Characterization of the temperature activation of pectin methylesterase in green beans and tomatoes

Low-temperature blanching of vegetables activates the enzyme pectin methylesterase (PME), which demethylates cell wall pectins and improves tissue firmness. This temperature activation of PME has been investigated by measuring the formation of methanol in intact tissue of green beans and tomatoes. Rates of methanol formation at temperatures of 35-65 degrees C were obtained by measuring the release of methanol from thin slices of tomato pericarp or green bean pod material. Activation energies of 112 and 97 kJ mol(-1) were calculated for PME activity in green beans and tomatoes, respectively. These activation energies indicate that the rate of pectin demethylation at 65 degrees C will be nearly 100 times that at 25 degrees C. PME activity was also determined titrimetrically using a solubilized form of the enzyme and purified pectin at temperatures from 30 to 60 degrees C. Under these conditions, much lower activation energies of 37 and 35 kJ mol(-1) were obtained for green beans and tomatoes, respectively. Methanol accumulation during heating of whole intact green beans was also determined and yielded an activation energy similar to that obtained with sliced beans. Whole green beans held at room temperature did not accumulate any methanol, but sliced or homogenized beans did. If whole beans were first heated to 45 degrees C and then cooled, methanol accumulation was observed at room temperature. These results indicate that two factors contribute to the observed high rate of pectin de-esterification during low-temperature blanching: (1) An irreversible change, causing PME to become active, occurs by heating to > or = 45 degrees C. (2) The high activation energy for pectin de-esterification means that the rate of de-esterification increases substantially with increasing temperature.     

Role of cations in the catalysis of thermostable pectinmethylesterase extracted from Marsh grapefruit pulp

The de-esterification of high methoxyl pectin by thermostable pectinmethylesterase (TS-PME) from Marsh grapefruit was studied at pH 7.5, in a temperature range between 25 degrees and 50 degrees C and in the presence of various cations. Arrhenius plots were constructed for CaCl(2) (5 to 20 mM), SrCl(2) (5 to 20 mM), and spermidine (2.5 to 10 mM) added reactions. Enthalpy (DeltaH()) and entropy (DeltaS()) of activation changed with cation type and concentration. The presence of cations modified the free energy of the resulting enzyme/substrate complex. The entropy of activation was positive at all concentrations of CaCl(2) studied, and negative in the same concentration range with SrCl(2). Reactions with spermidine showed negative entropy of activation at concentrations <5 mM and positive values of entropy at higher concentrations.     

Transacylation and de-esterification reactions of pectin as catalyzed by pectinesterases from tomato and citrus

The optimal conditions for the de-esterification reaction of tomato pectinesterase (PE) and citrus PE was 0.1-0.2 M NaCl and at pH 7.5-8.5, 65 degrees C, almost identical to those for the transacylation reaction as observed by turbidity (absorbance at 400 nm) change. Among the PEs tested, pea pod PE presented the most remarkable catalysis on the transacylation reaction, and 1.5% pectin solution was determined to be suitable for this reaction. Low methoxy pectin with a DE (degree of esterification) of 31% displayed a slow turbidity increase, revealing that the extent of DE was influential on the transacylation. Besides citrus pectin, apple pectin was also proved to progress transacylation reaction by PEs from tomato and citrus sources as apparently observed by turbidity method.     

Translational diffusion coefficients of volatile compounds in various aqueous solutions at low and subzero temperatures

Translational diffusion coefficients (D(12)) of volatile compounds were measured in model media with the profile concentration method. The influence of sample temperature (from 25 to -10 degrees C) was studied on translational diffusion in sucrose or maltodextrin solutions at various concentrations. Results show that diffusivity of volatile compounds in sucrose solutions is controlled by temperature, molecule size, and the viscosity of the liquid phase as expected with the Stokes-Einstein equation; moreover, physicochemical interactions between volatile compounds and the medium are determinant for diffusion estimation. At negative temperature, the winding path induced by an ice crystal content of >70% lowered volatile compound diffusion. On the contrary, no influence on translational diffusion coefficients was observed for lower ice content.     

