| Cod is one of the commercially important fish species in international markets,which distributed extensively in our country and possess a high economic value.Large quantities of by-products have been generated during the production process ofcod with the fast development of aquatic processing industries. Fish bone wastesmake up15%of the raw fish materials. Cod bone contains many essential nutrientsincluding high quality collagen and various mineral elements. Thus cod bone is anexcellent natural source of protein and calcium. In this study, the un-denaturedacid-soluble collagen and denatured hot-water extracted collagen was isolated fromthe bones of pacific cod (Gadus macrocephalus). The characterization and functionalproperties of both collagen samples were investigated. The hydrolyzed collagenpeptides were prepared by appropriate protease as efficacy factor. Utilizing culturedMG-63cells, the osteogenic mechanism of collagen polypeptides with differentmolecular weights was studied in vitro. The optimum condition for the preparation ofactive calcium from cod bone was determined through orthogonal tests and thebiological availability was investigated. Furthermore, the preventive effects of bonecollagen polypeptides and active calcium on osteoporosis of ovariectomized rats wasstudied in vivo. This paper aimed to broaden the research field of fish bone resource,provide experimental evidence for developing fish bone health food and aquaticproduct processing industry. The main results were as follows:1. The pacific cod bone was used as research materials and the main compositionswere determined. The proximate analysis of fish bone exhibited that protein content inbone reached14.5%. The content of essential amino acids made up26.02%of thetotal amino acids. The calcium and phosphorous content was20.78%and10.09%, theratio of which was approximately2:1. Tightly and firmly combined hydroxyapatite crystals characterized by the flat tabular shape were found under SEM observation.After decalcification process, large amounts of collagen fibrils in fish bone wereobserved using Van Gieson staining technology.2. The studies of the extraction methods and physicochemical properties ofcollagen from cod bone were undertaken. Acid-soluble collagen (ASC) was isolatedunder low temperature. Amino acid analysis found that ASC was rich in Gly, whilethe amount of His, Tyr and Ile was relatively low. Electrophoresis revealed twodifferent α (α1and α2) chains, β-component and γ-component. FTIR and X-raydiffraction measurement showed ASC kept in helix structure. Lyophilized ASC had aporous network microstructure with complicated lace-like fibers. Hot-water extractedcollagen (HEC) was prepared from pacific cod bone. The influences of differentextraction parameters, including concentration of hydrochloric acid, acid soaking time,extraction temperature and time on the extraction rate of bone HEC were investigated.On the base of single-factor experiment, the optimum parameters of extraction forHEC were obtained by orthogonal experiment as follows: the concentration ofhydrochloric acid4%, acid soaking time18h, the extraction temperature70℃andtime8h, the yield was98.02%. Four HEC samples were prepared, including45℃for4h，45℃for8h，70℃for4h and70℃for8h. Amino acid analysis showed there wasno significant difference among the four samples. The bloom strength was decreasedsignificantly with the increasing extraction temperature and time. FTIR and SEMobservation showed HEC lost the natural helical structure.3. Cod bone collagen was hydrolyzed by trypsin treatment to obtain peptides whichcould promote the alkaline phosphatase activity of MG-63cells. The optimumparameters of hydrolysis conditions were obtained by orthogonal experiment asfollows: hydrolysis temperature at50℃,hydrolysis time at90min,enzyme-to-substrate ratio ([E]/[S])1.5%. The ALP increasing rate under thiscondition was43.64%. The polypeptides were subjected to ultrafiltration using cut-offmembranes to obtain collagen peptides with different molecular weight: BCH1(2000Da<Mw<6000Da) and BCH2(Mw<2000Da). Utilizing cultured MG-63cells,the osteogenic mechanism of bone collagen hydrolysates (BCH) was investigated in vitro. Results showed that both two samples had no cytotoxic activity. Resultsindicated that the expression of BMP-2mRNA in MG-63cells was significantlyincreased after being treated with200μg/ml of BCH1and BCH2(P<0.01). Theseresults suggested that collagen peptides could promote the proliferation anddifferentiation of osteoblast through the regulation of BMP-2expression. BCH1andBCH2could increase the OPG mRNA level (P<0.01) and meanwhile remarkablydecrease the RANKL mRNA content (P<0.01), exhibiting a certain dose-relatedmanner. BCH2with low molecular weights showed a better effect to increase the ratioof OPG/RANKL mRNA (P<0.01). These results revealed that collagen peptides couldregulate the expression of OPG and RANKL of osteoblast in order to indirectlyinfluence the osteoclast differentiation and maturation, thus explained one mechanismof how collagen polypeptides could improve the bone metabolism.4. The optimum parameters of extraction for active calcium were obtained byorthogonal experiment as follows: the extraction temperature100℃, the extractiontime120min, the acid mixing ratio1:2, the acid concentration15%, the calcium yieldwas96.15%under this condition. The research on bioavailability of CCM calciumsample was carried out in growing rats. Results showed that there were no side effectsof calcium samples. Compared with the control group, CCM low dose groupexhibited a significantly higher femur calcium content (P<0.05), CCM middle andhigh dose as well as calcium carbonate group remarkably promote the femur calciumcontent (P<0.01). There is no statistical difference of bone calcium content betweenCCM high dose group and calcium carbonate group, which possessing the samecalcium level in samples. Calcium metabolism experiment results suggested thecalcium absorption of CCM high dose group was significantly higher than that ofcalcium carbonate group (P<0.05). These results proved that CCM active calciumpossessed excellent bioavailability and could be a natural nutritional supplement ofcalcium.5. The effect of collagen peptides, active calcium and peptide-calcium mixture onprevention of osteoporosis in ovariectomized rats was investigated. There was nosignificant difference in viscera index and common serum biochemical index in each group, and no uterine endometrial hyperplasia was observed. These results indicatedthat long-term administration of these three samples has less risk of endometrialhyperplasia and displayed a high security. Results showed that all the three samplescould mitigate the increase of ALP and TNF-α in serum. Compared with OVX group,BCH high dose, CCM and peptide-calcium mixture at all dose levels exhibited mostsignificant inhibitory effect to TNF-α (P<0.05), which indicated the tested samplescould decrease the high bone remodeling (an increase in bone resorption and boneformation) caused by ovariectomy and help to restore the balance. The BMD of femurneck, in animals treated with CCM high dose and peptide-calcium mixture of alldoses levels was higher than that of the OVX group (P<0.05). A significant increasein femoral calcium content was observed in CCM and peptide-calcium mixture bothat low dose groups (P<0.05) and high dose groups (P<0.01). These results indicatedCCM and peptide-calcium mixture treatment may decrease bone turnover and preventagainst the bone mineral loss in cancellous bone after ovariectomy.Histomorphometry analysis further revealed the CCM and peptide-calcium mixturetreatment could significantly prevent trabecular bone loss and keep bettermicroarchitecture as compared to the OVX group. RT-PCR results showed theRANKL mRNA levels of each group were significantly decreased as compared to theOVX group, indicating that BCH and CCM could be effective in the prevention ofbone loss by modeling the expression of OPG/RANKL system. These resultssuggested that the collagen peptides and active calcium treatment could counteractovariectomy-induced bone loss and reduce the risk of osteoporotic fractures. |
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