【作者】 何志平；

【导师】 茅林春；

【作者基本信息】 浙江大学 ， 食品科学， 2011， 博士

【摘要】 近年来,许多研究人员发现水果蔬菜的摄入量与预防许多疾病、延缓衰老之间存在着相关性,他们认为水果蔬菜的保健作用可能主要来源于它们的抗氧化作用。浙江山核桃是一种多年生木本植物,具有超强的抗氧化能力,是我国特有的经济林树种。本研究的目的是研究和评价山核桃的抗氧化活性以及影响山核桃抗氧化活性的因素,探索山核桃中抗氧化活性的物质基础,也对山核桃中蛋白质的开发和脂氧合酶基因进行了初步研究。本研究以以浙江山核桃、湖南山核桃、美国山核桃和大别山山核桃为原料,采用DPPH自由基清除能力、超氧阴离子去除能力和还原力为抗氧化指标,评价和比较不同品种山核桃的抗氧化能力；以福林酚和单宁为指标,比较四个品种山核桃中酚类物质的差异。以HPLC为手段,检测浙江山核桃的酚类物质的构成。结果表明在四个品种中,大别山山核桃和临安山核桃具有较强的抗氧化能力,对消费者和种植者来说具有较高的保健价值和经济价值。相关性分析结果表明酚类物质含量和山核桃的DPPH自由基清除能力、超氧阴离子去除能力和还原力均有较高的相关性(R2=0.9674；R2=0.9985；R2=0.9739),这说明山核桃中抗氧化活性物质主要为酚类化合物。HPLC检测结果表明浙江山核桃中含有最高的酚为没食子酸,达到6690μg/g脱脂山核桃；其次为儿茶素和鞣花酸,为2880和2738.5μg/g脱脂山核桃；香草酸也有一定含量,为829μg/g脱脂山核桃。本文以浙江山核桃为原料,采用DPPH自由基清除能力、超氧阴离子去除能力和还原力为抗氧化指标,评价烘烤、微波、水煮和蒸煮对山核桃抗氧化活性的影响。以总酚、单宁为指标,比较四个热处理对山核桃中酚类物质的影响。与原料相比,烘烤、微波、水煮和蒸煮处理分别导致山核桃多酚下降了4.47%、7.37%、47.90%、66.73%；单宁下降了33.65%、4.79%、46.23%和69.94%。烘烤和微波处理的山核桃的还原力、DPPH自由基清除能力、超氧阴离子清除能力和酚类物质以及单宁的含量均比水煮和蒸煮处理山核桃要高很多,相关性分析表明酚类物质是山核桃抗氧化的主要物质(R2=0.9875,R2=0.9933,R2=0.8917)。水煮和蒸煮过程中,酚类物质可能在水的作用下透过了细胞壁和山核桃的木质壳,最终溶解在热处理的水中,从而促进了酚类物质的损失。从营养的角度考虑,烘烤和微波处理的山核桃具有较高的保健功效。本研究以浙江山核桃为原料,采用DPPH自由基清除能力、超氧阴离子去除能力和还原力为抗氧化指标,评价贮藏时间、贮藏形式(带壳与不带壳)、贮藏温度、包装形式对山核桃抗氧化活性的影响。以福林酚和单宁为指标,评价上述贮藏条件对酚类物质变化的影响。以酸价、碘价和过氧化值为指标,评价上述贮藏条件对山核桃油脂品质的影响。结果表明对于山核桃的油脂品质来讲,常温贮藏油脂变质较快,即使是充氮包装,也不能有效延缓油脂的变质速度,因而山核桃适宜在低温下贮藏。对于低温贮藏来讲,核仁裸放油脂变质速度比带壳贮藏快,因而核仁在低温下也需要密封贮藏。贮藏8周后,山核桃的酚类物质有上升,但抗氧化性能却略有下降,这可能是由于山核桃在贮藏过程中酚类物质的构成发生了变化,一些抗氧化性能活性较强的酚类有所下降所导致的。本研究以以蛋白质提取率为指标,采用碱提酸沉法制备山核桃分离蛋白,对碱提酸沉工艺进行优化。以山核桃蛋白为原料,以氮溶指数、等电点(pH4.0)、起泡性、起泡稳定性、乳化性、乳化稳定性、最低凝胶浓度、持水力和持油力为指标,评价山核桃蛋白的功能特性。本研究对碱提酸沉法制备山核桃分离蛋白的工艺进行了优化,产品最终的蛋白质含量为78.26%。功能性实验表明山核桃核仁分离蛋白氮溶指数等电点(pH4.0)最小,仅为9.78%。同时pH为4时,蛋白质的起泡性也最小,但起泡稳定性最高,乳化性和乳化稳定性均急剧下降,也达到最小值。在pH4时,山核桃核仁分离蛋白的最低凝胶浓度最低(6%)。山核桃蛋白的持水力和持油力低于脱脂山核桃粉,分别为1.67±0.06g/g和1.89±0.02g/g。本研究以山核桃叶和外果皮为原料,采用DPPH自由基能力、还原力、抑制超氧阴离子能力为抗氧化指标,评价不同溶剂提取物的抗氧化活性。以福林酚为指标,评价不同溶剂提取物的酚类物质含量。结果表明经甲醇、蒸馏水、丙酮、异丙醇萃取后获得的山核桃叶提取液均具有一定的抗氧化活性。进一步的优化实验表明,60%甲醇溶液的提取物具有最高的DPPH自由基清除能力,总酚得率最高,达到10.63±0.31 mg/g山核桃叶。相关性分析表明,总酚含量与超氧阴离子自由基抑制率、还原力之间存在显著正相关(r=0.89,p<0.05；r=0.90,p<0.05)而与DPPH自由基清除率相关性不显著(r=0.69,p>0.05)。这说明核桃叶中多酚是核桃叶抑制超氧阴离子自由基和提供还原力的主要成分,而且核桃叶还存在有清除DPPH自由基的其他成分。在质量浓度为lmg/mL时,外果皮提取物还原力由强到弱依次：水提取物>VE>80%乙醇提取物>乙酸乙酯提取物；对DPPH自由基的清除能力依次为：VE>乙酸乙酯提取物>水提取物>80%乙醇提取物；对油脂抗氧化能力由强到弱依次为：VE>水提取物>80%乙醇提取物>乙酸乙酯提取物。山核桃青果皮提取物的抗氧化活性不但与总酚含量有关,而且与总酚含量的组成有着密切关联。山核桃脂氧合酶基因在山核桃脂肪氧化中具有重要的作用。本研究以山核桃为试验材料,成功克隆到山核桃LOX基因,全长为496 bP,并在GenBank进行了注册(注册号为HM747942)。同源性分析表明山核桃LOX基因与番茄、粗柠檬等LOX基因的同源性均较高。更多还原

【Abstract】 Recently, many epidemiological studies suggest that there was a correspondence between the consumption of fruits and vegetables and the prevention of the diseases and aging. This was attributed to antioxidants from vegetables and fruits. Chinese hickory (Carya cathayensis Sarg.), a perennial woody plants, is primarily natives of China. The nut from Carya genus are founded to possess high antioxidant activities. In the current study, antioxidant activities in vitro of Chinese hickory and the influence factors on the variation of them were evaluated, and involved antioxidant compounents were identified and measured.Tthe research of isolated protein from Chinese hickory and the cloning of LOX gene was also carried out in this paper.In this study, the antioxidant activity of methanol extracts from four species of the Carya genus (C dabieshanensis, C. cathayensis, C. hunanensis, C. illinoensis) was compared by various antioxidant assays, including the reducing power, DPPH radical scavenging