| In this study, strains with high CLA producing ability were screened from traditional fermentation cream products, isomer composition of CLA were analyzed by gas chromatography method, and then fermentation medium and fermentation conditions were optimized to improve their productions. The main results of this paper are as follows:1. Lactic acid bacteria (LAB) strains with CLA producing ability from traditional fermentation cream products were isolated primitively by the bromocresol purple plate medium and then determining the CLA content of fermentation liquid with UV-spectrophotometry. The strains with the highest CLA producing ability were identified by analyzing their colony characteristics, cell morphology, biochemistry and physiological characteristics, and the phylogenetic tree generated by MEGA 6 software, basing on 16S rDNA sequence. Thirty CLA producing strains were isolated from traditional cream products. When 2%(v/v) inocula were added to the MRS broth supplemented with 0.06%(v/v) LA, of eleven strains screened from the cultured butter of Xianghuang Banner, HS4 with the highest producing capacity was identified as Lactobacillus casei, whose yield of CLA was 23.59±0.99μg/mL, of nine strains screened from the cultured cream of Zhenglan Banner, LX5 with the highest producing capacity was identified as Lactobacillus paracasei, whose yield of CLA was 20.51±0.55μg/mL, of ten strains screened from the cultured butter of Abaga Banner, AS8 with the highest producing capacity was identified as Enterococcus faecium, whose yield of CLA was 13.22±0.52μg/mL.2. Gas chromatography method was used to analyze isomer composition of CLA. The peak area of c9,t11-CLA and t10, c12-CLA which were produced by strain HS4 were about 0.75% and 0.71%, and the relative amount of c9, t11-CLA and t10, c12-CLA were about 37.94% and 35.76% in fatty acid of fermentation broth. The peak area of c9, t11-CLA and t10, c12-CLA which were produced by strain LX5 were about 0.75% and 0.72%, and the relative amount of c9,t11-CLA and t10,c12-CLA were about 35.09% and 33.40%. The peak area of c9, t11-CLA and t10, c12-CLA which were produced by strain AS8 were about 0.78% and 0.70%, and the relative amount of c9,t11-CLA and t10, c 12-CLA were about 31.67% and 28.75%.3. The different fermentation mediums were optimized firstly by LB medium, MRS medium and skim milk medium, and finally the MRS medium was defined as the most optimum culture medium for strain HS4, LX5, and AS8. Compositions of MRS medium of three strains were studied by single factor experiment and orthogonal experiment. The optimum medium of strain HS4 and AS8 consisted of peptone lOg/L, beef extract lOg/L, yeast extract 7g/L, glucose 2.5%, sodium acetate 7g/L, ammonium citrate 2g/L, dipotassium hydrogen phosphate 3g/L, manganese sulfate 2g/L, magnesium sulfate 10g/L, and tween-80(0.10%); the optimum medium of strain LX5 consisted of peptone lOg/L, beef extract 15g/L, yeast extract 7g/L, glucose 2.5%, sodium acetate 7g/L, ammonium citrate 2g/L, dipotassium hydrogen phosphate 3g/L, manganese sulfate 2g/L, magnesium sulfatelOg/L, tween-80(0.10%).4. The fermentation conditions of strain HS4, LX5, and AS8 were studies by single factor experiment and response surface method (RSM). The optimum fermentation conditions of strain HS4 were culture temperature 33℃, culture time 37h, LA additional quantity 0.08%, inoculum volume 2.72%. The optimum fermentation conditions of strain LX5 were culture temperature 33℃, culture time 36h, LA additional quantity 0.07%, inoculum volume 2.70%. The optimum fermentation conditions of strain AS8 were culture temperature 33℃ culture time 36h, LA additional quantity 0.09%, inoculum volume 2.74%. In the optimized conditions, the CLA productions of three strains were respectively up to 43.88±0.75μg/mL,39.91±0.38μg/mL,28.67±0.99μg/mL, which were found to be 1.86 times,1.94 times,2.17times than those values without optimization separately. |
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