Application of the INFOGEST Standardized Method to Assess the Digestive Stability and Bioaccessibility of Phenolic Compounds from Galician Extra-Virgin Olive Oil.

The INFOGEST standardized method was applied to assess the potential bioaccessibility and bioaccessibility of the phenolic compounds from a Galician extra-virgin olive oil (EVOO). The in vitro digestion model involves three steps and generates two fractions after each one: an aqueous fraction (namely, water phase (Wp)) and an oily fraction (namely, oily phase (Op)). The results showed that secoiridoids were the most abundant family in the Galician EVOO polar fraction, representing 98% of the total phenolic compounds. After oral digestion, phenolic acids and simple phenols were mainly detected in Wp, while lignans and flavonoids were mostly found in Op. After gastric digestion, extensive hydrolysis of secoiridoids was observed to generate free tyrosol, hydroxytyrosol, and hydroxytyrosol acetate. The instability of secoiridoids after intestinal digestion was again responsible for the release of simple phenols, which were mainly recovered in Wp together with flavonoids. In contrast, lignans were stable to duodenal conditions and remained in Op.

Effect of olive ripening degree on the antidiabetic potential of biophenols-rich extracts of Brava Gallega virgin olive oils.

The diet management is imperative to anticipate risk factors that favour the development of diseases; indeed, the intake of virgin olive oil could be an alternative natural source of α-glucosidase enzyme inhibitors, which delay the digestion rate of carbohydrates. Consequently, the impact of diabetes mellitus (DM) could be diminished. Extra Virgin Olive Oils (EVOO) were elaborated from Galician autochthonous variety 'Brava Gallega' with olives selected at three different degree of ripeness (ripening index, RI: 1.4, 3.0, 5.5) in order to assess the effect of maturation on overall chemical composition, sensory quality, and enzyme inhibition. The phenolic profile of the EVOOs determined by LC-ESI-IT-MS exhibited quantitative differences as ripening advanced; for example oleocanthal, tyrosol, luteolin and apigenin concentrations were higher in the overripe olive oil (RI 5.5). Anyway, the phenolic extracts (from every tested RI) were more active than acarbose. In particular, those obtained from the most mature olives displayed the most powerful inhibitory activity (IC50 value of 143 µg of dry extract/mL). In addition, the significant effect of these compounds (i.e. luteolin, apigenin, tyrosol and oleocanthal) on the inhibitory activity of the olive oil extracts was demonstrated. Our results suggest that, regardless of RI, the inhibitory activity of 'Brava Gallega' olive oils could represent a valuable strategy for reinforcing the health claim of olive oil for phenolic compounds.

Tetraconazole alters the methionine and ergosterol biosynthesis pathways in Saccharomyces yeasts promoting changes on volatile derived compounds.

The influence of antifungal tetraconazole residues (either as an active substance or as a commercial formulation product) on the fermentative activity of Saccharomyces cerevisiae yeast was evaluated in pasteurized Garnacha red must by using laboratory-scale fermentation assays. The presence of this fungicide promoted a slight decrease in glucose consumption. Volatile fermentative-derived compounds were evaluated in deep. Statistically significant changes were found in methionol (with a mean decrease of around 24%), fatty acids (with increments ranged from 23% to 66%), and ethyl esters (with increases ranged from 23% to 145%) contents when grape musts were enriched with the commercial formulation at both contamination levels assayed. Based on protein mass fingerprinting analysis, it was possible to relate these variations on volatiles content with changes in the activity of several enzymes (Met3p, Met14p, Adh2p, Hmg1p, Erg5p, Erg6p, Erg11p, and Erg20p) involved in the secondary metabolism of yeasts.

Mepanipyrim residues on pasteurized red must influence the volatile derived compounds from Saccharomyces cerevisiae metabolism.

The impact of mepanipyrim (Mep) and its corresponding commercial formulation (Mep Form) on Saccharomyces cerevisiae metabolites was assessed, separately, by using laboratory-scale wine fermentation assays on pasteurized red must. The presence of Mep did not alter the fermentation course. With regard to volatiles formed at the intracellular level by fermenting yeast cells, Mep residues affected mainly the acetate and ethyl ester biochemical pathways. In particular, the target acetates showed a notorious increment, >90%, in presence of commercial Mep Form at the higher dose assayed. The addition of Mep and Mep Form, at both tested levels, highly increased ethyl caprylate (between 42 and 63%) and ethyl caprate (between 36 and 60%) contents as the same as their respective fatty acid precursors. No important effects were observed on colour and non-volatile pyranoanthocyanins, probably due to the low anthocyanin content characteristic of pasteurized musts.

