丝瓜褐变过程酚类物质的分析与研究

发布时间：2018-04-28 22:42

本文选题：丝瓜 + 酚类物质　；参考：《福建农林大学》2017年硕士论文

【摘要】：丝瓜药食两用,以其柔嫩多汁的果实深受人们的喜爱,在中国各地普遍种植。丝瓜果肉易出现褐变,影响其商品价值,揭示丝瓜褐变机理成为丝瓜育种和采后的研究重点。酚类物质与果蔬的褐变密切相关,是果蔬酶促褐变的主要条件之一。为探究引起丝瓜酶促褐变主要酚类物质的组分,从中筛选出抗褐变品种,本文以11个丝瓜品种为试材,优化了丝瓜酚类物质的提取方法,建立了丝瓜超高效液相色谱快速检测体系,分析了丝瓜酚类物质组分及其含量。通过体外模拟酚类物质的酶促褐变过程,结合超高效液相色谱法,确定了引起酶促褐变的主要酚类组分,并筛选出耐褐变丝瓜品种。对丝瓜酚类化合物生物合成中的关键酶LcPAL1、LcPAL2基因表达模式进行了分析。本研究为丝瓜抗褐变育种提供理论依据与方法。主要结果如下:1.优化了丝瓜酚类物质提取方法,结果显示:以乙醇为提取剂,料液比1g:20mL,强酸(pH3～4)环境下进行冰浴超声粉碎,丝瓜酚类物质提取效果最好。2.建立了超高效液相色谱法分析丝瓜酚类物质组分及其含量,结果如下:液相色谱柱为 ACQUITYUPLCBEHC18(2.1mm×100mm,1.7 μm),流动相为甲醇(A)/0.3%乙酸水溶液(B),柱温32℃,进样量2 μL,流速为0.25 mL·min-1。采用梯度洗脱,洗脱程序0～0.5 min,95%B;0.5～17.5min,95%～75%B;17.5～18min,75%～95%B。检测波长选取283nm。14种酚类化合物标准品在18 min内完全分离,样品中检测出14种酚酸。线性范围在0.05～4.0 mg· L-1,检出限(S/N=3)为0.001～0.049mg·L-1,决定系数均大于0.999,精密度、重复性、稳定性相对标准偏差均小于5%。平均回收率在95.36%～105.58%,相对标准偏差在 0.80%～3.64%。3.通过丝瓜酶促褐变体外模拟反应,确定了引起丝瓜酶促褐变的酚类物质为:绿原酸、对羟基苯甲酸和4-甲基儿茶酚,其中绿原酸为主要褐变底物。根据褐变底物的含量筛选出了耐褐变丝瓜品种C1、C7 和 C9。4.对丝瓜酚类化合物生物合成中的关键酶LcPAL1、LcPAL2基因进行表达分析,结果表明,2个基因都具有组织表达特异性,在叶中的表达量最高,根中的表达量也显著高于茎、花和果,LcPAL1基因的表达顺序为叶根果花茎,LcPAL2基因的表达顺序为叶根茎花果。丝瓜LcPAL1、LcPAL2基因在不同品种间呈差异表达,其中有棱丝瓜的表达量显著低于肉丝瓜。丝瓜果肉在25℃鲜切处理和4℃低温冷藏过程中LcPAL1、LcPAL2基因均表现为先上升再下降的表达趋势,结合所测PAL酶活和总酚含量相关性,推测丝瓜LcPAL1、LcPAL 基因在鲜切和冷藏过程中会调控苯丙氨酸解氨酶合成酚类物质,促进丝瓜酶促褐变的发生。
[Abstract]:The loofah is popular with its tender and juicy fruit. It is widely cultivated in all parts of China. It is easily browning and affects its commodity value. It is revealed that the browning mechanism of the loofah has become the focus of the research on the breeding and post harvest of the silk gourd. The phenolic substances are closely related to the browning of fruits and vegetables, and it is one of the main conditions for the enzymatic browning of fruits and vegetables. In order to explore the components of the main phenolic substances that cause the browning of the loofah, the anti browning varieties were screened. In this paper, 11 varieties of Luffa were used as the test materials to optimize the extraction method of the phenolic compounds of the Luffa. The rapid detection system for the liquid chromatography of the Luffa high performance liquid chromatography was established, and the components and content of the phenolic compounds were analyzed. The enzymatic browning process, combined with super high performance liquid chromatography, was used to determine the main phenolic components that cause enzymatic browning, and to screen out browning gourd varieties. The key enzyme LcPAL1 and LcPAL2 gene expression patterns in the biosynthesis of Luffa phenolic compounds were analyzed. This study provides a theoretical basis and method for the anti Browning breeding of silk gourd. The main results are as follows: 1. the extraction method of Luffa phenols was optimized. The results showed that using ethanol as the extraction agent, the material and liquid ratio 1g:20mL, strong acid (pH3 ~ 4) under the environment of ice bath ultrasonic pulverization, the extraction effect of Luffa phenolic substances was best.2. established by ultra high performance liquid chromatography to analyze the composition and content of silk gourd substances. The results are as follows: liquid phase color The spectrum column is ACQUITYUPLCBEHC18 (2.1mm x 100mm, 1.7 mu m), the flow phase is methanol (A) /0.3% acetic acid water solution (B), the column temperature is 32 c, the sample volume is 2 u L, the flow rate is 0.25 mL min-1., the gradient elution is used, the elution program is 0 ~ 0.5 min, 95%B, 0.5 to 95% ~ 18 min was completely separated and 14 kinds of phenolic acids were detected. The linear range was from 0.05 to 4 mg. L-1, the detection limit (S/N=3) was 0.001 to 0.049mg. L-1, the determination coefficient was greater than 0.999, the precision, repeatability, the relative standard deviation of stability were less than the average recovery of 5%. in 95.36% to 105.58%, and the relative standard deviation was 0.80% to 3.64%.3. through the silk gourd. Enzymatic browning in vitro simulated reaction to determine that the phenolic substances that cause the browning of the loofa are chlorogenic acid, para hydroxy benzoic acid and 4- methyl catechol, in which chlorogenic acid is the main browning substrate. According to the content of browning substrates, C1, C7 and C9.4. are the key enzymes in the biosynthesis of Luffa phenolic compounds, LcPA L1, LcPAL2 gene expression analysis, the results show that the 2 genes all have the specificity of tissue expression, the highest expression in the leaves, the amount of expression in the root is significantly higher than the stem, flower and fruit, the LcPAL1 gene expression sequence is the leaf root fruit stem, the LcPAL2 gene expression sequence is Ye Genjing flower and fruit. The LcPAL1, LcPAL2 gene of the silk gourd is in the different varieties. The expression of gourd was significantly lower than that of meat gourd. The LcPAL1, LcPAL2 gene of the pulp at 25 centigrade and 4 degrees centigrade in cold storage, showed a tendency to increase first and then decrease, and combined with the correlation between the PAL enzyme activity and the total phenol content, the LcPAL1 of the loofah was conjectured and the LcPAL gene was in the process of fresh cut and cold storage. It can regulate phenylalanine ammonia lyase to synthesize phenols, and promote the occurrence of enzymatic browning of Luffa.

【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S642.4

【参考文献】