Studies On Fed-batch Fermentation, Antifungal And Anticancer Activities Of Sophorolipids

Biosurfactants are the metabolic products of some microorganisms and have many advantages including low toxicity, good biodegradability and biocompatibility compared with synthetic surfactants by chemical methods. In order to meet the demands for environment protection, biosurfactants will play more and more role than chemically synthetic surfactants in our daily life.Sophorolipids are glycolipid biosurfactants produced by several selected variety of yeast strains. Due to their properties of low toxicity, high biodegradability biocompatibility and high yields, sophorolipids have great application prospects in petroleum industry, environmental industry, cosmetics, food, detergent industries and pharmaceutical sector. Recently, sophorolipids have been proved to have good antimicrobial, anticancer activities and even the anti-HIV activity, which will broaden the applications of sophorolipids in pharmaceutical sector.The main research aspects and results are as following:1. Sophorolipid production by fed-batch fermentationAfter feeding 5% rapeseed oil three times, the yield of sophorolipids in fed-batch fermentation was increased by 32% than batch fermentation in 300 mL flask. In 5 L fermentor, after 288 h of fed-batch fermentation, the yield of sophorolipids was 68.2 g/L and was increased by 109.2% more than batch fermentation in 300 mL flask and 58.2% than fed-batch fermentation in 300 mL flask. After the rotatory speed was improved from 400 rpm to 500 rpm, the yield of sophorolipids 5 L fermentor reached 71.1 g/L at 196 h, the fermentation period shortened 96h, the productivity of sophorolipids was increased from 0.24 g/L/h to 0.37 g/L/h.2. Inhibition of sophorolipids to bacteriumThe inhibition effects of sophorolipids to some bacterium including Escherichia coli, Bacillus cereus, Staphylococcus aureus, and Streptococcus mutans were studied. It was found that sophorolipids showed strong antibacterial activities against gram-positive bacteria.15 mg/L sophrolipids could fully inhibit the growth of B. cereus, and 40 mg/L sophrolipids could fully inhibit the growth of S. aureus. Sophorolipids showed no antibacterial activities against gram-negative bacteria E. coli. This is the first report that sophorolipid had strong inhibition effect to Streptococcus mutans which could cause decayed tooth.50 mg/L sophrolipids could fully inhibit the growth of Streptococcus mutans.3. Inhibition of sophorolipids to DermatophytesThe inhibition of sophorolipids to three common clinical dermatophytes, Trichophyton rubrum, Trichophyton gypseum, and Microsporum canis were investigated. We compared the inhibition of acidic sophorolipids with that of lactonic ones to the three dermatophytes. Lactonic or acidic sophorolipids showed inhibition on the growth of all the three dermatophytes and showed different inhibition towards each of them. To Trichophyton rubrum, the inhibition of lactonic sophorolipids was a little better than acidic ones. Toward Trichophyton gypseum, when at low concentrations, the inhibition of lactonic sophorolipids was much better than that of acidic ones. However, when at high concentrations, the inhibition of acidic sophorolipids was much better than lactonic ones. Toward Microsporum canis, the inhibition of lactonic sophorolipids was much better than acidic ones.For the inhibition of hypha extension, lactonic sophorolipids could inhibit the extension of hypha much better than the acidic ones. When lactonic sophorolipids concentration was 0.5 mg/mL, the inhibition ratio on hypha extension of Trichophyton rubrum, Trichophyton gypseum and Microsporum canis was 53.8%,62.5% and 68.2%, respectively. The MIC50 of lactonic sophorolipids to the three dermatophytes was 0.0625,0.125,0.0625 mg/mL, respectively. MIC90 of lactonic sophorolipids to Trichophyton rubrum, Trichophyton gypseum and Microsporum canis was 0.125,0.25, 0.125 mg/mL respectively. MFC of lactonic sophorolipids to Trichophyton rubrum, Trichophyton gypseum and Microsporum canis was 0.5,0.5,0.25 mg/mL respectively.The TEM observation results indicated that, after being treated by lactonic sophorolipids, three dermatophytes