The involvement of phenolic-rich extracts from Galician autochthonous extra-virgin olive oils against the α-glucosidase and α-amylase inhibition.

'Brava' and 'Mansa de Figueiredo' extra-virgin olive oils (EVOOs) are two varieties identified from north-western Spain. A systematic phenolic characterization of the studied oils was undertaken by LC-ESI-IT-MS. In addition, the role of dietary polyphenols from these EVOOs has been evaluated against the inhibition of key enzymes (α-glucosidase and α-amylase) in the management of diabetes mellitus (DM). Oleuropein and ligstroside derivatives comprised 83% and 67% of the total phenolic compounds in 'Brava' and 'Mansa de Figueiredo' EVOOs, respectively. The main secoiridoids from oleuropein were DOA (3,4-DHPEA-EDA, 59 and 22 mg kg-1, respectively) and the main isomer of OlAgl (3,4-DHPEA-EA, 74 and 23 mg kg-1). The main secoiridoids from ligstroside were D-LigAgl (p-HPEA-EDA or oleocanthal, 23 and 167 mg kg-1) and the main isomer of LigAgl (p-HPEA-EA, 214 and 114 mg kg-1). For α-glucosidase, both EVOO extracts displayed stronger inhibitory activity (IC50 values of 60 ±â€¯8 and 118 ±â€¯9 µg mL-1, respectively) than the commercial inhibitor acarbose (IC50 = 356 ±â€¯21 µg mL-1). Nevertheless, for α-amylase, only 'Brava' extracts showed anti-α-amylase capacity. A daily VOO intake lower than the requirements of EFSA seem to be enough to reach both 50% for α-glucosidase and 25% for α-amylase inhibition. These findings support the potential health benefits derived from Galician EVOOs that might be probably linked to the outstanding high concentration levels of phenolic acids and flavonoids.

Evaluation of the neuroprotective and antidiabetic potential of phenol-rich extracts from virgin olive oils by in vitro assays.

In this work, phenol-rich extracts from 'Cornicabra' and 'Picual' virgin-olive oils (EVOOs) were examined, for the first time, to establish their capacity to inhibit key enzymes involved in Alzheimer's disease (AD) (acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and 5-lipoxygenase (LOX)), major depressive disorder (MDD) and Parkinson's disease (PD) (monoamine oxidases: hMAO-A and hMAO-B respectively), and diabetes mellitus (DM) (α-glucosidase and α-amylase). 'Cornicabra' displayed the best inhibitory activity against all enzymes, when compared to 'Picual': BuChE (IC50 = 156 ±â€¯4 and 308 ±â€¯33 mg mL-1), LOX (IC50 = 26 ±â€¯0.5 and 37 ±â€¯3 mg mL-1), hMAO-A (IC50 = 20 ±â€¯2 and 37 ±â€¯0.2 mg mL-1), hMAO-B (IC50 = 131 ±â€¯7 and 215 ±â€¯13 mg mL-1) and α-glucosidase (IC50 = 154 ±â€¯17 and 251 ±â€¯31 mg mL-1), respectively. The behaviour observed can be associated with the higher content of secoiridoids, lignans and phenolic acids in 'Cornicabra' EVOO.

Characterisation of extra virgin olive oils from Galician autochthonous varieties and their co-crushings with Arbequina and Picual cv.

