Saline Samples Research Articles

Anti-fibrosis attributes: UHPLC-MS/MS-based pharmacokinetics profiling of a novel autotaxin inhibitor with excellent in vivo efficacy in rat.

3,4-Difluorobenzyl(1-ethyl-5-(4-((4-hydroxypiperidin-1-yl)-methyl)thiazol-2-yl)-1H-indol-3-yl)carbamate (NAI59), a small molecule with outstanding therapeutic effectiveness to anti-pulmonary fibrosis, was developed as an autotaxin inhibitor candidate compound. To evaluate the pharmacokinetics and plasma protein binding of NAI59, a UPLC-MS/MS method was developed to quantify NAI59 in plasma and phosphate-buffered saline. The calibration curve linearity ranged from 9.95 to 1990.00ng/mL in plasma. The accuracy was -6.8 to 5.9%, and the intra- and inter-day precision was within 15%. The matrix effect and recovery, as well as dilution integrity, were within the criteria. The chromatographic and mass spectrometric conditions were also feasible to determine phosphate-buffered saline samples, and it has been proved that this method exhibits good precision and accuracy in the range of 9.95-497.50ng/mL in phosphate-buffered saline. This study is the first to determine the pharmacokinetics, absolute bioavailability, and plasma protein binding of NAI59 in rats using this established method. Therefore, the pharmacokinetic profiles of NAI59 showed a dose-dependent relationship after oral administration, and the absolute bioavailability in rats was 6.3%. In addition, the results of protein binding showed that the combining capacity of NAI59 with plasma protein attained 90% and increased with the increase in drug concentration.

Novel studies of characterization, antioxidant, anticoagulant and anticancer activity of purified polysaccharide from Hypsizygus ulmarius mushroom

A water-soluble polysaccharide (HUP-2) was isolated from H. ulmarius using hot water extraction and purified using ion exchange chromatography (DEAE cellulose-52) and gel filtration (Sepharose-6B). The structure characteristics of HUP-2 were investigated using high performance liquid chromatography (HPLC), Fourier transform infrared (FT-IR), nuclear magnetic resonance (NMR) spectrum, and gas chromatography-mass spectrometry (GC-MS), and XRD analysis revealed that it formed a semi-crystalline structure. HUP-2 is a monosaccharide with a molecular weight of 25.296 kDa. The HUP-2 is assemble with 1.96% rhamnose, 16.51% mannose, 22.76% galactose, and 19.43% glucose. It could be found that the main backbone chain of HUP-2 consisted N-acetylglycosidic. The polysaccharide's DPPH radical scavenging activity ranged from 17.08 to 56.90%. The radical scavenging activity of ABTS varied from 8.02 to 71.44% at 0.5–2.5 mg/ml, whereas superoxide radical scavenging activity was 8.02–63.03%. The results of HUP-2 demonstrated an extension (p < 0.01) of APTT relative to the saline sample at concentrations more than 5 mg/mL. It exhibited a high antioxidant capacity as well. HUP-2 had significant inhibitory and cytotoxic effects in PC3 cells. HUP-2 arrested the cell cycle in the G0/G1 phase over all cells except PC3 cells. HUP-2 appears to have a wide range of biological applications, making it a good candidate for usage in functional foods, according to these studies.

Application of an In-situ Solvent Formation Microextraction Technique Using the Functionalized Ionic Liquids (FILs) as a Green Extractant for Cadmium Determination at Trace Levels in Real and Saline Samples

An in-situ solvent formation microextraction technique as another attractive mode of homogeneous liquid-liquid microextraction, was developed successfully for the sensitive extraction of cadmium ion. In this technique use green extracting solvent such as ionic liquids that are environment lover. So, because using ionic liquids, this technique is much safer than other extraction methods which use toxic common organic solvents. Functionalized ionic liquid of 3-(2-(bis(2-(tert-butoxy)-2-oxoethyl)amino)ethyl)-1-methylimidazolium bromide was used as extractant and organic phase which has double role including complexing agent and extractant, so to extraction of cadmium ion, not need to any complexing agent. To optimization of extraction conditions, several analytical parameters affected on microextraction efficiency including sample pH, functionalized ionic liquid dosage, counter-ion amount and centrifugation conditions were studied. To show abilities of the method, the figures of merits including limit of detection and quantification, relative standard deviation, linear dynamic range and enrichment factor were calculated and included 0.23 µg/L, 0.76 µg/L, 1.8%, 10-1500 µg/L and 75, respectively. It was found from results that the method was applied successfully for the determination of Cd (II) ion in several real, and saline samples. Because of use environmental solvents such as ionic liquids, and very small dosage of its, it can be said the method has low toxicity and classified as a green extraction/preconcentration technique. One the other hand, the presented method opens a new window in the analytical applications due to its costless, ease in experimental setup and use of non toxic solvents as an extractant.

