The Breeding Of Pullulan-producing Strains And Optimization Of Fermentation Condition

Pullulan,an extracelluar polysaccharide is obtained by fermentation using fungs, Aureobasidium pullulans.It is transparent,colourless,tasteless,odourless,tenacious,resistant to oil and grease and unaffentde by small thermal variations.So it can apply in many territory,such as,medicine,food,cosmetics,nicotiana tabacum,agriculture.And also it can be degradated by microbion easily,can not contaminate environment.It is multifunction material of having the largest economic value and exploitation potentiality.In extra-country pullulan has been produced in industry,but in our country pullulan industrialized production was not found now,because it is hard to found a strain with high pullulan production,so it is necessary for pullulan industrialized production to obtain a strain with high pullulan production.In recently years protoplast fusion was applied to many microbion,but it is not applied to Aureobasidium pullulans.This study selected the method for pllulan producing strain with high yield by using the died parental strain protoplasts fusion is deserved to generalize.Meanwhile it discussed the exploration about the conditions of fermentation in the dissertation.First prepare the protoplast with the Aureobasidium Pullulans P and a lower colourant strain of UV₁ which was conserved in our lab as original strain.After the strains was cultured in liquid basal medium for 24 hours,Later,the strain was washed by distilled water for one or twice and then suspended,The concentration of suspension was 10⁸ cells per mL.The suspension 2mL was added in the same volumeβ-mercaptoethanol,and incubated at 37℃for 30 min.The cells were collected by centrifugation about 6000 rpm,and then it was put in mixed enzymes(snailuse: celluluse=1:1) liquid and digested at 37℃for one hour.Protoplasts were observed by microscope (OLYMPUS) in certain time.In this time the preparation of the protoplasts were 100%.the regeneration of protoplasts in RMI and RM2 were more higher than others,especially in RM2, the regeneration of protoplasts were 64.6%.So we can see that this study not only rising the preparation of the protoplasts,but also rising the regeneration of protoplasts.Second research on fusion of inactivated parental protoplasts:The UV₁ protoplast suspension 2ml was incubated at 60℃for 20 min,Inactivation frequency of protoplasts was 100%.The UV inactivation condition was that Aureobasidium Pullulans P protoplast suspension 4mL at 30W ultraviolet light 30cm away for 3min.The above inactivated parental protoplasts were put together, the cells were collected by centrifugation at 6000 rpm for 10min,through away the above and then it was added 40%PEG6000 and new type of Ca₃(PO₄)₂ at 37℃for 5～10min.The protoplasts were collected by centrifugation at 6000 rpm for 10min,through away the above.The protoplasts were washed by 0.6mol/L KCl.The protoplasts were suspended in 0.6mol/L KCl.And protoplast suspension diluted properly was cultured in regenerate medium RM1.The fusion of inactivated parental protoplasts can regenerate,because of their injure can supplement each other.So the strains which were grow up in regenerated medium are fusants.The fusants were transferred for ten generation and conserved.The fusants were screened by first and second screening,and RH with high yield was screened in the fusants.Last study on optimizing fermentation condition of fusion RH.During the fermentation condition was optimized,first single factor screening method was designed containing carbon source,nitrogen source,inorganic salt,seed quality,temperature,pH,fermentation time and so on. Then orthogonal experiment was designed containing above factors.The relations among each factor were evaluated to attain the optimal nutritional factors and fermentation condition and result was analyzed by SPSS software.Finally the RH optimal condition is that 10%sucrose,0.2%yeast extract,0.04%NH₂SO₄,0.01%MgSO₄·7H₂O,0.2%KH₂PO₄,0.2%NaCl.28℃,cycle of fermentation is 120h,pH 6.0,amount of inoculation is 5%.After fermentation the contents of pullulan is 73.6g/L in broth,increased 9 times than primary strains.The selection method for pllulan producing strain with high yield by using the died parental strain protoplasts fusionis deserved to generalize.