The current trend of the olive oil market is the production of high quality extra from traditional minor olive varieties with peculiar and differentiated characteristics (especially with respect to the aromatic and phenolic composition). In this way, the interest of Galician oil producers (NW Spain) in recovering old autochthonous Local olive fruits has increased substantially in recent years. In order to investigate the potential of the Local olives by either producing high quality monovarietal oils or mixing with the most widespread olives in Galicia (Arbequina and Picual cv.), quality indices, and fatty acid composition as well as volatile and phenolic profiles were determined and compared. All EVOOs studied in this work can be considered as "extra virgin olive oil" due to quality indices fell within the ranges established in legislation. Picual and Local olive oils as well as those resulting from their co-crushing reach values which are required by EU legislation to add the specific health claim on the oil label. Co-crushing Picual:Local (80:20) provided a significant enhancement of grass and apple nuances and a decrease of banana notes with respect to Picual oils. The co-crushing process improved sensory and health properties of Picual extra virgin olive oils. The effect of co-crushing on phenolics, ester volatiles and banana nuances cannot be easily modulated, contrary to quality indices and fatty acid composition, both changing linearly in strict correlation with the fruit mass ratio.

Quality of extra virgin olive oils produced in an emerging olive growing area in north-western Spain.

Systematic studies of physico-chemical and stability-related properties, and chemical composition, of extra virgin olive oils (EVOOs) obtained from drupes cropped in specific regions are of special agricultural interest. This is particularly so with new production areas, where careful selection of the most suitable olive varieties for EVOO production is required. This paper reports the first comprehensive chemical characterisation of EVOOs obtained from three different olive varieties (viz., Picual, Morisca and Manzanilla de Sevilla) grown in a new cultivation area in Galicia (NW Spain). The Morisca variety was that providing the highest industrial oil yield (21%). However, the three types of EVOO exhibited no statistically significant differences in standard quality-related indices other than acidity. Morisca EVOO was that with the lowest content in oleic acid (mean=68%) and highest content in linoleic acid (mean=13%). Also, Morisca EVOO exhibited the highest sterol levels (mean=1,616 mg/kg) and Picual EVOO the lowest (mean=1,160 mg/kg). Picual EVOO contained greater amounts of the phenolic compounds luteolin and pinoresinol than both Morisca and Manzanilla de Sevilla EVOOs. Finally, Manzanilla de Sevilla EVOO exhibited differential attributes, with banana and olive fruit aromatic series prevailing predominantly over bitter-like, pungent-like and leaf series.

Improvements in the malaxation process to enhance the aroma quality of extra virgin olive oils.

The influence of olive paste preparation conditions on the standard quality parameters, as well as volatile profiles of extra virgin olive oils (EVOOs) from Morisca and Manzanilla de Sevilla cultivars produced in an emerging olive growing area in north-western Spain and processed in an oil mill plant were investigated. For this purpose, two malaxation temperatures (20/30 °C), and two malaxation times (30/90 min) selected in accordance with the customs of the area producers were tested. The volatile profile of the oils underwent a substantial change in terms of odorant series when different malaxation parameters were applied.

The phenolic chemistry and spectrochemistry of red sweet wine-making and oak-aging.

A natural sweet wine (NSW) was made with dried grapes from Vitis vinifera L. cv Garnacha Tintorera. A fortified sweet wine (FSW) was also obtained: the maceration-alcoholic fermentation of Garnacha Tintorera must was stopped by addition of ethanol 96% (v/v). UV/Vis spectrophotometry and HPLC/DAD-ESI/MS were applied to determine, respectively, the evolution of colour and phenolic compounds in Garnacha Tintorera based-sweet wines during aging. In sweet wines, aging decreased a(∗) (red/green), colour saturation and lightness and increased b(∗) (yellow/blue), and hue angle. Most of the phenolic compounds determined, such as anthocyanins, esters of hydroxycinnamic acids, flavan-3-ols monomers, oligomers and polymers decreased in both sweet wines during aging. On the contrary, hydroxybenzoic and hydroxycinnamic acids and vitisins increased after one year of aging. Despite that both terminal and extension subunit compositions show very small changes, mean degree of polymerisation of proanthocyanidins decline slightly as aging progressed in both sweet wines.

Ultrasound-assisted emulsification-microextraction for the determination of phenolic compounds in olive oils.

