Richard G. Maroun

Effect of the Drying Process on the Intensification of Phenolic Compounds Recovery from Grape Pomace Using Accelerated Solvent Extraction

In light of their environmental and economic interests, food byproducts have been increasingly exploited and valorized for their richness in dietary fibers and antioxidants. Phenolic compounds are antioxidant bioactive molecules highly present in grape byproducts. Herein, the accelerated solvent extraction (ASE) of phenolic compounds from wet and dried grape pomace, at 45 °C, was conducted and the highest phenolic compounds yield (PCY) for wet (16.2 g GAE/100 g DM) and dry (7.28 g GAE/100 g DM) grape pomace extracts were obtained with 70% ethanol/water solvent at 140 °C. The PCY obtained from wet pomace was up to two times better compared to the dry byproduct and up to 15 times better compared to the same food matrices treated with conventional methods. With regard to Resveratrol, the corresponding dry pomace extract had a better free radical scavenging activity (49.12%) than the wet extract (39.8%). The drying pretreatment process seems to ameliorate the antiradical activity, especially when the extraction by ASE is performed at temperatures above 100 °C. HPLC-DAD analysis showed that the diversity of the flavonoid and the non-flavonoid compounds found in the extracts was seriously affected by the extraction temperature and the pretreatment of the raw material. This diversity seems to play a key role in the scavenging activity demonstrated by the extracts. Our results emphasize on ASE usage as a promising method for the preparation of highly concentrated and bioactive phenolic extracts that could be used in several industrial applications.

Cytokine expression profile of sensitized human T lymphocytes following in vitro stimulation with amoxicillin

Since the withdrawal of penicillin determinants from the market, in addition to the hazard of re-exposing the patient to the drug, skin testing for the diagnosis of penicillin allergy has become less accurate and less standardized. The assay currently used, the lymphocyte transformation test (LTT), lacks sufficient sensitivity, and requires the use of radioactive material. The objective of this study was to establish an accessible and reliable method for the safe diagnosis of penicillin allergy. Peripheral blood mononuclear cells (PBMC) were isolated from 18 patients who were allergic to penicillin and 12 control subjects using the Ficoll-Hypaque method. The isolated, sensitized cells were stimulated in vitro with amoxicillin (1 mg/mL). Stimulation with phytohemagglutinin A (PHA) was used as the positive control. Transcriptional expression of specific cytokines (IL-2, -4, -5 and -13, TGF-beta, TNF-alpha and IFN-gamma) was assessed by RT-PCR. IFN-gamma expression was also evaluated by ELISPOT. Secreted levels of IL-2, -5 and IFN-gamma were measured by ELISA. All of these assays were performed two or five days, post-stimulation. This study of the in vitro diagnosis of penicillin allergy by the measurement of cytokine concentration in the supernatants of sensitized lymphocytes cultures involved the largest number of patients to-date. The Delta values (difference in cytokine concentration in the supernatants before and after stimulation) were compared between cases and controls using different statistical tests (Students t test and the Mann-Whitney rank test). Of the various tests performed in this study, measurement of secreted cytokines using ELISA was the most sensitive and specific (80% and 100% respectively). In vitro stimulation of human lymphocytes sensitized to amoxicillin is a safe and useful test for the diagnosis of penicillin allergy if the ELISA is used to measure cytokine expression. The advantages are that it can be performed by many laboratories since kits to determine cytokines are widely available, and it can be done without the need for particularly specialized equipment.

Self-association and Domains of Interactions of an Amphipathic Helix Peptide Inhibitor of HIV-1 Integrase Assessed by Analytical Ultracentrifugation and NMR Experiments in Trifluoroethanol/H2O Mixtures

EAA26 (VESMNEELKKIIAQVRAQAEHLKTAY) is a better inhibitor of human immunodeficiency virus, type 1, integrase than its parent Lys-159, reproducing the enzyme segment 147–175 with a nonpolar-polar/charged residue periodicity defined by four helical heptads (abcdefg) prone to collapse into a coiled-coil. Circular dichroism, nuclear magnetic resonance, sedimentation equilibrium, and chemical cross-linking were used to analyze EAA26 in various trifluoroethanol/H2O mixtures. In pure water the helix content is weak but increases regularly up to 50–60% trifluoroethanol. In contrast the multimerization follows a bell-shaped curve with monomers in pure water, tetramers at 10% trifluoroethanol, and dimers at 40% trifluoroethanol. All suggest that interhelical interactions between apolar side chains are required for the coiled-coil formation of EAA26 and subsist at medium trifluoroethanol concentration. The NH temperature coefficients measured by nuclear magnetic resonance show that at low trifluoroethanol concentration the amide groups buried in the hydrophobic interior of four α-helix bundles are weakly accessible to trifluoroethanol and are only weakly subject to its hydrogen bond strengthening effect. The increased accessibility of trifluoroethanol to buried amide groups at higher trifluoroethanol concentration entails the reduction of the hydrophobic interactions and the conversion of helix tetramers into helix dimers, the latter displaying a smaller hydrophobic interface. The better inhibitory activity of EAA26 compared with Lys-159 could arise from its better propensity to form a helix bundle structure with the biologically important helical part of the 147–175 segment in integrase.

