Involvement of phenolic compounds in the middle lamella
The “phytase-mineral and mineral-pectin interaction” and “lignification” theories in HTC defect in legumes suggest formation of new interactions or bonds in the middle lamella polymers that make cell separation difficult during cooking. Researchers have proposed that phenolic compounds other than lignin in the middle lamella may also form insoluble complexes with pectin and proteins that could impair cell separation (Srisuma et al., 1989).
Srisuma et al. (1989) observed increased free hydroxycinnamic acids during HTHH storage of beans with no increase in lignin content. The increase in free hydroxycinnamic acids was associated with increased hardening (Srisuma et al., 1989). The free hydroxycinnamic acids could have been synthesized de novo from free aromatic amino acids liberated from hydrolysis of proteins during HTHH storage (Srisuma et al., 1989). Hohlberg & Stanley (1987) reported an increase of aromatic amino acids as a result of storage proteins hydrolysis during HTHH storage of beans. Aromatic amino acids such as phenylalanine and tyrosine are immediate precursors of hydroxycinnamic acids (C6-C3 molecules) biosynthesis via phenylalanine and tyrosine ammonia lyases (Whetten et al., 1998). It is suggested that the increase in free phenolic acids observed during HTHH storage could promote protein-phenol interaction in the middle lamella therefore resulting in increased protein hydrophobicity (Srisuma et al., 1989). Free phenolic acids have a high affinity for interacting with proteins. The increased protein hydrophobicity could inhibit water imbibition restricting water uptake and impairing cell separation during cooking (Srisuma et al., 1989; Machado, Ferruzzi & Nielsen, 2008; Pirhayati, Soltanizadeh & Kadivar, 2011). It is proposed that free phenolic acids provides phenolic compounds for cross-linking to pectin in middle lamella and/or proteins that could result in HTC defect development (Srisuma et al., 1989; Garcia et al., 1998a). However, there seem to be no experimental study investigating the role of phenol-protein interaction in HTC defect development.
Garcia et al. (1998a) observed that phenolic acids bound to the water soluble pectin fraction were three times higher in HTC beans (Phaseolus vulgaris) than in control beans. Maurer et al. (2004) also observed more phenolic compounds in the soluble pectin fraction of HTC beans when compared to control. The observation of more ferulic acid bound to soluble pectin in HTC beans could inhibit cell separation during cooking as a result of crosslinking (Garcia et al., 1998). Ferulic acid has been implicated in cross-linking cell wall polysaccharides leading to increased inter cell adhesion (Brett & Waldron, 1996)). Hydroxycinnamic acids are reported to cross-link plant cell wall polymers, especially polysaccharides and lignin (Ralph, Bunzel, Marita, Hatfield, Lu, Kim, Schatz, Grabber & Steinhart, 2004). Ferulic acids esterified to pectin can form diphenyl or ether bonds between the hydroxyl groups of phenolic compounds and the hydroxyl groups on polysaccharides (Shiga, Cordenunsi & Lajolo, 2011). According to Garcia, Filisetti, Udaeta & Lajolo (1998b), presence of more ferulic acid bound to soluble pectin, if involved in cross-links with other polysaccharides could ultimately lead to changes in cell adherence therefore leading to HTC defect by imparing cell separation upon cooking. As reported, resistance to softening even in the presence of chelating agents e.g. EDTA, suggests that crosslinking of pectic polymers via calcium ions is not the only factor limiting cell cell separation during thermal treatment of plant parenchyma cells (Parker & Waldron, 1995; Waldron, Ng, Parker & Parr, 1997; Marry, Roberts, Jopson, Huxham, Jarvis, Corsar, Robertson & McCann, 2006). According to Waldron et al. (1997), lack of thermally induced cell separation in plant tissues often suggests “secondary thickening and associated lignification”. However in nonlignified, thin walled plant tissues, failure to soften or extremely slow softening during cooking of parenchyma-rich plant tissues has been reported (Waldron et al., 1997). The mechanism leading to thermal stability of these tissues has been suggested to be linked to the presence of ferulic acid dimers that crosslink cell wall polymers such as pectin (Parker & Waldron, 1995; Waldron et al., 1997). This crosslinking is suggested to result from the activity of cell wall peroxidase (Biggs & Fry, 1987; Wallace & Fry, 1995; Brett & Waldron, 1996). Although increased phenolic acid bound to pectin has been observed during HTHH storage of beans (Garcia et al., 1998b; Maurer et al., 2004), there are no studies showing the proposed possible crosslinks with other polysaccharides in the middle lamella.
The role of starch and protein in HTC development
According to Hentges et al. (1991) starch could contribute to HTC phenomena because of alterations observed in starch. In a study to investigate possible changes on starch as a result of the HTC phenomena in common beans (Phaseolus vulgaris), Garcia & Lajolo (1994) observed more birefringence in starch granules of HTC beans. Differential scanning calorimetry (DSC) thermograms showed a 10% increase in starch gelatinisation temperature between the control (64.2 oC) and HTC beans (72.9 oC). Such an increase could be attributed to an increase in starch granule crystallinity or lower water availability which is necessary for gelatinization (Yousif et al., 2007). Chemically hardened kidney beans were shown to exhibit high transition temperatures and enthalpy of gelatinisation of starch (Kaur & Singh, 2007). However, Hohlberg & Stanley (1987) found no differences for melt temperature, gelatinisation energy in isolated starch as a result of storage time or conditions. In the parenchyma cells in the cotyledon of cowpea seed, starch granules are embedded in a proteinaceous matrix (Sefa-Dedeh & Stanley, 1979). It has been suggested that during storage at high temperature and relative humidity there is a decrease in the solubility and thermal stability of protein (Hohlberg & Stanley, 1987; Liu et al., 1992a). These could be due to enzymatic (proteases) hydrolysis (Liu et al., 1992a). Thus, during cooking, protein coagulation/gelation is expected to occur before starch gelatinisation due to the reduction in the protein thermal transition temperature of stored cowpea seeds. This would lead to formation of a protein network around the starch granules, which would act as a water barrier thus leading to reduction of water available for starch gelatinization (Yousif et al., 2007). Liu et al. (1992a) observed that the thermal denaturation temperature (Tm) of unstored cowpea seeds was higher than 100 oC with no coagulation observed while after 18 months of storage, the Tm was 56 oC. A significant decrease in the protein denaturation enthalpy of common black beans has been observed on HTC beans (Garcia-Vela & Stanley, 1989).
1 INTRODUCTION AND PROBLEM STATEMENT
2 LITERATURE REVIEW
2.1 Physical, Structure and chemical composition of cowpeas
2.2 Physico-chemical changes during cooking of cowpeas
2.3 Hard-shell defect
2.4 Hard-to-cook phenomenon
2.5 Prevention of HTC defect
2.6 Gaps in knowledge
3.1 Hard-to-cook phenomenon in cowpeas: effect of accelerated storage on cooking and physico-chemical characteristics of cowpeas
3.2 Micronisation and hot-air roasting of cowpeas as pre-treatments to control the
development of hard-to-cook phenomenon
3.3 Micronisation and hot-air roasting in controlling hard-to-cook phenomenon in cowpeas: structural and physico-chemical changes
4 GENERAL DISCUSSION
4.1 Critical review of experimental design and methodologies
4.2 The effect of high temperature and high relative humidity storage on HTC defect development in cowpeas
4.3 The effects of micronisation and hot-air roasting of cowpeas as pre-treatments in the control of hard-to-cook phenomenon during HTHH storage
5. CONCLUSIONS AND RECOMMENDATIONS