Study On Mutation And Protoplasts Fusion Of Natamycin-producing Strains And The Optimization Of Its Fermentation Process

Food antisepsis is the important content of food industry,and also a question which the food science workers most pay attention.The food deterioration will cause not only the nutritional value lose but also the food poisoning.At present,the antiseptic measure commonly used is adding food antiseptic.The natural antiseptic is the new product which domestic and foreign proposing,seeking and developing the recently years.It was not only harmLess to the human health,but also had certain nutritional value.The natural antiseptic is the development direction in future.So far,dozens of kind of natural food antiseptics has been developed overseas with the complete laws and regulations.The natural food antiseptic's research start later in inland,was still in the initial stage at present.The domestic scholar has also done the massive work for the adaptation social development request.The natural antiseptic with the characteristic of strong inhibition,safe and non-toxic, water-solubility,and using broadly and so on is batter than synthesis antiseptics.Natamycin is also a natural antiseptic with market demands.But the primary cause limited its production is the low fermentation output.In this article a systematic investigation was carried out to improve the natamyin-producing starin by using of mutation breeding,protoplast fusion technique,in which,strains presereening,mutation breeding,protoplast preparation fusion and regeneration, protoplast mutation,and fermentation condition optimization were adopted for high natamycin-producing.1) The HPLC method of natamycin detection in fermentation solution was developed. The optimum determination conditions were detection wavelength:303nm,mobile phase: methanol:water:60:40,flow speed:1.00mL/min,keeping time:6min,log-linear regression equation Y=61036x+31764,R~2=0.9998.The limnimal detectability was 0.25μg/mL when the signal noise ratio was 3.Averaged recovery was 100.23%and averaged RSD was 0.753%.2) Firstly the suitable slant agar medium are screening for S.natalensis and S. chattanoogensis.The result shows that 61~# and 42~# agar medium are suitable for S.natalensis and 61# agar medium for S.chattanoogensis.The natamyci-producting strains were purified with agar block method.SN114 strain and SC21 strain was chosen respectively and its each yield of natamycin in shake flasks was 0.669g/L and 1.098g/L.The MIC of streptomycin sulfate was 0.2μg/mL and 10μg/mL respectively.3) SN11 was flashed 3 times with pulsed electrical flash and SC216 was mutanted 50min with diethyl sulfate.High producing mutants were selected with the streptomycin sulfate resist plate according to the suggested biosynthesis pathway and metabolic regulation mechanism of natamycin.The SN 114 resistant mutant was attained and its yield of natamycin was 1.090g/L,1.6 times of orginal strain.The SC216 resistant mutant was attained and its yield of natamycin was 1.262g/L,1.31 times of orginal strain.4) The protoplast preparation and regeneration technique of SN114 strain and SC216 strain were established.The optimum conditions of protoplast preparation and regeneration technique of SN114 were shake-cultured for 44h with 1.0%Gly and lysised with 4 mg/mL lysozyme,keeping warm at 35℃for 60min.That which of SC216 were shake-cultured for 52h with 1.2%Gly and lysised with 8mg/mL lysozyme,keeping warm at 35℃for 60min.5) With preliminary study on protoplast mutation breeding of SN114 with UV and SC216 with pulsed electrical flash,highest producer SN 1146 and SC2164 was obtained,the yield of natamycin was 1.62g/L and 1.98g/L and the MIC of streptomycin sulfate was 0.3μg/mL and 15μg/mL respectively.6) The fusion of sigle-parental inactivated protoplasts with screening of streptomycin sulfate was studied.One highest producer,Sr-1,could produce 2.45g/L natamycin,and its heredity property was stable.The crasis was identified.7) The culture medium composition and the fermentation condition of shake flask were optimizated in this dissertation.The effect of sigle carbon and sigle nitrogen source,mix carbon and mix nitrogen source,precursors and pH value on fermentation were studied.The optimal formula of medium through the response surface test was that glucose 33g/L, maltodextrin 7.7g/L,trypeone 26g/L,yeast extract powder 8.2g/L and orginal pH value 7.0. The optimum fermentation conditions were 2%inoculum volum,20mL/250mL medium volume,220r/min rotation speed and 29℃fermentation temputure.The yield of natamycin is 3.17g/L after the optimum of culture medium and fermentation conditions.8) Base on the above optimum condition,the batch fermentation was performed in 5-liter fermentor.And the optimal fermentation conditions were that 4L/min ventilatory capacity, 450r/min rotational speed speed,dissolves oxygen not be lower than 20%,fermentation period 86h and pH should control nearby 5.5,and the yield of natmycin is 2.92g/L.9) The natamycin purification using HPD200A macroporous resin was studied.The mecrocrystalline of natamycin was obtained and its purity reached to 86.6%with 83% recovery rate.