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. K_m and V_max 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. E_a and Z values were found to be 196.8 kJmol⁻¹ (r² = 0.996) and 2.16 °C (r² = 0.995), respectively.     

Evaluation of processing qualities of tomato juice induced by thermal and pressure processing

Effects of processing conditions including hot-break processing (92 °C for 2 min), cold-break processing (60 °C for 2 min) and hydrostatic pressure treatments (100-500 MPa) at different temperatures (4, 25 and 50 °C) for 10 min on quality aspects of tomato juice were investigated. Both hot- and cold-break processing induced significant changes in color, viscosity and radical-scavenging capacity of tomato juice compared with control (fresh tomato juice); moreover, hot-break processing induced a specific range of reduction of pectin methylesterase (PME) and polygalacturonase (PG) activities. Pressure treatments at and below 200 MPa at 4 and 25 °C maintained the color, extractable total carotenoids and lycopene, and radical-scavenging capacity; further, those at 500 MPa at 4 and 25 °C improved all the quality attributes the most except inactivation of PME in this study. The residual activity of PME showed the lowest after treating by 200 MPa at 25 °C; however, the PME activity was enhanced by treatments at 300-500 MPa and various temperatures. The residual activity of PG decreased gradually to 72% with pressure elevated from 100 to 400 MPa at 4 and 25 °C, further, that declined quickly to 10% after 500 MPa treatments. This research clearly shows that it is possible to selectively produce good tomato juice products by high pressure processing at ambient temperature.     

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.     

Extraction, thermal stability and kinetic behavior of pectinmethylesterase from hawthorn (Crataegus pubescens) fruit

Pectinmethylesterase (PME) extracted from hawthorn (Crataegus pubescens) fruit was evaluated for its thermal stability and kinetic behavior. The enzyme extraction process was established after studying different NaCl concentrations (0.5-3.0 moles/L). A maximum PME extraction of 51.61 units/mg protein was obtained using 2 moles/L NaCl. Kinetic parameters (K_m and V_max) were determined using a commercial citrus pectin and C. pubescens pectin as substrates. The effects of NaCl concentration, pH and temperature on PME activity were investigated. PME showed higher affinity for C. pubescens pectin (K_m and V_max of 2.84 mg/mL protein, and 64.10 units/mg protein, respectively) than for citrus pectin. C. pubescens PME extract showed maximum activity at 0.4 moles/L NaCl, pH 7.5, and 55 °C. The E_a and Q₁₀ for thermal activation were 36.27 kJ/mol and 2.01 (20-30 °C), respectively. About 50% of the activity still remained after heating for 25 min at 60 °C, and it was completely inactivated by incubation at 80 °C for 10 min. The Q₁₀ and E_a values for thermal inactivation reaction were 20.06 (70-80 °C) and 146.16 kJ/mol, respectively. These results provide useful information about the factors that affect the activity of C. pubescens PME, and might be used as a starting point for texture control during post-harvest handling and processing of this fruit.     

Effect of high-pressure/high-temperature processing on chemical pectin conversions in relation to fruit and vegetable texture

Heat sterilization of plant derived food products entails considerable organoleptic and nutritional quality losses. For instance, texture loss of fruits and vegetables occurs, next to turgor pressure losses, mainly due to chemical changes in the cell-wall pectic polysaccharides. High-pressure sterilization, i.e. the combination of high temperature (?90 °C) with high pressure (?500 MPa), could present a positive alternative assuring safety while minimizing quality losses. In this study, the potential of high-pressure sterilization in preserving fruit and vegetable texture was evaluated by investigating the effect of combined high-pressure/high-temperature (HP/HT) treatments on two texture related chemical pectin conversions in model sytems. First, a protocol was developed to perform reproducible kinetic studies at HP/HT under constant processing conditions. Subsequently, apple pectin solutions at pH 6.5 were subjected to different HP/HT combinations (500, 600 and 700 MPa/90, 110 and 115 °C) and the extent of chemical demethoxylation and β-eliminative depolymerization was determined. At atmospheric pressure, both zero-order reaction rate constants increased with increasing temperature. At all temperatures, demethoxylation showed a higher rate constant than β-elimination. However, a temperature rise resulted in a stronger acceleration of β-elimination than of demethoxylation. When combining high temperature with high pressure, β-elimination was retarded or even stopped, whereas demethoxylation was stimulated. These results are very promising in the context of the texture preservation of high-pressure sterilized fruits and vegetables, as β-elimination is accepted to be one of the main causes of thermal softening and low methoxylated pectin can enhance tissue strength by forming cross-links with calcium ions present.     

