Unlocking the potential of polysaccharide-degrading enzymes for polyphenols’ recovery from plant biomass

PIAZZA, DANA M; GENTILE, JULIA; MEINI, MARÍA ROCÍO

Rosario

Workshop; V Encuentro & II Workshop de la Red Argentina de Tecnología Enzimática (Red TEz); 2023

Red Argentina de Tecnología Enzimática

Introduction and objectives. Agro-industrial byproducts are a sustainable source of natural additives that can replace the synthetic ones in the food industry. Polyphenols are natural antioxidants with multiple health-promoting properties. They are secondary plant metabolites with a wide range of structures and thus, solubilities. In our previous study a novel extraction process of these compounds was developed using enzymes (tannase, cellulase, and pectinase) that specifically liberates target polyphenols in the appropriate hydroalcoholic mixture. In this way, the aim of this study was to test this extraction process using an enzyme preparation that would be used in an industrial scale. For these, we used Viscozyme L from Novozymes, a multi-enzyme complex used in the food industry, which contains a wide range of carbohydrases, including arabanase, cellulase, beta-glucanase, hemicellulase and xylanase. Methods. Cellulase, pectinase and xylanase activities of Viscozyme L were determined in media containing ethanol concentrations between 0% and 30% v/v. Polyphenols’ extracts were obtained from dried and milled grape pomace (GP; Vitis vinifiera) of the Cabernet Sauvignon variety. The extractions were carried out at 40 ℃ for 2 h, using Viscozyme L in different ethanol concentrations. The total phenolic content (Folin-Ciocalteu), antioxidant activity (TEAC), and polyphenolics profile (HPLC-DAD) of the extracts were determined. Also, a qualitative analysis of the extracts was carried out by mass spectrometry (HPLC-UV-MS). Results. Cellulase, pectinase, and xylanase activities of the enzyme preparation were retained up to 17, 79, and 60 %, respectively, in the maximum concentration of ethanol tested. The best results in terms of total polyphenols and antioxidant activity were obtained using Viscozyme L in 20 % and 30 % v/v ethanol. Extracts with polyphenols’ concentrations between 0.56 – 2.12 g Gallic Acid Equivalents/100 g GP were obtained; and antioxidant activity between 2.86 – 13.18 mmol Trolox Equivalents/100 g GP. Although the extracts presented high content of total polyphenols and great antioxidant activity, it was seen that these results were mainly because of the solvent. However, the detailed analysis of the polyphenolics profile showed that this enzymatic cocktail selectively enriched the extracts in high-value phenolic compounds such as gallic acid, p-coumaric acid, syringic acid, and malvidin-3-O-glucoside. Also, by mass spectrometry, it was seen that Viscozyme L allowed the recovery of caffeic acid, p-coumaric acid and quercetin, that were not present in the control extractions. Conclusions. The results obtained demonstrate that the polyphenols’ extraction process developed using enzymes of high purity is also applicable with Viscozyme L, a multi-enzyme complex used in the food industry. This method allows the extracts’ enrichment on specific polyphenols of great value. This selectivity is the result of the combination of the specificity of enzymes, their ability to act in a range of concentrations of organic solvent, and the solubility of the desired polyphenols. We are currently working on the recombinant production of fungal feruloyl esterase and tannase enzymes using Pichia pastoris. We aim to produce hydroxycinnamic acids and gallic acid from different agro-industrial by-products, such as brewery waste, olive pomace, tea waste, and wheat bran. By doing this, we will be able to confirm the effectiveness and wide applicability of the developed extraction process.