Texture classification and in situ cation measurements by SEM-EDX provide detailed quantitative insights into cell wall cation interaction changes during ageing, soaking, and cooking of red kidney beans.

Hard-to-cook (HTC) is a textural defect that delays the softening of common bean seeds during cooking. While this defect is commonly associated with conventionally stored beans, soaking/cooking of beans in CaCl2 solutions or sodium acetate buffer can also prolong the cooking time of beans due to formation of Ca2+ crosslinked pectin retarding bean softening during cooking. In this study, the role of the cell wall-bound Mg2+/Ca2+ content and the degree of pectin methyl esterification (DM) was quantified, as important factors for bean texture-related changes stipulated in the pectin-cation-phytate hypothesis, the most plausible hypothesis of HTC development. Evaluation of texture changes during cooking of conventionally aged beans (35 °C and 83% RH for up to 20 weeks), beans soaked/cooked in CaCl2 solutions (0.01 to 0.1 M) or soaked in 0.1 M sodium acetate buffer (pH 4.4) revealed large bean-to-bean variations. Therefore a texture-based classification approach was used to better capture the relation between texture characteristics and cell wall polymer, in particular pectin, related changes. While cell wall-bound Ca2+ and pectin DM did not change/were not related to the texture variation during cooking of fresh beans, increased cell wall-bound Ca2+ and decreased pectin DM were associated with prolonged conventional storage of beans and their texture changes during subsequent cooking (due to pectin cross linking, retarding its solubilization during cooking). Exogenously added Ca2+ from pre-treating beans in CaCl2 solutions promoted to a great extent the cell wall-bound Ca2+ during soaking but even more so during cooking, complementing the harder texture associated with these beans during cooking (compared to conventionally stored and fresh beans). Similarly, free Ca2+ endogenously generated by phytase-catalysed phytate hydrolysis (beans treated by acetate buffer) promoted crosslinking of pectin by Ca2+ (cell wall-bound Ca2+), delaying softening of beans during cooking.

Effect of bio-chemical changes due to conventional ageing or chemical soaking on the texture changes of common beans during cooking.

To establish the HTC defect development, the cooking kinetics of seeds of ten bean accessions (belonging to seven common bean market classes), fresh and conventionally aged (35 °C, 83% RH, 3 months) were compared to those obtained after soaking in specific salt solutions (in 0.1 M sodium acetate buffer at pH 4.4, 41 °C for 12 h, or 0.01 M CaCl2 at pH 6.2, 25 °C for 16 h and subsequently cooking in CaCl2 solution, or deionised water). The extent of phytate (inositol hexaphosphate, IP6) hydrolysis was evaluated to better understand the role of endogenous Ca2+ in the changes of the bean cooking kinetics. A significant decrease in the IP6 content was observed after conventional ageing and after soaking in a sodium acetate solution suggesting phytate hydrolysis (release of endogenous Ca2+). These changes were accompanied by an increase in the cooking time of the beans. Smaller changes in cooking times after soaking in a sodium acetate solution (compared to conventionally aged beans) was attributed to a lower ionisation level of the COOH groups in pectin (pH 4.4, being close to pKa value of pectin) limiting pectin Ca2+ cross-linking. In beans soaked in a CaCl2 solution, the uptake of exogenous cations increased the cooking times (with no IP6 hydrolysis). The change in cooking time of conventionally aged beans was strongly correlated with the extent of IP6 hydrolysis, although two groups of beans with low or high IP6 hydrolysis were distinguished. Comparable trends were observed when soaking in CaCl2 solution (r = 0.67, p = 0.14 or r = 0.97, p = 0.03 for two groups of beans with softer or harder texture during cooking). Therefore a test based on the Ca2+ sensitivity of the cooking times, implemented through a Ca2+ soaking experiment followed by cooking can be used as an accelerated test to predict susceptibility to HTC defect development during conventional ageing. On the other hand, a sodium acetate soaking experiment can be used to predict IP6 hydrolysis of conventionally aged bean accessions and changes of cooking times for these bean accessions (with exception of yellow bean-KATB1).

