【作者】 邵元龙；

【导师】 董英；

【作者基本信息】 江苏大学 ， 食品科学， 2009， 博士

【摘要】 我国是芝麻生产大国,资源丰富,仅芝麻饼粕的年产量就在50万吨以上。芝麻饼粕中含有40～60%的蛋白质,0.5～1.0%的木脂素,但目前主要作为饲料或肥料使用,深加工和综合利用的研究尤显不足。本课题旨在通过发酵芝麻饼粕提高其抗氧化活性,充分利用芝麻饼粕资源。用酱油曲霉发酵芝麻饼粕,对发酵基质组成和乙醇提取条件进行了优化,研究了木脂素提取物抗氧化活性和降低高血脂的作用;对发酵芝麻饼粕提取物中产生的特征物质进行分离纯化和鉴定,确定其为芝麻素酚,并对芝麻素酚的抗氧化活性进行了研究。主要研究结果如下:（1）优化了酱油曲霉发酵芝麻饼粕提高抗氧化活性发酵基质组成和木脂素提取条件。发酵基质组成的优化结果为:芝麻饼粕中添加辅料葡萄糖、硫酸铵和麦麸量分别为:1.9%、1.6%和2.6%;于28℃条件下固体发酵120 h;木脂素提取条件的优化结果为:乙醇浓度60%、液料比16 mL/g、时间120 min、温度50℃、转速150 r/min。优化的芝麻饼粕发酵提取物清除DPPH自由基活性为87.9%,未发酵提取物为53.4%,发酵的提取物中总酚含量为2.23%,未发酵的1.85%。（2）对芝麻饼粕发酵提取物（SCFE）体外抗氧化活性进行了研究,其活性优于芝麻饼粕提取物（SCE）。主要结果为:浓度为0.1～1.0 mg/mL时,SCFE的总抗氧化能力（TAC）和还原能力均随浓度的增大而逐渐增强,浓度高于0.5mg/mL时,增强效果显著;浓度为0.5 mg/mL和1.0 mg/mL时,SCFE对DPPH自由基清除率分别为72.7%和87.5%,而SCE为53.7%和70.6%;浓度为1.0 mg/mL时,SCFE对O₂^-·的清除率比SCE清除值高20%;浓度为1.0 mg/mL时,SCFE对自发和H₂O₂诱导的鼠肝脏微粒体脂质过氧化抑制率分别为84.4%和67%,SCE的抑制率分别为76.2%和52.3%;在0.1～1.0 mg/mL时,SCFE对亚油酸脂质氧化的抑制率逐渐增强,优于SCE;0.1%的芝麻木脂素提取物SCFE抑制冷藏熟肉糜脂质过氧化效果与0.02%的Vc作用相当。（3）通过动物试验,对SCFE预防和调节小鼠高血脂的作用进行了研究。结果发现SCFE既能有效降低小鼠的血脂水平,改善血脂代谢,减少动脉粥样硬化的发生几率,又能有效提高小鼠体内的抗氧化水平,增强抗氧化酶活性,减少过氧化脂质含量,且SCFE的作用效果优于SCE。主要结果为:诱导模型小鼠体内的抗氧化能力和抗氧化酶活性性显著下降,氧化的代谢产物量显著提高;灌胃SCFE和SCE后,显著降低总甘油三酯（TG）、总胆固醇（TC）、低密度脂蛋白胆固醇（LDL-C）及动脉硬化指数（AI₁,AI₂）,显著提高高密度脂蛋白胆固醇（HDL-C）含量;此外还显著提高小鼠血清的总抗氧化能力（TAC）和超氧化物歧化酶的活性（SOD）,降低脂质过氧化物丙二醛（MDA）含量,在剂量范围内,呈现显著的量效关系,给药SCFE的小鼠血清中各指标均显著优于SCE;比较了预防组和调节组中相同指标的相关系数,发现SCFE和SCE对高脂高胆固醇小鼠的预防效果要优于调节的效果。高血脂小鼠血脂水平和动脉硬化指数与其氧化-抗氧化水平密切相关,二者相互促进。（4）对SCFE中的特征物质P_X进行分离纯化和鉴定。SCFE经过乙酸乙酯萃取,硅胶柱层析得到纯化的P_X,得率分别为27.19%和1.43%;硅胶柱色谱分离纯化P_X用石油醚/乙酸乙酯=3/1和1/1两种比例洗脱剂洗脱;硫酸显色后,薄层层析（TLC）中P_X的迁移率R_f为0.20,紫外扫描为单一峰;P_X两个特征吸收波长分别为228 nm和287 nm;HPLC中其保留时间为13.5 min;LC-MS定性检测,分子量为370.0。该特征物质被鉴定为芝麻素酚,分子组成为:C₂₀H₁₈O₇。（5）对芝麻素酚体外抗氧化活性进行了研究。芝麻素酚具有抑制大鼠肝脏微粒体自发及H₂O₂诱导的脂质过氧化作用。在抑制自发性肝脏微粒体脂质过氧化作用时,芝麻素酚浓度从10μg/mL上升至100μg/mL时,SOD酶活性从130.7u/mgprot提高到198.3 u/mgprot,MDA抑制率从21.7%提高到61.8%;在亚油酸-吐温乳化系统和亚油酸-乙醇系统中,芝麻素酚均具有抑制亚油酸脂质氧化的作用,在乳化系统中,芝麻素酚的抑制效果优于BHT;浓度为20μg/mL时,芝麻素酚清除DPPH自由基的效果与5μg/mL BHT和Vc相当。通过生物转化芝麻饼粕中木脂素的研究,为芝麻饼粕深度加工和利用奠定了基础。利用微生物发酵技术,对芝麻饼粕中木脂素进行了生物转化,并分离纯化得到具有强抗氧化活性的特征物质芝麻素酚,为进一步开发利用芝麻饼粕提供理论依据。更多还原

【Abstract】 The resource of sesame is very abundant in our country,and the by-product of sesame processing is more than 50 thousand tons annually.The sesame cake contains 40～60%protein and 0.5～1.0%lignans,and is always used as feed or muck,and the research of deep processing and comprehensive utilization of sesame cake is far away from success.In this paper,the final objective is to improve the antioxidant activity of sesame cake by fermentation and to make full use of sesame cake resources.We optimized the composition of fermentation substrate and the condition of ethanol extraction in fermenting sesame cake with Aspergillus sojae,the antioxidant activity in vitro and decreasing