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.

Endoscopic fenestration combined with catheterization in the treatment of a giant colonic fecalith causing bowel obstruction: a case report.

Obstruction of the colon caused by a fecalith is not a rare condition, but endoscopic attempts at removal of the fecalith are often unsuccessful because of the size of the fecalith and its extremely hard stone-like consistency. We report a case of bowel obstruction of over two weeks' duration caused by a giant colonic fecalith. Conservative treatments including insertion of a gastric tube and enemas failed to resolve the obstruction. After an initial unsuccessful attempt at fecalith removal by colonoscopy using a snare, we successfully resolved the bowel obstruction over the course of subsequent colonoscopies with endoscopic fenestration of the fecalith and placement of a transrectal gastric tube for directed instillation of the enema fluid, and we were able to avoid surgical intervention in this case.

Effects of modernized fermentation on the microbial community succession and ethyl lactate metabolism in Chinese baijiu fermentation.

Modernized mechanization and intelligent transformation are the trends of Chinese baijiu industry development. However, these changes affect the operational environment, which lead to the decreased metabolism of esters during baijiu fermentation, especially for ethyl lactate. Ethyl lactate is an important flavor compound in most types of baijiu, which is mainly produced by esterification of lactic acid and ethanol. However, considerably less is known about the dynamic microbial succession related to ethyl lactate metabolism during the modernized baijiu fermentation process. In this study, we investigated the lactic acid bacteria, yeast and mold community succession and ethyl lactate metabolism during baijiu fermentation. Results showed that fermentation mode had a significant effect on lactic acid and ethyl lactate metabolism (p < 0.001). Specifically, the accumulation of lactic acid in modernized process was 50% lower than that in traditional process (23.22 ± 7.41 g/kg versus 33.92 ± 2.32 g/kg fermented grains). The accumulation of ethyl lactate in the modernized baijiu fermentation process (9.46 ± 1.78 g/kg fermented grains) was significantly lower than that in traditional baijiu fermentation process (37.10 ± 5.86 g/kg fermented grains). Moreover, Illumina Miseq sequencing showed 11 lactic acid bacteria OTUs, 6 yeast OTUs and 4 mold OTUs were abundant during fermentation. Compared with traditional baijiu fermentation process, modernized process led to more Lactobacillus and Candida, but less Pichia, Aspergillus, Rhizomucor and Rhizopus during fermentation. Based on the redundancy analysis and correlation network analysis, our study showed the metabolism of ethyl lactate mainly related to the activities of Pichia, Aspergillus, Rhizomucor and Rhizopus, especially for Rhizomucor and Rhizopus. This study revealed that the regulation of Pichia, Aspergillus, Rhizomucor Rhizopus and Candida might be an effective orientation for adjusting the metabolism of ethyl lactate during modernized baijiu fermentation.

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.

The effects of dynamic bacterial succession on the flavor metabolites during Baijiu fermentation.

The succession of microbial community significantly affect the flavor formation of traditional fermented foods and beverages. Chinese liquor (Baijiu) fermentation is a typical spontaneous solid-state fermentation process driven by natural microbiota. The type of process used to make liquor-craft or industrial-alters the operational environment and the aromatic qualities of the product contributed by various microbial consortia. But differences in microbial community assembly and temporal succession are often overlooked. In this study, we investigated bacterial community dynamics, substrate consumption, and metabolite production during both craft and industrial liquor-making processes (CLP and ILP, respectively). We found that the compositions of bacterial communities were different, even though no significant difference (p > 0.05) was observed in bacterial species between CLP and ILP at the beginning of fermentation. During ILP, glucose was used more rapidly by microflora, leading in turn to a higher ethanol production rate during the early stage of fermentation. The higher rate of ethanol production in ILP shortened the lifetime of bacteria such as Weissella, Pediococcus, Leuconostoc, and Bacillus during the early stage of fermentation. Lactobacillus sp. became dominant earlier in ILP than in CLP. Finally, the change in bacterial community dynamics led to changes in aroma compounds. Using CLP and ILP as a model system, our results illustrate the dynamic nature of Baijiu fermentations and microbial succession patterns therein. This can be applied to optimize the fermentation processes and flavors attributes of this and other fermented foods.