Application of state diagrams to understand the nature and kinetics of (bio)chemical reactions in dry common bean seeds: A scientific guide to establish suitable postharvest storage conditions.

Storage is a fundamental part of the common bean postharvest chain that ensures a steady supply of safe and nutritious beans of acceptable cooking quality to the consumers. Although it is known that extrinsic factors of temperature and relative humidity (influencing the bean moisture content) control the cooking quality deterioration of beans during storage, the precise interactions among these extrinsic factors and the physical state of the bean matrix in influencing the rate of quality deteriorative reactions is poorly understood. Understanding the types and kinetics of (bio)chemical reactions that influence the cooking quality of beans during storage is important in establishing suitable storage conditions to ensure quality stability. In this review, we integrate the current insights on glass transition phenomena and its significance in describing the kinetics of (bio)chemical reactions that influence the cooking quality changes during storage of common beans. Furthermore, a storage stability map based on the glass transition temperature of beans as well as kinetics of the main (bio)chemical reactions linked to cooking quality deterioration during storage was designed as a guide for determining appropriate storage conditions to ensure cooking quality stability.

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.

Novel insights into the role of the pectin-cation-phytate mechanism in ageing induced cooking texture changes of Red haricot beans through a texture-based classification and in situ cell wall associated mineral quantification.

Utilization of common beans is greatly hampered by the hard-to-cook (HTC) defect induced by ageing of the beans under adverse storage. Large bean-to-bean variations exist in a single batch of beans. Therefore, a texture-based bean classification approach was applied in this detailed study on beans with known textures, to gain in-depth insights into the role of the pectin-cation-phytate mechanism in relation to the texture changes during subsequent cooking of Red haricot fresh and aged beans. For the first time, a correlation between the texture (exhibited after cooking) of a single bean seed before ageing (fresh) and its texture after ageing was established. Furthermore, scanning electron microscopy coupled with energy dispersive spectrometry (SEM-EDS) based in situ cell wall associated mineral quantification revealed that the cell wall associated Ca concentration was significantly positively correlated with the texture of both fresh and aged cooked Red haricot bean cotyledons, with ageing resulting in a significant enrichment of Ca at the cell wall. These additional Ca cations originate from intracellular phytate hydrolysis during ageing, which was shown to affect the texture distribution of aged beans during cooking significantly. The relocation of the mineral cations from the cell interior to the cell wall occurs mainly during storage rather than subsequent soaking of the cotyledons. In addition, the pectin-cation-phytate hypothesis of HTC was further confirmed by demethylesterification of the cell wall pectin and increased pectin-Ca interactions upon ageing of the cotyledons, finally leading to HTC development of the cotyledon tissue.

Phytate and mineral profile evolutions to explain the textural hardening of common beans (Phaseolus vulgaris L.) during postharvest storage and soaking: Insights obtained through a texture-based classification approach.

During adverse postharvest storage of Red haricot beans, the inositol phosphate content, particularly InsP6, decreased significantly, along with a significant increase in InsP5. Using a texture-based classification approach, the InsP6 content in cotyledons was shown an indicator for the extent of hard-to-cook (HTC) development during bean aging. This textural defect development was predominated by storage-induced InsP6 degradation, rather than phytate interconversions during soaking. Ca cations, released during storage, did not leach out significantly during subsequent soaking, suggesting that they were bound with the cell wall pectin in cotyledons, while Mg cations were mostly leached out into the soaking water due to their weak binding capacity to the pectin, and the cell membrane damages developed during HTC. Results obtained herein provide evidence for the pectin-cation-phytate mechanism in textural hardening (and its distribution after cooking) of common beans, and call for a more detailed Ca-relocation study during postharvest storage, soaking and cooking.

Cell wall polysaccharide changes and involvement of phenolic compounds in ageing of Red haricot beans (Phaseolus vulgaris) during postharvest storage.

