Green Method Comparison and Optimization of Anthocyanin Recovery from "Sangiovese" Grape Pomace: A Critical Evaluation of the Design of Experiments Approach

Abstract

Grape pomace is the main by-product obtained from wine production that is still enriched in bioactive compounds. Within a framework of waste/by-product reuse through a sustainable approach, various green methods were utilized in this work to recover anthocyanins from the pomace resulting from "Sangiovese" grape vinification. Ultrasound- and Microwave-Assisted Extractions (UAE and MAE) were coupled with the use of green solvents, such as acidified water, an ethanol/water mixture, and Natural Deep Eutectic Solvents (NaDES), and their efficacy was compared with that of a conventional method based on a methanol/acidified water mixture. The Total Anthocyanin Index ranged from 36.9 to 75.2 mg/g DW for UAE, and from 54.4 to 99.6 mg/g DW for MAE, while resulting in 47.1 mg/g DW for conventional extraction. A Design of Experiments (DoE) approach was applied to MAE, the most efficient technique. Temperature, time, and the solid-to-liquid ratio were set as X variables, while malvidin-3-O-glucoside content and antioxidant activity were used as response variables, measured by High-Performance Liquid Chromatography with Diode Array Detection (HPLC-DAD) and 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay, respectively. The correlation between temperature and time and the antioxidant activity of the extract was positive, while it was found to be negative when considering malvidin-3-O-glucoside concentration as a response variable. Thus, the optimal conditions in temperature, time and solid-to-liquid ratio were different depending on the chosen variable. The results underline the importance of selecting an accurate response when using the response surface methodology approach.

Keywords: Design of Experiments; MAE; MODDE; NaDES; UAE; anthocyanins; grape pomace; green extraction; response surface model.

Figure 1

Summary of fit for the Design of Experiments models used; (a) 50% acidified ethanol; (b) acidified water; (c) NaDES mixture with 30% water.

Figure 2

Scaled and centered coefficient plots for the three refined models obtained with MAE coupled with the three green solvents. (a) 50% acidified ethanol; (b) acidified water; (c) NaDES mixture with 30% water. Temp: temperature; Tim: time; S/L: solid-to-liquid ratio.

Figure 3

Response surface plots. (a) 50% ethanol acidified with 1% HCl; (b) water with 1% citric acid; (c) NaDES (choline chloride/citric acid = 2:1; 30% water).

Figure 4

Assessment results of greenness of procedures optimized for oenin extraction from grape pomace: (a) 50% acidified ethanol; (b) acidified water; (c) NaDES mixture. The central, circular field corresponds to the final assessment score. It is surrounded by 10 labeled, wedge-shaped fields corresponding to each AGREEprep criterion. The color of each field, including the central field, is mapped to the particular score (0–1) through a “traffic lights” colour map (red-yellow-green), with red assigned to 0.0 and green to 1.0.

Figure 5

(a) HPLC-DAD profile of anthocyanin standards; (b) HPLC-DAD profile of grape pomace extract obtained with 50% ethanol optimized using the oenin content as Y variable; (c) HPLC-DAD profile of grape pomace extract obtained with 50% ethanol optimized using the antioxidant activity measured by DPPH assay as Y variable.