Plant pectin methylesterase and its inhibitor from kiwi fruit: Interaction analysis by surface plasmon resonance

Two surface plasmon resonance (SPR)-based interaction analysis methods were successfully implemented to explore the binding between plant PME and kiwi PMEI. In a first method, plant PMEs were immobilised on a chip surface via amine coupling. This experimental setup allowed studying the effect of pH and ionic strength on the PME–PMEI interaction kinetics. Strong binding was obtained at pH < 7 and at low salt concentrations, whereas both pH ? 8 and [NaCl] of ca. 1.0 M effectively caused dissociation. In a second method, kiwi PMEI was immobilised on a chip surface to which streptavidin had been covalently attached. Hereto, PMEI was biotinylated by means of a NHS-biotin reagent. With this immobilisation strategy, the effect of (partial) thermal or high pressure-induced denaturation of PME on its affinity towards PMEI was investigated. A notable degree of enzyme inactivation was required before interaction characteristics were significantly altered. Any incomplete inactivation of PME resulted in binding to the PMEI surface.     

Process stability of Capsicum annuum pectin methylesterase in model systems, pepper puree and intact pepper tissue

Process stability studies towards temperature and/or pressure on pepper pectin methylesterase (PME) were carried out in different systems (purified form, crude extract, pepper pieces and puree) at pH 5.6. Within the temperature range studied (22–80 °C, 5 min), pepper PME in pure form and crude extract was gradually inactivated showing a biphasic inactivation behaviour, indicating the presence of isoenzymes of different thermostability. Pepper samples heated for 15 min showed a maximum of residual PME activity around 55 °C. Isothermal inactivation of pepper PME in purified form and crude extract at pH 5.6 could be described by a biphasic inactivation model for the temperature range studied (62–76 °C). A stable behaviour towards high-pressure/temperature treatments (400–800 MPa/25–60 °C) was observed for crude extract and purified pepper PME. PME in pepper puree samples revealed to be very pressure stable. Mild temperatures combined with pressure treatments seem to increase the extractability from PME in pepper tissue, probably due to the effect on the cell structure.     

Inhibition of tomato pectin methylesterase by partially purified kiwi pectin methylesterase inhibitor protein

The inhibition of tomato pectin methylesterase (PME) by a recently discovered kiwi pectin methylesterase inhibitor (PMEI) is described. PME was consequently purified by CM Sephadex C-50, Concanavalin A-Sepharose 4 B and Mono S chromatography, and PMEI by Q-Sepharose and Sephacryl S-200 chromatography. Inhibition of tomato PME activity under optimal conditions (0.125 m NaCl, pH 7.5) by partially purified kiwi PMEI (MW of 27 kD, pI ≥ 3.67) was independent of the PMEI/PME ratio between 36 and 61% of the maximal uninhibited activity. the non-competitive inhibition observed was optimal in the pH range of 5 to 7. PMEI was inactivated by heating to 120°C, and showed actinidin-like activity towards N-α-benzyloxycarbonyl-L-lysine p -nitrophenyl ester (CBZ-lys-ONp) and azocasein which was partially inhibited by the protease inhibitor leupeptin.     

Combined osmodehydration and high pressure processing on the enzyme stability and antioxidant capacity of a grapefruit jam

A combined osmodehydration process and high pressure treatment (OD–HHP) was developed for grapefruit jam preservation. The inactivation kinetics of pectin methylesterase (PME) and peroxidase (POD) in the osmodehydrated (OD) jam treated by combined thermal (45–75 °C) and high pressure (550–700 MPa) processes were fitted using special cases of first-order kinetics, the fractional conversion and biphasic models and the Weibull distribution function. No complete inactivation was achieved by any combination of temperature and pressure, and 27–40% and 51–70% of PME and POD, respectively, were pressure-stable fractions. Two PME fractions with different pressure stabilities were observed and kinetic models successfully explained that behavior. POD was found to be very baroresistant and only the labile fraction could be inactivated. The extent of enzyme inactivation was lower in the OD jam in comparison with other food matrices showing a protective effect against the high pressure treatment. The antioxidant capacity was not affected by any treatment. The proposed high pressure preservation processing was able to improve the enzymatic stability of jam obtained by osmotic dehydration without affecting the bioactive content.     

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.     