Cell wall material was isolated from selected non-aged and aged Red haricot bean cotyledons using a texture-based classification approach. Pectin-depleted residual cell wall fractions were obtained by sequential pectin extraction and were characterized to investigate in situ cell wall related molecular changes upon ageing during adverse storage of the beans. Particularly, involvement of phenolic compounds in cell wall strengthening during the ageing process, resulting in the hard-to-cook defect, was evaluated. Results show that ageing induces substantial changes at a cell-wall-structural level in the Aged sample compared to the Non-aged sample, with mainly vanillin, 4-hydroxybenzoic acid and 4-hydroxybenzaldehyde covalently bound with sugar side-chains of pectin and/or involved in lignification-like mechanisms. FT-IR spectroscopy coupled with chemometric analysis reveals that lignin-like phenolic-cell wall polymers, which are known to reinforce cell wall structure, are present in the cell wall polysaccharide network of the Aged sample, and are therefore contributing factors to the hard-to-cook development during Red haricot bean ageing.

(Bio)chemical reactions associated with ageing of red kidney beans (Phaseolus vulgaris) during storage probed by volatile profiling: The role of glass transition temperature.

During storage, common beans are susceptible to ageing leading to quality changes, in particular their cooking quality. In this study, kinetics of evolution of volatile compounds was assessed in order to gain insight into possible reactions occurring during ageing of beans. The evolution of volatile compounds of red kidney beans stored at varying conditions of temperature and moisture content relative to their glass transition temperature (Tg) were evaluated. Storage conditions highly influenced the evolution of volatile compounds whereby more volatile compounds and higher concentrations were detected in beans stored at higher temperature and moisture content. The volatile marker compounds identified are typical for protein degradation and lipid oxidation reactions, although for beans stored at the highest moisture contents (12.8 and 14.5%) the compounds obtained do not allow to exclude microbial activity. The rate of evolution of selected volatile marker compounds was highly correlated (benzaldehyde (r = 0.58), acetic acid (r = 0.75), 1-propanol,2-methyl (r = 0.84) and 2-butanone (r = 0.89)) with storage above Tg signifying that the rate and extent of these (bio)chemical reactions can be largely controlled by storing the beans at temperatures not exceeding 20 °C above their Tg. Volatile profiling showed to be an important approach to monitor quality changes of beans during storage by assessing the nature, rate and extent of (bio)chemical reactions occurring.

Kinetics of phytate hydrolysis during storage of red kidney beans and the implication in hard-to-cook development.

In this study, the kinetics of phytate (inositol hexaphosphate, InsP6) hydrolysis by endogenous phytase in red kidney beans stored at varying conditions of temperature (25-42 °C) and moisture content (6.9-14.5%) was determined and the potential role in hard-to-cook (HTC) development was evaluated. In addition, the concept of glass transition temperature (Tg) was assessed and correlated against the rate of phytate hydrolysis. Under the conditions studied, phytate hydrolysis during storage was mainly influenced by storage temperature and time with limited influence of storage moisture content whereby the highest and lowest storage temperatures (42 °C and 25 °C) resulted in the highest and lowest hydrolysis rates (0.058 ± 0.003 and 0.003 ± 0.001 week-1). Hydrolysis of phytate resulted in formation of lower inositol phosphates, inositol pentaphosphate (InsP5) representing an intermediate whose concentration increased with storage time and temperature. The relationship between the rate of InsP6 hydrolysis and storage above the overall Tg (T - Tg) was moisture content dependent implying that this difference did not fully explain InsP6 hydrolysis. Nevertheless, for each moisture content, the rates of InsP6 hydrolysis during storage were strongly correlated (r > 0.98, p < 0.05) with rates of HTC development signifying that InsP6 hydrolysis facilitates HTC development in beans.

Calcium transport and phytate hydrolysis during chemical hardening of common bean seeds.

