Study On The Conditions Of CLA Biosynthesis By Whole Cells Of The Mixed Strains

Conjugated linoleic acid(CLA)defines a mixture of positional and geometric conjugated isomers of the essential fatty acid linoleic acid(LA)with conjugated double bonds.CLA has recently attracted much concern as a novel type of biologically beneficial functional fatty acids.Chemical synthesis of CLA could produce more byproducts which are very difficult to purify,therefore,this limits CLA’s application.Compared with chemical synthesis of CLA,microbial synthesis of CLA has the following advantages,such as the mild reaction conditions and single isomer with biological activities.Therefore,the microbial biosynthesis of CLA has been the the mainstream in the field.Based on the the technology of coculture and whole cell,whole cells of mixed strains were applied to produce CLA,which can not only enhance the ability that Lactic acid bacteria(LAB)strains produce enzyme,but avoid the inbibitional effect of LA on LAB strains.The conversion rate of LA to CLA was improved significently in the paper.In this paper,c9,t11-CLA,t10,c12-CLA and t,t-CLAmixture biosynthesized by whole cells of mixed LAB strains were the three kind of main products through the method of GC-MS.And the UV was selected to conduct quantitative analysis.Seven species,lactobacillus plantarum(Lp),lactobacillus acidophilus(La),streptococcus thermophilus(SS),lactobacillus bulgaricus(Lbu)，lactobacillus fermentum(Lf),lactobacillus casei(Lc),lactobacillus breris(Lb),were used as experimented strains to compare the ability of CLA production by whole cells of singal LAB strain and mixed LAB strains.Among them,the whole cells of La and Lp showed the higher CLA-producing ability than others.When the proportion of La and La was 1:1,it showed the highest CLA production.Then the research described in this dissertation concentrates on the CLA biosynthesized by whole cells of La and Lp at different conditions including nutritional conditions,induction culture conditions,permeabilization conditions and reaction conditions.Nutritional conditions of the biosynthesis of CLA by whole cells of the mixed bacteria strains were studyed systematically by using single factor test and orthogonal test.The optimal nutritional conditions for the whole cells converting LA to CLA were as follows:glucose concentration was 20 g/L,peptone concentration was 15 g/L,beef extract concentration was 5 g/L,yeast extract concentration was 5 g/L,anhydrous sodium acetate concentration was 7.5 g/L,tween-80 concentration was 1.5 g/L,diammonium citrate concentration was 3 g/L,K2HPO4 concentration was 3.93 g/L,MgS04 concentration was 0.58 g/L,MnSO4 concentration was 0.2 g/L.In view of the best nutritional conditions,the concentration of CLA reached the maximum biosynthesis 81.4μg/mL.Induction culture conditions of the biosynthesis of CLA by whole cells of the mixed bacteria strains were studyed systematically by using single factor test and orthogonal test.The optimal induction culture conditions for the whole cells converting LA to CLA were as follows:LA additional quantity 5%,inoculum size 1.5%,pH 6.0,incubation temperature 37℃ incubation time 16 h,added time of induction of LA 8 h.In view of the best induction culture conditions,the concentration of CLA reached the maximum biosynthesis 145.2μg/mL.The optimal induction culture conditions for permeabilization conditions were obtained when wholes cells were treated with 2%CTAB at 30 ℃ for 15min.Based on the above conditions CLA production was 194.8 μg/mL.Reaction conditions of the biosynthesis of CLA by whole cells of the mixed bacteria strains were studyed systematically by using single factor test and response surface design method.The optimal reaction conditions for the whole cells converting LA to CLA were as follows:In the Na2HPO4/KH2PO4 buffer system(0.1 mol/mL),substrate LA concentration0.7 mg/mL,whole cells concentration 4mg/mL,reaction system pH 6.5,reaction temperature 37.5 ℃,reaction time 16 h,BHT additional quantity 0.4%.Based on the best induction culture conditions,the concentration of CLA reached the maximum biosynthesis 492.4 μg/mL and the corresponding conversion rate was 70.3%.Compared with basic conditions,nutritional conditions and permeabilization conditions were relatively conducive to the biosynthesis of t,t-CLA mixture,induction culture conditions were realative to favour to the biosynthesis of t10,c12-CLA,and reaction conditions contribute to the biosynthesis of c9,t11-CLA.