摘要
油菜是我国重要的油料作物之一，菜籽饼粕中所含菜籽蛋白是一种非常优良的蛋白质和巨大的植物蛋白资源。但对菜籽蛋白、菜籽多肽食用功能及生物活性方面的深入研究尚鲜见报导。本课题以脱壳冷榨双低菜籽饼粕为原料，系统研究了菜籽分离蛋白各组分，并对清蛋白进行酶解，获得三个菜籽肽级分RSP-1、RSP-2和RSP-3。研究了菜籽肽粗品RSP-R及三个级分的体内对肿瘤生长的抑制作用和对免疫能力的影响、诱导人宫颈癌Hela细胞凋亡作用及其对血管紧张素Ⅰ转换酶(ACE)的抑制作用。主要结果如下：
1 菜籽各分离蛋白组分的研究
清蛋白和谷蛋白为华杂3号菜籽饼粕中主要的分离蛋白，分别占总分离蛋白的36.8％和31.6％。其它依次为谷蛋白(29.1％)和醇溶蛋白(2.5％)。SDS-PAGE结果显示出各组分蛋白的亚基成分，其中清蛋白组成较为简单。氨基酸检测表明菜籽分离蛋白氨基酸组成独特，平衡良好；必需氨基酸含量接近50％。同时菜籽清蛋白具有相当高的保水性和吸油性以及良好的乳化性和乳化稳定性，由于所选用的饼粕为低温冷榨原料，在加工过程中蛋白未受热变性，因而保持了蛋白固有特性，可广泛应用于食品、饮料中。
2 菜籽肽的制备
在菜籽肽的制备方面，采用双酶水解，一方面明显地提高了水解度，另一方面有效地降低了菜籽肽的苦味。解决了苦味肽在生产中阻碍多肽推广应用这一长期存在问题，并避免了进行后期脱苦的繁琐的生产工序。
以水解度为指标，兼顾考虑水解物的口感，选择碱性蛋白酶(alcalase)和复合风味酶(flavourzyme)分步水解制备菜籽清蛋白水解物。运用响应面分析法建立回归模型，确定最佳酶解条件为pH8.0，温度50℃，底物浓度4.87％，酶用量0.38AU／g。在此条件下水解1h，酶解液水解度可达14.72％左右，分步酶解的工艺条件为：alcalase反应进行1h后，加入50LAPU／g复合风味酶flavourzyme，50℃下酶解2h，水解度可达28％，得到的菜籽肽苦味极淡。
以水作为洗脱液，用葡聚糖凝胶Sephadex G-25层析分离菜籽肽，获得三个级分(RSP-1、RSP-2和RSP-3)。三个级分的蛋白质含量分别为71.76％、79.23％和86.14％；总糖含量分别为22.64％、13.88％和5.18％。凝胶渗透色谱法测得各分子量分别为：≥5000、1052和563Dal。各级分经液相色谱法测得所含氨基酸种类和含量各不相同。
朴服辉华中农义大学2004月博士学位论文
3菜籽肤抗氟化活性
通过腹腔注射菜籽肤50、100 mg/kg·d 15d，可以提高大鼠机体抗氧化能力，使血
清中MDA含量显著减少;离体实验结果表明，菜籽肤具有较强的还原能力及抑制·0H
的作用;RSP一R和RSP一1对活性氧产生有抑制作用，抑制效果的最佳浓度为0.5m留ml:
菜籽肤粗品及各级分对体外温育和H八诱导的小鼠肝匀浆初A生成有较强的抑制作
用，其中粗品RSP.R和级分RSP一1的作用最好，且有剂量效应关系;而对Fe2+诱导的
MDA生成的抑制作用稍差。对H众诱导的大鼠红细胞氧化溶血的影响均表现不明显。
4菜籽肤有抑制S，ao肿瘤生长的作用;其抑制机制一方面通过菜籽肤抗氧
化性和提高小鼠的免疫能力来发挥作用。另一方面可能是因为菜籽肤可
诱导肿瘤细胞凋亡。
100，150m叭g’d的RsP能抑制移植性肿瘤S:。的生长，同时可明显提高荷瘤
小鼠的免疫器官重量，RSP一R对巨噬细胞吞噬能力和迟发型超敏反应强度的作用，
只有高剂量组表现明显.RSP一R对HC:‘和HC.含量的提高没有显著影响。另外，RSP
使荷瘤小鼠血清中的SOD值升高，MDA值降低。说明RSP可能是通过其抗氧化性和
提高小鼠的免疫能力来抑制肿瘤生长。
首次揭示菜籽肤有诱导肿瘤细胞凋亡作用，MTT实验证明，菜籽肤对宫颈癌Hela
细胞增殖有明显抑制作用。并呈时间一效应和剂量一效应关系。
菜籽肤处理后的Hela细胞倒置显微镜下表现较为疏松，贴壁不牢，而且细胞触
角伸长、变形，圆形细胞增多。电镜下可看到细胞微绒毛消失，有较多的球状突起，
细胞体积变小，染色质轻度浓缩，凝聚;核物质致密，呈斑块状，或呈新月形边集
于核膜，细胞胞浆空泡化化。荧光双染色后也出现凋亡细胞典型特征:细胞核为致
密浓染的橘红色荧光，核染色质凝集深染、细胞皱缩和核固缩，形状为条状、新月
状甚至碎片状。TUNEL检测发现阳性特征，并且所出现的凋亡特征均和菜籽肤处理
浓度呈正相关，说明菜籽肤诱导细胞凋亡可能是其抗癌作用的重要机制之一。
单细胞电泳、DNA电泳检测、流式细胞仪分析及RT一PCR结果表明，菜籽肤可以
降解Hela细胞DNA、引起细胞S期阻滞和下调B。1一2基因可能是其诱导宫颈癌细胞
凋亡的主要原因。
5首次证明菜籽肤有降血压活性
Alcalase碱性蛋白酶单酶水解菜籽清蛋白可获得具有抑制血管紧张素I转换酶
(ACE)活性的菜籽肤，实验所选取的时间段内是水解1小时所得的菜籽肤抑制ACE
菜辛争几人的制杏及其生物亏舌性的研究
活性最高，为42 .68%。通过Alcalase碱性蛋白酶和Flav。址刃价e双酶水解可有效地
提高菜籽肤抑制血管紧张素I转换酶(ACE)的活性，当加入第二种酶水解2小时时，
AcE活性可被抑制69.13%。sePhadexG一25分离所得菜籽肤各级分均具有不同程度
的
