The catalytic activity of a lysozyme-based oenological preparation toward the specific substrates... more The catalytic activity of a lysozyme-based oenological preparation toward the specific substrates i) glycol chitosan, as a non-cellular synthetic substrate, and ii) Oenococcus oeni, as a microbial substrate for muramidase activity, was investigated in wine-like acidic medium (tartaric buffer, pH 3.2). The good reproducibility and reliability of results revealed that both substrates are useful for lysozyme kinetics studies. Both the chitinolytic and muramidase activity of lysozyme were affected by uncompetitive substrate inhibition. However, the O. oeni effect occurred at very high concentration with respect to the typical wine bacterial content, even when microbial population reaches its maximum growth level during malolactic fermentation. The optimum pH and temperature for lysozyme activity were very far from the usual wine handling conditions, but residual enzyme activity was observed at pH 3.2 and 20 o C, confirming that the oenological application of lysozyme is a useful practice.
Relazione presentata al Convegno: La Ricerca viticola ed Enologica: opportunità di svuluppo e pro... more Relazione presentata al Convegno: La Ricerca viticola ed Enologica: opportunità di svuluppo e promozione del territorio, Velletri, 17 dicembre 2004 Il percorso di affermazione commerciale, di vini ottenuti da vitigni autoctoni, prevede l'individuazione di caratteri distintivi, indispensabili per la identificazione varietale e per la valorizzazione e tutela del prodotto. I composti fenolici (Mattivi, 1991; Pigella et al. 1998) ed i composti correlati con l'aroma (Versini, 1991; Di Stefano, 1996), oltre ad influenzare la qualità dei vini, risultano determinanti nel definirne l'impronta varietale. I vitigni Nero d'Avola e Fiano, vitigni tradizionali del sud d'Italia, dopo alcuni anni di sperimentazione condotta sul territorio laziale da ARSIAL e Istituto Sperimentale per l'Enologia di Velletri, sono rientrati a far parte dei vitigni considerati, nell'ambito del Piano Regionale per la Ristrutturazione e la Riconversione dei Vigneti del Lazio (Deliberazione de...
Australian Journal of Grape and Wine Research, 2014
ABSTRACT Background and AimsSeveral studies have investigated the effectiveness of several proteo... more ABSTRACT Background and AimsSeveral studies have investigated the effectiveness of several proteolytic enzymes in free form as a treatment alternative to bentonite fining for wine stabilisation. This work tested the feasibility of stem bromelain, free or immobilised on a chitosan support, to reduce white wine protein haze potential.Methods and ResultsThe kinetic behaviour of stem bromelain, in free form and covalently immobilised on chitosan beads, was studied in 12 unfined white table wines fortified with a tripeptide chromogenic substrate. The effectiveness of the protease on wine hazing potential was determined by treating the wines in a laboratory-scale stirred reactor, and the heat test was applied to evaluate the decrease in nephelometric turbidity units. Immobilised protease was active towards the synthetic substrate and in reducing hazing potential of the wine. The effect of wine components on bromelain proteolytic behaviour, as investigated by principal component analysis, was much more noticeable for the free enzyme (in decreasing order of importance: pH, total phenolic substances, free and total sulfur dioxide) than for the immobilised form.Conclusions An amount of 10 g/L biocatalyst (bromelain immobilised on chitosan beads), after a 24-h treatment in a laboratory-scale stirred reactor, revealed a high capacity to reduce wine haze potential (approximately 70%), which was unaffected by wine composition.Significance of the StudyImmobilised bromelain appears a suitable treatment alternative to bentonite fining for white wine haze stabilisation.
Ochratoxin A (OTA), a potent nephrotoxic and human carcinogenic agent, is found fre quently in ag... more Ochratoxin A (OTA), a potent nephrotoxic and human carcinogenic agent, is found fre quently in agricultural products such as cereals, coffee, cocoa beans, dried fruits, beers, grape musts and wines. OTA is produced by several molds belonging to species o f Aspergillus and Penicillus. Several studies showed that A. carbonarius is the main OTA-producer in grapes and musts, particu larly during post-harvest. Cell-wall degrading enzymes (CWDE), such as chitinases and glucanases, could reduce fungal contamination on several plant structures and could be used in food technology to reduce occurrence of these contaminants. The Antarctic fungus Lecanicillium muscarium CCFEE 5003 is a strong producer of CWDE, chitinases in particular. In this work, w hite and black grape bunches were inoculated with spore suspension of A. carbonarius to simulate natural contamina tion. Grapes were then treated with crude CWDE solution from L. muscarium in order to study possible inhibition o f the OTA-producer in post-harvest conditions. Presence of contaminants on untreated grapes (control) was very high (> 2000 cfu/ml). On treated bunches residual spores were 95 and 89% less than on the control, in white and red grape, respectively. Light microscopy showed that the enzyme solution caused various spore damages such as cell-wall disruption and protoplast formation and bursting.
