【作者】 高天；

【导师】 赵玉红；

【作者基本信息】 东北林业大学 ， 食品科学， 2007， 硕士

【摘要】 我国是养鹿业大国，鹿骨是养鹿业的一种副产物，具有加工与应用潜力。本文以鹿骨为原料，提取其中的胶原蛋白并研究了鹿骨胶原蛋白的一些特性，然后利用酶工程技术，水解鹿骨中的胶原蛋白，制备具有抗氧化功能的活性肽。首先，以鹿骨为原料提取酸溶性胶原蛋白（ASC）和酶促溶性胶原蛋白（PSC），并且对ASC和PSC的部分理化性质进行测定。经UV扫描分析，鹿骨的ASC和PSC的在234nm、230nm处有一个强吸收峰，符合胶原蛋白特性；鹿骨的ASC和PSC样品的红外图谱显示出蛋白质的特征吸收，而且两样品图谱基本吻合，说明两者微观结构有很大的相似性；鹿骨的ASC和PSC热收缩温度分别为55℃、58℃；经DSC和电泳测定结果表明，样品较好的保持了胶原蛋白的三螺旋结构；鹿骨的ASC和PSC样品的氨基酸组成为典型的胶原蛋白的氨基酸组成。其次，采用热水浸提法提取鹿骨胶。通过水平试验确定时间、温度、料液比对鹿骨胶提取率的影响；采用正交实验对鹿骨胶的浸提工艺条件进行优化，得到最适提取工艺参数为：温度95℃、时间3h、料液比为1∶15，此时的提取率67.9％。分别采用Alcalase碱性蛋白酶、中性蛋白酶水解、胰蛋白酶水解鹿骨胶，并确定最优水解条件。首先通过单因素试验确定温度、pH、酶用量对鹿骨胶水解度的影响。然后，采用正交实验对水解条件进行优化，得到最佳水解条件：Alcalase碱性蛋白酶水解鹿骨胶的最优组合为温度65℃、pH9.5、酶用量4500U／g；中性蛋白酶水解鹿骨胶的最优组合为温度为50℃、pH8.5、酶用量7000U／g；胰蛋白酶水解鹿骨胶的最优组合为温度50℃、pH8.5、酶用量7000U／g。在三种酶的最适条件下水解鹿骨胶，水解时间分别为3.5min、5min、10min、15min、20min、30min、45min、60min、90min、120min。然后研究不同水解度水解物的抗氧化性。研究表明，三种酶解物都显示出较强的·OH和O₂^-清除能力，明显高于未水解的鹿骨胶对两种自由基的清除能力（P＜0.01）：随着水解度的增加，水解物对·OH和O₂^-的清除率也提高；Alcalase碱性蛋白酶水解物对·OH和O₂^-的清除效果优于其它两种水解物（P＜0.01）。三种水解物均具有螫合Fe²⁺的能力，且螯合能力强于未水解的鹿骨胶（P＜0.01）；总体来说随着水解度的增加，三种水解物对Fe²⁺的螯合力在升高；且Alcalase碱性蛋白酶水解物的螯合能力最强（P＜0.01）。在硫代巴比妥酸实验中，水解物抗脂质过氧化的能力明显高于未水解的鹿骨胶（P＜0.01）；随着水解度的增高，抗脂质过氧化的能力也在升高；Alcalase碱性蛋白酶水解物抗脂质过氧化的能力强于其它两种水解物（P＜0.01）。由以上可知，水解物的抗氧化能力随着水解度升高而增强，水解物的抗氧化性强于未水解的鹿骨胶，而且Alcalase碱性蛋白酶水解物比其它两种水解物具有更强的抗氧化能力；鹿骨胶水解物可作为自由基清除剂和金属离子螯合剂抑制油脂的氧化。然后，研究了水解物功能特性。结果表明鹿骨胶水解物具有很好的溶解性，在pH值2-10的范围内NSI指数明显高于鹿骨胶；水解物具有低粘度的特性；鹿骨胶水解物的吸水性好于鹿骨胶，而鹿骨胶显示了更好的保水性。最后，采用抗氧化性较好的Alcalase碱性蛋白酶水解物，研究其在大豆油和橄榄油中的抗氧化性。结果表明水解物具有一定的抗氧化性，而且随着胶原抗氧化肽添州量的增加，其抗氧化能力也增加；另外，水解物和V_E的复合抗氧化剂抗氧化效果好于单一抗氧化剂。Alcalase碱性蛋白酶鹿骨胶水解物显示出对猪肉氧化的抑制作用，且随着浓度的增加抑制率逐渐上升。更多还原

【Abstract】 Chian is a major country of deer processing. Deer bone is one of the most plentiful byproducts in deer processing industry. In this study, the collagen of deer bone was hydrolyzed with proteinase to get antioxidative peptide.Acide-soluble collagen （ASC） and pepsin-solubilized collagen （PSC） were isolated from deer bone. The maximum absorption of ASC and PSC were 234nm and 230nm on ultraviolet spectrophotometry. By infrared spectrum we know that ASC and PSC have simiIar structure. The shrinkage temperature of ASC and PSC were 55℃and 58℃respectively. ASC and PSC are typicallyⅠcollagen.The gelatin of deer bone was extracted by hot water. The optimal conditions were found by single factor experiments and orthogonal tests, which was temperature 95℃, time 3h, ratio of decalcified deer bone to water 1:15. And the highest extration yield of deer bone gelatin was 67.9%.Deer bone gelatin was hydrolyzed with Alcalase, neutrase or trypsinase. Single factor experiment and orthogonal tests were adopted to find the optimal hydrolysis condition. That of Alcalase was temperature 65℃, pH 9.5, the amount of Alcalase 4500U/g. For neutrase or trypsinase, the optimal hydrolysis condition was temperature 50℃, pH 8.5, the amount of Alcalase 7000U/g.The gelatin hydrolysates prepared by Alcalase, neutrase or trypsinase exhibited the stronger power to arrest·OH and O₂^-radicals than nonhydrolyzed gelatin. Radical-scavenging activity increased with hydrolysis time. Alcalase-hydrolyzed gelatin had strongest power to arrest·OH and O₂^-radicals. Fe²⁺-chelating activity of hydrolysates was influenced by hydrolysis time and sorts of proteinase. On the whole, Fe²⁺-chelating activity increased with hydrolysis time. The gelatin hydrolysates of Alcalase had strongest Fe²⁺-chelating activity. And Fe²⁺chelating activity Of nonhydrolyzed gelatin was largely lower than both hydrolysates. The hydrolysates exhibited significant inhibition of lipid peroxide formation. However, Alcalasehydrolyzed gelatin exhibited stronger antioxidant activity at any of the hydrolysis time. Nonhydrolyzed gelatin showed slight inhibitory effect on lipid oxidation, lower than hydrolysates heavily. The hydrolysates of deer bone gelatin can work as a radical stabilizer and metal ion chelator to inhibit lipid oxidation.At last, hydrolysates had better solubility and hygroscopicity than gelatin. And gelatin has better Moisture retention than hydrolysates.The study showed that hydrolysates of Alcalase can inhibit oxidation of soybean oil and olive oil. The antioxidant activity increased with the amount of hydrolysates. Antioxidant containing hydrolysates and V_E showed stronger antioxidant activity than single antioxidant.更多还原