High-pressure-induced rheological changes of low-methoxyl pectin plus micellar casein mixtures

The influence of high-pressure treatment (HPT) (200-800 MPa, 5 or 20 min, at 20 degrees C) on the rheological properties of solutions of amidated low-methoxyl pectin (LMP) and its mixtures with micellar casein (MC) has been investigated in the presence and absence of sucrose. The storage modulus G' of LMP gels containing 0-55 wt % sucrose and 0.1-1 wt % LMP was found to increase significantly following HPT at >or=400 MPa. Various concentrations of LMP in the presence of different amounts of MC (0.5-12 wt %) showed contrasting types of rheological behavior. In the presence of a low concentration of LMP (<0.3 wt %), HPT was found to induce a sol-gel transformation at relatively high LMP/MC molar ratios (<4 wt % MC), to reduce values of G' and the loss modulus G' ' at intermediate LMP/MC ratios (4-10 wt % MC), and to increase the values of G' and G' ' at low LMP/MC ratios (>10 wt % MC). In contrast, in the presence of a higher amount of LMP (>0.5 wt %), it was observed that HPT enhances the values of both the storage and the loss moduli over the whole range of MC concentrations.     

Mode of de-esterification of alkaline and acidic pectin methyl esterases at different pH conditions

Highly esterified citrus pectin was de-esterified at pH 4.5 and 8.0 by a fungal pectin methyl esterase (PME) that was shown to have an acidic isoelectric pH (pI) and an acidic pH optimum and by a plant PME that was characterized by an alkaline pI and an alkaline pH optimum. Interchain and intrachain de-esterification patterns were studied by digestion of the pectin products with endo-polygalacturonase and subsequent analysis using size exclusion and anion-exchange chromatography. No effect of pH was observed on the de-esterification mode of either of the two enzymes. Acidic, fungal PME converted pectin according to a multiple-chain mechanism, with a limited degree of multiple attack at the intrachain level, both at pH 4.5 and at pH 8.0. A multiple-attack mechanism, with a high degree of multiple attack, was more appropriate to describe the action mode of alkaline, plant PME, both at pH 4.5 and at pH 8.0.     

Isolation, characterization, and pectin-modifying properties of a thermally tolerant pectin methylesterase from Citrus sinensis var. Valencia

The thermally tolerant pectin methylesterase (TT-PME) was isolated as a monocomponent enzyme from sweet orange fruit (Citrus sinensis var. Valencia). It was also isolated from flower and vegetative tissue. The apparent molecular weight of fruit TT-PME was 40800 by SDS-PAGE and the isoelectric point estimated as pI 9.31 by IEF-PAGE. MALDI-TOF MS identified no tryptic-peptide ions from TT-PME characteristic of previously described citrus PMEs. TT-PME did not absolutely require supplemented salt for activity, but salt activation and pH-dependent activity patterns were intermediate to those of thermolabile PMEs. Treatment of non-calcium-sensitive pectin with TT-PME (reducing the degree of methylesterification by 6%) increased the calcium-sensitive pectin ratio from 0.01 to 0.90, indicating a blockwise mode of action. TT-PME produced a significantly lower end-point degree of methylesterification at pH 7.5 than at pH 4.5. Extensive de-esterification with TT-PME did not reduce the pectin molecular weight or z-average radius of gyration, as determined by HPSEC.     

Changes in molecular weight of transacylated pectin catalyzed by tomato and citrus pectinesterases as determined by gel permeation chromatography

The changes in molecular masses of pectin in 0.5% pectin-pectinesterase (PE) mixtures (2 units/mL) incubated at various temperatures, pH values, and NaCl levels for 30 min were observed by a Toyopearl TSK HW-65 (F) gel permeation chromatography. The molecular mass of pectin was remarkably increased from 103 to 266 kDa when the incubation temperature of pectin-tomato PE was increased from 25 to 45 degrees C. A further increase in molecular mass was observed when a pectin-citrus PE mixture was incubated at 65 degrees C. The values of pH and NaCl levels were also crucial to the transacylation activity of PEs. Reaction at pH 7.5 with tomato PE and citrus PE remarkably expanded the molecular mass of pectin to 410 and 670 kDa, respectively. The NaCl level of 0.3-0.5 and 0.3 M was favorable for the transacylation reaction of tomato PE and citrus PE, respectively. Only high methoxylpectin was the suitable substrate for PE to conduct the transacylation reaction.     

