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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
Article

Abstract

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.

Keywords

Soybean Kunitz trypsin inhibitor Two-dimensional liquid chromatography Purification Quantification 

Notes

Acknowledgements

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)

References

  1. Anuonye JC, Badifu GIO, Inyang CU, Akpapunam MA, Odumudu CU, Mbajika VI (2007) Protein dispersibility index and trypsin inhibitor activity of extruded blends of acha/aoybean: a response surface analysis. Am J Food Technol 2:502–511CrossRefGoogle Scholar
  2. Araba M, Dale NM (1990) Evaluation of protein solubility as an indicator of overprocessing soybean meal. Poult Sci 69:76–83CrossRefGoogle Scholar
  3. Chen Y, Xu Z, Zhang C, Kong X, Hua Y (2014) Heat-induced inactivation mechanisms of Kunitz trypsin inhibitor and Bowman-Birk inhibitor in soymilk processing. Food Chem 154:108–116. doi: 10.1016/j.foodchem.2013.12.092 CrossRefGoogle Scholar
  4. Cruz ACB et al (2013) Bioinsecticidal activity of a novel Kunitz trypsin inhibitor from Catanduva (Piptadenia moniliformis) seeds. Plant Physiol Biochem 70:61–68. doi: 10.1016/j.plaphy.2013.04.023 CrossRefGoogle Scholar
  5. Dipietro CM, Liener IE (1989) Soybean protease inhibitors in foods. J Food Sci 54:606. doi: 10.1111/j.1365-2621.1989.tb04663.x CrossRefGoogle Scholar
  6. Feng J, Liu X, Xu ZR, Wang YZ, Liu JX (2007) Effects of fermented soybean meal on digestive enzyme activities and intestinal morphology in broilers. Poult Sci 86:1149–1154CrossRefGoogle Scholar
  7. Friedman M, Brandon DL (2001) Nutritional and health benefits of soy proteins. J Agric Food Chem 49:1069–1086. doi: 10.1021/jf0009246 CrossRefGoogle Scholar
  8. Goetz H, Kuschel M, Wulff T, Sauber C, Miller C, Fisher S, Woodward C (2004) Comparison of selected analytical techniques for protein sizing, quantitation and molecular weight determination. J Biochem Biophys Methods 60:281–293. doi: 10.1016/j.jbbm.2004.01.007 CrossRefGoogle Scholar
  9. Grieshop CM, Kadzere CT, Clapper GM, Flickinger EA, Bauer LL, Frazier RL, Fahey GC (2003) Chemical and nutritional characteristics of United States soybeans and soybean meals. J Agric Food Chem 51:7684–7691. doi: 10.1021/jf034690c CrossRefGoogle Scholar
  10. Guo P, Piao X, Ou D, Li D, Hao Y (2007) Characterization of the antigenic specificity of soybean protein beta-conglycinin and its effects on growth and immune function in rats. Arch Anim Nutr 61:189–200. doi: 10.1080/17450390701318358 CrossRefGoogle Scholar
  11. Hong KJ, Lee CH, Kim SW (2004) Aspergillus oryzae GB-107 fermentation improves nutritional quality of food soybeans and feed soybean meals. J Med Food 7:430–435. doi: 10.1089/jmf.2004.7.430 CrossRefGoogle Scholar
  12. Hsieh J-F, Chen S-T (2007) Comparative studies on the analysis of glycoproteins and lipopolysaccharides by the gel-based microchip and SDS-PAGE. Biomicrofluidics 1. doi: 10.1063/1.2399892
  13. Huisman J, Vanderpoel AFB, Mouwen J, Vanweerden EJ (1990) Effect of variable protein contents in diets containing Phaseolus vulgaris beans on performance, organ weights and blood variables in piglets, rats and chickens. Br J Nutr 64:755–764. doi: 10.1079/bjn19900077 CrossRefGoogle Scholar
  14. Jansman AJM, Huisman J, Beelen GM, Wiebenga J (1998) Effects of soya trypsin inhibitors on faecal nutrient digestibility and nitrogen retention in young piglets. In: Jansman AJM, Hill GD, Huisman J, VanderPoel AFB (eds) Recent advances of research in antinutritional factors in legume seeds and rapeseed. EAAP European Association for Animal Production Publication, vol 93, pp 331–334Google Scholar
  15. Julka S, Kuppannan K, Karnoup A, Dielman D, Schafer B, Young SA (2012) Quantification of Gly m 4 protein, a major soybean allergen, by two-dimensional liquid chromatography with ultraviolet and mass spectrometry detection. Anal Chem 84:10019–10030. doi: 10.1021/ac3024685 CrossRefGoogle Scholar
