芝麻素高效提取检测技术的建立与高芝麻素种质的筛选

doi:10.3864/j.issn.0578-1752.2022.11.003

中国农业科学 ›› 2022, Vol. 55 ›› Issue (11): 2109-2120.doi: 10.3864/j.issn.0578-1752.2022.11.003

• 作物遗传育种·种质资源·分子遗传学 • 上一篇下一篇

芝麻素高效提取检测技术的建立与高芝麻素种质的筛选

钟艳平^1,²(),师立松¹,周瑢¹,高媛¹,何延庆¹,方圣²,张秀荣¹,王林海¹,吴自明²(),张艳欣¹()

¹中国农业科学院油料作物研究所/农业农村部油料作物生物学与遗传育种重点实验室,武汉 430062
²江西农业大学作物生理生态与遗传育种教育部重点实验室,南昌 330045

收稿日期:2022-01-20 接受日期:2022-03-14 出版日期:2022-06-01 发布日期:2022-06-16
通讯作者: 吴自明,张艳欣
作者简介:钟艳平,E-mail： yanpzhu1996@163.com。
基金资助:
农业农村部芝麻种质资源精准鉴定项目(19211121);农业农村部农作物种质资源保护与利用专项(19210163);科技部、财政部国家科技基础条件平台项目(NCGRC-2021-016);中国农业科学院科技创新工程(CAAS-ASTIP-2016-OCRI);江西省现代农业产业技术体系建设专项(JXARS-18)

Establishment of High Efficient Extraction and Detection Technology of Sesamin and Screening of High Sesamin Germplasm

ZHONG YanPing^1,²(),SHI LiSong¹,ZHOU Rong¹,GAO Yuan¹,HE YanQing¹,FANG Sheng²,ZHANG XiuRong¹,WANG LinHai¹,WU ZiMing²(),ZHANG YanXin¹()

¹Oil Crops Research Institute, Chinese Academy of Agricultural Sciences/Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture and Rural Affairs, Wuhan 430062
²Key Laboratory of Crop Physiology, Ecology and Genetics Breeding, Ministry of Education, Jiangxi Agricultural University, Nanchang 330045

Received:2022-01-20 Accepted:2022-03-14 Online:2022-06-01 Published:2022-06-16
Contact: ZiMing WU,YanXin ZHANG

摘要/Abstract

摘要：

【目的】在芝麻种子中建立一种芝麻素高效提取检测技术,并将该技术应用于芝麻素含量变异检测和筛选高芝麻素含量的优异种质,为推动高芝麻素基础研究和育种奠定基础。【方法】以芝麻种子为原料,进行钢珠直径、粉碎时间、捣碎振幅和种子量等单因素试验,以80%的乙醇为提取溶剂,利用超声波的机械破碎和空化作用,对芝麻进行萃取;结合高效液相色谱法（HPLC）测定芝麻种子中的芝麻素含量;在单因素试验的基础上,进行Box-Behnken四因素三水平的响应面试验,建立芝麻素含量为响应值的二次多项式回归方程模型,并绘制响应曲面图和等高线图,分析影响芝麻素含量的主要因素及各因素间的交互作用,确定芝麻素含量检测的最优提取条件。利用该参数条件对1 151份地方资源和创新种质进行芝麻素含量的测定,筛选芝麻素含量≥9 g·kg^-1的高芝麻素种质,并送至农业农村部油料及制品质量监督检验测试中心进行鉴定。【结果】建立的回归模型极显著（P<0.05）,失拟项不显著（P=0.1768）,该模型拟合程度较好,可用于分析和预测芝麻素含量。4个因素对芝麻素含量影响的大小依次为：捣碎振幅>钢珠直径>粉碎时间>种子量,其中,捣碎振幅与钢珠直径两因素之间的交互作用显著,捣碎振幅与粉碎时间的交互作用接近显著水平;超声波辅助提取芝麻素的最佳条件为：钢珠直径6.5 mm、粉碎时间225 s、捣碎振幅1 335 r/min、种子量0.20 g,在此条件下提取的芝麻素含量为4.601 g·kg^-1,与模型预测值4.633 g·kg^-1高度相符;从1 151份地方资源和创新种质中筛选出15份高芝麻素含量种质,最高含量为14.36 g·kg^-1,平均含量为12.35 g·kg^-1。【结论】建立了芝麻素高效提取技术,与传统方法相比,本方法只需0.2 g种子就能提取芝麻素,不仅提高了芝麻素的提取效率,还降低了样品的损耗,操作简单,能够精确检测芝麻种子中的芝麻素含量。

关键词: 芝麻素, 高效, 提取工艺, 响应面分析法, 种质筛选

Abstract:

