A metagenomic analysis of the relationship between microorganisms and flavor development in Shaoxing mechanized huangjiu fermentation mashes.

Complex microbial metabolism is responsible for the unique flavor of Shaoxing mechanized huangjiu. However, the relationship between the microorganisms present during fermentation and the formation of specific flavor components is difficult to understand. In this study, gas chromatography-mass spectrometry and high-performance liquid chromatography were used to identify flavor components, and a metagenomic sequencing approach was used to characterize the taxonomic and functional attributes of the Shaoxing mechanized huangjiu fermentation microbiota. The metagenomic sequencing data were used to predict the relationship between microorganisms and flavor formation. The chromatographic analysis identified amino acids, alcohols, acids, phenols and esters as major flavor components, and six microbial genera (Saccharomyces, Aspergillus, Saccharopolyspora, Staphylococcus, Lactobacillus, and Lactococcus) were most closely related to the production of these flavor components. This study helps clarify the different metabolic roles of microorganisms in flavor formation during Shaoxing huangjiu fermentation.

Adaptive Evolution Relieves Nitrogen Catabolite Repression and Decreases Urea Accumulation in Cultures of the Chinese Rice Wine Yeast Strain Saccharomyces cerevisiae XZ-11.

Urea is the major precursor of ethyl carbamate in Chinese rice wine. Although efforts have been made to decrease urea accumulation, few methods can be applied to industrial food production due to potential safety concerns. In this study, adaptive laboratory evolution (ALE) followed by high-throughput screening was used to identify low urea-accumulating strains derived from the industrial Chinese rice wine yeast strain Saccharomyces cerevisiae XZ-11. Three evolved strains were obtained that had 47.9%, 16.6%, and 12.4% lower urea concentrations than the wild-type strain. Comparative genomics analysis revealed that genes involved in carbon and nitrogen metabolism evolved quickly. Transcription levels of genes involved in urea metabolism were dramatically upregulated after ALE. This work describes a novel and safe strategy to improve nitrogen utilization of industrial yeast strains involved in food fermentation. The identified genomic variations may also help direct rational genetic engineering of nitrogen metabolism processes to achieve other goals.

Mutant Potential Ubiquitination Sites in Dur3p Enhance the Urea and Ethyl Carbamate Reduction in a Model Rice Wine System.

Ubiquitination can significantly affect the endocytosis and degradation of plasma membrane proteins. Here, the ubiquitination of a Saccharomyces cerevisiae urea plasma membrane transporter (Dur3p) was altered. Two potential ubiquitination sites, lysine residues K556 and K571, of Dur3p were predicted and replaced by arginine, and the effects of these mutations on urea utilization and formation under different nitrogen conditions were investigated. Compared with Dur3p, the Dur3pK556R mutant showed a 20.1% decrease in ubiquitination level in yeast nitrogen base medium containing urea and glutamine. It also exhibited a >75.8% decrease in urea formation in yeast extract-peptone-dextrose medium and 41.3 and 55.4% decreases in urea and ethyl carbamate formation (a known carcinogen), respectively, in a model rice wine system. The results presented here show that the mutation of Dur3p ubiquitination sites could significantly affect urea utilization and formation. Modifying the ubiquitination of specific transporters might have promising applications in rationally engineering S. cerevisiae strains to efficiently use specific nitrogen sources.

Genome-wide mapping of nucleosome positions in Saccharomyces cerevisiae in response to different nitrogen conditions.

Well-organized chromatin is involved in a number of various transcriptional regulation and gene expression. We used genome-wide mapping of nucleosomes in response to different nitrogen conditions to determine both nucleosome profiles and gene expression events in Saccharomyces cerevisiae. Nitrogen conditions influence general nucleosome profiles and the expression of nitrogen catabolite repression (NCR) sensitive genes. The nucleosome occupancy of TATA-containing genes was higher compared to TATA-less genes. TATA-less genes in high or low nucleosome occupancy, showed a significant change in gene coding regions when shifting cells from glutamine to proline as the sole nitrogen resource. Furthermore, a correlation between the expression of nucleosome occupancy induced NCR sensitive genes or TATA containing genes in NCR sensitive genes, and nucleosome prediction were found when cells were cultured in proline or shifting from glutamine to proline as the sole nitrogen source compared to glutamine. These results also showed that variation of nucleosome occupancy accompany with chromatin-dependent transcription factor could influence the expression of a series of genes involved in the specific regulation of nitrogen utilization.

