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(Bio)chemical insights into the hard-to-cook development of Red haricot beans upon postharvest ageing: Integrating a texture-based bean classification and in situ analytics

Nowadays, the benefits of legume-based foods in the human diet in both developed and developing countries have been recognized. Common beans (Phaseolus vulgaris L.) are the most consumed legume crop worldwide, with rather cheap raw materials of high nutritional value. However, the full benefit of common beans can be greatly hampered by a textural defect, the hard-to-cook (HTC) defect, which results in poor cooking quality of the seeds (delayed softening during cooking and /or hard texture despite prolonged cooking). The HTC defect often develops when beans are stored at high temperature and high relative humidity (RH) conditions, and is greatly attributed to the bean cotyledon, particularly (bio)chemical/structural changes of the biopolymers therein. Among the proposed postulations, the pectin-cation-phytate and phenolic involved hypotheses are the most plausible ones to explain the pathways underlying the HTC development. However, the mechanisms have not been fully elucidated so far. Therefore, the goal of this PhD research was to obtain in-depth (bio)chemical insights into the two most plausible mechanisms underlying storage-induced HTC development of common beans, using Red haricot beans as a model case.

A texture-based bean classification and sample selection approach was applied,  based on the texture values of cooked half-beans/cotyledons, throughout the whole research. The aim of this strategy was to minimize the large bean-to-bean variations within one sample, and to investigate how the texture-related physicochemical properties evolve with the textural development (texture distribution) of fresh and aged beans. Using this approach, detailed analyses on texture-related physicochemical properties in raw beans with known cooking textures (after a certain cooking time), thus the evaluation of the evolution of these properties in relation to the textural changes (texture distribution) of the fresh/aged beans were achieved.

First, pectin changes during Red haricot beans stored at 35 °C and 83% RH for 3 months were investigated to understand the cell wall/pectin changes during HTC development from a microstructural point of view. Using the texture-based bean classification and sample selection, bean samples with different texture levels (Non-aged, Aged and Very-hard aged samples) were generated from the fresh (non-aged) and stored (aged) beans. Consequently, cell wall strength of the cotyledons was evaluated, showing the aged samples (HTC seeds) exhibiting 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 extractability, cell wall autofluorescence and immunolabeling of pectic epitopes in the residual cell materials with JIM7, LM9 and 2F4 were examined. Upon ageing, the aged samples showed increased Ca2+-pectin interactions and feruloylated galactans, these pectic complexes being accumulated primarily at the intercellular spaces. The contribution of both the pectin-cation-phytate hypothesis and the involvement of phenolics in HTC development at the cotyledon during storage of common beans was suggested. Subsequent comprehensive characterization and a cell wall associated phenolic profiling of the pectin-depleted residual cell wall fractions after sequential pectin extractions from cotyledons of the selected Non-aged and Aged samples revealed that ageing induces substantial changes at a cell-wall-structural level, with mainly vanillin, 4-hydroxybenzoic acid and 4-hydroxybenzaldehyde covalently bound with sugar side-chains of pectin and/or involved in lignification-like mechanisms. Fourier transform infrared spectroscopy (FT-IR) coupled with chemometric analysis on the residual cell wall fractions further suggested that lignin-like phenolic-cell wall polymers are present in the cell wall polysaccharide network of the Aged sample, and are therefore contributing factors to the HTC defect observed during bean ageing.

In the context of the pectin-cation-phytate mechanism, detailed phytate and total mineral profiling showed that the inositol phosphate content in cotyledons, particularly InsP6, decreased significantly, along with a significant increase in InsP5, releasing Ca and Mg cations. The HTC development was predominated by storage-induced InsP6 degradation, rather than phytate interconversions during soaking. Scanning electron microscopy coupled with energy dispersive spectrometry (SEM-EDS) based in situ cell wall associated mineral quantification revealed that Ca cations, released by the InsP6 hydrolysis, were bound with the cell wall pectin in aged bean cotyledons, facilitated by demethylesterification of the cell wall pectin and relocation of the cations from the cell interior to the cell wall during storage (rather than soaking). While Mg cations, which also migrate and may bind to the cell wall pectin during storage of the beans, were mostly leached out into the soaking water during subsequent soaking due to their weak binding capacity to the pectin, thus contributing less to the HTC defect of the aged beans.

In addition, the role of the pectin-cation-phytate mechanism in relation to the texture changes during subsequent cooking (wide texture distributions) of Red haricot fresh and aged beans were evaluated, using the texture-based bean classification and sample selection approach. 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, SEM-EDS based in situ mineral microanalysis on individual (raw) beans with known textures suggested 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 (InsP6) hydrolysis during ageing, which was shown to affect the texture distribution of aged beans significantly, being an indicator for the extent of HTC development during bean aging.

Overall, the findings of this PhD work push the boundary further towards gaining novel and in-depth insights into the pectin-cation-phytate and the phenolic involved mechanisms of the HTC development upon common bean ageing. A methodological approach integrating a texture-based bean classification, and detailed in situ analytics was central to this. Moreover, influencing factors (in view of the pectin-cation-phytate hypothesis) on the cooking texture changes (texture distributions after cooking) in raw fresh and aged Red haricot beans were evaluated for the first time. The insights obtained can be useful for bean breeders and producers to explore targeted solutions towards underutilization of the HTC affected beans.

Date:28 Sep 2018 →  5 Dec 2022
Keywords:aging, processing, functional properties, legume based products
Disciplines:Biomaterials engineering, Biological system engineering, Biomechanical engineering, Other (bio)medical engineering, Environmental engineering and biotechnology, Industrial biotechnology, Other biotechnology, bio-engineering and biosystem engineering, Other chemical sciences, Nutrition and dietetics , Agricultural animal production, Food sciences and (bio)technology, Microbiology, Systems biology, Laboratory medicine
Project type:PhD project