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.     

Change in particle size of pectin reacted with pectinesterase isozymes from pea (Pisum sativum L.) sprout

Four pectinesterase (PE) isozymes were isolated by CM-Sepharose CL-6B chromatography from etiolated pea (Pisum sativum L.) sprouts and then reacted with citrus pectin (degree of esterification = 68%, 30-100 kDa) to observe the change in pectin particle size using a laser particle size analyzer. After incubation of a pectin-PE mixture (pH 6.5) at 30 degrees C for 4 h, PE 1 was observed to catalyze the transacylation reaction most remarkably, increasing the particle size from approximately 50-70 to approximately 250-350 nm, followed by PE 3, PE 2, and PE 4.     

Antioxidant activity and emulsion-stabilizing effect of pectic enzyme treated pectin in soy protein isolate-stabilized oil/water emulsion

The antioxidant activity of pectic enzyme treated pectin (PET-pectin) prepared from citrus pectin by enzymatic hydrolysis and its potential use as a stabilizer and an antioxidant for soy protein isolate (SPI)-stabilized oil in water (O/W) emulsion were investigated. Trolox equivalent antioxidant capacity (TEAC) was found to be positively associated with molecular weight (M(w)) of PET-pectin and negatively associated with degree of esterification (DE) of PET-pectin. PET-pectin (1 kDa and 11.6% DE) prepared from citrus pectin after 24 h of hydrolysis by commercial pectic enzyme produced by Aspergillus niger expressed higher α,α-diphenyl-β-picrylhydrazyl (DPPH) radical scavenging activity, TEAC, and reducing power than untreated citrus pectin (353 kDa and 60% DE). The addition of PET-pectin could increase both emulsifying activity (EA) and emulsion stability (ES) of SPI-stabilized O/W emulsion. When the SPI-stabilized lipid droplet was coated with the mixture of PET-pectin and pectin, the EA and ES of the emulsion were improved more than they were when the lipid droplet was coated with either pectin or PET-pectin alone. The amount of secondary oxidation products (thiobarbituric acid reactive substances) produced in the emulsion prepared with the mixture of SPI and PET-pectin was less than the amount produced in the emulsion prepared with either SPI or SPI/pectin. These results suggest that PET-pectin has an emulsion-stabilizing effect and lipid oxidation inhibition ability on SPI-stabilized emulsion. Therefore, PET-pectin can be used as a stabilizer as well as an antioxidant in plant origin in SPI-stabilized O/W emulsion and thus prolong the shelf life of food emulsion.     

Novel cross-linked alcohol-insoluble solid (CL-AIS) affinity gel from pea pod for pectinesterase purification

Alcohol-insoluble solids (AIS) from pea pod were cross-linked (CL-AIS) and used as an affinity gel matrix to isolate pectin esterases (PEs) from tendril shoots of chayote (TSC) and jelly fig achenes (JFA), and the results were compared with those isolated by ion-exchange chromatography with a commercial resin. CL-AIS gel matrix in a column displayed poor absorption and purification fold of PE; however, highly methoxylated CL-AIS (HM-CL-AIS), by exposing CL-AIS to methanolic sulfuric acid to increase the degree of esterification (DE) to 92%, facilitated the enzyme purification. The purified TSC PE and JFA PE by the HM-CL-AIS column were proofed as a single band on an SDS-PAGE gel, showing that the HM-CL-AIS column was a good matrix for purification of PE, either with alkaline isoelectric point (pI) (TSC PE) or with acidic pI (JFA PE).     

