CN110646525A - 一种凤丹种皮生长发育过程中的脂肪酸含量检测方法 - Google Patents

一种凤丹种皮生长发育过程中的脂肪酸含量检测方法 Download PDF

Info

Publication number
CN110646525A
CN110646525A CN201910859178.4A CN201910859178A CN110646525A CN 110646525 A CN110646525 A CN 110646525A CN 201910859178 A CN201910859178 A CN 201910859178A CN 110646525 A CN110646525 A CN 110646525A
Authority
CN
China
Prior art keywords
fatty acid
paeonia ostii
temperature
content
growth
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201910859178.4A
Other languages
English (en)
Inventor
丁怡婷
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
SHANGHAI SONGJIANG NO 1 HIGH SCHOOL
Original Assignee
SHANGHAI SONGJIANG NO 1 HIGH SCHOOL
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by SHANGHAI SONGJIANG NO 1 HIGH SCHOOL filed Critical SHANGHAI SONGJIANG NO 1 HIGH SCHOOL
Priority to CN201910859178.4A priority Critical patent/CN110646525A/zh
Publication of CN110646525A publication Critical patent/CN110646525A/zh
Pending legal-status Critical Current

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/30—Control of physical parameters of the fluid carrier of temperature
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G01N30/8624—Detection of slopes or peaks; baseline correction
    • G01N30/8631—Peaks
    • G01N30/8634—Peak quality criteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N2030/042—Standards
    • G01N2030/045—Standards internal
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N2030/067—Preparation by reaction, e.g. derivatising the sample

Landscapes

  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Quality & Reliability (AREA)
  • Engineering & Computer Science (AREA)
  • Medicines Containing Plant Substances (AREA)

Abstract

本发明涉及一种凤丹种皮生长发育过程中的脂肪酸含量检测方法，包括种子收集、种皮分离、提取总脂、脂肪酸甲酯化、GC‑MS检测。本发明完善了凤丹种皮在各个生长时期的脂肪酸含量的变化，为凤丹在大批量炼油时选取哪一个生长时期作为专业的数据参考，也为凤丹脂肪酸的进一步开发和代谢机理研究提供了可靠的基础数据。

