Process-Structure-Function Relations of Pectin in Food.

Pectin, a complex polysaccharide rich in galacturonic acid, has been identified as a critical structural component of plant cell walls. The functionality of this intricate macromolecule in fruit- and vegetable-based-derived products and ingredients is strongly determined by the nanostructure of its most abundant polymer, homogalacturonan. During food processing, pectic homogalacturonan is susceptible to various enzymatic as well as nonenzymatic conversion reactions modifying its structural and, hence, its functional properties. Consequently, a profound understanding of the various process-structure-function relations of pectin aids food scientists to tailor the functional properties of plant-based derived products and ingredients. This review describes the current knowledge on process-structure-function relations of pectin in foods with special focus on pectin's functionality with regard to textural attributes of solid plant-based foods and rheological properties of particulated fruit- and vegetable-derived products. In this context, both pectin research performed via traditional, ex situ physicochemical analyses of fractionated walls and isolated polymers and pectin investigation through in situ pectin localization are considered.

The impact of extraction with a chelating agent under acidic conditions on the cell wall polymers of mango peel.

The objective of this research was to evaluate whether mango peel is a potential source of functional cell wall polymers. To reach this objective, the native pectin polymers (NPP) extracted as alcohol insoluble residue from mango peel, were characterised in terms of uronic acid content, degree of methoxylation, neutral sugar content, and molar mass and compared to citric acid (pH 2.5, 2h at 80°C) extracted polymers, mimicking industrial pectin extraction conditions. Water-solubilised NPP were highly methoxylated having two populations with a Mw of 904 and 83kDa and a degree of methoxylation of 66%. Citric acid extraction with a yield higher than H2SO4 extraction resulted in a very branched pectin with an extremely high DM (83%) and a high molar mass. Comparing the Fourier Transform Infra-Red spectroscopy of extracted and native WSF showed that citric acid remained partially associated to the extracted pectin due to its chelating properties.

Pectin modifications and the role of pectin-degrading enzymes during postharvest softening of Jonagold apples.

This study aimed at understanding softening in Jonagold apple (Malus×domestica Borkh.) fruits, by investigating pectin modifications and the evolution of pectin-modifying enzymes during postharvest storage and ripening. Jonagold apples were harvested at commercial maturity and stored at different temperatures and controlled atmosphere conditions for 6 months, followed by exposure to ambient shelf life conditions (20 °C under air) for 2 weeks. The composition of the pectic material was analysed. Furthermore, the firmness and the ethylene production of the apples were assessed. Generally, the main changes in pectin composition associated with the loss of firmness during ripening in Jonagold apples were a loss of side chains neutral sugars, increased water solubility and decreased molar mass. Also, the activities of four important enzymes possibly involved in apple softening, ß-galactosidase, α-arabinofuranosidase, polygalacturonase and pectin methylesterase, were measured. Pectin-related enzyme activities highly correlated with ethylene production, but not always with pectin modifications.

A Review on the Relationships between Processing, Food Structure, and Rheological Properties of Plant-Tissue-Based Food Suspensions.

Nowadays, there is much interest in controlling the functional properties of processed fruit- and vegetable-derived products, which has stimulated renewed research interest in process-structure-function relations. In this review, we focus on rheology as a functional property because of its importance during the entire production chain up to the moment of consumption and digestion. This review covers the literature of the past decade with respect to process-structure-rheology relations in plant-tissue-based food suspensions. It became clear that the structure of plant-tissue-based food suspensions, consisting of plant-tissue-based particles in an aqueous serum phase, is affected by many unit operations (for example, heat treatment) and that also the sequence of unit operations can have an effect on the final structural properties. Furthermore, particle concentration, particle size, and particle morphology were found to be key structural elements determining the rheological properties of these suspensions comprising low amounts of starch and serum pectin. Since the structure of plant-tissue-based products was shown to be changed during processing, rheological parameters of these products were simultaneously altered. Therefore, this review also comprises a discussion of the effect on rheological properties of the most relevant processing steps in the production of plant-tissue-based products. Linking changes in rheology due to processing with process-induced alterations in structural characteristics turned out to be quite intricate. The current knowledge on process-structure-function relations can form the basis for future improved and novel food process and product design.