Magnet-assisted electrochemical immunosensor based on surface-clean Pd-Au nanosheets for sensitive detection of SARS-CoV-2 spike protein

Tracking and monitoring of low concentrations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can effectively control asymptomatic transmission of current coronavirus disease 2019 (COVID-19) in the early stages of infection. Here, we highlight an electrochemical immunosensor for sensitive detection of SARS-CoV-2 antigen marker spike protein. The surface-clean Pd-Au nanosheets as a substrate for efficient sensing and signal output have been synthesized. The morphology, chemical states and excellent stable electrochemical properties of this surface-clean heterostructures have been studied. Functionalized superparamagnetic nanoparticles (MNPs) were introduced as sample separators and signal amplifiers. This biosensor was tested in phosphate buffered saline (PBS) and nasopharyngeal samples. The results showed that the sensor has a wide linear dynamic range (0.01 ng mL−1 to 1000 ng mL−1) with a low detection limit (0.0072 ng mL−1), which achieved stable and sensitive detection of the spike protein. Therefore, this immunosensing method provides a promising electrochemical measurement tool, which can furnish ideas for early screening and the reasonable optimization of detection methods of SARS-CoV-2.

Cerebrospinal Fluid from Healthy Pregnant Women Does Not Harbor a Detectable Microbial Community.

ABSTRACTCerebrospinal fluid (CSF) circulating in the human central nervous system has long been considered aseptic in healthy individuals, because normally, the blood-brain barrier can protect against microbial invasions. However, this dogma has been called into question by several reports that microbes were identified in human brains, raising the question of whether there is a microbial community in the CSF of healthy individuals without neurological diseases. Here, we collected CSF samples and other samples, including one-to-one matched oral and skin swab samples (positive controls), from 23 pregnant women aged between 23 and 40 years. Normal saline samples (negative controls), sterile swabs, and extraction buffer samples (contamination controls) were also collected. Twelve of the CSF specimens were also used to evaluate the physiological activities of detected microbes. Metagenomic and metatranscriptomic sequencing was performed in these 116 specimens. A total of 620 nonredundant microbes were detected, which were dominated by bacteria (74.6%) and viruses (24.2%), while in CSF samples, metagenomic sequencing found only 26 nonredundant microbes, including one eukaryote, four bacteria, and 21 viruses (mostly bacteriophages). The beta diversity of microbes compared between CSF metagenomic samples and other types of samples (except negative controls) was significantly different from that of the CSF self-comparison. In addition, there was no active or viable microbe in the matched metagenomic and metatranscriptomic sequencing of CSF specimens after subtracting those also found in normal saline, DNA extraction buffer, and skin swab specimens. In conclusion, our results showed no strong evidence of a colonized microbial community present in the CSF of healthy individuals.IMPORTANCE The microbiome is prevalent throughout human bodies, with profound health implications. However, it remains unclear whether it is present and active in human CSF, which has been long considered aseptic due to the blood-brain barrier. Here, we applied unbiased metagenomic and metatranscriptomic sequencing to detect the presence of a microbiome in CSF collected from 23 pregnant women with matched controls. Analysis of 116 specimens found no strong evidence to support the presence of a colonized microbiome in CSF. Our findings will strengthen our understanding of the internal environment of the CSF in healthy people, which has strong implications for human health, especially for neurological infections and disorders, and will help further disease diagnostics, prevention, and therapeutics in clinical settings.