have some obvious changes in their microstructures. The cell wall became thicker and loose, the cytoplasm agglomerated, the membranes of organelles were disappearing, and no integrated organelles and clear nuclear zone were found in the cytoplasm.4. Purification and Structure elucidation of sophorolipidAfter being analysed by HPLC, it was found that the crude sophorolipids produced by Wickerhamiella domercqiae var. sophorolipid are a mixture composed of more than ten molecules. Ten sophorolipid molecules were separately collected by preparative HPLC. The structures of the ten sophorolipid molecules were elucidated by MS analysis. It was found that all the sophorolipid molecules are sophorolipids with C18 fatty acid. Their structures differ in acetylation degree of sophorose, unsaturation degree of hydroxyl fatty acid and lactonization or ring opening.5. The anticancer effects of sophorolipids with different structures on human esophageal cancer cellThe anticancer effects of sophorolipids with different structures on human esophageal cancer cell KYSE 109 and KYSE 450 were investigated. It was found that the differences of sophorolipid sructure in acetylation degree of sophorose, unsaturation degree of hydroxyl fatty acid, and lactonization or not can affect the anticancer activity of sophorolipid.(1) The results indicated that the inhibition of diacetylated lactonic sophorolipid to esophageal cancer cells (totally inhibition at 30μg/mL concentration) was stronger than momoacetylated lactonic sophorolipid (totally inhibition at 60μg/mL concentration), which confirmed that anticancer activity of SLs was affected by their acetylation degree in sophorose moiety.(2) Our results showed that sophorolipid with different unsaturation degree of hydroxyl fatty acid also had different cytotoxic effects on esophageal cancer cells. Sophorolipid having one double bond in fatty acid part had the strongest cytotoxic effect (totally inhibition at 30μg/mL concentration) on esophageal cancer cells, sophorolipid with two double bonds had a little weaker anticancer effect (totally inhibition at 60μg/mL concentration), while sophorolipid with no double bond had the weakest cytotoxic effect (only 20% of cells were inhibited at 60μg/mL concentration) among the three sophorolipid molecules. This was the first study to reveal the relationship of bioactivities of natural sophorolipid molecules with different unsaturation degree in hydroxyl fatty acid and their structures.(3) No matter acidic SL with one or two double bond in fatty acid part, with momoacetylated group or diacetylated groups in sophorose part, they have little anticancer effect against esophageal cancer cells.6. The inhibition mechanism of sophorolipids with different structures on human esophageal cancer KYSE450In our previous studies, the inhibition mechanism of diacetylated lactonic sophorolipid with a C18 momounsaturated fatty acid on the human liver cancer cells H7402 has been proved to induce cell apoptosis. The inhibition mechanism of two sophorolipid molecules C18:1 MLSL and C18:1 DLSL on human esophageal cancer cell KYSE 450 was investigated by reverse phase contrast microscopy, cell staining, fluorescence microscopy, flow cytometer, and TUNEL assay in this study.It was found that, after being treated with C18:1 MLSL and C18:1 DLSL, cell gradually shrank, turned round, membrane blebbing stood out. Chromatin condensation and margination, nuclear fragmentation and apoptotic bodies were observed. Cell cycle distribution change was observed and the sub-G1 population appeared. These changes can demonstrate the apoptosis of KYSE 450 induced by sophrolipids and the inhibition mechanism of sophrolipids to different cancer cells was all apoptosis.The apoptosis level of KYSE 450 induced by sophrolipid with different structure of the same concentration was different. It was found that C18:1 DLSL could induce apoptosis of KYSE 450 at a greater extent than C18:1 MLSL by morphological changes, cell cycle distribution changes and apoptosis rate of KYSE 450, which agree with the results that the inhibition of diacetylated lactonic sophorolipid to esophageal cancer cells was stronger than momoacetylated lactonic sophorolipid.