A reliable, sensitive and effective method based on ultrasound-assisted emulsification-microextraction (USAEME) coupled to HPLC-DAD has been developed to identify and quantify several target phenolic compounds from extra virgin olive oils (EVOO). This approach is based on the emulsification of a microvolume of polar organic extractant in a non-polar liquid sample by ultrasound radiation and further separation of both liquid phases by centrifugation. The percentage of methanol/water (v/v) in the extractant, the volume of extractant, and the extraction time as three effective parameters on the extraction were optimised by a central composite design (Box-Behnken response surface) method. The optimised method presented recoveries in EVOO between 91% and 115% for the target analytes (except vanillin with 65%) and a satisfactory precision with relative standard deviations (RSD%) lower than 8.4% for repeatability and reproducibility. The method showed good linearity and limits of detection and quantification were in the range 0.001-0.14 and 0.004-0.47mg/kg, respectively. After method validation, it was successfully applied to the analysis of three EVOO samples. All target compounds were detected in all analysed samples. Tyrosol and hydroxytyrosol were the major phenolic compounds, followed by pinoresinol and luteolin.

Influence of new generation fungicides on Saccharomyces cerevisiae growth, grape must fermentation and aroma biosynthesis.

The influence of ten new generation fungicides (ametoctradin, benthiavalicarb-isopropyl, boscalid, cyazofamid, dimethomorph, fenhexamid, kresoxim-methyl, mepanipyrim, metrafenone, and pyraclostrobin) on the fermentative activity of Saccharomyces cerevisiae yeast was initially evaluated in pasteurised red must. The presence of ametoctradin, dimethomorph and mepanipyrim seemed to affect sugars-to-ethanol yield in the stationary phase. The same fermentation experiments were carried out for these three fungicides in ecological red must from Vitis vinifera cv. Tempranillo. When ecological must was unfiltered, the fermentative activity of yeasts was unaffected by the presence of these selected fungicides. However, when ecological must was filtered beforehand, a slight decrease of biomass and ethanol production (in terms of biomass-to-ethanol yield and sugars-to-ethanol yield, respectively), as well as a decrease in fruity aroma, were registered with respect to the control wine.

Garnacha Tintorera-based sweet wines: detailed phenolic composition by HPLC/DAD-ESI/MS analysis.

Sweet wines are traditionally elaborated in Galicia (the N.W. corner of Spain). The denomination of origin (DO) Valdeorras, one of the five DOs in Galicia, wants to promote the production and marketing of new sweets wines. The first one is made with dried red grapes Vitis vinifera L. Garnacha Tintorera (GNSW); this cultivar is a teinturier cultivar which has excellent potential to produce wines from raisined grapes. The second one, a fortified sweet wine aged in oak barrels (GFSW). Additionally a dry young wine (GBW) was produced from the same variety. Their aroma profiles and chromatic characteristics (determined by simple spectrophotometric methods) have been previously established. Now, proanthocyanidins, flava-3-ol monomers, anthocyanins, phenolic acids, flavonols and resveratrol were determined by HPLC, for the same three wines. The highest concentration of total proanthocyanidins (PAs) was evaluated in the GBW (525mgL(-1)), which was about 2-fold the concentration in the GNSW (236mgL(-1)) and about more 10-fold the concentration in the GFSW (44mgL(-1)). No apparent difference in the aDP (mean degree of polymerisation) was observed for the GBW (1.9) and the GNSW (2.1), whereas a slightly lower value was obtained for the GFSW (1.5). Total anthocyanin concentration was described as follow as GBW: 390mgL(-1)≫GNSW: 57mgL(-1)>GFSW: 25mgL(-1), which indicates that sweet wines were polymerised in great extent. Only vitisin A and B were found the main concentration in GFSW when compared to GBW by the ageing process. In sweet wines, phenolic acids (hydroxybenzoic and hydroxycinnamic acids) and flavonols were lowest when compared to GBW and resveratrol not was found in sweet wines.

Evolution of colour and phenolic compounds during Garnacha Tintorera grape raisining.

The off-vine drying is one of the most important steps in the production of a high quality naturally sweet wine. However, only few studies have analyzed the changes in colour and phenolic compounds of the grapes throughout the process. In this work, UV/Vis spectrophotometry and HPLC/DAD-ESI/MS were applied to determine, respectively, the evolution of colour and phenolic compounds in the red grapes of Garnacha Tintorera during the raisining process. The total water loss in 83 days was about 62% and the sugar concentration rose from 225 to 464 g L(-1). Browning and low-medium molecular weight compounds increase during the dehydration process. In CIELab coordinates, raisining decreases a(*) (red/green) and lightness and increases b(∗) (yellow/blue), colour saturation and hue angle. In general, most of the phenolic compounds determined (anthocyanins, flavonols, esters of hydroxycinnamic acids, flavan-3-ol monomers and proanthocyanidins) increase but to a lesser extent than expected because of the water loss of the grapes during drying. The lower increase was observed for esters of hydroxicinnamic acids suggesting these compounds could undergo strong enzymatic degradation. Despite this, both the terminal and the extension subunit compositions show little changes, while the mean degree of polymerization of proanthocyanidins decreases as raisining progresses.

