Monday, April 30, 2007

Novel nisin purification method using a two-phase aqueous micellar systems

Angela F. Jozala, André M. Lopes, Priscila G. Mazzola, Larissa M. Sato, Stephanie G. Liu, Thereza C. V. Penna, and Adalberto Pessoa Jr. Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutics Sciences, Avda. Professor Lineu Prestes, 580, Bloco 16, São Paulo, 05508-900, Brazil

Nisin is a natural additive for conservation of food, pharmaceutical and dental products, and can be used as therapeutic agent. This work aims to evaluate a novel method to purify nisin, diminishing production costs and increasing purity. Aqueous two-phase micellar systems (ATPMS) are considered to be promising for bioseparation and purification purposes. In ATPMS phase separation can be induced by relatively small changes in the solution conditions. Nisin activity in two different Triton X114 concentrations (2 and 4%) was verified by the agar diffusion assay utilizing L. sake as a sensitive indicator microorganism. Seven different systems were prepared using standard nisin (SN, 105 AU) into 5mL of distillated water (DW) (i) SN; (ii) SN in 4% Triton; (iii) SN centrifuged in 4% Triton; (iv) SN in 2% Triton; (v) SN centrifuged in 2% Triton; (vi) 5mL DW in 4% triton and (vii) 5 mL DW in 2% Triton. The phase diagram of Triton X114 was measured by the cloud-point method. Buffered solutions of the surfactant, were prepared in graduated test tubes, each solution was placed in a thermo-regulated device at 28oC, for 2h. After equilibrium, the two coexisting micellar phases were withdrawn and nisin activity in each micellar phase was determined. Preliminary verification showed that Triton did not interfere in antimicrobial activity. After phase-separation higher nisin activity was detected in the micelle-rich phase, suggesting preferential partitioning of the biomolecule,  this preliminary investigation the results showed that nisin after partitioning effect stayed in the micelle-rich phase, encouraging further studies using ATPMS.