A study on the properties of some plant milk-clotting enzymes

This study investigated some native Egyptian plants as potential sources for Milk-Clotting Enzyme (MCE), to find a viable rennet substitute for cheese making. Seven plants, (Zingiber officinale, Aloe variegata, Pergularia tomentosa, Solanum elaeagnifolium, Moringa oleifera, Ananas comosus, and Actinidia deliciosa) were screened for potent MCE activity. Pergularia tomentosa extracts demonstrated the highest activity, yielding 53.90 U/mL in clottinga activity of 385 U/mg and the optimal MCA/PA ratio of 23.33 U/mL, Partial purification using fractionation by ammonium sulfate, in particular, the maximum MCE activity (108.17 U/mL) was obtained at the 40–50% saturation fraction, specific activity 470.30, and a 1.31-fold purification rate. Subsequent purification using DEAE-Sepharose, Sephadex G-100, and Sephadex G-200 chromatography resulted in a single active peak (at fractions 37 and 36). The overall purification achieved an 8.54-fold increase with 1.22 recoveries. The purified enzyme exhibited maximum MCA at pH 6.0 and 50°C MCA was enhanced by CaCl₂ up to 10 mM but sharply declined with NaCl concentrations over 16%. The MCE maintained stability between 50°C and 60°C. Stability over 15 days was significantly better at 4°C (6.3%) compared to room temperature (51.53%). The study concluded that the MCE extracted from Pergularia tomentosa can be successfully utilized in place of microbial rennet while making Damietta and Tallaga cheese without producing visible flaws while being stored.