Antibiotic resistance in bacterial communities of the oyster Crassostrea rivularis from different salinity zones in Qinzhou Bay, Beibu Gulf, China

The oyster is one of the most abundantly harvested shellfish in the world. To explore the impact of salinity on antibiotic-resistant bacteria (ARB) and the microbial community associated with farmed oysters, oysters were taken from high-, medium-, and low-salinity zones (labeled HS, MS, and LS, respectively) in Qinzhou Bay of Beibu Gulf, China. ARB were tested with the Kirby–Bauer method. Species of ARB were confirmed by 16 S rDNA analysis. Microbial communities were analyzed by high-throughput sequencing technology. The results indicate that HS-derived ARB (&amp;gt;60%) resisted β-lactams and aminoglycosides and that LS-derived strains resisted macrolide and tetracyclines. All strains resisted 4 or more antibiotics. A total of 542 operational taxonomic units were detected in the samples, with Shewanella, Vibrio, and Endozoicomonas being the dominant genera (&amp;gt;80%), although distributed differently among the different salinity samples. The oyster microbial richness ranked as MS &amp;gt; LS &amp;gt; HS. This study provides an important reference for future efforts to explain factors or mechanisms underlying correlations between ARB, the microbiome, and salinity and thus the potential health of oysters in this region.

Monitoring soil salinization and its spatiotemporal variation at different depths across the Yellow River Delta based on remote sensing data with multi-parameter optimization.

Soil salinization is recognized as a key issue negatively affecting agricultural productivity and wetland ecology. It is necessary to develop effective methods for monitoring the spatiotemporal distribution of soil salinity at a regional scale. In this study, we proposed an optimized remote sensing-based model for detecting soil salinity in different depths across the Yellow River Delta (YRD), China. A multi-dimensional model was built for mapping soil salinity, in which five types of predictive factors derived from Landsat satellite images were exacted and tested, 94 in-situ measured soil salinity samples with depths of 30-40cm and 90-100cm were collected to establish and validate the predicting model result. By comparing multiple linear regression (MLR) and partial least squares regression (PLSR) models with considering the correlation between predictive factors and soil salinity, we established the optimized prediction model which integrated the multi-parameter (including SWIR1, SI9, MSAVI, Albedo, and SDI) optimization approach to detect soil salinization in the YRD from 2003 to 2018. The results indicated that the estimates of soil salinity by the optimized prediction model were in good agreement with the measured soil salinity. The accuracy of the PLSR model performed better than that of the MLR model, with the R2 of 0.642, RMSE of 0.283, and MAE of 0.213 at 30-40cm depth, and with the R2 of 0.450, RMSE of 0.276, and MAE of 0.220 at 90-100cm depth. From 2003 to 2018, the soil salinity showed a distinct spatial heterogeneity. The soil salinization level of the coastal shoreline was higher; in contrast, lower soil salinization level occurred in the central YRD. In the last 15years, the soil salinity at depth of 30-40cm experienced a decreased trend of fluctuating, while the soil salinity at depth of 90-100cm showed fluctuating increasing trend.

Electrochemical determination of epirubicin in the presence of topotecan as essential anti-cancer compounds using paste electrode amplified with Pt/SWCNT nanocomposite and a deep eutectic solvent

Epirubicin (EP) and topotecan (TP) are two major anti-cancer compounds for the treatment of breast cancer with serious side effects. Hence herein, a carbon paste electrode (CPE) amplified with Pt/SWCNT nanocomposite and a deep eutectic solvent (CPE/DES/Pt-SWCNT) were proposed as an analytical tool for the monitoring of EP in the presence of TP in the real samples. Amplification of sensor was improve EP oxidation signal about 2.73 times. Under the optimized conditions, EP determined by using differential pulse voltammetry (DPV) technique with linear dynamic range of 0.001–500 μM with limit of detection (LOD) of 0.8 nM. The CPE/DES/Pt-SWCNT offered influential ability for monitoring of EP in injection and dextrose saline samples with a recovery range of 97.4%–104.9%.

An approach to determine solution properties in micro pipes by near-field microwave microscopy

In this article, we propose a quantitative, non-destructive and noninvasive approach to obtain electromagnetic properties of liquid specimens utilizing a home-designed near-field microwave microscopy. The responses of aqueous solutions can be acquired with varying concentrations, types (CaCl2, MgCl2, KCl and NaCl) and tip–sample distances. An electromagnetic simulation model also successfully predicts the behaviors of saline samples. For a certain type of solutions with varying concentrations, the results are concaves with different bottoms, and the symmetric graphs of concave extractions can clearly identify different specimens. Moreover, we obtain electromagnetic images of capillaries with various saline solutions, as well as a Photinia × fraseri Dress leaf.

Identification of bacterial communities in extreme sites of Pakistan using high throughput barcoded amplicon sequencing.

