Barriers impairing mineral bioaccessibility and bioavailability in plant-based foods and the perspectives for food processing.

Plant-based foods gain more importance since they play a key role in sustainable, low-meat and healthy diets. In developing countries, these food products, especially legumes and cereals, are important staple foods. Nevertheless, the question arises on how efficient they are to deliver minerals and if it is useful to encourage their consumption to reduce the prevalence of mineral deficiencies? This review paper focuses on the discrepancy between the mineral content and the amount of minerals that can be released and absorbed from plant-based foods during human digestion which can be attributed to several inherent factors such as the presence of mineral antinutrients (phytic acid, polyphenols and dietary fiber) and physical barriers (surrounding macronutrients and cell wall). Further, this review paper summarizes the effects of different processing techniques (milling, soaking, dehulling, fermentation, germination and thermal processing) on mineral bioaccessibility and bioavailability of plant-based foods. The positive impact of these techniques mostly relies on the fact that antinutrients levels are reduced due to removal of fractions rich in antinutrients and/or due to their leaching into the processing liquid. Although processing can have a positive effect, it also can induce leaching out of minerals and a reduced mineral bioaccessibility and bioavailability.

Understanding the Relations Among the Storage, Soaking, and Cooking Behavior of Pulses: A Scientific Basis for Innovations in Sustainable Foods for the Future.

The world faces challenges that require sustainable solutions: food and nutrition insecurity; replacement of animal-based protein sources; and increasing demand for convenient, nutritious, and health-beneficial foods; as well as functional ingredients. The irrefutable potential of pulses as future sustainable food systems is undermined by the hardening phenomenon that develops upon their storage under adverse conditions of temperature and relative humidity. Occurrence of this phenomenon indicates storage instability. In this review, the application of a material science approach, in particular the glass transition temperature concept, is presented to explain phenomena of storage instability such as the occurrence of hardening and loss of viability under adverse storage conditions. In addition to storage (in)stability, application of this concept during processing of pulses is discussed. The state-of-the-art on how hardening occurs, that is, mechanistic insights, is provided, including a critical evaluation of some of the existing postulations using recent research findings. Moreover, the influence of hardening on the properties and processing of pulses is included. Prevention of hardening and curative actions for pulses affected by the hardening phenomenon are described in addition to the current trends on uses of pulses and pulse-derived products. Based on the knowledge progress presented in this review, suggestions for the future include: first, the need for innovation toward implementation of recommended solutions for the prevention of hardening; second, the optimization of the identified most effective and efficient curative action against hardening; and third, areas to focus on for elucidation of mechanisms of hardening, although existing analytical methods require advancement.

Influence of Pectin Structural Properties on Interactions with Divalent Cations and Its Associated Functionalities.

Pectin is an anionic cell wall polysaccharide which is known to interact with divalent cations via its nonmethylesterified galacturonic acid units. Due to its cation-binding capacity, extracted pectin is frequently used for several purposes, such as a gelling agent in food products or as a biosorbent to remove toxic metals from waste water. Pectin can, however, possess a large variability in molecular structure, which influences its cation-binding capacity. Besides the pectin structure, several extrinsic factors, such as cation type or pH, have been shown to define the cation binding of pectin. This review paper focuses on the research progress in the field of pectin-divalent cation interactions and associated functional properties. In addition, it addresses the main research gaps and challenges in order to clearly understand the influence of pectin structural properties on its divalent cation-binding capacity and associated functionalities. This review reveals that many factors, including pectin molecular structure and extrinsic factors, influence pectin-cation interactions and its associated functionalities, which makes it difficult to predict the pectin-cation-binding capacity. Despite the limited information available, determination of the cation-binding capacity of pectins with distinct structural properties using equilibrium adsorption experiments or isothermal titration calorimetry is a promising tool to gain fundamental insights into pectin-cation interactions. These insights can then be used in targeted pectin structural modification, in order to optimize the cation-binding capacity and to promote pectin-cation interactions, for instance for a structure build-up in food products without compromising the mineral nutrition value.

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.

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.

Quantitative assessment of molecular, microstructural, and macroscopic changes of red kidney beans (Phaseolus vulgaris L.) during cooking provides detailed insights in their cooking behavior.

