Ariel R. Fontana

Grape pomace as a sustainable source of bioactive compounds: extraction, characterization, and biotechnological applications of phenolics.

Grape pomaces (GPs) are characterized by high contents of phenolics due to an incomplete extraction during the winemaking process. These phenolics are secondary plant metabolites with potential beneficial effects on human health because of their antioxidant activity and antimicrobial, antiviral, and anti-inflammatory properties. Therefore, GP constitutes an inexpensive source for the extraction of phytochemicals that can be used in the pharmaceutical, cosmetic, and food industries. As a result of the increased attention to sustainability of agricultural practices, efforts have been made to use GP in different fields of industry. Thus, it is necessary to have efficient extraction techniques to achieve good recoveries of compounds. In this respect, sensitive and selective analytical methods have been tried for the characterization of phenolic extracts. This review summarizes the most recent developments in the extraction of polyphenols from GPs. Furthermore, the techniques used for characterization of extracts are explained, with emphasis on sample preparation, separation, and analysis of phenolics. Finally, the possible applications of GP extracts in diverse biotechnological fields are also discussed.

Characterization of polyphenols and evaluation of antioxidant capacity in grape pomace of the cv. Malbec

Low molecular weight polyphenols (LMW-PPs) and anthocyanins, along with the antioxidant capacity, were assessed in grape pomace extract (GPE) of red grape (Vitis vinifera L.) cv. Malbec. Twenty-six phenolics (13 LMW-PPs and 13 anthocyanins) were characterized and quantified by HPLC-MWD and UPLC-ESI-MS. The maximum concentrations of LMW-PPs corresponded to the flavanols (+)-catechin and (-)-epicatechin, whereas malvidin-3-glucoside was the most abundant anthocyanin. Piceatannol, a stilbene analogue to resveratrol with higher antioxidant activity, was firstly identified and quantified in GPE of the cv. Malbec. The antioxidant activity for Malbec GPE determined by oxygen radical absorbance capacity (ORAC) assay was 2,756 μmol TEg(-1) GPE. Therefore, the data reported sustain the use of winemaking by-products as a cheap source of phenolic compounds suitable for biotechnological applications, as a strategy for sustainable oenology.

High-throughput method based on quick, easy, cheap, effective, rugged and safe followed by liquid chromatography-multi-wavelength detection for the quantification of multiclass polyphenols in wines

In this work, a reliable, simple, fast, inexpensive and robust sample preparation approach for the determination of multiclass polyphenols in wine samples is proposed. The polyphenols selected for this work were gallic acid, (+)-catechin, (-)-epicatechin, caffeic acid, syringic acid, coumaric acid, ferulic acid, trans-resveratrol, quercetin and cinnamic acid. The method is based on QuEChERS (quick, easy, cheap, effective, rugged and safe) extraction technique coupled with dispersive solid-phase extraction (d-SPE) clean-up. Under optimized conditions, the analytes were extracted from 5mL wine samples (previously acidified with 1% formic acid) using 2.5mL acetonitrile. For phase separation, 1.5g NaCl and 4g anhydrous MgSO4 were added. Then, a 1mL aliquot of the partitioned supernatant was cleaned-up using d-SPE with a combination of 150mg CaCl2, 50mg primary-secondary amine (PSA) and 50mgC18 as sorbents. A 250μL aliquot of the obtained cleaned extract was concentrated to dryness and taken up with the initial mobile phase previous to liquid chromatography-multi-wavelength detection (LC-MWD). The proposed method provided limits of detection (LODs) ranging from 0.004 to 0.079μgmL(-1) and an inter-day variability below 12% RSD for all analytes in red and white wine samples. Considering external calibration (red wines) and matrix-matched calibration (white wines) as quantification techniques, the overall recoveries (accuracy) of the method ranged between 75.0% and 119.6% for red and white wine samples, respectively. The developed method was applied for the determination of polyphenols in 10 wines produced in Argentina. Nine phenolic compounds were determined, at concentrations above detectable levels in the method. The maximum concentrations corresponded to (-)-epicatechin in white wines, while gallic acid and (+)-catechin were the most abundant in red wines.

