Carrot Pectin Methylesterase Research Articles

Interactions between citrus pectin and Zn2+ or Ca2+ and associated in vitro Zn2+ bioaccessibility as affected by degree of methylesterification and blockiness

Insight into the effect of the degree and pattern of methylesterification of citrus pectin on the Ca2+ and Zn2+ adsorption capacity and the associated Zn2+ bioaccessibility was established. Pectins with comparable methylesterification degrees (DM) but different patterns of methylester distribution (blockwise or random) were generated. Blockwise distributions of methylesters were produced through controlled enzymatic means (using carrot pectin methylesterase) while random patterns were obtained through alkaline demethylesterification (using NaOH). The pattern of methylesterification was estimated as the absolute degree of blockiness (DBabs), which is the ratio of non-methylesterified units present in blocks to the total number of galacturonic acid units. Determination and modeling (based on the Langmuir adsorption model) of the adsorption isotherms of these structurally modified pectins for Zn2+ or Ca2+ allowed quantification of their maximum cation binding capacity and interaction energy. Decreasing pectin DM and increasing DBabs were shown to promote the Zn2+ or Ca2+ binding capacity of pectin, with the maximum binding capacity being mainly determined by the DM and interaction energy by DBabs. The influence of cation type was evident, with higher maximum binding capacities and interaction energies exhibited for Zn2+ compared to Ca2+, mainly due to the higher electronegativity of the former. Increased Zn2+ binding to pectin was associated with lower Zn2+ bioaccessibility and no influence of low levels of added Ca2+ was observed. However, enzymes and bile salts added during in vitro simulated digestion were shown to bind substantial amounts of Zn2+ ions.

Pectin nanostructure influences pectin-cation interactions and in vitro-bioaccessibility of Ca2+, Zn2+, Fe2+ and Mg2+-ions in model systems

This study explores the effect of pectin structure on mineral bioaccessibility in model systems. Commercial citrus and sugar beet pectin (CP and SBP, respectively) were demethylesterified by the action of carrot pectin methylesterase and characterisation of the resulting samples revealed different degrees of methylesterification (DM). Additionally, SBP exhibited a higher protein content, degree of acetylation, degree of feruloylation, degree of branching and molar mass compared to CP. In vitro-simulated digestion (stomach and small intestine phases) of mineral-enriched pectin solutions showed that decreasing pectin DM results in a decrease in mineral-bioaccessibility due to pronounced cation-pectin interactions. For a given DM, SBP digests exhibited higher mineral-bioaccessibilities than CP digests possibly due to structural differences of pectin in these sources. Furthermore, increasing the mineral-enrichment prior to in vitro-digestion resulted in a higher mineral-bioaccessibility while increasing the pectin concentration decreased the mineral-bioaccessibility. Generally, Mg2+-ions were the most bioaccessible followed by Ca2+, Zn2+, then Fe2+-ions.

Evaluation of cation-facilitated pectin-gel properties: Cryo-SEM visualisation and rheological properties

To provide more insight into the cation facilitated pectin-gel structure-function relationship, the microstructure of pectin-gels was explored using cryo-scanning electron microscopy (Cryo-SEM) in combination with physiochemical and rheological analysis. Pectin with different degrees of methylesterification, DM, was obtained from commercial citrus pectin (CP) and sugar beet pectin (SBP) by action of carrot pectin methylesterase. Pectin characterisation confirmed that pectin properties other than DM were unaltered and pectin from the two sources exhibited different structures. Cryo-SEM visualisation of pectin-gels prepared with different cations revealed that the network density increased with decreasing DM and increasing pH, pectin and cation-concentration. At a given DM, the more linear and less acetylated pectin (CP) exhibited a more intertwined fibrous network compared to the highly branched and more acetylated pectin (SBP). Furthermore, gel-microstructure was influenced by type of cation with Zn2+, Fe2+ and Ca2+-pectin gels showing a more intertwined network compared to Mg2+-pectin gels. Finally, rheological properties of gels complementing Cryo-SEM observations showed that as the network density increased, the gel-stiffness (G′) increased.

