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作物学报 ›› 2010, Vol. 36 ›› Issue (12): 2062-2072.doi: 10.3724/SP.J.1006.2010.02062

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

大豆异黄酮组分HPLC快速分析技术及其在豆腐加工中的应用

王春娥1,2,赵团结1,盖钧镒1,*   

  1. 1 南京农业大学大豆研究所 / 国家大豆改良中心 / 作物遗传与种质创新国家重点实验室,江苏南京 210095;2 九江学院生命科学学院,江西九江 332000
  • 收稿日期:2010-03-04 修回日期:2010-08-04 出版日期:2010-12-12 网络出版日期:2010-10-09
  • 通讯作者: 盖钧镒, E-mail: sri@njau.edu.cn, Tel: 025-84395405
  • 基金资助:

    本研究由国家重点基础研究发展规划(973计划)项目(2006CB1017, 2009CB1184, 2010CB1259), 国家高技术研究发展计划(863计划)项目(2006AA1001, 2009AA1011), 国家自然科学基金项目(30671266), 国家科技支撑计划项目(2006BAD13B05-7), 高等学校创新引智计划项目(B08025), 国家重点实验室开放基金项目(ZW2009005), 江苏博士后科研计划项目(0901045C), 江西科技支撑计划项目(2009BNB06700)和江西教育科技项目(GJJ10672)和江西农业科技项目(Ny0901)资助。

Establishment of A Rapid HPLC Method for Quantifying Isoflavone Components and Its Application in Tofu Processing

WANG Chun-E,ZHAO Tuan-Jie,GAI Jun-Yi   

  1. 1 National Key Laboratory for Crop Genetics and Germplasm Enhancement / Soybean Research Institute, Nanjing Agricultural University / National Center for Soybean Improvement, Nanjing 210095, China; 2 College of Life Science, Jiujiang University, Jiujiang 332000, China
  • Received:2010-03-04 Revised:2010-08-04 Published:2010-12-12 Published online:2010-10-09
  • Contact: GAI Jun-Yi, E-mail: sri@njau.edu.cn, Tel: 025-84395405

摘要: 大豆异黄酮育种与大豆制品加工研究需要进行大批量样品12种异黄酮组分的快速定量分析。在前人研究基础上利用Agilent 1100高效液相色谱(HPLC)系统,以大豆品种南农95C-13为材料,研究大豆苷元,大豆苷,乙酰基大豆苷,丙二酰基大豆苷,染料木苷元,染料木苷,乙酰基染料木苷,丙二酰基染料木苷,黄豆苷元,黄豆苷,乙酰基黄豆苷,丙二酰基黄豆苷等12种标准品外标法快速定量技术。从样品制备与色谱条件对分析12种异黄酮组分的准确度和分离度入手,确定分析流程,以(科丰1号×南农1138-2)的184个重组自交系(NJRIKY)为材料,研究豆腐加工中总量和各组分的变化特点。(1)样品以80%甲醇水溶液50℃超声1 h提取;色谱条件为,检测波长254 nm, 柱温36℃,流速2.0 mL min–1, 进样量 10 μL, 流动相0.1%(V/V)乙酸水溶液(A)和100%甲醇(B),0~2 min,27% B (V/V)→2~3 min,27%~38% B→3~10 min, 38% B→10~12 min,38%~39% B→12~14 min, 39% B→14~15 min, 39~27% B梯度洗脱;在15 min内将12个组分良好分离,各组分峰面积与其相应浓度均呈良好线性关系(R2为0.9976~0.9999);加标回收率均大于99%,变异系数低于2%。(2)NJRIKY群体籽粒、豆乳、豆腐异黄酮总量和组分的大量分析验证了HPLC快速技术的效果。籽粒异黄酮总含量(3 695.00 μg g–1)在豆乳加工中平均85.15%转入豆乳 (3 146.12 μg g–1),14.85% (548.88 μg g–1)进入豆渣。传统豆腐加工通过硫酸钙絮凝,只有17.32% (639.89 μg g–1)转入豆腐,67.83% (2 506.23 μg g–1)留在黄浆水中。豆乳中12种组分含量比籽粒稍低,均以丙酰基染料木苷含量最高;而豆腐中乙酰基染料木苷和乙酰基黄豆苷缺失,以染料木苷元与大豆苷元含量较高。大豆科丰1号与南农1138-2杂交重组后家系间的异黄酮遗传变异增大,增加了对其遗传改良的潜力。

关键词: 大豆, 异黄酮组分, 高效液相色谱(HPLC), 梯度洗脱, 豆乳, 豆腐

Abstract: A rapid, precise, and stable quantifying method of isoflavone components is the key to quality soy–food processing and genetic improvement of quality soybeans. A quick procedure used Agilent 1100 high performance liquid chromatograph (HPLC) system with the diode array detection (DAD) and Zorbax SB-C18 packed column (5 μm, 4.6 ID × 150 mm) using external standards of isoflavone components, including daidzein,daidzin, 6”-O-acetyldaidzin, 6”-O-malonyldaidzin,genistein, genistin,6”-O-acetylgenistin, 6”-O-malonylgenistin, glycitein, glycitin,6”-O-acetylglycitin and 6”-O-malonylglycitin was established for measuring the 12 isoflavones in soybean seeds and its processing products. The procedure includes the following key points: 80% methanol aqueous solution under ultrasonication for 1 h at 50℃ was chosen; for separation of the 12 isoflavone components within 15 min, the mobile phase of 0.1% acetic acid (V/V) aqueous solvent A and 100% methanol solvent B with the flow rate of 2.0 mL min–1, injection volume at 10 μL, column temperature at 36℃ and detection wavelength at 254 nm were selected; and the linear gradient extraction of 0–2 min, 27% B (V/V)→2~3 min, 27~38% B→3–10 min, 38% B→10–12 min, 38–39% B→12–14 min, 39% B→14–15 min, 39%–27% B was adopted. The procedure was linear (R2 = 0.9976–0.9999), precise (CV or RSD ranged from 0.90% to 3.35% for 2 232 samples from NJRIKY), accurate [recoveries were more than 99.00% for the different concentrations of the 12 isoflavones (CV of 0.22%–1.40%)], robust (inter-day CV of 0.24%–3.95%) and rapid (less than 15 min for 12 isoflavones resolved). The procedure was verified to be effective by a large sample determination of isoflavone components in soybean seed, soymilk and tofu using the soybean population of NJRIKY. The data indicated that the isoflavones were 3 695.00 μg g–1 in seed, among them 14.85% (548.88 μg g–1) were transferred to the residual, 85.15% (3 146.12 μg g–1) to soymilk, but only 17.32% (639.89 μg g–1) to tofu while 67.83% (2 506.23 μg g–1) to whey under the traditional tofu processing with CaSO4 as coagulant. The 12 isoflavone components in soymilk was somewhat less than those in seeds with 6”-O-Malonylgenistin the highest in the both, while in tofu 6”-O-Acetylgenistin and 6”-O-Acetylglycitin were deficient but with high contents of genistein and daidzein. In addition, there was an enlarged genetic variation of the 12 isoflavone components among the lines, indicating the increased genetic potential for quality improvement due to recombination between Kefeng 1 and Nannong 1138-2.

Key words: Soybean, Isoflavone component, High performance liquid chromatograph (HPLC), Gradient elution, Tofu, Soymilk

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