Bioconversion of grain-based starch into extracellular polymeric substances (EPSs) and anticancer efficacy evaluation

Abstract

This project aims at exploring a novel anticancer compound (i.e., extracellular polymeric substances-EPSs) through biotransformation of crop starches by a marine protist (Thraustochytrid striatum) and using a novel 3D platform to evaluate the mimic in vivo anticancer efficacy of the EPSs in comparison with the traditional 2D platform. In addition to glucose as a control, five different types of starches from corn and wheat were studied as substrates for T. striatum fermentation to produce EPSs. The EPSs were fractionalized, purified, and characterized in structure and composition to identify its functioning components. The anticancer efficacy of the EPSs was assessed on both HepG2 (human hepatocellular carcinoma cells) and HeLa (human cervical cancer cells). Raw starchy substrates were successfully converted into EPSs by T. striatum by using its own amylolytic enzymes. The starch-degrading enzyme complex was found to mainly include α-amylase and α-glucosidase, which was able to degrade corn and wheat starches (normal and waxy) into glucose and maltose in one-pot at room temperature without separate liquefaction and saccharification. The crude enzyme activities were dependent on the cell growth stage of T. striatum. The amylolytic enzymes of T. striatum could be a promising robust starch-degrading enzyme systems that has a potential commercial value for starch industry. Among starchy substates examined, the EPSs yield varied with the growth of T. striatum cells. The composition analysis showed that the predominant carbohydrates of EPSs were mannose, glucose, and galactose. The monosaccharide compositions of EPSs derived from different starches were consistent, but the proportion of each monosaccharide differed significantly. In addition, the structure analysis of FTIR and NMR showed that the different-sourced EPSs also had different chemical structures. Overall, the main chain of EPSs consisted of α-mannose and α-glucose. The sulfated-galactose was found in EPSs as the main structure of (1→3) linked 4-Osulfate-β-D-galactose, which was also found in red algae EPSs. The starch-based EPSs differed from the glucose-based counterpart. Such structural difference in EPSs could be because the EPSs synthesis involved substrate-regulated metabolism pathways. The variation of starchy substrate affected the antiproliferative activity of EPSs against HepG2 and HeLa, i.e., EPSs’ anticancer activity is starchy substrate related. Wheat-EPSs exhibited consistent and antiproliferation effect on HepG2, yet slightly inhibitory effect to HeLa cells. The inhibition of cancer cell proliferation could be attributed to the sulfated polysaccharides involving ROS-mediated apoptosis. However, more research will be needed to explore the mechanisms governing the EPSs’ anticancer activity. A dosage over 200 μg/mL showed a significant antiproliferation effect on cancer cells. However, EPSs derived from other starchy substrates had worse performance of antiproliferation on cancer cells, which could be attributed to their different compositional and/or structural characteristics from wheat-EPSs.