Comprehensive Reviews in Food Science and Food Safety
Lysozyme, a muramidase enzyme from egg whites (EC 3.2.1.17), is widely used in soluble form to co... more Lysozyme, a muramidase enzyme from egg whites (EC 3.2.1.17), is widely used in soluble form to control lactic acid bacteria in different foods. Moreover, hen egg white lysozyme is a hydrolytic enzyme that can be used to the control malolactic fermentation (MLF) during winemaking. MLF is only desirable in red and in some white wine, this suggest that MLF is at fault and needs to be controlled in all other types of wine. Lysozyme exhibits selective antimicrobial activity based on the hydrolysis of peptidoglycan cell wall constituents in lactic acid bacteria. In the last decade, several studies identified allergic reactions due to the presence of lysozyme in food. Given this relatively high incidence of lysozyme sensitization, and in accordance with the recently changed EC food legislation (1266/2010/CE), the use of lysozyme as an additive has to be declared on the ingredient label. To overcome this problem, the immobilization of the enzyme on insoluble supports, which allows the enzym...
ABSTRACT Films of chitosan from two different sources, microbial (Aspergillus niger) and animal (... more ABSTRACT Films of chitosan from two different sources, microbial (Aspergillus niger) and animal (shell fish, providing three different molecular weights), were prepared by a solvent casting technique, and then characterised and tested for use as a covalently linked bromelain carrier in wine-like medium.Membrane morphology, microstructure, thermal and mechanical properties were investigated, showing that glycerol addition increases elongation at break and reduces tensile strength. Films based on low molecular weight chitosan blended with 25% glycerol, showed the best balance between stress strain and flexibility.The highest bromelain immobilization yield (41%) was observed on A. niger chitosan blended with glycerol, 25%, whereas for all shell fish chitosan membranes it appeared lower (27–33%).With regard to the proteolytic properties of immobilized bromelain measured towards a tripeptide chromogenic substrate, the enzyme immobilized on neat low molecular weight chitosan films turned out to be the most efficient biocatalyst in terms of maximum velocity of the enzyme-catalyzed reaction (Vmax) and the Michaelis–Menten constant (KM). Nevertheless, taking into account both mechanical and kinetic properties, low molecular weight chitosan blended with 25% glycerol should be considered the most suitable for application in wine-making.
When the vintage is poor, different must concentration techniques are used for overcoming grape c... more When the vintage is poor, different must concentration techniques are used for overcoming grape compositional deficiency, such as evaporative or freeze concentration, and membrane processes. The membrane filtration works at low temperature, preserving must components from degradation. The aim of the present work was to tailor reverse osmosis and nanofiltration technologies applied to grape juice testing several membranes for different grape varieties from southern Italy. The main oenological parameters, including sugar content, acidity, pH, color and total phenol content, were evaluated in comparison with the addition of rectified concentrated must. Quality characteristics of all musts showed to be improved by means of membrane processes, which could be successfully implemented in the cases of premature harvest carried out for obtaining wines with enhanced acidity.
The oxidation of dihydroxybenzene and hydroxycinnamate compounds generates unstable quinones whic... more The oxidation of dihydroxybenzene and hydroxycinnamate compounds generates unstable quinones which undergo further reactions to combine nucleophilic compounds. These reactions are limited when sulfur dioxide (SO 2 ) is used as wine preservative, as SO 2 plays an important role in reducing quinones back to their phenol form. In no sulfite-added wine, oxidation chemistry undoubtedly results changed. Therefore, in this work, the phenolic profile of a white wine without adding sulfites, after twelve-month storage, was investigated by HPLC-DAD-MS/MS and compared to a control stored with sulfur dioxide. A total of twenty-six phenolics were detected including hydroxycinnamate derivatives and minor compounds resulting from oxidation processes, i.e. oxidized caffeic acid dimers, whose structures were tentatively proposed on the basis of their UV, MS, and MS/MS typical fragmentation. The experimental evidence of these latter is described for the first time in wine stored without added sulfites, and it may contribute to get new insight in the field of wine oxidation processes.