Isomerisation of carrot ß-carotene in presence of oil during thermal and combined thermal/high pressure processing.

The effect of thermal processing (85-130°C) and combined thermal/high pressure processing (100°C combined with 0.1 to 700 MPa and 700 MPa combined with 85-115°C) on ß-carotene isomerisation in an olive oil/carrot emulsion and pure olive oil phase enriched with carrot ß-carotene was investigated. Thermal processing always resulted in an increase in the contribution of the cis-isomers, with the increase being more pronounced at higher temperatures. In the oil/carrot emulsion, less ß-carotene isomerisation was observed during combined thermal/high pressure processing compared to thermal processing. This effect was attributed to strengthening of the carrot cell walls under high pressure, thereby hindering the transfer of ß-carotene to the oil phase and lowering its susceptibility to isomerisation. In an oil phase enriched with ß-carotene, ß-carotene isomerisation was not influenced by the applied pressure at 100°C and became almost temperature insensitive at 700 MPa.

Relation between particle size and carotenoid bioaccessibility in carrot- and tomato-derived suspensions.

To study the effect of particle size on the relative all-E-ß-carotene and all-E-lycopene bioaccessibility in carrot- and tomato-derived suspensions, respectively, an in vitro digestion approach including oil was used. Adding olive oil (2%) during digestion, especially as an oil-in-water emulsion, resulted in a substantial increase in carotenoid uptake in the micellar phase. Carotenoid bioaccessibility decreased with average particle size. Only particles smaller than an individual cell resulted in high bioaccessibility values, pointing out the importance of the cell wall as the main barrier for carotenoid uptake. The relation obtained between particle size and bioaccessibility was used to predict the carotenoid bioaccessibility in carrot- and tomato-derived purées. These predictions indicated that carotenoid bioaccessibility in plant-based food suspensions is not only determined by the cell wall integrity (related with particle size) but is also affected by interactions between the structural compounds of the complex food matrix.

Lycopene degradation, isomerization and in vitro bioaccessibility in high pressure homogenized tomato puree containing oil: effect of additional thermal and high pressure processing.

In the present study, the effect of equivalent thermal and high pressure processes at pasteurization and sterilization intensities on some health related properties of high pressure homogenized tomato puree containing oil were investigated. Total lycopene concentration, cis-lycopene content and in vitro lycopene bioaccessibility were examined as health related properties. Results showed that pasteurization hardly affected the health related properties of tomato puree. Only the formation of cis-lycopene during intense thermal pasteurization was observed. Sterilization processes on the other hand had a significant effect on the health related properties. A significant decrease in total lycopene concentration was found after the sterilization processes. Next to degradation, significant isomerization was also observed: all-trans-lycopene was mainly converted to 9-cis- and 13-cis-lycopene. High pressure sterilization limited the overall lycopene isomerization, when compared to the equivalent thermal sterilization processes. The formation of 5-cis-lycopene on the other hand seemed to be favoured by high pressure. The in vitro lycopene bioaccessibility of high pressure homogenized tomato puree containing oil was decreased during subsequent thermal or high pressure processing, whereby significant changes were observed for all the sterilization processes.

Carrot ß-carotene degradation and isomerization kinetics during thermal processing in the presence of oil.

The effect of thermal processing (85-130 °C) on the stability and isomerization of ß-carotene in both an olive oil/carrot emulsion and an olive oil phase enriched with carrot ß-carotene was studied. During processing, degradation of total ß-carotene took place. Initially, total ß-carotene concentration decreased quickly, after which a plateau value was reached, which was dependent on the applied temperature. In the oil/carrot emulsion, the total ß-carotene concentration could be modeled by a fractional conversion model. The temperature dependence of the degradation rate constants was described by the activation energy and was estimated to be 45.0 kJ/mol. In the enriched oil phase, less degradation took place and the results could not be modeled. Besides degradation, ß-carotene isomerization was studied. In both matrices, a fractional conversion model could be used to model total isomerization and formation of 13-Z- and 15-Z-ß-carotene. ß-Carotene isomerization was similar in both the oil/carrot emulsion and enriched oil phase as the simultaneously estimated kinetic parameters (isomerization reaction rate constant and activation energy) of both matrices did not differ significantly. The activation energies of isomerization were estimated to be 70.5 and 75.0 kJ/mol in the oil/carrot emulsion and enriched oil phase, respectively.

