不同老化时间大豆种子活力测定及其酚类化合物的分析

发布时间：2018-04-20 13:17

  本文选题：大豆 + 老化　； 参考：《南昌大学》2017年硕士论文

【摘要】：大豆(Glycine max(L.)Merr)是一种富含油脂和蛋白质的豆科植物,是世界重要粮食作物之一。然而,大豆种子属于短命种子,随着储藏时间的延长,大豆种子发生劣变,种子活力下降,发芽率和发芽势降低,从而导致大豆的利用价值和种用价值降低。因此,寻找有效的方法检测大豆种子活力具有积极的生产实践意义。本文采用电喷雾萃取电离质谱(Extractive electrospray ionization mass spectrometry,EESI-MS)技术直接检测不同活力大豆种子水提液,获得其正离子模式下指纹图谱,结合主成分分析(Principal component analysis,PCA)、聚类分析(Cluster analysis,CA)和判别分析(Discriminant analysis,DA)等多变量分析方法对其进行区分,实现不同活力大豆种子的快速评价。同时,采用高效液相色谱(High performance liquid chromatography,HPLC)和EESI-MS技术分析大豆种子老化过程中酚类化合物的动态变化。主要研究内容及结果如下:1、采用人工加速老化法对种子进行处理,获得不同活力水平的大豆种子,老化后的种子分别进行标准发芽试验,以未经老化处理的种子为对照,获得大豆种子老化过程中各项发芽指标和种子活力的变化规律。结果表明,未经老化处理的大豆种子的发芽指标和活力指数与经老化处理后的大豆种子之间的差异极显著(P0.01)。随着老化处理时间的延长,种子的各项发芽指标和活力指数均呈下降趋势,老化至第8 d时,种子的各项发芽指标和活力指数均为0,说明种子活力随着老化处理时间的延长而下降,直至完全丧失。种子活力指标与老化处理时间相关性分析表明,活力指标与老化时间都呈极显著的负相关关系。2、为了对不同活力的大豆种子进行快速鉴别,本文采用EESI-MS,在无需复杂样品预处理前提下,对不同老化处理时间的大豆种子水提液进行直接质谱分析,获得EESI-MS指纹图谱。PCA的前3个主成分因子的累计贡献率为81.50%。CA和DA的正确率都为100%,对外部验证样本进行DA,正确率为90%。通过质谱鉴定的物质有γ-氨基丁酸、谷氨酰胺和乙酰基染料木苷,实验结果表明,随着大豆种子老化处理时间的延长,γ-氨基丁酸含量逐渐增加,谷氨酰胺与大豆异黄酮中的乙酰基染料木苷含量逐渐减少。EESI-MS结合多变量分析能对不同活力的大豆种子进行有效区分。3、本文采用HPLC对经老化处理过程中大豆种子的19种酚类化合物进行了定性定量分析,结果表明,大豆种子在老化处理过程中19种酚类化合物的含量均发生了明显的变化。其中,原儿茶酸、芦丁和桑黄素的含量随老化处理时间的增加而减少,分别从183.60μg/g、14.03μg/g和55.03μg/g降至138.10μg/g、12.37μg/g和38.60μg/g;黄豆苷元、黄豆黄素、染料木素和黄芩黄素的含量随着老化处理时间的增加而增加,分别从12.57μg/g、痕量(Trace;Tr)、9.10μg/g和0.83μg/g增加至91.97μg/g、6.60μg/g、76.10μg/g和3.53μg/g;咖啡酸、表儿茶素、阿魏酸、黄豆苷、染料木苷和白藜芦醇的含量随老化处理时间的增加呈先上升后下降的趋势,其中阿魏酸的含量在老化处理2 d时最大,其余5种酚类化合物的含量均在老化处理4 d时最大;绿原酸、杨梅素、肉桂酸、山奈酚、对-硝基苯甲酸和鹰嘴豆芽素A的含量随着老化处理时间的增加呈现出不规则的变化,其中绿原酸、杨梅素和山奈酚的变化趋势相同,肉桂酸、对-硝基苯甲酸和鹰嘴豆芽素A的变化趋势一致。19种酚类化合物的总含量随老化处理时间的增加呈先上升后下降的趋势,在老化处理第4 d时达到最大值6830.88μg/g。4、进一步采用EESI-MS对不同老化时间的大豆种子醇提取物进行直接质谱分析,获得其正离子模式下指纹图谱,并对四种典型异黄酮进行定性定量分析。结果表明,不同老化处理时间的大豆种子黄豆苷元、染料木素、黄豆苷和染料木苷的含量呈极显著差异。黄豆苷元和染料木素含量随老化时间的增加而增加;黄豆苷和染料木苷含量则随老化时间的增加呈现先上升后下降的趋势,在老化处理4 d时达到最大值。此结果与HPLC实验结果中黄豆苷元、染料木素、黄豆苷和染料木苷含量变化趋势相同。同时,对不同老化处理时间大豆种子的醇提物指纹谱图进行PCA分析,结果表明,不同活力大豆种子醇提物中的物质差异显著,能够被明显区分在PCA二维空间的不同的区域。本文采用EESI-MS与HPLC作为研究平台,对不同活力大豆种子进行快速鉴定,并对大豆种子老化过程中酚类物质的动态变化作了较系统的研究,建立了快速、灵敏、有效大豆种子样品质谱检测新方法,为大豆种子的贮藏及种质资源的研究与利用提供理论依据。
[Abstract]:Glycine max (L.) Merr is a kind of bean plant rich in oil and protein. It is one of the most important food crops in the world. However, soybean seeds belong to the short life seeds. With the prolongation of storage time, the seeds of soybean are deteriorated, the vigor of the seeds descends, the germination rate and the germinating potential are reduced, thus the use value and the value of the soybean are reduced. Therefore, it is of great practical significance to find effective methods to detect the vigor of soybean seed. This paper uses electrospray ionization mass spectrometry (Extractive electrospray ionization mass spectrometry, EESI-MS) to directly detect the water extract of soybean seeds with different vigor, and obtain the fingerprint of its positive ion mode and combine the principal component. Principal component analysis (PCA), cluster analysis (Cluster analysis, CA) and discriminant analysis (Discriminant analysis, DA) are used to distinguish them and realize the rapid evaluation of different vigor soybean seeds. The main research contents and results are as follows: 1, the seeds were treated with artificial accelerated aging method, and the soybean seeds with different vigor were obtained. The aged seeds were tested for standard germination respectively, and the aging of the seeds was compared to the aging of soybean seeds. The results showed that the difference between the germination index and the vigor index of the untreated soybean seeds was very significant (P0.01). The germination index and the vigor index of the seeds decreased with the prolongation of the aging treatment time. When aging to eighth D, the germination index and vigor index of the seeds were 0, indicating that the seed vigor decreased with the prolonging of the aging treatment time, until completely lost. The correlation analysis between the vigor