1. Introduction
Discovering new end uses of agro-byproducts through the establishment of green and sustainable technologies for their valorization is a challenging task of pivotal importance [
1]. Over the last years, the surge in the demand and consumption of wine (75% of the annual grape production) worldwide, and therefore the intensification of the winemaking process, has resulted in out-producing and discarding a huge volume of grape and wine byproducts (leaves, skin, stalk, stems, grape marc, seeds, wine lees, vine shoots, etc.) [
1].
The byproduct accumulated at the bottom of vessels containing wine after the completion of alcoholic or malolactic fermentation, decanting, clarification, tartaric stabilization, filtration, storage, or other typical procedures of winemaking, as well as the residue recovered by filtration or centrifugation of this product, are referred to as wine lees [
2,
3]. Traditionally, wine lees are used during wine aging for improving the sensory attributes of wine, most importantly for the enhancement of the sensorial profile of wine and color stability [
4].
Wine lees, although produced in lower quantities than the main winery byproduct, represent about 5% (
w/
w) of total grape mass used for vinification and an estimated annual residue for 2019 of about 0.88–2.66 million tones worldwide. This solid vinification byproduct presents a rich source of bioactive compounds [
2,
5], including tartaric acid, inorganic compounds, proteins, insoluble carbohydrates, phenolic compounds, yeast cells and other non-soluble residual grape material (seeds, skins. etc.). (Poly)phenolic compounds in wine lees are endowed with a high antiradical, antioxidant and metal chelating potential allowing them to act through biochemical mechanisms in order to delay oxidation phenomena preventing lipid oxidation and formation of peroxides [
6,
7]. The chemical profile of wine lees is highly depended on the
Vitis variety, the agroclimatic conditions in vines, the geographical origin, oenological techniques and the time of wine aging [
8,
9]. The commercial potentials of wine byproducts and their rich-in-natural-compounds extracts are gaining attention. Wine lees and other wine residues can be used as (a) ingredients of novel functional foods [
1,
10], (b) fortification agents that furnish high nutritional value and stability to the final products, (c) antioxidant and antimicrobial factors, (d) industrial enzymes [
11,
12], (e) substrates for novel mushroom cultivars [
13], (f) bioactive microencapsulated components [
14] and biopolymers [
2].
As the cornerstone in the pipeline of obtaining high-added value compounds and final products of excellent quality, the selection of an extraction technique of minimum environmental impact is of utmost significance [
1]. The main pitfalls of conventional extraction approaches are extended extraction time, high required volumes of extraction solvents, low selectivity and, in certain cases, non-thermoprotective nature [
15]. On the other hand, non-conventional extraction techniques (ultrasound-assisted extraction, pressurized liquid extraction, etc.) are engaging attention due to their short extraction time, reduced amounts of hazardous organic solvents and replacement with green innovative solvents (ionic liquids and deep eutectic solvents), maximization of extraction yields and reduced use of water and energy, lower risks and high reproducibility [
15,
16]. In addition, a large variety of bioactive components (i.e., (poly)phenols, carotenoids, polysaccharides, lignans, alkaloids, etc.) of different physicochemical properties (i.e., structure, polarity, volatility, etc.) are extracted from several matrices, among them wine and grape byproducts [
1,
17,
18].
Considered a non-conventional extraction technique, microwave-assisted extraction (MAE) shares the advantages of the other non-traditional approaches. However, the two main drawbacks of this methodology are the poor extraction yields of volatile compounds due to the higher temperatures and the limited number of available extraction solvents, since a MAE solvent must absorb the microwave energy [
15]. Apart from extraction solvent, other critical factors regarding MAE efficiency are extraction time, the ratio of solvent to material, microwave power and extraction temperature [
15].
By apprehending the market dynamics and future perspectives of high-added value products, the aim of the present study was (a) to optimize the extraction process of bioactive compounds using MAE and experimental design and (b) to employ spectrophotometric, spectroscopic and microbiological analysis for the assessment of the biological activity, antimicrobial activity and chemical profile of the wine lees’ extracts. The overall goal was to evaluate the current findings, proposing an efficient and eco-friendly procedure for wine lees exploitation.