In this study, two chemical bean seed hardening methods were used to investigate the changes in cooking behavior associated with Ca2+ transport and phytate hydrolysis to better understand their role in the pectin-cation-phytate hypothesis. The texture evolution of fresh and hardened red kidney beans was evaluated, hardening being induced by soaking or in a CaCl2 solution (0.01 M, 0.05 M, 0.1 M) or sodium acetate buffer (0.1 M, pH 4.4, 41 °C). The beans soaked in a CaCl2 solution at higher concentrations or in sodium acetate buffer for a longer time exhibited a delayed cooking behavior. This study also explored the bio-chemical changes (calcium content in different bean substructures, phytate content and the pectin degree of methylesterification (DM) in the cotyledons) occurring in the beans during chemical hardening and cooking. The Ca2+ concentrations in the whole beans and cotyledons of beans soaked and cooked in CaCl2 solutions significantly increased while inositol hexaphosphate IP6 content showed no significant changes. This indicates that the delayed texture drop in this case results from the influx of exogenous Ca2+ in the cotyledons and seed coats during cooking while the IP6 was not hydrolyzed and did not release endogenous Ca2+. For beans soaked in sodium acetate buffer, phytate profiling showed increased hydrolysis of IP6 with longer soaking time, suggesting the migration of endogenous Ca2+ released from phytate hydrolysis contributing to the delayed cooking of these beans. These results indicate that both an exogenous Ca2+ influx during soaking and cooking and an endogenous Ca2+ replacement resulting from phytate hydrolysis can play an important role in the hardening of beans. In neither of the cases, a significant change in pectin DM was observed during chemical hardening, therefore limiting the delayed cooking to the role of Ca2+ transport. The outcome of both cases is inline with the basic principles of the pectin-cation-phytate hypothesis whereby pectin DM changes are hardly involved and different mechanisms of release/transport are involved.

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.

An integrated kinetic and polymer science approach to investigate the textural stability of red kidney beans during post-harvest storage and subsequent cooking.

Evaluation of food quality and stability during storage and processing necessitates understanding the kinetics of food functional property changes and the underlying reactions. In this study, textural stability of beans during storage and subsequent cooking was evaluated through an integrated kinetic approach. Red kidney beans stored for different periods at various conditions of temperature (25 °C - 42 °C) and moisture content (6.9% - 14.5%) relative to their glass transition temperature (Tg) (above and around the Tg) were studied in detail. Consequently, kinetics of softening during subsequent cooking were investigated and the dependence of these rate constants on storage time (representing the hard-to-cook (HTC) development rate) as a function of storage temperature and moisture content was evaluated. All parameters investigated, moisture content, temperature and storage time, had a significant (p < 0.05) influence on the cooking rate of beans. It was revealed that the HTC development rate of beans during storage increases with increase in temperature and moisture content, these parameters showing a synergistic effect. In addition, the rate of HTC development during storage of beans was controlled by the difference between storage temperature and Tg, showing the important role of glass transition in textural stability of beans during storage.

Insight into pectin-cation-phytate theory of hardening in common bean varieties with different sensitivities to hard-to-cook.

In this study, a detailed quantitative analysis of the mechanisms linked with pectin-cation-phytate hypothesis of hard-to-cook development (HTC) was evaluated to assess the plausibility of this hypothesis. Several common bean varieties with varying sensitivities to HTC were characterized for pectin, cell wall bound calcium and inositol hexaphosphate (InsP6) content before and after ageing. Ageing resulted in a significant decrease in InsP6 content (resulting in calcium release) in all varieties. Despite not significantly changing during ageing, the cell wall bound calcium content significantly increased in most aged bean varieties upon short cooking indicating enhanced internal cation migration during the early phase of cooking in contrast to during ageing and soaking. Among the parameters evaluated in this study, the relative changes in InsP6 content significantly correlated with the change in cooking times as well as changes in cell wall bound calcium content. Results obtained in this study suggest that in some bean varieties, pectin-cation-phytate hypothesis is the predominant mechanism by which hardening occurs during storage while in other varieties, the role of other factors such as phenolic crosslinking as suggested in literature cannot be ruled out.

