Low-Temperature Crystallization for Separating Monoacetylated Long-Chain Sophorolipids: Characterization of Their Surface-Active and Antimicrobial Properties

Pseudohyphozyma (formerly Rhodotorula) bogoriensis synthesizes long-chain-length (22-carbon chain) sophorolipids (22:0-SL) that are variously acetylated at the 6′- or/and 6″-carbons of the sophorose unit. In this paper, we describe a low-temperature crystallization protocol that preferentially separates the 6′-monoacetylated 22:0-SL (6′-Ac₁-22:0-SL) from a parental mixture of 22:0-SL containing a majority (64.7%) of the 6′,6″-diacetylated moiety (6′,6″-Ac₂-22:0-SL), as deduced from high-pressure-liquid-chromatography evaporative-light-scattering-detection (HPLC-ELSD) and LC/Q-TOF-MS analyses. Tensiometry measurements using the Wilhelmy plate method yielded minimum-surface-tension (SFT_min) and critical-micelle-concentration (CMC) values of 34.6 ± 1.0 mN m⁻¹ and 0.014 mM, respectively, for 6′-Ac₁-22:0-SL (CRYSTAL) fraction and 34.9 ± 1.0 mM m⁻¹ (SFT_min) and 0.018 (CMC) for the hexane precipitate (Hx-PRCP) fraction containing a high concentration (89.2%) of the diacetylated 6′,6″-Ac₂-22:0-SL after crystal removal. In contrast, the SFT_min and CMC of the well-studied 16-18 carbon (C_16-18)-SL of Starmerella bombicola were 35–37.2 mN m⁻¹ and 0.05-(>0.3) mM, respectively. Individually, the purified CRYSTAL and Hx-PRCP fractions exhibited a similar degree of strong growth-inhibition activity against Cutibacterium (formerly Propionibacterium) acnes as determined by an agar-plate zone of inhibition assay. Study on the growth inhibition of oral health-related bacteria, i.e., Streptococcus mutans and Lactobacillus acidophilus, showed that, depending on the bacteria and strains tested, the CRYSTAL fraction was either slightly better than or equally effective as the Hx-PRCP fraction in inhibiting cell growth.     

Antimicrobial and SEM Studies of Sophorolipids Synthesized Using Lauryl Alcohol

In recent years, researchers have developed biosurfactants for industrial, pharmaceutical and medical applications revealing the promising biological activities of these biomolecules. One of the best studied microbial surfactants are glycolipids, especially sophorolipids (SLs) produced by selected non-pathogenic yeast species of Candida. They are biodegradable, non-toxic and are environmentally friendly. Sophorolipid production was carried out using glucose as the hydrophilic source and lauryl alcohol C_12–14, as the hydrophobic source using Candida bombicola ATCC 22214. Primary characterization of the SL obtained using lauryl alcohol (SLLA) was done by FTIR which depicted the presence of alkyl sophorosides/SLs. Antimicrobial activity testing revealed that SLLA showed complete inhibition against gram negative bacteria, Escherichia coli (ATCC 8739) Pseudomonas aeruginosa (ATCC 9027) at 30 and 1 μg/ml at a contact time of 2 and 4 h respectively. Whereas for gram positive bacteria Staphylococcus aureus (ATCC 6358), Bacillus subtilis (ATCC 6633), complete inhibition was observed at 6 and 1 μg/ml respectively at a contact time of 4 h. The formed SLLA showed noteworthy inhibition against the pathogenic yeast Candida albicans (ATCC 2091) at 50 μg/ml with a contact time of 4 h. These values are remarkably low compared to reported values of oleic acid SLs and linolenic acid SLs which were studied for antimicrobial properties. Scanning electron microscopy analysis of the treated cells revealed the changes in morphology and topography of the microorganisms.     

Structured Estolides: Control of Length and Sequence

Using ester-forming reactions such as carbodiimide coupling and a modified Yamaguchi symmetrical anhydride method, a variety of estolides based on 17-hydroxy oleic and 17-hydroxy stearic acid have been prepared. These hydroxy fatty acids are produced in good yields from hydrolysis of sophorolipids, which are in turn derived from fermentation of fats and oils. Since the estolides are formed one unit, or ester bond, at a time, their length and sequence can be precisely controlled. The key to this control is the use of protecting groups at either the carboxylic or hydroxy end of the starting hydroxy fatty acids. Two mono-protected dimers, for example, when combined in a fragment-condensation approach, give a tetramer with no “contamination” from estolides of other lengths. This methodology opens the way to functionalized estolides, and several variants were prepared: hybrid estolides, containing non-fatty acid moieties such as amino acids; polymerizable estolides, containing a norbornene unit; and non-linear estolides that extend from a branched core such as glycerol or pentaerythritol. With the benzoyl chloride-mediated symmetrical anhydride method, yields for individual coupling steps ranged from 75 to 93%. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.     

