluation Of Walnut Protein Peptides To Improve Osteoporosis Activity And Preparation And Structural Characterization Of Calcium Chelating Peptides

In this study,walnut kernel(Juglans regia L.)from Yunnan Province was used as raw material.The walnut kernels from which the seed coat was removed and defatted by petroleum ether and simulated in vitro digestion to obtain a preliminary walnut protease extract(WPH).The degree of hydrolysis,amino acid composition,molecular weight distribution and calcium binding capacity of WPH were determined.The effects of walnut protein hydrolysate(WPH)on calcium absorption and bone quality of osteoporosis rats were evaluated by using retinoic acid-induced osteoporosis animal model.The calcium binding ability of walnut protein hydrolysate(WPH)was used as the index to isolate and purify the peptide sequence of the purified component.Meanwhile,the peptide calcium chelate was prepared and its structure was identified.The main results are as follows:1.Walnut protein hydrolysate(WPH)was obtained by enzymolysis of peeled and defatted walnut kernel in vitro.The degree of hydrolysis and protein recovery rate of WPH were 11.19%and 81.72%,respectively.The amino acid composition of WPH was analyzed by automatic amino acid analyzer.The results showed that WPH was rich in glutamic acid(111.18 mg/g).High performance liquid chromatography analysis showed that the molecular weight distribution of peptides in WPH was mainly below 1000 Da.2.An animal experimental model of osteoporosis induced by retinoic acid was established,and the osteoprotective effect of WPH on osteoporotic rats at different doses was evaluated.The results of serum parameters showed that the content of calcium and phosphorus in serum of WPH group was significantly increased,the content of bone gla protein,the activity of alkaline phosphatase and tartrate resistant acid phosphatase were significantly decreased compared with model group.The results of bone parameters showed that the bone diameter,bone dry weight coefficient,and bone wet weight coefficient of the WPH group were significantly improved compared with the model group,and the bone biomechanical properties,bone calcium content,and bone phosphorus content were also significantly restored.Bone mineral density,cortical bone thickness,trabecular bone area,and bone density electronic images all showed relief of osteoporosis symptoms in the WPH group.The HE staining and TRAP staining images show that the trabecular structure and quantity of WPH group were significantly restored,the cortical bone thickness was increased,and the bone microstructure of osteoporosis rats was significantly improved.Meanwhile,the intervention of WPH reduced the number of osteoclasts and inhibited the increase of osteoclast activity.The results show that WPH can improve the absorption and utilization of calcium in vivo,regulate the balance of bone metabolism,and has a positive effect on promoting bone growth.3.Sephadex G-25 and RP-HPLC were used to separate and purify WPH,and the calcium binding capacity of the separated components was determined.The maximum calcium binding capacity of the separated components in the gel was93.68?g/mg This component was further separated by RP-HPLC,and the maximum calcium binding capacity in the separated component was measured to be 107.89?g/mg.UPLC-Q-Orbitrap-MS~2 was used to identify the peptide sequence of the purified fraction,and 15 peptide fragment sequences were obtained.Two peptides LQVLEK and LPHHLD were selected to prepare peptide calcium chelate.The structure of the peptide calcium chelate was identified by UV-VIS,SEM and XRD.The results showed that the peptide chelated with calcium ion.Mass spectrometry combined with molecular dynamics analysis showed that amino nitrogen atom and carboxyl oxygen atom at the terminal of peptide provided binding sites for calcium ion,and carbonyl oxygen atom in polypeptide was the main site for chelating with calcium ion.