硒处理对西兰花中硫代葡萄糖苷合成及代谢影响的研究

发布时间：2018-05-03 03:14

本文选题：西兰花 + 萝卜硫素　；参考：《华中农业大学》2017年博士论文

【摘要】：西兰花(Brassica oleracea.var.italic)因富含抗癌和抗氧化的活性物质(如萝卜硫素)深受广大消费者喜爱。萝卜硫素是脂肪族硫代葡萄糖苷的降解产物之一,也是迄今报道的蔬菜中抗癌能力最强的活性物质。硒元素是人体生长必需的微量元素,可激活人体生长发育过程中的关键酶,同时甲基化的含硒化合物已被证实是预防肿瘤的有效形式。鉴于西兰花同时具有大量合成硫代葡萄糖苷和富集硒元素的特性,近些年受到越来越多的关注。本研究首先以西兰花芽菜为研究对象,对其营养价值进行全面分析,设计四因素三水平响应面实验,确定了萝卜硫素的最佳提取工艺,并探明了高含量萝卜硫素的西兰花芽菜的最佳生长和储藏期。其次为了探究硒处理对西兰花中硫代葡萄糖苷合成的影响及相关机理,本研究采用一定浓度的硒酸盐处理西兰花芽菜、幼叶以及成熟花蕾。最后结合代谢组学的推论,在高浓度硒的培养液中添加适量的硫酸盐观察西兰花芽苗的生长表型并检测西兰花毒理反应,结果表明一定浓度的硫酸盐对高浓度硒的毒性具有缓解作用,本研究同样对其作用原理进行了深层次的探究。相关研究结果具体如下:1.西兰花芽菜中营养物质丰富,每100 g新鲜西兰花芽菜中大约含水分90.0 g,含蛋白质4.3-7.6 g,脂肪1.3-3.1 g。每1 g冻干芽菜中含钾17.0-30.0 mg,钙6.0-10.0 mg,钠1.5-2.6 mg,锌0.2-0.3 mg,镁4.0-6.6 mg,铁0.6-2.0 mg。西兰花芽菜中同时还含有丰富的氨基酸参与蛋白的合成。通过单因素实验和响应面实验建立二次回归模型确定西兰花芽菜中萝卜硫素提取的最佳工艺参数为:固液比1:30,加入抗坏血酸量4 mg/g冻干样品,提取时间1.5 h,提取温度65℃。基于最佳提取条件测定了发芽后2-9天的西兰花芽菜,可确定西兰花芽菜在生长期第5天时萝卜硫素含量最高,同时比较了冷冻储藏和冷藏储藏对萝卜硫素含量的影响,结果显示冷冻储藏前5天的西兰花芽菜均含有较高含量的萝卜硫素。2.硒处理发芽后的西兰花芽菜于第5天收获,测定西兰花中的主要营养成分,结果显示硒处理能显著地影响西兰花芽菜中对人体有益的初级和次级代谢产物的含量,除了总多酚含量在硒处理后略有下降,抗坏血酸,花青素以及总黄酮含量均略有上升。此外,本研究发现硫代葡萄糖苷的代谢同样也受到了硒处理的影响。实验结果表明,硒处理虽然不影响西兰花芽菜中总硫代葡萄糖苷的合成,但会影响降解硫苷的黑芥子酶的活性,并且黑芥子酶活性的变化与最终萝卜硫素的含量变化趋势保持一致。本实验中选取了三种不同品种的西兰花芽菜,结果均显示硒处理能影响黑芥子酶的活性从而影响萝卜硫素的生成。3.通过代谢组学的方法对硒处理的西兰花芽菜及空白对照的西兰花芽菜进行分析比较,结果表明硒处理对西兰花芽菜中的初级代谢产物产生了一定的影响。实验中得出的500多种差异化合物经过单变量统计分析和多变量统计分析相结合的方法并加以验证,最终筛选出约50种VIP1且p0.05的化合物。其中丝氨酸、D-赤藓糖内酯、松三糖、酪氨酸、苏-β-羟基天冬氨酸、异麦芽糖、核酮糖-5-磷酸以及部分其他氨基酸在处理后含量显著增加,处理后3-(1-吡唑基)-L-丙氨酸、戊二酸、半乳糖酸以及5,6-二氢尿嘧啶等代谢物含量显著下降。通过对差异代谢物的分析表明,β-丙氨酸和谷胱甘肽代谢途径受硒处理影响较大,由此推测硒处理可能会影响部分参与合成硫代葡萄糖苷的氨基酸的合成从而影响硫代葡萄糖苷的合成,同时硒处理后的西兰花芽菜可能需要吸收一定浓度的硫酸盐来抵抗高浓度硒的影响。4.硒处理后的嫩叶和成熟西兰花中的总硫代葡萄糖苷以及不同种类的硫代葡萄糖苷的含量显著的下降,但是西兰花芽菜不受其影响。通过实验证实硒处理虽然显著的增加了硒含量但并没有减少嫩叶和成熟西兰花中的硫含量。这一结果排除了硫吸收减少从而降低硫代葡萄糖苷合成的可能性。进一步通过测定与硫苷合成相关氨基酸的含量以及合成硫代葡萄糖苷相关基因的表达量,结果表明硒处理能够降低相关氨基酸的含量以及下调相关基因的表达量。为了验证上述结果,本研究选取成熟西兰花作为TMT标记的样本开展蛋白组学分析,结果显示硒处理后的西兰花中约384种蛋白呈下调表达,其中大部分的蛋白为参与硫代葡萄糖苷前体氨基酸合成的酶,对这些差异蛋白的KEGG代谢通路富集分析,结果表明,硒处理主要影响了合成硫代葡萄糖苷的氨基酸的合成从而进一步影响硫代葡萄糖苷的合成。5.过量的硒处理会抑制西兰花芽苗的正常生长,结合代谢组学的推论,发现在西兰花芽苗的生长环境中添加适量的硫酸盐则会缓解硒的毒性。本研究设计了西兰花芽苗在硒和硫不同组合的处理,结果表明在硫酸盐的保护下西兰花芽苗的生长表型明显的好于不含硫酸盐保护的芽苗。同时对芽苗体内总硒、可溶性硒以及不可溶性硒的含量进行了检测,结果同样表明在硫酸盐的保护下芽苗中这三种硒的含量均少于不含硫酸盐保护的芽苗。硒在植物体内的毒性主要是由于硒蛋白所引起,因此本研究对硒蛋白的含量进行测定,结果表明在含有硫酸盐的培养液中生长的芽苗硒蛋白含量显著的低于不含硫酸盐保护的芽苗中的硒蛋白含量。此外,硒参与芽苗代谢同样会产生大量的ROS从而破坏细胞膜的结构进而影响芽苗的生长,本研究通过DAB和NBT染色发现硫酸盐的保护能减少ROS产物的生成,同时通过测定细胞膜的渗透率发现硫酸盐处理的芽苗渗透率较低,这一结果表明细胞膜损伤较小。本研究进一步测定能够清除植物组织中的超氧自由基以及过氧化氢的酶的活性,结果表明芽苗生长液中加入硫酸盐时能增强芽苗中相关酶的活性从而起到缓解含硒化合物对西兰花芽苗的毒害作用。
[Abstract]:Brassica oleracea.var.italic is very popular with consumers for its anti-cancer and antioxidant active substances such as sulforaphane. Sulforaphane is one of the degradation products of aliphatic glucosinolates, and it is also the most potent active substance in vegetables reported to date. Selenium is a necessary trace element for human growth. It can activate key enzymes in human growth and development, and methylation of selenium containing compounds has been proved to be an effective form of cancer prevention. In recent years, broccoli has attracted more and more attention in the light of the characteristics of a large number of Thioglucosides and selenium enrichment in recent years. The optimum extraction process of sulforaphane was determined by four factors and three level response surface experiments, and the best growth and storage period of high content sulforaphane was explored. Secondly, the effect of selenium treatment on the synthesis of glucosinolates in broccoli and the related mechanism were studied. A certain concentration of selenate was used to treat broccoli, young