Enzymatic modification of pectin to increase its calcium sensitivity while preserving its molecular weight

A commercial high-methoxy citrus pectin was treated with a purified salt-independent pectin methylesterase (PME) isozyme isolated from Valencia orange peel to prepare a series of deesterified pectins. A series of alkali-deesterified pectins was also prepared at pH 10 under conditions permitting beta-elimination. Analysis of these pectins using high-performance size exclusion chromatography (HPSEC) with on-line multiangle laser light-scattering, differential viscometer, and refractive index (RI) detectors revealed no reduction in weight-average molecular weight (M(w); 150000) in the PME-treated pectin series, whereas a 16% reduction in intrinsic viscosity (IV) occurred below a degree of esterification (DE) of 47%. In contrast, alkali deesterification rapidly reduced both M(w) and IV to less than half of that observed for untreated pectin. PME treatment of a non-calcium-sensitive citrus pectin introduced calcium sensitivity with only a 6% reduction in the DE. Triad blocks of unesterified galacturonic acid were observed in (1)H nuclear magnetic resonance spectra of this calcium-sensitive pectin (CSP). These results demonstrate that the orange salt-independent PME isozyme utilizes a blockwise mode of action. This is the first report of the preparation of a CSP by PME treatment without significant loss of the pectin's M(w) due to depolymerization.     

Separation and characterization of a salt-dependent pectin methylesterase from Citrus sinensis var. Valencia fruit tissue

A pectin methylesterase (PME) from sweet orange fruit rag tissue, which does not destabilize citrus juice cloud, has been characterized. It is a salt-dependent PME (type II) and exhibits optimal activity between 0.1 and 0.2 M NaCl at pH 7.5. The pH optimum shifted to a more alkaline range as the salt molarity decreased (pH 8.5-9.5 at 50 mM NaCl). It has an apparent molecular mass of 32.4 kDa as determined by gel filtration chromatography, an apparent molecular mass of 33.5 kDa as determined by denaturing electrophoresis, and a pI of 10.1 and exhibits a single activity band after isoelectric focusing (IEF). It has a K(m) of 0.0487 mg/mL and a V(max) of 4.2378 nkat/mg of protein on 59% DE citrus pectin. Deblocking the N-terminus revealed a partial peptide composed of SVTPNV. De-esterification of non-calcium-sensitive pectin by 6.5% increased the calcium-sensitive pectin ratio (CSPR) from 0.045 +/- 0.011 to 0.829 +/- 0.033 but had little, if any, effect on pectin molecular weight. These properties indicate this enzyme will be useful for studying the PME mode of action as it relates to juice cloud destabilization.     

Cationic antimicrobial (ε-polylysine)-anionic polysaccharide (pectin) interactions: influence of polymer charge on physical stability and antimicrobial efficacy

The cationic biopolymer ε-polylysine (ε-PL) is a potent food-grade antimicrobial that is highly effective against a range of food pathogens and spoilage organisms. In compositionally complex systems such as foods and beverages, cationic ε-PL molecules may associate with anionic substances, leading to increased turbidity, sediment formation, and reduced antimicrobial activity. This study therefore characterized the interactions between cationic ε-PL and anionic pectins with different degrees of esterification (DE) and then investigated the influence of these interactions on the antimicrobial efficacy of ε-PL. The nature of the interactions was characterized using isothermal titration calorimetry (ITC), microelectrophoresis (ME), and turbidity measurements. High (DE 61%), medium (DE 51%), and low (DE 42%) methoxyl pectins interacted with ε-PL molecules through electrostatic forces, forming either soluble or insoluble complexes with various electrical charges, depending on the relative mass ratio of pectin and ε-PL. The interaction of pectin with ε-PL increased as the negative charge density on the pectin molecules increased, that is, with decreasing DE. The antimicrobial efficacy of ε-PL against two acid-resistant spoilage yeasts (Zygosaccharomyces bailii and Saccharomyces cerevisiae) decreased progressively in the presence of increasing levels of all three pectins. Nevertheless, the low DE pectin decreased the antimicrobial efficacy of ε-PL much more dramatically, likely due to strong electrostatic binding of ε-PL onto low DE pectin molecules reducing its interaction with anionic microbe surfaces. This study provides knowledge that will facilitate the rational application of ε-PL as an antimicrobial in complex food systems.     

