Enzymatically and chemically de-esterified lime pectins: characterisation, polyelectrolyte behaviour and calcium binding properties.

A series of pectins with different levels and patterns of methyl esterification was produced by treatment of a very highly methylated pectin with acid, alkali, plant pectin methyl esterase and fungus pectin methyl esterase. The intrinsic pK values, as well as the free fractions of monovalent and calcium counterions, were determined on pectin salt-free solutions. The variations of pK(a) versus the ionisation degree were found to depend on the de-esterification process but a unique value of 2.90+/-0.15 was estimated for the intrinsic pK value. Calcium binding properties of chemically and enzymatically de-esterified pectins were investigated and experimental results were compared to Manning's theoretical values. A progressive dimerisation process for pectins with a blockwise distribution of carboxyl groups in the presence of calcium ions is hypothesised.     

Impact of pectin esterification on the antimicrobial activity of nisin‐loaded pectin particles

The relationship between pectin structure and the antimicrobial activity of nisin‐loaded pectin particles was examined. The antimicrobial activity of five different nisin‐loaded pectin particles, i.e., nisin‐loaded high methoxyl pectin, low methoxyl pectin, pectic acid, dodecyl pectin with 5.4 and 25% degree of substitution were tested in the pH range of 4.0–7.0 by agar‐diffusion assay and agar plate count methods. It was found that the degree of esterification of carboxyl group of galacturonic acid in pectin molecule is important for the antimicrobial activity of nisin‐loaded pectin particles. Nisin‐loaded particles prepared using pectic acid or the pectin with low degree of esterification exhibit higher antimicrobial activity than nisin‐loaded high methoxyl pectin particles. Pectins with free carboxyl groups or of low degree of esterification are the most suitable for particles preparation. Moreover, nisin‐loaded pectin particles were active at close to neutral or neutral pH values. Therefore, they could be effectively applied for food preservation. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:245–251, 2017     

Intramolecular distribution of carboxyl groups in low methoxyl pectins — A review

Low methoxyl pectins (LMP) have been manufactured since the 1940s primarily for use as gelling agents. At the time, it was noted that low methoxyl pectates (LMPs) prepared by different methods had different gelling properties and this was attributed to the way in which the free carboxyl groups were distributed along the chain following deesterification. Various workers have shown that LMP prepared by enzymic deesterification is more heterogeneous with respect to the degree of esterification (DE) than LMPs of the same DE prepared by acid deesterification. This, together with enzyme studies, has been taken as a sign that pectinesterase works in a sequential fashion and, similarly, acid deesterification is a random process.More recently, the preparation of crude enzyme-deesterified LMP, which is used as a thickener in canned goods, has been described. Although enzyme-deesterified LMPs appear to form weak gels with calcium ions at room temperature (acid-deesterified LMP/calcium gels are reported to be stronger) they are superior when incorporated into canned goods which receive a severe heat treatment.This review briefly describes the preparation of LMP and that work on LMP which provides information about the distribution of carboxyl groups in the pectate molecule. Since it is established that this distribution affects the gelling properties of the pectin a sound understanding of the chemical aspects may assist in understanding the mechanism of gelation.     

Interchain heterogeneity of enzymatically deesterified lime pectins

Two series of pectins with different levels and patterns of methyl esterification were produced by treatment of a very highly methylated lime pectin with a fungus- or plant-pectin methylesterase. The interchain distribution of free carboxyl groups was investigated by size exclusion and ion exchange chromatography. "Homogeneous" populations with respect to molar mass or charge density were thereby obtained, and their composition, molar mass, and calcium binding properties were investigated. The composition varies from one size exclusion chromatography fraction to another, the highest molar mass fraction being richer in rhamnogalacturonic sequences and exhibiting a slightly higher degree of methylation (DM). Separation of pectins by ion exchange chromatography revealed a narrow charge density distribution for pectins deesterified by fungus-pectin methylesterase, in agreement with a multichain mechanism. Conversely, pectins deesterified by plant-pectin methylesterase exhibited a very large charge density distribution suggesting a processive mechanism. The interchain polydispersity with regard to DM was however shown to have no impact on calcium binding properties of the different fractions. The progressive dimerization through calcium ions with decreasing DM of pectins deesterified by plant-pectin methylesterase seems to be the result of a peculiar intrachain pattern of methyl esterification that can be attributed to a multiple attack mechanism.     

