and, therefore, these methods should only be used as a rough indicator of glycolipid production.
Thin layer chromatography (TLC) is a simple method that allows the detection of glycolipids and can also provide information on possible structural types of glycolipids present in the sample. TLC detection should be carried out before purification procedures to evaluate the presence of glycolipids and should be used afterwards to determine purity.
Further analyses are required for both quantification and/or identification of structural features, mainly using high-performance liquid chromatography-mass spectrometry (HPLC-MS) (Marqués et al. 2009) and nuclear magnetic resonance (NMR) (Kügler et al. 2014). Among these techniques, mass spectrometry offers the greatest amount of information with regard to purity and structural conformation of carbohydrate and fatty acid components of the glycolipids.
In fact, HPLC is a method that allows the separation of glycolipids and when coupled with an evaporative light scattering detector (ELSD) or mass spectrometry provides valuable information related to the identification and quantification of glycolipids. HPLC-UV can also be used for analysis when the test compounds have been derivatized to p-bromophenacyl esters (Mata-Sandoval et al. 1999).
In the specific case of trehalose lipids many techniques can be used for the identification and characterization. Similar to other glycolipids, trehalose lipid content can be estimated using the colorimetric anthrone method. Also TLC has been extensively used in the detection of trehalose lipids. A few solvent systems have been used to separate trehalose lipids, although the most frequently used is chloroform:methanol:water (65:15:2) for the mobile phase (Christova et al. 2015) and to reveal them, using a chemical developer, frequently made of acetic acid: anisaldehyde: sulfuric acid, under 150°C airstream for 2–4 min. Trehalose lipids appear in the form of spots (Kügler et al. 2014), with the monomycolates near the point of application of the sample and the mark of dimycolates a little further away.
High-performance liquid chromatography (HPLC) and reverse phase (HPLC-RC) have been used for the identification of trehalose lipids (Janek et al. 2018), especially with mass spectrometry HPLC (HPLC-MS) it is possible to disclose the glycoside linkage between two of the sugar moieties (Patil and Pratap 2018). Moreover reversed-phase HPLC (HPLC-RC) was also used for the identification of these compounds.
Mass spectrometry (MS) provides detailed structural information on the molecular mass of the compounds under investigation. Tandem MS (MS/MS) results in the fragmentation of structures thus allowing the identification of individual isomers without the need for separation. Moreover, when combined with HPLC, it provides the most sensitive method for identification and quantification of trehalose lipids. A drawback is that it requires a high level of purification as salts and free non-polar lipids can induce suppression of ion signals under MS conditions. Glycolipids can be analyzed on all types of mass spectrometers, with electrospray ionization (ESI-MS) and matrix assisted laser desorption ionization (MALDI) (Yagi-Utsumi 2019).
Electrospray ionization provides excellent trehalose lipid ionization when used for direct infusion or HPLC-MS. Using this technique virtually no fragmentation occurs in the primary molecules under investigation. Ionized molecules are detected by a mass analyzer according to their mass to charge ratio (m/z) and can be fragmented using collision-induced dissociation (CID) to provide valuable information about each structure and their isomers (Yagi-Utsumi 2019).
MALDI is a soft ionization mass spectrometry technique that allows the identification of intact compounds. Basically, samples to be analyzed are mixed with a matrix and dried on a platform, onto which a laser is fired with various degrees of energy, thus forming gaseous ions, which can then be observed in a time of flight analyzer (Yagi-Utsumi 2019).
The characterization can be done by breaking down the structure separating the fatty acids from the carbohydrate or just by analysing the entire molecule. Mass spectrometry or mass spectrometry in conjugation gas chromatography (GS) (Christova et al. 2015) can be used.
To achieve a full structural determination, nuclear magnetic resonance spectroscopy (NMR) is utilized and is the most powerful method able to identify functional groups as well as the position of linkages within the carbohydrate and lipid molecules. Using a series of NMR experiments the exact location of each functional group can be obtained and information about the structural isomers is also possible (Sato et al. 2019).
Fourier-transform infrared spectroscopy (FTIR) is applied for the characterization of trehalose lipids, once these molecules form polymeric aggregates in aqueous media above the critical micellar concentration (CMC). The type of aggregate structure can play a biological role and is determined by the shape of the contributing molecules which is determined by their primary chemical structure and is influenced by pH, concentration of mono- and divalent cations, among others (Brandenburg and Seydel 1988).
1.8 Surface-Active Properties
Biosurfactants are surface-active compounds, capable of reducing surface and interfacial tension at the interfaces between liquids, solids and gases, allowing them to mix or disperse readily as emulsions in water or other liquids.
Surfactant-related parameters are surface tension, maximum surface excess value, minimum surface area occupied by the surfactant molecule at the air–water interface at saturated adsorption, the minimum concentration of the surfactant required to reduce surface tension and the Gibbs free energy of adsorption (Marqués et al. 2009). The interfacial tension, the emulsification index, and the hydrophilic lipophilic balance (HLB) are other important surface active parameters. The interfacial tension is usually measured by tensiometry. The emulsification index is the direct indication of the amount of surfactant. Afterwards the emulsification index can be monitored during the course of fermentation to evaluate the production of trehalose lipids, by mixing an equal volume of cell free broth and liquid paraffin (Cooper and Goldenberg 1987).
Moreover, surface tension has a tendency to decrease with the increase of glycolipid concentration. The structural diversity of glycolipids structures originate different surfactant properties. Generally they are able to reduce the surface tension of water to 43–24.1 mN m-1 and present CMC values of 0.7–37 mg L-1 (Marqués et al. 2009; Tuleva et al. 2009; Yakimov et al. 1999).
The trehalose lipids are able to reduce the surface tension of the water, for instance from 72 to 34 mN m-1 (Janek et al. 2018). This is in agreement with most trehalose lipids produced from Rhodococcus, with strong surface activity by lowering water surface below 30 mN m-1, lowering the interfacial tension against hexadecane up to 1 mN m-1. An example, is a purified trehalose lipid, produced by a novel marine bacterium Rhodococcus sp. PML026 using sunflower oil as a hydrophobic substrate, being able to reduce the surface tension of water to 29 mN m-1 with a CMC value of 250 mg L-1 (White et al. 2013).
Marqués et al. (2009) analyzed the chemical and physical properties of a glycolipid synthesized by Rhodococcus sp. 51T7. They demonstrated that this biosurfactant was a trehalose tetraester (THL) consisting of six components: one major and five minor. The hydrophobic moieties ranged in size from 9 to 11 carbons. The critical micelle concentration (CMC) was 37 mg L-1 and the interfacial tension against hexadecane was 5 mN m-1. At pH 7.4 the trehalose lipid CMC/critical aggregation concentration (CAC) was 50 mg L-1 and at pH 4 it was 34 mg L-1 (Marqués et al. 2009). A break at around 40 mg L-1 was consistent with the CMC/CAC obtained from surface tension measurements. An increase in pH (4–7.4), CAC values increased as the more negatively charged carboxylate group of trehalose lipid tends to complicate aggregation. When NaCl was added at pH 7 trehalose lipid behaved in a non-ionic way and the CAC was unaffected. A phase diagram showed effective emulsification with water and paraffin or isopropyl myristate. A composition of 11.3–7.5–81.8% (isopropyl myristate–trehalose lipid–water) was stable for at least three months. The HLB was 11 and the phase behaviour of the glycolipid revealed the formation of lamellar and hexagonal liquid-crystalline textures (Marqués et al. 2009).
Depending on the Rhodococcus sp. the different trehalose lipids produced changes in interfacial tensions, critical micelle concentrations and emulsifier index