Effect of high pressure/high temperature processing on cell wall pectic substances in relation to firmness of carrot tissue

Thermal processing for food preservation results in undesired softening of fruits and vegetables. To explore the potential of high pressure sterilization in food processing, the effects of combined high pressure/high temperature (HP/HT) treatments on carrot pectic polysaccharides and the related textural properties were investigated and compared with that of samples thermally processed at atmospheric pressure. Disks of fresh carrot (Daucus carota var. Yukon) tissue were subjected to three different treatments (80 °C–0.1 MPa, 100 °C–0.1 MPa and 80 °C–600 MPa) for varying time intervals. Subsequently, the residual texture and microstructural changes of the carrots were evaluated. Alcohol-insoluble residues were prepared from the samples and sequentially fractionated with water, cyclohexane-trans-1,2-diamine tetra-acetic acid (CDTA) and Na₂CO₃ solutions. Thermal treatments at 0.1 MPa caused extensive tissue softening. This was marked by increased cell separation, an increase in water soluble pectin (WSP) paralleled by a decrease in chelator (CSP) and sodium carbonate (NSP) soluble pectin. HP/HT treated carrots showed minimal softening and negligible changes in intercellular adhesion. This was accompanied by a significant reduction in the degree of methyl esterification of pectin, low WSP in contrast to the high CSP and NSP fractions, minor changes in the different pectin fractions during treatment, and a substantial amount of pectin in the fractionation residue. There was a clear difference between HP/HT and thermally processed carrot pectin; HP/HT showing pronounced texture preservation.     

Defining the stability interfaces of apple juice: Implications on the optimisation and design of High Hydrostatic Pressure treatment

In this article an experimental approach is applied to determine the impact of High Hydrostatic Pressure (HHP) processing (350 to 550 MPa at 20 °C and for 1 to 25 min of holding time) on the survival of Issatchenkia orientalis and the spoilage of apple juice (with 300 ppm added ascorbic acid) during different storage conditions, i.e., 4 to 12 °C and 0 to 36 days of storage. Probabilistic modelling approaches based on logistic regression models were developed in order to describe quantitatively the spoilage/no spoilage and survival/death interfaces. For a microbially stable processed apple juice treated at 400 MPa, 10 °C and a holding time of 15 min the degradation kinetics of vitamin C were described quantitatively during subsequent storage at 4, 8, 12 °C. The rate of vitamin losses were highly reduced after the first 13 days of storage. The stability of the apple juice with respect to browning and cloudiness was evaluated by studying qualitatively the activity of polyphenol oxidase (PPO), and pectin methyl esterase (PME) enzymes at combined treatments of HHP and temperature (10 to 50 °C, HHP at 750 MPa and holding time from 1 to 25 min). The highest achieved reduction of PPO and PME was 51.47% and PME 81.44%, respectively.

Industrial relevance

This paper demonstrates an approach based on quantitative probabilistic and qualitative studies for defining the stability interfaces of apple juice. Its applicability contributes on the design and optimisation of High Hydrostatic Pressure treatments.     