Structural studies of the solutions of anionic polysaccharides. IV. Study of pectin solutions by light-scattering

Aqueous solutions of pectins, with a degree of esterification (DE) varying from 0 to 95%, have been studied by light-scattering. A set of samples of similar molecular sizes were prepared by methylation of sodium pectate with diazomethane. The solutions were subjected to ultracentrifugation to ensure the removal of small amounts of gel fraction. The second virial coefficient of pectin was positive and constant for DE values varying between 43 and 95%, but increased by a factor of three when the DE was reduced to zero. This increase is at least partly due to coulombic electrostatic interactions. All of the pectins investigated behaved as semi-rigid-chain polymers. The chain flexibility was a maximum in the DE range 43 to 58%.In pectin solutions with a DE higher than 58% attractive forces exist between pectin macromolecules due to the presence of ester groups. It is expected that the interactions between ester groups contribute to both the chain rigidity and the gel-forming ability of pectins.     

Microcalorimetric studies on the lyophilic properties of pectic substances

The exothermic effects observed on wetting pectins with water and aliphatic alcohols were studied using a microcalorimeter.The heat released on wetting 1 g pectin with water was found to be 171 ± 7·5 J g^?1. It was experimentally established that 1 g of dry pectin exothermically bonded up to 0·57 g of water.By using the Gibbs-Helmholtz-Young equation which relates the heat released by wetting to the area of the wetted surface, it was estimated that the surface accessible to water in 1 g of pectin was 1·46 × 10³ m² g^?1. The heat of hydration was independent of the degree of esterification of the pectin. The experimental results revealed that there were about six molecules of energetically bonded water per monomer unit of pectin.A specific interaction between methanol and the methoxyl groups of pectin was observed on wetting pectins with methanol and dependence was established between the released heat and the degree of esterification. No similar dependence was reported for the remaining aliphatic alcohols.     

Complexation of ferric oxide particles with pectins of ordered and random distribution of charged units

Complexation between ferric oxide particles and pectins with degree of methylation 50% but having ordered or random arrangement of free carboxyl groups is investigated by electric light scattering and electrophoresis. The influence of charge distribution in pectin chain on the electrical properties of oppositely charged oxide particles and stability of their suspensions is examined as a function of pectin concentration. Although the difference in charge density of pectin samples is ~5%, we found small but measurable difference in the behavior of both oxide/pectin complexes. This is attributed to condensation of counterions near the chains of pectin with ordered distribution of charges, leading to a decrease in the effective charge density and to a corresponding decrease in the contour length of the adsorbing pectin chains. Two parameters are sensitive to the conformation of the adsorbed chains in suspensions, stabilized by pectin adsorption (at particle charge reversal). The electro-optical effect is higher for the complex with less charged pectin, which is explained with larger amount of chains, adsorbed in more coiled conformation than the chains of pectin with random distribution of free carboxyl groups. The addition of small amounts of CaCl(2) has no significant influence on the thickness of the layer from the less charged pectin, in agreement with a more compact conformation of the chains in this adsorbed layer. In contrast, the thickness of the layer from pectin with random distribution of charged groups decreases with increasing concentration of CaCl(2), indicating a more loose structure of this layer.     

Distribution of methoxyl groups in apple pectic substances

The distribution of methoxyl groups in apple pectic substances was investigated by means of fractionation on ion-exchange and gel-filtration columns and by means of degradation of pectin fractions by pectin lyase and pectate lyase. Pectin fragments thus obtained were fractionated by gel-permeation chromatography and high-pressure liquid chromatography. It was concluded that a heterogeneous intermolecular distribution of the methoxyl groups exists with peaks at degrees of esterification of about 50%, 70% and 95%. The intramolecular distribution of the methoxyl groups cannot be distinguished from a random distribution. Since plant pectin esterases cause a blockwise de-esterification, it is unlikely that the biosynthesis of apple pectic substances passes through a stage of 100% esterification after which partial de-esterification by pectin esterase occurs.     