  16. Maurer M, Muller AC, Wagner C, Huber ML, Rudashevskaya EL, Wagner SN, Bennett KL (2013) Combining filter-aided sample preparation and pseudoshotgun technology to profile the proteome of a low number of early passage human melanoma cells. J Proteome Res 12:1040–1048. doi: 10.1021/pr301009u CrossRefGoogle Scholar
  17. Oddepally R, Sriram G, Guruprasad L (2013) Purification and characterization of a stable Kunitz trypsin inhibitor from Trigonella foenum-graecum (fenugreek) seeds. Phytochemistry 96:26–36. doi: 10.1016/j.phytochem.2013.09.010 CrossRefGoogle Scholar
  18. Onesti S, Brick P, Blow DM (1991) Crystal structure of a Kunitz-type trypsin inhibitor from Erythrina caffra seeds. J Mol Biol 217:153–176. doi: 10.1016/0022-2836(91)90618-g CrossRefGoogle Scholar
  19. Pacheco WJ, Stark CR, Ferket PR, Brake J (2014) Effects of trypsin inhibitor and particle size of expeller-extracted soybean meal on broiler live performance and weight of gizzard and pancreas. Poult Sci 93:2245–2252. doi: 10.3382/ps.2014-03986 CrossRefGoogle Scholar
  20. Palliyeguru MWCD, Rose SP, Mackenzie AM (2011) Effect of trypsin inhibitor activity in soya bean on growth performance, protein digestibility and incidence of sub-clinical necrotic enteritis in broiler chicken flocks. Br Poult Sci 52:359–367. doi: 10.1080/00071668.2011.577054 CrossRefGoogle Scholar
  21. Pedersen MH et al (2008) Soybean allergen detection methods—a comparison study. Mol Nutr Food Res 52:1486–1496. doi: 10.1002/mnfr.200700394 CrossRefGoogle Scholar
  22. Perez-Maldonado RA, Mannion PF, Farrell DJ (2003) Effects of heat treatment on the nutritional value of raw soybean selected for low trypsin inhibitor activity. Br Poult Sci 44:299–308. doi: 10.1080/0007166031000085463 CrossRefGoogle Scholar
  23. Reitsma BH, Yeung ES (1987) Reversed-phase high-performance liquid chromatography of soybean trypsin inhibitor with optical activity and ultraviolet absorbance detection. J Chromatogr A 405:295–303. doi: 10.1016/S0021-9673(01)81770-2 CrossRefGoogle Scholar
  24. Romarheim OH, Aslaksen MA, Storebakken T, Krogdahl A, Skrede A (2005) Effect of extrusion on trypsin inhibitor activity and nutrient digestibility of diets based on fish meal, soybean meal and white flakes. Arch Anim Nutr 59:365–375. doi: 10.1080/17450390500352897 CrossRefGoogle Scholar
  25. Sun P, Li D, Dong B, Qiao S, Ma X (2008a) Effects of soybean glycinin on performance and immune function in early weaned pigs. Arch Anim Nutr 62:313–321. doi: 10.1080/17450390802066419 CrossRefGoogle Scholar
  26. Sun P, Li D, Li Z, Dong B, Wang F (2008b) Effects of glycinin on IgE-mediated increase of mast cell numbers and histamine release in the small intestine. J Nutr Biochem 19:627–633. doi: 10.1016/j.jnutbio.2007.08.007 CrossRefGoogle Scholar
  27. Torbica AM, Zivancev DR, Nikolic ZT, Dordevic VB, Nikolovski BG (2010) Advantages of the lab-on-a-chip method in the determination of the Kunitz trypsin inhibitor in soybean varieties. J Agric Food Chem 58:7980–7985. doi: 10.1021/jf100830m CrossRefGoogle Scholar
  28. Zarkadas LN, Wiseman J (2005) Influence of processing of full fat soya beans included in diets for piglets. I. Performance. Anim Feed Sci Technol 118:109–119. doi: 10.1016/j.anifeedsci.2004.09.010 CrossRefGoogle Scholar
  29. Zhou J-Y, Liao H, Zhang N-H, Tang L, Xu Y, Chen F (2008) Identification of a Kunitz inhibitor from Albizzia kalkora and its inhibitory effect against pest midgut proteases. Biotechnol Lett 30:1495–1499. doi: 10.1007/s10529-008-9699-0 CrossRefGoogle Scholar
  30. Zhou T, Qiao S, Ma X, He P (2015) Detection and analysis of main antinutritional factors content in soybean products. Chinese Journal of Animal Nutrition 27:221–229Google Scholar

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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