Optimized Single-Step Recovery of Lipophilic and Hydrophilic Compounds from Raspberry, Strawberry and Blackberry Pomaces Using a Simultaneous Ultrasound-Enzyme-Assisted Extraction (UEAE)

Abstract

An ultrasound-enzyme-assisted extraction (UEAE) was optimized to extract, simultaneously, the hydrophilic and lipophilic compounds from three berry pomaces (raspberry, strawberry and blackberry). First, an enzyme screening designated a thermostable alkaline protease as the most suitable enzyme to recover, in an aqueous medium, the highest yields of polyphenols and oil in the most efficient way. Secondly, the selected enzyme was coupled to ultrasounds (US) in sequential and simultaneous combinations. The simultaneous US-alkaline enzyme combination was selected as a one-single-step process and was then optimized by definitive screening design (DSD). The optimized parameters were: US amplitude, 20% (raspberry pomace) or 70% (strawberry and blackberry pomaces); pH, 8; E/S ratio, 1% (w/w); S/L ratio, 6% (w/v); extraction time, 30 min; temperature, 60 °C. Compared to conventional extractions using organic solvents, the UEAE extracted all the polyphenols, with around 75% of the active polyphenols (measured by the DPPH^● method) and up to 75% of the initial oil from the berry pomaces. Characterized lipophilic compounds were rich in polyunsaturated fatty acids (PUFAs), tocols and phytosterols. The polyphenolics were analyzed by UPLC-MS/MS; characteristic ellagitannins of the Rosaceae family (sanguiin H-6 or agrimoniin, sanguiin H-10, …) and ellagic acid conjugates were found as the major components.

Keywords: DSD optimization; alkaline protease; antioxidant activity; berry pomaces; oil; polyphenols; ultrasound-enzyme-assisted extraction.

Figure 1

Enzyme screening for the simultaneous recovery of polyphenols and oil in water from raspberry pomace. A mix of carbohydrases (Viscozyme L), one acid protease (P0107) and one thermostable alkaline protease (Alcalase 2.4 L FG) were evaluated, alone or sequentially combined. The enzyme-to-solid ratio (E/S) was set at 2% (w/w) and extractions were conducted under the optimum conditions given by the suppliers. Control extractions were also realized with the same extraction parameters but without enzyme. The dashed line represents the yields obtained with conventional extractions taken as references (hexane for oil recovery and methanol:acetone:water (7:7:6, v/v/v) for total and active polyphenols recoveries).

Figure 2

Effect of alkaline protease and US combinations on the simultaneous recovery of oil and polyphenols from raspberry pomace. A control extraction was carried out in the same extraction conditions without enzyme or US. Extractions with only the enzyme (EAE) and only US (UAE) were carried out as a comparison under the same conditions. Two sequential extractions were screened: a US pre-treatment followed by enzyme extraction and vice versa (EAE → UAE; UAE → EAE) as well as a simultaneous extraction with the enzyme used in situ with a US treatment (UEAE). Extraction parameters were fixed at: extraction time = 1 h or 30 min pre-treatment followed by 30 min extraction; US amplitude (if relevant) = 70%; pH = 8; E/S ratio (if relevant) = 2% (w/w); S/L ratio = 5% (w/v); T° = 50 °C; and size particle = < 250 µm. Alkaline protease from Bacillus licheniformis was used as the enzyme system (Alcalase 2.4 L FG, ≥2.4 U/g). The dashed line represents the yields obtained with conventional extractions (hexane for oil recovery and methanol:acetone:water (7:7:6, v/v/v) for total and active polyphenols recoveries). Mean values are expressed in efficiency (%) compared to conventional extractions with standard deviations (n = 3). Different letters indicate significant differences calculated by ANOVA (p < 0.05).

Figure 3

Representation of the distribution of the calculated loss functions for the 13 essays carried out during DSD experimental design for the optimization of UEAE procedure. Experiment 14 is a combination of experiments 3 and 8 and was designated as the optimized extraction. The represented loss function allows one to give one score for each experiment taking into account the obtained extraction yields (oil and polyphenols) and their corresponding deviations. A loss function tending to 0% is synonymous with the best extraction parameters, with the highest yields and the lowest standard deviations, while a loss function of 100% corresponds to the worst experiment.

Figure 4

Synergistic effect of US and alkaline protease combination (UEAE) on the simultaneous recovery of lipophilic and hydrophilic compounds from raspberry pomace in an aqueous medium. Optimized extraction parameters were determined at: US amplitude = 70%; pH = 8; S/L ratio = 6% (w/v); E/S ratio = 1% (w/w); extraction time = 30 min; extraction temperature = 60 °C. Alkaline protease from Bacillus licheniformis (Alcalase 2.4 L FG, ≥2.4 U/g) was used as an enzyme system and particle size < 250 µm. Controls without enzyme or US (control extraction), with the enzyme alone (EAE) and with US alone (UAE) were carried out as a comparison under the same extraction parameters. The dashed line represents the yields obtained with conventional extractions (hexane for oil recovery and methanol:acetone:water (7:7:6, v/v/v) for total and active polyphenols recoveries). Mean values are expressed in efficiency (%) compared to conventional extractions with standard deviations (n = 3). Different letters indicate significant differences calculated by ANOVA (p < 0.05).