Particle size distribution of orange juice cloud after addition of sensitized pectin

The effect of the addition of commercial pectin to orange juice that was de-esterified by alkaline treatment or pectinmethylesterase was evaluated. Pectin at various degrees of esterification (DE) was added to reconstituted frozen orange juice concentrate, and the extent of cloud loss was determined after centrifugation. Of the five pectin treatments, percent transmittance (% T) of juice remained <5%, except for the pectin treatment with a residual methoxyl content (% DE) of <5%. Addition of 100--600 ppm of 5% DE pectin to juice resulted in % T between 36 and 73%. Despite the absence of clarification, increases in cloud particle size were observed and depended on the % DE, the amount of pectin added, and the method used to modify the pectins.     

Characterization of pectinesterase inhibitor in jelly fig (Ficus awkeotsang Makino) achenes

Pectinesterase inhibitor (PEI) extract prepared from intact jelly fig (Ficus awkeotsang Makino) achenes was separated by membrane (MWCO 3 and 10 kDa) and fractionated by a Sepharose G-50 gel permeation chromatography. Results from Sepharose G-50 gel permeation chromatography and concanavalin A Sepharose chromatography revealed PEI as polypeptides with molecular weights ranging from 3.5 to 4.5 kDa. Incubation of a PE (1 unit/mL)-PEI (2 mg/mL) mixture for 1 min decreased the PE activity by approximately 50%. On the basis of the results of Lineweaver-Burk double-reciprocal plots, Michaelis constant (K(m)) and V(max) values for jelly fig achenes PE (pH 6.0, 30 degrees C) were 0.78 mM -OCH3 and 1.09 microequiv of -COOH/min, respectively. In addition, PEI competitively inhibited both citrus and jelly fig achenes PEs.     

Addition of pectin and soy soluble polysaccharide affects the particle size distribution of casein suspensions prepared from acidified skim milk

Pectins are negatively charged polysaccharides employed as stabilizers in acidified milk dispersions, where caseins aggregate because of the low pH and serum separation needs to be prevented. The objective of this research was to study the effect of charge on the stabilizing functionality of the polysaccharide in acid milk drinks. Unstandardized pectins with various charges (as degree of esterification, DE) as well as soybean soluble polysaccharide (SSPS) were tested for their stabilizing behavior as a function of pH and concentration. Skim milk was acidified by glucono-delta-lactone and then homogenized in the presence of polysaccharide at different pH values (in the range from 4.2 to 3.0). Measurements of particle size distribution demonstrated that pectins with a DE of 71.4, 68.6, and 67.4 stabilized milk at pH > 4.0. Pectins with a lower DE (63.9%) needed a higher concentration (0.4%) at the same pH to show a monomodal distribution of particle sizes. Pectins with lower DE (<50%) did not stabilize the dispersions. Although this difference in behavior was attributed mainly to the pectin charge, the efficiency in stabilizing the casein dispersion decreased with decreasing pectin size. For example, the high methoxyl pectin (HMP) with 63.9 DE was smaller in size than the HMPs with a higher charge. Pectins showed a pH-dependent stabilization effect, as at pH < 4.0 the dispersions contained aggregates. When SSPS was used to stabilize acid milk, at pH < 4.0, it showed a better stabilization behavior than HMP. When SSPS and pectin were used in combination, the particle size distribution of the acid milk dispersion was pH-dependent, and results were similar to those for samples containing pectin alone. This suggested that in the mixture, pectin dominated the behavior over SSPS, even when an excess of SSPS was added to the dispersions before homogenization.     

