J. F. Bello

Studies on the occurrence of non-enzymatic browning during storage of citrus juice

Abstract In freshly produced commercial citrus juice, aseptically filled in TetraBrik cartons, it has been demonstrated that nonenzymatic browning was mainly due to carbonyl compounds formed from l -ascorbic acid degradation. Contribution from sugar–amine reactions is negligible as evident from the constant total sugar content of degraded samples. The presence of amino acids and possibly other amino compounds, however, enhance browning. Although formation of 5-hydroxymethyl-furaldehyde (5-HMF) has been detected in degraded juice samples, its presence could not be used as an index of browning. 5-HMF has been found to be unreactive in the browning process in citrus juices and its contribution to browning in this type of products is insignificant, if not negligible. Increasing the l -ascorbic acid concentration extends the nutritional value of the product but also increases the severity of browning. Hence where l -ascorbic acid is added as an antioxidant in foods, care should be taken so that the amount added is just proportional to the level of oxygen present or to the amount of oxidizable substances.

Possible additives for extension of shelf-life of single-strength reconstituted citrus juice aseptically packaged in laminated cartons

It has been demonstrated that both aerobic and anaerobic oxidation of L-ascorbic acid occur in freshly-produced commercial orange juice in aseptically-filled TetraBrik cartons. The aerobic oxidation proceeds during the initial stage of the degradation process at a rate which is dependent on the L-ascorbic acid concentration, dissolved oxygen level and temperature. However, when the oxygen level reaches equilibrium, an anaerobic process follows at a much slower rate.Comparative studies on food additives, such as sodium metabisulphite, sodium tripolyphosphate, D-sorbitol and carboxymethyl cellulose, which may be used for extending the shelf-life of citrus juices, showed sodium metabisulphite to be the most effective. However, use of L-ascorbic acid derivatives is recommended.

Kinetic behaviour and reaction mechanism of the hydrolysis of p-nitrophenyl palmitate in mixed micelles with Triton X-100 catalyzed by lipase from Candida rugosa

Abstract The physicochemical properties of lipase from Candida rugosa in the hydrolysis of micellized p -nitrophenyl palmitate, such as thermal stability, enzyme concentration and the effect of ionic strength on the rate of catalysis, have been characterized. As regards the specificity for a series of p -nitrophenyl esters ( p -NPC n ), n = 2, 4, 8, 12 and 16 being the number of carbon atoms of the hydrophobic tail, the lipase from Candida rugosa proved to be non-specific, although it did hydrolyze them at different rates, depending on n and the physicochemical nature of the substrate (mixed micelles with surfactant or simple solution). At Triton X-100 levels above the critical micelle concentration (c.m.c.), the kinetic behaviour of the hydrolysis of p -nitrophenyl palmitate in Triton X-100 mixed micelles catalyzed by Candida rugosa lipase was consistent with the Michaelis—Menten rate equation under three different experimental conditions: (i) the molar fraction of substrate held constant and the Triton X-100 concentration varied; (ii) the bulk substrate concentration held constant and the Triton X-100 concentration varied, and (iii) the Triton X-100 concentration held constant and the bulk substrate concentration varied. Kinetic analysis performed in the above conditions revealed that the simple model described by Verger et al. [ J. Biol. Chem. , 248 (1973), 4023] correctly interprets the kinetic behaviour of the commercial lipase from Candida rugosa used in the study and highlights the advantage that this classic mechanism may have in current lipase modelling in biocatalysis.

Non‐enzymatic Browning in Single‐Strength Reconstituted Citrus Juice in TetraBrik Cartons

In freshly produced commercial citrus juice, aseptically filled in a TetraBrik carton, it has been demonstrated that non‐enzymatic browning was mainly due to carbonyl compounds formed from l‐ascorbic acid degradation. At different storage temperatures (4−37 °C), this, browning showed first‐order kinetics with higher rate constants as temperature was increased. An assessment of the degree of browning as a function of time in the presence of additives (sodium metabisulfite, sodium tripolyphosphate, (carboxymethyl)cellulose and sorbitol) showed that the addition of sodium metabisulfite substantially reduced browning, particularly at the higher storage temperatures of 76 and 105 °C where a greater accumulation of 5‐(hydroxymethyl)furfuraldehyde was observed.

Human placental alkaline phosphatase covalently immobilized on a cross-flow microfiltration polyvinylidene difluoride membrane. Part I. Physicochemical effectors

Abstract Human placental alkaline phosphatase has been chemically immobilized on a hydrophilic cross-flow microfiltration membrane made of polyvinylidene difluoride (PVDF) chemically labelled with 1-carbonylimidazole groups. Physicochemical characterization of this immobilized biocatalyst focused especially on attributes such as the immobilization isotherms (Langmuir type) of the enzyme to the support, the stability of the catalytic activity, the effects of pH and temperature on this activity and the existence of limitations of external diffusion for H+, substrate and/or products. Regarding enzyme stability and its dependence on different experimental conditions, patterns of hysteresis or memory are proposed.

Human placental alkaline phosphatase covalently immobilised on a cross-flow microfiltration polyvinylidene difluoride membrane Part II. Activity vs. [substrate] kinetic behaviour

Abstract Human placental alkaline phosphatase was chemically immobilized on a hydrophilic cross-flow microfiltration membrane made of polyvinylidene difluoride (PVDF) chemically labelled with 1-carbonylimidazole groups and characterized physicochemically (in Part I, J. Mol. Catal . (Vol. 93, 85–104)) The intrinsic kinetic behaviour (rate vs. substrate concentration) in the absence of diffusional constraints was analysed graphically and numerically, by non-linear regression and use of the F statistical test for model discrimination, postulating a minimum rational rate equation of 2:2 degree in substrate concentration. According to the results found, a mechanism for the catalytic kinetic action has been postulated.

Kinetic aspects of human placental alkaline phosphatase enzyme membrane

The crosslinking of alkaline phosphatase of human placenta with human serum albumin has been optimized. During the physico-chemical characterization of this immobilized biocatalyst, special attention was paid to attributes such as the irreversibility of the enzyme support bonding, the stability of the catalytic activity, and the effects of pH and temperature on this activity. Regarding stability, patterns of denaturation are proposed, to account for inactivation curves over time and under storage/operation conditions. These patterns, in some cases, indicate the existence of different populations of immobilized enzyme molecules, with a different degree of sensitivity to denaturation. The activity vs pH profiles are clearly modified by the immobilization process. This is because the pH of the free homogeneous solution, measurable with a pH-meter, differs from the real pH of the immediate microenvironment of the immobilized enzyme molecules due to the effects of proton accumulation in the microenvironment (in the reaction catalysed by alkaline phosphatase, protons are produced), to limitations to the free diffusion of H+ and to the possible partition effects of H+ due to polar interactions with residues or molecules of the enzyme membrane. In the experimental working conditions, the apparent optimum temperatures are centered at 40 degrees C, inactivation (thermal denaturation) occurring above this temperature. In the temperature range 10-40 degrees C, the kinetic control over the overall activity of the immobilized enzyme was observed, causing the Arrhenius profiles to be linear.