Comparison of the structural features of apple and citrus pectic substances

Pectic substances were extracted from Alcohol Insoluble Solids from lemon peel (albedo) and fractionated by ion exchange chromatography and gelfiltration. The pectin molecules contained rhamnose, arabinose, galactose, glucose and galacturonic acid residues; xylose residues were almost absent. Degradation with purified pectolytic enzymes and subsequent gelfiltration of the resulting pectin fragments showed that the neutral sugar side chains were present in ‘hairy regions’ (blocks of neutral sugar side chains). The distribution of the methoxyl groups was studied by HPLC analysis of enzyme-degraded pectins. Some influence of native pectinesterase on the distribution of the methoxyl groups was found. The results are compared with those of similarly extracted and purified apple pectic substances.     

Quantification of blockiness in pectins—A comparative study using vibrational spectroscopy and chemometrics

The gelling properties of pectins are related not only to the degree of esterification (DE), but also to the distribution of the ester groups. In this study, we have examined an experimentally designed series of 31 pectins originating from the same mother pectin and de-esterified using combinations of two different enzymatic mechanisms. The potential of using infrared (IR), Raman, and near infrared (NIR) spectroscopies combined with chemometrics for reliable and rapid determination of the DE and distribution patterns of methyl ester groups in a designed set of pectin powders was investigated. Quantitative calibration models using partial least squares (PLS) regression were developed and compared. The calibration models for prediction of DE obtained on extended inverse signal correction (EISC)-treated spectra of all three spectroscopic methods yielded models with cross-validated prediction errors (RMSECV) between 1.1%p and 1.6%p DE and correlation coefficients of 0.99. A calibration model predicting degree of random de-esterification (R) and block de-esterification (B) was developed for each spectroscopic method, yielding RMSECV values between 4.4 and 6.7 and correlation coefficients (r) between 0.79 and 0.92. Variable selection using interval PLS (iPLS) significantly improved the prediction of R for IR spectroscopy, yielding RMSECV of 3.5 and correlation coefficients of 0.95. All three spectroscopic methods were able to distinguish the spectral patterns of pectins with different enzyme treatments in simple classification models by principal component analysis (PCA). Extended canonical variate analysis revealed one specific signal in the Raman (1045 cm⁻¹) spectrum and one significant area (1250-1400 cm⁻¹) in the IR spectrum which are able to classify the pectin samples according to the four different enzyme treatments. In both Raman and IR spectra, the signal intensity decreased in the sequence R-B > B > B-R > R > re-methylated pectin.     

An efficient procedure for studying pectin structure which combines limited depolymerization and 13C NMR

A protocol for partial thermally-induced depolymerization of differently methoxylated pectin samples is described. The resulting macromolecules have been fully characterized with various complementary techniques, such as size exclusion chromatography (SEC), potentiometry, viscometry and ¹³C NMR. Optimum conditions afford samples at 50–80% yield with weight-average molecular weights in the 4 to 20 kDa range. The major fraction of these polysaccharides adopts the random-coil conformation and such samples are suitable for ¹³C NMR structural studies at room temperature. The methoxyl distributions of two apple pectin samples with a degree of esterification (DE) between 54 and 74% and a citrus pectin (DE, 72%) were shown to be random in nature, whereas that of a lightly methoxylated apple pectin (DE 39%) was partially blockwise. The carbon relaxation parameters of the depolymerized pectins attain asymptotic values for M_W > 4 kDa. The M_W values estimated from intrinsic viscosity data with the Mark-Houwink relationship reported for native pectins are in good agreement with those obtained by either end-group analysis (NMR) or SEC. Thus, all the physicochemical data indicate that the secondary structure of the isolated chains of depolymerized pectin is closely related to that of the parent polymers. Finally, pectinmethylesterase activity towards the depolymerized pectins was similar to that of the untreated samples. Received: 10 July 1997 / Accepted: 12 November 1997     

