本文選題：西蘭花 + 蘿卜硫素　； 參考：《華中農(nóng)業(yè)大學(xué)》2017年博士論文

【摘要】：西蘭花(Brassica oleracea.var.italic)因富含抗癌和抗氧化的活性物質(zhì)(如蘿卜硫素)深受廣大消費(fèi)者喜愛。蘿卜硫素是脂肪族硫代葡萄糖苷的降解產(chǎn)物之一,也是迄今報(bào)道的蔬菜中抗癌能力最強(qiáng)的活性物質(zhì)。硒元素是人體生長必需的微量元素,可激活人體生長發(fā)育過程中的關(guān)鍵酶,同時(shí)甲基化的含硒化合物已被證實(shí)是預(yù)防腫瘤的有效形式。鑒于西蘭花同時(shí)具有大量合成硫代葡萄糖苷和富集硒元素的特性,近些年受到越來越多的關(guān)注。本研究首先以西蘭花芽菜為研究對(duì)象,對(duì)其營養(yǎng)價(jià)值進(jìn)行全面分析,設(shè)計(jì)四因素三水平響應(yīng)面實(shí)驗(yàn),確定了蘿卜硫素的最佳提取工藝,并探明了高含量蘿卜硫素的西蘭花芽菜的最佳生長和儲(chǔ)藏期。其次為了探究硒處理對(duì)西蘭花中硫代葡萄糖苷合成的影響及相關(guān)機(jī)理,本研究采用一定濃度的硒酸鹽處理西蘭花芽菜、幼葉以及成熟花蕾。最后結(jié)合代謝組學(xué)的推論,在高濃度硒的培養(yǎng)液中添加適量的硫酸鹽觀察西蘭花芽苗的生長表型并檢測(cè)西蘭花毒理反應(yīng),結(jié)果表明一定濃度的硫酸鹽對(duì)高濃度硒的毒性具有緩解作用,本研究同樣對(duì)其作用原理進(jìn)行了深層次的探究。相關(guān)研究結(jié)果具體如下:1.西蘭花芽菜中營養(yǎng)物質(zhì)豐富,每100 g新鮮西蘭花芽菜中大約含水分90.0 g,含蛋白質(zhì)4.3-7.6 g,脂肪1.3-3.1 g。每1 g凍干芽菜中含鉀17.0-30.0 mg,鈣6.0-10.0 mg,鈉1.5-2.6 mg,鋅0.2-0.3 mg,鎂4.0-6.6 mg,鐵0.6-2.0 mg。西蘭花芽菜中同時(shí)還含有豐富的氨基酸參與蛋白的合成。通過單因素實(shí)驗(yàn)和響應(yīng)面實(shí)驗(yàn)建立二次回歸模型確定西蘭花芽菜中蘿卜硫素提取的最佳工藝參數(shù)為:固液比1:30,加入抗壞血酸量4 mg/g凍干樣品,提取時(shí)間1.5 h,提取溫度65℃。基于最佳提取條件測(cè)定了發(fā)芽后2-9天的西蘭花芽菜,可確定西蘭花芽菜在生長期第5天時(shí)蘿卜硫素含量最高,同時(shí)比較了冷凍儲(chǔ)藏和冷藏儲(chǔ)藏對(duì)蘿卜硫素含量的影響,結(jié)果顯示冷凍儲(chǔ)藏前5天的西蘭花芽菜均含有較高含量的蘿卜硫素。2.硒處理發(fā)芽后的西蘭花芽菜于第5天收獲,測(cè)定西蘭花中的主要營養(yǎng)成分,結(jié)果顯示硒處理能顯著地影響西蘭花芽菜中對(duì)人體有益的初級(jí)和次級(jí)代謝產(chǎn)物的含量,除了總多酚含量在硒處理后略有下降,抗壞血酸,花青素以及總黃酮含量均略有上升。此外,本研究發(fā)現(xiàn)硫代葡萄糖苷的代謝同樣也受到了硒處理的影響。實(shí)驗(yàn)結(jié)果表明,硒處理雖然不影響西蘭花芽菜中總硫代葡萄糖苷的合成,但會(huì)影響降解硫苷的黑芥子酶的活性,并且黑芥子酶活性的變化與最終蘿卜硫素的含量變化趨勢(shì)保持一致。本實(shí)驗(yàn)中選取了三種不同品種的西蘭花芽菜,結(jié)果均顯示硒處理能影響黑芥子酶的活性從而影響蘿卜硫素的生成。3.通過代謝組學(xué)的方法對(duì)硒處理的西蘭花芽菜及空白對(duì)照的西蘭花芽菜進(jìn)行分析比較,結(jié)果表明硒處理對(duì)西蘭花芽菜中的初級(jí)代謝產(chǎn)物產(chǎn)生了一定的影響。實(shí)驗(yàn)中得出的500多種差異化合物經(jīng)過單變量統(tǒng)計(jì)分析和多變量統(tǒng)計(jì)分析相結(jié)合的方法并加以驗(yàn)證,最終篩選出約50種VIP1且p0.05的化合物。