超高压处理对鲜橙汁中果胶酶及过氧化物酶活性的影响

为推进国内非冷冻浓缩橙汁加工业的发展,对新型超高压杀菌技术对橙汁中酶钝化的效果进行研究,采用200～600MPa超高静压处理鲜榨橙汁,使用紫外分光光度法、滴定法分别测定鲜橙汁中过氧化物酶(POD)和果胶酶(PME)的活性,进行两种酶在常温下超高压钝化酶一级动力学拟合研究。结果表明：在常温条件下200MPa处理10min使两种酶轻微激活,在300～600MPa条件下,随压力和处理时间的增大,两种酶钝化反应明显,且符合一级动力学模型,且果胶酶对压力钝化更加敏感。     

A review of pectin methylesterase inactivation in citrus juice during pasteurization

BackgroundPectin, naturally found in citrus, plays a key role in the quality of the obtained juices. Pectin methylesterase enzyme (PME) influences the cloud stability, viscosity, color, mouth feeling and flavor of the juices by de-esterification of pectin. Iinactivation of PME is introduced as a pasteurization index in citrus juices, due to its higher thermal resistance than the spoilage microorganisms.Scope and approachInactivation of PME using different thermal (conventional, microwave and ohmic heating) and non-thermal (pulsed electric field, high pressure processing and high pressure carbon dioxide) processes is important in juice production. The aim of this study was to review the effect of these processing methods on the PME inactivation in different citrus juices.Key finding and conclusionUsing non-thermal methods in combination with moderate thermal methods can be more effective in PME inactivation with minimum loss in citrus juice quality.     

Kinetics of Tomato Pectin Methylesterase Inactivation by Temperature and High Pressure

ABSTRACT: In this study we investigated the inactivation of endogenous pectin methylesterase (PME) in tomato juice during combined high-hydrostatic pressure (ambient to 800 MPa) and moderate temperature (60 to 75 °C) treatments under isobaric and isothermal processing conditions. PME inactivation rates increased with increasing processing temperature, with the highest rate obtained during processing at 75 °C and ambient pressure. Inactivation rates were dramatically reduced as soon as processing pressure was raised. High inactivation rates were again attained when processing pressure exceeded a value of about 700 MPa. Such a behavior was described by considering two parallel mechanisms of inactivation, each one following first order kinetics with its own kinetic parameters.     

Freezing of strawberries by immersion in CaCl2 solutions

The present investigation studied the freezing of strawberries by immersion in CaCl₂ solutions, analysing drip loss, pectin content and the degree of esterification of the pectins, total and cell-wall bonded calcium contents, the ratio bonded calcium/total calcium, and textural parameters. In addition, the effect of immersion in pectin methylesterase (PME) solutions prior to immersion freezing (IF) was analysed. The firmness of thawed fruit decreased by approximately 74% with respect to fresh strawberries, and neither IF-CA (freezing by immersion in CaCl₂ solution) nor PME–IF-CA (immersion in PME solution + freezing by immersion in CaCl₂ solution) provided significant benefit in maintaining firmness when compared to slow freezing (SF). However, IF provided a significant benefit in reducing drip loss of thawed strawberries when compared to SF, but pre-treatment with PME did not provide any additional benefit.     

High-pressure treatment of cloudy apple juice

A factorial design with four factors (pressure, holding time, temperature and waiting period between crushing of apple and high-pressure (HP) treatment) was built up to study the effect of HP treatment on pectin methylesterase activity (PME, EC 3.1.1.11) in cloudy apple juice. PME activity was measured by the methanol release during the storage of samples at 4 °C for 1 month. Only the holding time of the treatment and three interactions between factors (holding time×temperature, pressure×waiting and holding time×waiting) had significant effects on the PME level. Increasing the pressure or the holding time at moderate temperatures (15-40 °C) increased the activity. PME purified from apple was pressure stable in the range 100-600 MPa at 25 °C. The soluble pectin content was not changed after HP treatment. The particles size increased with the temperature reached during the treatment, in agreement with a diffusion-limited-aggregation model but were independent of pressure and holding time. In a separate experiment we found a positive correlation between the PME activity and the residual content of the catechins suggesting that polyphenoloxidase (PPO, EC 1.14.18.1) and oxidized polyphenols play a role in the PME activity level after HP treatment.     