Quantitative changes at different length scales (molecular, microscopic, and macroscopic levels) during cooking were evaluated to better understand the cooking behavior of common beans. The microstructural evolution of presoaked fresh and aged red kidney beans during cooking at 95 °C was quantified using light microscopy coupled with image analysis. These data were related to macroscopic properties, being hardness and volume changes representing texture and swelling of the beans during cooking. Microstructural properties included the cell area (Acell), the fraction of intercellular spaces (%Ais), and the fraction of starch area within the cells (%As/c), reflecting respectively cell expansion, cell separation, and starch swelling. A strong linear correlation between hardness and %Ais (r = -0.886, p = 0.07), along with a significant relative change in %Ais (∼5 times), suggests that softening is predominantly due to cell separation rather than cell expansion. Regarding volume changes, substantial cell expansion (Acell increased by ∼1.5 times) during the initial 30 min of cooking was greatly associated with the increase in the cotyledon volume, while the significance of cell separation became more prominent during the later stages of cooking. Furthermore, we found that the seed coat, rather than the cotyledon, played a major role in the swelling of whole beans, which became less pronounced after aging. The macroscopic properties did not correlate with %As/c. However, the evolution of %As/c conveyed information on the swelling of the starch granules during cooking. During the initial phase, the starch granule swelling mainly filled the cells, while during the later phase, the further swelling was confined by the cell wall. This study provides strong microscopic evidence supporting the direct involvement of the cell wall/ middle lamella network in microstructural changes during cooking as affected by aging, which is in line with the results of molecular changes.

From static to semi-dynamic in vitro digestion conditions relevant for the older population: starch and protein digestion of cooked lentils.

In the context of adequately feeding the rising older population, lentils have an important potential as sources of (plant-based) protein as well as slowly digestible bio-encapsulated starch and fibre. This study evaluated in vitro digestion of protein and starch in lentils under conditions representing the gastrointestinal tract of older adults. Both static and semi-dynamic simulations were applied to analyze the effect of specific gastrointestinal conditions (healthy versus older adult) on macronutrient digestion patterns. Gastric proteolysis was strongly dependent on applied gastric pH (gradient), leading to a lower extent of protein hydrolysis for simulations relevant for older adults. Fewer and smaller (lower degree of polymerization, DP) bioaccessible peptides were formed during gastric proteolysis under older adult compared to healthy adult conditions. These differences, developed during the in vitro gastric phase, were compensated during small intestinal digestion, yielding similar final proteolysis levels regardless of the applied simulation conditions. In contrast, in the presence of saliva, amylolysis was generally accelerated under older adult conditions. Moreover, the current work highlighted the importance of considering saliva (or salivary amylase) incorporation in simulations where the applied gastric pH (gradient) allows salivary amylase activity. Under both healthy and older adult conditions, in vitro starch hydrolysis bio-encapsulated in cotyledon cells of cooked lentils was attenuated, compared to a white bread reference.

Farm to Fork Stability of Phytochemicals and Micronutrients in Brassica oleracea and Allium Vegetables.

Brassica oleracea and Allium vegetables are known for their unique, family specific, water-soluble phytochemicals, glucosinolates, and S-alk(en)yl-l-cysteine sulfoxides, respectively. However, they are also important delivery systems of several other health-related compounds, such as carotenoids (lipid-soluble phytochemicals), vitamin C (water-soluble micronutrient), and vitamin K1 (lipid-soluble micronutrient). When all-year-round availability or transport over long distances is targeted for these often seasonal, locally grown vegetables, processing becomes indispensable. However, the vegetable processing chain, which consists of multiple steps (e.g., pretreatment, preservation, storage, preparation), can impact the nutritional quality of these vegetables corresponding to the nature of the health-related compounds and their susceptibility to (bio)chemical conversions. Since information about the impact of the vegetable processing chain is scattered per compound or processing step, this review targets an integration of the state of the art and discusses needs for future research. Starting with a discussion on substrate-enzyme location within the vegetable matrix, an overview is provided of the impact and potential of processing, encompassing a wide range of (nonenzymatic) conversions.

Stability and bioaccessibility of micronutrients and phytochemicals present in processed leek and Brussels sprouts during static in vitro digestion.

Vegetables are frequently processed before consumption. However, vegetable functionalization continues beyond ingestion as the human digestive tract exposes vegetable products to various conditions (e.g. elevated temperature, pH alterations, enzymes, electrolytes, mechanical disintegration) which can affect the stability of micronutrients and phytochemicals. Besides the extent to which these compounds withstand the challenges posed by digestive conditions, it is equally important to consider their accessibility for potential absorption by the body. Therefore, this study investigated the impact of static in vitro digestion on the stability (i.e. concentration) and bioaccessibility of vitamin C, vitamin K1, glucosinolates, S-alk(en)yl-l-cysteine sulfoxides (ACSOs) and carotenoids in Brussels sprouts (Brassica oleracea var. gemmifera) and leek (Allium ampeloprasum var. porrum). Water-soluble compounds, glucosinolates and ACSOs, remained stable during digestion while vitamin C decreased by >48%. However, all water-soluble compounds were completely bioaccessible. Lipid-soluble compounds were also stable during digestion but were only bioaccessible for 26-81%.