hyperlipidemia in vivo were studied.A characteristic ingredient was isolated and purified from sesame cake fermented extract and named as sesaminol through structural analysis.The antioxidant activity of sesaminol was studied.The major results are as follows:（1） The fermentation substrate composition and extraction condition of increasing antioxidant activity were optimized in sesame cake by Aspergillus sojae.The optimized fermentation substrate composition were as follows:amount of glucose, ammonium sulfate and wheat bran in substrate were 1.9%,1.6%and 2.6% respectively,and fermentation temperature and time were 28℃and 120 h respectively.The optimized extraction conditions were:ethanol concentration 60%, ratio of solution to material 16 mL/g,time 120 min,temperature 50^C and rotary speed 150 r/min.The peak area and height of characteristic material from optimum sesame cake fermentation extraction（SCFE） was increased by 88 and 51 times than that of sesame cake extraction（SCE）,respectively.The DPPH radical scavenging activity was increased from 53.4%to 87.9%,and the total phenol content was increased from 1.85%to 2.23%.（2） The antioxidant activity of SCFE in vitro was studied,which is better than SCE.The total antioxidant capacity（TAC） and reduction ability of SCFE accentuated with concentration increasing in the range of 0.1～1.0 mg/mL,and the activity significantly increased at 0.5 mg/mL.The DPPH radical scavenging activity of SCFE at 0.5 mg/mL and 1.0 mg/mL was 72.7%and 87.5%,respectively,while that of SCE was 53.7%and 70.6%,respectively.The O₂^-·scavenging ability of SCFE at 1.0 mg/mL was 20%higher than that of SCE.The inhibition ratio of SCFE on the lipid peroxidation in liver homogenate spontaneously and H202 induced at 1.0 mg/mL was 84.4%and 67%,respectively,while that of SCE was 76.2%and 52.3%,respectively. The antioxidant effect of SCFE was higher than that of SCE on preventing lipid peroxidation of linoleic acid,which was increased in a dose-dependent way within the concentration range of 0.1 ¹.0 mg/mL.The antioxidant activity of SCFE at 0.1%was comparable to that of Vc at 0.02%on inhibition of refrigerated cooked ground pork oxidant.（3） Simulation test of prevention and regulation of SCFE on hyperlipidemian rat were studied.The result showed that SCFE could effectively decrease blood lipid level of SD rats with hyperlipidemia,improve blood lipid metabolism,reduce the formation of atherosclerosis.Meanwhile,SCFE effectively increased antioxidative level of rat body,improved antioxidase activities,reduced peroxidative lipid contents, and the effect of SCFE was much better than SCE.Main features were:the antioxidant activity and antioxidase decreased significantly,and the oxidation