Studies On The Mutation Breeding Of Bacteria Producing Conjugated Linoleic Acid And High-efficiency Transformation Technology

Conjugated linoleic acid(CLA)refers to a mixture of positional and geometric isomers of linoleic acid(C18:2,cis-9,cis-12 octadecadienoic acid),CLA has been proven to have extensive health benefits in anticarcinogenic,antiadipogenic,antiatherogenic and ant-inflammatory aspects by many studies.It was one of important functional lipids discovered by human recently 20 years.Research of this paper included bacteria with high ability of fermentation CLA screening from lactobacillus,identification,and induced mutation by ion implantation,producing cell of strain A6-1F by optimized condition of mutantA6-1F synthesize linoleic isomerase,enzymatic characteristics of strain A6-1F and yield of CLA produced by cell of strain A6-1 in the shaking bath,ultrasonic bath and SC-CO₂, and mechanisms of CLA highly fermented by cell of strain A6-1 in the ultrasonic bath and SC-CO₂.The main results of this paper are as follows:The objective of the research is to select the high produce of CLA from aerobic and aerotolerant bacterium in the broth fermentation.Stains of A6-1 and SL-3 showed the higher ability of CLA formation in 40 lactic acid bacteria strains tested.Both of A6-1 and SL-3 strains were identified as Lactobacillus plantarum by API 50 system identification.The product of CLA in the broth,which was cultured in MRS including 0.5%LA at 37℃for 24 h, was analyzed by Gas Chromatography,the result indicated that the product was a mixture of CLA isomer,there were 61.554μg/mL of c-9,t11 CLA and 14.235μg/mL of t-10,c12 CLA in the fermented broth of A6-1 strain,and 46.161μg/mL of c-9,t11 CLA and 8.7399μg/mL of t-10,cl2 CLA in the fermented broth of SL-3 strain.Effects of the fermentation conditions of transformation linoleic acid into conjugated lonoleic acid by L.planturum A6-1 were investigated.The results indicated,effect of amount of inoculums,reaction temperature,initial pH,incubation time,concentration of LA on the formation of CLA,The optimal condition were as follow:amount of inoculums 2%,reaction temperature 37℃,initial pH 7.0,incubation time 32h,concentration of LA in the MRS broth 1.0mg/mL.91.4μg/mL of CLA were produced under optimum conditionBiological effects of strain of A6-1 implanted with N⁺ ions used 50keV of energy and doses as1×l0¹³,3×10¹³,5×l0¹³,8×10¹³,10×10¹³,30×l0¹³,50×l0¹³,80×l0¹³,100×l0¹³ ion/cm²,survival rate of strain of A6-1 implanted by 30×l0¹³ions/cm² and 50×l0¹³ions/cm² of doses were 20～35%,30×l0¹³ions/cm² of dose is suitable dose to mutation。25 mutants from strains selected had high ability of fermentation CLA,mutants strain named A6-1F had the best stability through 8 time generation and highest yield was 162.5μg/mL,its potency was higher than 69.8%of the control group's.The condition of mutant A6-1F to synthesize linoleic isomerase was studied.The optimum condition were as fellows:0.4mg/mL of LA in the seed broth,culture-age 20 h of seed broth,amount of inoculums 4%,initial pH 7.0,concentration of LA in the MRS broth 0.75mg/mL,incubation time 32h at temperature 36±1℃,activity of isomerase were 337.4U/mL under optimum condition.Yield of CLA produced by cell which was permeabilized by 10%ethanol,2%toluene, 30%acetonum not was higher than tant of cell freezed and thawed,yield of CLA produced by cell of freed ang thaved was higher than 20%that of cell untreated by any way.The optimum reaction condition of A6-1F were as fellows:cell concentration 25%,pH 7.0,concentration of LA in buffer 2.0mg/mL,reaction 2h at temperature 36±1℃,yield of CLA was 275.7μg/mL under optimum condition.Yield of CLA produced by cell in the different condition was as fellow:in the SC-CO2＞ultrasonic bath＞shaking bath.Yield of CLA was 0.623mg/mL,ratio of transformation was41.5%in buffer including 15%of cell,1.5mg/mLof LA,reaction time 30 min,under SC-CO₂.