The kinetics of inactivation of pectin methylesterase and polygalacturonase in tomato juice by thermosonication

The ultrasonic inactivation kinetics of polygalacturonase (PG) and pectin methylesterase (PME) in tomato juice were studied at a frequency of 20 kHz, amplitude of 65 μm and temperatures between 50 and 75 °C. Thermal treatments at the same temperatures were conducted to separate the effects of heat and ultrasound. The thermal inactivation of PG was described by a fractional conversion model with PG 1 remaining stable, whereas the inactivation of PG by combined ultrasonic and heat treatment (thermosonication) was best described by first order biphasic kinetics, with both PG1 and PG2 inactivated at different rates. The thermal and thermosonication inactivation of PME was described well by first order kinetics. The inactivating effect of combined ultrasound and heat was synergistic. Thermosonication enhanced the inactivation rates of both PME and PG. The inactivation rate of PME was increased by 1.5–6 times and the inactivation rate of PG2 by 2.3–4 times in the temperature range 60–75 °C, with the highest increase corresponding to the lowest temperature.     

超高压技术对草莓汁果胶甲酯酶钝化作用的研究

研究不同条件超高压处理及超高温瞬时灭菌(UHT)对调配草莓汁果胶甲酯酶(PME)钝化作用的影响。以完成调配的草莓汁为对照,分别考察处理压力、保压时间、协同温度以及处理次数对样品的影响。结果表明,不同超高压处理及UHT处理与对照组相比都能导致PME酶活性降低(p<0.05)。200、400MPa(30℃,20min)处理对PME钝化效果与UHT处理相当,达到一定压力(600MPa)钝化效果明显强于UHT处理;超高压处理时间越长,PME钝化效果越明显;超高压协同温度30~60℃处理对PME钝化无显著差异;超高压处理时间一定,增加处理次数对PME钝化作用不明显。贮藏30d后,不同处理PME酶活性基本无变化。      

Pectin methylesterase in Citrus bergamia R.: purification, biochemical characterisation and sequence of the exon related to the enzyme active site

Three forms of pectin methylesterase (PME) were purified, from bergamot fruit (Citrus bergamia R.), to homogeneity by ion-exchange and affinity chromatography. The isoforms, named PME I, PME II and PME III, according their elution order on a heparin–sepharose column, were characterized for their relative molecular mass, activity kinetic parameters and thermostability. The molecular mass was estimated to be 42 kDa for the three forms, and the apparent K_m values for citrus pectin were 0.9 mg/ml for PME I and 0.5 mg/ml for PME II and PME III. The optimum pH values lie within the range 6.5–9.0, depending on salt concentration. Thermal behaviours of the three PME isoforms were studied in a temperature range from 65 °C to 80 °C with the less abundant PME I isoform showing a higher heat resistance. Moreover, the complete exon 2 sequence of PME gene was acquired (GenBank accession no. DQ458770) using a PCR-based approach on well-known Citrus genomic DNA present in the NCBI database.     

Quality-Related Enzymes in Plant-Based Products: Effects of Novel Food-Processing Technologies Part 3: Ultrasonic Processing

High-power ultrasound is a versatile technology which can potentially be used in many food processing applications including food preservation. This is part 2 of a series of review articles dealing with the effectiveness of nonthermal food processing technologies in food preservation focusing on their effect on enzymes. Typically, ultrasound treatment alone does not efficiently cause microbial or enzyme inactivation sufficient for food preservation. However, combined with mild heat with or without elevated pressure (P ≤ 500 kPa), ultrasound can effectively inactivate enzymes and microorganisms. Synergistic effects between ultrasound and mild heat have been reported for the inactivation of both enzymes and microorganisms. The application of ultrasound has been shown to enhance the rate of inactivation of quality degrading enzymes including pectin methylesterase (PME), polygalacturonase (PG), peroxidase (POD), polyphenol oxidase (PPO), and lipoxygenase (LOX) at mild temperature by up to 400 times. Moreover, ultrasound enables the inactivation of relatively heat-resistant enzymes such as tomato PG1 and thermostable orange PME at mild temperature conditions. The extent to which ultrasound enhances the inactivation rate depends on the type of enzyme, the medium in which the enzyme is suspended, and the processing condition including frequency, ultrasonic intensity, temperature, and pressure. The physical and chemical effects of cavitation are considered to be responsible for the ultrasound-induced inactivation of enzymes, although the dominant mechanism depends on the structure of the enzyme.     