activity and the superoxide anion scavenging activity. Total phenolic (TP) and condensed tannins (CT) were determined in all samples spectrophotometrically. At last, individual phenolic compounds were identified and quantified by HPLC methods. Among the four species, C. dabieshanensis and C. cathayensis possess the higher antioxidant activity and nutritional value and more health benefits than C. hunanensis and C. illinoensis. In addition, strong correlations of total phenolic content and condensed tannins content with reducing power, DPPH radical scavenging activities and superoxide anion scavenging activity was also found in this work. This showed that the main components contributions to the overall antioxidant capacity are phenolic compounds. Gallic acid(6690μg/g defatted kernel), catechin(2880μg/g defatted kernel) and ellagic acid(2738.5μg/g defatted kernel) were the main phenolic compounds identified by HPLC in Chinese hickory.The effect of thermal process(boiling, pressured-steam heating, microwave baking, roasting) on the phenolic compounds, condensed tannin and antioxidant activities of methanol extracts of chinese hickory kernels was investigated in this work. Several methods for determining antioxidant activities of the samples were employed, including the reducing power, DPPH radical scavenging activity and the superoxide anion scavenging activity. Total phenolic (TP) and condensed tannins (CT) were determined in all samples spectrophotometrically. Comparing with the raw materials, total phenolic content was reduced by 4.47%,7.37%,47.90% and 66.73% and CT reduced by 33.65%,4.79%,46.23% and 69.94% after roasting, microwave baking, boiling, and pressured-steam heating treatment, respectively. In this research, antioxidant activities of Chinese hickory were obviously decreased after boiling and pressured-steam heating, while little influence after roasting and microwaving. A strong correlations between TP and reducing power, DPPH radical scavenging activities and superoxide anion scavenging activity (R2=0.9875, R2=0.9933, R2=0.8917, respectively) was observed. The reduction of phenolic compounds after boiling and pressured-steam heating processing is mainly due to the fact that those compounds consequently leach into the liquid medium. The results of this work indicated Chinese hickory after roasting and microwave baking possessing higher functional benefit than other thermal process to the view of nutritional.The effect of storage conditions on the phenolic compounds, antioxidant activities of methanol extracts of Chinese hickory kernels and oil quality was investigated in this work. Several methods for determining antioxidant activities of the samples were employed, including the reducing power and DPPH radical scavenging activity. Total phenolic (TP) were determined in all samples spectrophotometrically. The oil quality were determined according to GB/T 5009.37-2003 and GB/T 5532-1995. The oxidative rancidity of the oil from Chinese hickory storing at room temperature even flushed with nitrogen is faster than at low temperature. At low temperature, the oxidative rancidity of unshelled chinese hickory is faster than inshelled samples. The total phenolic content of Chinese hickory is increased while the antioxidant activities decreased. This maybe interpreted than the composition of the phenolic compound is changed after storge.Chinese hickory (Carya cathayensis