Aroma biogenesis and distribution between olive pulps and seeds with identification of aroma trends among cultivars.

The two constitutive parts of four cultivars (Arbequina, Picual, Local and Manzanilla de Sevilla) grown in Spain were separately analysed in order to establish the role of pulp and seed in the biogenesis of extra virgin olive oil (EVOO) aroma through the lipoxygenase (LOX) pathway. C6 and C5 volatile compounds responsible of EVOO aroma were produced by endogenous enzymes in both parts of olive fruits and the differences can be attributed to different enzymes distribution in pulp and seed. According to results, C6 and C5 volatile compounds have mainly their biogenesis in pulp (80-90%) vs. seed (20-10%), independently of the cultivar considered. A linear discriminant analysis was used to establish discriminant aroma compounds between pulp and seed related to the maturity index. A decrease in trans-2-hexen-1-al and an increase in 1-hexanol with ripeness were observed independently of the cultivar considered. Finally, Partial Least Squares (PLS) regression analysis between pulp and seed aroma compounds allowed to establish those volatile compounds that better describe each cultivar.

Garnacha Tintorera-based sweet wines: chromatic properties and global phenolic composition by means of UV-Vis spectrophotometry.

Valdeorras (the N.W. corner of Spain) wants to promote the production and marketing of new sweet wines. The present work represents the first study on sweet wines manufactured with red grapes Vitis vinifera L. Garnacha Tintorera, a teinturier cultivar. Two different red sweet wines were elaborated: the first one was made with dried grapes; Vitis vinifera L. Garnacha Tintorera has excellent potential to produce wines from raisined grapes; the second one, a fortified sweet wine aged in oak barrels. Different red Garnacha Tintorera-based wines (a dry base wine, GBW; a naturally sweet wine, GNSW; and a fortified sweet wine, GFSW) were characterized. Chromatic characteristics and phenolic compounds were established by spectrophotometric methods in order to assess the technology of Garnacha Tintorera-based sweet wines. High molecular weight brown polymers, produced during the grape drying process and isolated from sweet wines by the dialysis process, were responsible for the brown colour of sweet wines. As a consequence, yellowness of sweet wines was also higher which was confirmed by colorimetric indexes. With respect to phenolic content, GFSW presented the lowest content because the maceration-alcoholic fermentation was stopped through the addition of alcohol before the diffusion of red pigments from skins to must was complete. GNSW presented the highest phenolic content due to the concentration effect resulting from evaporation of water from the grapes. Anthocyanins of sweet wines were polymerised in great extent. The percentage of polymerised tannins was sufficient to guarantee the aging process of sweet wines.

Evolution of the aromatic profile in Garnacha Tintorera grapes during raisining and comparison with that of the naturally sweet wine obtained.

The postharvest dehydration is one of the most important steps in obtaining a high quality naturally sweet wine and it can play an important role in modulating the production and the release of volatile compounds. However, only a few studies have analysed the changes in the free and bound volatile compounds of grapes throughout the process. In this work, GC-MS was applied to determine the aromatic composition of Garnacha Tintorera grapes subjected to off-vine dehydration or raisining at several points during the process. The total water loss in 83 days was about 62% and the sugar concentration rose from 225 to 464 g/L. Within the free volatile compounds, isoamyl alcohols, benzaldehyde and guaiacol registered the largest increase above the concentration effect due to water loss; while within the bound volatile compounds were isoamyl alcohols, ethyl vanillate and benzoic acid. The aromatic profile of the raisins obtained were mainly caramelised, floral, phenolic and burned.

Evolution of flavonoids in Mouratón berries taken from both bunch halves.

