【作者】 张宾；

【导师】 汪东风；

【作者基本信息】 中国海洋大学 ， 食品科学， 2010， 博士

【摘要】 大豆乳清是工业上采用碱提、酸沉等工艺制备大豆分离蛋白的副产物。目前,我国每年排放的大豆乳清废弃液在300万吨以上,这既造成了大量的资源浪费又造成了环境的污染。而大豆乳清液中含有大量的功能性成分,如其中的胰蛋白酶抑制剂具有多种生理功能,在生物农药、医药等领域均有着广泛的应用前景。对于胰蛋白酶抑制剂的分离、纯化工作,目前仅在实验室水平上采取水浸、酸提、盐析、凝胶过滤层析等较为繁杂的方法纯化制得,耗时长、产率和纯度也不高；还有利用琼脂糖凝胶亲和纯化制得,但其材料价格昂贵且活化时具有较大毒性。黄曲霉在自然界中分布很广,能侵染饲料、花生、油料等各种食物,而黄曲霉侵染后所产生的黄曲霉毒素是化合物中毒性最强的有害物质之一。目前,关于黄曲霉对食物浸染的控制研究还仅仅局限于采用通风、控制湿度和温度等物理方法,及添加有毒的化学抑菌剂等方法,而在花生、玉米及其他农产品中添加天然生物抑菌剂,以减弱黄曲霉侵染作用的研究还鲜有报道。。本文利用壳聚糖、大豆及野生大豆为原料,将制备的壳聚糖树脂-胰蛋白酶亲和介质,应用于胰蛋白酶抑制剂的分离、纯化中,并研究胰蛋白酶抑制剂结构性质及其抗黄曲霉侵染活性,为开发控制花生、玉米等食品中黄曲霉毒素含量的生物制剂提供理论支持和应用基础。1)壳聚糖树脂制备条件的优化：当壳聚糖溶液2.0%(w/v),搅拌速度300 r/min,戊二醛终浓度3.6%(v/v),还原剂NaBH4终浓度0.06 mol/l时,制备的壳聚糖树脂尺寸均一,球状规整,平均粒径约为416μm,适合于作为固定化酶的载体。经FTIR, XRD和DSC分析发现,交联反应主要是通过壳聚糖糖环上的氨基与戊二醛的醛基结合形成Shicff碱结构,连接分子间或分子内的壳聚糖分子进而形成球状的壳聚糖树脂。此外,戊二醛和NaBH4的添加量直接影响着制备树脂的孔度值、交联度及树脂表面的微观结构等。2)壳聚糖树脂-胰蛋白酶制备条件的优化：以制备的壳聚糖树脂为载体,采用低毒、高效的环氧氯丙烷为活化剂,胰蛋白酶为偶联剂,制备了壳聚糖树脂-胰蛋白酶亲和介质。其最佳制备工艺条件为,环氧氯丙烷终浓度15%,活化温度60℃,偶联温度30℃,活化过程中加入适量的NaBH4和1,4-二氧六环。正是由于胰蛋白酶的固定化防止了蛋白肽链的折叠缠绕,保护了其三级结构或结构域不被过高的温度或环境所破坏,因此拥有较高的温度和pH耐受性,且具有良好的贮藏稳定性和操作稳定性。3)胰蛋白酶抑制剂亲和纯化：采用盐析和等电点沉淀法从大豆及野生大豆乳清液中提取胰蛋白酶抑制剂粗品,然后以制备的壳聚糖树脂-胰蛋白酶为亲和介质进行一步亲和纯化,制得大豆胰蛋白酶抑制剂。纯化后的胰蛋白酶抑制剂经SDS-PAGE分析,呈现单一谱带,相当分子量为8.2 kDa,且亲和纯化倍数、比活力及活性回收率均较高。经其复合物分析、全水解氨基酸分析及氨基酸残基的化学修饰等方法证明,纯化制备的抑制剂属于Bowman-Birk型胰蛋白酶抑制剂。4)抗黄曲霉侵染活性：大豆及野生大豆胰蛋白酶抑制剂对黄曲霉生长具有较强的抑制活性,其IC50值分别为1.6μM和1.2州。当其浓度达到1.8μM时,可极大程度的抑制黄曲霉孢子萌发和菌丝体的生长。此外,通过黄曲霉分泌的α-淀粉酶和蛋白酶的活性及其对底物(淀粉、酪蛋白)的利用情况,发现抑制剂抑制黄曲霉生长的具体机理是通过抑制α-淀粉酶和蛋白酶的活性,进而限制黄曲霉分解碳水化合物所需营养物质的产生,阻断黄曲霉毒素的分泌和产生。5)壳聚糖-胰蛋白酶抑制剂复合膜的制备及应用：以壳聚糖、胰蛋白酶抑制剂粗提取物为原料,甘油为增塑剂,即当壳聚糖浓度1.8%,抑制剂粗提取物浓度0.2%,甘油浓度1.2%,干燥温度45℃时,制备的复合膜具有较强抗黄曲霉活性。且复合膜表面及内部严紧致密,结构均一,分子间靠形成更多的氢键维持其稳定的结构。将该复合膜液涂膜于花生上,发现此复合膜对黄曲霉的侵染具有较强的抑制作用。我国是世界花生的生产、消费和出口的主要国家,对于黄曲霉毒素污染问题目前仍无最佳防治技术。本研究在充分利用大豆乳清液资源和壳聚糖资源的基础上,采用制备的壳聚糖-胰蛋白酶亲和介质,纯化制备胰蛋白酶抑制剂,并将其粗提取物应用于花生的抗黄曲霉侵染中,为花生、玉米等谷物贮藏过程中的生物防治黄曲霉技术提供理论支持和具体应用基础。更多还原