[Higher alcohols metabolism by Saccharomyces cerevisiae: a mini review].

Higher alcohols are one of the main by-products of Saccharomyces cerevisiae in brewing. High concentration of higher alcohols in alcoholic beverages easily causes headache, thirst and other symptoms after drinking. It is also the main reason for chronic drunkenness and difficulty in sobering up after intoxication. The main objective of this review is to present an overview of the flavor characteristics and metabolic pathways of higher alcohols as well as the application of mutagenesis breeding techniques in the regulation of higher alcohol metabolism in S. cerevisiae. In particular, we review the application of metabolic engineering technology in genetic modification of amino transferase, α-keto acid metabolism, acetate metabolism and carbon-nitrogen metabolism. Moreover, key challenges and future perspectives of realizing optimization of higher alcohols metabolism are discussed. This review is intended to provide a comprehensive understanding of metabolic regulation system of higher alcohols in S. cerevisiae and to provide insights into the rational development of the excellent industrial S. cerevisiae strains producing higher alcohols.

Automated software-assisted diagnosis of esophageal squamous cell neoplasia using high-resolution microendoscopy.

BACKGROUND AND AIMS: High-resolution microendoscopy (HRME) is an optical biopsy technology that provides subcellular imaging of esophageal mucosa but requires expert interpretation of these histopathology-like images. We compared endoscopists with an automated software algorithm for detection of esophageal squamous cell neoplasia (ESCN) and evaluated the endoscopists' accuracy with and without input from the software algorithm. METHODS: Thirteen endoscopists (6 experts, 7 novices) were trained and tested on 218 post-hoc HRME images from 130 consecutive patients undergoing ESCN screening/surveillance. The automated software algorithm interpreted all images as neoplastic (high-grade dysplasia, ESCN) or non-neoplastic. All endoscopists provided their interpretation (neoplastic or non-neoplastic) and confidence level (high or low) without and with knowledge of the software overlay highlighting abnormal nuclei and software interpretation. The criterion standard was histopathology consensus diagnosis by 2 pathologists. RESULTS: The endoscopists had a higher mean sensitivity (84.3%, standard deviation [SD] 8.0% vs 76.3%, P = .004), lower specificity (75.0%, SD 5.2% vs 85.3%, P < .001) but no significant difference in accuracy (81.1%, SD 5.2% vs 79.4%, P = .26) of ESCN detection compared with the automated software algorithm. With knowledge of the software algorithm, the specificity of the endoscopists increased significantly (75.0% to 80.1%, P = .002) but not the sensitivity (84.3% to 84.8%, P = .75) or accuracy (81.1% to 83.1%, P = .13). The increase in specificity was among novices (P = .008) but not experts (P = .11). CONCLUSIONS: The software algorithm had lower sensitivity but higher specificity for ESCN detection than endoscopists. Using computer-assisted diagnosis, the endoscopists maintained high sensitivity while increasing their specificity and accuracy compared with their initial diagnosis. Automated HRME interpretation would facilitate widespread usage in resource-poor areas where this portable, low-cost technology is needed.

Transformation of Microbial Negative Correlations into Positive Correlations by Saccharomyces cerevisiae Inoculation during Pomegranate Wine Fermentation.

