Preparation And Physiological Activity Evaluation Of Bovine Bone Collagen Hydrolysates

Bovine bone accounts for about 20%of the body weight of cattle,which is one of the main by-products of beef industry.It contains about 20%protein,mainly type I collagen,making it a good source of collagen for preparing collagen peptides.This thesis first optimized the pretreatment conditions to extract bovine bone collagen(BBC),and then enzymatically hydrolzyed collagen to prepare BBC hydrolysates(BBCH);the hydrolysate was purified by cation exchange chromatography and reversed phase liquid high performance chromatography and peptide seuqnces were characterized by LC-MS/MS;glucose uptake property was assessed using rat L6 cells,and other activities were preducted using an on-line software.This thesis was attempted to develop BBCH with potential physiological activity,and thus provided a scientific basis for value-added application of livestock by-products.The main findings are as follows:1.The influence of bovine bone pretreatment on the physical and chemical properties of BBC.The proximate composition of bovine bone was determined.Bovine bone was pretreated by three methods:high temperature and high pressure(121?,0.1MPa),high temperature(100?),acid-alkali extraction(10%acetic acid,0.08 mo L/L sodium hydroxide),collagen ws extracted and the physicochemical properties were studied.The total protein content of bovine bone was 22.50±2.6 g/100g,collagen content was 20.50±2.9 g/100g,fat content was 20.89±2.8 g/100g,ash content was36.01±3.1 g/100g.Collagen prepared by high temperature and high pressure pretreatment showed the best yield of BBC of 63.67±1.9%,the particle size of404.30±6.1 nm,the thermal denaturation temperature of 85?,hydroxyproline content of 4.95±0.8 g/100g and the total amino acid content of 41.43±1.8 g/100g.Two clear?chains were observed by SDS-PAGE analysis.Through the secondary structure detection,it seems that the three strands of collagen obtained by high temperature and high pressure pretreatment remain intact while the hydrogen bond is broken.2.Effects of pretreatment on yield and properties of BBCH.BBC obtained above were hydrolyzed by typsin,and the characteristics of BBCH were compared.Collagen peptides prepared by high temperature and high pressure pretreatment had a total protein content of 81.62±3.1 g/100g,the protein recovery and collagen recovery of which are 73.6±1.3%and 76.71±6.3%,respectively,which were higher than that pretreated only by high temperature pretreatment.The particle size of BBCH prepared afer high temperature and high pressure pretreatment is 237.4±5.3 nm,68.76%of the peptides has the molecular weight less than 1500 Da,and the content of total amino acid is 58.26±1.5 g/100g.Most peptides identified by LC-MS/MS were derived from?₁ and?₂ chains of bovine-derived type?collagen,with the characteristic peptide repeat motifs of collagen.3.Preparation and functinal study of BBCH.BBCH were prepared using single enzyme or a combination of two enzymes from four commercial proteases(alkaline protease,neutral protease,trypsin,and animal proteolytic enzymes).The enzymatic hydrolysis effect was evaluated by combining the yield,recovery rate,molecular weight,the L6 cell proliferation effect and glucose uptake activity.The results show that the yield and hydrolysis degree of the product after enzymatic hydrolysis are slightly lower than that of the compound enzyme.In the L6 cell model,the cell proliferation effect of the single enzyme BBCH is better than that of the compound enzyme group at a concentration of 1 mg/m L,and the BBCH obtained by trypsin has the best proliferation effect;meanwhile,the single enzyme BBCH promotes glucose absorption of L6 cells,BBCH obtained by trypsin is also the most significance.4.Purification of BBCH with glucose uptake activity.Cation exchange chromatography and C₁₈ reversed-phase high-performance liquid chromatography were used to fractionate BBCH.The gucose uptake activity was studied in L6 cells.After stepwise fractionation,the most active components were determined,and their peptide sequences were identified and characterized by reversed-phase liquid chromatography-tandem mass spectrometry(RPLC-MS/MS).The results showed that the peptides were derived mainly from the?₁(P02453)and?₂(P02465)chains of collagen.These peptides were predicted to have angiotensin converting enzyme(ACE)inhibitory and dipeptidylpeptidase-IV(DPP IV)inhibitory activities.In summary,after extraction,enzymatic hydrolysis and purification,bovine bone can be produced into BBCH,which was able to promote glucose update in L6 cells and may have hypoglycemic effect,further studies are warranted to understand the in vivo efficacy and mechanisms of action.This study provides ideas for the high-value utilization of bovine bone.