Emnrnary-Electroanalytical methods based on biosensor technology and differential pH measurement ... more Emnrnary-Electroanalytical methods based on biosensor technology and differential pH measurement for the determination of L-lactic and L-malic acids in wine were developed. Lactic acid was determined according to two procedures consisting of the use of two lactate electrochemical biosensors, one based on a Clark 0, probe and the other based on the H202 probe. The two probes were assembled employing a polymeric membrane where the enzyme lactate oxidase was previously immobilized. Both sensors exhibited high storage and operational stabilities, and good reproducibility when used in wine matrix. Measurement of lactate was carried out using two different flow-through cells assembled with the lactate probes. The low detection limit of these probes and the relatively high concentration of lactate in wine samples required a serial dilution in the range 1: 100 to 1: 200, thus eliminating all potential electrochemical or enzyme interferences present in the sample. Malic acid was determined using a differential commercially available pH-meter and an enzymatic procedure. For this analysis, we employed the malic enzyme (EC 1.1.1.40) and the NADP+ cofactor. The pH variation due to the action of this enzyme on malic acid was found to be proportional to the malic acid present in the sample. Experimental parameters such as pH, temperature, and co-factor concentration were optimized, resulting in malate determination in less than 1 min with good reproducibility. Various samples of wine and wine musts were assayed for lactate with the two biosensor procedures, and for malate with differential pH-metry. The results when compared with those obtained with the commonly used spectrophotometric procedure correlated well.
In this work, a purified acid urease preparation was covalently immobilized on Eupergit C 250 L a... more In this work, a purified acid urease preparation was covalently immobilized on Eupergit C 250 L and stabilized with glycine. Its average activity was found to be 69 +/- 16% of the initial one after 34-day storage at 4 degrees C . The kinetics of urea degradation in a model wine solution by immobilized enzyme was confirmed to be of pseudo-first-order with respect to the urea concentration in the liquid bulk, its apparent pseudo-first-order kinetic rate constant (k(Ii)) being about one-fourth of that (k(If)) pertaining to the free enzyme. In the operating conditions tested, the reaction kinetics was estimated as unaffected by the contribution of the external film and intraparticle diffusion mass transfer resistances. Because in the presence of the high-inhibitory tannins extracted from grape seeds in the range of 3-620 g of GAE m(-3) the loss in k(Ii) was quite smaller than that in k(If), the biocatalyst tested here is likely to overcome the present limits to the application of free acid urease in wine treatment.
The specific activity of a whole cell acid urease preparation was tested in five wines manufactur... more The specific activity of a whole cell acid urease preparation was tested in five wines manufactured in the Apulia region of Italy in the 2003 vintage at both short and long treatment times, thus confirming the validity of the pseudo-first-order kinetic model to describe urea removal in real wines. The ratio between the experimental pseudo-first-order kinetic rate constant (k Ie ) for any real wine tested and that (k I ) referred to a model wine solution having the same composition and pH reduced from about 0.21 to 0.02 as the overall content of phenolic compounds (P) increased from 109 to 853 g m -3 of gallic acid equivalent (GAE). The specific inhibitory effect of such compounds was explained by accounting for the equilibrium constant (K P ) of the reaction of polyphenols with acid urease, which was found to be about 21 g of GAE m -3 for the real wines tested, whereas it ranged from about 16 to 45 g of GAE m -3 when the model wine solution was enriched with tannins extracted from grape seeds or skins, respectively. A sequential experimental procedure consisting of accelerated acid urease tests at high doses of enzyme followed by accelerated ethyl carbamate tests on the resulting acid urease treated wine was recommended to assess preliminarily the technoeconomic feasibility of the acid urease hydrolytic process for the wine of concern.
A flow injection analysis procedure was used for the determination of L-lactic acid during the pr... more A flow injection analysis procedure was used for the determination of L-lactic acid during the production of mozzarella cheese. The apparatus consists of an electrochemical flow-through walljet cell assembled with a platinum sensor covered with the immobilized lactate oxidase enzyme and connected to an amperometer. This system was used to monitor the concentration of L-lactic acid produced by lactose fermentation catalyzed by selected starters during cheese manufacture. Lactate was detected in the range (5 × 10 -6 )-(1 × 10 -4 ) mol/L with a detection limit of 2 × 10 -6 mol/L. L-Lactate has been measured in raw milk and during the manufacture of cow and water buffalo mozzarella cheese. The starter used was a commercially available strain of Streptococcus thermophilus. Real time analysis of lactate allowed a control of the curd-ripening evolution at different pasteurization temperatures of the milk. Values of lactic acid were compared with pH variation during the process. This method proved to be more sensitive than the pH measurement procedure for the control of the continuous production of lactic acid particularly near the "stretching point" when very slight pH variations were observed.