其中絲氨酸、D-赤蘚糖內(nèi)酯、松三糖、酪氨酸、蘇-β-羥基天冬氨酸、異麥芽糖、核酮糖-5-磷酸以及部分其他氨基酸在處理后含量顯著增加,處理后3-(1-吡唑基)-L-丙氨酸、戊二酸、半乳糖酸以及5,6-二氫尿嘧啶等代謝物含量顯著下降。通過對(duì)差異代謝物的分析表明,β-丙氨酸和谷胱甘肽代謝途徑受硒處理影響較大,由此推測(cè)硒處理可能會(huì)影響部分參與合成硫代葡萄糖苷的氨基酸的合成從而影響硫代葡萄糖苷的合成,同時(shí)硒處理后的西蘭花芽菜可能需要吸收一定濃度的硫酸鹽來抵抗高濃度硒的影響。4.硒處理后的嫩葉和成熟西蘭花中的總硫代葡萄糖苷以及不同種類的硫代葡萄糖苷的含量顯著的下降,但是西蘭花芽菜不受其影響。通過實(shí)驗(yàn)證實(shí)硒處理雖然顯著的增加了硒含量但并沒有減少嫩葉和成熟西蘭花中的硫含量。這一結(jié)果排除了硫吸收減少從而降低硫代葡萄糖苷合成的可能性。進(jìn)一步通過測(cè)定與硫苷合成相關(guān)氨基酸的含量以及合成硫代葡萄糖苷相關(guān)基因的表達(dá)量,結(jié)果表明硒處理能夠降低相關(guān)氨基酸的含量以及下調(diào)相關(guān)基因的表達(dá)量。為了驗(yàn)證上述結(jié)果,本研究選取成熟西蘭花作為TMT標(biāo)記的樣本開展蛋白組學(xué)分析,結(jié)果顯示硒處理后的西蘭花中約384種蛋白呈下調(diào)表達(dá),其中大部分的蛋白為參與硫代葡萄糖苷前體氨基酸合成的酶,對(duì)這些差異蛋白的KEGG代謝通路富集分析,結(jié)果表明,硒處理主要影響了合成硫代葡萄糖苷的氨基酸的合成從而進(jìn)一步影響硫代葡萄糖苷的合成。5.過量的硒處理會(huì)抑制西蘭花芽苗的正常生長,結(jié)合代謝組學(xué)的推論,發(fā)現(xiàn)在西蘭花芽苗的生長環(huán)境中添加適量的硫酸鹽則會(huì)緩解硒的毒性。本研究設(shè)計(jì)了西蘭花芽苗在硒和硫不同組合的處理,結(jié)果表明在硫酸鹽的保護(hù)下西蘭花芽苗的生長表型明顯的好于不含硫酸鹽保護(hù)的芽苗。同時(shí)對(duì)芽苗體內(nèi)總硒、可溶性硒以及不可溶性硒的含量進(jìn)行了檢測(cè),結(jié)果同樣表明在硫酸鹽的保護(hù)下芽苗中這三種硒的含量均少于不含硫酸鹽保護(hù)的芽苗。硒在植物體內(nèi)的毒性主要是由于硒蛋白所引起,因此本研究對(duì)硒蛋白的含量進(jìn)行測(cè)定,結(jié)果表明在含有硫酸鹽的培養(yǎng)液中生長的芽苗硒蛋白含量顯著的低于不含硫酸鹽保護(hù)的芽苗中的硒蛋白含量。此外,硒參與芽苗代謝同樣會(huì)產(chǎn)生大量的ROS從而破壞細(xì)胞膜的結(jié)構(gòu)進(jìn)而影響芽苗的生長,本研究通過DAB和NBT染色發(fā)現(xiàn)硫酸鹽的保護(hù)能減少ROS產(chǎn)物的生成,同時(shí)通過測(cè)定細(xì)胞膜的滲透率發(fā)現(xiàn)硫酸鹽處理的芽苗滲透率較低,這一結(jié)果表明細(xì)胞膜損傷較小。本研究進(jìn)一步測(cè)定能夠清除植物組織中的超氧自由基以及過氧化氫的酶的活性,結(jié)果表明芽苗生長液中加入硫酸鹽時(shí)能增強(qiáng)芽苗中相關(guān)酶的活性從而起到緩解含硒化合物對(duì)西蘭花芽苗的毒害作用。
[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.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：TS255.3

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;Factors Influencing Glucoraphanin and Sulforaphane Formation in Brassica Plants:A Review[J];Journal of Integrative Agriculture;2012年11期

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本文編號(hào)：1836721