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.

Secreted protein extract analyses present the plant pathogen Alternaria alternata as a suitable industrial enzyme toolbox.

Lignocellulosic plant biomass is the most abundant carbon source in the planet, which makes it a potential substrate for biorefinery. It consists of polysaccharides and other molecules with applications in pharmaceutical, food and feed, cosmetics, paper and textile industries. The exploitation of these resources requires the hydrolysis of the plant cell wall, which is a complex process. Aiming to discover novel fungal natural isolates with lignocellulolytic capacities, a screening for feruloyl esterase activity was performed in samples taken from different metal surfaces. An extracellular enzyme extract from the most promising candidate, the natural isolate Alternaria alternata PDA1, was analyzed. The feruloyl esterase activity of the enzyme extract was characterized, determining the pH and temperature optima (pH 5.0 and 55-60 °C, respectively), thermal stability and kinetic parameters, among others. Proteomic analyses derived from two-dimensional gels allowed the identification and classification of 97 protein spots from the extracellular proteome. Most of the identified proteins belonged to the carbohydrates metabolism group, particularly plant cell wall degradation. Enzymatic activities of the identified proteins (ß-glucosidase, cellobiohydrolase, endoglucanase, ß-xylosidase and xylanase) of the extract were also measured. These findings confirm A. alternata PDA1 as a promising lignocellulolytic enzyme producer. SIGNIFICANCE: Although plant biomass is an abundant material that can be potentially utilized by several industries, the effective hydrolysis of the recalcitrant plant cell wall is not a straightforward process. As this hydrolysis occurs in nature relying almost solely on microbial enzymatic systems, it is reasonable to infer that further studies on lignocellulolytic enzymes will discover new sustainable industrial solutions. The results included in this paper provide a promising fungal candidate for biotechnological processes to obtain added value from plant byproducts and analogous substrates. Moreover, the proteomic analysis of the secretome of a natural isolate of Alternaria sp. grown in the presence of one of the most used vegetal substrates on the biofuels industry (sugar beet pulp) sheds light on the extracellular enzymatic machinery of this fungal plant pathogen, and can be potentially applied to developing new industrial enzymatic tools. This work is, to our knowledge, the first to analyze in depth the secreted enzyme extract of the plant pathogen Alternaria when grown on a lignocellulosic substrate, identifying its proteins by means of MALDI-TOF/TOF mass spectrometry and characterizing its feruloyl esterase, cellulase and xylanolytic activities.

Molecular analysis of lungs from pigs immunized with a mutant transferrin binding protein B-based vaccine and challenged with Haemophilus parasuis.

The molecular analysis of pigs vaccinated with a mutant transferrin-binding protein B (Y167A) from Haemophilus parasuis was compared with that performed for unvaccinated challenged (UNCH) and unvaccinated unchallenged (UNUN) pigs. Microarray analysis revealed that UNCH group showed the most distinct expression profile for immune response genes, mainly for those genes involved in inflammation or immune cell trafficking. This fact was confirmed by real-time PCR, in which the greatest level of differential expression from this group were CD14, CD163, IL-8 and IL-12. In Y167A group, overexpressed genes included MAP3K8, CD14, IL-12 and CD163. Proteomics revealed that collagen α-1 and peroxiredoxins 2 and 6 were overexpressed in Y167A pigs. Our study reveals new data on genes and proteins involved in H. parasuis infection and several candidates of resistance to infection that are induced by Y167A vaccine. The expression of proinflammatory molecules from Y176A pigs is similar to their expression in UNUN pigs.

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.

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.

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.

CHACO outreach project: the development of a primary health care-based medical genetic service in an Argentinean province.

Dissemination of knowledge in genetics to be applied in medicine has created a growing need for capacity building in health care workers. The CAPABILITY ARGENTINA outreach project protocol was designed as a model to introduce genetics in areas without genetic services. Our aim was for genetic health care to become part of primary care in an Argentine province lacking genetic services. The program was innovative as professionals from the referral center (Garrahan Hospital S.A.M.I.C.) traveled to remote areas to train professionals through problem-based education. A logical framework was designed for a local needs assessment. Teaching materials (Powerpoint presentations, printed syllabus, and CD) and a web page were developed. A demonstration project was carried out in the Province of Chaco, Argentina. A total of 485 health workers were trained. The number of consultations increased significantly in participating areas comparing before and after the training period. To support this increase, a complementary project was set up from a public hospital sponsored from within Argentina to build a cytogenetic laboratory in the capital of the Province of Chaco. The model was improved for reproduction in other areas in Argentina. CAPABILITY ARGENTINA is a capacity building model for training of primary care professionals in genetics that may be applied to other medical specialties. The outcomes of the programme have a direct impact on clinical practice.

Health needs assessment for medical genetic services for congenital disorders in middle- and low-income nations.

Medical genetic services for the care and prevention of congenital disorders have received little attention in most middle- and low-income countries to date. In 2010, the World Health Organisation prioritized services for the care and prevention of birth defects in these nations, emphasising their importance in assisting such countries to reach their Millennium Development Goals. Health Needs Assessment is an inclusive, rational, epidemiological-assisted approach for providing information to plan, introduce and beneficially change health care services to improve the health of populations. It is intrinsic to much of the development of health care systems in industrialised nations. Its use by middle- and low-income countries to introduce and develop medical genetic services commensurate with their needs and circumstances would be beneficial. An approach to applying Health Needs Assessment in these circumstances is described.

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.

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.

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.