Extracts from ‘kesinai’ (Streblus asper) leaves were investigated as a potential source of enzymes that can serve as an alternative to calf rennet in cheese making. Different types of extraction buffers were investigated namely sodium acetate buffer (pH 4.2-5.0), phosphate buffer (pH 6.0-7.0) and Tris-HCl buffer (pH 7.0-9.0). Finally, the milk-clotting enzyme was extracted using 100 mM Tris-HCl buffer (pH 7.4) with and without 5.0 mg/mL polyvinylpyrrolidone, 0.015 mL/mL Triton X-100 and 2 mM sodium metabisulphite. Purification was carried out using acetone precipitation, and ion-exchange and size-exclusion chromatographic techniques. Results showed that 100 mM Tris-HCl buffer (pH 7.4) was the most efficient extraction buffer among the buffers used in the extraction study. After the final purification step of size-exclusion chromatography, the enzyme was purified 3.3-fold with 42.3% of recovery. The enzyme showed an optimum temperature and pH at 60°C and pH 7.4, respectively. The enzyme was stable up to 70°C for one hour and the partially purified enzyme retained 83% and 96% of its original activity at pH 6.0 and 8.0, respectively. The molecular weight of the partially enzyme was estimated to be 75.8 kDa on SDS-PAGE. The milk-clotting activity of ‘kesinai’ enzyme was found to be lower than that of commercial Mucor rennet.
Phytates have been considered as a threat in human diet due to its antinutrients behaviour which
known as strong chelators of divalent minerals such as Ca2+, Mg2+, Zn2+ and Fe2+. Phytic acid has a potential for binding positively charged proteins, amino acids, and/or multivalent cations or minerals in foods. The resulting complexes are insoluble, difficult for humans to hydrolyze during digestion, and thus, typically are nutritionally less available for absorption. The reduction of this phytates can be achieved through both enzymatic and nonenzymatic removal. Enzymatic degradation includes addition of either isolated form of wild-type or recombinant exogenous phytate-degrading enzymes microorganisms in the food matrix. Non-enzymatic hydrolysis of phytate occurred in the final food during food processing or physical separation of phytate-rich parts of the plants seed. The application of phytase with respect to breadmaking process, probiotics, animal feed supplement and transgenic crops are emphasised in this paper.