Phenolic compounds in different olive varieties were determined by HPLC analysis over 2 years. De... more Phenolic compounds in different olive varieties were determined by HPLC analysis over 2 years. Demethyloleuropein was found in only two (Coratina and Leccino) of the eight varieties studied, so it could be used as varietal marker. Elenolic acid glucoside and hydroxytyrosol can be considered indicators of maturation for olives. In fact, as the olives ripen, their tenor increases whereas oleuropein decreases.
The specific activity (π A ) of a whole cell acid urease preparation was assessed in model wine s... more The specific activity (π A ) of a whole cell acid urease preparation was assessed in model wine solutions at different levels of malic (M) and lactic acids, metabisulfite, ethanol, and pH by performing a central composite design. M and then pH were found to be the most controlling variables, their effects being practically coincident but of opposite sign. For urea concentrations up to ≈1 mol m -3 the ammonium formation rate was assumed of the pseudo-first-order with respect to urea, this being confirmed by two independent validation tests performed at 20°C for as long as 24 h. In the case of real wines the effective pseudo-first-order kinetic rate constants were found to be smaller than those pertaining to the model solutions having the same wine composition and pH by a factor varying from 10 to 1000, this affecting significantly the specific urease treatment costs per liter of wine treated.
ABSTRACT “Natural” amines in plants, endogenous constituents of tissues, are involved in differen... more ABSTRACT “Natural” amines in plants, endogenous constituents of tissues, are involved in different physiological processes such as fruit development and senescence. Their levels can vary depending on variety, ripening, and storage conditions. Moreover, microbial contamination results in the increase of the “biogenic” amine content of fruits and vegetables. Electrochemical biosensors for the determination of the amine content in fruits have been assembled using diamine oxidase and polyamine oxidase covalently immobilized onto polymeric membranes. Both enzymes in the presence of their substrates produced H2O2 that was detected at a platinum electrode polarized at +650 mV versus Ag/AgCl. The best analytical conditions for the two enzyme electrodes have been optimized. The main positive effect of packaging fresh fruits with polymeric films is the provision of a barrier to the gas diffusion; the reduced O2 and increased CO2 concentrations slow the respiration rate. The films also allow the maintenance of high relative humidity with reduction of water loss. To investigate the postharvest life of apricots and sweet cherries in modified atmosphere storage at 0 °C, the concentration of amines in fruits packaged using different polymeric films was monitored. Keywords: Modified atmosphere packaging (MAP); polymeric film; electrochemical biosensor; amine; diamine oxidase; polyamine oxidase; sweet cherry; apricot fruit; postharvest
A purified acid urease preparation was covalently immobilized onto either Eupergit C 250 L or glu... more A purified acid urease preparation was covalently immobilized onto either Eupergit C 250 L or glutaraldehyde-cross-linked chitosan-derivative beads (i.e., Chitopearls BCW-3003 and BCW-3010). The kinetics of urea degradation in two target Italian white (i.e., Grechetto and Sauvignon Blanc) wines, as well as in a model wine solution, by using the above Eupergit C 250 L-, BCW-3003-, or BCW-3010-based biocatalysts, was confirmed to be of the pseudofirst order with respect to the urea concentration in the liquid bulk and not limited by urea mass transfer. In Grechetto and Sauvignon Blanc wines, the corresponding kinetic rate constants were quite similar, being about 7, 18, or 17% of that observed for free enzyme in the model wine solution, respectively. Owing to their minor sensitivity to the phenolic content of the wines tested, the chitosan-based biocatalysts might be potentially employable in the make up of packed-bed cartridges to continuously remove urea from commercial wines.
The influence of potential inhibitors, naturally present in wine, on the activity of stem bromela... more The influence of potential inhibitors, naturally present in wine, on the activity of stem bromelain was investigated in order to evaluate the applicability of this enzyme for protein stabilization in white wine. Bromelain proteolytic activity was tested against a synthetic substrate (Bz-Phe-Val-Arg-pNA) in a model wine system after adding ethanol, sulfur dioxide (SO 2 ), skin, seed, and gallic and ellagic tannins at the average range of their concentration in wine. All the inhibitors of stem bromelain activity tested turned out to be reversible. Ethanol was a competitive inhibitor with a rather limited effect. Gallic and ellagic tannins have no inhibitory effect on stem bromelain activity, while both seed and skin tannins were uncompetitive inhibitors. The strongest inhibition effect was revealed for sulfur dioxide, which was a mixed-type inhibitor for the enzyme activity. This study provides useful information relative to a future biotechnological application of stem bromelain in winemaking.