Effect of de-methylesterification on network development and nature of Ca 2+-pectin gels: Towards understanding structure–function relations of pectin

Pectins of varying degree and pattern of methylesterification were produced through controlled de-esterification of highly esterified citrus pectin, using carrot pectin methylesterase (PME) (P-pectins), Aspergillus aculeatus PME (F-pectins) or sodium hydroxide saponification (C-pectins). Estimation of the degree of methylesterification (DM) and quantification of the pattern of methylester distribution in terms of absolute degree of blockiness (DB abs) enabled the characterisation of pectins. Characterised pectins were used for the preparation of Ca 2+-pectin gels with varying calcium ion (Ca 2+) concentration. The rheological characteristics of produced gels were evaluated by means of small-amplitude oscillatory tests. During gel formation, gel strength was monitored so as to allow assessment of network development. Based on the evaluation of mechanical spectra, the nature of the cured gels was established. Depending on Ca 2+ concentration as well as DM and DB abs, gels prepared from specific C-pectins (48 ≥ DM ≥ 26%; 9 ≤ DB abs ≤ 37%) and F-pectins (64 ≥ DM ≥ 29%; 9 ≤ DB abs ≤ 50%) showed a striking decrease of the gel strength with time, while gel networks produced from other pectins either displayed a continuous increase of the gel strength or verged towards pseudo-equilibrium within the observation time of 5 h. Furthermore, the DM, DB abs and Ca 2+ concentration influenced the evolution of Ca 2+-pectin networks from “structured liquids” to “strong gels”. Based on the experimental results, specific mechanisms of Ca 2+ interactions with pectins were (also) considered.

Effect of debranching on the rheological properties of Ca 2+–pectin gels

Water-soluble pectin (WSP) extract was subjected to controlled carrot pectin methylesterase treatment, thereby producing de-esterified pectin (DEP). Both WSP and DEP were incubated with a mixture of endo-arabinanase and α-L-arabinofuranosidase to yield partially debranched pectins (WSP DBr and DEP DBr respectively). Pectin samples were characterised in terms of degree of methylesterification (DM), neutral sugar content, and degree of branching (DBr). The characterised pectins were used for the preparation of pectin gels with high and low calcium ion (Ca 2+) concentrations. The rheological characteristics of the produced gels were evaluated by means of small-amplitude oscillatory tests. These characteristics include network development of the gel, gel strength (G′), gel elastic character and gel type. Partial debranching of pectin resulted in a reduction of the arabinose content (by approximately 50%) and caused a slight decrease in polymer DBr. Gels produced from semi-dilute solutions of partially debranched pectins showed network development profiles similar to those prepared from semi-dilute solutions of the corresponding non-debranched polymers. Yet, the former gels showed lower G′ values, poor gel elastic character and a “weaker” nature as compared to the latter. Next to Ca 2+ cross-links, the presence of long arabinose-containing side chains is suggested to play an important role in the rheological characteristics of Ca 2+–pectin gels.

Size Exclusion Chromatography To Gain Insight into the Complex Formation of Carrot Pectin Methylesterase and Its Inhibitor from Kiwi Fruit As Influenced by Thermal and High-Pressure Processing

A size exclusion chromatography (HPSEC) method was implemented to study complex formation between carrot pectin methylesterase (PME) and its inhibitor (PMEI) from kiwi fruit in the context of traditional thermal and novel high-pressure processing. Evidence was gained that both thermal and high-pressure treatments of PME give rise to two distinct enzyme subpopulations: a catalytically active population, eluting from the size exclusion column, and an inactive population, aggregated and excluded from the column. When mixing a partly denatured PME sample with a fixed amount of PMEI, a PME-PMEI complex peak was observed on HPSEC, of which the peak area was highly correlated with the residual enzyme activity of the corresponding PME sample. This observation indicates complex formation to be restricted to the active PME fraction. When an equimolar mixture of PME and PMEI was subjected to either a thermal or a high-pressure treatment, marked differences were observed. At elevated temperature, enzyme and inhibitor remained united and aggregated as a whole, thus gradually disappearing from the elution profile. Conversely, elevated pressure caused the dissociation of the PME-PMEI complexes, followed by a separate action of pressure on enzyme and inhibitor. Remarkably, PMEI appeared to be pressure-resistant when compressed at acidic pH (ca. 4).

