An effective and simple procedure to isolate abundant quantities of biologically active chemopreventive lunasin-protease inhibitor concentrate (LPIC) from soybean

Authors: Krishnan, Hari; Wang, Thomas - Tom

Submitted to: Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/4/2015
Publication Date: 1/9/2015
Publication URL: http://handle.nal.usda.gov/10113/60248
Citation: Krishnan, H.B., Wang, T.T. 2015. An effective and simple procedure to isolate abundant quantities of biologically active chemopreventive lunasin-protease inhibitor concentrate (LPIC) from soybean. Food Chemistry. 177:120-126.

Interpretive Summary: Soybeans are a rich source of protein. Several studies have demonstrated that diets containing soybean protein contribute to human health beyond the nutritional aspects. Among soybean seed proteins, lunasin and Bowman-Birk proteinase inhibitor (BBI) have received particular attention because these proteins have chemopreventive activity against different types of cancer. Lunasin and BBI are traditionally purified by ammonium sulfate precipitation, organic solvent extraction, gel filtration, column chromatography, or high performance liquid chromatography. However, these methods are cumbersome, time consuming and cost-prohibitive. To overcome these constrains we have developed a novel method to isolate a soybean protein fraction (LPIC) that is enriched in lunasin and protease inhibitors. Our simple cost-effective procedure avoids the use of time-consuming anion-exchange chromatography and size-exclusion chromatography and the entire isolation procedure can be completed in less than 2 h. Our method can be easily scaled up to yield kilograms of highly enriched lunasin/BBI preparation. This novel isolation procedure can be exploited as a simplified method for the preparation of large quantities of LPIC which can be used as a cancer chemoprotective agent.

Technical Abstract: Lunasin is a 5-kDa soybean bioactive peptide with demonstrated anti-cancer and anti-inflammatory properties. The use of lunasin as a chemopreventive agent in large-scale animal studies and human clinical trials is hampered by the paucity of large quantities of lunasin. Recently, purification methods have been developed to obtain gram quantities of lunasin. However, these methods are cumbersome, time consuming and cost-prohibitive. To overcome these constrains we have developed a novel method to isolate a soybean protein fraction that is enriched in lunasin and protease inhibitors. This procedure involves extraction of soybean flour with 30% ethanol followed by preferential precipitation of lunasin and protease inhibitors by calcium. The calcium precipitated protein fraction, which we termed as Lunasin Protease Inhibitor Concentrate (LPIC), contains three abundant proteins with molecular weights of 21 kDa, 14 kDa and 5 kDa. Immunoblot and mass spectrometry analyses confirmed the 21 kDa as Kunitz trypsin inhibitor, the 14 kDa as soybean 2S albumin precursor and the 5 kDa protein as lunasin. These three proteins accounted for greater than 70% of the total protein content of LPIC. This simple procedure yields 3.2 g of LPIC from 100 g of soybean flour and the entire isolation procedure can be completed in less than 2 h. Treatment of THP-1 human monocyte cell lines with LPIC resulted in suppression of lipopolysaccharide-stimulated cytokine expression, demonstrating that the LPIC isolated by our simple procedure is biologically active.