Characteristics of enzymatically-deesterified pectin gels produced in the presence of monovalent ionic salts

Pectin methylesterases (PMEs) from Valencia orange (p-PME) and Aspergillus aculeatus (f-PME) were used to produce pectin gels in the presence of 0.2 M monovalent ionic salts. At pH 5.0, pectin gels were induced following enzymatic deesterification of high methoxy pectin, with greater deesterification observed using p-PME compared to f-PME. Under these conditions, the deesterification limit of f-PME ended up with a pectin of DE 30.5–31.9 which did not gel at the PME reaction completion, while p-PME reduced the pectin's DE to 16.0–17.2, resulting in gel formation. The pectin gel induced by KCl was significantly stronger than the NaCl-induced gel, but LiCl did not induce pectin gelation. The gel strength was influenced by both DE and species of monovalent cation. The KCl-induced gels released less water than NaCl-induced gels. A synergistic effect on gel strength was observed from the pectin treated with a combination of (p + f)-PMEs, producing even more stable gels. These results indicated that the pectin gelation of our system would be enhanced both by using larger monovalent cation and by lowering the DE value, which would presumably be attributed to the different action patterns recognized for p- and f-PMEs. This pectin gelation system could provide a useful alternative to acid-sugar or calcium cross-linked gels in food and other industrial applications.     

Optimization of low-temperature blanching for retention of potato firmness: Effect of previous storage time on compression properties

Low-temperature blanching (LTB) of potatoes (cv. Kennebec), both without further processing and prior to cooking or freezing + cooking, significantly increased firmness retention as measured from compression parameters. The increase in firmness with respect to that of unblanched potatoes diminished in the order: blanched at 60 °C for 60 min and cooked > blanched at 60 °C for 60 min frozen and cooked > blanched at 60 °C for 60 min. Potato tubers were kept in refrigerated storage, and firmness, PME activity and dry matter (DM) content were periodically sampled over a period of 80 days. In the early stages of storage, PME activity lost 40% of its original value after 60 min at 60 °C, indicating that the contribution of starch breakdown products to the firmness of cooked and frozen cooked potatoes predominated over the effect of enzyme activity. With increasing time in storage, PME activity measured in the fresh tissue increased by 95% of its original value after 35 days; this resulted in changes in the pectic polymers which made for a firmer texture and different PME behaviour versus LTB temperature and time. A central composite rotatable design was used to study the effects of variation in levels of temperature (52.93–67.07 °C) and time (31.72–88.28 min) on compression parameters and PME activity. Stationary points showing maximum mechanical resistance had critical temperatures and times in the ranges of temperature (58–60 °C) and time (66–75 min) used for each independent variable. Results show a high correlation between PME activity and tissue firmness, suggesting that the contribution of the changes in the composition of the cell wall to the firmness of frozen cooked potatoes increased with increasing time in storage and reached a maximum in the intermediate stages of storage (35 days). Engineering stress (_u) proved to be the most appropriate compression parameter for detecting the firming effect that the PME activity produced on the frozen-cooked potato tissues as a consequence of LTB under these conditions.     

Firming of fruit tissues by vacuum-infusion of pectin methylesterase: Visualisation of enzyme action

Apple pieces were vacuum-impregnated with either a pectin methylesterase (PME) and calcium solution or with water prior to pasteurization. Pasteurized apple pieces impregnated with PME and calcium showed a significantly higher firmness. Moreover, solid state ¹³C NMR spectroscopy of apple cell wall residues revealed an increase of their molecular rigidity. Exogenous PME addition involved a decrease from 82% to 45% of apple pectin degree of methyl-esterification. Microscopic observations of apple slices immunolabelled with antibodies specific for pectins showed that (i) demethyl-esterification was more intense in the cell wall region lining intercellular spaces (demonstrating a key role for these intercellular channels in the enzyme penetration in the tissue during vacuum-infusion) and that (ii) the number of calcium-dimerized deesterified homogalacturonan chains increased. The results corroborate the hypothesis that vacuum-impregnated PME action liberates free carboxyl groups along pectin chains that could interact with calcium, increasing the rigidity of pectins and finally the mechanical rigidity of apple tissue.     

