| Biosurfactant are high surface-active metabolites secreted by microorganism in the special culture conditions, mainly including the glycolipids and lipopeptides. The Mannosylerythritol Lipids (MELs) are the kinds of glycolipid, it has attracted considerable interest in recent years not only due to their excellent interfacial properties, biodegradabilitiy, nontoxicity, but also it has the function of anti-tumor, anti-virus and variety of functions, therefore, it has a very wild range of application and prospects.At present, the study of the Pseudozyma is mainly focused on the two categories including bio-control and the metabolites. MELs are secreted in abundance by strains of the genus Pseudozyma. Many studies about the MEL produced by Pseudozyma abroad, they pay attention on the fermentation conditions and the structural features of MEL, less research on its antibacterial property, And only few reports about MEL at home. In our laboratory, a strain CNm2012 has potential as bio-control was found and isolated, which has been identified and named Pseudozyma aphidis CNm2012, the study found that it has possibility become bio-control agent in the control of powdery mildew on mulberry. In this paper, the antimicrobial activity of the metabolites produced by P.aphidis CNm2012 was studied and investigated the effect of the carbon and the nitrogen source on it. The research provides an important reference on the further application of the strain CNm2012.(1) The paper choosed the potato dextrose broth, the Sabouraud’s dextrose broth and the special medium containing oil to ferment P.aphidis CNm2012. Then centrifuging the culture medium, the produced metabolites were extracted from the culture supernatant with an equal volume of ethyl acetate. The inhibition effects of extract from the above fermentation mediums on three species of pathogenic bacteria including Escherichia coli, Serratia marcescens, Staphylococcus aureus and three species of pathogenic fungi including Beauveria bassiana, Phomopsis sp., Penicillium sp. were studied. The results showed that the crude extracts under the fermented condition of potato dextrose broth and Sabouraud’s dextrose broth were not inhibitory to bacteria. However, when the P.aphidis CNm2012 grown on soybean oil as the sole carbon source, the extract showed strong antibacterial activities not only against the gram positive bacteria, but also against the gram negative bacteria, furthermore, the extract showed antifungal effect against Beauveria bassiana.(2) The effect of metabolites on the surface tension of the culture supernatant by the P.aphidis CNm2012 grown on soybean oil as the sole carbon source was assessed by the droplet collapse method and oil spreading method. An aliquot of the supernatant was dropped on a strip of Parafilm M as a hydrophobic surface. Fresh culture medium was used as negative control, the tween-80 was used as positive control to estimate whether the culture supernatant of P.aphidis CNm2012 has surfactants. The results showed that the diameter of the droplet of the supernatant was 9.0 mm, which was greater than that of a fresh culture medium (5.0 mm), and it is the same as the fresh culture medium mixed with Twain-80, it indicated the culture supernatant contained bio-surfactant, which highly reduced the surface tension of the culture medium. The liquid paraffin can appear the oil expulsion circle when the culture supernatant of the P.aphidis CNm2012 was dropped on it. These result indicated that P.aphidis CNm2012 produced biosurfactant which has high surface activities. Theextract was identified using high-performance liquid chromatography combined with mass spectrometry (HPLC-MS) and gas chromatography combined with mass spectrometry (GC-MS). The molecule ions and fragment ions can be derived based on the given structure of MEL. All these explained that the biosurfactant which was contained in the extract were MELs, and four types of MEL (MEL-A, MEL-B, MEL-C, and MEL-D) may be included in the crude extract.(3) The extract was analyzed by TLC on silica plates with a solvent system consisting of chloroform:methanol:ammonium hadroxide (65:15:2, v/v/v), it appears 5 points in the thin layer plate, the Rf value was 0.98,0.85,0.73,0.67,0.47, respectively, afterwards, theextract was further purified by column chromatograph, the bacteriostatic experiment has been done with the purified compounds, we discovered only sample 3 has antimicrobial activity, then the compound was used for LC-MS and GC-MS test, the results of LC-MS analysis showed that the MW of molecular ion was 780.4. The results of GC-MS analysis showed that n-Hexadecanoic acid and 9,12-Octadecadienoic acid were found in sample 3. It can speculate that the sample 3 was MEL-D at the utmost. We found the MEL-D can not only against the gram positive bacteria, but also against the gram negative bacteria and fungi. Analysis of the antibacterial effect of the sample 3, the results proved that the antibiotic rate of MEL-D was declined when increasing of the processing temperature and the processing time, and the antibacterial activity of the MEL-D on the bacteria was better than the fungi.(4) Reportedly, the metabolites have quite different under the different fermentation conditions of the genus Pseudozyma. The effect of the carbon and nitrogen source on the antibacterial avtivity of the metabolite by Pseudozyma aphidis CNm2012 were optimized through singer-factor test, it was found the strain produce the antibiotic substance mainly under the induction of the carbon source, the bran was better than that of soybean oil, the inhibition zone diameter up to 48.3 mm. |
