Optimization of linoleic acid emulsion preparation to reduce substrate losses after filter-sterilization

Introduction: Probiotic strains have demonstrated the capacity to convert linoleic acid (LA) into conjugated linoleic acid (CLA) isomers, a group of conjugated fatty acids well-characterized for its bioactive properties1. In vitro studies focused on CLA microbial production normally test the capacity of potential CLA-producing strains by culturing them in the presence of the precursor substrate, which in this case is LA2. In most assays the LA is added as a stock solution prepared with pure LA and an emulsifier, usually Tween 80, at specific concentrations, dissolved in distilled water. Afterwards the mixture is sterilized through filtration3,4. However, this preparation procedure leads to LA losses requiring higher amounts of LA to achieve the intended concentration. Objectives: The main aim of this work was to optimize the LA emulsification strategy in order to obtain a more efficient and cost-effective procedure. To do so the following specific objectives were pursued: Test the impact of treating LA emulsion with Ultra-Turrax prior to filtration; Test the impact of treating LA emulsion with Sonicator prior to filtration; Test the profitability of the more suitable treatment procedure when applying a smaller pore sized membrane that assures microbiological sterility. Results: Filtration of LA emulsion directly through a 0.45 μm-pore size membrane (F4) led to a 17.09% LA loss whereas the introduction of a blending step, either using a sonicator (C) or an Ultra-Turrax (A and B) led to lower losses of 13.01%, 7.17% and 7.40%, respectively. Since there were no significant differences between treatments A and B (p>0.05) with Ultra-Turrax, which demonstrated the lowest LA losses, treatment B was chosen to be further tested with a smaller pore size filter of 0.20 μm (D). The application of a smaller pore size membrane also contributed to lower losses; LA reductions were from 10.07% (F2) to 3.71% (D). Conclusions: In conclusion, a previous dispersion with Ultra-Turrax, independent of the filter pore size, demonstrated to be the best method to reduce substrate losses in filter-sterilization of LA emulsions. This brings new insights for more cost-effective CLA producing assays.