【作者】 高丹丹；

【导师】 曹郁生；

【作者基本信息】 南昌大学 ， 食品科学与工程， 2010， 博士

【摘要】 棉籽蛋白氨基酸组成齐全,比例合理,营养价值接近豆类蛋白质,是一种十分重要的植物蛋白质资源。但目前对棉籽蛋白的食用功能及棉籽蛋白活性肽的生物活性方面的深入研究尚未见到较详细的报导。本课题以脱酚无毒棉籽饼粕为原料,提取棉籽蛋白,加入生物酶进行酶解,对水解物的抗氧化能力和血管紧张素转换酶(ACE)抑制活性进行分析,利用超滤、凝胶色谱、反相高效液相色谱、等方法进行产物分离纯化,并用质谱对得到的活性肽进行了结构鉴定。本论文系统研究了酶解工艺的各主要参数、产物的抗氧化活性和ACE抑制活性、抗氧化肽和ACE抑制肽的分离纯化和结构表征,为开发功能性产品提供理论依据和技术支撑。主要结果如下：1.利用碱法提取棉籽蛋白,正交法优化提取条件。采用SDS-PAGE测定棉籽蛋白的亚基成分,结果显示减法提取的蛋白组成较简单。氨基酸分析结果显示棉籽蛋白氨基酸组成独特,平衡良好。同时棉籽蛋白具有较好的保水性和吸油性,以及良好的乳化性和乳化稳定性,适合应用于食品中。2.以棉籽蛋白为底物,分别用商品酶Alcalase碱性蛋白酶、Flavourzyme风味蛋白酶、Neutrase中性蛋白酶、胰蛋白酶、木瓜蛋白酶和胃蛋白酶对其进行水解,测定了各酶解物的水解度和抗氧化能力。结果表明Neutrase中性蛋白酶水解物的抗氧化能力最强。在Neutrase中性蛋白酶水解棉籽蛋白单因素试验的基础上,采用响应面设计进行了试验优化,得到最优的酶解条件为：温度39.5℃、水解pH为7.5、水解时间5.5h,在此水解条件下,水解度可达到38.09%。3.利用Sephadex G-25凝胶色谱将棉籽蛋白Neutrase中性蛋白酶水解物(CPH)分成四个组分Fra.Ⅰ～Ⅳ,通过抗氧化活性分析,发现Fra.Ⅲ具有最强的抗氧化活性。采用半制备RP-HPLC将Fra.Ⅲ分成8个组分,以DPPH清除率为指标筛选出具有较强抗氧化活性的组分Fra.Ⅲ-5,经RP-HPLC鉴定为单一峰；采用MALDI-TOF-TOF二级质谱,鉴定抗氧化活性肽Fra.Ⅲ-5的相对分子量为857.4Da,最后经分析软件得出该肽的氨基酸组成及序列为HDDAPRF (His-Asp-Asp-Ala-Pro-Arg-Phe)。4.对棉籽蛋白的六种蛋白酶水解产物的ACE抑制能力进行了比较,结果表明木瓜蛋白酶水解产物的ACE抑制能力最高(85.61%)。在单因素试验的基础上,经响应面分析优化,得到最佳酶解条件为：水解温度为43.5℃,E／S为1.04%,pH值为7.5,该条件下制备的棉籽蛋白酶解物的水解度达到25.69%,ACE抑制率达到88.15%。利用超滤技术将木瓜蛋白酶水解物分离为四个组分,分别为：UF-Ⅰ(>30kDa), UF-Ⅱ(30～10kDa), UF-Ⅲ(10～5kDa), UF-Ⅳ(<5kDa),其中UF-Ⅳ(＜5kDa)的ACE抑制能力最高(IC50=0.792±0.079mg／mL)；利用Sephadex G-25凝胶色谱将超滤组分中的UF-Ⅳ分成四个组分Fra.Ⅰ-Ⅳ,发现Fra.Ⅱ具有最高的ACE抑制能力(0.159±0.007mg/mL)。利用半制备RP-HPLC将Fra.Ⅱ分成9个组分,发现组分2有最强的ACE抑制能力。经RP-HPLC再次鉴定组分2为单一组分。经过MALDI-TOF-TOF二级质谱分析鉴定ACE抑制肽Fra.Ⅱ-2的相对分子量为763.4Da,氨基酸序列为FPAIGMK (Phe-Pro-Ala-Ile-Gly-Met-Lys)。5.在传统方法的基础上,结合纸层析的方法改进食物源ACE抑制肽体外活性的检测方法,建立了一种在可见光区显色的马尿酸显色方法。首先确定了显色反应的最大吸收波长为459nm。并在459nm处,对反应条件进行优化,最后确定最佳的显色温度为40℃,最佳显色时间为30min,最佳的显色剂比例是0.5%。用卡托普利和棉籽ACE抑制肽作为样品进行检测验证,表明该方法能得到可信的结果,比传统方法具有更好的重复性、精确性,适合于ACE抑制肽的体外活性测定。更多还原