index and the aging time showed that the vigor index and the aging time had a very significant negative correlation of.2, in order to be different vitality. The rapid identification of soybean seeds was made by using EESI-MS. Under the precondition of no complex sample pretreatment, the soybean seed water extract with different aging time was analyzed by direct mass spectrometry. The cumulative contribution rate of the first 3 principal component factors of the EESI-MS fingerprint.PCA was 81.50%.CA and DA, both of which were 100%, and the external verification samples were verified. DA, the correct rate of 90%. was identified by mass spectrometry: gamma aminobutyric acid, glutamine and acetyldyestuside. The experimental results showed that the content of gamma aminobutyric acid increased gradually with the prolongation of aging treatment time of soybean seeds, and the content of glutamine and acetyl dye in soybean isoflavones gradually decreased by.EESI-MS binding. Multivariate analysis can effectively distinguish.3 from soybean seeds with different vitality. In this paper, 19 kinds of phenolic compounds in soybean seeds were analyzed qualitatively and quantitatively by HPLC. The results showed that the content of 19 phenolic compounds in the aging process of soybean seeds all changed obviously. The content of acid, rutin and saoflavin decreased with the increase of aging treatment time, from 183.60 mu g/g, 14.03 mu g/g and 55.03 mu g/g to 138.10 mu g/g, 12.37 mu g/g and 38.60 mu g/g, and the content of daioside, flavin, genistein and baicalein increased with the increase of aging treatment, from 12.57 Mu g/g, trace (Trace; Tr), respectively. 9.10 mu g/g and 0.83 mu g/g increased to 91.97 mu g/g, 6.60 mu g/g, 76.10 mu g/g and 3.53 mu g/g; caffeic acid, epicatechin, ferulic acid, daidzein, geniside and resveratrol increased first and then decreased with the aging treatment time, and ferulic acid content was the largest in aging treatment 2 D, and the other 5 phenolic compounds contained The amount of chlorogenic acid, myricetin, cinnamic acid, kaempferol, and the content of A with chlorogenic acid, myricetin and kaempferol were the same, and the changes of chlorogenic acid, myricetin and kaempferol were the same, cinnamic acid, P - Nitrobenzoic Acid and olecranon A of A were changed. The total content of.19 phenols increased first and then decreased with the increase of aging treatment time, reaching the maximum value of 6830.88 mu g/g.4 at fourth D aging treatment, and further using EESI-MS to analyze the alcohol extracts of soybean seeds with different aging time, and obtain the fingerprint of their positive ion mode. The qualitative and quantitative analysis of four typical isoflavones was carried out. The results showed that the content of Huang Dougan, genistein, geniside and genistein increased with the aging time, while the content of daidzein and genistein increased with the aging time. The trend of increasing first and then decreasing, reaching the maximum when the aging treatment was 4 d. This result was the same as the content of daidzein, genistein, daidzein and genistein in the experimental results of HPLC. At the same time, the fingerprint spectrum of the alcohol extracts of soybean seeds with different aging treatment time was analyzed by PCA. The results showed that the different vitality was great. The substance difference in soybean seed alcohol extracts is significant and can be clearly distinguished in different regions of PCA two-dimensional space. This paper uses EESI-MS and HPLC as the research platform for rapid identification of different vigor soybean seeds, and systematically studies the dynamic transformation of phenolic substances in the aging process of soybean seeds, which is rapid and sensitive. A new method for mass spectrometry of effective soybean seed samples is provided to provide theoretical basis for soybean seed storage and research and utilization of germplasm resources.

