【作者】 章绍兵；

【导师】 王璋；

【作者基本信息】 江南大学 ， 食品科学， 2008， 博士

【摘要】 油菜在全世界范围内都是一种重要的油料作物,我国现在的油菜种植面积及油菜籽（简称菜籽）产量均居世界之首。菜籽含有丰富的油脂和蛋白质。菜籽蛋白具有平衡性强的必需氨基酸组成模式,几乎不存在限制性氨基酸,是一种优质的蛋白质。传统的制油工艺都是以取油为主要目的,得到的菜籽粕由于经过高温处理以及含有抗营养因子仅能作为饲用或肥料。在人类蛋白质资源日益缺乏的今天,提高菜籽蛋白的利用价值具有重要意义。新兴的水酶法制油工艺能够同时提取油脂和蛋白质,而且可以避免使用有机溶剂减少大气污染。本文以甘蓝型“双低”脱皮菜籽为研究对象,采用水酶法从菜籽中提取油及生物活性肽,主要研究内容及实验结果如下:论文首先研究了菜籽内源硫苷酶及硫苷的湿热稳定性,确定将菜籽置于水中煮沸5min,此时硫苷酶基本失活而硫苷的降解率为11.28%。以乳化油得率为考察指标,通过对不同酶制剂的筛选以及复配实验,最终确定将果胶酶、纤维素酶和β-葡聚糖酶按4∶1∶1（v/v/v）复配后处理湿磨菜籽浆。扫描电镜实验结果直观地揭示细胞壁多糖复合酶水解菜籽浆释放油脂的作用机理。通过单因素优化实验确定复合酶的最优水解条件为:固液比1∶5,加酶量3%（v/w）,酶解时间4h。对湿磨水酶法提油工艺所得乳状液的部分性质进行了研究,包括不同pH值下乳状液油滴表面电位、蛋白质结合量和界面膜的微观结构,在此基础上提出了水酶法提油过程中碱提的作用机理:通过提高体系的pH值,使油滴界面膜上蛋白质分子带电量增加,分子间静电斥力增强,蛋白质分子易从油滴表面脱吸,导致界面膜变薄,从而有利于蛋白酶的作用和油滴聚集,提高了清油得率。进一步研究了水酶法体系中蛋白质水解度与其乳化能力的关系,得出结论:当水解度＞10%时,蛋白质乳化能力降低可释放出被其乳化的油脂。为最大程度破乳获得清油和水解蛋白,在细胞壁多糖复合酶水解菜籽浆后,将体系pH值调至碱性并加入碱性蛋白酶Alcalase 2.4L进一步水解菜籽浆。响应面回归分析结果表明,实验范围内碱提pH对清油得率和水解度的影响并不显著（p＞0.05）,而对水解蛋白得率影响显著（p＜0.05）;随着加酶量和酶解时间的增加,清油和水解蛋白得率及水解度均显著（p＜0.05）增高;加酶量和酶解时间对水解蛋白得率具有显著的（p＜0.05）交互作用。通过对单因素以及响应面实验结果的优化,确定碱提和Alcalase 2.4L水解的最佳条件分别为:碱提pH10,温度60℃,时间45min;Alcalase 2.4L加酶量1.3%（v/w）,酶解时间160min。在优化的复合酶水解、碱提和Alcalase 2.4L水解条件下,菜籽清油和水解蛋白得率分别为74.2～75.1%和80.9～82.5%,蛋白质水解度为18.9～21.0%。通过增加洗渣以及二次破乳（低温静置—离心法和冷冻解冻—离心法）步骤可显著提高清油及水解蛋白得率。在优化的水酶法工艺条件下最终可获得88～90%的清油和94～97%的水解蛋白。和索氏抽提油相比,水酶法提取油颜色略深,酸价较高,但过氧化值低,两者的皂化价、碘价及脂肪酸组成接近。水酶法提取的菜籽油中总不饱和脂肪酸含量占90%左右,亚麻酸和亚油酸之比约为1∶2.5,因此它是一种品质优良的油脂。采用DA201-C型大孔吸附树脂对菜籽蛋白水解液进行纯化及脱苦。动态吸附与解吸实验结果表明,将菜籽蛋白水解液pH值调整为4后上柱,使用相同pH值的去离子水洗脱除盐后,再用85%乙醇溶液解吸,蛋白质回收率为66.7%。所得菜籽粗肽（crude rapeseed peptides,CRPs）味较苦,糖和灰分含量显著降低,蛋白质含量从纯化前47.0%增加至纯化后73.5%,抗营养因子硫苷和植酸均未检出。为分离出CRPs中的苦味组分,在水洗脱盐后采用不同浓度的乙醇溶液（25%,55%和85%）分步洗脱,可得到3个菜籽肽组分:RP25,RP55和RP85,其所占相对比例分别为64～66%,29～32%和5～7%。RP25颜色最浅,蛋白质含量最高（81.04%）,单宁含量最低（0.91%）,没有苦味,因此可作为理想的食品配料。采用体外模型分别研究了菜籽肽的抗氧化和抗血栓活性。实验结果表明,菜籽肽具有广谱清除自由基的能力和抑制脂质过氧化活性,并随浓度的增加而增强。RP25、RP55和CRPs清除DPPH·的ED₅₀值分别为499、41和72μg/mL;RP55和CRPs清除O₂^-·的ED₅₀分别为0.91和1.70mg/mL,两者的清除能力均显著（p＜0.05）高于RP25;在相同浓度下RP25清除·OH的能力显著（p＜0.05）高于RP55和CRPs,RP25和RP55清·OH的ED₅₀分别为2.53和6.79mg/mL。RP55和CRPs抑制脂质过氧化的ED₅₀（?）分别为4.06和4.69 mg/mL,两者的抑制能力均显著（p＜0.05）高于RP25。浓度为5.0mg/mL时,RP55抑制脂质过氧化的能力和抗坏血酸无显著差异（p＞0.05）。将RP55添加到曲奇中可以显著（p＜0.05）降低油脂的氧化程度。RP55的高抗氧化活性与其特殊的氨基酸组成、单宁和深褐色物质含量密切相关。此外,菜籽肽在一定浓度时具有显著（p＜0.05）抑制凝血酶催化的纤维蛋白形成的能力。通过分离重组实验,证实RP55的高抗氧化活性来自单宁和肽的共同作用。采用阴离子交换树脂、凝胶色谱和反相高效液相色谱对RP55进行分离纯化,最终得到1个具有较高抗氧化活性的菜籽肽单体（RP55-E2-G5-R8-F1）,其抑制DPPH·的ED₅₀为0.063mg/mL。经电喷雾—四极杆—飞行时间串联质谱分析该肽的相对分子质量为487.2,氨基酸序列为Pro-Ala-Gly-Pro-Phe。此外,还通过串联质谱分析得到相对分子质量分别为661.4和683.3的2个菜籽抗氧化肽的结构,其氨基酸序列分别为Arg-Asn-Leu（Ile）-Pro-Tyr和Tyr-Pro-Leu（Ile）-Tyr-Glu。3个抗氧化肽的氨基酸组成或/和序列信息符合已报道的抗氧化肽的结构特征。更多还原

【Abstract】 Rapeseed is one of the most important oilseeds and the production of rapeseed in China ranks first now in the world.Rapeseed contains rich oil and protein.The composition of amino acids in rapeseed protein is well-balanced.Conventional industrial processing of rapeseed involves pressing and hexane extraction,which yields two products-the oil and a low-valued meal that is mainly used as animal feed or fertilizer.It is necessary to effectively utilize the rapeseed protein for edible use when the protein sources for human are increasingly scarce.Aqueous enzymatic extraction （AEE） has emerged as a novel oil extraction technique by which oil and protein can be obtained simultaneously without use of organic solvent.In this dissertation,AEE was applied to "double-low" rapeseeds（Brassica napus） for simultaneous production of free oil and bioactive peptides.The results are as follows:The wet-heating inactivation of endo-myrosinases in intact rapeseeds and the stability of glucosinolates during the heat treatment were investigated.Results indicate that boiling intact rapeseed for 5min can inactivate myrosinases effectively and 11.28% of glucosinolates is degraded due to heating.Through screening the enzymes,the formula of pectinase,cellulase andβ-glucanase（4:1:1,v/v/v） was used to treat the rapeseed slurries.The experimental results of SEM threw light on the mechanisms of the AEE.The optimal conditions for the complex enzyme hydrolysis are as follows:the ratio of solid to liquid 1:5,enzymes/rapeseeds 3%（v/w） and hydrolysis time 4 h.The properties of emulsions from the AEE during different pH conditions was studied,including the zeta potential,the amount of bound protein and the microstructures of the surface membrane of oil droplet.The mechanisms of alkaline extraction were proposed:the pH value of the slurries is raised which would lead to an increase in the charges of the proteins on the surface of the oil droplet.As a result,the electrostatic repulsive power among the protein molecules is enhanced and then they are easily desorbed from the surface.Thus,the membrane would become thin which is in favor of the protein hydrolysis and the aggregation of oil droplets.Further,the relationship between the degree of protein hydrolysis（DH） and its emulsifying capacity was investigated.A conclusion was drawn that notable amounts of free oil could be obtained due to the decrease of the protein emulsifying capacity as the DH rose above 10%.Following the carbohydrase treatment,sequential treatments were carried out consisting of alkaline extraction and an alkaline protease（Alcalase 2.4L） hydrolysis to simultaneously produce free oil and protein hydrolysates.Response surface methodology（RSM） was used to study and optimize the effects of alkaline extraction pH,Alcalase 2.4L concentration and hydrolysis time.Increasing Alcalase 2.4L concentration and hydrolysis time significantly（p＜0.05） increased free oil,protein hydrolysates yields and the DH while the alkaline extraction pH had a significant （p＜0.05） effect on the protein hydrolysates yield.The optimal conditions for the alkaline extraction and Alcalase 2.4L hydrolysis are as follows:alkaline extraction pH 10, temperature 60℃,extraction time 45 min;Alcalase 2.4L concentration 1.3%（v/w）, hydrolysis time 160min.Under these conditions,the yields of free oil and protein hydrolysates were 74.2-75.1%and 80.9～82.5%,respectively,and the protein DH was 18.9～21.0%.Through washing the wet precipitate and a stepwise demulsification procedure consisting of storage-centrifugation and freezing-thawing followed by centrifugation,notable amounts of free oil and protein hydrolysates could be obtained. The total yields of free oil and protein hydrolysates were 88～90%and 94～97%, respectively.Compared with Soxlet-extracted oil,the content of free fatty acid of enzyme-extracted oil is higher while the peroxide value is lower.The color of enzyme-extracted oil is slightly darker than that of Soxlet-extracted oil.The iodine value,sapo（?）ificati（?）n value and fatty acid composition between them are similar.The total content ot the unsaturated fatty acids in the