Physicochemical Properties Of Surfactants Secreted By Bacillus Sp. HG224 And Its Application In Extraction Of Saponins From Dioscorea Zingiberensis C.h. Wright

Diosgenin is an important precursor for the synthesis of hundreds of steroid hormone drugs.The traditional acid hydrolysis process of diosgenin has bottlenecks such as high pollution,low yield and high cost,which seriously threatens the water quality of the Middle Route Project of South-to-North Water Transfer.After many years of scientific and technological research,we proposed a new green technology for the water extraction and enrichment of saponin from Dioscorea zingiberensis C.H.Wright（DZW）by biological method and converting saponin to diosgenin.The new process abandoned traditional acid hydrolysis and significantly reduced pollution and production costs.Further studies have found that the sodium dodecyl sulfate（SDS）can greatly increase the water extraction rate of saponin,but the addition of SDS will introduce new pollution,and is not conducive to the subsequent biotransformation of DZW saponin.Therefore,there is an urgent need to find an environmentally friendly biosurfactant.With the screening index od reducing the surface tension of water-soluble system,a strain of Bacillus sp.HG224 which can secrete surfactant was obtained.And the physicochemical properties of the substance and its effect on the extraction of DZW saponin were studied.The following results were obtained.（1）Isolation and preliminary identification of surface active substances secreted by Bacillus sp.HG224A surface active substance was isolated from the fermentation supernatant of Bacillus sp.HG224 by acid precipitation and organic solvent extraction.Thin-layer chromatography,Fourier transform infrared spectroscopy and high-resolution liquid chromategraphy-mass spectrometry were used to determine that the substance was a mixed lipopeptide surfactants composed of four C13^C15 Surfactin homologues.（2）Study on the physicochemical properties of lipopeptide surfactants secreted by Bacillus sp.HG224The critical micelle concentration of the lipopeptide surfactants is 30 mg/L based on the surface tension-lipopeptide concentration curve.Through the analysis of emulsifying properties and oil draining ring,it was found that the emulsification index of the material could be stably maintained above 60%,and the diameter of the oil drain ring was 8.2 cm.It meant the lipopeptide surfactants had a good emulsifying properties and oil discharging performance.The charge detection analysis found that the lipopeptide surfactants was an anionic surfactant.Furthermore,the stability of the the lipopeptide surfactants was tested.And it was found that the surface activity and emulsifying properties of the substance were stable in a wide range of 0 to 100°C and pH 4¹2 and high salt concentration（0¹8%NaCl,w/v）.（3）Optimization of fermentation process of lipopeptide surfactants secreted by Bacillus sp.HG224By optimizing the fermentation parameters and medium components of Bacillus sp.HG224,it was found that when the inoculum size was 3%（v/v）,the initial pH was 7.5,the culture temperature was 31°C,and the liquid volume was 30 mL/250 mL,the carbon source in the medium was sucrose,the nitrogen source was soybean cake powder,and copper ions were added,the yield of lipopeptides could be greatly increased;further the orthogonal experiment of three main affecting factors such as the concentration of nitrogen source,carbon source and copper ion in the medium was carried out,and the optimized concentration was sucrose 20 g/L,soybean cake powder60 g/L,Cu²⁺0.5 mg/L.Using the above optimized fermentation conditions,the lipopeptide production was as high as 5.74 g/L,which was 17.39 times before optimization（0.33 g/L）.The diameter of the oil drain of the fermentation broth was 12.4cm,which was 4.2 cm more than before optimization;further using the isolated and purified lipopeptide surfactants for extraction of saponin test,it verified that the substance can effectively promote the extraction of saponin from DZW.（4）Application of lipopeptide surfactants secreted by Bacillus sp.HG224 in promoting the extraction of DZW saponinsThe DZW saponins was extracted by the fermentation liquid containing lipopeptide surfactants secreted by Bacillus sp.HG224.The results showed that the water extraction rate of DZW saponins was 45.16%higher than that of direct water extraction,indicating that the method of extracting DZW saponins by fermentation liquid had a good application prospect.Using different concentrations of lipopeptide surfactants to treat the aqueous suspension of dried DZW,it was found that the aqueous solution of 90mg/L lipopeptide surfactants increased the water extraction rate of DZW saponins by11.27%,compared with direct water extraction.It was further confirmed that the lipopeptide surfactants had a promoting effect on the water extraction of DZW saponins.The HPLC and HR-LC-MS analysis confirmed that after treated with lipopeptide surfactants,the content of three less polar spiroxane saponins in the aqueous phase of the aqueous suspension of dried DZW was significantly higher than that of direct water extraction.It was further confirmed that the lipopeptide surfactant had an effect of promoting water extraction of DZW saponins.