Updated component analysis method for naturally occurring sophorolipids from Starmerella bombicola

Abstract

Sophorolipids (SLs) are promising glycolipid biosurfactants as they are easily produced and functional. SLs from microorganisms are comprised of mixtures of multiple derivatives that have different structures and properties, including well-known acidic and lactonic SL (ASLs and LSLs, respectively). In this study, we established a method for analyzing all SL derivatives in the products of Starmerella bombicola, a typical SL-producing yeast. Detailed component analyses of S. bombicola products were carried out using reversed-phase high-performance liquid chromatography and mass spectrometry. Methanol was used as the eluent as it is a good solvent for all SL derivatives. With this approach, it was possible to not only quantify the ratio of the main components of ASL, LSL, and SL glycerides but also confirm trace components such as SL mono-glyceride and bola-form SL (sophorose at both ends); notably, this is the first time these components have been isolated and identified successfully in naturally occurring SLs. In addition, our results revealed a novel SL derivative in which a fatty acid is bonded in series to the ASL, which had not been reported previously. Using the present analysis method, it was possible to easily track compositional changes in the SL components during culture. Our results showed that LSL and ASL are produced initially and that SL glycerides accumulate from the middle stage during the fermentation process. KEY POINTS: • An easy and detailed component analysis method for sophorolipids (SLs) is introduced. • Multiple SL derivatives were identified different from known SLs. • A novel hydrophobic acidic SL was isolated and characterized.

Keywords: Starmerella bombicola; Biosurfactant; Component analysis; Glycolipid; LC–MS analysis; Sophorolipid.

Fig. 1

Chemical structures of well-known sophorolipid (SL) derivatives (a) Acidic sophorolipid (ASL), (b) lactonic sophorolipid (LSL), (c) sophorolipid di-glyceride (SLG-A), (d) sophorolipid tri-glyceride (SLG-B), and (e) 2-O-fatty acyl-1,3-di-O-sophorolipid glycerol (SLG-C: 2SL + 1FA tri-glyceride)

Fig. 2

Charged aerosol detector high-performance liquid chromatography (CAD-HPLC) analysis of the collected microbial products of S. bombicola by jar fermentation from rapeseed oil using a C18 silica gel column and methanol/water eluent

Fig. 3

Putative chemical structures of the main components of three compounds newly found in S. bombicola products by CAD-HPLC and liquid chromatography- mass spectrometry (LC–MS) analysis. (a) Compound X (bola-form sophorolipid), (b) compound Y (sophorolipid mono-glyceride), and (c) compound Z

Fig. 4

The 400-MHz proton nuclear magnetic resonance (¹H-NMR) spectrum of compound Z, isolated from S. bombicola

Fig. 5

Partial heteronuclear multiple bond coherence (HMBC) spectrum of compound Z, isolated from S. bombicola, F1 axis: Carbon 13 NMR (¹³C-NMR) spectrum ranging from 165 to 185 ppm; F2 axis: ¹H-NMR spectrum ranging from 0.6 to 6.0 ppm

Fig. 6

Component analysis of the culture medium of S. bombicola by CAD-HPLC using a C18 silica gel column and methanol/water eluent. Using rapeseed oil as a carbon resource at a) 24, b) 48, and c) 72 h, or using rice bran oil at d) 24, e) 48, and f) 72 h after the start of fermentation, and g) Quantification of the SL derivatives