【Abstract】 Soybean is one of the rich resources in China, and many various bioactive components are present in the soybean whey wastewater byproduct of soy protein concentrate. However, there are more 3 million tons whey wastewater abandoned every year, which have caused the waste of resources and environment pollution, such as the functional trypsin inhibitors and flavonoids. Trypsin inhibitor (TI) mainly distributing in cultivated and wild-type soybean or other plant seeds, possesses various specialized functions, which has been applied in pharmacological and medical fields owning to the ability to prevent or suppress carcinogen-induced transformation, as detected in various in vitro and in vivo model systems. However, very few studies on the purification of TI are available and often the extraction procedures are long and tedious, such as extracted with water or acid, salting out, gel filtration chromatography and affinity purification with sepharose gel.Aspergillus flavus is one of the major spoilage moulds of intermediate moisture foods that can infect several agricultural crops such as peanuts, cotton, tree nuts, maize, rice, peppers, spices, and figs, resulting in the production of one of the most toxic and potent carcinogenic metabolites known to mammals, aflatoxin. Therefore, many research approaches are being used to reduce and eliminate A. flavus contamination with some physical or chemical methods. However, there are rarely questioned about cultivated and wild soybean TI anti-A. flavus activity.Therefore, the objectives of this study is to purify TI from the cultivated and wild soybean seeds with a quick, effective and scalable affinity chromatography, and further seek to determine if the trypsin inhibitor resistant to A. flavus spore germination and mycelial growth.1) Chitosan resin was prepared by reverse suspension cross-linking method with glutaraldehyde as the cross-linking agent. The influences of several parameters (stirring rate, cross-linking and chemical modifying agent) on the chitosan resin were investigated. The results showed that chitosan resin demonstrated a spherical shape and showed the appropriate pile-up density and porosity degree at levels of 300 r/min stirring rate,3.6% glutaraldehyde (v/v) and 0.06 M NaBH4. The results of FTIR, XRD and DSC showed that the cross linking reaction was took place between the amino groups of chitosan and aldehyde groups of glutaraldehyde to generate the Shicff structure, further to form the chitosan beads. The concentrations of glutaraldehyde and NaBH4 played an important role in the porosity values, cross-linking degree and microstructure of the prepared chitosan resins.2) Chitosan resin-trypsin was prepared with the chitosan resin as the carrier, epichlorohydrin as the activator and trypsin as the coupling agent. When the concentration of epichlorohydrin arrived at 15%, activating temperature at 60℃with NaBH4 and 1,4-Dioxane added, and coupling temperature at 30℃, the prepared resin-trypsin obtained the higher trypsin activity. Furthermore, the trypsin activity assay indicated that chitosan resin-trypsin could tolerate relatively high temperature (65℃) and wide pH range (5.0-9.0), compared with free trypsin (45℃, pH 6.0-8.0), caused by the immobilization of trypsin inside the porous resins increased the enzyme stability by reducing the overlapping, preserving tertiary structure and protecting the enzyme from conformational changes.3) With prepared chitosan resin-trypsin as matrix on affinity chromatography column, a Bowman-Birk TI from cultivated and wild soybean was purified (adsorption capacity of chitosan resin-trypsin for TI was about 1.33 mg/g wet matrix), with homogeneous MW of 8.2 kDa estimated by SDS-PAGE. The affinity purification factor and recovery rate of TI were much improved over established procedures. And it was further demonstrated that the purified TI belonged to the family of Bowman-Birk TI, based on the evidences of its amino acid composition, two independent binding sites for trypsin and chymotrypsin, and lysine residue as the active site for trypsin-inhibitory.4) Studying on the inhibition of A. flavus showed that the effect of TI from the cultivated and wild soybean on A. flavusα-amylase and proteases activity and aflatoxin B1 production depended on TI’s concentrations, and with inhibition IC50 at 1.6μM and 1.0μM, respectively. The resistance to A. flavus infection was partially due to the inhibition ability of TI for the endogenous a-amylase and proteases activity, thereby limiting the availability of the hydrolyzed reducing sugar for fungal growth and suppressing aflatoxin B1 biosynthesis.5) chitosan-based blend films were prepared from chitosan, TI extract and glycerol solutions, the properties of which were also investigated, including thickness, mechanical property, water vapor transmission, optical transmittance, and solubility. In addition, the resulting blend films were characterized by SEM, XRD and FTIR. The result of SEM images showed the surface and cross section of the blend films had more smooth and dense morphology than pure chitosan film. XRD and FTIR spectra indicated that the possible interaction force among the components might be the hydrogen bonds of N-H…O=C and O-H…O=C. Furthermore, the facts that the germination and growth of A. flavus were strongly inhibited by the blend films indicated that it might be useful as potential bio-control packaging against A. flavus during the peanuts and other cereals storage.Nowadays, China has become one of the major peanut production, consumption and export countries in the world. However, the peanut export volume has been hovering around 200 million dollars, due to the pollution of aflatoxins in recent years. According to determination of aflatoxins for 1685 copies of peanuts and 1172 copies of peanut oil samples from the 22 provinces by Ministry of Health Food Supervision and Inspection Institute, there were 26.3 percent and 47.3 percent of peanuts and peanut oil, which showed much more aflatoxins than the standard limit. Therefore, the objectives of this study is to purify TI from the cultivated and wild soybean seeds with a quick, effective and scalable chitosan resin-trypsin affinity chromatography, and further seek to determine if TI inhibit A. flavus spore germination and mycelial growth by inhibiting a-amylase and proteases, to provide a theoretical basis for the biological control of A. flavus technology during the peanut, corn or other grains storage.更多还原