Impact of refrigerated storage on (bio)chemical conversions of health-related compounds in pretreated, pasteurized Brussels sprouts and leek.

Vegetable processing often consists of multiple processing steps. Research mostly focused on the impact of individual processing steps on individual health-related compounds. However, there is a need for more holistic approaches to understand the overall impact of the processing chain on the health potential of vegetables. Therefore, this work studied the impact of pretreatment (relatively intact versus pureed vegetable systems), pasteurization and subsequent refrigerated storage (kinetic evaluation) on multiple health-related compounds (vitamin C, vitamin K1, carotenoids, glucosinolates and S-alk(en)yl-L-cysteine sulfoxides (ACSOs)) in Brussels sprouts and leek. It could be shown that differences introduced by different types of pretreatment were not nullified during pasteurization and refrigerated storage. Clearly, enzymatic conversions controlled during pretreatment resulted in different health-related compound profiles still observable after pasteurization. Moreover, about -42% and -100% relative concentration differences of ACSOs and dehydroascorbic acid, respectively, were detected immediately after pasteurization, while glucosinolates concentrations decreased by about 47% during refrigerated storage. All other compounds were stable during pasteurization and refrigerated storage.

Bean softening during hydrothermal processing is greatly limited by pectin solubilization rather than protein denaturation or starch gelatinization.

The rate liming step of bean softening during cooking was evaluated. Red kidney beans (fresh/non-aged and aged) were cooked at different temperatures (70-95 °C) and their texture evolution established. Softening of beans (loss of hard texture) with cooking and increasing cooking temperature was evident at ≥ 80 °C more so for non-aged than aged beans, evidencing hard-to-cook development during storage. Beans at each cooking time and temperature were subsequently classified into narrow texture ranges and bean cotyledons in the most frequent texture class evaluated for the extent of starch gelatinization, protein denaturation and pectin solubilization. During cooking, starch gelatinization was shown to precede pectin solubilization and protein denaturation, with these reactions progressing faster and to a greater extent with increasing cooking temperature. At 95 °C for instance (practical bean processing temperature), complete starch gelatinization and protein denaturation is attained earlier (∼10 and 60 min cooking, respectively and at comparable time moments for both non-aged and aged beans) than plateau bean texture (∼120 and 270 min for non-aged and aged beans)/plateau pectin solubilization. The extent of pectin solubilization in the cotyledons was consequently most correlated (negatively, r = 0.95) with and plays the most significant role (P < 0.0001) in directing the relative texture of beans during cooking. Ageing was shown to significantly retard bean softening. Protein denaturation plays a less significant role (P = 0.007) while the contribution of starch gelatinization is insignificant (P = 0.181). Thermo-solubilization of pectin in bean cotyledons is therefore the rate limiting step of bean softening towards attaining a palatable texture during cooking.

Pre-duodenal lipid digestion of emulsions: Relevance, colloidal aspects and mechanistic insight.

The digestion of lipids in the human body has several health and nutritional implications. Lipid digestion is an interfacial phenomenon meaning that water-soluble lipases need to first adsorb to the oil-water interface before enzymatic conversions can start. The digestion of lipids mainly occurs on colloidal structures dispersed in water, such as oil-in-water (o/w) emulsions, which can be designed during food formulation/processing or structured during digestion. From a food design perspective, different in vitro studies have demonstrated that the kinetics of lipid digestion can be influenced by emulsion properties. However, most of these studies have been performed with pancreatic enzymes to simulate lipolysis in the small intestine. Only few studies have dealt with lipid digestion in the gastric phase and its subsequent impact on intestinal lipolysis. In this aspect, this review compiles information on the physiological aspects of gastric lipid digestion. In addition, it deals with colloidal and interfacial aspects starting from emulsion design factors and how they evolve during in vitro digestion. Finally, molecular mechanisms describing gastric lipolysis are discussed.

Importance of adapted digestion conditions to simulate in vitro lipid digestion of broilers in different life stages.

