pH-dependent stability of major betalains in the encapsulated beetroot extracts (Beta vulgaris L.)

Betalain is a water-soluble pigment contained in Caryophyllales plants. It not only holds potential as a natural food colorant but also offers various health benefits, acting as an antioxidant. This study focused on analyzing the pH-dependent stability of encapsulated betalain pigments extracted from red beetroot (Beta vulgaris L.) using methods such as absorption spectroscopy, HPLC, and LC-MS. The major pigments identified were vulgaxanthin I, betanin, isobetanin, and neobetanin, alongside minor components, including three betaxanthin species and a degradation product known as betalamic acid. Spectrophotometric analyses revealed that above pH 8, the betalain peak at 435 nm decreased and red-shifted to a peak at 549 nm, a shift that could be reversed through neutral pH treatment. At pH 11, a new broad peak appeared at 410 nm and was identified as betalamic acid. To assess the pH-dependency of each betalain, the targeted betalains were separated and quantified through HPLC after incubation across a wide pH range of 2-11 and during storage. After 3 days of storage in highly alkaline conditions (pH 10-11), major betalains, with the exception of neobetanin, underwent significant degradation. Conversely, these pigments displayed relative stability in acidic conditions. In contrast, neobetanin showed vulnerability to acidic conditions but exhibited tolerance to alkaline pH levels of 10-11. The degradation product, betalamic acid, demonstrated a similar susceptibility to alkaline pH as betanins. In conclusion, the significant stability decrease under highly alkaline conditions results not only from the hydrolytic reaction of betalains but also from the degradation of betalamic acid itself. PRACTICAL APPLICATION: Encapsulation methods are used to enhance the stability of betalains against temperature variations; however, the effects of pH, especially when considering individual betalain species, are not well understood. Despite betalains exhibiting similar features and being suitable for a wide pH range from acid to alkaline conditions, they are significantly affected by alkaline pH levels exceeding 10, as well as by storage duration. This study demonstrated the application of encapsulation to pH-dependent stability, and the findings offer valuable insights and a fresh perspective on betalains as red biocolorants, extending their potential application to a wide range of pH-controlled food products.