3. Materials and Methods
3.1. Reagents and Standards
Folin–Ciocalteu’s (FC) phenol reagent was supplied from Merck KGaA (Darmstadt, Germany). Gallic acid (3,4,5-trihydroxybenzoic acid) was obtained from Alfa Aesar GmbH&Co (Karlsruhe, Germany). Trolox (6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) and potassium persulfate were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid ammonium salt) (ABTS●+) was purchased from Tokyo Chemical Industry Co. LTD (Tokyo, Japan). 2,4,6-tris(2-pyridyl)-S-triazine (TPTZ) and iron (III) chloride hexahydrate were obtained from Sigma Chemical (St. Louis, MO, USA). All reagents used were of analytical grade and were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France), Merck KGaA (Darmstadt, Germany) and Carlo Erba Reagents (Val de Reuil, France).
3.2. Wine Sees Sample-Set and Sample Preparation
Wine lees samples (N = 28) of white and red grape varieties were collected during the vinification period in autumn 2016 from different wineries from the Attica and Peloponnese regions in Greece. Samples with their general characteristics are presented in
Table 6. Until further treatment, samples were kept in glass bottles in −20 °C in darkness.
After sample collection, all samples were centrifuged at 2100 G for 10 min at 10 °C in order to separate the wine lees’ sediment from the supernatant, which was discarded. The sediment was then dried in an oven at 40 °C until reaching a stable weight to remove sample moisture and to suspend any enzymatic and microbial activity. Dried material was homogenized and powdered in a laboratory mill (Type ZM1, Retsch GmbH, Haan, Germany). Dry material and samples were placed in glass jars and vials at −20 °C until further process and analysis. The moisture of wine lees sediments varied from 40–80% as shown in
Table S4. The flowchart of the developed experimental platform is presented in
Figure 3.
3.3. Microwave-Assisted Extraction (MAE) Instrumentation and Process
Microwave-assisted extraction (MAE) was performed using a CEM Focused Microwave Synthesis System, Model Discover (CEM Corporation, Matthews, NC, USA) in open-vessel mode with a reflux system adjusted over the open cell.
Ethanol, water and a 1:1 v/v mixture of ethanol:water were the extraction solvents used. One gram (1 g) of dried wine lees sediment was used for the extraction. The solvent volume was not the same in all experimental runs since solvent-to-material ratio was one of the factors under optimization of the experimental design models. After MAE, the extract was filtered with Whatman paper filter and evaporated to dryness by rotary evaporation at 50 °C. Then, the dry residue of the extract was collected with 5 mL of the extraction solvent. Aliquots of the extracts were used for the (a) spectrophotometric, (b) IR and (c) antimicrobial analyses.
3.4. Experimental Design (DOE) Models
The screening and the optimization of the extraction factors were carried out on the dry residue of the wine lees sediment by implementing a two-level full factorial design, 23, and a symmetrical 16-run three-level Box–Behnken design (BBD), respectively. The examined extraction factors were the (a) microwave (MW) power, X1 (W); (b) extraction time, X2 (min) and (c) solvent/material ratio, X3 (mL g−1). The extraction yield of (poly)phenols, expressed as mg of gallic acid equivalents (GAE) per gram of dry sediment, was chosen as the measured response of the two DOE models.
In order to settle on the optimal extraction conditions that allow the recovery of (poly)phenols from samples of both high and low concentration, the wine lees sample used for the DOE process belonged to the white variety Savvatiano (Sample 7,
Table 4), since white varieties contain lower concentrations of (poly)phenols than red varieties [
28]. In addition, the Savvatiano variety is the most representative variety of the current sample set as it includes the majority of white wine samples.
In order to assure that DOE models provide unbiased results, the real values of the extraction variables, which are expressed in different physical units (i.e., Watt, minutes, volume-to-weight, etc.), were transformed to coded normalized dimensionless values (x
1, x
2, x
3) [
52]. Real and normalized values of the extraction factors for the two DOE models are presented in
Table S5.
