A gel permeation chromatography ( GPC ) coupled with solid phase extraction and gas chromatography-mass spectrometric ( GPC-SPE-GC/MS ) method was developed to analyze seven kinds of organophosphate esters ( OPEs ) , including tri-n-butylphosphate, tri ( 2-chloroethyl ) phosphate, triphenyl phosphate, tris ( 2-butoxyethyl ) phosphate, tri-o-tolylphosphate, tri-m-tolylphosphate, and tri-p-tolylphosphate) in human serum. The recoveries of cleanup methods between GPC-silica/alumina column and H2 SO4-silica/sulfuric acid gel column were compared. The purification method with the GPC-silica/alumina column didn’t destroy the structure of organophosphate esters ( OPEs ) and could effectively remove some protein and lipid matrix influence in serum. The developed method was verified using the spiked blank and the spiked serum, the good recoveries and reproducibilities were achieved. The recoveries of all of OPEs in spiked blank (n=3) were all more than 75%. The recoveries of d12-TCEP and d15-TPhP in human serum samples (n=9) were 86. 3%±21. 6% and 103. 1%±16. 5%, respectively. In human serum samples (n=9), the detection ratios for TnBP, TCEP, TPhP, TBEP and m-TTP were more than 90% in all of the serum samples, p-TTP was only 30%, o-TTP was not detectable. The concentrations of TnBP, TCEP, TPhP, TBEP and m-TTP in serum were 3. 4-46. 5 ng/g lipid, 248. 6-958. 2 ng/g lipid, n. d. -4. 2 ng/g lipid, n. d. -49. 9 ng/g lipid and n. d.-23. 1 ng/g lipid, respectively.

In order to enrich the library of domestic research about new red fluorescent marker in lactic acid bacteria (LAB), we described a new fusion expression system in Lactobacillus plantarum WCFS1 based on the pSIP vector. This system contained red fluorescent protein mCherry as a marker and bile salt hydrolase gene (bsh) as a reporter gene. Moreover, in this study, four different promoters (PsppA, PldhL, P32 and PslpA) were used to regulate the expression of the fusion protein mCherry-BSH, completing the inducible and constitutive expression in lactic acid bacteria. The recombinant protein mCherry-BSH presented activity of red fluorescence and bile salt hydrolase (BSH). The successful construction of the fusion expression system in LAB using a red fluorescent protein mCherry provides favorable conditions for the distribution, intestinal colonization and survival rate of lactic acid bacteria, so as to reveal the function mechanism of its probiotic characteristics; and the system also could lay the foundation for researches on protein expression, cellular localization and properties identification of active protein in lactic acid bacteria.
Lactobacillus plantarum ; Luminescent Proteins ; Probiotics