a b s t r a c tTwo methodologies were compared to encapsulate nisin in liposomes of partially purified soybean phosphatidylcholine: reversed-phase and hydration film. In the hydration film method, both probe-type and bath-type ultrasound were evaluated. The size of liposomes was evaluated by light scattering analysis and residual antimicrobial activities by agar diffusion assay using Listeria monocytogenes ATCC 7644 as indicator strain. The size of liposomes prepared by reversed-phase, hydration film using probe-type and bath-type ultrasound were 190, 181 and 148 nm with residual antimicrobial activities after encapsulation of 25%, 50% and 100%, respectively. The methodology of film hydration using bath-type ultrasound was chosen for assessment of its physicochemical characteristics. Nisin had entrapment efficiency of 94.12%. Measured Zeta potentials for unfiltered and filtered (0.22 lm) liposomes were À55.8 and À54.5 mV, respectively. The antimicrobial activity of free nisin, encapsulated nisin and filtered was evaluated for a period of 24 days. It was observed that the free nisin remained 100% of residual activity while the liposomes containing nisin were losing their antimicrobial activity over time reaching 25% residual activity after 10 days. The size (132-149 nm) and pH (4.5) remained constant over time. It was observed by microscopy that the liposomes maintained their spherical morphology. The stability observed by size and pH was not the same regarding antimicrobial activity and Zeta potential, indicating that the liposomes should be applied shortly after its preparation.

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Partially ordered stacks of phospholipid bilayers on a flat substrate can be obtained by the evaporation of a spread droplet of phospholipid-in-chloroform solution. When exposed to an aqueous buffer, numerous micrometric buds populate the bilayers, grow in size over minutes, and eventually detach, forming the so-called liposomes or vesicles. While observation of vesicle growth from a hydrated lipid film under an optical microscope suggests numerous events of vesicle fusion, there is little experimental evidence for discriminating between merging of connected buds, i.e., a shape transformation that does not imply bilayer fusion and real membrane fusion. Here, we use electroformation to grow giant unilamellar vesicles (GUVs) from a stack of lipids in a buffer containing either (i) nanometric liposomes or (ii) previously prepared GUVs. By combining different fluorescent labels of the lipids in the substrate and in the solution, and by performing a fluorescence analysis of the resulting GUVs, we clearly demonstrate that merging of bulges is the essential pathway for vesicle growth in electroformation.

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