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Acta Agronomica Sinica ›› 2021, Vol. 47 ›› Issue (2): 368-375.doi: 10.3724/SP.J.1006.2021.04110

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Establishment of HPLC-RID method for the determination of soluble sugars in peanut seed

LI Wei-Tao(), GUO Jian-Bin, YU Bo-Lun, XU Si-Liang, CHEN Hai-Wen, WU Bei, GONG Ting-Feng, HUANG Li, LUO Huai-Yong, CHEN Yu-Ning, ZHOU Xiao-Jing, LIU Nian, CHEN Wei-Gang, JIANG Hui-Fang*()   

  1. Oil 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, Hubei, China
  • Received:2020-05-16 Accepted:2020-09-14 Online:2021-02-12 Published:2020-09-28
  • Contact: JIANG Hui-Fang E-mail:965301631@qq.com;peanutlab@oilcrops.cn
  • Supported by:
    Crop Germplasm Resources Protection Project(2017NWB033);Plant Germplasm Resources Sharing Platform(NICGR2017-36);China Agriculture Research System(CARS-13-花生种质资源评价);Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences(CAAS-ASTIP-2013-OCRI)

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Abstract:

Edible peanut is one of the important directions of peanut breeding in China and even in the world. However, the lack of efficient detection method of soluble sugar content related to edible quality in peanut kernel limits the progress of edible peanut breeding. In this study, a method of extracting soluble sugar from peanut kernel by 80% ethanol and water bath was established. The method simplified the pre-treatment steps of samples and speeded up the extraction progress compared with the GB method. The accuracy and repeatability tests showed that this method had high repeatability accuracy and effectiveness. Then, the samples extracted by this method and GB method were both tested by HPLC-RID, it was found that the soluble sugar in mature peanut kernel was mainly sucrose, with little glucose and fructose in 20 peanut varieties. There were no significant differences between these two methods. The results of 20 peanut varieties using the method and GB method showed that the sucrose content ranged from 16.19 mg g-1 to 83.81 mg g-1, 15.60 mg g-1 to 81.38 mg g-1, and with an average of 30.41 mg g-1, 30.20 mg g-1. In summary, the results not only further verified the practicability of the method established in this study, but also showed that there was a huge difference in the sucrose content between these peanut varieties.

Key words: HPLC-RID, peanut kernel, fructose, glucose, sucrose

Table 1

Peanut samples used in the study"

品种
Variety		 来源
Source		 类型
Type		 种皮颜色
Seed coat color
豫花9805 Yuhua 9805 河南 Henan 普通型 Virginia 粉红色 Pink
冀花甜1号 Jihuatian 1 河北 Hebei 多粒型 Valencia 粉红色 Pink
冀花甜2号 Jihuatian 2 河北 Hebei 多粒型 Valencia 红色 Red
中花12 Zhonghua 12 湖北 Hubei 珍珠豆型 Spanish 红色 Red
中花21 Zhonghua 21 湖北 Hubei 珍珠豆型 Spanish 粉红色 Pink
桂花17 Guihua 17 广西 Guangxi 珍珠豆型 Spanish 粉红色 Pink
桂花22 Guihua 22 广西 Guangxi 珍珠豆型 Spanish 粉红色 Pink
桂花红35 Guihuahong 35 广西 Guangxi 珍珠豆型 Spanish 红色 Red
中花9号 Zhonghua 9 湖北 Hubei 珍珠豆型 Spanish 黑色 Black
桂花红166 Guihuahong 166 广西 Guangxi 珍珠豆型 Spanish 红色 Red
白沙1016 Baisha 1016 广东 Guangdong 珍珠豆型 Spanish 粉红色 Pink
伏花生 Fuhuasheng 山东 Shandong 珍珠豆型 Spanish 粉红色 Pink
粤油9号 Yueyou 9 广东 Guangdong 珍珠豆型 Spanish 粉红色 Pink
闽花5号 Minhua 5 福建 Fujian 珍珠豆型 Spanish 粉红色 Pink
仲恺花1号 Zhongkaihua 1 广东 Guangdong 珍珠豆型 Spanish 粉红色 Pink
湘花1号 Xianghua 1 湖南 Hunan 珍珠豆型 Spanish 粉红色 Pink
豫花10号 Yuhua 10 河南 Henan 普通型 Virginia 粉红色 Pink
冀9402 Ji 9402 河北 Hebei 普通型 Virginia 粉红色 Pink
中花6号 Zhonghua 6 湖北 Hubei 珍珠豆型 Spanish 粉红色 Pink
徐花13 Xuhua 13 江苏 Jiangsu 中间型 Intermediat 粉红色 Pink

Fig. 1

Chromatograms of HPLC A: standards for fructose, glucose and sucrose; B: the sample of Zhonghua 12. a: fructose; b: glucose; c: sucrose."