3.5. Spectrophotometric Analyses
3.5.1. Total Phenolic Content (TPC)
The total phenolic content (TPC) of each sample was determined by applying a micromethod of Folin–Ciocalteu’s (FC) colorimetric assay [
53]. The results were expressed as mg of gallic acid equivalents (GAE) per 1 g of dried sediment, using a standard curve with a range of 25–500 mg L
−1 gallic acid (y = 0.001 × x + 0.003, R
2 = 0.997). The photometric measurements were performed at 750 nm.
3.5.2. Scavenging Activity on 2,2′-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) Radical (ABTS●+)
For each sample, the antiradical activity was determined as described in previous works [
53]. Trolox, a water-soluble form of vitamin E, was used as a standard compound, and the antiradical activity of each sample was expressed as mg of Trolox equivalents (TE) per 1 g of dried sediment, using a standard curve with a concentration range of 0.20–1.5 mM (y = 0.282 × x − 0.002, R
2 = 0.995). Sample measurements were conducted at 734 nm.
3.5.3. Ferric Reducing/Antioxidant Power Assay (FRAP)
The Ferric reducing/antioxidant power for each sample was evaluated based on the reduction in Fe(III) in the form of ferric-2, 4,6-tripyridyl-s-triazine complex to Fe(II), as described by Lantzouraki et al. [
53]. A standard curve was prepared using various concentrations (600–2000 μM) of FeSO
4·7H
2O stock solutions. The results are expressed as mg of Fe (II) per 1g of dried sediment (y = 0.00027 × x − 0.011, R
2 = 0.997). The analysis was carried out at 593 nm.
3.6. Fourier Transform Infrared Spectroscopy (FTIR)
FT-IR spectra were collected with an Alpha-P spectrometer (Bruker, Billerica, MA, USA), the Alpha FT-IR wine analyzer (Bruker Optics) on a diamond ATR crystal covered with a flow through cell, facilitating sample injection. The Alpha-P instrument has a potassium bromide (KBr) beam splitter and a 2 × 2 mm temperature controllable ATR diamond crystal sample plate, which was set at 40 °C. The instrument was fitted with OPUS software (OPUS version 7.2 for Microsoft Windows, Bruker Optics, Billerica, MA, USA). No further sample preparation was performed for spectral analysis and volumes of 5 mL were used. The spectrum of each sample and background were obtained from 4000 to 375 cm−1 and the average of 64 scans at a resolution of 8 cm−1 with a scanner velocity of 7.5 kHz was recorded. One background measurement was taken before each sample measurement. The ALPHA Wine Analyzer comes with a starter calibration that was assembled by the accredited (DAkkS) Institute Heidger (Kesten, Germany) and contains more than 1700 wines from wine producing countries worldwide. The organic acid and sugar contents were measured for each wine lees sample using the “ALPHA wine analyzer” apparatus and the starter calibration curves dedicated to each of the determined compounds.
3.7. Antimicrobial Activity
3.7.1. Tested Microorganisms
Four Gram (+) bacteria (
Bacillus cereus clinical isolate,
Micrococcus flavus ATCC 10240,
Staphylococcus aureus ATCC 6538 and
Listeria monocytogenes NCTC 7973), four Gram (−) bacteria (
Escherichia coli ATCC 35210,
Enterobacter cloacae human isolate,
Pseudomonas aeruginosa ATCC 27853 and
Salmonella typhimurium ATCC 13311) and three resistant bacteria, Methicillin-resistant
Staphylococcus aureus (MRSA),
Escherichia coli and
Pseudomonas aeruginosa PAO1, were used for testing of antibacterial activity of wine less products. The microorganisms are deposited in the Mycological Laboratory, Department of Plant Physiology, Institute for Biological Research “Siniša Stanković”, University of Belgrade, Belgrade, Serbia. The bacterial suspensions were adjusted with sterile saline to a concentration of 1.0 × 10
5 CFU mL
−1. The inocula were prepared daily and stored at 4 °C until use. Dilutions of the inocula were cultured on solid medium to verify the absence of contamination and to check the validity of the inoculum. All experiments were performed in duplicate and repeated three times. The isolation and determination of clinical bacteria is described in
Supplementary Materials [
54,
55].