Different action patterns for apple pectin methylesterase at pH 7.0 and 4.5

The mechanism of action of purified apple pectin methylesterase on pectin (degree of methoxylation: DM 75) and methoxylated homogalacturonans (DM 70 and 90) was studied at pH 7.0 (optimal pH of the enzyme) and at pH 4.5 (close to the pH of apple juice). Different interchain distributions of the free carboxyl groups were obtained at pH 7.0 and 4.5: high-performance ion exchange chromatography indicated a typical single chain mechanism at pH 7.0, but a mechanism differing from the single and multiple chain ones at pH 4.5. However, the same intrachain distribution of the newly demethoxylated galacturonic acid residues was observed for both pHs by 1H NMR. The high content of consecutive de-esterified or consecutive esterified galacturonic acid residues suggested that apple PME acted with a multiple attack mechanism on the pectic substrate. The degree of multiple attack of the enzyme was greater than or equal to 10-11.     

Functional characterization of the gels prepared with pectin methylesterase (PME)-treated pectins

High- and low-methoxyl pectins were treated with pectin methylesterase (PME) and the functional properties of the resulting pectin gels were characterized. The degree of esterification of high- and low-methoxyl pectins decreased from 74.5% to 6.3% and 40.0% to 6.5%, respectively while not changing their molecular weight. Also, the addition of glucono-delta-lactone (GDL) dramatically affected the gel strength and the pH reduction by the GDL led to the increased syneresis of the pectin gels, which was also observed in the PME-treated samples. When flavor compounds were incorporated into the pectin gels, the flavor release from the gels increased with decreasing the degree of esterification due to increased hydrophilic properties.     

Conformations and interactions of pectins: II. Influence of residue sequence on chain association in calcium pectate gels

In the accompanying paper (Morris et al., 1982) we present evidence of Ca²⁺-induced association of poly-d-galacturonate sequences from pectin into dimers of 2₁ chain symmetry, with co-operative (“egg-box”) binding of Ca²⁺ on specific sites along the interior faces of each chain. We now investigate the role in calcium pectate gel networks of other structural features, in particular methyl esterification and 1,2-linked l-rhamnosyl residues in the polymer backbone. Acid hydrolysis of citrus, apple and sunflower pectins gave polygalacturonate blocks with a relatively narrow molecular weight distribution, and average chainlength of ~25 residues in each case. Since the known relative stabilities of glycosidic linkages would lead to chain cleavage predominantly at l-rhamnose, this result indicates that the length of polygalacturonate sequences between rhamnose interruptions is approximately constant within and between the pectins studied. Calcium pectate gel strength is reduced dramatically by the incorporation of these chain segments when they are de-esterified, but not when they are esterified. This interference with the development of a network structure that resists applied stress, provides further support for our model of junction zone formation from sequences of contiguous deesterified residues, with Ca²⁺-mediated chain dimers providing the primary associations that can offer resistance to deformation.Samples with different levels and patterns of esterification were prepared by enzymic (blockwise) and chemical (random) de-esterification of almost fully methyl esterified pectin. In the former series, the extent of Ca²⁺ binding (as monitored by circular dichroism) increased almost linearly with the fraction of free carboxyl groups, whereas the latter showed a non-linear relationship of a form consistent with the requirement of this binding for blocks of contiguous non-esterified residues and, in the presence of excess univalent cations, binding was negligible when more than ~40% of the carboxyl groups were esterified. Statistical calculations of sequence length distribution at different degrees of random de-esterification show the best fit with experimental data when binding is assumed to require sequences with seven or more consecutive free carboxyl groups along the participating face of the chain. For 2₁ chain symmetry, this corresponds to a sequence length of 14 residues, in excellent agreement with previous independent studies of Ca²⁺ binding to oligogalacturonates.In the absence of competing univalent counterions, circular dichroism changes are similar in form but so large in magnitude that site-binding of Ca²⁺ must now go beyond the half-stoichiometry at which it is arrested in their presence. Ca²⁺ binding monitored by circular dichroism, and gel strength (yield stress) measured mechanically, both show a similar dependence on the pattern as well as the level of esterification, as expected for network formation by co-operative binding of Ca²⁺ within interchain junction zones.To fit this binding data quantitatively, it is necessary to postulate a two-stage process. (1) Initial dimerization, probably corresponding to the “strong associations” indicated by evidence from competitive inhibition (see above), for which a critical minimum sequence of seven residues is again required but esterified residues can now be accommodated within individual sites provided that they are paired with a free carboxylate on the complementary chain. (2) Subsequent Ca²⁺-induced aggregation of these preformed dimers, which can occur irrespective of the pattern of esterification on the external faces; the evidence from mechanical measurements shows that these contribute little to gel strength at high stress.     