Evaluation of high pressure (HP) treatment for rapid and uniform pH reduction in carrots

Acidification (pH < 4.6) of marginally low acid foods allows them to be treated like high acid foods and, hence, has the potential to improve quality and reduce energy costs by lowering the severity of processing conditions. The objective of this study was to evaluate high pressure (HP) treatment for rapid and uniform pH reduction of low acid foods using carrot. Three organic acids (citric, malic and glucono-delta-lactone) were used in the study. Conventional acidification tests were carried out at atmospheric pressure at different temperatures (36–48 °C) and different treatment times (0–36 min). HP treatments were given at room temperature (maximum process temperature <32 °C) with different pressures (200–300 MPa) and treatment times (0–14 min). Time dependent acid infusion and the resulting pH reduction were used to evaluate the acidification kinetics. Results showed that the pH reduction rates were described by a first order kinetic model. No significant differences (p > 0.05) in acidification rates were observed between the acidifying agents either in conventional or HP acidification process. Pressure (HP acidification) and temperature (conventional acidification) significantly (p < 0.05) reduced decimal reduction time (D) for pH drop. The associated D values were 2.4–4.4 times higher in conventional (slower) as compared with HP acidification. For conventional acidification, the z values (temperature sensitivity) were 34–44.8 °C and for HP acidification, the z values (pressure sensitivity) were 206–222 MPa. HP acidification provided more rapid and uniform pH reduction as compared to conventional method.     

High-pressure destruction kinetics of Clostridium sporogenes ATCC 11437 spores in milk at elevated quasi-isothermal conditions

The high-pressure sterilization establishment requires data on isobaric and isothermal destruction kinetics of baro-resistant pathogenic and spoilage bacterial spores. In this study, Clostridium sporogenes 11437 spores (10⁷ CFU/ml) inoculated in milk were subjected to different pressure, temperature and time (P, T, t) combination treatments (700–900 MPa; 80–100 °C; 0–32 min). An insulated chamber was used to enclose the test samples during the treatment for maintaining isobaric and quasi-isothermal processing conditions. Decimal reduction times (D values) and pressure and temperature sensitivity parameters, Z_T (pressure constant) and Z_P (temperature constant) were evaluated using a 3 × 3 full factorial experimental design. HP treatments generally demonstrated a minor pressure pulse effect (PE) (no holding time) and the pressure hold time effect was well described by the first order model (R² > 0.90). Higher pressures and higher temperatures resulted in a higher destruction rate and a higher microbial count reduction. At 900 MPa, the temperature corrected D values were 9.1, 3.8, 0.73 min at 80, 90, 100 °C, respectively. The thermal treatment at 0.1 MPa resulted in D values 833, 65.8, 26.3, 6.0 min at 80, 90, 95, 100 °C respectively. By comparison, HP processing resulted in a strong enhancement of spore destruction at all temperatures. Temperature corrected Z_T values were 16.5, 16.9, 18.2 °C at 700, 800, 900 MPa, respectively, which were higher than the thermal z value 9.6 °C. Hence, the spores had lower temperature sensitivity at elevated pressures. Similarly, corrected Z_P values were 714, 588, 1250 MPa at 80, 90, 100 °C, respectively, which illustrated lower pressure sensitivity at higher temperatures. By general comparison, it was concluded that within the range operating conditions employed, the spores were relatively more sensitive to temperature than to pressure.     

Physiological and biochemical response of harvested plum fruit to oxalic acid during ripening or shelf-life

The effect of oxalic acid application on plum fruit (Prunus salicina cv. ‘Damili’) ripening properties during storage or shelf-life was determined. The fruits were dipped for 3 min in solutions containing 5 mmol/L oxalic acid and then were packed into polyethylene bags and stored at 25 °C for 12 days, or at 2 °C for 20 days and subsequently at 25 °C for 12 days. Ethylene production, fruit firmness, contents of pectin and anthocyanin, specific activities of polygalacturonase (PG), pectin methylesterase (PME) and phenylalanine ammonia lyase (PAL), and chlorophyll fluorescence (Fv/Fm) were measured. The application of oxalic acid reduced ethylene production and delayed softening of plum fruit. The inhibition of softening was associated with decreased PG and PME activities; that is, the retardation of pectin solubilization/degradation. During storage or shelf-life, flesh reddening and anthocyanin synthesis were significantly inhibited in oxalic acid-treated plum fruit, accompanied with decreased PAL activity. Furthermore, it was found that variable:maximalchlorophyll fluorescence (Fv/Fm), an indicator of ripening, senescence or stress injury of fruit and vegetable, decreased much more slowly in oxalic-treated plum fruits than in control fruits. Thus, oxalic acid treatment can be an effective means to extend the shelf life of plum fruit.     