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.

Quantifying the influence of thermal process parameters on in vitro ß-carotene bioaccessibility: a case study on carrots.

This study describes a detailed and systematic investigation on the effect of thermal processing in terms of temperature and time (kinetic study) on ß-carotene in vitro bioaccessibility in carrots. ß-Carotene in vitro bioaccessibility increased with increasing processing temperature and time until steady-state conditions were reached after prolonged heating. The bioaccessibility values in steady-state conditions were temperature dependent. The experimental bioaccessibility data could adequately be modeled with a fractional conversion model. For the first time, modeling of processing-induced bioaccessibility changes is reported in literature. The results of the present kinetic study were used to estimate the impact of industrially relevant thermal processes on ß-carotene bioaccessibility in carrots by simulation. It was shown that, to achieve a high ß-carotene bioaccessibility, processing of carrots is essential (i.e., on the one hand, intense thermal processing or, on the other hand, mild thermal processing combined with intense mechanical processing).

Advances in understanding pectin methylesterase inhibitor in kiwi fruit: an immunological approach.

In order to gain insight into the in situ properties and localisation of kiwi pectin methylesterase inhibitor (PMEI), a toolbox of monoclonal antibodies (MA) towards PMEI was developed. Out of a panel of MA generated towards kiwi PMEI, three MA, i.e. MA-KI9A8, MA-KI15C12 and MA-KI15G7, were selected. Thorough characterisation proved that these MA bind specifically to kiwi PMEI and kiwi PMEI in complex with plant PME and recognise a linear epitope on PMEI. Extract screening of green kiwi (Actinidia deliciosa) and gold kiwi (Actinidia chinensis) confirmed the potential use of these MA as probes to screen for PMEI in other sources. Tissue printing revealed the overall presence of PMEI in pericarp and columella of ripe kiwi fruit. Further analysis on the cellular level showed PMEI label concentrated in the middle lamella and in the cell-wall region near the plasmalemma. Intercellular spaces, however, were either completely filled or lined with label. In conclusion, the developed toolbox of antibodies towards PMEI can be used as probes to localise PMEI on different levels, which can be of relevance for plant physiologists as well as food technologists.

Particle size reduction leading to cell wall rupture is more important for the ß-carotene bioaccessibility of raw compared to thermally processed carrots.

The amount of nutrients that can be released from food products (i.e., nutrient in vitro bioaccessibility) is often studied as it is a starting point for investigating nutrient bioavailability, an indicator for the nutritional value of food products. However, the importance of mastication as a particle size reduction technique is poorly understood and is often neglected during in vitro procedures determining bioaccessibility. Therefore, the aim of the present work was to study the effect of mechanical breakdown on the ß-carotene bioaccessibility of carrot samples, having different textural/structural characteristics (as a result of thermal processing). In the first part of this study, the all-E-ß-carotene bioaccessibility of carrot particles of different sizes (ranging from cell fragments up to large cell clusters), generated from raw as well as from gently and intensely cooked carrot samples, was determined. In the second part of the study, the effect of human mastication on the particle size reduction of raw as well as of gently and intensely cooked carrot samples was investigated in order to allow identification and validation of a technique that could mimic mastication during in vitro procedures. Results showed a strong dependency of the all-E-ß-carotene bioaccessibility on the particle size for raw and gently cooked carrots. After intense cooking, on the other hand, a considerable amount of all-E-ß-carotene could be released from cell fragments (smaller than a cell) as well as from small and large cell clusters. Hence, the importance of mechanical breakdown, and thus also of (in vitro) mastication, is dependent on the carrot sample that is considered (i.e., the extent to which the carrot sample has been thermally processed prior to the particle size reduction). Structural changes occurring during mechanical and thermal processing are hereby key factors determining the all-E-ß-carotene bioaccessibility. The average particle size distribution curves of raw and cooked carrots, which were chewed by 15 persons, could be mimicked by mixing 50 g of carrots using a Grindomix (Retsch) at 2500 rpm during 5 s. Based on this scientific knowledge, the identified in vitro mastication technique was successfully integrated in the in vitro digestion procedure determining the all-E-ß-carotene bioaccessibility of carrot samples.