Rapeseed is increasingly becoming a major crop in China. Meal proteins have a well-balanced composition in regard to FAO requirements. But there are little reports on esculent function and bioactivity of rapeseed protein and peptide.Defatted and dehulled meal of double-zero rapeseed variety (HuaZa-3) was used as material in this study. A research on preparing rapeseed protein fractionation and part of their functional properties were performed. Based on this, rapeseed albumin was sequentially hydrolyzed with alcalase and flavourzyme and was separated into three purified three grades-RSP-1, RSP-2 and RSP-3.The bioactivities of crude rapeseed peptide(RSP-R) and its purified grades were investigated, including the antioxidant activities, inhibition effect of tumor growth and influence on immune ability , induce the apoptosis of Hela cells, inhibiting effect on angiotension I-converting enzyme(ACE) activity of SHR. (in Vitro).This dissertation researched RSP systematically from preparation to bioactivities. The
main results are shown as follows:
1 Classification of rapeseed protein isolate
Albumins and globulins were found to be the predominant proteins in Hua-Za 3 meal, comprising 36.8% and 31.6% of total proteins. While glutelins and prolamins were 29.1% and 2.5%,respectively. The molecular weight of fractionation was shown by SDS-PAGE and the structure of albumins is simple. Amino acid profile of the isolates indicated that the essential amino acid of each protein fraction nearly reach to 50%. Albumins have better function, including water absorption, oil absorption, emulsifying activity and emulsion stability. In addition, the albumin maintains their native capability because of low-temperature pressing, So albumin can be widely applied in the food and beverage industry.
2 Preparation of rapeseed peptides
Double-enzyme can not only increase the DH ,but remove the bitter of RSP.In our experiment,Rapeseed Albumin was hydrolyzed with sequentially alcalase and flavourzyme. Degree (DH) were used as response values in analysis of response surface regression (RSREG), considering the mouth-sense , the optimum conditions of alcalase
enzymatic hydrolysis have been determined by mono-factor analysis and response surface methodology as follows, pH :8.0, hydrolyzing temperature: 50.1C,enzyme concentration: 0.38AU per gram of substrate, concentration of substrate:4.87%. Flavourzyme is used by step for 2h after reacted lh with alcalase, the degree of hydrolysis of rapeseed albumin can go up to 28%.