Deficiency of adenosine kinase activity affects the degree of pectin methyl-esterification in cell walls of Arabidopsis thaliana

Pectin methyl-esterification is catalysed by S-adenosyl-l-methionine (SAM)-dependent methyltransferases. As deficiency in adenosine kinase (ADK; EC 2.7.1.20) activity impairs SAM recycling and utilization, we investigated the relationship between ADK-deficiency and the degree of pectin methyl-esterification in cell walls of Arabidopsis thaliana. The distribution patterns of epitopes associated with methyl-esterified homogalacturonan in leaves and hypocotyls of wild-type (WT) and ADK-deficient plants were examined using immunolocalization and biochemical techniques. JIM5 and LM7 epitopes, characteristic of low esterified pectins, were more irregularly distributed along the cell wall in ADK-deficient plants than in WT cell walls. In addition, epitopes recognized by JIM7, characteristic of pectins with a higher degree of methyl-esterification, were less abundant in ADK-deficient leaves and hypocotyls. Since de-esterified pectins have enhanced adhesion properties, we propose that the higher abundance and the altered distribution of low methyl-esterified pectin in ADK-deficient cell walls lead to the leaf shape abnormalities observed in these plants.     

Structural characterization, degree of esterification and some gelling properties of Krueo Ma Noy (Cissampelos pareira) pectin

Pectins extracted from Krueo Ma Noy (Cissampelos pareira) leaves mainly consisted of galacturonic acid with trace amount of neutral sugars. The dominant structure of Krueo Ma Noy pectin was established as a 1,4-linked -D-galacturonan by a combination of carboxyl reduction and methylation analysis, and confirmed by FT-IR spectroscopy. The degree of esterification of Krueo Ma Noy pectins was 41.7 and 33.7% for crude and dialyzed pectins, respectively. Krueo Ma Noy pectin has an average molecular weight of 55 kDa, radius of gyration of 15.2 nm and intrinsic viscosity of 2.3 dl/g. Krueo Ma Noy pectin exhibited gelling properties in aqueous solutions at 0.5% (w/v) at 5 °C. Gels were formed at concentrations of 1.0% (w/v) and above even at room temperature. The gel strength, melting point, and melting enthalpy of Krueo Ma Noy pectin increased with polysaccharide concentration.     

Pectin Esterification Degree in the Bioavailability of Non-heme Iron in Women

Pectins are a type of soluble fiber present in natural and processed foods. Evidence regarding the effect of esterification degree of pectins on iron absorption in humans is scarce. In the present study, the effect of pectins with different degrees of esterification on non-heme iron absorption in women was evaluated. A controlled experimental study was conducted with block design, involving 13 apparently healthy, adult women. Each subject received 5 mg Fe (FeSO₄) without pectin (control) or accompanied by 5 g citrus pectin, two with a low degree of esterification (27 and 36%), and one with a high degree of esterification (67 to 73%), each on different days. Each day, the 5 mg Fe doses were marked with radioactive ⁵⁹Fe or ⁵⁵Fe. Radioactivity incorporated into erythrocytes was determined in blood samples 14 days after the marked Fe doses were consumed. On days 18 and 36 of study, 30 and 20 mL blood samples were obtained, respectively, and blood sample radioactivity incorporated into erythrocytes was determined. Body iron status was determined from blood taken on day 18. Whole body blood volume was estimated for calculate iron bioavailability; it was assumed that 80% of absorbed radioactivity was incorporated into the Hb. All women participants signed an informed consent of participation at baseline. Iron bioavailability (mean geometric ±1 SD) alone (control) was 18.2% (12.3–27.1%), iron + pectin27 was 17.2% (10.2–29.2%), iron + pectin36 was 15.3% (9.5–24.6%), and iron + pectin67 was 19.5% (10.0–38.0%). No statistically significant differences between iron bioavailability (repeated measures ANOVA, p = 0.22) were observed. Pectin esterification degree does not influence the bioavailability of non-heme iron in women.     