The catalytic activity of a lysozyme-based oenological preparation toward the specific substrates... more The catalytic activity of a lysozyme-based oenological preparation toward the specific substrates i) glycol chitosan, as a non-cellular synthetic substrate, and ii) Oenococcus oeni, as a microbial substrate for muramidase activity, was investigated in wine-like acidic medium (tartaric buffer, pH 3.2). The good reproducibility and reliability of results revealed that both substrates are useful for lysozyme kinetics studies. Both the chitinolytic and muramidase activity of lysozyme were affected by uncompetitive substrate inhibition. However, the O. oeni effect occurred at very high concentration with respect to the typical wine bacterial content, even when microbial population reaches its maximum growth level during malolactic fermentation. The optimum pH and temperature for lysozyme activity were very far from the usual wine handling conditions, but residual enzyme activity was observed at pH 3.2 and 20 o C, confirming that the oenological application of lysozyme is a useful practice.
Relazione presentata al Convegno: La Ricerca viticola ed Enologica: opportunità di svuluppo e pro... more Relazione presentata al Convegno: La Ricerca viticola ed Enologica: opportunità di svuluppo e promozione del territorio, Velletri, 17 dicembre 2004 Il percorso di affermazione commerciale, di vini ottenuti da vitigni autoctoni, prevede l'individuazione di caratteri distintivi, indispensabili per la identificazione varietale e per la valorizzazione e tutela del prodotto. I composti fenolici (Mattivi, 1991; Pigella et al. 1998) ed i composti correlati con l'aroma (Versini, 1991; Di Stefano, 1996), oltre ad influenzare la qualità dei vini, risultano determinanti nel definirne l'impronta varietale. I vitigni Nero d'Avola e Fiano, vitigni tradizionali del sud d'Italia, dopo alcuni anni di sperimentazione condotta sul territorio laziale da ARSIAL e Istituto Sperimentale per l'Enologia di Velletri, sono rientrati a far parte dei vitigni considerati, nell'ambito del Piano Regionale per la Ristrutturazione e la Riconversione dei Vigneti del Lazio (Deliberazione de...
Australian Journal of Grape and Wine Research, 2014
ABSTRACT Background and AimsSeveral studies have investigated the effectiveness of several proteo... more ABSTRACT Background and AimsSeveral studies have investigated the effectiveness of several proteolytic enzymes in free form as a treatment alternative to bentonite fining for wine stabilisation. This work tested the feasibility of stem bromelain, free or immobilised on a chitosan support, to reduce white wine protein haze potential.Methods and ResultsThe kinetic behaviour of stem bromelain, in free form and covalently immobilised on chitosan beads, was studied in 12 unfined white table wines fortified with a tripeptide chromogenic substrate. The effectiveness of the protease on wine hazing potential was determined by treating the wines in a laboratory-scale stirred reactor, and the heat test was applied to evaluate the decrease in nephelometric turbidity units. Immobilised protease was active towards the synthetic substrate and in reducing hazing potential of the wine. The effect of wine components on bromelain proteolytic behaviour, as investigated by principal component analysis, was much more noticeable for the free enzyme (in decreasing order of importance: pH, total phenolic substances, free and total sulfur dioxide) than for the immobilised form.Conclusions An amount of 10 g/L biocatalyst (bromelain immobilised on chitosan beads), after a 24-h treatment in a laboratory-scale stirred reactor, revealed a high capacity to reduce wine haze potential (approximately 70%), which was unaffected by wine composition.Significance of the StudyImmobilised bromelain appears a suitable treatment alternative to bentonite fining for white wine haze stabilisation.
Ochratoxin A (OTA), a potent nephrotoxic and human carcinogenic agent, is found fre quently in ag... more Ochratoxin A (OTA), a potent nephrotoxic and human carcinogenic agent, is found fre quently in agricultural products such as cereals, coffee, cocoa beans, dried fruits, beers, grape musts and wines. OTA is produced by several molds belonging to species o f Aspergillus and Penicillus. Several studies showed that A. carbonarius is the main OTA-producer in grapes and musts, particu larly during post-harvest. Cell-wall degrading enzymes (CWDE), such as chitinases and glucanases, could reduce fungal contamination on several plant structures and could be used in food technology to reduce occurrence of these contaminants. The Antarctic fungus Lecanicillium muscarium CCFEE 5003 is a strong producer of CWDE, chitinases in particular. In this work, w hite and black grape bunches were inoculated with spore suspension of A. carbonarius to simulate natural contamina tion. Grapes were then treated with crude CWDE solution from L. muscarium in order to study possible inhibition o f the OTA-producer in post-harvest conditions. Presence of contaminants on untreated grapes (control) was very high (> 2000 cfu/ml). On treated bunches residual spores were 95 and 89% less than on the control, in white and red grape, respectively. Light microscopy showed that the enzyme solution caused various spore damages such as cell-wall disruption and protoplast formation and bursting.