Table 2

Preparation of sucrose standard solution"

标准液
Standard solution (mL)		 标准液浓度
Concentration of standard solution
(mg mL-1)		 糖储备液
Sugar storage solution
(mL)		 80%乙醇
80%
ethanol (mL)
10 0.5 0.25 9.75
10 1.5 0.75 9.25
10 2.5 1.25 8.75
10 3.5 1.75 8.25
10 4.5 2.25 7.75
10 5.5 2.75 7.25
10 6.5 3.25 6.75

Table 3

Sucrose content under different treatment time and extraction liquid"

提取液
Extracting solution
(mL)		 水浴时间
Water bath time
(min)		 蔗糖含量
Sucrose content
(mg g-1)
10 30 16.33
10 40 16.12
10 50 16.58
15 30 16.25
15 40 16.16
15 50 16.31
20 30 15.88
20 40 16.70
20 50 16.41

Table 4

Results of ANOVA analysis"

变异来源
Variable		 平方和
Sum of squares		 自由度
DF		 均方
Mean square		 F值
F-value		 P值
P-value
80%乙醇80% ethanol (mL) 0 1 0 0.004 0.949
水浴时间Water bath time (min) 0.118 1 0.118 1.937 0.213
误差 Error 0.364 6 0.061
总计 Total 2392.996 9

Table 5

Results of precision test"

序号
Number		 蔗糖含量
Sucrose content (mg g-1)
1 16.78
2 16.73
3 16.96
4 16.78
5 16.97
6 16.88
7 16.72
8 16.80
9 16.97
10 16.82
均值±标准差Mean±SD 16.84±0.10
相对标准偏差
Relative standard deviation (RSD %)		 0.59

Table 6

Result of repetitive test"

序号
Number		 蔗糖含量
Sucrose content (mg g-1)
1 16.66
2 16.91
3 16.79
4 16.90
5 16.97
6 16.72
均值±标准差Mean±SD 16.83±0.12
相对标准偏差
Relative standard deviation (RSD %)		 0.73

Table 7

Results of spike recovery test"

蔗糖储备液
Sucrose storage solution (mL)		 实测蔗糖含量
Content of measured sucrose (mg g-1)		 理论蔗糖含量
Content of theoretical sucrose (mg g-1)		 加标量
Amount of sucrose content (mg)		 回收率Recovery rate (%)
0 16.65 — — —
0.5 26.34 26.65 10 96.90
1.0 36.02 36.65 20 96.85
1.5 46.35 46.65 30 99.03
平均值Mean — — — 97.59

Table 8

Determination of sucrose content by HPLC-RID and GB in peanut kernel (mg mL-1)"

名称
Variety		 均值±标准差Mean±SD 名称
Variety		 均值±标准差Mean±SD
HPLC-RID GB 5009.8-2016 HPLC-RID GB 5009.8-2016
豫花9805 Yuhua 9805 16.58±0.44 15.60±0.75 白沙1016 Baisha 1016 19.59±0.03 20.55±1.57
冀花甜1号 Jihuatian 1 83.81±0.59 81.38±3.67 伏花生 Fuhuasheng 27.82±0.02 28.70±0.56
冀花甜2号 Jihuatian 2 51.61±0.87 48.96±1.33 粤油9号 Yueyou 9 27.56±1.03 27.35±0.18
中花12 Zhonghua 12 16.19±0.20 17.39±0.73 闽花5号 Minhua 5 24.72±0.31 25.14±1.15
中花21 Zhonghua 21 34.42±1.18 32.69±0.82 仲恺花1号 Zhongkaihua 1 26.10±0.54 24.89±0.83
桂花17 Guihua 17 25.62±0.79 25.82±1.36 湘花1号 Xianghua 1 22.61±0.05 23.08±1.54
桂花22 Guihua 22 20.33±0.09 21.37±1.33 豫花10 Yuhua 10 26.77±0.76 25.82±0.58
桂花红35 Guihuahong 35 26.85±0.30 25.72±0.59 冀9402 Ji 9402 60.26±0.90 59.52±1.73
中花9号 Zhonghua 9 31.62±0.05 31.19±0.62 中花6号 Zhonghua 6 21.26±0.69 22.76±2.46
桂花红166 Guihuahong 166 23.12±0.26 23.62±1.08 徐花13 Xuhua 13 21.32±0.25 22.36±3.24
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