| Sika deer tendon,as traditional Chinese medicine,has the effects of strengthening tendons and bones,nourishing liver and kidney,nourishing Yang,and dispelling wind and dampness,used in the treatment of bone metabolism diseases.Deer tendon is rich in collagen and essential amino acids,which is a high-quality resource for the preparation of bioactive peptides.To explore the influence of extraction and processing technology on the properties of deer tendon collagen,and make full use of emerging technologies to develop functional food or medicine of deer tendon collagen peptide,which is of great significance to increase the added value of deer by-products and improve the living standards of patients with osteoporosis.Based on this,this study firstly extracted collagen from deer tendon as experimental raw material,and compared the differences of enzymatic hydrolysis effects of seven common commercial proteases on collagen protein of deer tendon with the indexes of degree of hydrolysis,molecular weight distribution and promoting bone cell proliferation activity,so as to screen the optimal enzymatic hydrolysis conditions.Secondly,the osteoporosis model was established by tail suspension,and the effect of collagen protein peptides on osteoporosis was evaluated by taking the content of serum markers of bone transformation,bone mechanical parameters and bone microstructure as indicators.Finally,collagen protein was pretreated by ultrasound,and the synergistic activity of collagen hydrolysates in the intervention of osteoporosis was evaluated by cell model.The structure of collagen protein was characterized by modern analytical technologies.The synergistic mechanism of ultrasound pretreatment on the intervention of collagen protein hydrolysates in osteoporosis was clarified by establishing the structure-activity relationship between ultrasonic pretreatment time,collagen structure and activity of hydrolysates.The main research contents and results are as follows:（1）Preparation of deer tendon collagen hydrolysatesTaking the hot water boiling time as the index,the acid treatment process was orthogonal optimized.The optimal process conditions were as follows:citric acid concentration 7%,solid-liquid ratio 1:25 and soaking time 36 h.Under these conditions,the deer tendon collagen protein was obtained by hot water boiling for 41 min.By comparing the degree of hydrolysis,molecular weight distribution,protein extraction rate and proliferation promoting activity of MC3T3-E1 cells of deer tendon collagen hydrolysates by seven enzymes（pepsin,papain,compound protease,flavor protease,trypsin,neutral protease and alkaline protease）,the optimum enzymatic hydrolysis conditions were selected as follows:alkaline protease was the optimum enzyme,enzyme addition was 1000 U/g,enzymatic hydrolysis temperature was 40℃ and enzymatic hydrolysis time was 24 h.Based on these conditions,the extraction rate of collagen hydrolysis was 72.07%±2.52%,the degree of hydrolysis was 23.94%±0.19%,and the cell proliferation rate was 145.48%±9.09%.（2）Intervention of deer tendon collagen hydrolysates on osteoporosisThe osteoporosis model of tail suspension was established.The effects of deer tendon collagen hydrolysate on osteoporosis were evaluated by measuring the contents of alkaline phosphatase（ALP）and type Ⅰ collagen terminal peptide（CTX-I）,bone hardness,bone formation and calcification in serum and heart of mice.The results showed that deer tendon collagen hydrolysates in each dose group could increase the content of osteogenic markers in serum and heart and reduce the production of bone resorption markers.In terms of bone mechanics,compared with the injury group,the horizontal and vertical stiffness of mouse femur in the medium and high dose groups were significantly increased（p<0.05）.In addition,deer tendon collagen hydrolysate and estradiol could significantly increase the number and density of bone trabeculae,increase the thickness of cortical bone,reduce the spacing of bone trabeculae and promote bone calcification,which was conducive to bone formation.Therefore,deer tendon collagen polypeptide could significantly intervene osteoporosis and improve bone hardness and bone morphology in a dose-dependent manner.（3）Synergistic effect of ultrasonic pretreatment on the intervention of deer tendon collagen hydrolysates on osteoporosisThe acid extracted collagen was pretreated by ultrasound.The effects of ultrasonic pretreatment on the activity of deer tendon collagen hydrolysate were evaluated by investigating the chemical,in vivo and in vitro antioxidant activity and promoting cell proliferation and differentiation.The results showed that compared with the collagen hydrolysates prepared by enzymatic hydrolysis after hot water treatment of deer tendon（HWPHs）,the hydrolysis prepared by ultrasound for 60 min（CPHs（60 min））significantly improved the DPPH free radical scavenging ability（22.47%）,FRAP reducing ability（11.56%）and cell proliferation promoting ability（15.67%）.In addition,CPHs（60 min）could inhibit the intracellular calcium influx,protect oxidative damaged cells and increase cell survival rate by reducing the production of ROS in cells.Therefore,ultrasonic pretreatment of collagen could significantly improve the antioxidant activity and bone proliferation of collagen.The collagen extracted by acid method was pretreated at different times by ultrasonic technology.The protein structure was characterized by circular dichroism,IR and fluorescence spectra by polyacrylamide gel electrophoresis（SDS-PAGE）,and the microstructure of the protein was observed by scanning electron microscope（SEM）.Gel permeation chromatography（GPC）was used to study the molecular weight distribution of hydrolysates after ultrasonic pretreatment,and the effects of ultrasonic pretreatment time on collagen structure and hydrolysate characteristics were systematically studied.The results showed that ultrasound at an appropriate time could partially destroy the triple helix structure of collagen,unfold the protein,loosen the structure,expose more hydrophobic groups,increase the contact site between enzyme and substrate,improve the enzymatic hydrolysis efficiency and release more small molecule active peptides.Ultrasound at long time coluld promote the interaction between protein molecules and hydrophobic groups to form aggregation,which was molecular rearrangement or hiding hydrophobic groups that hinder enzymatic hydrolysis,resulting in the reduction of small molecular peptides and the activity of hydrolysates.Therefore,appropriate ultrasonic time could change the structure of collagen,improve its degree of hydrolysis,and then improve the cell proliferation and antioxidant activity of the hydrolysates. |
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