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.

Modified Rhamnogalacturonan-Rich Apple Pectin-Derived Structures: The Relation between Their Structural Characteristics and Emulsifying and Emulsion-Stabilizing Properties.

In the context of the increasing interest in natural food ingredients, the emulsifying and emulsion-stabilizing properties of three rhamnogalacturonan-rich apple pectin-derived samples were assessed by evaluating a range of physicochemical properties. An apple pectin (AP74) was structurally modified by a ß-eliminative reaction to obtain a RG-I-rich pectin sample (AP-RG). Subsequent acid hydrolysis of AP-RG led to the generation of pectin material with partially removed side chains (in particular arabinose depleted) (AP-RG-hydrolyzed), thus exhibiting differences in rhamnose, arabinose, and galactose in comparison to AP-RG. All samples exhibited surface activity to some extent, especially under acidic conditions (pH 2.5). Furthermore, the viscosity of the samples was assessed in relation to their emulsion-stabilizing properties. In a stability study, it was observed that the non-degraded AP74 sample at pH 2.5 exhibited the best performance among all the apple pectin-derived samples evaluated. This emulsion presented relatively small oil droplets upon emulsion production and was less prone to creaming than the emulsions stabilized by the (lower molecular weight) RG-I-rich materials. The AP-RG and AP-RG-hydrolyzed samples presented a slightly better emulsion stability at pH 6.0 than at pH 2.5. Yet, neither pectin sample was considered having good emulsifying and emulsion-stabilizing properties, indicated by the presence of coalesced and flocculated oil droplets.

The Impact of Drying and Rehydration on the Structural Properties and Quality Attributes of Pre-Cooked Dried Beans.

Fresh common beans can be made 'instant' to produce fast-cooking beans by first soaking and cooking the beans before drying to create a shelf-stable product that can be rehydrated at the time of use. This study investigated the interplay between the drying process (air, vacuum and freeze drying), the microstructure and functional attributes of rehydrated pre-cooked beans. The microscopic study revealed that the three different drying techniques resulted in distinctly different microstructures, with the freeze drying process resulting in highly porous materials, while the air- and vacuum-dried samples underwent shrinkage. Additionally, the rehydration behavior (modeled using empirical and diffusion models) demonstrates that the high rehydration rate of freeze-dried beans is due to capillarity, while rehydration, in the case of air- and vacuum-dried beans, is primarily diffusion-controlled. Irrespective of the drying technique, the high rehydration capacity supports little to no structural collapse or damage to the cell walls. The color and texture of the rehydrated beans did not differ greatly from those of freshly cooked beans. The total peak area of the volatiles of rehydrated beans was significantly reduced by the drying process, but volatiles characteristic of the cooked bean aroma were retained. This new understanding is beneficial in tailoring the functional properties of pre-cooked dry convenient beans requiring short preparation times.

Thermal treatment of common beans (Phaseolus vulgaris L.): Factors determining cooking time and its consequences for sensory and nutritional quality.

Over the past years, the shift toward plant-based foods has largely increased the global awareness of the nutritional importance of legumes (common beans (Phaseolus vulgaris L.) in particular) and their potential role in sustainable food systems. Nevertheless, the many benefits of bean consumption may not be realized in large parts of the world, since long cooking time (lack of convenience) limits their utilization. This review focuses on the current insights in the cooking behavior (cookability) of common beans and the variables that have a direct and/or indirect impact on cooking time. The review includes the various methods to evaluate textural changes and the effect of cooking on sensory attributes and nutritional quality of beans. In this review, it is revealed that the factors involved in cooking time of beans are diverse and complex and thus necessitate a careful consideration of the choice of (pre)processing conditions to conveniently achieve palatability while ensuring maximum nutrient retention in beans. In order to harness the full potential of beans, there is a need for a multisectoral collaboration between breeders, processors, and nutritionists.