Aseptically packaged UHPH-treated apple juice: Safety and quality parameters during storage

Ultra-high pressure homogenization (UHPH) at 300 MPa and 4 °C inlet temperature were used to preserve apple juice, and shelf-life evaluation of aseptically packaged juice was investigated. After processing Tetra Brik containers were stored at temperatures of 4, 10, 20 and 30 °C during 60 days. In this article, the effect of processing on the spoilage inactivation was evaluated after processing and during the storage trial. Non-germinated and germinated spores were found in the UHPH-treated juice, being an inactive population during storage. Patulin content was also not modified by UHPH processing, but a significant decrease was observed during storage at 30 °C (P < 0.05). Polyphenoloxidase (PPO) and pectinmethylesterase (PME) activity was not found after UHPH-processing and during storage.A kinetic study of post-processing quality loss was conducted. Vitamin C, chlorogenic acid, total polyphenols and color change were measured during storage study and were used to model the UHPH-treated apple juice shelf-life. Loss of vitamin C was correlated with the hydroxymethylfurfural (HMF) accumulation (0.59, P < 0.05). A limiting quality parameter was polyphenolic content. UHPH-treated apple juice stored at 4 °C was found to show a shelf-life for about 21 months by preserving the color characteristics of the juice with low HMF accumulation. From 15 °C changes in quality parameters were more evident.     

Application of high pressure argon treatment to maintain quality of fresh-cut pineapples during cold storage

High pressure (HP) argon (Ar) treatment was applied to preserve fresh-cut pineapples at 4 °C. The effects of treatment temperature (2–10 °C), time (30–120 min) and pressure (0.5–4.5 MPa) on the efficiency of pressurized Ar treatment was studied to determine the optimum conditions. Temperature in the range of 2–6 °C did not significantly affect the efficacy of the pressurized Ar treatment. A combination of pressure–time of pressurized Ar treatment at 1.6–2.2 MPa for 43–65 min was found to be the optimum processing conditions for the preservation of pineapple slices. Samples treated at optimum condition (1.8 MPa, 60 min) were stored at 4 °C for 20 days to evaluate the effect of HP Ar treatment on nutritional components, microbial growth, sensory quality, membrane permeability and microstructure. A shelf life extension of 6 days was achieved by applying HP Ar treatment for fresh-cut pineapples during cold storage in air or in modified atmospheric packaging.     

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.     

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.     

Comparison of enzymatic de-esterification of strawberry and apple pectin at elevated pressure by fungal pectinmethylesterase

Water soluble pectin was isolated from strawberries. Sugar composition, degree of esterification and molar mass were compared with commercial apple pectin. Both pectins were subjected to enzymatic de-esterification by recombinant Aspergillus aculeatus pectinmethylesterase (PME). Rate of enzymatic de-esterification at elevated pressures (0.1–500 MPa) and temperatures (20–60 °C) was assayed by measuring the release of methanol as a function of time. Optimal activity was observed at 200–300 MPa combined with 45–55 °C. At all conditions investigated, both pectins were de-esterified at similar initial rates. However, after prolonged enzymatic treatment at atmospheric pressure and 30 °C, apple pectin was de-esterified to a significantly lower degree of esterification (7%) than strawberry pectin (32%). The mode of action of A. aculeatus PME was investigated by enzymatic fingerprinting of de-esterified pectin chains. The enzyme de-esterified according to a “multiple chain, multiple attack” mechanism, irrespective of the substrate.Industrial relevanceThis article demonstrates that both strawberry and apple pectin de-esterification by recombinant Aspergillus aculeatus PME is accelerated by high hydrostatic pressure. Since de-esterification of pectin in fruits gives rise to a texture improvement, this enzyme can be used to minimize texture damage during high-pressure processing of fruits and fruit-based products. It was also shown that this enzyme de-esterifies strawberry and apple pectin according to a “multiple chain, multiple attack” mechanism. The resulting pattern of esterification might have an influence on textural properties of fruits.     