β-Cyclodextrin-Assisted Extraction of Polyphenols from Vine Shoot Cultivars

This work optimized the β-cyclodextrin (β-CD)-assisted extraction process of polyphenols from vine shoots. The efficiency of β-CD was compared to that of ethanol in terms of the quantity and antioxidant capacity (AC) of the extracted polyphenols. Response surface methodology permitted the optimization of the β-CD concentration, time, and temperature. The optimal polyphenol content (PC) [5.8 mg of gallic acid equivalent (GAE)/g of dry matter (DM)] and AC [3146 micromolar trolox equivalent per milliliter (μMTE)] were initially obtained with Syrah cultivar after an extraction of 48 h at 66.6 °C with a 37.7 mg/mL aqueous β-CD solvent. The same PC (5.8 mg of GAE/g of DM) was reached with 50% ethanol/water solvent after 1.65 h. However, a lower AC was found with ethanol (2000 μMTE) compared to β-CD. A comparison of the PC and AC of four different vine shoot cultivars was realized. Our results clearly show the capacity of β-CD to amplify polyphenol extraction from vine shoots.

Changes in polyphenol profiles and color composition of freshly fermented model wine due to pulsed electric field, enzymes and thermovinification pretreatments.

This work compares the effects of three pretreatments techniques: pulsed electric fields (PEFs), enzymes treatment (ET) and thermovinification (TV) on the improving of extraction of main phenolic compounds, color characteristics (L(∗)a(∗)b(∗)), and composition (copigmentation, non-discolored pigments) of freshly fermented model wine from Cabernet Sauvignon variety. The pretreatments produced differences in the wines, with the color of the freshly fermented model wine obtained from PEF and TV pretreated musts being the most different with an increase of 56% and 62%, respectively, compared to the control, while the color only increased by 22% for ET. At the end of the alcoholic fermentation, the contents of anthocyanins for all the pretreatments were not statistically different. However, for the content of total phenolics and total flavonols, PEF and TV were statistically different, but ET was not statistically different. The contents of flavonols in musts pretreated by PEF and TV were significantly higher comparing to the control. An increase of 48% and 97% was noted respectively, and only 4% for ET. A similar result was observed for the total phenolics with an increase by 18% and 32% respectively for PEF and TV, and only 3% for ET comparing to the control. The results suggest that the higher intensity and the difference of color composition between the control and pretreated freshly fermented model wines were not related only to a higher content of residual native polyphenols in these freshly fermented model wines. Other phenomena such as copigmentation and formation of derived pigments may be favored by these pretreatments.

Anaerobic digestion of grape pomace: Biochemical characterization of the fractions and methane production in batch and continuous digesters.

In this study, we have estimated the biogas and methane production from grape pomace (variety Cabernet Franc). The physical and chemical characteristics of the raw material were determined, and the structural polysaccharides were identified and analyzed by the Van Soest method. Batch anaerobic digestions were carried out to assess the methane production of the grape pomace, pulp and seeds. The obtained cumulative methane productions are 0.125, 0.165 and 0.052 Nm(3) kg COD(-1) for grape pomace, pulps and seeds, respectively. The effect of grinding on the methane potential of the substrates, as a mechanical pretreatment, was evaluated. We found that it increased the anaerobic biodegradability for grape pomace, pulp and seeds by 13.1%, 4.8% and 22.2%, respectively. On the other hand, the methane potential of the grape pomace was determined in a laboratory pilot plant (12L) continuously mixed with an organic loading rate of 2.5 kg COD m(3) d(-1) and a hydraulic retention time of 30 days. The corresponding biogas production was 6.43 × 10(-3) Nm(3) d(-1), with a methane content of 62.3%. Thus, the pilot plants efficiency compared to that achieved in the batch process was 81.2%. Finally, a significant correlation was found between the biochemical content and methane production.

Could an anisotropic molecular mechanics/dynamics potential account for sigma hole effects in the complexes of halogenated compounds?