metabolin increased significantly in hyperlipidemian rat.When received intragastric administration with SCFE and SCE,they significantly decreased levels of triglyceride （TG）,total cholesterol（TC）,low density lipoprotein cholesterol（LDL-C） and atherosclerosis index（AI）,and significantly increased high density lipoprotein cholesterol（HDL-C）.In addition,they significantly increased total antioxidation capacity（TAC）,superoxide dismutase（SOD） activities of rat serum,decreased malonyldialdehyde（MDA） contents and displayed significant dose-dependence.The effect of SCFE on decreasing blood lipids in hyperlipidemia rat serum index was much better than that of SCE at the same concentration.Comparing the coefficient of correlation of prevention array with regulation array,the former was better.The blood lipid level and atherosderosis index of rats with hyperlipidemia correlated closely with oxidation and antioxidation levels,both of which were cause and effect mutually and promoted each other.（4） The characteristic ingredient P_x was isolated,purified and identified in SCFE. It was obtained by ethyl acetate abstraction and silica gel column chromatography. The condition of silica gel column chromatography was as follows:petroleum benzin/acetoacetate eluted fraction（3:1 and 1:1,v/v）.The UV scan of P_x showed one peak by thin layer chromatography（TLC）,and the transport ratio of P_x after sulphuric acid carbonization was 0.20.The UV scanning spectrum max wave of P_x was 228 nm and 287 nrn,respectively.The retention time was 13.5 rain in HPLC.The molecular weight was analyzed by LC-MS and showed to be 370.0.The material was identified as sesaminol and the molecular formula is C₂₀H₁₈O₇.（5） The antioxidant activity of sesaminol was studied.Sesaminol has inhibitory function on lipid peroxidation in liver homogenate spontaneously and H₂O₂ induced, the superoxide dismutase（SOD） activity increased from 130.7 u/mgprot to 198.3 u/mgprot and maleic dialdehyde（MDA） inhibition ratio increased from 21.7%to 61.8%in liver homogenate spontaneously when sesaminol concentration increased from 10μg/mL to 100μg/mL.Sesaminol prevented lipid peroxidation both in linoleic acid-tween emulsion system and linoleic acid-ethanol system.The inhibitory effect of sesaminol was better than BHT in emulsion system.The DPPH radical scavenging activity of sesaminol at 20μg/mL was equivalent with BHT and Vc at 5μg/mL.The deep processing and exploitation of sesame cake were studied through biotransformation of the sesame cake lignans.We carried out the biotransformation of sesame lignan and isolated a characteristic ingredient which named sesaminol,and the result will provide a theoretical basis to deeply develop and utilize sesame cake.更多还原