Description

一种凤丹种皮生长发育过程中的脂肪酸含量检测方法
技术领域
本发明属于凤丹籽油技术领域，特别涉及一种凤丹种皮生长发育过程中的脂肪酸含量检测方法。
背景技术
凤丹籽油的不饱和脂肪酸含量90％以上，亚麻酸含量超过40％。凤丹的干燥根皮，具有清热凉血、活血散瘀之功效，其主要成分牡丹酚有抗炎、镇静、降温、解热、镇痛、解痉等中枢抑制作用及抗动脉粥样硬化、利尿、抗溃疡等作用。但是，在何时(哪一个生长发育时期)进行大批量的采摘和提取凤丹籽油的质量最好，凤丹籽油中所含脂肪酸(重要营养价值所在)最多的问题上，并没有相关的研究。
发明内容
本发明所要解决的技术问题是提供一种凤丹种皮生长发育过程中的脂肪酸含量检测方法，该方法完善了凤丹种皮在各个生长时期的脂肪酸含量的变化，为凤丹在大批量炼油时选取哪一个生长时期作为专业的数据参考，也为凤丹脂肪酸的进一步开发和代谢机理研究提供了可靠的基础数据。
本发明提供了一种凤丹种皮生长发育过程中的脂肪酸含量检测方法，包括：
(1)在凤丹人工授粉(day after fertilization，DAF)后21天开始采集凤丹种子，不同时间节点采集的种子人工分离种皮、胚乳和胚，分开保存，处理好的样品液氮冻存；
(2)提取总脂并甲酯化，采用GC-MS对样品做定量和定性分析。
所述步骤(1)中的不同时间节点为35DAF、49DAF、63DAF、70DAF、77DAF、84DAF、91DAF、98DAF、105DAF。
所述步骤(2)中的甲酯化温度为80-90℃，甲酯化时间为1-2h。
所述步骤(2)中的GC-MS的参数为：色谱柱为HP-88型毛细管柱；升温程序为130℃保留5min，4℃/min升温至220℃用于12min，20℃/min升温至240℃，最后保留8min；分流比为20:1；进样量为1μL。
有益效果
本发明完善了凤丹种皮在各个生长时期的脂肪酸含量的变化，为凤丹在大批量炼油时选取哪一个生长时期作为专业的数据参考，也为凤丹脂肪酸的进一步开发和代谢机理研究提供了可靠的基础数据。
附图说明
图1a和b为凤丹种皮生长发育过程中五种脂肪酸的含量变化图；
图2为凤丹种皮发育过程中总脂肪酸(TFA)和不饱和脂肪酸(UFA)百分含量变化。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例1
(一)种子收集
样品采集在凤丹人工授粉后(day after fertilization，DAF)21天开始，每隔2周采集一次。样品采集分为九个时间节点，分别为35DAF、49DAF、63DAF、70DAF、77DAF、84DAF、91DAF，98DAF、105DAF。
(二)种皮的分离
不同时间节点采集的种子进行人工分离种皮、胚乳和胚分开保存，处理好的样品液氮冻存后放入-80℃冰箱的保存。
(三)提取总脂
首先，进行称量：a.称量研磨管和小钢珠的重量；b.称量顶空样品瓶的重量；c.种皮的鲜重。
其次，取三粒种子约0.6g用液氮研磨，粉末均分成3个平行样，放入研磨管。
随后，再次称重：称重研磨管的重量。通过计算：粉末重量＝放入粉末后的研磨管重量-空的研磨管的重量，得出了粉末的重量。在粉末中加入3mL氯仿。必要地，研磨之后进行震荡：a.涡旋振荡1min；b.35℃的水浴中以180rpm震荡1h；c.涡旋振荡1min。之后，在顶空样品瓶中加入1mL氯仿，并涡旋振荡1min。加入1.8mL的超纯水，以保持最终氯仿和甲醇和水的体积比1:1:0.9。
再后，以5000rpm离心15min，抽提液分成两层，下层为有机相，抽提的总脂溶于有机相。中间层的残渣重复步骤。
最后，有机相放于一个预先称重的顶空样品瓶中，并且氮气吹干。
(四)脂肪酸甲酯化
首先，吹干的黑玻璃管中抽提的脂类溶于含4％硫酸的甲醇溶液。
随后，涡旋振荡1min，并充氮气，以防氧化。
之后，将酯化瓶盖旋紧于85℃水浴1h(进行酯化反应)然后，振荡2min，并加入2mL水，结束酯化反应。加入2mL正己烷(与水的密度不同，水的密度大，正己烷会溶解脂肪酸的成分，为了离心后的抽取有效成分)，并混匀。以4000rpm离心10min，并收集上清液至样品瓶。
然后，氮气浓缩至正己烷完全挥发。样品重新溶于1mL正己烷，并取100μL甲酯化的样品溶于100μL的正己烷中，那么，样品稀释了两倍，最终上机检测的样品中的内标C19的浓度为100mg/L。
(五)应用GC-MS检测脂肪酸甲酯及分析
色谱柱为HP-88型毛细管柱(100m×0.25mm×0.2μm)；升温程序为130℃保留5min，4℃/min升温至220℃用于12min，20℃/min升温至240℃，最后保留8min；分流比为20:1；进样量为1μL。脂肪酸的定性分析采用标准品(32种脂肪酸标准品，Sigma)和执行谱库检索两种方法进行，质谱数据库参考NIST MS Search 2.0。脂肪酸定量分析采用内标法，根据色谱峰的积分面积进行计算。
(六)所得数据整合和分析
1、应用内标法计算得到脂肪酸的浓度，计算出每个样品中脂肪酸含量。
2、作图并分析，五种主要脂肪酸含量变化的结果(图1a)表明35DAF时，种皮的油酸含量较高为0.94mg/g，亚油酸含量为0.91mg/g，ALA含量仅为0.13mg/g。在49DAF和63DAF两个时期，油酸含量呈现下降趋势，油酸含量分别降低为0.79mg/g；而亚油酸和ALA含量呈小幅上升趋势，亚油酸和ALA分别升高为1.50mg/g和0.72mg/g。在70DAF时，油酸、亚油酸和ALA含量分别骤然增加至2.25mg/g，3.54mg/g和4.88mg/g，较前一个时期分别增加了3倍、2倍和近7倍，但是在77DAF时又骤然分别下降至1.56mg/g、2.39mg/g和4.42mg/g，较前一个时期分别降低4倍，3倍和近11倍。不饱和脂肪酸在种皮整个发育过程中含量的变化趋势呈倒V型变化(图1b)，在70DAF时含量达到最高。在84DAF时，不饱和脂肪酸含量大幅下降；在91DAF时期，油酸和亚油酸出现反弹，呈现上升趋势，较84DFA时分别增长了4倍和近5倍。而饱和脂肪酸棕榈酸和硬脂酸在70DAF也有增加，但是硬脂酸较前一个发育时期63DAF时偏低，因此，硬脂酸在种皮发育过程中含量变化比较复杂。
3、作图并分析，种皮发育过程中总脂肪酸(Total fatty acid，TFA)和不饱和脂肪酸(Unsaturated fatty acid，UFA)百分含量变化(图2)结果表明在35DAF至84DAF发育过程中，总脂肪酸(TFA)和不饱和脂肪酸(UFA)的含量变化趋势大致相同。TFA在70DAF达到最高，含量为11.02mg/g，同时UFA含量也达到最高为97％，表明不饱和脂肪酸含量的变化对于影响总脂含量的变化是主导作用的。在91DAF至119DAF中，TFA含量急剧下降，至84DAF时降低为70DAF的16倍，饱和脂肪酸和不饱和脂肪酸含量均大量减少，但是UFA在总脂肪酸含量的变化幅度不大，表明凤丹种皮中不饱和脂肪酸含量占比较高，整个发育过程中UFA百分含量均在80％以上。
由以上结果可知，在70DAF凤丹种皮进行采摘质量最好。