【Abstract】 Cotton is not only the most important fiber crop in the world but also one of the best potential sources for plant proteins after soybean. Cottonseed protein is generally regarded as potential sources of nutrients for human and animals due to their good balance in essential amino acids composition, and low level of antinutritional factors. It has also been a subject for numerous investigations. To our knowledge, however, few investigations have been done on the antioxidant and ACE inhibitory activities of cottonseed protein hydrolysate (CPH). Therefore, the antioxidant and ACE inhibitory activities of cottonseed protein hydrolysates were investigated in this study. The antioxidant peptide and ACE inhibitory peptide were separated and analyzed by using ultrafiltration, gel filtration chromatography, RP-HPLC and MALDI-TOF-TOF. The main results are as follows.1. The cottonseed protein isolates was prepared according to the alkaline method, and the extraction conditions were optimized by orthogonal test. The molecular weight, physical properties of cottonseed protein was investigated. Cottonseed protein has better function, including water absorption, oil absorption, emulsifying properties and emulsion stability. So cottonseed protein can be applied to the food industry.2. Six proteolytic enzymes, including Alcalase, Flavourzyme, Trypsin, Neutrase, Papain and Pepsin, were employed to hydrolyze cottonseed protein to produce the hydrolysates with antioxidant activity and ACE inhibitory activity. The result indicated that the cottonseed protein hydrolysate (CPH) produced by Neutrase presented stronger scavenging activity against free radicals. To achieve the maximum hydrolysis degree in the enzymolysis of cottonseed, response surface methodology was applied to determine the optimum conditions based on the single factor test. The optimum conditions were:enzymolysis temperature,39.5℃; time,5.5h; pH,7.5. Under these conditions, the hydrolysis degree reached 38.09%.3. Cottonseed protein hydrolysate (CPH) was prepared by Neutrase and was fractionated into four fractions (Fra.Ⅰ, Fra.Ⅱ, Fra.Ⅲ, and Fra.Ⅳ) by gel filtration on Sephadex G-25, the antioxidant potential of the CPH fractions and the CPH were investigated by using the classical methods, including the inhibiting effect on the autoxidation of linoleic acid, the scavenging ability to DPPH, hydroxyl radical, and superoxide radical. The results revealed that fractionⅢhas the highest antioxidant and free radicals scavenging activities. The peptide purified by using consecutive chromatographic methods had a molecular mass of 857.4 Da and amino acid sequence was identified as HDDAPRF (His-Asp-Asp-Ala-Pro-Arg-Phe) by MALDI-TOF-TOF analysis.4. Six proteolytic enzymes, including Alcalase, Flavourzyme, Trypsin, Neutrase, Papain and Pepsin, were employed to hydrolyze cottonseed protein to produce the hydrolysates of ACE inhibitory activity. Papain was the best one among six enzymes according to the ACE inhibitory activity. On the basis of single factor test, the optimum conditions for Papain reaction were determined by response surface methodology. Temperature 38.9℃, E/S ratio 1.04%, and pH 7.5 were found to be the optimal conditions to obtain high ACE inhibitory activity close to 88.15% and DH of the cottonseed protein was 25.69%. The result indicated that the cottonseed protein hydrolysate (CPH) produced by Papain had the highest ACE inhibitory activity (85.61%), and the CPH was separated into four ranges of molecular weight (UF-Ⅰ,> 30 kDa; UF-Ⅱ,30-10 kDa; UF-Ⅲ,10-5 kDa; UF-Ⅳ,< 5 kDa) by using an ultrafiltration (UF) membrane bioreactor system. Among them, UF-Ⅳshowed the highest ACE inhibitory activity (IC50=0.792 mg/mL). UF-Ⅳwas further fractionated with Sephadex G-25 gel filtration chromatography into four fractions (Fra.Ⅰ, Fra.Ⅱ, Fra.Ⅲand Fra.Ⅳ) that were composed of peptides of >2.43 kDa,2.43～0.82 kDa, 0.82 -0.35 kDa, and <0.35 kDa, respectively. Fra.Ⅱexhibited the strongest ACE inhibitory ability (IC50=0.159 mg/mL) with the yield of 41.63%. FPAIGMK (Phe-Pro-Ala-Ile-Gly-Met-Lys) had a molecular mass of 763.4 Da was separated by two step RP-HPLC from Fra.Ⅱ, and was identified by MALDI-TOF-TOF spectrometry.5. A modified spectrophotometric assay was developed for determination of angiotensinⅠ-converting enzyme (ACE) inhibitory activity of peptides derived from plant protein. This method relies on classical paper chromatography determination of hippuric acid (HA) content in the urine, which was based on the specific colorimetric reaction of HA with 4-(dimethylamino) benzaldehyde in the presence of pyridine and acetic anhydride. In this study, the maximum absorbance of HA was measured at 459 nm according to the modified method. The absorbance is reduced when the angiotensinⅠ-converting enzyme inhibitor is added, and the change of color in the reaction solution was measured by spectrophotometer for analyzing the ACE inhibitory activity. The method was more sensitive, more accurate and reproducible than the classical method, and it could be used for the screening of ACE inhibitory peptides derived from food proteins.更多还原