Microbial Metabolomic Study Of The Bioconversion Of Conjugated Linoleic Acid By Lactobacillus Plantarum Using GC-MS

Metabolomics is a new branch of science,which focuses on the metabolic intermediates and end-products of cell or organism.Therefore,it has been applied into many areas,including the study of Lactobacillus plantarum,which has benefits on human health and is regarded as a safe suppliment in food.In this study,a preparation method of L.plantarum for metabolomic analysis was set up.Then this method was used to investigate the metabolic feature of L.plantarum ZS2058 and its linoleate isomerase gene deletion mutants as well as L.plantarum ST-III,in order to explain the difference during the bioconvision of conjugated linoleic acid(CLA).Firstly,this study set up a metabolomics analysis method for L.plantarum ZS2058.The quenching and extraction procedures with different solvents and operational temperatures are evaluated,and the recovery of this method is verified.Eight quenching methods that used readily available organic solvents(methanol and ethanol)or cryoprotectants(glycerol and dimethyl sulfoxide)were also assessed quantitatively,and their effects on cell membrane integrity and metabolic inactivation were identified with propidium iodide and energy charge assays,respectively.The combination of 20% methanol and 0.9% sodium chloride(-4oC),which yielded a propidium iodide labelling rate of 3.06±0.16% and an energy charge value of 0.849±0.003,outperformed most of its counterparts and was adopted for further use.The abilities of four extraction solvent systems,including methyl tert-butyl ether/methanol/water,methanol/water,acetonitrile/water and acetonitrile/methnol/water,to retrieve cellular metabolites were then evaluated.Here,acetonitrile/methnol/water was found to be the most efficient extraction solvent system.Furthermore,the selected sample pre-treatment protocol was subjected to analytical validation using a series of selected representative metabolites and yielded a linear range of 0.00128 to 100 ?g/mL with coefficients of 0.9953 to 0.9999 and recovery levels of 90% to 110%.Secondly,we applied the above method to expore the metabolic feature among L.plantarum ZS2058,L.plantarum ZS2058Δmcra,L.plantarum ZS2058Δdh,L.plantarum ZS2058Δdc and L.plantarum ST-III related to the metabolism of linoleic acid(LA).The results of metabolomics analysis implied that the intracellular metabolic feature of L.plantarum ZS2058 and L.plantarum ST-III showed significant difference.This study screened 28 and 46 diffirentiating compounds through multivariate statistical analysis between L.plantarum ZS2058 and L.plantarum ST-III grown in MRS or LA+MRS,resepectvely.The differentiating compounds included amino acids,sugars and nucleotides.Lastly,in order to further research the fatty acids of the five L.plantarum shown as the above,we study the fatty acids composition of them at the culture time of 0 h,5 h,10 h,14 h,18 h and 24 h.The results revealed the content of intracellular LA and 10-hydroxy-cis-12-octadecenoic acid(10-HOE)in L.plantarum ZS2058 wouldn’t accumulate significantly.However,the intracellular and extracellular content of LA and 10-HOE showed relatively high in L.plantarum ZS2058Δdh,L.plantarum ZS2058Δdc and L.plantarum ST-III.And when the LA in cystolic increased,other fatty acids such as oleic acid(OA)would incerse.For L.plantarum ZS2058,the occurrence time of intracellular 10-HOE was early than that outside the cell.For L.plantarum ST-III,the occurrence time of intracellular and extracellular 10-HOE was same.