Claims (4)

1.一种凤丹种皮生长发育过程中的脂肪酸含量检测方法，包括：
(1)在凤丹人工授粉后21天开始采集凤丹种子，不同时间节点采集的种子人工分离种皮、胚乳和胚，分开保存，处理好的样品液氮冻存；
(2)提取总脂并甲酯化，采用GC-MS对样品做定量和定性分析。
2.根据权利要求1所述的检测方法，其特征在于：所述步骤(1)中的不同时间节点为35DAF、49DAF、63DAF、70DAF、77DAF、84DAF、91DAF、98DAF、105DAF。
3.根据权利要求1所述的检测方法，其特征在于：所述步骤(2)中的甲酯化温度为80-90℃，甲酯化时间为1-2h。
4.根据权利要求1所述的检测方法，其特征在于：所述步骤(2)中的GC-MS的参数为：色谱柱为HP-88型毛细管柱；升温程序为130℃保留5min，4℃/min升温至220℃用于12min，20℃/min升温至240℃，最后保留8min；分流比为20:1；进样量为1μL。
CN201910859178.4A 2019-09-11 2019-09-11 一种凤丹种皮生长发育过程中的脂肪酸含量检测方法 Pending CN110646525A (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910859178.4A CN110646525A (zh) 2019-09-11 2019-09-11 一种凤丹种皮生长发育过程中的脂肪酸含量检测方法

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910859178.4A CN110646525A (zh) 2019-09-11 2019-09-11 一种凤丹种皮生长发育过程中的脂肪酸含量检测方法

Publications (1)

Publication Number Publication Date
CN110646525A true CN110646525A (zh) 2020-01-03

