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A new method for simultaneous determination of 23 kinds of per-and polyfluoroalkyl substances(PFASs)(13 kinds of perfluoro carboxylic acids,4 kinds of perfluoro sulfonic acids,and 6 kinds of new substitutes)in plant leaf tissue by ultra-high performance liquid chromatography-tandem mass spectrometry(UHPLC-MS/MS)using automatic online solid phase extraction(SPE)to remove the matrix interference components in plant crude extracts was developed.The plant leaf samples were extracted twice with 1%formic acid-methanol solution,then evaporated to dry,redissolved with 70%methanol solution,and directly injected for analysis.After 23 kinds of target PFASs were purified automatically by online SPE with a WAX column,the six-way valve was switched to rinse PFASs onto an alkaline mobile phase system-compatible C18 analytical column.Then,the 23 kinds of target PFASs were separated within 16 min by gradient elution using a binary mobile phase system of methanol/water(Containing 0.4%ammonium hydroxide).Tandem mass spectrometry was performed in multiple reaction monitoring(MRM)mode for online detection of various PFASs,and quantification was carried out by internal standard method.The results of the method validation showed that satisfactory average recoveries of 23 kinds of PFASs in plant leaf samples(64.2%-125.5%),precision(relative standard deviations(RSDs)of 0.7%-12.8%),linearity(R2＞0.990),and sensitivity(the detection limits(S/N=3)were in the range of 0.02-0.50 μg/kg)were achieved.Finally,this method was used to detect PFASs in the marine green tide algae(Enteromorpha prolifera)and several tree leaves,and a total of 6 kinds of PFASs were detected,in which PFBA was the main contaminant.Compared with the reported offline SPE methods,the proposed online SPE technique significantly simplified the sample pretreatment process and provided an automatic,simple,and environment-friendly method for the routine monitoring of legacy and emerging PFASs in plant tissues.

RESUMEN

In view of the present situation that edible marine shellfishes are combinedly contaminated by different kinds of lipophilic toxins, common lipophilic shellfish toxins in marine shellfishes were simultaneously detected by liquid chromatography-tandem mass spectrometry, and the safety risk of commercial marine shellfish was evaluated using the risk assessment method based on combined contamination of various toxins. Under the optimum conditions, satisfactory recoveries (63. 3％ - 88. 8％ ), precision ( relative standard deviations RSD≤14. 5％ ) and sensitivity (limit of detection in the range of 0. 5-2. 7 ng / g) of the method were achieved for all the analytes. Among the 105 commercially available shellfish samples, 42. 86％ of the samples had at least a kind of toxin. The highest average content was 47. 6 μg / kg of DTX1, which was the most serious contaminant for marine shellfishes. The total Expose Risk Index (∑ERI) was calculated based on Tolerable Daily Intake (TDI) and Acute Reference Dose (ARfD) of each toxin to evaluate the safety risk of commercial marine shellfish. The results showed that the ratio of commercially available marine shellfish with safety risk was 19. 05％ and the food safety risk of scallop was the highest. In summary, a new method based on the combined contamination of lipophilic shellfish toxins was successfully developed for risk assessment of the commercial marine shellfish. The proposed method is more harsh compared with the European Food Safety Authority (EFSA) regulation and can make shellfish consumers better to avoid the risk of poisoning.

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Recombinant plasmid pICG was constructed by replacing the internal fragment of a-acetohydroxyacid synthase (AHAS) gene (ILV2) with a copy of gamma-glutamylcysteine synthetase gene (GSH1) and copper chelatin gene (CUP1) from the industrial brewing yeast strain YSF31. YSF31 was transformed with plasmid pICG linearized by Kpn I and Pst I. A recombinant strain with high-glutathione and low-diacetyl production was selected. The results of fermentation in 100-L bioreactor showed that the lagering time of beer produced for recombinant strain T2 was shortened by 3 days and the shelf life of the beer was prolonged about 50%. It may be more acceptable for the commercial application, as it does not contain foreign DNA.

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The yeast fusant ZFF-28, which is high in biomass production and rich in selenium, was constructed after mutagenesis and protoplasts fusion between yeast strains. The total selenium content of ZFF-28 is 1.8 and 1.0 times higher than that of the parental strains Saccharomyces cerevisiae ZY-67 and Saccharomyces kluyveri SZY-198 respectively. Using single factor tests and a L16(4(3) x 2(1)) orthogonal design, the cultivation conditions was optimized as: 50mL culture in 250mL shake flasks in molasses containing 6% sugar and 60microg/mL Se at 28 degree C for 25h at 220 r/min, with the initial pH adjusted to 6.0 - 6.5. Under the optimized conditions, the biomass (dry weight) reached 8.2g/L and the Se content of the cells reached 2050microg/g, with organic and inorganic Se contents being 91% and 9% respectively.

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The enzyme activity of ?- Acetolactate Decaroboxylases (ALDC) from different microbes was studied, the results demonstrated that it was quite different among them. There were diversities of their enzyme reaction velocities. It was clear that the enzyme activity was affected by the pH of the enzyme reaction system, for example, the optimum pH of ALDC from Lactococcus lactis was 6. 6, while for Aerobacter Aerogenes it was 5. 8. Addition leucine,valine and isoleucine into enzyme reaction system obviously affected the enzyme activity of ALDC from different microbes.

RESUMEN

Self-cloning is a favorable technique in modification of industrial microorganisms especially food microorganisms because of its bio-safety. A survey and outlook of research in breeding of self-cloning industrial brewing yeast were introduced in this study.