Purification of lipopeptide biosurfactant extracts obtained from a complex residual food stream using Tricine-SDS-PAGE electrophoresis

Abstract

Protocols to identify lipopeptide biosurfactant extracts contained in complex residual streams are very important, as fermented agri-food matrices are potential sources of these valuable compounds. For instance, corn steep liquor (CSL), a secondary stream of the corn wet-milling industry, is composed of a mixture of microbial metabolites, produced during the corn steeping process, and other natural metabolites released from corn, that can interfere with the purification and analysis of lipopeptides. Electrophoresis could be an interesting technique for the purification and further characterization of lipopeptide biosurfactant extracts contained in secondary residual streams like CSL, but there is little existing literature about it. It is necessary to consider that lipopeptide biosurfactants, like Surfactin, usually are substances that are poorly soluble in water at acidic or neutral pH, forming micelles what can inhibit their separation by electrophoresis. In this work, two lipopeptide biosurfactant extracts obtained directly from CSL, after liquid–liquid extraction with chloroform or ethyl acetate, were purified by applying a second liquid extraction with ethanol. Following that, ethanolic biosurfactant extracts were subjected to electrophoresis under different conditions. Lipopeptides on Tricine-SDS-PAGE (polyacrylamide gels) were better visualized and identified by fluorescence using SYPRO Ruby dye than using Coomassie blue dye. The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of lipopeptide isoforms separated by electrophoresis revealed the presence of masses at 1,044, 1,058, and 1,074 m/z, concluding that Tricine-SDS-PAGE electrophoresis combined with MALDI-TOF-MS could be a useful tool for purifying and identifying lipopeptides in complex matrices.

Regulation, Biosynthesis, and Extraction of Bacillus-Derived Lipopeptides and Its Implications in Biological Control of Phytopathogens

“…Identifying lipopeptides at the protein level can be challenging but is essential for understanding their functions and potential applications. When biosurfactant extracts are derived from intricate mixtures following liquid-liquid extraction using organic solvents or precipitation, using mass spectrometry immediately after extraction might not be the most favorable approach [136]. This is primarily due to the potential interference of numerous substances within the extract, resulting in spectra with a high abundance of signals.…”

“…Within these spectra, the masses corresponding to lipopeptide biosurfactants are obscured by those of other metabolites, leading to the observation of weak signals for lipopeptides [137]. In this context, the utilization of electrophoresis as a preliminary step before mass spectrometry analysis may present an intriguing method for both purifying and identifying lipopeptides, particularly within intricate sample matrices [136]. Thus, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a widely used technique for the detection and separation of proteins and peptides, and it can also be applied to the analysis of lipopeptides [138].…”

“…This method can reveal the presence and approximate molecular weight of lipopeptides in a sample. Additionally, by transferring the separated lipopeptides to a membrane and employing specific antibodies or stains, it is possible to further characterize and confirm the identity of lipopeptides, making SDS-PAGE a valuable tool for the analysis and detection of these compounds in complex samples [136].…”

Techno-economics analysis of biosurfactants productions from pseudomonas aeruginosa OPEFB-based with variations in purifications method

“…The high demand for biosurfactant production and high production costs have caused many studies to focus on the optimization of production design, extraction process, purification, and characterization [6]. Research by Moldes [7] has identified the performance of the solvent extraction method in the purification process of surfactants, which is this method helps to purify and characterize biosurfactants in complex metrics. Another method that can be used in the purification process is adsorption/desorption chromatography.…”