Comprehensive Reviews in Food Science and Food Safety
Lysozyme, a muramidase enzyme from egg whites (EC 3.2.1.17), is widely used in soluble form to co... more Lysozyme, a muramidase enzyme from egg whites (EC 3.2.1.17), is widely used in soluble form to control lactic acid bacteria in different foods. Moreover, hen egg white lysozyme is a hydrolytic enzyme that can be used to the control malolactic fermentation (MLF) during winemaking. MLF is only desirable in red and in some white wine, this suggest that MLF is at fault and needs to be controlled in all other types of wine. Lysozyme exhibits selective antimicrobial activity based on the hydrolysis of peptidoglycan cell wall constituents in lactic acid bacteria. In the last decade, several studies identified allergic reactions due to the presence of lysozyme in food. Given this relatively high incidence of lysozyme sensitization, and in accordance with the recently changed EC food legislation (1266/2010/CE), the use of lysozyme as an additive has to be declared on the ingredient label. To overcome this problem, the immobilization of the enzyme on insoluble supports, which allows the enzym...
ABSTRACT Films of chitosan from two different sources, microbial (Aspergillus niger) and animal (... more ABSTRACT Films of chitosan from two different sources, microbial (Aspergillus niger) and animal (shell fish, providing three different molecular weights), were prepared by a solvent casting technique, and then characterised and tested for use as a covalently linked bromelain carrier in wine-like medium.Membrane morphology, microstructure, thermal and mechanical properties were investigated, showing that glycerol addition increases elongation at break and reduces tensile strength. Films based on low molecular weight chitosan blended with 25% glycerol, showed the best balance between stress strain and flexibility.The highest bromelain immobilization yield (41%) was observed on A. niger chitosan blended with glycerol, 25%, whereas for all shell fish chitosan membranes it appeared lower (27–33%).With regard to the proteolytic properties of immobilized bromelain measured towards a tripeptide chromogenic substrate, the enzyme immobilized on neat low molecular weight chitosan films turned out to be the most efficient biocatalyst in terms of maximum velocity of the enzyme-catalyzed reaction (Vmax) and the Michaelis–Menten constant (KM). Nevertheless, taking into account both mechanical and kinetic properties, low molecular weight chitosan blended with 25% glycerol should be considered the most suitable for application in wine-making.
When the vintage is poor, different must concentration techniques are used for overcoming grape c... more When the vintage is poor, different must concentration techniques are used for overcoming grape compositional deficiency, such as evaporative or freeze concentration, and membrane processes. The membrane filtration works at low temperature, preserving must components from degradation. The aim of the present work was to tailor reverse osmosis and nanofiltration technologies applied to grape juice testing several membranes for different grape varieties from southern Italy. The main oenological parameters, including sugar content, acidity, pH, color and total phenol content, were evaluated in comparison with the addition of rectified concentrated must. Quality characteristics of all musts showed to be improved by means of membrane processes, which could be successfully implemented in the cases of premature harvest carried out for obtaining wines with enhanced acidity.
The oxidation of dihydroxybenzene and hydroxycinnamate compounds generates unstable quinones whic... more The oxidation of dihydroxybenzene and hydroxycinnamate compounds generates unstable quinones which undergo further reactions to combine nucleophilic compounds. These reactions are limited when sulfur dioxide (SO 2 ) is used as wine preservative, as SO 2 plays an important role in reducing quinones back to their phenol form. In no sulfite-added wine, oxidation chemistry undoubtedly results changed. Therefore, in this work, the phenolic profile of a white wine without adding sulfites, after twelve-month storage, was investigated by HPLC-DAD-MS/MS and compared to a control stored with sulfur dioxide. A total of twenty-six phenolics were detected including hydroxycinnamate derivatives and minor compounds resulting from oxidation processes, i.e. oxidized caffeic acid dimers, whose structures were tentatively proposed on the basis of their UV, MS, and MS/MS typical fragmentation. The experimental evidence of these latter is described for the first time in wine stored without added sulfites, and it may contribute to get new insight in the field of wine oxidation processes.
