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An early mechanism for the health benefits of dietary plant phenols is their antioxidant activity in the upper digestive tract. Indeed, these non-essential micronutrients abundant in fruits and vegetables can efficiently fight the iron-induced peroxidation of dietary lipids in the gastric compartment, a recognized form of postprandial oxidative stress. In this work, this phenomenon is investigated through a simple model based on nano-emulsions of trilinoleylglycerol, which permits a direct spectroscopic monitoring and mechanistic insights sustained by extensive kinetic analysis. Polyphenols belonging to the main dietary classes are tested, in particular, flavonols, anthocyanins, flavanols and oligomeric procyanidins. Overall, the common polyphenols tested are good inhibitors of lipid peroxidation induced by metmyoglobin (heme iron) in the early stage of digestion (pH 5-6). For instance, under our peroxidation conditions (2 μM heme, 0.7 mM linoleic acid equivalent, 4.5 mM Brij®35), IC50 concentrations in the range 0.4-1.9 μM were estimated for the set of polyphenols, with oligomeric procyanidins being less inhibitory than the flavanol monomers. However, the polyphenols are ineffective at lower pH (pH 4) when the hematin cofactor is dissociated from its protein (globin). On the other hand, a moderate protection against lipid peroxidation induced by free iron (e.g., released by the oxidative degradation of hematin) persists. This protocol, which combines simplicity and nutritional relevance, could provide a basis for standard tests aimed at assessing the antioxidant capacity of foods and food additives.

Lipid oxidation takes place in the gastric tract after the ingestion of a Western diet rich in ω-6 polyunsaturated fatty acids (PUFA) and red meat (heme iron). The incorporation of oxidation products such as 4-hydroxy-2-nonenal (4-HNE) into low-density lipoproteins is further correlated to endothelial dysfunction. Gastric postprandial stress could thus be reduced by antioxidant phytomicronutrients. The aim of this study was to investigate dietary lipid oxidation and its inhibition by apple polyphenols under different matrix forms (fresh fruit, puree, extract) under in vitro gastric digestion conditions. A deep insight was given into the two factors pH and pepsin governing the metmyoglobin-initiated lipid oxidation of sunflower oil-in-water emulsions simulating the physical state of dietary lipids. Our results first showed that pepsin accelerated lipid oxidation at pH 5 through the formation of a micro-metmyoglobin form likely displaying a higher accessibility to lipids. Spectroscopic studies further highlighted the formation of a reversible unfolded metmyoglobin form at pH 3 which was shown to be more pro-oxidant in the absence of pepsin. At nutritional levels, the three apple matrices inhibited less efficiently the accumulation of lipid-derived conjugated dienes and 4-HNE at pH 5 when pepsin was present whereas at pH 3 the opposite was true. High initial bioaccessibilities of monomeric phenolic compounds were evidenced for both puree (57-74%) and the phenolic extract (79-96%) compared to fresh apple (1-14%) supporting their greater antioxidant capacity. By contrast, the bioaccessibility of dimer B2 was low for all matrices suggesting non-covalent binding to apple pectins.

A beta-glucosidase enzyme activity was enriched from skins of ripe grape berry by cell wall fractionation, hydrophobic interaction and cation-exchange chromatographies. This enriched enzyme extract contained several beta-glycosidase activities hydrolyzing a wide range of synthetic and natural monoglycosides and diglycosides, as well as a beta-fructosidase activity. The enzyme extract was further characterized by two-dimensional gel electrophoresis coupled to peptide mass fingerprinting of eight spots using MALDI-TOF mass spectrometry. No beta-glucosidase but a beta-fructosidase associated to the relevant spot at 66 kDa/pI 5.1 was identified. Taken together all results issued from the biochemical characterization, the substrate specificity and the mass spectrometry-based identification of this enriched enzyme extract, we propose that this protein could be a specific beta-fructosidase isoform associated with a broad spectrum of beta-glycosidase activities in grape berry skin and involved in cell wall modifications which occur during the ripening-induced thickness of the grape.

Six hundred ten yeast colonies isolated from various vineyards in Chile were screened for the presence of a beta-glucosidase activity as well as the resistance to glucose and ethanol inhibition. Among them, Debaryomyces vanrijiae was found to produce high levels of an extracelular beta-glucosidase which was tolerant to glucose (K(i) = 439 mM) and ethanol inhibitions. The enzyme (designated DV-BG) was purified to apparent homogeneity, respectively, by gel filtration, ion-exchange, and chromatofocusing techniques. Its molecular weight was 100 000, and its pI 3.0, optimum pH, and temperature activities were 5.0 and 40 degrees C, respectively, and had a V(max) of 47.6 micromol min(-)(1) mg(-)(1) and a K(m) of 1.07 mM. The enzyme was active against different beta-d-glucosides including glucosidic flavor precursors. The disaccharidic flavor precursors were not substrates for the enzyme. When added to a Muscat grape juice, the concentration of several monoterpenes increased as the consequence of its hydrolytic activity.

Aspergillus oryzae was found to secrete two distinct beta-glucosidases when it was grown in liquid culture on various substrates. The major form had a molecular mass of 130 kDa and was highly inhibited by glucose. The minor form, which was induced most effectively on quercetin (3,3',4',5,7-pentahydroxyflavone)-rich medium, represented no more than 18% of total beta-glucosidase activity but exhibited a high tolerance to glucose inhibition. This highly glucose-tolerant beta-glucosidase (designated HGT-BG) was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and anion-exchange chromatography. HGT-BG is a monomeric protein with an apparent molecular mass of 43 kDa and a pI of 4.2 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing polyacrylamide gel electrophoresis, respectively. Using p-nitrophenyl-beta-D-glucoside as the substrate, we found that the enzyme was optimally active at 50 degreesC and pH 5.0 and had a specific activity of 1,066 micromol min-1 mg of protein-1 and a Km of 0.55 mM under these conditions. The enzyme is particularly resistant to inhibition by glucose (Ki, 1. 36 M) or glucono-delta-lactone (Ki, 12.5 mM), another powerful beta-glucosidase inhibitor present in wine. A comparison of the enzyme activities on various glycosidic substrates indicated that HGT-BG is a broad-specificity type of fungal beta-glucosidase. It exhibits exoglucanase activity and hydrolyzes (1-->3)- and (1-->6)-beta-glucosidic linkages most effectively. This enzyme was able to release flavor compounds, such as geraniol, nerol, and linalol, from the corresponding monoterpenyl-beta-D-glucosides in a grape must (pH 2.9, 90 g of glucose liter-1). Other flavor precursors (benzyl- and 2-phenylethyl-beta-D-glucosides) and prunin (4',5,7-trihydroxyflavanone-7-glucoside), which contribute to the bitterness of citrus juices, are also substrates of the enzyme. Thus, this novel beta-glucosidase is of great potential interest in wine and fruit juice processing because it releases aromatic compounds from flavorless glucosidic precursors.