| As a large consumer of livestock and poultry products,a large number of livestock and poultry bones are produced as byproducts of livestock and poultry industry in China every year.Current processing of chicken cartilage in general there are a series of problems of large production yield but little use value and poor deep processing technology,which makes large amounts of nutrients found in chicken cartilage,such as type II collagen,chondroitin sulfate,hydroxyproline,etc.difficult to use and develop.In this dissertation,fresh chicken cartilage was used as raw material to study the directional preparation of chicken cartilage hydrolysates with property of anti-rheumatoid arthritis,so as to promote the application of chicken cartilage as a health foods and provide new ideas for high-valued chicken cartilage.Development and utilization costs provide new ideas.The main research contents of this paper are as follows:1.To study the effect of different types of enzymes on the directionally prepared enzymatic hydrolysate of chicken cartilage with anti-rheumatoid arthritis activityFive common commercial enzymes including papain,trypsin,37071 enzyme,neutral protease and complex protease were selected to hydrolyze chicken cartilage to analyze the effect of these enzymes on the directionally prepared enzymatic hydrolysate with anti-rheumatoid arthritis activity,based on the evaluation methods of protein recovery,hydrolysis extent,chondroitin sulfate content,hydroxyproline content,inhibition rate of collagenase activity,free amino acid level,total amino acid level,and molecular weight distribution.The results showed that tryptic has the highest protein recovery of 81.51%and highest inhibitory rate of collagenase activity of 70.04%(5mg/mL)among the five hydrolyzed products.Therefore,the products hydrolyzed by tryptic was selected to conduct animal experiments to verify its anti-rheumatic arthritis efficacy in vivo.2.To study the therapeutic efficacy of chicken cartilage hydrolysates on rheumatoid arthritis and to compare the efficacy of principally effective ingredients in the enzymatic hydrolysateThe main components of enzymatic hydrolysates in chicken cartilage are collagen polypeptides and chondroitin sulfate.The main functional components,obtained from ultrafiltration using a 10 KDa membrane,of pancreatic enzyme hydrolysates are high sugar component with relative mass>10 KDa and polypeptide with relative mass<10 KDa.The rat model of osteoarthritis induced by papain was used to investigate the therapeutic effects of the three samples on osteoarthritis in rats.The following conclusions were obtained:The enzymatic hydrolysate group consisted of 6 patients in grade 1,3 in grade 2 and 1 in grade s,and scored 1.50±0.71,the high glucose group consisted of 1in grade 0,7 in grade 1,1 in grade 2 and 1 in grade 3,and scored 1.20±0.79,and the peptide group consisted of 7 in grade 1 and 2 in grade 2 when semi-quantitative graded using OOCHAS method established by the international OA association for the study of Cartilage histopathology in terms of cartilaginous tissue of rat in each group.Compared with the sham group,the scores of cartilage tissue in the model group were significantly different(P<0.01).Compared with the model group,the scores of cartilage tissue in group C were significantly different(P<0.05).In addition,the results of blood inflammatory markers in rats such as IL-8,IL-10,IL-1(3,TNF-a and liver and kidney biochemical markers such as creatinine(CRE)?urea nitrogen(UREA)?total bilirubin(TBIL),Alanine transaminase(ALT),aspartate aminotransferase(AST),alkaline phosphatase(ALP),as well as joint pathological indicators and matrix metalloproteinase-13(MMP-13)with a strong pathogenic association with rheumatoid arthritis showed that compared with the model group,the levels of IL-8,MMP-13,IL-1?,IL-10 and TNF-a in the serum in the enzymatic hydrolysate group decreased and 1?,IL-10,TNF-a were statistically significant in the rat serum inflammatory factor test;the levels of IL-8,MMP-13,IL-1?,IL-10 of rats decreased and IL-8 was statistically significant in high blood glucose;The levels of IL-8,IL-1?,IL-10,and-TNF-a decreased and L-1? and IL-10 were significantly different in the blood of the rats in the peptide group.But there was no statistically significant difference in the serum and hepatorenal biochemical indexes such as ALT,AST,UREA,CRE,TBIL,and ALP between the sham and model groups,indicating that the model is successful and excludes the influence of sham operation factors.Moreover,compared with the model group,UREA and CRE in the high glucose group increased statistically,while ALT,AST,GGT,TBIL,and ALP were not statistically significant.However,high-sugar batches can easily cause UREA,CRE increased in rats because of their high sugar content,while considering the weight changes of rats in each experimental group,the level of ALT,AST,GGT,UREA,CRE,TBIL,ALP in the other two groups were not statistically significant compared with the model group.It can be seen that each sample in the experiment had no hepatic and renal toxicity in rats.And from the detection indicators,it was found that all three samples had therapeutic efficacy on rheumatoid arthritis.The overall efficacy was better in the peptide group(<10 KDa group)than in the high glucose group(>10 KDa group)and better in the high glucose group than enzymatic hydrolysate group.3.Isolation,Purification and Identification of Matrix Metalloproteinase Inhibiting PeptidesAfter separation and purification by Sephadex G-25,the five components(<10 KDa component)that have strong anti-rheumatoid arthritis effect were isolated and purified,namely F1,F2,F3,F4,and F5,in which the FI component had the strongest collagenase inhibitory activity of 56.75%(2 mg)and had the highest content.To further explore the active peptides in the F1 fraction,a total of six peptides were identified using UPLC-Q-TOF-MS/MS(ultrahigh-pressure liquid chromatography-time-of-flight mass spectrometry)and the corresponding sequences were GMPGAPG,GMPGAPG,PGEVCIGK,PGELPGQ,GLVGEPG,DGARGLPGAIGAPGPAGGA. |
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