Emnrnary-Electroanalytical methods based on biosensor technology and differential pH measurement ... more Emnrnary-Electroanalytical methods based on biosensor technology and differential pH measurement for the determination of L-lactic and L-malic acids in wine were developed. Lactic acid was determined according to two procedures consisting of the use of two lactate electrochemical biosensors, one based on a Clark 0, probe and the other based on the H202 probe. The two probes were assembled employing a polymeric membrane where the enzyme lactate oxidase was previously immobilized. Both sensors exhibited high storage and operational stabilities, and good reproducibility when used in wine matrix. Measurement of lactate was carried out using two different flow-through cells assembled with the lactate probes. The low detection limit of these probes and the relatively high concentration of lactate in wine samples required a serial dilution in the range 1: 100 to 1: 200, thus eliminating all potential electrochemical or enzyme interferences present in the sample. Malic acid was determined using a differential commercially available pH-meter and an enzymatic procedure. For this analysis, we employed the malic enzyme (EC 1.1.1.40) and the NADP+ cofactor. The pH variation due to the action of this enzyme on malic acid was found to be proportional to the malic acid present in the sample. Experimental parameters such as pH, temperature, and co-factor concentration were optimized, resulting in malate determination in less than 1 min with good reproducibility. Various samples of wine and wine musts were assayed for lactate with the two biosensor procedures, and for malate with differential pH-metry. The results when compared with those obtained with the commonly used spectrophotometric procedure correlated well.
In this work, a purified acid urease preparation was covalently immobilized on Eupergit C 250 L a... more In this work, a purified acid urease preparation was covalently immobilized on Eupergit C 250 L and stabilized with glycine. Its average activity was found to be 69 +/- 16% of the initial one after 34-day storage at 4 degrees C . The kinetics of urea degradation in a model wine solution by immobilized enzyme was confirmed to be of pseudo-first-order with respect to the urea concentration in the liquid bulk, its apparent pseudo-first-order kinetic rate constant (k(Ii)) being about one-fourth of that (k(If)) pertaining to the free enzyme. In the operating conditions tested, the reaction kinetics was estimated as unaffected by the contribution of the external film and intraparticle diffusion mass transfer resistances. Because in the presence of the high-inhibitory tannins extracted from grape seeds in the range of 3-620 g of GAE m(-3) the loss in k(Ii) was quite smaller than that in k(If), the biocatalyst tested here is likely to overcome the present limits to the application of free acid urease in wine treatment.
The specific activity of a whole cell acid urease preparation was tested in five wines manufactur... more The specific activity of a whole cell acid urease preparation was tested in five wines manufactured in the Apulia region of Italy in the 2003 vintage at both short and long treatment times, thus confirming the validity of the pseudo-first-order kinetic model to describe urea removal in real wines. The ratio between the experimental pseudo-first-order kinetic rate constant (k Ie ) for any real wine tested and that (k I ) referred to a model wine solution having the same composition and pH reduced from about 0.21 to 0.02 as the overall content of phenolic compounds (P) increased from 109 to 853 g m -3 of gallic acid equivalent (GAE). The specific inhibitory effect of such compounds was explained by accounting for the equilibrium constant (K P ) of the reaction of polyphenols with acid urease, which was found to be about 21 g of GAE m -3 for the real wines tested, whereas it ranged from about 16 to 45 g of GAE m -3 when the model wine solution was enriched with tannins extracted from grape seeds or skins, respectively. A sequential experimental procedure consisting of accelerated acid urease tests at high doses of enzyme followed by accelerated ethyl carbamate tests on the resulting acid urease treated wine was recommended to assess preliminarily the technoeconomic feasibility of the acid urease hydrolytic process for the wine of concern.
A flow injection analysis procedure was used for the determination of L-lactic acid during the pr... more A flow injection analysis procedure was used for the determination of L-lactic acid during the production of mozzarella cheese. The apparatus consists of an electrochemical flow-through walljet cell assembled with a platinum sensor covered with the immobilized lactate oxidase enzyme and connected to an amperometer. This system was used to monitor the concentration of L-lactic acid produced by lactose fermentation catalyzed by selected starters during cheese manufacture. Lactate was detected in the range (5 × 10 -6 )-(1 × 10 -4 ) mol/L with a detection limit of 2 × 10 -6 mol/L. L-Lactate has been measured in raw milk and during the manufacture of cow and water buffalo mozzarella cheese. The starter used was a commercially available strain of Streptococcus thermophilus. Real time analysis of lactate allowed a control of the curd-ripening evolution at different pasteurization temperatures of the milk. Values of lactic acid were compared with pH variation during the process. This method proved to be more sensitive than the pH measurement procedure for the control of the continuous production of lactic acid particularly near the "stretching point" when very slight pH variations were observed.
