Facile fabrication of carbon nanotube hollow microspheres as a fiber coating for ultrasensitive solid-phase microextraction of phthalic acid esters in tea beverages.

The efficient extraction of phthalic acid esters (PAEs) is challenging due to their extremely low concentration, complicated matrices and hydrophilicity. Herein, hollow microspheres, as an ideal coating, possess significant potential for solid-phase microextraction (SPME) due to their fascinating properties. In this study, multiwalled carbon nanotube hollow microspheres (MWCNT-HMs) were utilized as a fiber coating for the SPME of PAEs from tea beverages. MWCNT-HMs were obtained by dissolving the polystyrene (PS) cores with organic solvents. Interestingly, MWCNT-HMs well maintain the morphology of the MWCNTs@PS precursors. The layer-by-layer (LBL) assembly of MWCNTs on PS microsphere templates was achieved through electrostatic interactions. Six PAEs, di-ethyl phthalate (DEP), di-iso-butyl phthalate (DIBP), di-n-butyl phthalate (DBP), benzyl butyl phthalate (BBP), di-2-ethylhexyl phthalate (DEHP) and di-n-octyl phthalate (DOP), were selected as target analytes for assessing the efficiency of the coating for SPME. The stirring rate, sample solution pH and extraction time were optimized by using the Box-Behnken design. Under optimal working conditions, the proposed MWCNT-HMs/SPME was coupled with gas chromatography-tandem mass spectrometry (GC-MS/MS) to achieve high enrichment factors (118-2137), wide linearity (0.0004-10 µg L-1), low limits of detection (0.00011-0.0026 µg L-1) and acceptable recovery (80.2-108.5%) for the detection of PAEs. Therefore, the MWCNT-HM coated fibers are promising alternatives in the SPME method for the sensitive detection of PAEs at trace levels in tea beverages.

GAT1 Gene, the GATA Transcription Activator, Regulates the Production of Higher Alcohol during Wheat Beer Fermentation by Saccharomyces cerevisiae.

Uncoordinated carbon-nitrogen ratio in raw materials will lead to excessive contents of higher alcohols in alcoholic beverages. The effect of GAT1 gene, the GATA transcription activator, on higher alcohol biosynthesis was investigated to clarify the mechanism of Saccharomyces cerevisiae regulating higher alcohol metabolism under high concentrations of free amino nitrogen (FAN). The availability of FAN by strain SDT1K with a GAT1 double-copy deletion was 28.31% lower than that of parent strain S17, and the yield of higher alcohols was 33.91% lower. The transcript levels of the downstream target genes of GAT1 and higher alcohol production in the double-copy deletion mutant suggested that a part of the effect of GAT1 deletion on higher alcohol production was the downregulation of GAP1, ARO9, and ARO10. This study shows that GATA factors can effectively regulate the metabolism of higher alcohols in S. cerevisiae and provides valuable insights into higher alcohol biosynthesis, showing great significance for the wheat beer industry.

Identification of Core Regulatory Genes and Metabolic Pathways for the n-Propanol Synthesis in Saccharomyces cerevisiae.

The n-propanol produced by Saccharomyces cerevisiae has a remarkable effect on the taste and flavor of Chinese Baijiu. The n-propanol metabolism-related genes were deleted to evaluate the role in the synthesis of n-propanol to ascertain the key genes and pathways for the production of n-propanol by S. cerevisiae. The results showed that CYS3, GLY1, ALD6, PDC1, ADH5, and YML082W were the key genes affecting the n-propanol metabolism in yeast. The n-propanol concentrations of α5ΔGLY1, α5ΔCYS3, and α5ΔALD6 increased by 121.75, 22.75, and 17.78%, respectively, compared with α5. The n-propanol content of α5ΔPDC1, α5ΔADH5, and α5ΔYML082W decreased by 24.98, 8.35, and 8.44%, respectively, compared with α5. The contents of intermediate metabolites were measured, and results showed that the mutual transformation of glycine and threonine in the threonine pathway and the formation of propanal from 2-ketobutyrate were the core pathways for the formation of n-propanol. Additionally, YML082W played important role in the synthesis of n-propanol by directly producing 2-ketobutyric acid through l-homoserine. This study provided valuable insights into the n-propanol synthesis in S. cerevisiae and the theoretical basis for future optimization of yeast strains in Baijiu making.

