Non-Conventional Extraction of Bovine Lactoperoxidase From Whey

Abstract

Food spoilage is a global industrial issue, and consumers are vary of the ill-effects of chemical preservatives. Bovine Lactoperoxidase (LP- EC 1.11.1.7), a natural antimicrobial, is used as bio preservative. Industries are pursuing new purification methods as conventional techniques like chromatography and membrane separation suffer drawbacks. The present work investigates the use of non-conventional liquid- liquid extraction (LLE) techniques to extract and purify LP. The stability of LP was explored in various phase forming components of LLE, viz. polymers, surfactants, salts sugars, polyols, and alcohols. The surfactant systems showed compatibility, and the Reverse Micellar Extraction (RME) was studied to extract the LP from aqueous solution using systems formed by ionic and non-ionic surfactant mixtures to reduce the denaturation of LP. Tween series surfactants with Aerosol-OT (bis-(2-ethylhexyl) sulfosuccinate) showed better extraction than Triton and Span series. Complete extraction of LP occurred with the RM formed by 90mM Aerosol-OT/8 mM Tween 80 in isooctane and a maximum of 95.5% back extraction efficiency with 66% active LP recovered using pH of 10.5, 1M KCl, and 60 mM cetyltrimethylammonium bromide system. Further, selective RME of LP was extended to whey. A maximum of 86% LP was extracted from acid whey at pH 9.5 with the addition of 0.2M KCl, using 115 AOTmM / Tween 80 23mM surfactant blends in the organic phase. Active LP of 80% with 112% extraction efficiency was achieved with a stripping phase of 1.5M KCl at pH 10.5 and 60 mM CTAB in the organic phase.Further, Rhamnolipid-based RME was studied to avoid the adverse effect of synthetic surfactants. A novel back extraction strategy using pH-specific protonation – deprotonation of the Rhamnolipid headgroups was used during back extraction. The optimized extraction conditions resulted in 96.65% LP extraction and 85.71% active LP recovery with 8.4 fold purification. The recovered LP from acid whey studies was qualitatively analyzed using RP-HPLC. The antimicrobial activity of the extracted LP showed a good reduction in colony-forming units of S. aureus and specifically exhibited a bacteriostatic effect.