Galicia (N.W. Spain) is a Spanish region with several old-traditional winegrowing areas. There are autochthonous grapevine varieties, such as Vitis vinifera L. cv. Mouratón, considered a biodiversity resource in viticulture and an opportunity for Galician sustainable wine production. Therefore, it is necessary to assess the potential of traditional cultivars to produce quality red wines. In this work, anthocyanin and flavonol evolution was followed in red berries from V. vinifera L. cv. Mouratón. The novelty of this study is that grapes were separately collected from two different positions (tips and shoulders) within the cluster, over ripening to examine the effects of berry position within the fruit cluster on the flavonoid compounds. Derivatives of five anthocyanins (malvidin, peonidin, petunidin, delphinidin and cyanidin) and derivatives of six flavonols (quercetin, myricetin, kaempherol, laricitrin isorhamnetin and syringetin) were detected in both positions within the cluster. Dynamic of anthocyanins (from 819 mg/kg to 1206 mg/kg in tips; and from 786 mg/kg to 1077 mg/kg in shoulders) and dynamic of flavonols (from 25mg/kg to 41 mg/kg in tips; and from 18 mg/kg to 21 mg/kg in shoulders) confirmed their upward trends over ripening. Grapes located inside the shoulder bunch receive less sunlight radiation than those located inside the tip bunch and this fact could explain the different accumulation observed for both positions. These results can be useful for winemakers in order to obtain different final red wine quality.

Floral, spicy and herbaceous active odorants in Gran Negro grapes from shoulders and tips into the cluster, and comparison with Brancellao and Mouratón varieties.

Within the framework of a more and more competitive market, the opportunity to obtain different wines from the same variety cultivated in the same vineyard is becoming of increasing importance. In this study the presence of aroma compounds in Gran Negro (Vitis vinifera L.) grapes was investigated in order to obtain its aroma potential fingerprint taking into consideration the separation of apical (tips) and basal (shoulders) berries of the clusters. In the final stages of maturation, differences were searched in the probable alcohol content, total acidity of the must, as well as in the aromatic composition of skin and flesh from shoulder and cluster tip berries. A GC-MS method was used to determine the aromatic composition. The obtained results showed that there was variability for their aromatic composition. These results are promising for those wine cellars that are considering the separation of berries from tips and shoulders of the clusters for the elaboration of wines with different qualities. For the berries from the tips of the clusters, aromatic alcohols and volatile phenols were mainly found in the flesh (15 and 2 times higher than in the skin, respectively); whereas aldehydes and C6 alcohols were mainly in the skin (4 and 3 times higher than in the flesh, respectively). For this reason, it could be recommended to separate berry skin before enzymatic maceration of the berry flesh must. For the berries from the shoulders of the clusters, the group of volatile phenols showed 2 times more concentration in the skin than in the flesh; it could be recommended to maintain berry skin during enzymatic maceration of the must. Overall, the tips showed a 40% lower level of C6 alcohols (contributing to herbaceous nuances). These results from Gran Negro were compared with those of Brancellao and Mouratón cultivars.

Aroma profile of Garnacha Tintorera-based sweet wines by chromatographic and sensorial analyses.

The aroma profiles obtained of three Garnacha Tintorera-based wines were studied: a base wine, a naturally sweet wine, and a mixture of naturally sweet wine with other sweet wine obtained by fortification with spirits. The aroma fingerprint was traced by GC-MS analysis of volatile compounds and by sensorial analysis of odours and tastes. Within the volatiles compounds, sotolon (73 µg/L) and acetoin (122 µg/L) were the two main compounds found in naturally sweet wine. With regards to the odorant series, those most dominant for Garnacha Tintorera base wine were floral, fruity and spicy. Instead, the most marked odorant series affected by off-vine drying of the grapes were floral, caramelized and vegetal-wood. Finally, odorant series affected by the switch-off of alcoholic fermentation with ethanol 96% (v/v) fit for human consumption followed by oak barrel aging were caramelized and vegetal-wood. A partial least square test (PLS-2) was used to detect correlations between sets of sensory data (those obtained with mouth and nose) with the ultimate aim of improving our current understanding of the flavour of Garnacha Tintorera red wines, both base and sweet. Based on the sensory dataset analysis, the descriptors with the highest weight for separating base and sweet wines from Garnacha Tintorera were sweetness, dried fruit and caramel (for sweet wines) vs. bitterness, astringency and geranium (for base wines).