Halogenated compounds are gaining an increasing importance in medicinal chemistry and materials science. Ab initio quantum chemistry (QC) has unraveled the existence of a “sigma hole” along the CX (X = F, Cl, Br, I) bond, namely, a depletion of electronic density prolonging the bond, concomitant with a build‐up on its sides, both of which are enhanced along the F < Cl < Br < I series. We have evaluated whether these features were intrinsically built‐in in an anisotropic, polarizable molecular mechanics (APMM) procedure such as SIBFA (sum of interactions between fragments ab initio computed). For that purpose, we have computed the interaction energies of fluoro‐, chloro‐, and bromobenzene with two probes: a divalent cation, Mg(II), and water approaching X through either one H or its O atom. This was done by parallel QC energy‐decomposition analyses (EDA) and SIBFA computations. With both probes, the leading QC contribution responsible for the existence of the sigma hole is the Coulomb contribution Ec. For all three halogenated compounds, and with both probes, the in‐ and out‐of‐plane angular features of Ec were closely mirrored by the SIBFA electrostatic multipolar contribution (EMTP). Resorting to such a contribution thus dispenses with empirically‐fitted “extra”, off‐centered partial atomic charges as in classical molecular mechanics/dynamics.

Pulsed Electric Field-Assisted Cold Maceration of Cabernet franc and Cabernet Sauvignon Grapes

The effect of pulsed electric field treatment on the cold maceration (6 days at 6ºC) of Cabernet franc and Cabernet Sauvignon grapes was investigated. The parameters of grape juice pH, Brix, color intensity, anthocyanin and total polyphenol contents, and free radical scavenging activity were determined during the entire period of cold maceration. The high pulsed electric field treatment (5 kV/cm, 1 ms, 48 kJ/kg) enhanced significantly the extraction of flavonoid (quercetin 3-β-D-glucoside and epicatechin gallate) components after subsequent cold maceration of red grapes. The high treatment also enhanced the color intensity (75% for Cabernet franc and 68% for Cabernet Sauvignon) and increased the anthocyanin content of juice (from 87 to 172 mg/L for Cabernet franc and 168 to 269 mg/L for Cabernet Sauvignon). The moderate pulsed electric field treatments (400 and 800 V/cm, 50 to 100 ms) were less effective for polyphenol extraction but consumed less energy (3 to 40 kJ/kg). The wines obtained from treated Cabernet franc and Cabernet Sauvignon musts had higher phenolic content and color intensity during the alcoholic fermentation period than wines obtained from the untreated musts.

Substituent-modulated affinities of halobenzene derivatives to the HIV-1 integrase recognition site. Analyses of the interaction energies by parallel quantum chemical and polarizable molecular mechanics.

The C-X bond of halobenzenes (X = Cl, Br) has a dual character, its electron density being depleted in its prolongation and built-up on its sides. We have recently considered three protein or nucleic acid recognition sites of halobenzenes and quantified the energy gains that either electron-attracting substituents or electron-donating ones contribute due to such a character (El Hage et al., paper in revision). Nonadditivity was found to impact the total interaction energies. We focus here on one recognition site, that of the HIV-1 integrase, in which the halobenzene ring of the drug elvitegravir is sandwiched between a guanine and a cytosine base. We perform energy-decomposition analyses of the ab initio quantum-chemistry (QC) binding energies of the parent halobenzene ring and its derivatives with this G-C base pair. In these complexes, the nonadditivity of ΔE could be traced back mostly to the polarization contribution Epol. In view of large-scale applications to the entirety of the complex formed between the integrase, the viral DNA, and the whole drug, the analyses were performed in parallel with a polarizable molecular mechanics method, SIBFA. This method could faithfully reproduce most features of the QC energies. This is due to its use of QC-derived distributed multipoles and polarizabilities, which enable us to account for both nonisotropy and nonadditivity.

Specificity of LTR DNA recognition by a peptide mimicking the HIV-1 integrase α4 helix

HIV-1 integrase integrates retroviral DNA through 3′-processing and strand transfer reactions in the presence of a divalent cation (Mg2+ or Mn2+). The α4 helix exposed at the catalytic core surface is essential to the specific recognition of viral DNA. To define group determinants of recognition, we used a model composed of a peptide analogue of the α4 helix, oligonucleotides mimicking processed and unprocessed U5 LTR end and 5 mM Mg2+. Circular dichroism, fluorescence and NMR experiments confirmed the implication of the α4 helix polar/charged face in specific and non-specific bindings to LTR ends. The specific binding requires unprocessed LTR ends—i.e. an unaltered 3′-processing site CA↓GT3′—and is reinforced by Mg2+ (Kd decreases from 2 to 0.8 nM). The latter likely interacts with the ApG and GpT3′ steps of the 3′-processing site. With deletion of GT3′, only persists non-specific binding (Kd of 100 μM). Proton chemical shift deviations showed that specific binding need conserved amino acids in the α4 helix and conserved nucleotide bases and backbone groups at LTR ends. We suggest a conserved recognition mechanism based on both direct and indirect readout and which is subject to evolutionary pressure.