Comparative transcriptome analysis reveals the key regulatory genes for higher alcohol formation by yeast at different α-amino nitrogen concentrations.

Higher alcohols are important flavor substance in alcoholic beverages. The content of α-amino nitrogen (α-AN) in the fermentation system affects the formation of higher alcohols by Saccharomyces cerevisiae. In this study, the effect of α-AN concentration on the higher alcohol productivity of yeast was explored, and the mechanism of this effect was investigated through metabolite and transcription sequence analyses. We screened 12 most likely genes and constructed the recombinant strain to evaluate the effect of each gene on high alcohol formation. Results showed that the AGP1, GDH1, and THR6 genes were important regulators of higher alcohol metabolism in S. cerevisiae. This study provided knowledge about the metabolic pathways of higher alcohols and gave an important reference for the breeding of S. cerevisiae with low-yield higher alcohols to deal with the fermentation system with different α-AN concentrations in the brewing industry.

Identification by comparative transcriptomics of core regulatory genes for higher alcohol production in a top-fermenting yeast at different temperatures in beer fermentation.

Undesirable flavor caused by excessive higher alcohols restrains the development of the wheat beer industry. To clarify the regulation mechanism of the metabolism of higher alcohols in wheat beer brewing by the top-fermenting yeast Saccharomyces cerevisiae S17, the effect of temperature on the fermentation performance and transcriptional levels of relevant genes was investigated. The strain S17 produced 297.85 mg/L of higher alcohols at 20 °C, and the production did not increase at 25 °C, reaching about 297.43 mg/L. Metabolite analysis and transcriptome sequencing showed that the metabolic pathways of branched-chain amino acids, pyruvate, phenylalanine, and proline were the decisive factors that affected the formation of higher alcohols. Fourteen most promising genes were selected to evaluate the effects of single-gene deletions on the synthesis of higher alcohols. The total production of higher alcohols by the mutants Δtir1 and Δgap1 was reduced by 23.5 and 19.66% compared with the parent strain S17, respectively. The results confirmed that TIR1 and GAP1 are crucial regulatory genes in the metabolism of higher alcohols in the top-fermenting yeast. This study provides valuable knowledge on the metabolic pathways of higher alcohols and new strategies for reducing the amounts of higher alcohols in wheat beer.

Flavobacterium zaozhuangense sp. nov., a new member of the family Flavobacteriaceae, isolated from metolachlor-contaminated soil.

Strain ZZ-8T, a Gram-negative, aerobic, non-spore-forming, non-motile, yellow-pigmented, rod-shaped bacterium, was isolated from metolachlor-contaminated soil in China. The taxonomic position was investigated using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain ZZ-8T is a member of the genus Flavobacterium and shows high sequence similarity to Flavobacterium humicola UCM-46T (97.2%) and Flavobacterium pedocola UCM-R36T (97.1%), and lower (< 97%) sequence similarity to other known Flavobacterium species. Chemotaxonomic analysis revealed that strain ZZ-8T possessed MK-6 as the major respiratory quinone; and iso-C15:0 (28.5%), summed feature 9 (iso-C17:1 w9c/C16:0 10-methyl, 22.9%), iso-C17:0 3-OH (17.0%), iso-C15:0 3-OH (8.9%), iso-C15:1 G (8.6%) and summed feature 3 (C16:1 w7c/C16:1 w6c, 5.7%) as the predominant fatty acids. The polar lipids of strain ZZ-8T were determined to be lipids, a glycolipid, aminolipids and phosphatidylethanolamine. Strain ZZ-8T showed low DNA-DNA relatedness with F. pedocola UCM-R36T (43.23 ± 4.1%) and F. humicola UCM-46T (29.17 ± 3.8%). The DNA G+C content was 43.3 mol%. Based on the phylogenetic and phenotypic characteristics, chemotaxonomic data and DNA-DNA hybridization, strain ZZ-8T is considered a novel species of the genus Flavobacterium, for which the name Flavobacterium zaozhuangense sp. nov. (type strain ZZ-8T = KCTC 62315 T = CCTCC AB 2017243T) is proposed.

