Purification and Quantification of Kunitz Trypsin Inhibitor in Soybean Using Two-Dimensional Liquid Chromatography
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Purification and Quantification of Kunitz Trypsin Inhibitor in Soybean Using Two-Dimensional Liquid Chromatography Tianjiao Zhou 1 & Shuaijuan Han 1 & Zhen Li 2 & Pingli He 1
Received: 5 January 2017 / Accepted: 10 April 2017 # Springer Science+Business Media New York 2017
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 Electronic supplementary material The online version of this article (doi:10.1007/s12161-017-0902-6) contains supplementary material, which is available to authorized users. * Pingli He [email protected] 1
State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing 100193, People’s Republic of China
2
State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, People’s Republic of China
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
Introduction Soybean, with about 40% crude protein, is one of the major vegetative protein sources used in the food and feed industries. Soybean meal provides ∼70% of protein meal utilized in animal feed mixtures for poultry and swine. However, soybean meal contains a number of antinutritional factors, which can interfere with the digestion and absorption of nutrients, disturb normal metabolism and cause adverse physiological responses, and result in hypersensitivity and decrease the production performance of animals (Guo et al. 2007; Sun et al. 2008a, b). Trypsin inhibitors (TI) are important antinutritional factors in soybean seeds, which can lead to decrease of
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