leaves and mature buds. Finally, combined with the inference of metabonomics, a proper amount of sulfate was added to observe the growth phenotype of broccoli buds in the medium of high concentration selenium, and the toxicity of broccoli was detected. The results showed that the toxicity of a fixed concentration of sulphate to high concentration of selenium was found. In this study, a deep study was also made in this study. The results are as follows: 1. the nutrient substance in 1. broccoli is rich, and the moisture content of every 100 g fresh broccoli sprouts contains about 90 g, protein 4.3-7.6 g, and fat 1.3-3.1 g. per 1 g freeze-dried sprouts containing 17.0-30.0 mg, calcium 6.0-10.0 mg, Na 1.5-2 .6 mg, zinc 0.2-0.3 mg, magnesium 4.0-6.6 mg, iron 0.6-2.0 mg. New Zealand flower bud vegetables also contain rich amino acids to participate in the synthesis of protein. Through single factor experiment and response surface experiment, two regression models are established to determine the optimum extraction process parameters of sulforaphane in broccoli: the solid-liquid ratio is 1:30, and the anti bad blood acid content is 4 mg/g freeze-drying. The extraction time was 1.5 h and the extraction temperature was 65. Based on the optimum extraction conditions, the new broccoli was determined for 2-9 days after the germination. The content of sulforaphane was determined at the growth period of fifth days, and the effects of frozen storage and storage on the content of sulforaphane were compared. The results showed that broccoli for 5 days before frozen storage. The results showed that the selenium treatment could significantly affect the content of primary and secondary metabolites in broccoli, except that the content of total polyphenols decreased slightly after the treatment of selenium (.2.). The content of ascorbic acid, anthocyanin and total flavonoids increased slightly. In addition, this study found that the metabolism of glucosinolates was also affected by selenium treatment. The experimental results showed that the selenium treatment did not affect the synthesis of glucosinolates in the total glucosinolates in the broccoli, but the activity of the glucosinolase was degraded and the black mustard could be degraded. In this experiment, three different varieties of broccoli were selected. The results showed that selenium treatment could affect the activity of black mustard enzyme and influence the production of sulforaphane, and.3. was treated by the method of metabolomics to selenium treated broccoli and blank control. The results showed that the selenium treatment had a certain effect on the primary metabolites in the flower bud of sealand. 500 kinds of different compounds in the experiment were combined by univariate statistical analysis and multivariate statistical analysis, and about 50 kinds of VIP1 and P0.05 compounds were selected. The content of serine, D- erythractone, pine three sugar, tyrosine, Su - beta hydroxyl aspartic acid, ISO maltose, ribose -5- phosphoric acid and some other amino acids increased significantly after treatment. The metabolites of 3- (1- pyrazolyl) -L- alanine, glutaric acid, galactoacid and 5,6- two uracil decreased significantly after treatment. The analysis of metabolites showed that the metabolic pathways of beta alanine and glutathione were greatly influenced by selenium treatment. Therefore, it was speculated that selenium treatment might affect the synthesis of some amino acids involved in the synthesis of glucosinolates, thus