Roseomonas chloroacetimidivorans sp. nov., a chloroacetamide herbicide-degrading bacterium isolated from activated sludge.

A Gram-negative, aerobic, short rod-shaped, pink-pigmented, non-motile bacterium, designated BUT-13(T), was isolated from activated sludge of an herbicide-manufacturing wastewater treatment facility in Jiangsu province, China. Growth was observed at 0-5.5 % NaCl, pH 6.0-9.0 and 12-37 °C. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain BUT-13(T) is a member of the genus Roseomonas, and shows high sequence similarities to R. pecuniae N75(T) (98.0 %) and R. rosea 173-96(T) (97.5 %), and lower (<97 %) sequence similarities to all other Roseomonas species. Chemotaxonomic analysis revealed that strain BUT-13(T) possesses Q-10 as the predominant ubiquinone; summed feature 8 (C18:1 w7c and/or C18:1 w6c; 38.8 %), C18:0 (16.6 %), C16:0 (15.2 %), summed feature 3 (C16:1 ω6c and/or C16:1 ω7; 7.9 %) and C18:1 w9c (4.7 %) as the major fatty acids. The polar lipids were found to consist of two aminolipids, a glycolipid, a phospholipid, a phosphoglycolipid, phosphatidylcholine, phosphatidylethanolamine and diphosphatidylglycerol. Strain BUT-13(T) showed low DNA-DNA relatedness with R. pecuniae N75(T) (45.2 %) and R. rosea 173-96(T) (51.2 %). The DNA G+C content was determined to be 67.6 mol%. Based on the phylogenetic analysis, DNA-DNA hybridization and chemotaxonomic analysis, as well as biochemical characteristics, strain BUT-13(T) can be clearly distinguished from all currently recognised Roseomonas species and should be classified as a novel species of the genus Roseomonas, for which the name Roseomonas chloroacetimidivorans sp. nov. is proposed. The type strain is BUT-13(T) (CCTCC AB 2015299(T) = JCM 31050(T)).

Nitratireductor soli sp. nov., isolated from phenol-contaminated soil.

Strain ZZ-1(T), a Gram-negative, rod-shaped bacterium, motile by flagella, was isolated from phenol-contaminated soil. Strain ZZ-1(T) was found to grow at 15-37 °C (optimum 25-30 °C), at pH 6.0-10.0 (optimum pH 7.5) and with 0-8.0% (w/v) NaCl (optimum 0.5%). The isolate was found to be able to reduce nitrate to nitrite, but not to nitrogen. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain ZZ-1(T) is a member of the genus Nitratireductor, and shows high sequence similarities to Nitratireductor pacificus MCCC 1A01024(T) (98.5%) and lower (<97%) sequence similarities to all other Nitratireductor species. Chemotaxonomic analysis revealed that strain ZZ-1(T) possesses Q-10 as the predominant ubiquinone and Summed feature 8(C(18:1) ω6c and/or C(18:1) ω7c; 66.6%), C(19:0) ω8c cyclo (23.3%), C(18:0) (3.4%), iso-C(17:0) (2.3%) and C(17:0) (1.0%) as the major fatty acids. The polar lipids of strain ZZ-1(T) were determined to be diphosphatidylglycerol, phosphatidylcholine, phospholipids, aminolipids, a glycolipid and an aminophospholipid. The DNA G+C content was determined to be 64.1 mol%. Based on the draft genome sequence, the DNA-DNA hybridization estimate value between strain ZZ-1(T) and N. pacificus MCCC 1A01024(T) was 46.5 ± 3.0% and ANI was 75.9 %. The combination of phylogenetic analysis, phenotypic characteristics, chemotaxonomic data and DNA-DNA hybridization supports the conclusion that strain ZZ-1(T) represents a novel species of the genus Nitratireductor, for which the name Nitratireductor soli sp. nov. is proposed. The type strain is ZZ-1(T) (=JCM 30640(T) = MCCC 1K00508(T)).