【Objective】 Establish an efficient extraction and detection technology for sesamin content evaluation in sesame seeds, then apply it to examine sesamin content variation and identify high-sesamin content materials in sesame germplasm, which will lay the foundation for advancing basic research and breeding of sesame with high sesamin content. 【Method】 Using sesame seeds as raw materials, the single factor tests, including diameters of steel balls, crushing time, crushing amplitude, and seeds weight, were investigated. The seeds were extracted with 80% ethanol via ultrasonic mechanical crushing and cavitation, and the sesamin content was determined by High Performance Liquid Chromatography (HPLC). Based on the Box-Behnken center combined test design principle, we carried out the response surface test with four factors and three levels and established the quadratic polynomial regression equation model with sesamin content as response value. We drew the response surface diagram and contour diagram and determined the main factors affecting sesamin content and the interaction between factors. Finally, the optimal extraction conditions of sesamin were determined, and 1 151 local resources and innovative germplasm were analyzed. The accessions with high sesamin content≥9 g·kg^-1 were selected and identified at the Oil and Product Quality Supervision and Testing Center of the Ministry of Agriculture and Rural Affairs. 【Result】 The regression model established in this experiment was extremely significant (P<0.05), and the lack of fit was insignificant (P=0.1768). The model had a good fitting degree and could be used to predict sesamin content. The influence of the four factors on sesamin content was as follows: crusher amplitude>diameters of steel balls>crushing time>seeds weight. The interaction between the crusher amplitude and diameters of steel balls was significant, while that between crusher amplitude and crushing time was close to the significance level. The optimal conditions for ultrasonic-assisted extraction of sesamin were as follows: steel ball diameter of 6.5 mm, crushing time of 225 s, crushing amplitude of 1 335 r/min, and seeds weight of 0.20 g. Under these conditions, the sesamin content detected was 4.601 g·kg^-1, which was consistent with the predicted value of 4.633 g·kg^-1. Fifteen varieties with specific high sesamin content were identified from 1 151 sesame germplasm for breeding purposes. The highest content was 14.36 g·kg^-1, and the average content was 12.35 g·kg^-1.【Conclusion】 An efficient extraction technology of sesamin was established,compared with the traditional method, the method only needed 0.2 g seeds, which improved the extraction efficiency and reduced sample consumption, the operation was simple, and the sesamin content in sesame seeds could be accurately detected.

Key words: sesamin, high efficient, extraction process, response surface analysis methodology, screening germplasm

钟艳平,师立松,周瑢,高媛,何延庆,方圣,张秀荣,王林海,吴自明,张艳欣. 芝麻素高效提取检测技术的建立与高芝麻素种质的筛选[J]. 中国农业科学, 2022, 55(11): 2109-2120.

ZHONG YanPing,SHI LiSong,ZHOU Rong,GAO Yuan,HE YanQing,FANG Sheng,ZHANG XiuRong,WANG LinHai,WU ZiMing,ZHANG YanXin. Establishment of High Efficient Extraction and Detection Technology of Sesamin and Screening of High Sesamin Germplasm[J]. Scientia Agricultura Sinica, 2022, 55(11): 2109-2120.

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水平/因子 Level/Factor	钢珠直径 Diameter of steel ball (mm)	粉碎时间 Crusher time (min)	捣碎振幅 Crusher amplitude (r/min)	种子量 Seeds weight (g)
-1	5	180	1100	0.17
0	6	210	1300	0.20
1	7	240	1500	0.23

方差来源 Sources of variance	平方和 Sum of squares	自由度 df	均方 Mean square	F值 F value	P值 P value	显著性 Significance
模型Model	1.52	14	0.11	24.67	<0.0001	**
A	0.29	1	0.29	66.40	<0.0001	**
B	0.12	1	0.12	27.16	0.0001	**
C	0.35	1	0.35	78.63	<0.0001	**
D	0.12	1	0.12	26.22	0.0002	**
AB	3.540×10^-3	1	3.540×10^-3	0.81	0.3843
AC	0.11	1	0.11	25.34	0.0002	**
AD	0.010	1	0.010	2.32	0.1496
BC	0.017	1	0.017	3.94	0.0671
BD	1.936×10^-3	1	1.936×10^-3	0.44	0.5174
CD	7.023×10^-4	1	7.023×10^-4	0.16	0.6952
A²	0.20	1	0.20	46.65	<0.0001	**
B²	0.13	1	0.13	29.34	<0.0001	**
C²	0.30	1	0.30	69.45	<0.0001	**
D²	0.12	1	0.12	27.45	0.0001	**
残差Residual	0.061	14	4.389×10^-3
失拟误差 Lack of fit	0.053	10	5.348×10^-3	2.69	0.1768
纯误差 Pure error	7.965×10^-3	4	1.991×10^-3
总和 Correlation total	1.58	28
R²=0.9610 R²_adj=0.9221 CV=1.55%

芝麻素高效提取检测技术的建立与高芝麻素种质的筛选

Establishment of High Efficient Extraction and Detection Technology of Sesamin and Screening of High Sesamin Germplasm

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试验号 Test number	钢珠直径 Diameter of steel ball (mm)	粉碎时间 Crusher time (min)	捣碎振幅 Crusher amplitude (r/min)	种子量 Seeds weight (g)	芝麻素含量 Sesamin content (g∙kg^-1)
1	0	1	0	-1	4.431
2	0	0	-1	-1	4.142
3	0	0	-1	1	3.991
4	1	-1	0	0	4.264
5	0	0	0	0	4.488
6	1	0	0	1	4.182
7	-1	-1	0	0	4.005
8	0	1	1	0	4.332
9	1	1	0	0	4.545
10	1	0	-1	0	4.218
11	-1	0	0	1	4.019
12	0	-1	-1	0	3.902
13	-1	0	0	-1	4.170
14	1	0	1	0	4.370
15	0	0	0	0	4.584
16	-1	0	1	0	4.351
17	1	0	0	-1	4.535
18	0	-1	0	-1	4.197
19	0	-1	0	1	4.083
20	0	0	0	0	4.576
21	0	1	0	1	4.229
22	0	0	1	-1	4.457
23	0	1	-1	0	4.220
24	0	0	1	1	4.253
25	0	0	0	0	4.496
26	-1	0	-1	0	3.532
27	0	-1	1	0	4.277
28	-1	1	0	0	4.167
29	0	0	0	0	4.550

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