“丰香”草莓果实发育过程中主要品质特征成分的代谢谱分析
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摘要
糖含量和有机酸含量的高低以及糖酸比是影响果实品质风味的重要因素,与糖酸代谢相关的其他成分如花色苷、氨基酸、芳香物质也与果实的品质有着至关重要的作用。代谢组学技术在果实品质研究上应用,不仅可以揭示糖、酸、氨基酸、花色苷、香气物质等特征品质成分在果树体中的形成、转化及其相互关系的本质规律,阐明果实品质的形成机理,而且有助于利用现代生物技术进行水果品种改良和品质调控。
草莓(Fragaria ananassa Duch.)为多年生浆果类果树,其全基因组很小,生育周期短(约4个月),植株小并适于保护栽培,且属于糖直接积累型果实,去除了淀粉形态的介入对糖代谢研究的干扰,而且,前人在草莓糖代谢研究方面已有一定的工作积累。这些相关特点为开展果实主要品质特征成分代谢研究提供了适宜的研究体系并奠定了良好的研究基础,是进行果实品质特征成分代谢谱研究的良好试材。
本文以“丰香”草莓果实为试材,初步建立了代谢谱分析技术平台中有关样品制备、分析检测以及数据处理的技术和方法,并通过对不同生育期草莓果实中糖以及与糖代谢密切相关的重要果实品质特征成分(包括有机酸、花色素、氨基酸和芳香物质)的代谢谱分析,研究这些化学成分在果实发育过程中形成和转化规律。该课题研究不仅对于揭示与糖代谢有关的品质特征成分的复杂生理生化过程,探讨不同品质特征成分之间的相互转化关系,阐明草莓果实品质的形成机理具有重要的理论研究意义,而且对于促进和提高草莓品种的改良效率和品质调控也具有实际应用价值。实验取得以下主要结果:
在草莓果实样品中,丝氨酸、精氨酸、谷氨酸、组氨酸、天冬氨酸、脯氨酸含量较高,为主要氨基酸。在果实成熟过程中,氨基酸总量呈逐步下降的趋势,从幼果期的48.39μg. g~(-1)降到成熟期仅有8.62μg. g~(-1);含量较高丝氨酸、精氨酸、组氨酸等变幅较大;含量较低的缬氨酸、酪氨酸、亮氨酸等含量变化较小。
草莓果实中花色苷种类很少,最主要的花色苷组分为矢车菊色素和天竺葵色素,仅在果实转熟期出现少量的芍药花色素和锦葵色素,且含量仅为同时期花色苷总量的1.37%;在果实的整个生育阶段内,花色苷总量呈逐步增加的趋势,幼果期几乎检测不到花色苷;果实膨大后期,花色苷才开始迅速积累,天竺葵色素为完熟期最主要的花色苷,含量逐渐增加,矢车菊色素则从转熟期的0.1013 mg.g~(-1)迅速减少到完熟期的0.0512 mg.g~(-1)。
本实验建立了草莓果实有机相提取物的GC和GC-MS分析测定方法,供试样品基本达到基线分离,分离效果良好。从草莓果实有机相提取物中共分离得到195个峰,鉴定出128种化合物,其中,酯类物质含量最高,为草莓果实香气最主要的成分,橙花叔醇、蜂花醇等萜烯类也是“丰香”草莓果实中重要的香气成分。草莓果实石油醚相提取物中第4号样品(4月10日采集)与其他样品偏离较远,而环己烷相提取物中第4号样品(4月25日采集)与其他生育期供试样品存在较大差异。
本实验比较了不同衍生化方法的衍生效果,筛选并确定BSTFA:TMCS(99:1)的衍生化方法,实现了糖、有机酸、糖醇的同时衍生;并采用正交试验设计对衍生化条件进行了优化,选择处理组合17作为最优衍生条件,即100μL糖溶液(4mg.mL~(-1))加100μL盐酸羟胺溶液(25mg.mL~(-1)),40℃反应50min可肟化完全,加入60μL BSTFA:TMCS(99:1),室温下反应40min可硅烷化完全。
在草莓果实样品中,果糖、葡萄糖、总糖含量均随果实成熟呈上升趋势,在整个生育期中,葡萄糖含量相对较低,果糖占总糖比例较大。草莓果实中的酸主要为有机酸,其中柠檬酸占总酸量的60%以上,有机酸含量波动较大,在果实发育早期有机酸含量逐渐升高,并在4月10日(半红期)达到峰值,随着果实成熟,有机酸含量开始下降,果实生长后期有机酸含量下降可能是因为转化为碳水化合物或者被氧化形成酯类。
Fruit flavor are deeply influenced by the contents of sugar and organic acid, as well as the ratio of sugar to acid, and other components related to the metabolism of sugar and acid, such as anthocyanin, amino acid, aroma, also play a crucial role in the formation of fruit quality. The application of metabolomics in the research of fruit quality, is helpful for not only revealing the mechanism of formation, transformation and relationship of the major characteristic quality components in fruits such as sugar, organic acid, amino acid, anthocyanin, aroma and so on, but also for supplying the clue for fruit breeding and quality improvement using modern biotechnique methods.
