In the context of WP 7, Sphera Encapsulation received from the University of Seville (USE) a total of 11 crude extracts from halophilic bacteria and a purified compound from the University of Athens (UoA) characterised for biosurfactant activity.
The critical micelle concentration (CMC) and the emulsification performances were used as parameters to screen and compare the efficiency of the fractions to better understand the functional properties of these naturally occurring surfactants, and their potential applications in different industries such as the nutraceutical, cosmetics, agrochemical or pharmaceutical.
Among the biosurfactants (BSs) analyzed, those with a CMC below 0.1 mg/ml were selected. This threshold was selected since it was close to the one of most of the commercial surfactants (e.g., SDS: 0.142 ± 0.020 mg/mL and Sucrose ester: 0.139 ± 0.005 mg/mL). BSs from USE-15 (M63-6), USE-15 (M63-9), and USE-20 (BMB-9) crude extracts, and the purified compound NM1-5 from UoA, demonstrated significantly lower CMC values, even seven times lower than the commercial standards (e.g., respectively for BSs from USE-15 (M63-6), USE-15 (M63-9), and USE-20 (BMB-9), and the purified compound NM1-5: 0.027 mg/ml, 0.033 mg/ml, 0.0620 mg/ml, and 0.027 mg/ml ).
The selected BSs were evaluated in o/w emulsion (1:9 ratio respectively) at a concentration of the crude extract of 0,5 mg/ml, and the results were analyzed by considering the phase separation as an instability phenomenon and the particle size of the generated emulsions.
All the obtained emulsions were characterized by a particle size lower than 2 µm, but the ones stabilized by the BSs from USE-15 (M63-9), USE-20 (BMB-9), and NM1-5 showed a clear loss of stability over time. Indeed, after 7 days at room temperature, a layer of water was visible at the bottom of the tube with a layer of free oil accumulated on the top. In contrast, emulsions stabilized by the USE-15 (M63-6) crude extract exhibited a stable emulsion layer with a detectable small layer of free oil. Still an encouraging result since the presence of free oil can be correlated to the needs of using a higher concentration of the surfactant, information that is still under elaboration from USE.

It is important to underline that despite exhibiting similar CMC values, the tested BSs showed markedly different emulsification properties. Among them, the BS crude extract from USE-15 (M63-6) demonstrated the highest emulsification efficiency, making it a particularly promising candidate for further investigation. It will be important to gather more information about the molecular structures of the BS contained in the crude extract and their concentrations to clarify better how these BS work and their optimal concentration of use. These factors may significantly influence the observed functional properties. Therefore, further structural and compositional analyses will be essential to fully understand the mechanisms underlying their activity and to unlock their potential for industrial and environmental applications.