3.7.2. In Vitro Assays for Determination of Antibacterial Activity—Microdilution Method
The compounds were tested on antibacterial activity using a microdilution method [
55,
56]. Minimal inhibitory (MIC) and minimal bactericidal (MBC) concentrations of the tested samples were determined by serial dilutions of compounds dissolved in 5% DMSO-water solution in 96-well microtitre plates. Bacterial inoculum, 1.0 × 10
4 CFU mL
−1, was added to LB medium and compounds dissolved in 5% DMSO solution containing 0.1% Tween 80 (
v/
v) (1 mg mL
−1). Minimal inhibitory concentration (MIC) was defined as the lowest concentration that inhibited bacterial growth, without visible growth, at the binocular microscope. The lowest concentration with no visible growth was defined as the MBC, indicating 99.5% killing of the original inoculum. All wells were measured at a wavelength of 655 nm by Microplate manager 4.0 (Bio-Rad Laboratories, Hercules, California, USA) and compared with a blank and the positive control. Antibiotics used as a positive control were Streptomycin (Sigma P 7794) and Ampicillin (Panfarma, Belgrade, Serbia) (1 mg mL
−1 in sterile physiological saline), while 5% DMSO was used as a negative control. All experiments were performed in duplicate and repeated three times.
3.8. Data Analysis
Data processing was conducted and graphs were produced using the Statistica package (Version 12, TIBCO Software Inc., Palo Alto, CA, USA). All measurements were realized at 95% (
p-values ≤ 0.05) confidence level. Normal or non-normal distribution of the samples was confirmed by using the Shapiro–Wilks test. All data were normally distributed with the exception of BBD model data. However, since the number of samples/observations was larger than 30, normality can be assumed for the BBD dataset [
57]. In all cases, the pairwise multiple comparison of the samples’ averages to determine whether there is any statistical difference between them and which of them differ significantly was performed by one-way ANOVA and Tukey honestly significant difference post hoc analysis. The correlation of spectrophotometric results was carried out using Pearson’s correlation.
4. Conclusions
Wine lees are the byproduct accumulated at the bottom of vessels containing wine after the completion of winemaking processes, as well as the residue recovered by filtration or centrifugation of this byproduct [
2]. As the cornerstone in the pipeline of obtaining high-added value final products of excellent quality and beneficial biological activities, the selection of an extraction technique of reduced environmental impact is of utmost significance [
1].
In the present study, MAE process was optimized using two-level full-factorial and Box–Behnken design in order to achieve higher TPC values. Ethanol and water mixture at a 1:1 ratio was selected as the best extraction solvent, while extraction temperature was set at 85 °C. Extraction time at 35 min, MW power at 54 W and solvent/material ratio at 60 mL g−1 were determined as the optimal values of extraction factors. In general, white varieties showed lower FRAP and ABTS•+ values than red varieties, which contained higher amounts of (poly)phenols, tannins and anthocyanins.
The same trend was also presented in the antimicrobial assays, where red varieties exhibited strong antimicrobial activities especially against B. aureus strains. Based on the results, samples of the white variety Moschofilero were the ones with the most significant biological activities. On the other hand, samples of Merlot and Agiorgitiko red varieties demonstrated the most satisfactory antioxidant, antiradical and antimicrobial activity. However, factors other than grape variety, such as the vineyard location and the fermentation stage, also emerged as crucial factors affecting wine lees biological activities. Post-fermented red wine lees samples from Peloponnese showed higher TPC, ABTS•+ and FRAP values and lower MIC values against Gram-negative bacteria. On the other hand, red wine lees extracts obtained at pre-fermentation stage from Attica displayed high antibacterial activity against both Gram-negative and Gram-positive bacteria. Furthermore, the interpretation of FT-IR bands highlighted the presence of sugars (glucose, fructose and sucrose), amino acids, organic acids and para-substituted aromatic compounds in wine lees extracts.
By overviewing the results of the present study, MAE could emerge as a suitable alternative for the recovery of extracts with strong antioxidant and antimicrobial activity from winemaking by-products by replacing the laborious and time-consuming conventional extraction techniques.