The application of starter is a common practice to accelerate and steer the pomegranate wine fermentation process. However, the use of starter needs a better understanding of the effect of the interaction between the starter and native microorganisms during alcoholic fermentation. In this study, high-throughput sequencing combined with metabolite analysis was applied to analyze the effect of commercial Saccharomyces cerevisiae inoculation on the native fungal community interaction and metabolism during pomegranate wine fermentation. Results showed that there were diverse native fungi in pomegranate juice, including Hanseniaspora uvarum, Hanseniaspora valbyensis, S. cerevisiae, Pichia terricola, and Candida diversa Based on ecological network analysis, we found that S. cerevisiae inoculation transformed the negative correlations into positive correlations among the native fungal communities and decreased the Granger causalities between native yeasts and volatile organic compounds. This might lead to decreased contents of isobutanol, isoamylol, octanoic acid, decanoic acid, ethyl laurate, ethyl acetate, ethyl hexadecanoate, phenethyl acetate, and 2-phenylethanol during fermentation. This study combined correlation and causality analysis to gain a more integrated understanding of microbial interaction and the fermentation process. It provided a new strategy to predict certain behaviors between inoculated and selected microorganisms and those coming directly from the fruit.IMPORTANCE Microbial interactions play an important role in flavor metabolism during traditional food and beverage fermentation. However, we understand little about how selected starters influence interactions among native microorganisms. In this study, we found that S. cerevisiae inoculation changed the interactions and metabolisms of native fungal communities during pomegranate wine fermentation. This study not only suggests that starter inoculation should take into account the positive features of starters but also characterizes the microbial interactions established among the starters and the native communities. It may be helpful to select appropriate starter cultures for winemakers to design different styles of wine.

Quantitative studies of gully slope erosion and soil physiochemical properties during freeze-thaw cycling in a Mollisol region.

Gully erosion has been widely studied during the rainy season due to soil loss that seriously reduces arable area and decreases soil quality. However, very few publications have focused on gully slope erosion (GSE) during freeze thaw cycle (FTC). In this study, GSE on both active and stable gullies in Mollisol fields was investigated by 3D-photogrammetry. Soil bulk density (BD), soil moisture (SM), soil temperature (ST), daily maximum difference in soil temperature (MDT), saturated water (SW), field capacity (FC), soil organic carbon (SOC), soil total nitrogen (TN), water-stable soil aggregate (WA), vegetation cover rate (VC), root dry weight (RW), root length (RL), slope length (SL) and slope steepness (SS) were compared before- and after FTCs. The main results are as follows: (1) combined with both front and profile views, 3D photogrammetry can be used to monitor GSE; (2) GSE mainly occurred at the early stage of FTCs (approximately 80%) and was mainly determined by snowmelt of both the gully slope and farmland and was driven by the solar radiation in activity gully; (3) the high ST in surface soil layers (0-5 cm) of active gullies accelerated the GSE; (4) GSE on the active gully slope was 7.3-9.8 times greater than that on the stable gully slopes; (5) the plough pan as the important layer can effectively reduce GSE at upper slope positions in an active gully; (6) low values of VC, BD, SOC, RW, RL and macro-WA and high values of SL, SW and MDT in the middle of the gully slope typically accelerate the GSE; (7) the index SS*SL/VC can be used to predict GSE on Mollisol gully slopes. Generally, GSE was greatest after FTCs compare to the soil loss tolerance in the Mollisol region, especially in the middle slope position of the active gully, and should urgently be controlled.

Exploring the impacts of raw materials and environments on the microbiota in Chinese Daqu starter.

Many traditional fermented foods and beverages in both eastern and western countries are produced with the addition of starter cultures. Daqu is the undefined starter used for Chinese liquor, which contributed many fermentation functional fungal and bacterial communities to liquor fermentation process. However, the sources of these microbial communities and how these microorganisms formed in Daqu are still unclear. In this study, high-throughput sequencing combined with microbial source tracking analysis were applied to analyze the sources of the microbial communities in Daqu. We found fungal communities in Daqu were mainly originated from Daqu making environments (mainly tools and indoor ground), including Saccharomycopsis fibuligera, Pichia kudriavzevii, Rhizopus oryzae, Sterigmatomyces elviae, Aspergillus flavus/oryzae, Hyphopichia burtonii and Lichtheimia corymbifera. Meanwhile, most of bacterial communities in Daqu were from raw materials, including Gammaproteobacteria, Alphaproteobacteria and Bacilli. During Daqu-making process, the abundance of Gammaproteobacteria and Alphaproteobacteria that were not beneficial to liquor fermentation declined, but the abundance of the fermentation functional yeasts and Bacilli increased. Moreover, network analysis showed the bacteria in Daqu might be the drivers for the microbial succession during the Daqu-making process. This study shows that the Daqu production technology is a good way to screen fermentation functional microorganisms from complex environmental microbial communities.