Lycopene degradation and isomerization kinetics during thermal processing of an olive oil/tomato emulsion.

The stability of lycopene in an olive oil/tomato emulsion during thermal processing (80-140 °C) was studied. Initially, the degradation of total lycopene (all-E plus Z-forms) occurred quickly at temperatures above 100 °C. However, a nonzero plateau value, depending on the processing temperature, was attained after longer treatment times. Besides degradation, the isomerization of total-Z-lycopene as well as the individual isomerization of all-E-, 5-Z-, 9-Z-, and 13-Z-lycopene was studied in detail. After prolonged heating, the isomer conversion reached a temperature-dependent equilibrium state. The degradation of total lycopene and the isomerization could be described by a fractional conversion model. The temperature dependency of the corresponding reaction rate constants was quantified by the Arrhenius equation. The activation energy of degradation was estimated to be 28 kJ/mol, and the activation energy of overall (all-E and total-Z) isomerization was estimated to be 52 kJ/mol.

A pectin-methylesterase-inhibitor-based molecular probe for in situ detection of plant pectin methylesterase activity.

In the quest of obtaining a molecular probe for in situ detection of pectin methylesterase (PME), the PME inhibitor (PMEI) was biotinylated and the biotinylated PMEI (bPMEI) was extensively characterized. Reaction conditions for single labeling of the purified PMEI with retention of its inhibitory capacity were identified. High-performance size-exclusion chromatography (HPSEC) analysis revealed that the bPMEI retained its ability to form a complex with plant PME and that it gained the capacity to strongly bind an avidin species. By means of dot-blot binding assays, the ability of the probe to recognize native and high-temperature or high-pressure denatured plant PMEs, coated on an absorptive surface, was investigated and compared to the binding characteristics of recently reported anti-PME monoclonal antibodies. Contrary to the antibodies, bPMEI only detected active PME molecules. Subsequently, both types of probes were used for PME localization in tissue-printing experiments. bPMEI proved its versatility by staining prints of carrot root, broccoli stem, and tomato fruit. Applying the tissue-printing technique on carrot roots after thermal treatment demonstrated the complementarity of bPMEI and anti-PME antibodies, with the former selectively detecting the remaining active PME and the latter staining both native and inactivated PME molecules.

Effect of thermal processing on the degradation, isomerization, and bioaccessibility of lycopene in tomato pulp.

Thermal processing affects the nutritional value of food products. The nutritional value is not only determined by the content but also by the bioaccessibility of nutrients. The present study was performed to gain detailed insight into the influence of thermal processing on the degradation, isomerization, and bioaccessibility of lycopene isomers in tomato pulp, without adding any other ingredient. The bioaccessibility, which is defined as the fraction of the nutrient that can be released from the food matrix, was measured using an in vitro method. The results demonstrated the rather high thermal stability of lycopene. Although a treatment at 140 °C induced isomerization, the contribution of cis-lycopene to the total lycopene content remained small. Results also confirmed that thermal processing as such can improve the in vitro bioaccessibility of lycopene in tomato pulp, but the improvement was only significant upon treatments at temperatures of 130 and 140 °C. At such intense process conditions, one should be aware of the negative effect on other quality and nutrient parameters. Possibilities of thermal processing as such to improve the nutritional value of tomato pulp (without the addition of other ingredients) thus looks rather limited.

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 34.5kDa, 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.