Studies On The Screening Of Bacteria Producing Conjugated Linoleic Acid And The Properties Of Linoleate Isomerase

Conjugated linoleic acid (CLA) refers to a mixture of positional and geometric isomers of linoleic acid with conjugated double bonds. CLA has been proven to have extensive health benefits in anticarcinogenic, antiadipogenic, antiatherogenic, antidiabetogenic and anti-inflammatory aspects by many studies and it has become the focus of studies. The objective of this paper is to carry out comprehensive research in the field of biotransform CLA by dairy starters and their linoleate isomerase. The main results of this paper are as follows:CLA had the highest UV absorption at 233 nm and the absorbance was positive to samples' concentration. The standard UV absorbance curve of CLA was y=0.0678x+0.127 (x was the content of CLA, y was the absorbance at 233 nm), R²=0.981. One linear and two non-linear curve fits were employed to the data on quantization of CLA content analyzed by UV spectrophotometer and GC. The formula y=4.15+300.16/(l+10^2-96-0..04x) where x was the CLA content analyzed by UV spectrophotometer and y was the CLA content analyzed by GC was the most appreciated. Results showed that UV spectrophotometry combined with regression formula was an effective, quick and low cost method to analyze CLA content.Two strains each of P. freudenreichii ssp. shermanii and P. freudenreichii ssp. freudenreichii were tested for their ability to produce CLA in sodium lactate medium (SLM), De Man-Rogosa-Sharpe (MRS) medium and skim-milk. Data showed that both strains were able to produce CLA in three media supplemented with different concentrations of sunflower oil. Maximum production of CLA (78.79μg/mL) was observed after 36 h of incubation in MRS containing 12 mg/mL sunflower oil by P. freudenreichii ssp. shermanii. Moreover, the growths of both strains were inhibited by sunflower oil and a positive relationship between CLA production and ability to tolerate sunflower oil was observed. At the same time, it was also observed that the inhibitory effects on P. freudenreichii ssp. shermanii and P. freudenreichii ssp. freudenreichii in three media follow the order SLM＞skim-milk＞MRS and SLM＞MRS＞skim-milk, respectively. Micro aerobic conditions were in favor of increasing the amounts of CLA.The ability of CLA formation by 24 lactic acid bacteria strains was studied and 7 strains had the ability to produce CLA. The identification of strains by API system revealed that the CLA formation strains were Lactobacillus plantarum, Lactobacillus salivarius and Lactococcus lactis ssp. lactis. The highest CLA concentration (103.366μg/mL) was obtained by cultivating L. plantarum in skim-milk media with sunflower oil content of 6 mg/mL. The concentration of CLA under aerobic conditions was in agreement with that under micro aerobic conditions, but micro aerobic conditions were in favor of the stability of CLA.Linoleate isomerase from P. freudenreichii ssp. shermanii and L. plantarum was purified by 80% ammonium sulphate; gel filtration column chromatography ( Sephacryl S-200HR ) and weak anion-exchange column chromatography (DEAE Sepharose F.F.), achieving an overall purification of 31.6-fold and 36-fold, respectively. The purified L. plantarum linoleate isomerase was a single band of around 45 kDa on SDS-PAGE, but the isomerase from P. frendenreichii ssp. shermanii was two bands. The optimum pH for P. freudenreichii ssp. shermanii linoleate isomerase was 8.0 and the optimum temperature was about 30℃. The K_m and V_max of linoleic acid was 20.53μM and 0.44μg/mL.min. The optimum pH for L. plantarum linoleate isomerase was 7.5 and the optimum temperature was about 50℃. The K_m and V_max of linoleic acid was 17.85μM and 0.73μg/mL.min.The peptide mass fingerprints produced by P. freudenreichii ssp. shermanii and L. plantarum linoleate isomerase were studied by HPLC-ESI-MS system.Data from the HPLC-ESI-MS system showed that the amino acid sequencees of linoleate isomerase extracted from P. freudenreichii ssp. shermanii (sample1) were similar to that from L. plantarum. However, the amino acid sequences of P. freudenreichii ssp. shermanii linoleate isomerase (sample2) were less closely related with that of P. freudenreichii ssp. shermanii linoleate isomerase (samplel). Studies showed that the sequences of experimental linoleate isomerase were different from that of reported.In order to study the enzyme formation of CLA under the supercritical carbon dioxide circumstances a quadic regression model was established. The model fitted significantly well and the highest CLA content (0.93 mg/mg protein) was obtained when reaction time 1 h, temperature 46.4℃and pressure 25 MPa.Above all, L. plantarum had the higher ability to produce CLA than P. freudenreichii ssp. shermanii. Furthermore we observed the isomerase transformation of LA to CLA under supercritical carbon dioxide circumstances and could draw a conclusion that L. plantarum linoleate isomerase should be paid more attention to and studied further.