Turbidity-based assay for polygalacturonic acid depolymerase activity

A technically simple and inexpensive discontinuous turbidity assay for qualitative and/or quantitative assessments of polygalacturonic acid depolymerase activity is presented. The enzyme reaction is initiated by the addition of enzyme preparation to a reaction mixture containing 0.02% polygalacturonic acid (PGA) in acetate buffer. The progress of the reaction is monitored by terminating aliquots of the reaction mixture (via heat treatment at appropriate times), subsequently adding poly(diallyldimethylammonium chloride) (PDADMAC) for turbidity development (approximately 30 min), and then measuring the turbidity (typically at 420 nm) of the resulting PGA-PDADMAC complex-containing solution. PGA depolymerase activity causes a decrease in the observed turbidity of PGA-PDADMAC-containing solutions because the stability of the interpolyelectrolyte PGA-PDADMAC complex is a function of the degree of polymerization of the PGA. The rate of turbidity change is herein shown to be proportional to a relatively wide range of enzyme concentrations. The assay is demonstrated using a commercial pectinase preparation and tomato fruit extracts.     

Enzymatic modification of a model homogalacturonan with the thermally tolerant pectin methylesterase from Citrus: 1. Nanostructural characterization, enzyme mode of action, and effect of pH

Methyl ester distribution in pectin homogalacturonan has a major influence on functionality. Enzymatic engineering of the pectin nanostructure for tailoring functionality can expand the role of pectin as a food-formulating agent and the use of in situ modification in prepared foods. We report on the mode of action of a unique citrus thermally tolerant pectin methylesterase (TT-PME) and the nanostructural modifications that it produces. The enzyme was used to produce a controlled demethylesterification series from a model homogalacturonan. Oligogalacturonides released from the resulting demethylesterified blocks introduced by TT-PME using a limited endopolygalacturonase digestion were separated and quantified by high-pressure anion-exchange chromatography (HPAEC) coupled to an evaporative light-scattering detector (ELSD). The results were consistent with the predictions of a numerical simulation, which assumed a multiple-attack mechanism and a degree of processivity ～10, at both pH 4.5 and 7.5. The average demethylesterified block size (0.6-2.8 nm) and number of average-sized blocks per molecule (0.8-1.9) differed, depending upon pH of the enzyme treatment. The mode of action of this enzyme and consequent nanostructural modifications of pectin differ from a previously characterized citrus salt-independent pectin methylesterase (SI-PME).     

Pectinesterase inhibitor from jelly-fig (Ficus awkeotsang Makino) achenes reduces methanol content in carambola wine

Crude pectinesterase (PE) inhibitor (PEI) extracted from jelly-fig achenes (JFA) (Ficus awakeosang Makino) was added to carambola (Averrhoa carambola L.) puree to determine the change in methanol production during fermentation. Addition of pectin or microbial pectic enzyme to puree increased dose-dependently the methanol content in fermented products. Decreasing ratio (from 1:0 to 1:19, v:v) of pectic enzyme to diluted crude PEI solution in the puree-enzyme mixture decreased the PE activity remarkably. Except for transmittance (%T), addition of crude PEI to puree did not affect apparently the physical and chemical properties of wine; however, it reduced methanol content in the control from 256 to 58 ppm. The degree of esterification (DE) of pectin in starting puree was approximately 70%. It decreased to approximately 27% in the control group and reduced slightly to approximately 67% in fermented puree with crude PEI added after 14 days of fermentation. This reveals that crude PEI solution was potent in inhibiting intrinsic carambola PE activity and appeared to be a potential alternative for methanol reduction in wines.     

Effects of cryostabilizers, low temperature, and freezing on the kinetics of the pectin methylesterase-catalyzed de-esterification of pectin

The kinetics of the pectin methylesterase (PME)-catalyzed de-esterification of pectin was studied at 25 degrees C in the presence of sucrose, fructose, maltodextrin (DE = 16.5-19.5), and carboxymethylcellulose at different concentrations and in the presence of maltodextrin and sucrose at different concentrations in a temperature range between +25 and -4 degrees C in subcooled and frozen states. The objective was to determine whether the reaction is diffusion-controlled, to gain insight about the factors determining the diffusion of the reactants, and to determine the effect of the carbohydrates, low temperature, and freezing on the structural conformation of the enzyme. The results indicate that the PME-catalyzed de-esterification of pectin is diffusion-controlled. Nevertheless, the diffusion is not controlled by the macroviscosity of the reaction medium, but rather by the microviscosity experienced by the diffusants. Low temperature in the temperature range studied does not affect the structural conformation of the enzyme, while freezing seems to have some effect.     