【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S565.1

【参考文献】

中国期刊全文数据库 前10条

1 李水明;何曼文;王勇;;亚胺及其相关离子在区分赖氨酸和谷氨酰胺残基中的应用[J];质谱学报;2014年03期

2 欧阳永中;李操;周亚飞;周振;;电喷雾萃取电离质谱法用于掺假白酒的快速分析[J];化学学报;2013年12期

3 王凤鹏;曾全荣;叶尚臣;尹真;;基于激光散斑的大豆活力检测实验研究[J];应用激光;2013年04期

4 Nazim Hussain;Zahra Jabeen;LI Yuan-long;CHEN Ming-xun;LI Zhi-lan;GUO Wan-li;Imran Haider Shamsi;CHEN Xiao-yang;JIANG Li-xi;;Detection of Tocopherol in Oilseed Rape (Brassica napus L.) Using Gas Chromatography with Flame Ionization Detector[J];Journal of Integrative Agriculture;2013年05期

5 黄情;李云霞;魏先杰;吴辉;唐启源;;种子劣变与修复[J];种子;2013年04期

6 瞿德敬;王俊英;王俊平;王硕;;HPLC-ESI-MSn法鉴定大豆中12种大豆异黄酮的结构[J];现代食品科技;2013年04期

7 左玉;;多酚类化合物研究进展[J];粮食与油脂;2013年04期

8 李月明;郝楠;孙丽惠;张庆芳;叶雨盛;;种子活力测定方法研究进展[J];辽宁农业科学;2013年01期

9 郭远;杨柳;王园清;薛树鹏;张娇;崔岳;陈学珍;谢皓;;大豆种子发育过程中异黄酮含量的动态分析[J];农学学报;2012年11期

10 贾滨;张兴磊;丁健桦;杨水平;陈焕文;;电喷雾萃取电离质谱技术及其应用进展[J];科学通报;2012年20期

中国硕士学位论文全文数据库 前2条

1 张燕慧;白沙蒿种子对人工老化的生理生化响应研究[D];兰州大学;2016年

2 王海涛;大豆异黄酮的抑菌活性及其机制的研究[D];辽宁师范大学;2009年

，

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