affecting the synthesis of glucosinolates. Salt resists the effect of high concentration of selenium on.4. selenium treated tender leaves and mature broccoli, and the content of Glucosinolates of different types of glucosinolates significantly decreased, but the broccoli was not affected. The selenium content was significantly increased by real selenium treatment but did not reduce the tender leaves and mature West. Sulfur content in orchids. This result excludes the possibility of reducing sulfur absorption and reducing the possibility of Thioglucoside synthesis. Further by determining the content of amino acids related to glucosinolates synthesis and the expression of glucosinolates related genes, the results show that selenium treatment can lower the content of related amino acids and reduce the correlation of glucosinolates. In order to verify the above results, this study selected mature broccoli as a sample of TMT markers to carry out a proteomics analysis. The results showed that about 384 proteins were down regulated in the broccoli after selenium treatment, most of them were enzymes involved in the synthesis of amino acid precursors of glucosinolates, and KEG of these differential proteins. G metabolic pathway enrichment analysis, the results show that selenium treatment mainly affects the synthesis of amino acids of glucosinolates synthesis, thus further affecting the synthesis of glucosinolates,.5. excess selenium treatment will inhibit the normal growth of broccoli bud seedlings. Combined with the inference of metabonomics, it is found that a proper amount is added to the growth environment of broccoli buds. This study designed the treatment of different combinations of selenium and sulfur in New Zealand flower bud seedlings. The results showed that under the protection of sulfate, the growth phenotype of broccoli sprout was better than that of non sulfate protected shoots. Meanwhile, the total selenium, soluble selenium and insoluble selenium content in the bud seedlings were examined. The results also showed that the contents of these three kinds of selenium in the bud seedlings were less than those without sulfate protection. The toxicity of selenium in the plant was mainly caused by selenoprotein. Therefore, the content of selenoprotein was determined in this study. The results showed that the selenoprotein of the sprout seedlings in the culture medium containing sulfate contains the selenoprotein. The amount of selenoprotein was significantly lower than that in the bud seedlings without sulfate protection. In addition, selenium involved in the metabolism of bud seedlings also produced a large number of ROS to destroy the structure of cell membrane and then affect the growth of bud seedlings. This study found that the protection of sulfate can reduce the production of ROS products by using DAB and NBT staining, and by measuring the infiltration of cell membrane. The permeability of buds treated by sulfate was low. This result showed that the damage of cell membrane was small. This study further determined the activity of the superoxide radical and the enzyme of hydrogen peroxide in plant tissue. The results showed that the activity of the related enzymes in the bud seedlings could be relieved when the sprout seedling was added to the seedling growth liquid. Toxic effects of selenium containing compounds on sprout of broccoli.

【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TS255.3

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