Evaluation of storage stability of low moisture whole common beans and their fractions through the use of state diagrams.

A material science approach was explored towards understanding storage stability of common dry bean seeds. State diagrams of powders from distinct bean varieties were generated through determination of their glass transition temperatures (Tgs) using differential scanning calorimetry. Confronting the state diagrams with dry matter-temperature combinations during storage facilitated establishing the link between the relative position of the bean storage conditions along the Tg line and extent of hard-to-cook (HTC) development. Generally, Tg increases with dry matter content of the bean powders implying stability at increasingly higher temperatures attributed to the reduced plasticizing effect of water. Whereas Tg lines of powders of the different bean varieties were very similar, distinct differences were observed for the powders of bean substructures. At a given moisture content, the Tg of the cotyledon material was lower than that of the seed coat material and the Tg values of the whole bean powders were dominated by the cotyledon material. Cooking time analysis showed that whole beans stored above their Tg developed the HTC defect, this extent being correlated with the difference between storage temperature and Tg value. Considering the HTC development rate, (R-value, rate of change in cooking time with storage time over a period of 0-4 months or at 0 months of storage) the higher the difference between the storage temperature and the Tg value, the faster the change in cooking time during storage. Exploring the role of the major polymer components of bean cotyledon revealed that at a given moisture content, the cell wall material showed the lowest Tg values compared to the protein and starch isolates (Tg cell wall < Tg protein < Tg starch isolate). Confronting these values with the HTC development rates (change of cooking time with storage time) supports involvement of the cell wall material and probably protein changes in the development of this defect.

Microscopic evidence for pectin changes in hard-to-cook development of common beans during storage.

In this study, pectin changes during Red haricot bean storage under high temperature and high humidity conditions were investigated to understand the hard-to-cook (HTC) development from a microstructural point of view. First, to ensure repeatability of the microscopy results, a classification of the fresh and stored beans (aged at 35 °C and 83% relative humidity) into different hardening levels (the Non-aged, Aged and Very-hard aged sample) was performed based on the texture values of cooked half-cotyledons. Cell wall strength of the cotyledons was evaluated, showing that the aged samples (HTC seeds) exhibit stronger cell walls with more/stronger pectic cross-linkages than the Non-aged sample. After a sequential pectin extraction aiming at removing pectin fractions of different solubility, cell wall autofluorescence and immunolabeling of JIM7, LM9 and 2F4 epitopes in the residual materials were examined. Upon ageing, the samples exhibited an increased Ca2+-pectin and ferulic acid-pectin crosslinking, these pectic complexes being accumulated primarily at the intercellular spaces. The results suggest a contribution of both the pectin-cation-phytate hypothesis and the involvement of phenolic-pectin crosslinks in HTC development at the cotyledon during storage of common beans.

Arabinoxylan, ß-glucan and pectin in barley and malt endosperm cell walls: a microstructure study using CLSM and cryo-SEM.

The architecture of endosperm cell walls in Hordeum vulgare (barley) differs remarkably from that of other grass species and is affected by germination or malting. Here, the cell wall microstructure is investigated using (bio)chemical analyses, cryogenic scanning electron microscopy (cryo-SEM) and confocal laser scanning microscopy (CLSM) as the main techniques. The relative proportions of ß-glucan, arabinoxylan and pectin in cell walls were 61, 34 and 5%, respectively. The average thickness of a single endosperm cell wall was 0.30 µm, as estimated by the cryo-SEM analysis of barley seeds, which was reduced to 0.16 µm after malting. After fluorescent staining, 3D confocal multiphoton microscopy (multiphoton CLSM) imaging revealed the complex cell wall architecture. The endosperm cell wall is composed of a structure in which arabinoxylan and pectin are colocalized on the outside, with ß-glucan depositions on the inside. During germination, arabinoxylan and ß-glucan are hydrolysed, but unlike ß-glucan, arabinoxylan remains present in defined cell walls in malt. Integrating the results, an enhanced model for the endosperm cell walls in barley is proposed.