Development and evaluation of monoclonal antibodies as probes to assess the differences between two tomato pectin methylesterase isoenzymes

The enzyme pectin methylesterase (PME) was purified from red ripe tomatoes ( Lycopersicon esculentum) and through affinity chromatography two isoenzymes were fractionated (t1PME and t2PME). Further analysis of these two isoenzymes, both having a molar mass of 34.5 kDa, revealed a difference in the N-terminal sequence and in amino acid composition. t1PME was identified as the major isoenzyme of PME in tomato fruit. In this study the aim was to develop a toolbox, consisting of monoclonal antibodies, that allows to gain insight into the in situ localization of PME in plant based food systems like tomatoes. A panel of six interesting monoclonal antibodies was raised against both isoenzymes, designated MA-TOM1-12E11, MA-TOM1-41B2, MA-TOM2-9H8, MA-TOM2-20G7, MA-TOM2-31H1 and MA-TOM2-38A11. The differences in epitopes between these monoclonal antibodies were determined using affinity tests towards denatured PME, cross-reactivity tests and inhibition tests. Characterization of these antibodies indicated an immunological difference between t1PME and t2PME, also revealing a conserved epitope on t2PME, carrot PME and strawberry PME. Different epitopes are recognized by the generated antibodies making them excellent probes for immunolocalization of PME by tissue printing. In tomato, t1PME and t2PME showed a pronounced co-localization, especially in the pericarp and the radial arms of the pericarp. Three of the generated antibodies could be used for immunolocalization of PME in carrots ( Daucus carota L.), which was only present in the cortex and not in the vascular cylinder of carrots.

Extraction and characterisation of pectin methylesterase from black carrot (Daucus carota L.)

This study was carried out to determine some of the biochemical properties of pectin methylesterase (PME) from black carrot. The enzyme showed very high activity in a broad pH range of 6.5–8.5, with the optimum pH occurring at 7.5. The optimum temperature for maximal PME activity was found to be 55 °C. NaCl enhanced PME activity, particularly at 0.2 M. Km and Vmax values for black carrot PME using apple pectin as substrate were found to be 2.14 mg/ml (r2 = 0.988) and 3.75 units/ml, respectively. The enzyme was stable between the temperatures of 30–50 °C/5 min whereas it lost nearly all of its activity at 70 °C/5 min. Ea and Z values were found to be 196.8 kJmol−1 (r2 = 0.996) and 2.16 °C (r2 = 0.995), respectively.

Carrot pectin methylesterase and its inhibitor from kiwi fruit: Study of activity, stability and inhibition