In vitro digestion studies demonstrate large potential to gain more and quicker insights into the underlying mechanisms of feed additives, allowing the optimization of feed design. Unfortunately, current in vitro digestion models relevant for broiler chickens lack sufficient description in terms of protocols and standardisation used. Furthermore, no distinction is made between the different life phases of these animals (starter, grower, and finisher). Hence, our research aimed to establish adapted in vitro digestion conditions, corresponding to the 3 life phases in broilers, with specific focus on lipid digestion. The effect of 3 different bile salt concentrations of 2, 10, and 20 mM, and 3 different lipase activities of 5, 20, and 100 U/mL, on in vitro lipid digestion kinetics were evaluated using a full factorial design. These values were selected to represent starter, grower, and finisher birds, respectively. Our findings showed that the extent of lipid digestion was mainly influenced by lipase activity. The rate of lipid digestion was affected by an interplay between bile salt concentration and lipase activity, due to possible lipase inhibition at certain bile salt concentrations. Overall, this work resulted in 3 in vitro lipid digestion models representative for starter, grower, and finisher birds. In conclusion, this research showed the impact of adapted in vitro digestion conditions on lipid digestion kinetics and thus the need for these conditions relevant for each life phase of broilers.

The volatile profile of pasteurized leek (Allium ampeloprasum var. porrum) and Brussels sprouts (Brassica oleracea var. gemmifera) (products), as a witness to (bio)chemical reactivity, influenced by pretreatment and successive refrigerated storage.

Processing can affect (bio)chemical conversions in vegetables and can act on their volatile properties accordingly. In this study, the integrated effect of pretreatment and pasteurization on the volatile profile of leek and Brussels sprouts and the change of this profile upon refrigerated storage were investigated. Pretreatments were specifically selected to steer biochemical reactivities to different extents. Volatile profiles were analyzed by headspace-solid phase microextraction-gas chromatography-mass spectrometry. For both vegetables, it was observed that different pretreatments prior to a pasteurization step led to diverse volatile profiles. The differences in volatile profiles observed in the different samples were presumably attributed to the different degrees of enzymatic conversions, further conversions of enzymatically formed products and thermally induced reactivities. Interestingly, the observed initial relative differences between volatile profiles of differently pretreated pasteurized samples were still observed after a refrigerated storage of 4 weeks at 4 °C. In conclusion, refrigerated storage only limitedly affected the resulting volatile profile.

The rehydration attributes and quality characteristics of 'Quick-cooking' dehydrated beans: Implications of glass transition on storage stability.

Storage stability is an essential consideration for minimizing the deteriorative quality changes in foods post-processing. This study, for the first, time aimed to gain insight into the storage stability of quick-cooking 'convenient' dehydrated beans (Phaseolus vulgaris L.) using the glass transition (Tg) concept. Quick-cooking dehydrated beans were prepared by hydrothermal treatment of fresh beans followed by air-drying and are rehydrated prior to use. The impact of storage temperatures (25, 28, 35 and 42 °C) on the rehydration indices (rate constant and extent) and quality characteristics (colour, texture and volatile profile) of the beans were studied. The results indicate a decrease in the rehydration rate constants with increasing storage temperatures and duration. The rehydration ability also significantly decreased with increased storage duration (>28 °C) suggesting a strong inverse link with hardness. Although there was no overall colour change with storage, the formation of new volatiles associated with non-enzymatic chemical reactions occurred at elevated temperatures (28-42 °C). Identification of the critical water contents based on the Tg-moisture relation and the moisture sorption isotherm revealed that dehydrated beans of 10 % moisture content stored below 28 °C are in a glassy state. Overall, the quality characteristics are significantly influenced by storage and the utilization of the glass transition concept allows for identifying suitable storage conditions.

Utilizing Hydrothermal Processing to Align Structure and In Vitro Digestion Kinetics between Three Different Pulse Types.

Processing results in the transformation of pulses' structural architecture. Consequently, digestion is anticipated to emerge from the combined effect of intrinsic (matrix-dependent) and extrinsic (processed-induced) factors. In this work, we aimed to investigate the interrelated effect of intrinsic and extrinsic factors on pulses' structural architecture and resulting digestive consequences. Three commercially relevant pulses (chickpea, pea, black bean) were selected based on reported differences in macronutrient and cell wall composition. Starch and protein digestion kinetics of hydrothermally processed whole pulses were assessed along with microstructural and physicochemical characteristics and compared to the digestion behavior of individual cotyledon cells isolated thereof. Despite different rates of hardness decay upon hydrothermal processing, the pulses reached similar residual hardness values (40 N). Aligning the pulses at the level of this macrostructural property translated into similar microstructural characteristics after mechanical disintegration (isolated cotyledon cells) with comparable yields of cotyledon cells for all pulses (41-62%). We observed that processing to equivalent microstructural properties resulted in similar starch and protein digestion kinetics, regardless of the pulse type and (prolonged) processing times. This demonstrated the capacity of (residual) hardness as a food structuring parameter in pulses. Furthermore, we illustrated that the digestive behavior of isolated cotyledon cells was representative of the digestion behavior of corresponding whole pulses, opening up perspectives for the incorporation of complete hydrothermally processed pulses as food ingredients.