Kinetic study of high pressure microbial and enzyme inactivation and selection of pasteurisation conditions for Valencia Orange Juice

Keeping quality of fresh orange juice is highly dependent on pectinolytic enzyme activity and the growth of spoilage microorganisms. The inactivation kinetics of indigenous pectin methylesterase (PME) and of the two more pressure resistant species of spoilage lactic acid bacteria (LAB) Lactobacillus plantarum and L. brevis in freshly squeezed Valencia orange juice under high hydrostatic pressure (100–500 MPa) combined with moderate temperature (20–40 °C) was investigated. PME inactivation followed first order kinetics with a residual PME activity (15%) at all pressure–temperature combinations used. The values of activation energy and activation volume were estimated at each pressure and at each temperature, respectively. Values of 90 kJ/mol and ?30 mL/mol at reference pressure of 300 MPa and reference temperature of 35 °C were estimated respectively. The corresponding z_T and z_P values of LAB inactivation were also estimated at all conditions tested. Values of 19.5 °C and 95 MPa at reference pressure of 300 MPa and reference temperature of 30 °C were estimated respectively for L. plantarum, while the corresponding values for L. brevis were 40 °C and 82 MPa, respectively, at the same reference conditions. Pressure and temperature were found to act synergistically both for PME and LAB inactivation. The PME and LAB inactivation rate constants were expressed as functions of the temperature and pressure process conditions. These functions allow the determination of the pressure/temperature conditions that achieve the target enzyme and microbial inactivation at a selected processing time. The process conditions of 350 MPa at 35 °C for 2 min are proposed as effective for Valencia orange juice cold pasteurisation.     

Unravelling process-induced pectin changes in the tomato cell wall: An integrated approach

The activity of the pectin-modifying enzymes pectin-methylesterase (PME) and polygalacturonase (PG) in tomato fruit was tailored by processing. Tomatoes were either not pretreated, high-temperature blanched (inactivation of both PME and PG), or high-pressure pretreated (selective inactivation of PG). Subsequently, two types of mechanical disruption, blending or high-pressure homogenisation, were applied to create tomato tissue particle suspensions with varying degrees of tissue disintegration. Process-induced pectin changes and their role in cell-cell adhesion were investigated through in situ pectin visualisation using anti-pectin antibodies. Microscopic results were supported with a (limited) physicochemical analysis of fractionated walls and isolated polymers. It was revealed that in intact tomato fruit pectin de-esterification is endogenously regulated by physical restriction of PME activity in the cell wall matrix. In disintegrated tomato tissue on the other hand, intensive de-esterification of pectin by the activity of PME occurred throughout the entire cell wall. PG was selectively inactivated (i.e. in high-pressure pretreated tomatoes), with de-esterification of pectin by PME, which resulted in a high level of Ca²⁺-cross-linked pectin and a strong intercellular adhesion. In non-pretreated tomato suspensions on the other hand, combined PME and PG activity presumably led to pectin depolymerisation and, hence, reduced intercellular adhesion. However, because of the high amount of Ca²⁺-cross-linked pectin in these samples, cell-cell adhesion was still stronger than in the high-temperature blanched tomatoes, in which the absence of PME activity during suspension preparation implied few Ca²⁺-cross-linked pectic polymers and extensive cell separation upon tissue disruption.     

Purification and characterization of a papaya (Carica papaya L.) pectin methylesterase isolated from a commercial papain preparation

We purified a Carica papaya pectin methylesterase (CpL-PME; EC 3.1.1.11) from a commercial papain preparation. This CpL-PME was separated from the abundant cysteine endopeptidases activities using sequential hydrophobic interaction and cation-exchange chromatographies and then purified by affinity chromatography using Sepharose-immobilized kiwi PME inhibitor protein to obtain a single electrophoretically homogeneous protein. The enzyme was purified 92-fold with 38% yield, providing a specific activity of 1200 U/mg. The molecular weight was determined to be 35,135 by MALDI-TOF-MS in linear mode. MALDI-TOF-MS peptide mass fingerprinting following trypsin digestion indicated CpL-PME represents a novel Carica PME isoform. The CpL-PME required salt for activity, and it showed a broad activity range (pH 6–9) and moderate thermostability (optimum ca. 70 °C). A calcium-insensitive methylated lime pectin treated with CpL-PME to reduce degree of methylesterification by 6% converted the substrate to high calcium sensitivity, indicating a processive mode of action. These properties support further research to apply CpL-PME to tailor pectin nanostructure.