New method for a two-step hydrolysis and chromatographic analysis of pectin neutral sugar chains

A new method for the determination of the main neutral sugars in pectin has been developed. The sample preparation involves a mild chemical attack followed by an enzymatic hydrolysis. The completeness and nondestructive character of the method are demonstrated by comparison of the results obtained with different acids such as H2SO4, HCl, and trifluoroacetic acid (TFA) at different concentrations (2, 1, or 0.2 M) at two temperatures (80 or 100 degrees C). The chemical hydrolysis of pectin neutral sugar chains with strong acid (1 or 2 M) and high temperature (100 degrees C) shows that the liberation of the pectin sugars is not realized at the same rate for each sugar. Different optimum conditions are thus obtained. However, the chemical pectin hydrolysis with 0.2 M TFA at 80 degrees C is characterized by the liberation of pectin neutral sugar side chains without any degradation within 72 h of hydrolysis. Under these conditions, the liberation of some pectin sugars, essentially galactose, glucose, and rhamnose, was not complete. An enzymatic hydrolysis is necessary to obtain a complete release of all the sugars. The combination of the two treatments, a chemical hydrolysis realized with diluted acid (0.2 M) for 72 h at low temperature (80 degrees C) on one hand and an enzymatic hydrolysis on the other hand, allow a total liberation of pectin sugars. The quantitative analysis of the carbohydrates is realized with accuracy, high selectivity, and sensitivity with high-performance anion-exchange chromatography with pulsed-amperometric detection. The sugars can be analyzed without any derivatization with a limit of quantification of 0.1 mM.     

Activity and process stability of purified green pepper (Capsicum annuum) pectin methylesterase

Pectin methylesterase (PME) from green bell peppers (Capsicum annuum) was extracted and purified by affinity chromatography on a CNBr-Sepharose-PMEI column. A single protein peak with pectin methylesterase activity was observed. For the pepper PME, a biochemical characterization in terms of molar mass (MM), isoelectric points (pI), and kinetic parameters for activity and thermostability was performed. The optimum pH for PME activity at 22 degrees C was 7.5, and its optimum temperature at neutral pH was between 52.5 and 55.0 degrees C. The purified pepper PME required the presence of 0.13 M NaCl for optimum activity. Isothermal inactivation of purified pepper PME in 20 mM Tris buffer (pH 7.5) could be described by a fractional conversion model for lower temperatures (55-57 degrees C) and a biphasic model for higher temperatures (58-70 degrees C). The enzyme showed a stable behavior toward high-pressure/temperature treatments.     

Inactivation of heat-resistant pectinmethylesterase from orange by manothermosonication

Pectinmethylesterase of navel oranges shows two fractions greatly differing in thermostability. The most thermostable fraction accounts for approximately 10% of total activity. The thermal inactivation of this fraction follows first-order kinetics both in 5 mM, pH 3.5, citrate buffer and in orange juice at the same pH, showing a z value of 5.1 degrees C and an activation energy (E(a)) of 435 kJ mol(-)(1) K(-)(1). The heat resistance of the enzyme is approximately 25-fold higher in the juice than in citrate buffer. When ascorbic acid, sucrose, glucose, and fructose are added to the citrate buffer at the concentrations found in orange juice, the heat resistance of the enzyme increases 3-fold. The addition of pectin at 0.01% concentration multiplies it by a factor of 50. Manothermosonication (MTS), the simultaneous application of heat and ultrasound under moderate pressure (200 kPa), at 72 degrees C, increases the inactivation rate 25 times in buffer and >400 times in orange juice. MTS inactivation shows a higher z value (35.7 degrees C) and lower E(a) (56.9 kJ mol(-)(1) K(-)(1)) than simple heating.     

Reaction rate modeling in cryoconcentrated solutions: alkaline phosphatase catalyzed DNPP hydrolysis

The hydrolysis of disodium p-nitrophenyl phosphate catalyzed by alkaline phosphatase was chosen as a model to study the kinetics of changes in frozen food products. The initial reaction rate was determined in concentrated sucrose solutions down to -24 degrees C, and the enzymatic characteristics K(M) and V(max) were calculated. The experimental data were compared to the kinetics predicted by assuming that the reaction was viscosity dependent. Indeed, an analysis of the enzymatic reaction demonstrated that both the diffusion of the substrate and the flexibility of the enzyme segments were controlled by the high viscosity of the media. When the temperature was too low for the viscosity to be measured simply, the Williams-Landel-Ferry equation was used to predict the viscosity, taking, as reference temperature, the glass transition temperature (T(g)) corresponding to the concentration of the freeze-concentrated phase at the test temperature. Predicted values of the reaction rate were very close to the experimental ones in the studied temperature range.     