应用煎炸废油发酵制备槐糖脂生物表面活性剂

应用正交实验,优化了假丝酵母菌O-13-1以煎炸废油为油溶性碳源发酵制备槐糖脂的条件,并应用高效液相色谱-高分辨串联质谱(HPLC-MS/MS)鉴定了所产槐糖脂同系物的组成及结构。结果表明,经正交实验优化的O-13-1菌株以煎炸废油为底物制备槐糖脂的摇瓶发酵条件为:摇床转速200 r/min,双碳源质量浓度均为90 g/L,温度为25℃,接种量为体积分数12%。在此条件下,槐糖脂产量达到最大,为73.26 g/L;O-13-1菌株以煎炸废油为底物所合成槐糖脂由9种乙酰基取代的内酯型和酸型槐糖脂同系物构成,内酯型槐糖脂同系物所占质量分数达76%。基于摇瓶得到的以煎炸废油为油溶性碳源的优化发酵条件,O-13-1菌株在5 L自动发酵罐中转速500 r/min时发酵合成槐糖脂产量可达163.28 g/L。以煎炸废油为碳源发酵槐糖脂的原料成本较普通大豆油降低18%,在有效降低槐糖脂生产成本的同时,实现了煎炸废油的资源化利用。     

Acid Precipitation versus Solvent Extraction: Two Techniques Leading to Different Lactone/Acidic Sophorolipid Ratios

Sophorolipids (SL) prepared from biotransformation of refined bleached deodorized (RBD) palm olein by Starmerella bombicola (ATCC 22214) occur as a mixture of homologs. Characterization of SL was performed using high-performance liquid chromatography with electrospray ionization mass spectrometry detection (HPLC-ESI-MS) under negative ion mode. Two different purification methods were compared: precipitation and solvent extraction. SL purified by precipitation and solvent extraction consisted of five major homologs with m/z values of 687, 645, 689, 705, and 661. The fractions of lactone and acidic SL via precipitation were 70.24 ± 1.4% and 29.76 ± 1.4%, respectively. SL isolated by solvent extraction contained 44.78 ± 1.9% lactone and 55.22 ± 1.9% acidic SL. Therefore, the purification method strongly impacted the composition of the SL products.     

Sophorolipid Biosurfactants Activate Taste Receptor Type 1 Member 3-Mediated Taste Responses and Block Responses to Bitter Taste In Vitro and In Vivo

Sophorolipids (SL) are typically produced and secreted by select nonpathogenic yeast species (i.e., Candida) from renewable substrates. They are currently being used by industry on a limited basis in formulations for cleaning solutions as well as laundry and dishwashing detergents. Due to the nature of their chemical structure, it was hypothesized that SL would demonstrate taste-sensory properties. In this study, SL were produced via fermentation on a mixed substrate platform with glucose and either palmitic acid, stearic acid, or oleic acid using Candida (currently reclassified as Starmerella) bombicola ATCC 22214. The taste properties of SL were determined using a single-cell manual calcium imaging technique on cultured human fungiform taste papillae (HBO) cells. The results of those studies demonstrated that sweetener-responsive HBO cells also respond to SL, and these responses are mediated by the type 1 taste receptors 3 (T1R3), because they were blocked by lactisole (a T1R3 receptor-specific blocker). The involvement of the T1R3 receptor in SL recognition was confirmed via the chorda tympani nerve recording (CTNR) study in a (−/−) T1R3 knockout (KO) mouse model. We further demonstrated that SL are capable of blocking the bitter stimuli-elicited responses both in HBO cells and in the CTNR study. This is the first report demonstrating that SL have taste-sensory properties, which opens up numerous possibilities for practical applications of SL to ameliorate bitter tastes in foods and drugs and understand the potential source of dysgeusia in some patients.     

Microbial Oxidation of Medium Chain Fatty Alcohol in the Synthesis of Sophorolipids by Candida bombicola and its Physicochemical Characterization

Sophorolipids (SL) are glycolipid biosurfactants abundantly produced from different feedstocks by yeasts and have been widely developed for various applications. The amphipathic structures of sophorolipids imparts to them surfactant type properties. These biosurfactants are readily isolated in high yield and are nontoxic and biodegradable, which make them industrially interesting as surfactants or emulsifiers. Sophorolipid production was carried out using glucose as a hydrophilic source and lauryl alcohol C_12–14, as a hydrophobic source by Candida bombicola (ATCC 22214). Process economics and environmental attractiveness was increased by using a low cost substrate. Optimization studies were carried out where the effect of glucose concentration, pH, temperature and metal ions on sophorolipid production was studied. Optimum production of sophorolipid obtained using lauryl alcohol (SLLA) was achieved after 96 h, the initial pH of the medium was 6.0 with 10 % glucose at 30 °C. Primary characterization of SLLA was done by FTIR. The SLLA showed high physicochemical properties in terms of the surface activities when compared with synthetic surfactants including dodecyl tetraethylene glycol ether and sodium lauryl sulfate. The surface tension of SLLA thus obtained was found to be 24 mN/m with a critical micellar concentration (CMC) value of 0.68 mg/L. Antimicrobial testing showed that SLLA have potent antimicrobial activity against both gram negative bacteria, Escherichia coli (ATCC 8739) and gram positive cocci, Staphylococcus aureuss (ATCC 2079).     

3-Hydroxypicolinic Acid as an Effective Matrix for Sophorolipid Structural Elucidation Using Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry

Determination of the distribution of sophorolipid (SL) congeners controls their self-assembly structures and therefore their employment as glycolipid biosurfactants in cosmetics, environmental remediation, and personal care applications. The compatibility of 3-hydroxypicolinic acid (HPA) as a matrix for compositional analysis of SL using palm olein as carbon energy source by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) analysis under positive and negative ion modes is studied in this work. Examination in positive ion mode revealed ions that were not detected in negative ion mode. Molecular ions produced using HPA matrix were lactonic Glc-Glc-1′,4″-C18:1, anionic Glc-Glc-1′-C18:1, lactonic monoacetate of Glc-Glc-1′,4″-C18:1, and anionic monoacetate Glc-Glc-1′-C18:1 and the anionic diacetate of Glc-Glc-1′-C18:1. We also detected Glc-Glc-1′-C18:0, GlcOAc-Glc-1′-C18:0, GlcOAc-GlcOAc-1′,4″-C18:0, and GlcOAc—GlcOAc-1′-C18:0 which are congeners of SL with stearic acid moieties. 3-HPA has been established to be the superior matrix for elucidation of structures for SL using MALDI-TOF MS.