enzyme-extracted oil is about 90%. Therefore,the quality of the oil is high.The macroporous adsorption resins（type:DA201-C） were used to treat the aqueous phase.Aqueous phases were pooled and adsorbed onto macroporous adsorption resins to remove salts and sugars.Following extensive rinsing with deionized water （pH4）,desorption was achieved by washing with 85%ethanol（v/v） to obtain crude rapeseed peptides（CRPs）.The protein recovery was 66.7%and the protein content was enriched from 47.04 to 73.51%in the CRPs.No glucosinolates and phytic acid were detected in the CRPs.In a separate experiment,stepwise desorption was carried out with 25,55 and 85%ethanol to separate the bitter peptides from the other peptides. From the stepwise desorption,a non-bitter fraction RP25（containing 64～66%of total desorbed protein） had bland color and significantly higher protein content（81.04%） and hence was the more desirable product.The in vitro antioxidant and antithrombotic activities of crude rapeseed peptides （CRPs） and peptide fractions（RP25 and RP55） were determined.The results indicate that rapeseed peptides possess potent antioxidant activities and they are dose-dependent. The median effective dose(ED₅₀) values of CRPs,RP25 and RP55 forα,α-diphenyl-β-picrylhydrazyl（DPPH） radical scavenging were 72,499 and 41μg/mL, respectively.The ED₅₀ values for RP55 and CRPs for O₂^-·scavenging were 0.91 and 1.70mg/mL,respectively.The ED₅₀ values for RP25 and RP55 for hydroxyl radicals scavenging were 2.53 and 6.79mg/mL,respectively while the ED₅₀ values of RP55 and CRPs for inhibition of lipid peroxidation in a liposome model system were 4.06 and 4.69mg/mL,respectively.The difference between RP55 and ascorbic acid with respect to inhibition of lipid peroxidation was insignificant（p＞0.05） at a concentration of 5mg/mL.With addition of RP55 in the cookies,the lipid peroxidation was significantly （p＜0.05） inhibited during the storage.RP55 generally showed more potent antioxidant activities except for hydroxyl radicals scavenging ability than RP25 and CRPs at the same concentrations,which was thought to relate to the significantly higher contents of hydrophobic amino acid,tannin,and the brown color substances in RP55.Rapeseed peptides possess marked inhibitory activities on the thrombin-catalyzed coagulation of fibrinogen at certain concentrations.It can be established that both tannin and peptides contribute much to the potent antioxidant activities of RP55 according to the fractionation and reconstitution experimental results.A peptide（RP55-E2-G5-RS-F1） showing strong antioxidant activity was isolated from RP55 using consecutive chromatographic methods including ton-exchange chromatography,gel-filtration chromatography and RP-HPLC.Its ED₅₀ value for DPPH radical scavenging was 0.063mg/mL.The molecular mass（487.2） and the amino acid sequence of the purified peptide（Pro-Ala-Gly-Pro-Phe） were determined using electrospray ionization-quadrupole-time of flight（ESI-Q-TOF） mass spectrometry. Another two peptides（the molecular mass is 661.4 and 683.3,respectively） were also analyzed for their amino acid sequences using tandem mass spectrometry.The respective sequences were Arg-Asn-Leu（Ile）-Pro-Tyr and Tyr-Pro-Leu（Ile）-Tyr-Glu. The amino acid compositions and sequences of these peptides agreed with the reported characteristics of antioxidant peptides.更多还原