Effects of heat treatment and pectin addition on beta-lactoglobulin allergenicity

The specific effects of heat treatment and/or addition of low/high-methylated pectin (LMP/HMP) on the allergenicity of beta-lactoglobulin (beta-Lg) and its hydrolysis products were investigated through a two-step in vitro digestion approach. beta-Lg was first hydrolyzed by pepsin and then by a trypsin/chymotrypsin (T/C) mixture done in a dialysis bag with a molecular weight cutoff of 1000. The protein digestion was followed by SDS-PAGE electrophoresis performed on each digestion product, and their in vitro allergenicity was analyzed by immunoblotting. Such procedure was applied on beta-Lg samples mixed with the two kinds of pectin before or after heating (80 degrees C, 25 min) to determine the respective impact of heat treatment and pectin addition. Heat denaturation improved significantly the susceptibility of beta-Lg against the pepsin and the T/C. This effect, which was coupled to a reduction in immunoreactivity of the digested beta-Lg, appeared to be distinctively modulated by LMP and HMP. Through nonspecific interaction with the beta-Lg, pectin could reduce the accessibility of cleavage sites and/or epitope sequences. This mechanism of action is discussed in relation to the intra- and intermolecular interactions between beta-Lg and pectin initiated under the experimental conditions.     

柑橘果实发育中果胶酸钙、草酸钙和果胶动态的研究

以单性结实的龟井蜜柑和自花结实的鄂柑1号橘为试材,对整个果实发育期的子房(幼果)、果皮和果肉的果胶酸钙、草酸钙和果胶含量变化进行了测定。结果表明,1)两品种子房(幼果)果胶酸钙含量呈类似的下降趋势;草酸钙则相反,龟井花后趋下降,而鄂柑1号却明显上升;而且鄂柑1号子房(幼果)果胶酸钙、草酸钙和果胶含量均相对较高。2)在果实增大期内,两品种果皮和果肉的果胶酸钙含量均出现显著上升,对应果皮草酸钙含量虽有波动但居相对较高水平,而果肉草酸钙则趋明显下降。3)两品种果皮和果肉水溶性果胶含量均在增大期内呈显著上升,对应原果胶含量均相对较高,进入增大后期均明显下降。     

Characterization of a salt-independent pectin methylesterase purified from valencia orange peel

The pectin methylesterase (PME; EC 3.1.1.11) present in a commercial orange peel enzyme preparation was characterized to establish its identity among the multiple PME isozymes present in Valencia orange (Citrus sinensis L.) peel. We show the commercial enzyme corresponds to the major peak 2 PME previously separated by heparin-Sepharose chromatography (Cameron et al., J. Food Sci. 1998, 63, 253). Both PMEs have comparable elution profiles on cation-exchange and hydrophobic-interaction perfusion chromatography columns, molecular weights (ca. 34 kDa) and pI (pH 9.2), and biochemical properties, including a broad pH activity range and activity in the absence of added cations. An identical partial amino terminal peptide sequence was also obtained for the PMEs, which further demonstrated a structural identity with other plant PMEs. The biochemical and structural properties readily distinguish this Valencia orange PME from salt-dependent isozymes and further suggest that it is an ortholog to the salt-independent fruit-specific isozyme of tomato. This work provides a well-defined, enzymatically homogeneous, salt-independent (type 1) plant PME isozyme that is suitable for studying details of the enzyme's mode of action and for use in modifying methylester patterns for studying the structure-functional property relationships in pectin.     

Sucrose, glucose, and fructose extraction in aqueous carrot root extracts prepared at different temperatures by means of direct NMR measurements

Solutions obtained by heating carrot roots in water (stocks) are widely used in the food industry, but little information is available regarding the metabolites (intermediates and products of metabolism) found in the stock. The effect of treatment temperature and duration on the sugar composition of stocks was investigated directly by quantitative (1)H NMR spectroscopy, to understand the extraction mechanism when processing at 100 degrees C. Stocks prepared at three different temperatures (50, 75, and 100 degrees C) were investigated for up to 36 h. Three sugars (sucrose, glucose, and fructose) were detected and quantified. The concentrations of these three sugars reached a maximum after 9 h when the temperature of treatment was 50 or 75 degrees C. At 100 degrees C, the sucrose concentration reached a maximum after 3 h, whereas the concentration of glucose and fructose was still increasing at that time. Comparison of the kinetic composition of these carrot stocks with that of model sugar solutions leads to the proposal that the changes in stock composition result from sugar diffusion, sucrose hydrolysis, and hydroxymethylfurfural (HMF) formation.     

