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.