Hydrolysates were clarified by filtration to remove insoluble substrate fragments, and the filtrate was lyophilized and freeze-dried for further use. The hydrolysates(RSP-R) was graded by Sephadex G-25 column with distilled water as eluant .fractions were pooled into three major groups(RSP-l , RSP-2 and RSP-3).Protein content in RSP-l,RSP-2 and RSP-3 were 71.76%, 79.23% and 86.14%;crude polysaccharides were 22.64%, 13.88% and 5.18%. Molecular weight of each grade were bigger than 5000,1052 and 563 Dal. Contents of amino acids determined by HPLC in three grades differed from each other.
3 Antioxidant activities of rapeseed peptides
After injecting 50,100 mg/kg-d 15d for 15 d, MDA levels in serum in the RSP group were obviously lower than that of the control (P < 0. 05) ;So RSP could improve the antioxidant capacity in vivo. The results in vitro showed that RSP had higher reductive activity and can scavenge hydroxyl radicals.
RSP had powerful inhibiting activity on active oxygen within certain concentration. As for malondialdehyde(MDA) formation and H2O2-induced of mice liver ,RSP-R and RSP-1 had better inhibiting effects than RSP-2 and RSP-3 .exhibiting dosage-depended . but little inhibiting effect on MDA induced by Fe2+. There were not much influence of RSP on hemolysis of mice red blood cell. In a word, antioxidant activities of RSP-R and RSP-1 were better than others.
4.RSP can inhibit the growth of S180 tumor,the mechanism may have something to do with its antioxidant activities and increasing organize
immune capa
引文
1 何照范．粮油籽粒品质及其分析．农业出版社，1983
2 吴谋成，袁俊华．双低油菜产业化开发的研究与建议．作物研究，2000，14，(4)：1-4．
3 胡志和，庞广昌，王凤玲，陈庆森，阎亚丽，刘剑宏．萃取超滤技术制备食用菜籽蛋白．食品科学，1999(1)：34—37
4 郭尧君．生物化学与生物物理进展．SDS电泳技术的实验考虑及最新进展．1991，18(1)：32-37
5 郭兴凤，周瑞宝，汤坚等．菜籽蛋白的制备．郑州工程学院学报，2001，22(1)：60-62
6 曾晓波，吴谋成，李小定．菜籽肽对小鼠肿瘤生长抑制和免疫功能的影响．营养学报，2002，24(4)：405—407
7 熊志勇，夏伏建，陆师国．优质油菜籽粕蛋白质分类研究．武汉植物学研究．2001，19(3)：259-261．
8 A. A. Bestchart, R. Y. Fong and R. M. Saunders. Rice By-products: Comparative Extraction and precipitation of Nitrogen form U. S. and Spanish Bran and Grem. 1977,
9 A. Rao, H. E. Shallo, A. P. Ericson and R. L. Thomas. Characterization of soy protein concentrate produced by membrane ultrafiltration. 2002, 67(4): 1412-1418
10 Donna M. Klockeman, Romeo Toledo, and Kevin A. Sims. Isolation and Characterization of Defatted Canola Meal Protein. J. Agric. Food Chem. 1997, 45: 3867-3870.
11 E.C.Henley, J. M. Kuster. Protein Quality Evaluation by Protein Digestibility-Corrected Amino Acid Scoring. Food Technology. 1994,4: 74-77.
12 E Helmy Rahma, Steffi Dudek, Ralf Mothes, Eckhard Gomitz and K. Dieter Schwenke. Physicochemical characterization of mung bean protein isolates. 2000,80:477-483
13 Michele. Dalgalarrondo, Jean-Michel Robin and Jean-Louis Azanza. Subunit composition of the globulin fraction of rapeseed (Brassia Napus L.). Plant Science, 1986, 43,115-124
14 Javier Vioque.Raul Sanchez-Vioque,Alfonso Clemente. et al. Production and Characterization of an Extensive Rapeseed Protein Hydrolysate.JAOCS. 1999, 76(7): 819-823.
15 Youle, R. L. Huang, H. C. A. Occurrence of low M.W. and high cysteine containing albumin storage proteins in oilseeds of diverse apeices. Am. J. Bot. 1981.68:44-48.