An Up-Scalable and Cost-Effective Methodology for Isolating a Polypeptide Matrix Metalloproteinase-9 Inhibitor from <i>Lupinus albus</i> Seeds

• Main Authors: Joana Mota, Maria E. Figueira, Ricardo B. Ferreira, Ana Lima
• Format: Article
• Language: English
• Published: MDPI AG 2021-07-01
• Series: Foods
• Subjects: MMP-9; HT29; GRAS-safe; deflamin; white lupin; nutraceutical
• Online Access: https://www.mdpi.com/2304-8158/10/7/1663

One of the most challenging problems with food-borne bioactive compounds is that there are commonly no cost-effective, generally recognized as safe (GRAS) methods for obtaining gram quantities of their purified forms. Here we aimed at developing a method to isolate deflamin, an oligomeric protein from lupin seeds with anti-inflammatory and anticancer activity through matrix metalloprotease (MMP)-9 inhibition. Our goal was to develop a GRAS method that could be easily up-scalable whilst maintaining deflamin’s activity. A sequential precipitation methodology was developed, using an aqueous extraction, followed by heat denaturation, acid precipitation and solubilization in ethanol. A final precipitation with 90% ethanol yielded a purified protein which was sequenced through mass spectrometry and tested for its MMP inhibitory activity using the Dye-quenched (DQ) gelatin assay and the standard wound healing assay in HT29 cells. The developed method yielded a purified oligomer, which represented 0.1% (<i>w</i>/<i>w</i>) of total dry seed weight and was positively confirmed to be deflamin. It further showed to effectively reduce MMP-9 gelatinolytic activity as well as colon cancer cell migration, hence corroborating the effectiveness of our method. Overall, this is the first reported method for isolating an MMP-9 inhibitor from legume seeds, which is up-scalable to an industrial level, in a cost-effective manner.