Metagenomic sequencing reveals the relationship between microbiota composition and quality of Chinese Rice Wine.

Chinese Rice Wine (CRW) is a common alcoholic beverage in China. To investigate the influence of microbial composition on the quality of CRW, high throughput sequencing was performed for 110 wine samples on bacterial 16S rRNA gene and fungal Internal Transcribed Spacer II (ITS2). Bioinformatic analyses demonstrated that the quality of yeast starter and final wine correlated with microbial taxonomic composition, which was exemplified by our finding that wine spoilage resulted from a high proportion of genus Lactobacillus. Subsequently, based on Lactobacillus abundance of an early stage, a model was constructed to predict final wine quality. In addition, three batches of 20 representative wine samples selected from a pool of 110 samples were further analyzed in metagenomics. The results revealed that wine spoilage was due to rapid growth of Lactobacillus brevis at the early stage of fermentation. Gene functional analysis indicated the importance of some pathways such as synthesis of biotin, malolactic fermentation and production of short-chain fatty acid. These results led to a conclusion that metabolisms of microbes influence the wine quality. Thus, nurturing of beneficial microbes and inhibition of undesired ones are both important for the mechanized brewery.

The modification of Gat1p in nitrogen catabolite repression to enhance non-preferred nitrogen utilization in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, when preferred nitrogen sources are present, the metabolism of non-preferred nitrogen is repressed. Previous work showed that this metabolic regulation is primarily controlled by nitrogen catabolite repression (NCR) related regulators. Among these regulators, two positive regulators (Gln3p and Gat1p) could be phosphorylated and sequestered in the cytoplasm leading to the transcription of non-preferred nitrogen metabolic genes being repressed. The nuclear localization signals (NLSs) and nuclear localization regulatory signals (NLRSs) in Gln3p and Gat1p play essential roles in the regulation of their localization in cells. However, compared with Gln3p, the information of NLS and NLRS for Gat1p remains unknown. In this study, residues 348-375 and 366-510 were identified as the NLS and NLRS of Gat1p firstly. In addition, the modifications of Gat1p (mutations on the NLS and truncation on the NLRS) were attempted to enhance the transcription of non-preferred nitrogen metabolic genes. Quantitative real-time PCR showed that the transcriptional levels of 15 non-preferred nitrogen metabolic genes increased. Furthermore, during the shaking-flask culture tests, the utilization of urea, proline and allantoine was significantly increased. Based on these results, the genetic engineering on Gat1p has a great potential in enhancing non-preferred nitrogen metabolism in S. cerevisiae.

Effects of three permeases on arginine utilization in Saccharomyces cerevisiae.

Arginine plays an important role in cellular function and metabolism. Arginine uptake mainly occurs through three amino acid permeases, Alp1p, Gap1p and Can1p, which act as both transporters and receptors for amino acid utilization. In this study, seven mutants were constructed with different combinations of permease deficiencies that inhibit arginine utilization. Their effects on arginine metabolism were measured. The three amino acid permeases were also individually overexpressed in wild-type (WT), Δalp1Δgap1Δcan1 and Δnpr1 strains. The growth and arginine utilization of Δcan1, Δgap1Δcan1 and Δalp1Δgap1Δcan1 mutants were suppressed in YNB medium when arginine was the sole nitrogen source. Meanwhile, overexpression of Alp1p and Can1p enhanced growth and arginine utilization in WT, Δalp1Δgap1Δcan1 and Δnpr1. Besides, overexpression of Can1p caused a 26.7% increase in OD600 and 29.3% increase in arginine utilization compared to that of Alp1p in Δalp1Δgap1Δcan1. Transcription analysis showed that the effects of three amino acid permeases on the arginine utilization and the regulation of related genes, were tightly related to their individual characteristics. However, their overall effects were different for different combinations of mutants. The results presented here suggest some possible synergistic effects of different amino acid permeases on regulation of amino acid utilization and metabolism.

Comparative proteomic analysis of Saccharomyces cerevisiae under different nitrogen sources.