The role of mechanical collapse by cryogenic ball milling on the effect of high-pressure homogenization on the microstructural and texturizing properties of partially pectin-depleted tomato cell wall material.

In the current study, the effect of different particle size reduction techniques, namely high-pressure homogenization (HPH) and cryogenic ball milling (CBM), on the microstructural and texturizing properties of the tomato acid-unextractable fraction (AcUF) in suspension was studied. Partial pectin depletion was performed by nitric acid pectin extraction on the alcohol-insoluble residue. In the absence of the aforementioned mechanical treatments, the partially pectin-depleted material, i.e., the AcUF, showed a cellular morphology and a high texturizing potential. By short-time CBM in dry-state, the AcUF was extensively fractured and clumped, resulting in a collapsed structure with negligible texturizing potential and low water binding capacity. In contrast, HPH could disrupt the cell wall network (destroying the cellular morphology) resulting in a continuum of interacting material having very similar texturizing potential and a slightly higher water binding capacity than the AcUF before HPH. Furthermore, the potential of HPH to (re)functionalize the collapsed cryogenic ball milled AcUF by its shear-induced disruption was shown. Indeed, the debris-like cell wall remnants could to some extent be reopened by HPH, which resulted in a partial recovery of the original texturizing potential and an improved water binding capacity. However, the potential of HPH at 20 MPa to revert the detrimental effect of CBM decreased with increasing CBM treatment time.

The Volatile Profile of Brussels Sprouts (Brassica oleracea Var. gemmifera) as Affected by Pulsed Electric Fields in Comparison to Other Pretreatments, Selected to Steer (Bio)Chemical Reactions.

Pulsed electric fields (PEF) at low field strength is considered a non-thermal technique allowing membrane permeabilization in plant-based tissue, hence possibly impacting biochemical conversions and the concomitant volatile profile. Detailed studies on the impact of PEF at low field strength on biochemical conversions in plant-based matrices are scarce but urgently needed to provide the necessary scientific basis allowing to open a potential promising field of applications. As a first objective, the effect of PEF and other treatments that aim to steer biochemical conversions on the volatile profile of Brussels sprouts was compared in this study. As a second objective, the effect of varying PEF conditions on the volatile profile of Brussels sprouts was elucidated. Volatile fingerprinting was used to deduce whether and which (bio)chemical reactions had occurred. Surprisingly, PEF at 1.01 kV/cm and 2.7 kJ/kg prior to heating was assumed not to have caused significant membrane permeabilization since similar volatiles were observed in the case of only heating, as opposed to mixing. A PEF treatment with an electrical field strength of 3.00 kV/cm led to a significantly higher formation of certain enzymatic reaction products, being more pronounced when combined with an energy input of 27.7 kJ/kg, implying that these PEF conditions could induce substantial membrane permeabilization. The results of this study can be utilized to steer enzymatic conversions towards an intended volatile profile of Brussels sprouts by applying PEF.

Production and molecular characterization of tailored citrus pectin-derived compounds.

In this study, tailored-made citrus pectin-derived compounds were produced through controlled enzymatic and/or chemical modifications of commercial citrus pectin with different degrees of methylesterification (DM) and similar average molecular weight (MW). In the first treatment, degradation of the citrus pectin (CP) materials by endo-polygalacturonase (EPG) yielded pectins with average Mw's (between 2 and 60 kDa). Separation and identification of the oligosaccharide fraction present in these samples, revealed the presence of non-methylesterified galacturonic acid oligomers with degree of polymerization (DP) 1-5. In the second treatment, exploiting the combined effect of EPG and pectin lyase, compounds with MW between 2 and 21 kDa, containing methylesterified and non-methylesterified polygalacturonans (DP 1-6), were generated. Finally, CP was sequentially modified by chemical saponification and the action of EPG. A sample of DM 11% and MW 2.7 kDa, containing POS (DP 1-5), was produced. Diverse pectin-derived compounds were successfully generated for further studies exploring their functionality.

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.