Abstract Carrot pectin methylesterase (PME) and its inhibitor (PMEI) from kiwi fruit were successfully purified by affinity chromatography. Enzyme and inhibitor activity and stability and PME–PMEI complex formation, as influenced by intrinsic product factors (pH and NaCl) and extrinsic process factors (temperature and pressure), were studied. The effect of temperature- or pressure-induced denaturation of PME and PMEI on their respective activities was assessed by estimating inactivation kinetic parameters. PME inactivation obeyed first-order kinetics. The enzyme was rather heat-labile but pressure-stable. PMEI inactivation was best described by a model taking into account a processing-stable PMEI intermediate. The behavior of PME and the PME–PMEI complex at elevated temperature or pressure in the presence of pectin was explored by following methanol formation as a function of treatment time. PME catalytic activity was stimulated up to a certain temperature or pressure level before declining. No conclusive evidence was obtained for a temperature-induced dissociation of the PME–PMEI complex, whereas high pressure exposure caused the complex to separate. Industrial relevance PME activity control is a major point of interest in the quest of obtaining high quality plant-derived food products. The current study demonstrates that both traditional thermal processing and novel high hydrostatic pressure processing allow stimulation as well as inactivation of PME and, hence, directing the PME-catalyzed pectin hydrolysis. An alternative or additional approach to control endogenous PME activity (e.g. to obtain cloud-stable juices) is through enzyme inhibition using kiwi PMEI. In this context, pH and NaCl boundaries for application were established, the existence of a temperature- and pressure-stable PMEI intermediate was shown and the PME–PMEI complex was proven not to be dissociated at mild temperature and pressure levels. These observations endorse the possibility of inhibiting undesirable PME activity remaining after mild processing.

Model based process design of the combined high pressure and mild heat treatment ensuring safety and quality of a carrot simulant system

In this research the combined mild heat and high pressure (HP) treatment of two food processing targets is under study: one microbiological safety target (Escherichia coli K12) and one quality related target (carrot Pectin Methylesterase, PME). A polynomial non-monotonous model structure fulfilling a number of constraints was identified for describing the log-linear inactivation kinetics of E. coli (at T=5–45°C and P=200–500MPa). Similarly, a polynomial non-monotonous model structure is used in order to describe the evolution of the carrot PME inactivation kinetics at T=10–65°C and P=0.1–825MPa. Iso-rate contour plots are constructed integrating the microbial and enzymatic kinetics for the combined T and P treatments. Additionally, the effect of the pressure build-up time (specific to the experimental set-up at hand) on the processing targets was quantified based on the microbial and enzymatic activity load before the initiation of the experiment and after the stabilisation of the treatment conditions. When T–P kinetic diagrams were constructed with combinations of treatments (also at extrapolation regions) resulting in the same log reductions, i.e., iso-reduction contour plots, it was evident that carrot PME was more resistant than E. coli. According to the analysis of the T–P diagrams (incorporating the pressure build-up processing effects), a thermal process in a range of 55–80°C, and a combined low temperature (30–50°C)–high pressure (700–800MPa) process revealed to be equivalent.

Combined thermal and high pressure effect on carrot pectinmethylesterase stability and catalytic activity

Carrot pectinmethylesterase (PME, EC 3.1.1.11) was extracted and purified using affinity chromatography. The effect of pressure and temperature on the stability and catalytic activity of carrot PME was studied for model systems as well as shredded carrots. The purified enzyme was characterized by an estimated molecular weight of 32kDa and a pI above 9.3. At atmospheric pressure, carrot PME showed an optimal pH of 8.0 at 22.5°C. The enzyme was rather heat labile (inactivation above 50°C), however, it showed a notable pressure resistance (up to 600MPa) especially at low temperatures (<40°C). The catalytic activity of carrot PME was highly dependent on the temperature and pressure applied. In model and food systems (shredded carrots), optimal PME activity was registered at 50°C in combination with pressures of about 300–500MPa. Intact tissues revealed a pronounced PME activity at 60°C at all pressures studied.

Inactivation of plant pectin methylesterase by thermal or high intensity pulsed electric field treatments