Solvent effects on the molecular properties of pectins

Measurements revealed that LiAc/HAc buffer, when compared with other solvents, gave relatively low values of turbidity for five commercial pectins with various apparent molar masses and degrees of methyl esterification (DE). Therefore, HPSEC with on-line light scattering and viscosity detection was employed to compare LiAc/HAc buffer against NaNO(3) solution, a commonly used mobile phase for measuring the molecular properties of these pectins and an additional pectin prepared by microwave extraction. Microwave-extracted pectin was included in the study for its higher molar mass and DE compared with commercial pectins. Most commercial samples were more soluble and had a higher molar mass when dissolved in NaNO(3) than in LiAc/HAc buffer, whereas the microwave-extracted pectin was more soluble in LiAc/HAc buffer and had about the same molar mass. Furthermore, association fragments of pectin contained in samples were more dissociated by LiAc/HAc buffer than by NaNO(3). For the samples studied, weight-average molar masses ranged from about 41000 to 307000, weight-average intrinsic viscosities from about 0.86 to 9.76 dL/g, z-average radii of gyration from about 13 to 45 nm, and Mark-Houwink constants from about 0.62 to 0.94.     

Monovalent salt-induced gelation of enzymatically deesterified pectin

Pectin gels were induced by monovalent salts (0.2 M) concurrently with deesterification of high methoxy pectin using a salt-independent orange pectin methylesterase (PME). Constant pH was maintained during deesterification and gelation. If salt or PME was absent, the pectin did not form a gel. The gel strength was influenced by both pH and species of monovalent cation. At pH 5.0, the pectin gel induced by KCl was significantly stronger than the NaCl-induced gel. In contrast, a much stronger gel was produced in the presence of NaCl as compared to KCl at pH 7.0. LiCl did not induce pectin gelation at either pH. Molecular weights of pectins increased from 1.38 x 10(5) to 2.26 x 10(5) during NaCl-induced gelation at pH 7. One proposal to explain these pectin molecular weight changes is a hypothetical PME transacylation mechanism. However, these pectin molecular weight changes can also be explained by metastable aggregation of the enzymatically deesterified low methoxy pectin. We postulate that gelation was induced by a slow deesterification of pectin under conditions that would normally salt out (precipitate) low methoxy pectin in the absence of PME.     

Priming effects: Interactions between living and dead organic matter

In this re-evaluation of our 10-year old paper on priming effects, I have considered the latest studies and tried to identify the most important needs for future research. Recent publications have shown that the increase or decrease in soil organic matter mineralization (measured as changes of CO₂ efflux and N mineralization) actually results from interactions between living (microbial biomass) and dead organic matter. The priming effect (PE) is not an artifact of incubation studies, as sometimes supposed, but is a natural process sequence in the rhizosphere and detritusphere that is induced by pulses or continuous inputs of fresh organics. The intensity of turnover processes in such hotspots is at least one order of magnitude higher than in the bulk soil. Various prerequisites for high-quality, informative PE studies are outlined: calculating the budget of labeled and total C; investigating the dynamics of released CO₂ and its sources; linking C and N dynamics with microbial biomass changes and enzyme activities; evaluating apparent and real PEs; and assessing PE sources as related to soil organic matter stabilization mechanisms. Different approaches for identifying priming, based on the assessment of more than two C sources in CO₂ and microbial biomass, are proposed and methodological and statistical uncertainties in PE estimation and approaches to eliminating them are discussed. Future studies should evaluate directions and magnitude of PEs according to expected climate and land-use changes and the increased rhizodeposition under elevated CO₂ as well as clarifying the ecological significance of PEs in natural and agricultural ecosystems. The conclusion is that PEs - the interactions between living and dead organic matter - should be incorporated in models of C and N dynamics, and that microbial biomass should regarded not only as a C pool but also as an active driver of C and N turnover.     

