Food Analytical Methods

, Volume 10, Issue 10, pp 3350–3360 | Cite as

Purification and Quantification of Kunitz Trypsin Inhibitor in Soybean Using Two-Dimensional Liquid Chromatography

  • Tianjiao Zhou
  • Shuaijuan Han
  • Zhen Li
  • Pingli HeEmail author


Kunitz trypsin inhibitor (KTI) is one of the major antinutritional factors in soybean and results in inhibition of digestion of dietary protein. In this study, we developed a novel strategy to purify and quantify KTI from soybean using two-dimensional liquid chromatography. Lipids from ground soybean were removed using hexane after which the ground soybean was extracted with protein extraction buffer. The crude extract was first purified by weak anion exchange chromatography, and then the fraction containing KTI was further separated by size exclusion chromatography. The fraction containing KTI was collected and analyzed by SDS-PAGE and electrospray ionization mass spectrometry. Results indicated that purified KTI has a molecular mass of 20 kDa and a purity of ~98% with inhibitory activity of 2425 TIU/mg protein. This assay was used for the quantification of KTI in soybean samples. The assay showed concentrations with a range between 7.81 and 500.00 μg/mL and a limit of detection of 0.12 mg/g. The recoveries of KTI in spiked soybean samples were between 82.19% and 86.65%, and intra- and interday precisions (% CV) were less than 7.35% and 8.42%. The developed method was used to analyze soybean samples from different sources and soybean products derived from different processing techniques, which demonstrated that the developed procedure provided an accurate and sensitive tool for separation and quantification of intact KTI in soybean.


Soybean Kunitz trypsin inhibitor Two-dimensional liquid chromatography Purification Quantification 



The financial support from the National Basic Research Program of China (973 Program, 2013CB127305), the National Natural Science Foundation of China (31472126), and the 111 Project (B16044) is gratefully acknowledged.

Compliance with Ethical Standards

Conflict of Interest

Tianjiao Zhou declares that he has no conflict of interest. Shuaijuan Han declares that he has no conflict of interest. Zhen Li declares that he has no conflict of interest. Pingli He declares that he has no conflict of interest.

Ethical Approval

This article does not contain any studies with human participants performed by any of the authors.

Informed Consent

Not applicable.

Supplementary material

12161_2017_902_MOESM1_ESM.doc (12.8 mb)
ESM 1 (DOC 13115 kb)


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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Tianjiao Zhou
    • 1
  • Shuaijuan Han
    • 1
  • Zhen Li
    • 2
  • Pingli He
    • 1
    Email author
  1. 1.State Key Laboratory of Animal Nutrition, College of Animal Science and TechnologyChina Agricultural UniversityBeijingPeople’s Republic of China
  2. 2.State Key Laboratory of Plant Physiology and Biochemistry, College of Biological SciencesChina Agricultural UniversityBeijingPeople’s Republic of China

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