Liquid chromatography mass spectrometry (LCMS) analysis

The LC–MS analysis was conducted in negative ion mode using a Q-TOF Premier model from Waters. The dry biosurfactant was dissolved in an acetonitrile solution and subsequently injected into the LC–MS system, which consisted of a 1260 Infinity LC instrument coupled with a 6410 Triple Quadrupole MS from Agilent Technologies, USA. Mass spectra were acquired within the m/z range of 100–1000, following the procedures described by Goveas et al.³⁰ and Sharma et al.³¹. In this study, the LC–MS analysis method employed an MS detector with an ESI ( +) ion source and an MS analyzer in the form of a Q-Tof, enabling the generation of chromatographic peaks. Subsequently, the chromatographic peaks were subjected to analysis using the Masslynx program, as detailed by Sommeng et al.³².

The Box-Behnken experimental design was selected to investigate the impact of temperature, pH, and salinity on the stability of the biosurfactant activity. The design included three levels for each factor and was conducted in triplicate, resulting in a total of 45 experimental runs, as outlined by Kumar et al.³³. Both coded and actual variables were utilized and are presented in Table ​Table1.1. The statistical analysis was performed using MinitabTM 19.0. The main effects and two-factor interactions were estimated through variance analysis (ANOVA), which involved Fisher and Student’s t-tests with a significance level (α) of 0.05, following the approach described by Montgomery³⁴. The response variable utilized in this study was the emulsification index (EI24) of the biosurfactant. The EI24 was determined after 24 h, serving as an indicator of the biosurfactant’s emulsifying ability.

Box-Behnken 3³ factorial design with actual/coded values and results.

X₁ = temperature (^oC); X₂ = pH; X₃ = Salinity (%).