【作者】 谢正军；

【导师】 金征宇；

【作者基本信息】 江南大学 ， 粮食、油脂及植物蛋白工程， 2009， 博士

【摘要】 苜蓿叶蛋白是以苜蓿叶为原料,经压榨取汁、汁液中蛋白质分离和浓缩干燥而制备的蛋白质浓缩物(Alfalfa Leaf Protein Concentration,简称ALPC)。苜蓿叶蛋白属“功能性蛋白质”,主要由叶绿体内基质蛋白和细胞质内溶解性良好的细胞质蛋白等组成。被联合国粮农组织(FAO)认为是一种具有高开发价值的新型蛋白质资源。但目前国内外对苜蓿叶蛋白的研究与开发主要集中于蛋白质的提取和利用。但对其蛋白质的功能机理及其水解后的蛋白肽和氨基酸组成研究甚少,特别是水解肽的生物活性研究更是鲜见报道。因此利用苜蓿叶蛋白研究和开发具有某种功能的生物活性肽,就具有较好的理论研究和一定的现实意义。本论文首先以不使蛋白质变性为前提,采用碱提酸沉的基本路线,以压块苜蓿为材料,通过不同的加水量、调节pH值、浸泡时间等处理以寻求最佳的提取可溶性苜蓿叶蛋白浓缩物(Soluble Alfalfa Leaf Protein Concentration,简称SALPC)的工艺与参数。得到的最优提取条件为:料液比为1∶7(w/w),碱提pH为9.0,浸泡时间为50min,酸沉pH为4.0。影响蛋白提取率的因素主次顺序为:酸沉pH>碱提pH>浸泡时间>料液比。用优化后的工艺参数提取SALPC,其提取率为57.87%,蛋白质含量高达87.38%,明显高于资料报道的其它提取方法50%-65%的得率。SALPC在pH值6～10范围内有较好的氮溶解指数NSI,最大值为77.32%。其次以体外抗氧化活性及水解度DH为考察指标,研究了SALPC的酶解工艺,从5种商品蛋白酶中确定了最适水解酶为碱性蛋白酶Alcalase FG 2.4L。通过单因素试验确定其水解SALPC的较适宜的条件为:水解温度60℃,pH值为8.0,SALPC的底物浓度为5%(w/v),[E]/[S]为4.0%(w/w)。酶解后的苜蓿叶蛋白水解物(Alfalfa Leaf Protein Hydrolysate,简称ALPHs)相对分子质量主要分布在5000以下,其中相对分子质量小于3000的组分比例为93.03%,小于1000的组分比例为67.86%,对DPPH·有较好的清除率。尽管通过酶解工艺的优化而获得分子质量相对较为集中、生物活性较强的组分,但蛋白质酶解液的成分仍然复杂多样,有必要对ALPHs进行超滤分离。其次在碱提酸沉法和水解过程中产生了一些盐分,这些盐分可能会干扰ALPHs生理活性的分析和作用。因此有必要研究大孔吸附树脂对ALPHs的吸附性能和脱盐效果。结果表明:采用相对分子质量为3 000的聚砜膜对ALPHs进行超滤分离的最适工艺条件为:操作压力0.35 MPa ,操作温度25℃,ALPHs浓度35 g/L,超滤后的ALPHs中相对分子质量1000～500的组分含量由21.85%提高到27.91%,500～130的组分含量由21.64%提高到30.83%,130以下的组分含量从4.91%提高到7.61%。并且疏水性氨基酸的质量百分比从32.00%提高到33.98%。这些疏水性氨基酸的增加使得超滤后的ALPHs在各个浓度上对DPPH·清除率都有所提高。DA201-C大孔吸附树脂对ALPHs的静态吸附与解吸的效果优于其它4种树脂。动态吸附解吸试验表明:ALPHs以50 mg/mL、0.5 BV/h流速上样、1 BV/h的流速水洗、1.5 mL/min的75%的乙醇解吸,ALPHs的脱盐率为92.21%,处理后ALPHs中疏水性氨基酸的质量百分比从33.98%提高至36.69%,脱盐后ALPHs在各个浓度上对DPPH·清除率都有所提高。ALPHs在6种体外抗氧化模型中均表现出较强的抗氧化活性,且有较显著的量效关系。这说明ALPHs是良好的电子供体和氢供体,能明显抑制亚油酸体系的过氧化作用,具有清除O2-·、·OH及DPPH·能力,对金属Fe2+具有较好的螯合作用。其清除自由基的机制可能是通过肽类对过渡金属离子的螯合来实现的;当ALPHs的浓度为GSH的3～5倍时,可达到与GSH相当的抗氧化效率。小鼠体内抗氧化试验的结果表明,ALPHs能显著降低小鼠体内MDA的含量(p<0.01),显著提高血清和心脏,特别是肝脏组织中SOD和GSH-Px的活性(p<0.01),充分说明ALPHs对小鼠具有较好的抗氧化作用。利用Sephadex G-15凝胶色谱将ALPHs分成了4个组分A、B、C和D。并分别检测了各组分的还原能力和对不同自由基的捕获能力,结果发现组分ALPHs-D为抗氧化活性最高的组分,其余三组分的抗氧化能力依次为B>C>A。利用半制备RP-HPLC将ALPHs-D分成了10个组分,以自由基捕获率为考察指标筛选出ALPHs-D-6为较强抗氧化活性的组分,经RP-HPLC分析为单一峰。结合氨基酸组成分析和MALDI-TOF-TOF二级质谱分析鉴定出抗氧化活性肽ALPHs-D-6的相对分子量为521.2,氨基酸序列为EYDP(Glu-Tyr-Asp-Pro)。更多还原