Phenolic compounds in different olive varieties were determined by HPLC analysis over 2 years. De... more Phenolic compounds in different olive varieties were determined by HPLC analysis over 2 years. Demethyloleuropein was found in only two (Coratina and Leccino) of the eight varieties studied, so it could be used as varietal marker. Elenolic acid glucoside and hydroxytyrosol can be considered indicators of maturation for olives. In fact, as the olives ripen, their tenor increases whereas oleuropein decreases.
The specific activity (π A ) of a whole cell acid urease preparation was assessed in model wine s... more The specific activity (π A ) of a whole cell acid urease preparation was assessed in model wine solutions at different levels of malic (M) and lactic acids, metabisulfite, ethanol, and pH by performing a central composite design. M and then pH were found to be the most controlling variables, their effects being practically coincident but of opposite sign. For urea concentrations up to ≈1 mol m -3 the ammonium formation rate was assumed of the pseudo-first-order with respect to urea, this being confirmed by two independent validation tests performed at 20°C for as long as 24 h. In the case of real wines the effective pseudo-first-order kinetic rate constants were found to be smaller than those pertaining to the model solutions having the same wine composition and pH by a factor varying from 10 to 1000, this affecting significantly the specific urease treatment costs per liter of wine treated.
ABSTRACT “Natural” amines in plants, endogenous constituents of tissues, are involved in differen... more ABSTRACT “Natural” amines in plants, endogenous constituents of tissues, are involved in different physiological processes such as fruit development and senescence. Their levels can vary depending on variety, ripening, and storage conditions. Moreover, microbial contamination results in the increase of the “biogenic” amine content of fruits and vegetables. Electrochemical biosensors for the determination of the amine content in fruits have been assembled using diamine oxidase and polyamine oxidase covalently immobilized onto polymeric membranes. Both enzymes in the presence of their substrates produced H2O2 that was detected at a platinum electrode polarized at +650 mV versus Ag/AgCl. The best analytical conditions for the two enzyme electrodes have been optimized. The main positive effect of packaging fresh fruits with polymeric films is the provision of a barrier to the gas diffusion; the reduced O2 and increased CO2 concentrations slow the respiration rate. The films also allow the maintenance of high relative humidity with reduction of water loss. To investigate the postharvest life of apricots and sweet cherries in modified atmosphere storage at 0 °C, the concentration of amines in fruits packaged using different polymeric films was monitored. Keywords: Modified atmosphere packaging (MAP); polymeric film; electrochemical biosensor; amine; diamine oxidase; polyamine oxidase; sweet cherry; apricot fruit; postharvest
A purified acid urease preparation was covalently immobilized onto either Eupergit C 250 L or glu... more A purified acid urease preparation was covalently immobilized onto either Eupergit C 250 L or glutaraldehyde-cross-linked chitosan-derivative beads (i.e., Chitopearls BCW-3003 and BCW-3010). The kinetics of urea degradation in two target Italian white (i.e., Grechetto and Sauvignon Blanc) wines, as well as in a model wine solution, by using the above Eupergit C 250 L-, BCW-3003-, or BCW-3010-based biocatalysts, was confirmed to be of the pseudofirst order with respect to the urea concentration in the liquid bulk and not limited by urea mass transfer. In Grechetto and Sauvignon Blanc wines, the corresponding kinetic rate constants were quite similar, being about 7, 18, or 17% of that observed for free enzyme in the model wine solution, respectively. Owing to their minor sensitivity to the phenolic content of the wines tested, the chitosan-based biocatalysts might be potentially employable in the make up of packed-bed cartridges to continuously remove urea from commercial wines.
The influence of potential inhibitors, naturally present in wine, on the activity of stem bromela... more The influence of potential inhibitors, naturally present in wine, on the activity of stem bromelain was investigated in order to evaluate the applicability of this enzyme for protein stabilization in white wine. Bromelain proteolytic activity was tested against a synthetic substrate (Bz-Phe-Val-Arg-pNA) in a model wine system after adding ethanol, sulfur dioxide (SO 2 ), skin, seed, and gallic and ellagic tannins at the average range of their concentration in wine. All the inhibitors of stem bromelain activity tested turned out to be reversible. Ethanol was a competitive inhibitor with a rather limited effect. Gallic and ellagic tannins have no inhibitory effect on stem bromelain activity, while both seed and skin tannins were uncompetitive inhibitors. The strongest inhibition effect was revealed for sulfur dioxide, which was a mixed-type inhibitor for the enzyme activity. This study provides useful information relative to a future biotechnological application of stem bromelain in winemaking.
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