CAJ | 학술논문

为制备肽钙螯合物并探明肽钙螯合机理，对羊骨胶原多肽与 CaCl2的螯合工艺进行优化并对肽钙螯合物进行表征分析。采用Box-Behnken中心组合设计及响应面分析法，确定了最佳螯合参数：肽钙质量比2∶1，pH值7.66，53℃螯合50 min，螯合率达67.24%。红外光谱、电镜分析及能谱扫描结果表明胶原多肽与Ca2+分别在多肽内部的C=O处及肽链末端的-NH2和-COOH 处发生螯合，形成了铵盐和羧酸盐；多肽与 Ca2+螯合后由疏松的片状结构变成了致密的小颗粒聚集体结构。红外光谱及能谱扫描同时也证实所制备的胶原多肽中有部分钙螯合肽的存在，说明酶解可使钙由羟基磷灰石形式转变为可溶性离子钙。研究结果可为新型钙制剂生产及畜骨的高值利用提供了参考。
위제비태개오합물병탐명태개오합궤리，대양골효원다태여 CaCl2적오합공예진행우화병대태개오합물진행표정분석。채용Box-Behnken중심조합설계급향응면분석법，학정료최가오합삼수：태개질량비2∶1，pH치7.66，53℃오합50 min，오합솔체67.24%。홍외광보、전경분석급능보소묘결과표명효원다태여Ca2+분별재다태내부적C=O처급태련말단적-NH2화-COOH 처발생오합，형성료안염화최산염；다태여 Ca2+오합후유소송적편상결구변성료치밀적소과립취집체결구。홍외광보급능보소묘동시야증실소제비적효원다태중유부분개오합태적존재，설명매해가사개유간기린회석형식전변위가용성리자개。연구결과가위신형개제제생산급축골적고치이용제공료삼고。
Preparation and identification of calcium-chelated ossein polypeptides were carried out in this study. Ossein polypeptide used for chelating was the supernatant fraction of the alkaline protease hydrolysates of sheep bone powder (with a hydrolyzing degree of 28.12%) after centrifugation. The optimal chelating condition between ossein polypeptide and CaCl2 was obtained by Box-Behnken factorial design and response surface analysis. According to the results of 4-factor 3-level response surface experimental design, a regression model of the chelating rate in response to initial pH value, temperature, reaction time, mass ratio of polypeptide to CaCl2 was established using the software of Minitab 16. Variance analysis for the regression equation indicated that the model was extremely significant (P<0.01) and the lack-of-fit was not significant (P>0.05), so the model was reliable. In light of the model, within the set level of each factor, the theoretical maximum of the chelating rate reached 66.4%. After the verification tests, the optimal chelating condition was determined: the mass ratio of polypeptiede to CaCl2 of 2:1, the pH value of 7.66 and chelating at 53℃ for 50 min, under which a chelating rate of 67.24% was obtained. After reaction, the calcium-chelated polypeptide was precipitated by ethanol (>90%), collected after centrifugation (8000 r/min, 15 min), and then dehydrated by vacuum freeze drying. NaS replacing test together with ninhydrin reaction suggested that the product was the chelate of Ca2+ and polypeptides. The infrared spectrum of the polypeptide before and after chelating did not totally overlap, so chelating reaction actually occurred between peptides and Ca2+. The wave number of distinctive absorption peak representing –COOH shifted from 1 406.75 to 1 425.92 cm-1, that representing -NH2 shifted from 3 285.84 to 3 405.31 cm-1 and that representing C=O shifted from 2 969.54 to 2 972.06 cm-1, which indicated that the 3 chelating sites were C=O inside the polypeptide, –COOH and –NH2 at the terminal of the peptide chain. Scanning electronic microscopy (SEM) showed that before chelating, the structure of the ossein polypeptides was loose and sheet-shaped, with small amount of crystal-like substance (formed through naturally chelating) on the surface of each sheet-like structure; after chelating, the loose structure turned to be tight and exhibited as the aggregation of small salt-like particles. This further proved the formation of carboxylate and ammonia salt by chelating. The energy spectrum of the SEM picture demonstrated that there were 3 Ca peaks both for the ossein polypeptides and for the calcium-chelated polypeptide. Peak 2 had the largest area in the spectrum for polypeptides, while for calcium-chelated polypeptide peak 3 had the largest area. The energy spectrum also illustrated that there were 3 sites where Ca2+ could be chelated on the polypeptides, and before chelating with exogenous Ca2+, the polypeptides could be chelated with the endogenously released Ca2+ during hydrolyzing. Element analysis of the energy spectrum demonstrated that the atom percentage of Ca increased from 1.76% to 6.84%, and the element weight percentage of Ca increased from 4.76% to 15.98%. In conclusion, SEM picture, energy spectrum and element analysis proved that the sheep ossein polypeptide itself contains calcium that can be naturally chelated, which further indicated that the enzymatic hydrolysis can cause the release of the Ca2+from the hydroxyapatite accompanied by collagen degradation. Infrared spectrum illustrated 3 chelating sites on the peptide, and they are C=O, –COOH and –NH2, respectively. The chelating technique determined in present study will be useful for the future production of new-type supplementary calcium which is safer than other forms of calcium and easily absorbed by body, for the high-value use of the bones of the live stock and for the extension of sheep-breeding industry.