Strawberry (Fragaria ananassa Duch.) as one of perennial fruit trees, has small genome and short reproductive cycle (about 4 months). The characteristic of small plant is suitable for protected cultivation, furthermore, photosynthetic product accumulates in strawberry fruits just in the form of sugar, which is helpful for investigating sugar metabolism because of no disturbance of starch. So, strawberry is a suitable material for metabolomic research to investigate metabolic profiling of the major fruit quality components.
Fragaria×ananassa‘Fengxiang’fruits serve as the research system in this paper, and the metabolic profiling platform based on the fundmental techniques and methods involved in sample preparation, sample detection and data perform were implored, and that was further applied for the metabolic profiling analysis on the major quality components contained sugar, organic acid, anthocyanin, amino acid and aromatic substances during the development of strawberry fruits, which can help to reveal the complex physiological and biochemical metabolism of quality formation. The main results are as following:
The major components of amino acids in strawberry fruits were serine, arginine, glutamic acid, histidine, aspartic acid and proline. During the development of strawberry fruits, the total content of amino acids decreased gradually, from 48.39μg. g-1 in immature stage to 8.62μg. g-1 in mature stage. The major components of amino acids with higher contents differed significantly such as serine, arginine and histidine, and the amino acids with lower contents often gave small decrease.
There were few varieties of anthocyanins in strawberry fruits and the major anthocyanin components were Cy3glc and Pg3glc. Pn3glc and Mv3glc were discovered only at start-red stage and 1.37 percent of the whole anthocyanin content. The anthocyanin content increased gradually during fruit ripening. Few anthocyanins were detected in young fruits, but accumulated rapidly during fruit developing. Pg3glc was the major anthocyanin component and accumulated gradually at full-red stage, while the Cy3glc content decreased rapidly from 0.1013 mg.g~(-1) at start-red stage to 0.0512 mg.g~(-1) at full-red stage.
In this experiment, GC and GC-MS methods was well constructed for the identification of organic phase extract from strawberry fruit. The results showed that the sample could be well separated, and 195 peaks were detected and 128 compounds were identified. Esters were the major aromatic components, and terpenoids such as Nerolido and N-hentriacontanolhen-1 played an important role in flavor formation. Based on PCA and PLS analysis, petroleum ether extract from strawberry fruits of the fourth period (sample collected at April 10) was quite different from other growth periods. Cyclohexane extract of the seventh period (sample collected at April 25) was also different from other growth periods.
The effects of different derivatisation methods were compared in this paper, and simultaneous derivatisation of sugar, organic acid and alditol can be achived with the derivatisation solvent of BSTFA and TMCS (99:1, v/v). The derivatisation conditions were further optimized by orthogonal experimental design, and treatment no.17 was selected as the optimal derivatisation condition, which is 100μL of 25mg.mL~(-1) hydroxylamine hydrochloride, 100μL of 4mg.mL~(-1) sugar solution, treated at 40℃for 50min and further added 60μL of BSTFA and TMCS (99: 1) derivatized at room temperature for 40min.