Family

ID=68991770

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910859178.4A Pending CN110646525A (zh) 2019-09-11 2019-09-11 一种凤丹种皮生长发育过程中的脂肪酸含量检测方法

Country Status (1)

Country Link
CN (1) CN110646525A (zh)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105296142A (zh) * 2015-10-10 2016-02-03 上海辰山植物园 一种快速提取和分离牡丹种子中脂类成分的方法
CN108157167A (zh) * 2018-02-11 2018-06-15 青岛农业大学 提高油用牡丹‘凤丹’种子α-亚麻酸含量的方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105296142A (zh) * 2015-10-10 2016-02-03 上海辰山植物园 一种快速提取和分离牡丹种子中脂类成分的方法
CN108157167A (zh) * 2018-02-11 2018-06-15 青岛农业大学 提高油用牡丹‘凤丹’种子α-亚麻酸含量的方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
VIRENDRA S. RANA 等: "Fatty Acid and Non-Fatty Acid Components of the Seed Oil of Celastrus paniculatus willd", 《INTERNATIONAL JOURNAL OF FRUIT SCIENCE》 *
李喜悦 等: "牡丹种皮、种子及4种干果油脂的脂肪酸组成分析", 《食品安全质量检测学报》 *

Similar Documents

Publication Publication Date Title
Temime et al. Changes in olive oil quality of Chétoui variety according to origin of plantation
Moaddabdoost Baboli et al. Characteristics and composition of watermelon seed oil and solvent extraction parameters effects
Malainey et al. The fatty acid composition of native food plants and animals of western Canada
Gómez-González et al. Oil content and fatty acid profile of Spanish cultivars during olive fruit ripening
Suh et al. Direct transesterification of wet microalgal biomass for preparation of biodiesel
Ferrari et al. Macauba as promising substrate for crude oil and biodiesel production
CN105647639A (zh) 瓜蒌籽油的液压冷榨方法
Muthai et al. Quantitative variation of fatty acid composition in seed oil from baobab (Adansonia digitata L.) wild populations in sub-Sahara Africa
Alcaraz-Marmol et al. Determination of fatty acid composition in arils of 20 pomegranates cultivars grown in Spain
CN110646525A (zh) 一种凤丹种皮生长发育过程中的脂肪酸含量检测方法
CN110089419A (zh) 一种高油酸高产花生的育种方法
Yahaya et al. Extraction of oil from Jatropha seed kernels: optimization and characterization
Hemida et al. Influence of environmental factors on olive oil production and quality in the Northern Region of kingdom of Saudi Arabia
Uddin et al. Fatty acid composition of oil palm (Elaeis guineensis Jacq) fruits grown in Bangladesh
Almoselhy et al. Identification of Triacylglycerol Composition of Some Olive Oil Cultivars Cultivated in Some Different Areas in Egypt
Chanioti et al. Processing parameters on the extraction of olive pomace oil and its bioactive compounds: A kinetic and thermodynamic study
Kiritsakis et al. Effect of olive collection regime on olive oil quality
Agar et al. Compositional changes of fatty acids during the development of embryo in Pistacia vera
CN105001982A (zh) 富含二十碳五烯酸拟微球藻油脂超声波辅助溶剂提取方法
Dandan et al. Study on the effect of extraction process of Moringa instant tea on its sensory quality
da Silva Júnior et al. Seed oil content and fatty acid composition in natural populations of Tarenaya longicarpa (Cleomaceae) during the dry and rainy seasons in Northeast Brazil
EP2235150B1 (en) Method of obtaining a solid component rich in a petroselinic compound
Naresh Kumar et al. Seasonal variations in fatty acid composition of oil in developing coconut
Mawarni et al. Simulation of triacylglycerols separation from crude Calophyllum inophyllum oil with batchwise solvent extraction method
林賢治 et al. Seasonal Changes in the Contents of Eicosapentaenoic Acid-Containing Triglycerides in Hepatopancreas of Scallop.

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication

Application publication date: 20200103

RJ01 Rejection of invention patent application after publication