Environmentally friendly preparation of pectins from agricultural byproducts and their structural/rheological characterization

Apple pomace which is the main waste of fruit juice industry was utilized to extract pectins in an environmentally friendly way, which was then compared with chemically-extracted pectins. The water-based extraction with combined physical and enzymatic treatments produced pectins with 693.2 mg g⁻¹ galacturonic acid and 4.6% yield, which were less than those of chemically-extracted pectins. Chemically-extracted pectins exhibited lower degree of esterification (58%) than the pectin samples obtained by physical/enzymatic treatments (69%), which were also confirmed by FT-IR analysis. When subjected to steady-shear rheological conditions, both pectin solutions were shown to have shear-thinning properties. However, decreased viscosity was observed in the pectins extracted by combined physical/enzymatic methods which could be mainly attributed to the presence of more methyl esters, thus limiting polymer chain interactions. Moreover, the pectins which were extracted by combined physical/enzymatic treatments, showed less elastic properties under high shear rate conditions, compared to the chemically-extracted pectins.     

Some cultural conditions for maximum bioextraction of beet pulp pectin

The bioextraction of the beet pulp pectin by Kluyveromyces marxianus was inhibited by ferrous sulphate penta hydrate and potassium dihydrogen phosphate, but stimulated by magnesium sulphate hepta hydrate salt. The pectin yields were also influenced by the addition of some enzymatic activators and some natural additives such as yeast extract.

The characterization of both microbiologically and chemically extracted pectin samples indicated that the former had higher percentages of galacturonic acid, methoxyl groups and a higher degree of esterification and thus possesses superior qualities to chemically-extracted pectin.     

Characterization of pectin methyltransferase from soybean hypocotyls

Pectin methyltransferase (PMT) catalyzing the transfer of the methyl group from S-adenosyl-L-methionine (SAM) to the C-6 carboxyl group of galactosyluronic acid residues in pectin was found in a membrane preparation of etiolated hypocotyls from 6-d-old soybean (Glycinemax Merr.). The enzyme was maximally active at pH 6.8 and 35–40 °C, and required 0.5% (w/v) Triton X-100. The incorporation of the methyl group was significantly enhanced by addition of a pectin with a low (22%) degree of methyl-esterification (DE) as exogenous acceptor substrate. The apparent Michaelis constants for SAM and the pectin (DE22) were 0.23 mM and 66 μg · ml⁻¹, respectively. Attachment of the methyl group to the carboxyl group of the pectin via ester linkage was confirmed by analyzing radiolabeled product from incubation of the enzyme with [¹⁴C]methyl SAM and the acceptor pectin. Size-exclusion chromatography showed that both enzymatic hydrolysis with a pectin methylesterase and a mild alkali treatment (saponification) led to the release of radioactive methanol from the product. Enzymatic hydrolysis of the product with an endopolygalacturonase degraded it into small pectic fragments with low relative molecular mass, which also supports the idea that the methyl group is incorporated into the pectin. The soybean hypocotyls were fractionated into their cell wall components by successive extraction with water, EDTA, and alkali treatment. Among the resulting polysaccharide fractions, high PMT activity was observed when a de-esterified polysaccharide derived from the EDTA-soluble fraction (the pectic fraction) was added as an alternative acceptor substrate, indicating that the enzyme may be responsible for producing methyl-esterified pectin in vivo. Received: 10 September 1999 / Accepted: 11 October 1999