【Abstract】 Alfalfa leaf protein concentration (ALPC) extracted from alfalfa leaves by extracting, separating, concentrating and drying. ALPC as a sort of function protein mainly consists of matrix protein and cytoplasmic proteins with good solubility. It is considered by FAO (Food Agriculture Organization) as a new protein source valuable for further utilization.Researches and explorations of alfalfa leaf protein globally at present are still on the stage of extracting and application, without sufficient study on function, hydrolysates, and amino acid components. Bioactivities of hydrolyzed peptides are rarely reported. Therefore researches and development of bioactive peptides using alfalfa leaf protein is of theoretical and practical significance.Firstly, optimal process and parameters for extraction of soluble alfalfa leaf protein concentration (SALPC) from the compacted alfalfa leaves was studied by adding water, adjusting pH and marinating time, with a prerequisite of non-denaturalization of protein. The optimal condition for SALPC were a ratio 1:7 for material to liquid, an alkali pH9.0, a time of 50 minutes for marinating and an acidic pH4.0. The order of factors influencing protein extraction yield were the acidic pH > the alkali pH > marinating time > ratio of material to liquid. The extraction yield and protein content was 57.87% and 87.38% respectively at the optimal conditions. The SALPC nitrogen soluble index (NSI) was up to 77.32% at pH6.0～10.0Secondly, this project studied the enzymatic hydrolysis techniques. Five types of proteases were selected by comparing antioxidant activity and hydrolysis degree (DH) in vitro. The optimal protease is Alcalase FG 2.4L. The optimal hydrolytic conditions for SALPC was temperature 60℃, pH8.0, concentration of SALPC [S] 5% (w/v), [E]/[S] 4.0% by single-factor tests. The relative molecular weight of alfalfa leaf protein hydrolysates (ALPHs) was mostly under 5000, of which 93.03% under 3000 and 67.86%under 1000. ALPHs had good scavenging activity against DPPH radical.ALPHs were ultrafiltrated due to the multi-components in the SALPC produced with the optimal enzymatic process. The processing of hydrolysis inevitably brought in some salts that may influence the quality of the finished products and the analysis of the bioactivity of ALPHs. The effects of concentrations of ALPHs, pressure and temperature on ultrafiltration permeation were investigated. The results showed that the optimal conditions for ultrafiltration were pressure of 0.35MPa, temperature of 25℃, concentrations of ALPHs 35g/L . The relative molecular weight distribution of ALPHs in 1000～500、500～130 and under 300 increased respectively from 21.85%, 21.64% and 4.91% to 27.91%, 30.83% and 7.61%, after ultrafiltration using polysulfone membrane with 3000 relative molecule weight. The scavenging activity of ultrafiltered. ALPHs against DPPH radicals was increased because its hydrophobic amino acid content increased from 32.00% to 33.98%. The types of macroporous resin were selected by comparing their performances in adsorbing and desorbing, and the optimum absorbent material for ALPHs was DA201-C. Dynamical adsorption-desorption trials showed that desalt ratio of ALPHs reached 92.21% at 0.5 BV/h flow injecting with a 50 mg/mL concentration, 1 BV/h flow water washing and 1.5 mL/min of 75% ethanol desorbing. The hydrophobic amino acid content in ALPHs increased from 33.98% to 36.69% and the scavenging activity of ALPHs against DPPH radical was enhanced.The antioxidant activities of ALPHs evaluated using six different antioxidant tests in vitro had stronger and significant linear relationship with concentration. So ALPHs was a good electron and hydrogen donator. It can clearly inhibit peroxidation in linoleic acid system, with a strong scavenging activity against O2-·,·OH and DPPH·radicals. It can chelate well the Fe2+. ALPHs can get the same antioxidant effect when the concentration is as 3 to 5 time as glutathione.Good antioxidant effects of ALPHs in mice were proved sufficiently by trials on mice in vivo. that ALPHs was able to decrease significantly the content of MDA in mice (P<0.01), increase the activity of GSH-Px and SOD (P<0.01) in mice serum, heart and especially liver.ALPHs was divided into four fractions (A, B, C and D) by gel filtration on Sephadex G-15. The antioxidant and free radical-scavenging activities of the four fractions were measured using reducing power and DPPH/superoxide/hydroxyl radical scavenging assay. The antioxidant activity of ALPHs-D was the strongest and the other three were listed as B>C>A. The ALPHs-D was separated into 10 components in which the ALPHs-D-6 was a purified compound that had stronger antioxidant activity than others. The ALPHs-D-6 purified by using consecutive chromatographic methods had a molecular mass 521.2 Da and the amino acid sequence was identified as Glu-Tyr-Asp-Pro by amino acid analysis and MALDI-TOF-TOF MS/MS.更多还原