Sarg.) kernel protein was isolated from Chinese hickory kernel defatted flour by isoelectric precipitation. isolated protein was evaluated for chemical composition and selected functional properties including nitrogen solubility index, emulsifying capacity, emulsifying stability, foaming capacity, foaming stability, the least gelation concentration, the water absorption capacity and oil absorption capacity. The isolated protein contained over 72.0% dry weight (DW) of protein. Nitrogen solubility curves for isolated protein were pH-dependent in the pH range of 2.0-12.0 with minimum solubility observed at pH 4.0 (9.8%) and maximum solubility at pH 12.0 (89.9%). Minimum emulsifying capacity, emulsifying stability, foaming capacity and maximum foaming stability of isolated protein were also observed at pH 4.0. Isolated protein had a least gelation concentration of (LGC) of 6% (w/v) at pH 4.0. The water absorption capacity and oil absorption capacity was 1.67±0.06g/g and 1.89±0.02g/g, respectively.The phenolic content, antioxidant activities of the leaves and husks of Chinese hickory was investigated in this work. Several methods for determining antioxidant activities of the samples were employed, including the reducing power and DPPH radical scavenging activity. Total phenolic (TP) were determined in all samples spectrophotometrically. The effect of different solvent on antioxidant activities and total phenolic content and antioxidant activities of extracts of Chinese hickory leaves and husks were investigated in this work. Several methods for determining antioxidant activities of the samples were employed, including DPPH radical scavenging activity, reducing power and superoxide anion scavenging activity. Total phenolic was determined by folin phenol agent. The results showed that all extracts provided various antioxidant activities and methanol and water extracts possessed the highest effect. The optimum concentration of methanol water solution for the extract with highest DPPH elimination was 60%. The highest extraction yield of total phenolic was 10.63±0.31 mg gallic acid equivalents/g of fresh leaves in methanol extract. A correlations between total phenolic content and reducing power, superoxide anion scavenging activity and DPPH radical scavenging activity (r=0.89, p<0.05; r=0.90, p<0.05,r=0.69, p>0.05,respectively) was observed. This may be interpreted that there are some other compounds contributions to DPPH radical scavenging activity. The results show that the capacity of reducing power is in the order of water extracts>VE>80% ethanol extracts> ethyl extracts at the concentration of 1mg/mL. The capacity of scavenging effects on DPPH radicals is in the order of VE>ethyl extracts>water extracts>80% ethanol extracts. The order is VE>water extracts> 80% ethanol extracts>ethyl extracts inβ-carotene linoleate model system. The antioxidant activity of Carya cathayensis green husks may not depend only on the total phenolic compounds content but also on compositions of phenolic compounds.The LOX gene plays an important role in the oxidative rancidity of the oil from chinese hickory. The LOX gene was successfully cloning from the Chinese hickory with 496bp. The LOX gene was also registered at genebank (register number: HM747942). The homology analysis showed that the genome sequences affinity of the LOX gene among Chinese hickory, tomato and rough lemo are high.更多还原