A comparative study between new and traditional food preservation technologies was performed on pectin methylesterase (PME) inactivation from different plant sources. PME was extracted from carrots, tomatoes, bananas and oranges and was purified by affinity chromatography. Its inactivation was investigated during high intensity pulsed electric fields (HIPEF) and thermal treatments. Thermal treatment was performed at temperatures from 54 °C to 81 °C and up to 120 min treatment time. PME inactivation was adequately described by a first-order kinetic model for PME from bananas, carrots and tomatoes, whereas a biphasic model described adequately thermal inactivation of orange PME. The thermal stable fraction of orange PME was the least sensible to heat and carrot PME was the most thermally sensitive. HIPEF treatment consisted of 40-μs-square-wave pulses applied up to a total treatment time of 1.6 ms at 0.5 or 5 Hz and an electric field between 13.2 and 19.1 kV/cm. The higher the electric field, total treatment time or pulse frequency the higher the degree of PME inactivation from all sources. Maximum enzyme inactivation was: 87% for orange and tomato PME, 83% for carrot PME and 45% for banana PME at the most intense conditions. Industrial relevance Increasing studies show that using high intensity pulsed electric fields technology for fruit juices pasteurisation is feasible in the near future. A critical point on the fruit juice production and preservation is the pectin methyl esterase activity control. Consequently, the effect of HIPEF on PME activity has a great industrial relevance. This paper shows high inactivation percentages of PME from different plant sources with a HIPEF treatment carried out at moderate temperatures (55-65 °C) and offers an attractive alternative to the traditional heat treatment.

Thermal and high-pressure inactivation kinetics of carrot pectinmethylesterase: from model system to real foods

Pectinmethylesterase (PME) was extracted from carrots ( Daucus carrota) and subsequently purified by affinity chromatography on a CNBr-Sheparose-PME inhibitor (PMEI) column. Detailed kinetic studies on the inactivation of purified carrot PME by thermal and high-pressure processing were performed. Next to the model systems, the thermal and high-pressure inactivation of PME in real products (carrot juice and carrot pieces) was investigated. The inactivation was performed under isothermal and isothermal–isobaric conditions. Under all conditions investigated, a first-order kinetic model could describe the inactivation data. It was found that carrot PME is much more thermostable and pressure-stable in carrot pieces than in carrot juice or purified form.

Mild‐Heat and High‐Pressure Inactivation of Carrot Pectin Methylesterase: A Kinetic Study

ABSTRACT: Pectin methylesterase (PME) was extracted from carrots and purified by affinity chromatography. The thermal high‐pressure inactivation of the PME was investigated in a model system. Under these conditions, the (thermo) stable fraction is not inactivated and the isobaric‐isothermal inactivation followed a fractional‐conversion model. At lower pressures (&lt; 300 MPa) and higher temperatures (&gt; 50°C), an antagonistic effect of pressure and heat was observed. A 2nd‐ and 3rd‐degree polynomial model (derived from available thermodynamic model) was successfully used to describe the heat pressure dependence of the inactivation rate constants. From the purified carrot PME sample, the thermostable PME fraction was isolated. The thermal inactivation of this fraction followed first‐order kinetics.

Partial purification, characterization, and thermal and high-pressure inactivation of pectin methylesterase from carrots (Daucus carrota L.).

Pectin methylesterase (PME) from carrots (Daucus carrota L.) was extracted and purified by affinity chromatography on a CNBr-Sepharose 4B-PME inhibitor column. A single protein and PME activity peak was obtained. A biochemical characterization in terms of molar mass (MM), isoelectric points (pI), and kinetic parameters of carrot PME was performed. In a second step, the thermal and high-pressure stability of the enzyme was studied. Isothermal and combined isothermal-isobaric inactivation of purified carrot PME could be described by a fractional-conversion model.

Characterization of carrot pectin methylesterase.

The most alkaline form of pectin methylesterase was purified from ripe carrot roots and used for structural analysis. Determination of an N-terminal blocking group and of the primary structure allowed comparisons with other forms, and facilitated crystallographic determination of the three-dimensional structure. The mature enzyme has 319 residues and the N-terminal blocking group was shown to be a pyroglutamyl residue derived from a glutaminyl cyclization. Few other methylesterases have been isolated and assigned to exact mature forms, and together with the present enzyme, only two have been analyzed in three-dimensional structure. However, comparison of 39 forms, mainly from GenBank data, reveals clear relationships and identifies subgroups of this enzyme type, deviating in structure but centering around two functionally important and conserved Asp residues at positions 136 and 157 in the carrot enzyme.