Enzyme-assisted extraction of stabilized chlorophyll from spinach

Zinc complex formation with chlorophyll derivatives in spinach pulp was studied by adding 300 ppm Zn²⁺ for production of stable food colorant, followed by the heating at 110 °C for 15 min. Zinc complex formation increased at pH values of 7.0 or greater. Pectinex Ultra SP-L was selected for enzyme-assisted release of zinc-chlorophyll derivatives from spinach pulp. Effect of enzyme concentration (1-9%), treatment temperature (30-60 °C), and time (30-210 min) on total chlorophyll content (TCC) were optimized using response surface methodology. A quadratic regression model (R² = 0.9486) was obtained from the experimental design. Optimum treatment conditions were 8% enzyme concentration, 45 °C, and 30 min, which yielded a 50.747 mg TCC/100 g spinach pulp. Enzymatic treatment was followed by solvent extraction with ethanol at a solvent-to-sample ratio of 2.5:1 at 60 °C for 45 min for the highest TCC recovery. Pretreatment with enzyme and extraction in ethanol resulted in 39% increase in Zn-chlorophyll derivative yield.