The contents of fructose, glucose, total sugar increased gradually during fruit ripening, while the contents of fructose and glucose were low. Organic acid was the major acid component in strawberry fruits, and the content of citric acid was high up to 60 percent. Organic acid accumulated at early stage,and the maximum content was achived at April 10(half-red stage). Organic acid decreased after half-red stage which might be related to the transformation to carbohydrates or oxidization to esters.
草莓(Fragaria ananassa Duch.)为多年生浆果类果树,其全基因组很小,生育周期短(约4个月),植株小并适于保护栽培,且属于糖直接积累型果实,去除了淀粉形态的介入对糖代谢研究的干扰,而且,前人在草莓糖代谢研究方面已有一定的工作积累。这些相关特点为开展果实主要品质特征成分代谢研究提供了适宜的研究体系并奠定了良好的研究基础,是进行果实品质特征成分代谢谱研究的良好试材。
本文以“丰香”草莓果实为试材,初步建立了代谢谱分析技术平台中有关样品制备、分析检测以及数据处理的技术和方法,并通过对不同生育期草莓果实中糖以及与糖代谢密切相关的重要果实品质特征成分(包括有机酸、花色素、氨基酸和芳香物质)的代谢谱分析,研究这些化学成分在果实发育过程中形成和转化规律。该课题研究不仅对于揭示与糖代谢有关的品质特征成分的复杂生理生化过程,探讨不同品质特征成分之间的相互转化关系,阐明草莓果实品质的形成机理具有重要的理论研究意义,而且对于促进和提高草莓品种的改良效率和品质调控也具有实际应用价值。实验取得以下主要结果:
在草莓果实样品中,丝氨酸、精氨酸、谷氨酸、组氨酸、天冬氨酸、脯氨酸含量较高,为主要氨基酸。在果实成熟过程中,氨基酸总量呈逐步下降的趋势,从幼果期的48.39μg. g~(-1)降到成熟期仅有8.62μg. g~(-1);含量较高丝氨酸、精氨酸、组氨酸等变幅较大;含量较低的缬氨酸、酪氨酸、亮氨酸等含量变化较小。
草莓果实中花色苷种类很少,最主要的花色苷组分为矢车菊色素和天竺葵色素,仅在果实转熟期出现少量的芍药花色素和锦葵色素,且含量仅为同时期花色苷总量的1.37%;在果实的整个生育阶段内,花色苷总量呈逐步增加的趋势,幼果期几乎检测不到花色苷;果实膨大后期,花色苷才开始迅速积累,天竺葵色素为完熟期最主要的花色苷,含量逐渐增加,矢车菊色素则从转熟期的0.1013 mg.g~(-1)迅速减少到完熟期的0.0512 mg.g~(-1)。
本实验建立了草莓果实有机相提取物的GC和GC-MS分析测定方法,供试样品基本达到基线分离,分离效果良好。从草莓果实有机相提取物中共分离得到195个峰,鉴定出128种化合物,其中,酯类物质含量最高,为草莓果实香气最主要的成分,橙花叔醇、蜂花醇等萜烯类也是“丰香”草莓果实中重要的香气成分。草莓果实石油醚相提取物中第4号样品(4月10日采集)与其他样品偏离较远,而环己烷相提取物中第4号样品(4月25日采集)与其他生育期供试样品存在较大差异。
本实验比较了不同衍生化方法的衍生效果,筛选并确定BSTFA:TMCS(99:1)的衍生化方法,实现了糖、有机酸、糖醇的同时衍生;并采用正交试验设计对衍生化条件进行了优化,选择处理组合17作为最优衍生条件,即100μL糖溶液(4mg.mL~(-1))加100μL盐酸羟胺溶液(25mg.mL~(-1)),40℃反应50min可肟化完全,加入60μL BSTFA:TMCS(99:1),室温下反应40min可硅烷化完全。
在草莓果实样品中,果糖、葡萄糖、总糖含量均随果实成熟呈上升趋势,在整个生育期中,葡萄糖含量相对较低,果糖占总糖比例较大。草莓果实中的酸主要为有机酸,其中柠檬酸占总酸量的60%以上,有机酸含量波动较大,在果实发育早期有机酸含量逐渐升高,并在4月10日(半红期)达到峰值,随着果实成熟,有机酸含量开始下降,果实生长后期有机酸含量下降可能是因为转化为碳水化合物或者被氧化形成酯类。
Fruit flavor are deeply influenced by the contents of sugar and organic acid, as well as the ratio of sugar to acid, and other components related to the metabolism of sugar and acid, such as anthocyanin, amino acid, aroma, also play a crucial role in the formation of fruit quality. The application of metabolomics in the research of fruit quality, is helpful for not only revealing the mechanism of formation, transformation and relationship of the major characteristic quality components in fruits such as sugar, organic acid, amino acid, anthocyanin, aroma and so on, but also for supplying the clue for fruit breeding and quality improvement using modern biotechnique methods.