Environmental Microbiota Drives Microbial Succession and Metabolic Profiles during Chinese Liquor Fermentation.

Many microorganisms in the environment participate in the fermentation process of Chinese liquor. However, it is unknown to what extent the environmental microbiota influences fermentation. In this study, high-throughput sequencing combined with multiphasic metabolite target analysis was applied to study the microbial succession and metabolism changes during Chinese liquor fermentation from two environments (old and new workshops). SourceTracker was applied to evaluate the contribution of environmental microbiota to fermentation. Results showed that Daqu contributed 9.10 to 27.39% of bacterial communities and 61.06 to 80.00% of fungal communities to fermentation, whereas environments (outdoor ground, indoor ground, tools, and other unknown environments) contributed 62.61 to 90.90% of bacterial communities and 20.00 to 38.94% of fungal communities to fermentation. In the old workshop, six bacterial genera (Lactobacillus [11.73% average relative abundance], Bacillus [20.78%], Pseudomonas [6.13%], Kroppenstedtia [10.99%], Weissella [16.64%], and Pantoea [3.40%]) and five fungal genera (Pichia [55.10%], Candida [1.47%], Aspergillus [10.66%], Saccharomycopsis [22.11%], and Wickerhamomyces [3.35%]) were abundant at the beginning of fermentation. However, in the new workshop, the change of environmental microbiota decreased the abundances of Bacillus (5.74%), Weissella (6.64%), Pichia (33.91%), Aspergillus (7.08%), and Wickerhamomyces (0.12%), and increased the abundances of Pseudomonas (17.04%), Kroppenstedtia (13.31%), Pantoea (11.41%), Acinetobacter (3.02%), Candida (16.47%), and Kazachstania (1.31%). Meanwhile, in the new workshop, the changes of microbial community resulted in the increase of acetic acid, lactic acid, malic acid, and ethyl acetate, and the decrease of ethyl lactate during fermentation. This study showed that the environmental microbiota was an important source of fermentation microbiota and could drive both microbial succession and metabolic profiles during liquor fermentation.IMPORTANCE Traditional solid-state fermentation of foods and beverages is mainly carried out by complex microbial communities from raw materials, starters, and the processing environments. However, it is still unclear how the environmental microbiota influences the quality of fermented foods and beverages, especially for Chinese liquors. In this study, we utilized high-throughput sequencing, microbial source tracking, and multiphasic metabolite target analysis to analyze the origins of microbiota and the metabolic profiles during liquor fermentation. This study contributes to a deeper understanding of the role of environmental microbiota during fermentation.

Exploring the microbial origins of p-cresol and its co-occurrence pattern in the Chinese liquor-making process.