Development of a high-performance liquid chromatography method based on a core–shell column approach for the rapid determination of multiclass polyphenols in grape pomaces

A rapid and economically affordable reverse-phase chromatographic approach based on a core-shell column with high-performance liquid chromatography multi-wavelength detector (HPLC-MWD) is proposed for the quantification and quality control of multiclass polyphenols (PPs). The separation of 20 relevant polyphenols from grape pomace extracts (GPEs) was achieved in less than 12 min by using a Kinetex C18 column (3.0 mm × 100 mm, 2.6 μm) with a gradient system of ultrapure water (0.1% formic acid) and acetonitrile, a temperature of 35 °C and a flow rate of 0.8 mL min(-1). The maximum backpressure reached was 327 bar, meaning the developed method is adequate for standard HPLC instruments. The applicability of the method was demonstrated by the determination of PPs in GPEs of different red grape varieties. Cabernet Sauvignon GPE showed the highest content of studied PPs (9804.2 μg g(-1)GPE) followed by Bonarda GPE (7302.0 μg g(-1)GPE). Besides the methodological development for a high throughput routine quality control of GPEs, this is the first report of PPs content for Bonarda and Aspirant Bouchet GPE, so the results add knowledge for these grape varieties cultivated in Argentina.

Matrix solid-phase dispersion: a simple and fast technique for the determination of phenolic compounds in olive oil by liquid chromatography

A methodology was developed using matrix solid-phase dispersion, together with liquid chromatography with multiple wavelength detectors for the determination of 20 phenolic compounds in olive oil samples. Under optimized conditions, the analytes were extracted using 0.5 g of olive oil, 1.0 g of Florisil as a sorbent and 1 mL methanol–water (80 : 20 acidified with formic acid 0.5% (v/v)) as an eluting solvent. The proposed methodology provided detection and quantification limits of individual compounds in the ranges of 0.02–0.75 and 0.08–2.50 mg kg−1, respectively. The RSDs resulting from the analysis of six replicates of 0.5 g of a sample pool containing 2.5 mg kg−1 phenolic compounds were ranged between 2.1% and 14.8%. Considering matrix-matched calibration as a quantification technique, the average recoveries ranged from 74.8% to 95.0% with relative standard deviations between 1.5% and 9.3%. The developed methodology was applied for the determination of phenolic compounds in nine olive oils produced in Argentina, identifying seventeen analytes at concentrations above the detectable levels.

Malbec grape (Vitis vinifera L.) responses to the environment: Berry phenolics as influenced by solar UV-B, water deficit and sprayed abscisic acid

High-altitude vineyards receive elevated solar ultraviolet-B (UV-B) levels so producing high quality berries for winemaking because of induction in the synthesis of phenolic compounds. Water deficit (D) after veraison, is a commonly used tool to regulate berry polyphenols concentration in red wine cultivars. Abscisic acid (ABA) plays a crucial role in the acclimation to environmental factors/signals (including UV-B and D). The aim of the present study was to evaluate independent and interactive effects of high-altitude solar UV-B, moderate water deficit and ABA applications on Vitis vinifera cv. Malbec berries. The experiment was conducted during two growing seasons with two treatments of UV-B (+UV-B and -UV-B), watering (+D and -D) and ABA (+ABA and -ABA), in a factorial design. Berry fresh weight, sugar content, fruit yield, phenolic compounds profile and antioxidant capacity (ORAC) were analyzed at harvest. Previous incidence of high UV-B prevented deleterious effects of water deficit, i.e. berry weight reduction and diminution of sugar accumulation. High UV-B increased total phenols (mainly astilbin, quercetin and kaempferol) and ORAC, irrespectively of the combination with other factors. Fruit yield was reduced by combining water deficit and high UV-B or water deficit and ABA. Two applications of ABA were enough to induced biochemical changes increasing total anthocyanins, especially those with higher antioxidant capacity.

Phenolics profiling of pomace extracts from different grape varieties cultivated in Argentina

Grape pomace can be considered as an excellent and inexpensive source of phenolic compounds with potential bioactive properties. Therefore, the key aim of this study was to carry out a comparative study of different Argentinean grape varieties for selecting the most convenient residue during the recovery of compounds with antioxidant activities that will be useful in emerging industrial applications. Phenolics, including 17 non-anthocyanins and 13 anthocyanins, isolated from grape pomace (GP) obtained from the varieties Malbec, Cabernet Sauvignon, Cabernet Franc, and Merlot, were characterised via assessing different chemical profiles of the studied samples. The maximum concentrations of the non-anthocyanins corresponded to the following flavanols: (−)-epicatechin (5518 μg g−1), (+)-catechin (5340 μg g−1), procyanidin B1 (1820 μg g−1), and syringic acid (6665 μg g−1). Malvidin-3-glucoside was the most abundant anthocyanin (32 056 μg g−1). The compound OH-tyrosol was first identified and quantified in the GPE of Cabernet Franc and Malbec. Moreover, stilbene trans-resveratrol was found at the levels as high as 328 μg g−1 GPE in Malbec, thus highlighting the potentiality of the variety as a source of compounds with nutraceutical value. A discussion about the relationship between the high levels of syringic acid and the anthocyanin malvidin-3-glucoside has also been presented. Based on the results, new knowledge for understanding the possible synergic effects between the contents of compounds of different families in the extracts was presented. This information can be helpful for the technological application of the extracts and to justify different beneficial effects achieved when these extracts are used in experimental models.