Strawberry (Fragaria ananassa Duch.) as one of perennial fruit trees, has small genome and short reproductive cycle (about 4 months). The characteristic of small plant is suitable for protected cultivation, furthermore, photosynthetic product accumulates in strawberry fruits just in the form of sugar, which is helpful for investigating sugar metabolism because of no disturbance of starch. So, strawberry is a suitable material for metabolomic research to investigate metabolic profiling of the major fruit quality components.
Fragaria×ananassa‘Fengxiang’fruits serve as the research system in this paper, and the metabolic profiling platform based on the fundmental techniques and methods involved in sample preparation, sample detection and data perform were implored, and that was further applied for the metabolic profiling analysis on the major quality components contained sugar, organic acid, anthocyanin, amino acid and aromatic substances during the development of strawberry fruits, which can help to reveal the complex physiological and biochemical metabolism of quality formation. The main results are as following:
The major components of amino acids in strawberry fruits were serine, arginine, glutamic acid, histidine, aspartic acid and proline. During the development of strawberry fruits, the total content of amino acids decreased gradually, from 48.39μg. g-1 in immature stage to 8.62μg. g-1 in mature stage. The major components of amino acids with higher contents differed significantly such as serine, arginine and histidine, and the amino acids with lower contents often gave small decrease.
There were few varieties of anthocyanins in strawberry fruits and the major anthocyanin components were Cy3glc and Pg3glc. Pn3glc and Mv3glc were discovered only at start-red stage and 1.37 percent of the whole anthocyanin content. The anthocyanin content increased gradually during fruit ripening. Few anthocyanins were detected in young fruits, but accumulated rapidly during fruit developing. Pg3glc was the major anthocyanin component and accumulated gradually at full-red stage, while the Cy3glc content decreased rapidly from 0.1013 mg.g~(-1) at start-red stage to 0.0512 mg.g~(-1) at full-red stage.
In this experiment, GC and GC-MS methods was well constructed for the identification of organic phase extract from strawberry fruit. The results showed that the sample could be well separated, and 195 peaks were detected and 128 compounds were identified. Esters were the major aromatic components, and terpenoids such as Nerolido and N-hentriacontanolhen-1 played an important role in flavor formation. Based on PCA and PLS analysis, petroleum ether extract from strawberry fruits of the fourth period (sample collected at April 10) was quite different from other growth periods. Cyclohexane extract of the seventh period (sample collected at April 25) was also different from other growth periods.
The effects of different derivatisation methods were compared in this paper, and simultaneous derivatisation of sugar, organic acid and alditol can be achived with the derivatisation solvent of BSTFA and TMCS (99:1, v/v). The derivatisation conditions were further optimized by orthogonal experimental design, and treatment no.17 was selected as the optimal derivatisation condition, which is 100μL of 25mg.mL~(-1) hydroxylamine hydrochloride, 100μL of 4mg.mL~(-1) sugar solution, treated at 40℃for 50min and further added 60μL of BSTFA and TMCS (99: 1) derivatized at room temperature for 40min.
The contents of fructose, glucose, total sugar increased gradually during fruit ripening, while the contents of fructose and glucose were low. Organic acid was the major acid component in strawberry fruits, and the content of citric acid was high up to 60 percent. Organic acid accumulated at early stage,and the maximum content was achived at April 10(half-red stage). Organic acid decreased after half-red stage which might be related to the transformation to carbohydrates or oxidization to esters.
引文
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