In cultures containing multiple sources of nitrogen, Saccharomyces cerevisiae exhibits a sequential use of nitrogen sources through a mechanism known as nitrogen catabolite repression (NCR). To identify proteins differentially expressed due to NCR, proteomic analysis of S. cerevisiae S288C under different nitrogen source conditions was performed using two-dimensional gel electrophoresis (2-DE), revealing 169 candidate protein spots. Among these 169 protein spots, 121 were identified by matrix assisted laser desorption ionization-time of flight/time of flight mass spectrometry (MALDI-TOF/TOF). The identified proteins were closely associated with four main biological processes through Gene Ontology (GO) categorical analysis. The identification of the potential proteins and cellular processes related to NCR offer a global overview of changes elicited by different nitrogen sources, providing clues into how yeast adapt to different nutritional conditions. Moreover, by comparing our proteomic data with corresponding mRNA data, proteins regulated at the transcriptional and post-transcriptional level could be distinguished. Biological significance In S. cerevisiae, different nitrogen sources provide different growth characteristics and generate different metabolites. The nitrogen catabolite repression (NCR) process plays an important role for S. cerevisiae in the ordinal utilization of different nitrogen sources. NCR process can result in significant shift of global metabolic networks. Previous works on NCR primarily focused on transcriptomic level. The results obtained in this study provided a global atlas of the proteome changes triggered by different nitrogen sources and would facilitate the understanding of mechanisms for how yeast could adapt to different nutritional conditions.

Determination of trace heavy metals in environmental and biological samples by solution cathode glow discharge-atomic emission spectrometry and addition of ionic surfactants for improved sensitivity.

Solution cathode glow discharge-atomic emission spectrometry (SCGD-AES) was evaluated for its ability to determine toxic heavy metals, including cadmium (Cd), mercury (Hg), lead (Pb), and chromium (Cr), in environmental and biological samples. A significant enhancement in heavy metal signal was observed by addition of a small amount of cetyltrimethylammoniumchloride (CTAC, C16H33 (CH3)3NCl) to the samples. The net intensity of atomic emission lines of Cd, Hg, Pb, and Cr increased by 2.1-, 4.8-, 6.6-, and 2.6-fold, respectively, after addition of 0.15% CTAC to the test solutions. The effects of ionic surfactants (CTAC) compared with non-ionic surfactants, e.g., Triton x-45 and Triton x-100, on the sensitivity of Cd, Hg, Pb, and Cr were also investigated in the present study. The enhancement effect is in the order Triton x-45

Metabolic engineering of the regulators in nitrogen catabolite repression to reduce the production of ethyl carbamate in a model rice wine system.

Rice wine has been one of the most popular traditional alcoholic drinks in China. However, the presence of potentially carcinogenic ethyl carbamate (EC) in rice wine has raised a series of food safety issues. During rice wine production, the key reason for EC formation is urea accumulation, which occurs because of nitrogen catabolite repression (NCR) in Saccharomyces cerevisiae. NCR represses urea utilization by retaining Gln3p in the cytoplasm when preferred nitrogen sources are present. In order to increase the nuclear localization of Gln3p, some possible phosphorylation sites on the nuclear localization signal were mutated and the nuclear localization regulation signal was truncated, and the disruption of URE2 provided an additional method of reducing urea accumulation. By combining these strategies, the genes involved in urea utilization (DUR1,2 and DUR3) could be significantly activated in the presence of glutamine. During shake flask fermentations of the genetically modified strains, very little urea accumulated in the medium. Furthermore, the concentrations of urea and EC were reduced by 63% and 72%, respectively, in a model rice wine system. Examination of the normal nutrients in rice wine indicated that there were few differences in fermentation characteristics between the wild-type strain and the genetically modified strain. These results show that metabolic engineering of the NCR regulators has great potential as a method for eliminating EC during rice wine production.

Nitrogen regulation involved in the accumulation of urea in Saccharomyces cerevisiae.

Rice wine is a popular traditional alcoholic drink with a long history in China. However, the presence of the potential carcinogen ethyl carbamate (EC) raises a series of food safety concerns. Although the metabolic pathway of urea (the major precusor of EC) has been characterized in Saccharomyces cerevisiae, the regulation of urea accumulation remains unclear, making the efficient elimination of urea difficult. To demonstrate the regulatory mechanisms governing urea accumulation, three key nitrogen sources that can inhibit urea utilization for a commercial S. cerevisiae strain were identified. In addition, regulators of nitrogen catabolite repression (NCR) and target of rapamycin (TOR) pathways were identified as being involved in urea accumulation by real-time quantitative PCR. Based on these results, preferred nitrogen sources were found to repress urea utilization by converting them to glutamine or glutamate. Moreover, the results indicated that the manner of urea metabolism regulation was different for two positive regulators involved in NCR; Gln3p can be retained in the cytoplasm by glutamine, while Gat1p can be retained by glutamine and glutamate. Furthermore, this was confirmed by fluorescence location detection. These new findings provide new targets for eliminating EC and other harmful nitrogen-containing compounds in fermented foods.

