A Structural Study on Canola Oil-Fermented Microbial Emulsions Using BT1 Strain

Abstract

The demand for sustainable bioderived surfactants, such as microbial surfactants, is steadily increasing. While prior studies have mainly focused on developing new production methods for biosurfactants, identifying their chemical identities, or elucidating underlying metabolic mechanisms, research on the structure of emulsions formed by these surfactants and their internal fluid-fluid interfaces remain relatively scarce. In this study, we present a novel microbial emulsion produced by fermenting canola oil with BT1 strain and investigate its detailed structure. The extracted fermentation products exhibited water affinity and water-canola oil interfacial activity, which were characterized using relaxation nuclear magnetic resonance and pendant drop tensiometry, respectively. These fermentation products, functioning as biosurfactants, spontaneously generate water-in-oil-in-water (W/O/W) emulsions during fermentation by facilitating transitional phase inversion, thereby eliminating the need for complex multistep processes. The resulting emulsion droplets displayed intriguing interfacial coloration when observed under a reflective polarized optical microscope (POM). Typically, emulsions exhibiting similar patterns (e.g., Maltese crosses) in POM are classified as "liquid crystal emulsions". However, for the first time in this study, we present counterexamples that exhibit such interfacial patterns without possessing a crystalline structure. We discuss the origins of this phenomenon and emphasize the need for careful interpretation of similar systems. Cryogenic scanning electron microscopy suggests the adsorption of interface-active substances derived from fermentation products at the water-oil interfaces, while others may remain suspended in the aqueous continuous phase. Differential scanning calorimetry demonstrates their influence on the melting and crystallization behaviors of water and canola oil, which is associated with enhanced refrigeration stability of the resulting microbial emulsions.

1

Photographs of the fermentation batch: (a) in the beginning and (b) at the end of the process, along with (c) samples taken at different time points. (d) Precipitated fermentation products obtained through centrifugal washing with excess benzene. (e) Freeze-dried fermentation products. (f) OM, (g) POM, and (h) SEM images of the fermentation products at magnifications of (h1) × 1,000 (h2) × 2,000, and (h3, h4) × 3,000, accompanied by (i) EDS analysis results. The scale bars in (f–h) indicate 10 μm.

2

(a) Normalized transverse magnetization data for (a) fermentation products and (b) unfermented canola oil dispersed in deionized water at different concentrations, obtained through relaxation NMR (with “water” denoting the curve for deionized water without solutes). (c) The transverse relaxation time, T ₂, extracted from (a,b), presented as functions of solute concentrations.

3

Interfacial tension (γ, in mN/m) between the aqueous phase, in which fermentation products were predispersed at various concentrations, and the canola oil phase, measured using the pendant drop method.

4

Observations of the droplets within the naturally formed microbial emulsion using (a) inverted OM, (b) confocal laser scanning microscopy, (c) standard reflective OM, and (d) POM, and of those within the artificially produced canola oil-in-water emulsion using (e) standard reflective OM and (f) POM (scale bar: 10 μm).

5

Replication of W/O/W emulsions by adding varying amounts of extracted fermentation products: (a) photographs of the produced emulsion immediately after homogenization, and (b) the morphology of droplets within the emulsions observed with standard reflective OM (scale bar: 10 μm). The concentrations of the added fermentation extracts are indicated in yellow.

6

SAXS patterns of the naturally formed microbial emulsion at sample-to-detector distances of 5 m (black) and 1 m (red).

7

(a) Cryo-SEM images of fermented (left) and unfermented (right) emulsions before sectioning. (b) Cross-sectional images of isolated oil droplets in fermented (left) and unfermented (right) emulsions. (c) Magnified images for water–oil interfaces in the fermented emulsion. (d–i) Images of multiple isolated oil droplets (left) and their interfaces (right) in the fermented emulsions.

8

DSC cooling and heating curves of (a) the unfermented and (b) fermented emulsions. The parenthesized numbers above or below the arrows represent the order of temperature variations: (1) 25 °C to −80 °C, (2) −80 to 80 °C, and (3) 80 to 25 °C. (c) Comparison of the cooling curves between −40 °C to −53 °C. Comparison of the heating curves for both samples between (d) −10 to 5 °C and (e) −30 to 10 °C (solid green: fermented emulsion, dashed brown: unfermented emulsion, solid red: pure canola oil).

9

Observations of fermented (left) and unfermented (right) emulsions stored under refrigeration (2–4 °C): (a) photographs of the samples. (b) Inverted OM images.