Pressure and temperature combination for inactivation of soymilk trypsin inhibitors

High hydrostatic pressure (HHP) processing, an emerging technology for food preservation, in combination with thermal treatment (250/50, 550/19, 550/65, and 550/80 MPa/°C) was applied to soymilk made from previously soaked soybeans (in distilled water or 0.5% sodium bicarbonate solution). First order kinetics constants ranging from 0.081 to 0.217 min⁻¹, for residual trypsin, were estimated in soymilk from soaked soybeans at selected pressure–temperature combinations. Residual trypsin, at 550 MPa and 80 °C, was high at higher HHP holding times. The highest percentage of residual trypsin (76%) was estimated after a 15 min holding time. The use of sodium bicarbonate for soaking of soybeans synergistically decreased the trypsin inhibitor activity in soymilk in comparison with residual trypsin using distilled water alone.     

Effects of high pressure CO2 treatments on microflora, enzymes and some quality attributes of apple juice

The inactivation of microorganisms and enzymes of cloudy apple juice, and some physico-chemical properties of the juice were investigated after continuous high pressure carbon dioxide (HPCD) at 25 MPa and 43 °C, for 2 min and at 22 MPa and 60 °C for 3, 5 and 10 min, respectively. The coliform bacteria were completely inactivated in all the cases. Total aerobic bacteria was reduced by 3.72 log cycles, pectin methylesterase was reduced by 54.3% and polyphenol oxidase was completely inactivated after 10 min treatment at 22 MPa and 60 °C. The yeasts and molds were completely inactivated and the turbidity increased at 22 MPa and 60 °C regardless of time, while the L and a value reduced, but browning degree did not change. The particle size distributions of the juice changed after HPCD but were regained as treatment time was prolonged. The pH was reduced at 22 MPa and 60 °C for 3 or 5 min.     

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.     

Comparison of the inactivation kinetics of pectin methylesterases from carrot and peach by high-pressure carbon dioxide

The inactivation of pectin methylesterases (PMEs) from carrot and peach in buffer by high-pressure carbon dioxide (HPCD) at 55 °C was investigated. The two PMEs were effectively inactivated by HPCD, their residual activity (RA) decreasing with increasing pressures. The RA of the two PMEs exhibited a fast decrease firstly and reached a constant after a prolonged treatment time; their inactivation kinetics was adequately modelled by a fractional-conversion model. The non-zero RA(A_∞)of the two PMEs was 6–7%, with increasing pressures the kinetic rate constant, k, increased and the decimal reduction time, D, decreased for the HPCD-labile fraction of the two PMEs. The labile fraction of carrot PME was more susceptible to HPCD than that of peach PME; the activation volume, V_a, and Z_P (the temperature increase needed for a 90% reduction of D) was −1079.37 cm³/mol and 5.80 MPa for carrot PME, and −130.51 cm³/mol and 48.31 MPa for peach PME.     

Hydrolysis of apple pectin by the coordinated activity of pectic enzymes

The hydrolysis of pectin from apples, cv. Budimka fruit (Pyrus mahus L.), by individual and/or combined action of fungal polygalacturonase from Aspergillus niger (FPG), fungal pectin esterase from A. niger (FPE) and plant tomato pectin esterase (PPE) was studied. The optimum pH values for individual actions of FPG, FPE and PPE on 1% apple pectin were determined to be 4.5, 3.5 and 6.5, and the optimum temperatures were 40, 45, and in the range 45–50 °C, respectively. FPE was found to be more efficient for the hydrolysis of the apple pectin than was PPE from tomato. By measuring the initial velocities on 1% apple pectin it was confirmed that the PG expressed no effect on the PE activity. By using the combination of FPG (162 U/l) and FPE (60 U/l), e.g., in a respective ratio of 2.5, an efficient pectin degradation process, with a viscosity reduction of η/η₀ = 1.05, could be reached in less than 2 h. This process produced about 160 ppm of methanol in the pectin digest. The long term hydrolysis reaction of the apple pectin with FPG (162 U/l) and FPE (27 U/l) achieved a degree of hydrolysis of around 29% after 12 h and consisted mostly of trimers (28.4%).