Study On The Key Technologies In Preparation Of Antimicrobial Lipopeptide

A strain of Bacillus was isolated from the medicinal pant by our lab and mutated in the method of low energy ion beam to improve the yield of antimicrobial lipopeptides.Then the procession of fermentation and separation of this strain was optimized.Three kinds of defoamers were selected to solve the problem of generating lots of bulbs caused by the surfactin.The results showed that soybean oil could not only inhibit the generation of bulbs,but also increase the production of antimicrobial lipopeptides.Taking everything into consideration,soybean oil was chosen to be the defoamer in fermentation procession.But serval problems still existed in the industrial and commercialization of surfactin:soybean oil as defoamer would inhibit activity of surfactin and pose difficulty for the separation and purification procession;the character of surfactin as bio-substance was that it could be easy to be degraded in the unsuitable environmental situations.All of those problems prevented the acceleration of industrial and commercialization.The goal of this paper was to solve those problems with the research of separation,microcapsule and application.In this study,two-step method of silica gel chromatography was established to remove the defoamer in broth of antimicrobial lipopeptides for the first time.High-performance liquid chromatography(HPLC)was applied to detect the concentration of surfactin.Thin layer chromatography(TLC)-visible spectrophotometry was used to conduct quantitative analysis of triglyceride in eluent.Also,thin layer chromatography(TLC)was taken as the way to select the developing agent for triglyceride.The result was that when 200-300 mesh silica gels was used as the chromatography support beads,the total loading amount of silica gels was 12.5mg/g.Under the condition of the mixture(petroleum:ethyl acetate:ethyl acid=20:2:1,proportation of volume)as the fluid phase,flow rate of 3BV/h,98.0 percents of all the defoamer was obtained in the eluent.Secondly,when absolute ethyl alcohol was used as the fluid phase with a flow rate of 3BV/h,surfactin could be desorpted from the silica gel column,and its recovery reached 91.5%.The purification was greatly improved from 34.1%to 93.1%.The macroporous resin chromatograph was a simple and cheap separation method which could also reduced the production cost of surfactin.Among widely used twelve macroporous resins,X-5 resin had the highest adsorption capacity in static and dynamic tests,its non-polarity and configuration were suited for the lipopeptides(surfactin).Dynamic adsorption and desorption experiments were performed using a column packed with X-5 resin to obtain optimum conditions,with 30.0g/L resin concentration,0.5 BV/h feed flow rate,80%aqueous ethanol at 1 BV/h elute flow rate as eluant agent,the recovery of lipopeptides in final product was 78.4%?and purification as 70.2%.Because of its low cost,high efficiency,procedural simplicity and scale up easy,the adsorption-desorption method could be more efficient for the preparative separation and extraction of antibiotic lipopeptide than the conventional acid precipitation method,which could provide scientific references for antibiotic lipopeptide production.After the separation,piercing-solidifying method was applied to obtain the capsules of surfactin with sodium alginate as wall material and calcium chloride as solid solvent.On the basement of one-factor experiments,four factors including calcium chloride and sodium alginate concentration,ratio of core to wall,temperature were optimized by the method of response surface.The performance of all factors was evaluated by the microencapsulation efficiency.The results showed,under the condition of calcium chloride concentration 2%,temperature 49.0?,the proportion of sodium alginate to surfactin 1:2,sodium alginate concentration 2.4%,the microencapsulation efficiency reached 87.6%,the drug loading reached 12.5%.The release rate of surfactin from the microcapsule reached 91.7%,which indicated that it had good slow-release effect.The surfactin microcapsule was used in the preparation of walnut milk with fat floating rate and centrifugal sedimentation rate as evaluation.The result of one-factor experiments for the peeling procession of walnut were that,bath temperature 70 ?,time 1h,the concentrarion of NaOH 0.6mol/L.One-factor and orthogonal method were used to optimize the extract procession of protein in walnut.The optimum conditions were that stiring time 3.5min,walnut:water=1:10,the concentration of sodium bicarbonate 0.09g/L.After the study on the effects of surfactin microcapsules on steady of walnut milk,steady characteristic of walnut milk reached the best under the conditions of surfactin:monoglyceride=5:1,the concentration of emulsifier 0.03g/L,stirring time 3min.