Betalains
Betalains pigments are also water-soluble and usually localized in a unique organelle of plant cell: the vacuole. So far, betalain synthesis has only been observed in plants belonging to the taxonomic group of Centrospermae (red beet, etc.) [122]. The pH-dependent ionization of betalains provided the rationale for the release technique described by Mukundan et al. [94]: Betacyanins and betaxanthins are cations below pH 2.0, zwitterions at pH 2.0, monoanions between pH 2.0 and pH 3.5 and bisanions above pH 3.5 and 7.5 [114]. At pH 2, the zwitterionic state might permit diffusion and release from the vacuole. This behavior is similar to ion trapping where metabolites, such as alka- loids, are retained within the vacuole as a result of acquiring a net charge at the acidic vacuolar pH [112]. The observed release kinetics of betalains extends well beyond the low-pH treatment, which suggests that the observed mechanism involves an alteration in membrane properties and possibly preferential death of the pigment-containing cortical cells. This release kinetics is actually fortuitous in terms of recovery since betalains are most stable between pH 3.5–6.5 and have limited stability at pH 2 [98]. Therefore, the procedure of acidic exposure followed by release of the pigments into B5 medium [46] of pH 5.5 facilitates recovery under stable conditions. In this regard, the low-pH-mediated release of betalains is substantially different from that reported previously, where alterations of medium pH have been used to facilitate release of
secondary metabolites from plant tissue culture [64, 118]. For alkaloids in particular, the acid dissociation constant (pKa) is sufficiently close to the physiological pH for the medium pH to be altered without necessarily disrupting the tissue. Under these conditions, concepts of equilibrium partitioning can be useful to describe release [112], and the acidified medium pH must be maintained throughout the release period.