Filter-vial dispersive solid-phase extraction as a simplified clean-up for determination of ethylphenols in red wines

In-vial filtration with dispersive solid-phase extraction (d-SPE) clean-up of QuEChERS (quick, easy, cheap, effective, rugged and safe) extracts is proposed for the determination of ethylphenols (EPs) in red wines. Analytes were extracted from 5mL wine sample (previously alkalinized with 0.5% sodium hydroxide) using 5mL acetonitrile. For phase separation, 1.5g NaCl and 4g anhydrous MgSO4 were added. Then, a 0.5mL aliquot of the partitioned supernatant was cleaned-up using d-SPE and in-vial filtration with a combination of anhydrous CaCl2 (100mg) and primary-secondary amine (PSA, 25mg) as sorbents. The proposed method provided limits of quantification (LOQs) ranging from 0.024 to 0.04mgL-1. Considering matrix-matched calibration as quantification technique, the recoveries (accuracy) ranged between 73% and 116%. The method was applied for the determination of EPs in 15 commercial wines of Argentina, where 4-EP was quantified at concentrations ranging from 0.25 to 3.01mgL-1.

High-throughput modified QuEChERS method for the determination of the mycotoxin tenuazonic acid in wine grapes

A reliable, simple, fast, inexpensive and robust sample preparation approach for tenuazonic acid (TA) determination in grapes by liquid chromatography with ultraviolet detection (HPLC-UV) is proposed. The method is based on a modified QuEChERS (quick, easy, cheap, effective, rugged and safe) approach using ethyl acetate as extraction solvent. Its involves extraction of 2.5 g sample (plus 2.5 mL 1% formic acid acidified water) with 5 mL of ethyl acetate; drying/cleaning-up of extract with 0.25 g anhydrous CaCl2 and determination by HPLC-UV. The method was optimized and validated achieving a limit of quantification (LOQ) of 0.05 μg g−1. The overall recoveries were 96%, 82% and 97% for grape samples spiked at 0.05, 0.5 and 5 μg g−1, respectively. The method showed excellent reproducibility with RSDs for the above data ≤8% and Horwitz ratios <0.11. The procedure was applied to evaluate the occurrence of TA in grapes and it was quantified at concentrations ranging between 0.057 and 0.595 μg g−1. The methodological development allows a high throughput routine quality control of grapes, adding a valuable tool for the winemaking industry. The method could be applied in an average laboratory and help to understand possible effects related to Alternaria rotting on the final wines elaborated. As well, this is the first report of TA presence in grapes used for wine elaboration, so the results add new knowledge to a growing research area.

Natural occurrence and production of tenuazonic acid in wine grapes in Argentina

Abstract A survey was carried out to determine natural occurrence of tenuazonic acid (TA) in healthy and rotten wine grapes samples from different varieties (n = 37) collected during 2016 vintage in the region of DOC San Rafael (Argentina). In addition, inoculation experiments with three Alternaria alternata strains in wine grapes were done to elucidate TA production and its major influencing factors. The 16.2% (6/37) of total wine grape samples showed TA contamination with 4% (1/25) of incidence in healthy samples (77 μg·kg−1) and 42% (5/12) in rotten samples (10–778 μg·kg−1). Malbec, Cabernet Sauvignon, and Syrah varieties showed TA contamination, whereas Bonarda, Ancelota, Torrontés, Semillón, and Chenin did not. During inoculation experiments in wine grapes, two of three strains were able to produce TA among the evaluated conditions and the highest TA production was observed at 15°C and 25°C after 24 days of incubation. Nutritional composition of grapes results appropriate for A. alternata infection and TA production and, together with the adequate field conditions, favors TA natural occurrence in wine grapes.