Pulsed electric fields versus thermal treatment: equivalent processes to obtain equally acceptable citrus juices.

Pulsed electric field treatment has been claimed to produce more acceptable chilled citrus juices than those obtained by conventional thermal treatment. The pectin methylesterase activity and the acceptability of nine juices obtained from Clementine mandarins, Valencia oranges, and Ortanique fruits (hybrid of mandarin and orange), untreated, pasteurized (85 degrees C for 10 s), and treated by pulsed electric fields (25 kV/cm for 330 micros), were evaluated. The treatments, selected to reach a similar level of pectin methylesterase inactivation, produced juices that did not differ in acceptability from each other for the three varieties and in all cases were less acceptable than the untreated juice.

A theoretical equation describing the time evolution of the concentration of a selected range of substrate molecular weights in depolymerization processes mediated by single-attack mechanism endo-enzymes.

Monitoring the time evolution of the concentration of a selected range of molecular weights of substrate, referred to as "detectable" substrate, has been used to determine endo-enzymic activities in polysaccharide depolymerizing processes. In the methodologies based on the use of dye-labeled substrates, the "detectable" substrate extends from a given molecular weight threshold downward. On the contrary, in the fluorescent probe-flow injection analysis methodology, initially developed to determine (1 --> 3)-(1 --> 4)-beta-D-glucanase activities, the "detectable" substrate extends from a given molecular weight threshold upward. Assuming that the time evolution of the molecular weight distribution of the substrate follows the most probable distribution (the enzymic attack is random and its mechanism is single attack), a theoretical equation describing the time evolution of the concentration of "detectable" substrate (from a given molecular weight threshold upward or downward) has been deduced. This equation, Wd = Wo. (1 + alphat). e-alphat, where Wd is the concentration of "detectable" substrate, Wo is the initial concentration of the substrate, t is the depolymerization time, and alpha is a parameter correlated through a hyperbola with the initial concentrations of enzyme and substrate and the Michaelis-Menten constant, Km, has been tested against different (1 --> 3)-(1 --> 4)-beta-D-glucan/(1 --> 3)-(1 --> 4)-beta-D-glucanase systems using the fluorescent probe-flow injection analysis methodology and Calcofluor as the fluorescent probe. The most important predictions of the theoretical equation, which allow accurate determination of both endo-enzymic activities and kinetic constants, have been experimentally confirmed.

Monitoring of chemical and enzymatic hydrolysis of water-soluble proteins using flow-injection analysis with fluorescence detection and an aqueous eluant containing 2-p-toluidinylnaphthalene-6-sulfonate as the fluorescent probe.

The exposed hydrophobicity of proteins, which is due to the hydrophobic regions located on their surfaces, enhances the fluorescence intensity of the probe 2-p-toluidinylnaphthalene-6-sulfonate (2,6-TNS) by the formation of a complex. During the hydrolysis of a protein, the average exposed hydrophobicity of the substrate continuously changes with incubation time, and these changes are immediately reflected by a corresponding change in the fluorescence intensity of the 2,6-TNS/substrate complex. Therefore, 2,6-TNS seems to be a good probe to monitor the course of the depolymerization processes of proteins. In this work, bovine serum albumin and alpha-casein have been hydrolyzed both chemically and enzymatically, and the course of the reactions is monitored by using flow-injection analysis (FIA) with fluorescence detection and a buffered aqueous eluant containing 2,6-TNS as the fluorescent probe. Results indicate that the time evolution of the fluorescence intensity of the 2,6-TNS/substrate complex can be correlated with the initial concentration of the parent protein, in mass per unit volume, the hydrolytic activity added, and the time evolution of the mean chain length of the substrate. In addition, because the time elapsed between injection of the sample into the FIA system and measurement of the corresponding fluorescence intensity is only a few seconds, this methodology could be a useful tool for on-line monitoring of processes for the production of protein hydrolysates.

Determination of beta-amylase activity by a fluorimetric 2-p-toluidinylnaphthalene-6-sulfonate flow-injection analysis (2, 6-TNS-FIA) method, using amylose and amylopectin as substrates.

2-p-Toluidinylnaphthalene-6-sulfonate (2,6-TNS) is a compound which is barely fluorescent in pure water but whose fluorescence can be strongly enhanced if the environment becomes hydrophobic, i.e. by the addition of suitable substrates such as proteins or 1, 4-alpha-D-glucans. The enhancement of fluorescence results from the formation of a 2,6-TNS/substrate complex. For linear and ramified 1, 4-alpha-D-glucans, the fluorescence intensities of the complexes depend linearly on their concentrations but nonlinearly on their average molecular weights (AMW). Thus, the fluorescence detector acts simultaneously as a linear detector concerning the concentration of 1,4-alpha-D-glucan and as a nonlinear mass-selective detector concerning its AMW. These properties have been used for the development of a fluorimetric 2,6-TNS-FIA methodology for the determination of beta-amylase activity, using amylose and amylopectin as substrates. The experimental data points, corresponding to the concentration of "detectable" substrate vs depolymerization time, were fitted using a two-parameter exponential decay curve, and the depolymerization rates at time zero were calculated. The depolymerization rates at time zero vs the corresponding initial substrate concentrations were fitted using the Michaelis-Menten hyperbola and the enzymic constants k(3) and K(m) for amylose (5.93 x 10(-3) g/microKat. min and 1.49 g/L, respectively) and for amylopectin (7.40 x 10(-3) g/microKat+. min and 1.65 g/L, respectively) were determined.

Determination of depolymerization kinetics of amylose, amylopectin, and soluble starch by Aspergillus oryzae alpha-amylase using a fluorimetric 2-p-toluidinylnaphthalene-6-sulfonate/flow-injection analysis system.

This study reports on the determination of the depolymerization kinetics of amylose, amylopectin, and soluble starch by Aspergillus oryzae alpha-amylase using flow-injection analysis with fluorescence detection and 2-p-toluidinylnaphthalene-6-sulfonate as the fluorescent probe. The experimental data points, corresponding to the evolution of the concentration of "detectable" substrate with depolymerization time, were fit to a single exponential decay curve in the case of amylose and to a double exponential decay curve in the cases of amylopectin and soluble starch. For all the assayed substrates, the determined depolymerization rates at time zero correlated well with the initial enzyme and substrate concentrations through the usual Michaelis-Menten hyperbola. Therefore, this methodology allows the determination of alpha-amylase activity using any of these substrates. For amylopectin and soluble starch, the value of the total depolymerization rate at any depolymerization time was the result of the additive contribution of two partial depolymerization rates. In contrast, the total depolymerization rate for amylose was always a single value. These results, in conjunction with the relative time evolution of the two partial depolymerization rates (for amylopectin and soluble starch), are in good agreement with a linear molecular structure for amylose, a "grape-like" cluster molecular structure for amylopectin, and an extensively degraded grape-like cluster structure for soluble starch.