Separation and utilization of pectin lyase from commercial pectic enzyme via highly methoxylated cross-linked alcohol-insoluble solid chromatography for wine methanol reduction

The isolation and utilization of pectin lyase (PL) from commercial pectic enzyme for methanol reduction in wine production was investigated. PL can be separated from pectinesterase (PE) and polygalacturonase (PG) on HM-CL-AIS affinity chromatography at pH 4; however, it is difficult to further distinguish PE from PG. Some desirable physicochemical properties such as transmittance, lightness, redness, and lower total pectin content are found in the external enzyme adding groups (PL, PE and PG, and pectic enzyme groups) in comparison to the control group. Methanol contents in pectic enzyme and the PE and PG groups increase from 628 +/- 13 (control group) to 3103 +/- 16 and 1736 +/- 67 mg/L ethanol in the final products, respectively. Nevertheless, the adding of PL does not cause any increase in methanol content. The results present in this study suggest that the HM-CL-AIS column is a simple, inexpensive, convenient, and effective method for PL purification. Moreover, the partial purified PL is a potential replacement of commercial pectic enzyme for pectin depolymerizing, methanol content reducing, and wine quality improving in wine production.     

Physical and oxidative stability of fish oil-in-water emulsions stabilized with beta-lactoglobulin and pectin

The oxidation of fatty acids can be inhibited by engineering the surface of oil-in-water emulsion droplets to decrease interactions between aqueous phase prooxidants and lipids. The objective of this research was to evaluate whether emulsions stabilized by a multilayer emulsifier systems consisting of beta-lactoglobulin and citrus or sugar beet pectin could produce fish oil-in-water emulsions that had good physical and oxidative stability. Sugar beet pectin was compared to citrus pectin because the sugar beet pectin contains the known antioxidant, ferulic acid. A primary Menhaden oil-in-water emulsion was prepared with beta-lactoglobulin upon which the pectins were electrostatically deposited at pH 3.5. Emulsions prepared with 1% oil, 0.05% beta-lactoglobulin, and 0.06% pectins were physically stable for up to 16 days. As determined by monitoring lipid hydroperoxide and headspace propanal formation, emulsions prepared with the multilayer system of beta-lactoglobulin and citrus pectin were more stable than emulsions stabilized with beta-lactoglobulin alone. Emulsions prepared with the multilayer system of beta-lactoglobulin and sugar beet pectin were less stable than emulsions stabilized with beta-lactoglobulin alone despite the presence of ferulic acid in the sugar beet pectin. The lower oxidative stability of the emulsions with the sugar beet pectin could be due to its higher iron and copper concentrations which would produce oxidative stress that would overcome the antioxidant capacity of ferulic acid. These data suggest that the oxidative stability of oil-in-water emulsions containing omega-3 fatty acids could be improved by the use of multilayer emulsion systems containing pectins with low metal concentrations.     

Vacuum infusion of plant or fungal pectinmethylesterase and calcium affects the texture and structure of eggplant

The effect of vacuum infusion on eggplant quality of a commercial fungal (Aspergillus niger) and citrus pectinmethylesterase (PME) with calcium chloride (4000 ppm) was investigated after processing and during storage. Firmness of infused eggplants using fungal or citrus PME was significantly increased compared to controls (fresh noninfused and water-infused control) after processing and during storage for 7 days at 4 degrees C. Activity of fungal PME-infused eggplant increased almost 32 times, whereas activity of eggplant infused with Marsh grapefruit PME increased 2-fold. Degree of esterification of pectin of eggplants infused with fungal or citrus PME decreased slightly. Cryo-SEM showed that samples treated with fungal PME/ CaCl2 displayed more integrity among cells as compared with water-infused control. The change of pectin in the cell wall was visualized using monoclonal antibodies JIM5 (low-esterified pectin) and JIM7 (high-esterified pectin). JIM5 showed more binding than JIM7 with the cell walls of eggplant tissues from fungal PME/ CaCl2 treatment.