Microbial community structure in fermentation process of Shaoxing rice wine by Illumina-based metagenomic sequencing.

BACKGROUND: To understand the role of the community structure of microbes in the environment in the fermentation of Shaoxing rice wine, samples collected from a wine factory were subjected to Illumina-based metagenomic sequencing. RESULTS: De novo assembly of the sequencing reads allowed the characterisation of more than 23 thousand microbial genes derived from 1.7 and 1.88 Gbp of sequences from two samples fermented for 5 and 30 days respectively. The microbial community structure at different fermentation times of Shaoxing rice wine was revealed, showing the different roles of the microbiota in the fermentation process of Shaoxing rice wine. The gene function of both samples was also studied in the COG database, with most genes belonging to category S (function unknown), category E (amino acid transport and metabolism) and unclassified group. CONCLUSION: The results show that both the microbial community structure and gene function composition change greatly at different time points of Shaoxing rice wine fermentation.

Model and experimental investigations of aluminum oxide slurry transportation and vaporization behavior for nebulization inductively coupled plasma optical emission spectrometry.

We analyzed aluminum oxide (Al2O3) by slurry introduction inductively coupled plasma (ICP) optical emission spectrometry through modeling and experimentation. We also studied the relationship between the ICP nebulizer gas flow, the spray chamber geometry, and the particle size of Al2O3 in an attempt to minimize the need for correction factors by ensuring an efficient aerosol mass transport. A cut-off point for the particle size was implemented at approximately 7-10 µm for the sample introduction system. Based on modeling using a customized computer model and some experimental evidences, the maximum particle size for complete vaporization is approximately 7 µm. For a gas flow of 0.8 Lmin(-1), particles with a diameter of up to 8 µm can be evaporated with an efficiency of 68% and particles as large as 5 µm can be evaporated completely within the nebulization gas flow region. The Al2O3 sintering block was ground using a self-made alumina mortar combined with a mixer mill device for particle reduction. The sample slurry was prepared by directly dispersing powdered Al2O3 in an aqueous solution with an addition of 0.5 wt% poly (acrylate amine) (NH4PAA) as the dispersant. The accuracy of the results was compared with the data obtained through high-pressure digestion with acid and with the value of a certified reference material, NIST SRM 699 alumina.

[Direct determination of copper, iron and sodium in high-purity alumina by slurry introduction furnace atomic absorption spectrometry with Smith-Hieftje background correction].

Direct determination of copper, iron and sodium in high-purity alumina was performed by slurry-furnace atomic absorption spectrometry with Smith-Hieftje background correction (S-H-GFAAS). Instrument conditions of GFAAS such as pyrolysis, atomization and hollow cathode lamp current by S-H background correction were optimized, and the optimal experimental conditions were selected. Calibration was performed using aqueous standards method for sample analysis. The accuracy of the proposed method was shown for the case of Al2O3 (AKP-30), and compared with those obtained by furnace atomic absorption spectrometry subsequent to decomposition by sulfuric acid in PTFE pressure vessels. The results were in agreement well with values found in the literature by different methods. It is a simple, convenient and accurate method and it is suitable for the rapid analysis of trace element in alumina. The linear regression coefficients of the calibration curves were better than 0.999 0. The detection limits were 0.66, 2.5 and 0.13 ng x g(-1), respectively, with a relative standard deviation being not more than 5.2%.

[Research advance in slurry introduction for graphite furnace atomic absorption spectrometry].

Slurry introduction graphite furnace atomic absorption spectrometry combining the significant advantages of the solid and liquid sampling methods is already considered as a mature technique. It was widely utilized for metal determination in trace and even ultra trace analysis in organic and inorganic complicated matrix, even for routine analysis. Methodology of the analysis of various materials using slurry furnace atomic absorption spectrometry was reviewed in the present paper in late 10 years. Techniques of slurry preparation (liquid media, stabilizing agents, mass/volume ratio, particle size and slurry homogenization systems), the chemical modification, background correctors, calibration, precision and trueness of analysis were described in detail. The developing trend has also presented. Eighty one references were cited.