Study On Facilitating Calcium Absorption And Preventing Osteoporosis With Collagen Peptides From Carassiusauratus Skin

The fishery in our country has developed rapidly,more and more people's attention was attracted by the processing and comprehensive utilization of aquatic products.In this process,a lot of scrap,such as fish bones,fish scales and skin,were produced and wasted inevitably.In our experiment,the skin of Carassiusauratus was used as raw material.Gelatin was extracted with hot water and hydrolyzed by different enzymes to prepare carassiusauratus skin collagen peptides(CCPs)with high calcium-binding activities.Calcium deficiency model was built with low calcium diet,the effects of CCPs on calcium absorption and bone mineral density were investigated.Osteoporosis rats model induced by retinoic acid was built,the effects of CCPs on bone loss and bone metabolic disorders were investigated too.The sequences of CCPs with high calcium binding activity was identified by means of ultrafiltration,amino acid analysis and RP-HPLC,HPLC-ESI/MS/MS,mass spectrometry database information.The main results were as follows:1.Preparation of CCPs with high calcium-binding activitiesThe main component of Carassiusauratus skin were moisture and crude protein,the proportion was 66.21%,32.44%respectively.Crude protein accounted for 96.7%of dry matter content,indicating a very rich protein in Carassiusauratus skin.With the hot-water extraction method,gelatin is extracted 24h under the condition of 42°C.Protamex was chosen as the optimal enzyme to hydrolyze collagen from alkaline protease,trypsin,protamex.After optimization,its hydrolysis condition was:CCPs concentration of 3%,Promamex dosages of 3000U/g,p H7.0,temperature 50?and enzymatic hydrolysis time of 3 h.Under these conditions,the soluble calcium bound by DPs was 2.0mg/g and the degrees of hydrolysis reached 10.57%.2.Evaluate the ability of CCPs to promote calcium absorption and strong bones using low calcium diet mice modelCalcium deficiency model was built with low calcium diet.The results show that CCPs can promote calcium absorption in mice.500 mg/kg,1000 mg/kg CCPs+Ca CO₃could increase calcium apparent absorption rate,significantly higher than Ca CO₃group(P<0.05)and no significant differences were found among 500 mg/kg,1000 mg/kg CCPs+Ca CO₃group and 500 mg/kg CPP group(P>0.05).500 mg/kg CCPs could significantly increase femur and tibia length,dry weight index,bone calcium content,bone mineral density(BMD),bone mineral content(BMC)(P<0.05),no significant differences were found between 500 mg/kg CCPs+Ca CO₃group and CPP+Ca CO₃group(P>0.05).The microstructure of the tibia was observed by scanning electron microscopy,500 mg/kg,1000 mg/kg CCPs+Ca CO₃could improve the condition that the bone surface was porous and aperture was bigger,the surface gap was obviously reduced.HE staining was used to observe the trabecular structure,500 mg/kg,1000 mg/kg CCPs+Ca CO₃could increase the number and intensity of trabecular,promote the deposition of calcium in the bone,provide bone organic matter,improve osteoporosis caused by low calcium diet.3.Evaluate the ability of CCPs to strong bones using retinoic-acid induced osteoporosis rats modelOsteoporosis rats model induced by retinoic acid was built.The results showed that CCPs could prevent osteoporosis caused by retinoic acid.600 mg/kg,1200mg/kg CCPs could significantly increase the serum alkaline phosphatase activity(P<0.05),and1200mg/kg CCPs could significantly increase the content of osteocalcin(P<0.05).600mg/kg,1200mg/kg CCPs could significantly increase femur dry weight index,maximum load(P<0.05)and there were no significant differences among alendronate group(5mg/kg)and 600 mg/kg,1200mg/kg CCPs group(P<0.05).600 mg/kg,1200mg/kg CCPs could significantly increase femur and tibia bone calcium content,BMD,BMC(P<0.05).The bone calcium content of l600 mg/kg CCPs was significantly higher than alendronate group(P<0.05).600 mg/kg,1200mg/kg CCPs could increase the number and intensity of trabecular,decrease the number of active osteoclasts.4.Primary structure of CCPs fraction with the highest calcium-binding abilityFour different fractions of CCPs were fractionated by ultrafiltration membrane:Mw<1k Da,1-3k Da,3-5k Da,>5k Da,the main contributors to the calcium-binding ability of CCPs were the peptides with a molecular weight less than 1 k Da.The amino acid analysis showed that Gly,Pro,Hyp,Ala,Glx,Asx,His and Thr were relatively higher than others.Using HPLC/ESI/MS/MS technology,the sequences of CCPs were identified and confirmed by searching the online MS database.Thirty one short peptides(2?6peptide)were isolated from CCPs fraction(Mw<l k Da).Two peptides containing hydroxyproline((OH)P)were VFA(OH)PD,FVA(OH)PD Twenty nine peptides do not contain(OH)P were EEQQ,EEKAG,MPSSVP,MPSVSP,MTGPK,etc.Some amino acids such as acidic amino acids Glu,Asp,alkaline amino acids Arg,Lys,phosphorylated amino acids Thr,Ser that frequently appeared in the peptides may contributed to chelating with calcium.