Inhibitory activities of semicarbazide-sensitive amine oxidase and angiotensin converting enzyme of pectin hydroxamic acid

Solutions of 100 mL of 1% commercial pectin each with a different degree of esterification (DE), DE94, DE65, and DE25, were reacted with 100 mL of 2 M alkaline hydroxylamine (pH 12.0) at room temperature for 4 or 18 h. These pectin hydroxamic acids (PHAs; DE94T4, DE94T18, DE65T4, and DE25T4) were used to test the inhibitory activities against semicarbazide-sensitive amine oxidase (SSAO) and angiotensin-converting enzyme (ACE). Compared to different DE pectins (DE94, DE65, and DE25), the PHAs of DE94T4, DE94T18, DE65T4, and DE25T4 showed different inhibition activities against SSAO or ACE. Commercial pectins with different DE values showed negligible SSAO or ACE inhibitions. The order of SSAO inhibition was DE65T4 > DE94T18 approximately DE25T4 > DE94T4. However, the order of ACE inhibition was DE94T4 > DE94T18 > DE65T4 > DE25T4. The SSAO activity staining or ACE-hydrolyzed products on TLC chromatogram also confirmed the inhibitory activities of PHAs against SSAO or ACE.     

果胶对脂类和类胡萝卜素消化利用影响研究进展

果胶已经被证实可以影响脂类的消化,脂溶性的类胡萝卜素在消化阶段需要被脂滴包裹才能进入小肠形成胶束,因此果胶对类胡萝卜素的消化利用也会存在潜在影响。该文综述了近年来果胶对脂类和类胡萝卜素消化利用影响研究进展,主要分为果胶对消化液黏度的影响、对消化酶的影响、与钙离子的相互作用、与胆盐的结合作用以及对脂滴的包裹作用这5个方面。该文为后续分析如何提高果蔬中类胡萝卜素生物利用度提供理论依据。     

Ascorbic acid oxidation in sucrose aqueous model systems at subzero temperatures

The reduction of Tempol by ascorbic acid in concentrated sucrose solutions was measured by electron paramagnetic resonance (EPR) at temperatures ranging from 16 to -16 degrees C. This method allowed the determination of the rate constants (k) of this fast reaction, by recording the Tempol reduction as a function of time. The two reactants were initially separated and had to migrate for the reaction to occur. The experimental findings were compared with predicted values according to the equation for diffusion-controlled reaction proposed by Atkins. The experimental reaction rate constants were observed to be lower than the calculated ones. However, the experimental values were found to be controlled by the temperature and viscosity changes of the reaction media, as expected for a diffusion-controlled reaction.     

The microbial destruction of chitin,pectin, and cellulose in soils

The most intensive degradation of polysaccharides takes place upon low and moderate temperatures in typical chernozems and gray forest soils and upon high temperatures in brown desert-steppe soils. This regularity is related to the structure of soil microbial complexes. The soil water content exerts a more pronounced effect on chitin decomposition in comparison with cellulose and pectin decomposition. The most favorable conditions for pectin decomposition by microbes are created at the water content close to the field capacity. Model experiments indicate that the range of moisture, upon which the transformation of chitin by microbes is most active, is wider in clay and loamy soils than in sandy soils. Direct study of microorganisms in the investigated soils under microscope has shown that actinomycetes, bacteria, and fungi participate in the transformation of polysaccharides. The role of actinomycetes in chitin decomposition increases in parallel with the rise in the soil water content and temperature. The role of fungi in pectin decomposition becomes higher under higher moistening and lower temperatures. The use of the FISH method makes it possible to reveal differences in the structure and number of metabolically active representatives of Bacteria and Archaea chitinolytic and pectinolytic prokaryotic complexes in the investigated soils under the impact of different ecological factors.