The compound p-cresol is the major off-odor and toxic component of strong aroma-type Chinese liquor. To trace its origin, the p-cresol contents in the liquor-making process were detected by gas chromatography-mass spectrometry. The prokaryotic communities involved in the process were revealed by 16S rRNA gene MiSeq sequencing. The results showed that the microbial diversity and concentration of p-cresol in pit mud significantly increased with increased pit depth. Canonical correspondence analysis further revealed that p-cresol was positively correlated with the genera Aminobacterium, Clostridium, Sedimentibacter and Syntrophomonas. On investigating potential p-cresol producers, we obtained 11 species from pit mud samples using selective culture media. Clostridium butyricum, Clostridium tyrobutyricum, Clostridium aminovalericum and Eubacterium contortum were confirmed to possess the capacity for p-cresol production. Moreover, based on volatile compounds data, these strains were assigned to the same clusters characterized by the high abundance of butanoic acid and p-cresol. Furthermore, 24 pairs of significant correlations were identified from 14 genera using co-occurrence network analysis. Clostridium was the hub in pit mud and could be inhibited by increasing levels of Lactobacillus. These findings represented a step forward for controlling p-cresol in a complex microbial community of Chinese liquor.

Source tracking of prokaryotic communities in fermented grain of Chinese strong-flavor liquor.

The fermentation process of Chinese strong-flavor liquor involves numerous microbes originating from Daqu and pit mud. Daqu is the starter of fermentation, and pit mud acts as another source of inoculum of microbes in the liquor-making process. However, the contribution of microbes in pit mud and Daqu to fermented grain, and the sources of microbes in fermented grain are still waiting to be defined clearly. In this study, prokaryotic communities in fermented grain, pit mud and Daqu were identified via next generation sequencing of the V4 region of 16S rRNA gene. Principal-coordinate analysis indicated that Daqu had stronger influence on the prokaryotic communities in fermented grain at the prophase of fermentation, but pit mud influenced the fermented grain continuously during the whole fermentation process. Totally, 299 genera were detected in all fermented grain, pit mud and Daqu samples. Among them, 204 genera were detected in 3days' fermented grain. Ten genera (Lactobacillus, Leuconostoc, Staphylococcus, Gluconobacter, Acetobacter, Petrimonas, Clostridium, Ruminococcus, Methanobacterium and Methanobrevibacter) were dominant, and accounted for 84.31%-87.13% relative abundance of the total prokaryotic community in fermented grain. Venn analysis indicated Daqu was the main source of strict aerobes and facultative aerobes, which took up over 74% of prokaryotic communities in fermented grain. Conversely, pit mud was the sustained-release source of anaerobes, which accounted for over 14% of prokaryotic communities in fermented grain. In addition, part of anaerobes originated from both Daqu and pit mud. This study could help track the source of prokaryotic communities in fermented grain, and improve the quality and controllability in liquor production.

Quantitative analysis of high-resolution microendoscopic images for diagnosis of esophageal squamous cell carcinoma.

BACKGROUND & AIMS: High-resolution microendoscopy is an optical imaging technique with the potential to improve the accuracy of endoscopic screening for esophageal squamous neoplasia. Although these microscopic images can be interpreted readily by trained personnel, quantitative image analysis software could facilitate the use of this technology in low-resource settings. In this study, we developed and evaluated quantitative image analysis criteria for the evaluation of neoplastic and non-neoplastic squamous esophageal mucosa. METHODS: We performed an image analysis of 177 patients undergoing standard upper endoscopy for screening or surveillance of esophageal squamous neoplasia, using high-resolution microendoscopy, at 2 hospitals in China and at 1 hospital in the United States from May 2010 to October 2012. Biopsy specimens were collected from imaged sites (n = 375), and a consensus diagnosis was provided by 2 expert gastrointestinal pathologists and used as the standard. RESULTS: Quantitative information from the high-resolution images was used to develop an algorithm to identify high-grade squamous dysplasia or invasive squamous cell cancer, based on histopathology findings. Optimal performance was obtained using the mean nuclear area as the basis for classification, resulting in sensitivities and specificities of 93% and 92% in the training set, 87% and 97% in the test set, and 84% and 95% in an independent validation set, respectively. CONCLUSIONS: High-resolution microendoscopy with quantitative image analysis can aid in the identification of esophageal squamous neoplasia. Use of software-based image guides may overcome issues of training and expertise in low-resource settings, allowing for widespread use of these optical biopsy technologies.