Microorganisms thrive nearly everywhere including extreme environments where few other forms of life can exist. Geochemistry of extreme sites plays a major role in shaping these microbial communities and microbes thriving in such harsh conditions are untapped sources of novel biomolecules. To understand the structure and composition of such microbial communities, culture-independent bacterial diversity was characterised for two extreme sites in Pakistan, Khewra salt range and Murtazaabad hot spring. Barcoded amplicon sequencing technique was used to study the microbial communities. Physicochemical analysis of these sites was also conducted to study the dynamics of microbial communities under stressed conditions. Metagenomic sequencing of salt range soil samples yielded of 40,433 16S rRNA sequences, while hot spring sediments produced 76,449 16S rRNA sequence reads. Proteobacteria were predominant in saline soil while Firmicutes were most abundant in hot spring sediment. The taxonomic analysis of saline samples revealed 914 operational taxonomic units (OTUs) while that of hot spring sequences were clustered into 726 distinct OTUs. OTUs from genus Alkalibacillus were most abundant in hot spring sediments, whereas Haloarcula were more prevalent in saline soil. Some unidentified sequences were also present at each taxonomic level. Multivariate analysis indicated that electrical conductivity and pH are the major environmental factors involved in modelling microbial communities. This study revealed a poly-extremophilic microbial community in the Murtazaabad hot spring and characterised the unexplored halophilic microbial diversity of saline soil of Pakistan.

Structure engineering of silicon nanoparticles with dual signals for hydrogen peroxide detection

Fluorescent silicon nanoparticles (SiNPs) were synthesized by a one-step, simple, and green method with 3-Aminopropyltriethoxysilane (APTES) and ascorbic acid (AA) as reaction agents. Subsequently, the SiNPs and AgNPs nanocomplex (SiNPs@AgNPs) was constructed as the probe for hydrogen peroxide (H2O2) detection. The fluorescence of SiNPs was quenched due to the surface plasmonic-enhanced energy transfer between SiNPs and AgNPs. Meanwhile, the color tends to be yellow due to the existence of AgNPs. As the AgNPs were etched by H2O2, the fluorescence recovers and color fadings. Based on the well-designed structure, the “off–on” fluorescence sensing and “on–off” color sensing platforms for H2O2 were fabricated. The as-synthesized materials were characterized by Fourier transform infrared (FT-IR) spectroscopy, dynamic light scattering (DLS), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Fluorescence and UV–vis absorption spectra were used to evaluate the optical performance. The fabricated sensor exhibited a linear range of 1.0–100.0 μM, with a limit of detection of 0.36 μM for the fluorescence sensing of H2O2. Additionally, a linear range of 1.0–50.0 μM and a limit of detection of 0.45 μM were displayed for the detection of H2O2 by colorimetric assay. The feasibility in complex medium of the fabricated fluorescent and colorimetric dual-signal sensor was evaluated by the detection of H2O2 in phosphate buffer saline (PBS) and lake water samples.

Pentamethinium Salts Nanocomposite for Electrochemical Detection of Heparin.

This study presents a simple route to heparin detection and develops a voltammetric approach using supramolecular principles and nanomaterials. Nanocomposites, including gold nanoparticles (AuNPs) and γ-substituted pentamethinium salts (PMS) deposited on a glass carbon (GC) electrode surface (GC/AuNPs/PMS) and covered by a plasticized poly(vinyl chloride) (PVC) membrane, are proposed for heparin detection. The conductivity of the nonconducting PVC-plasticized membrane is guaranteed by AuNPs, and the selectivity is provided by the interaction between γ-substituted PMS and anionic analytes. In order to extend the linear range, it is necessary to apply a solvent compatible with PVC-plasticized membrane, namely tetrahydrofuran. The proposed voltammetric sensor showed a concentration dependence from 1.72 up to 45.02 IU mL−1 heparin and was used for heparin detection in saline and biological samples with recovery of 95.1–100.9%.

Applications of ambient electric arc ionization mass spectrometry in saline samples

Salinity tolerance of ambient electric arc ionization (AEAI) was evaluated by comparing electrospray ionization for various samples at NaCl concentrations from 0 to 1000 mmol/L. AEAI-mass spectrometry (AEAI-MS) exhibited an excellent signal intensity even at NaCl concentrations of 1000 mmol/L, while the ESI-MS had no signal because high salinity has a strong inhibitory effect on analytes. The sodium adduct was verified using LiCl instead of NaCl. AEAI-MS successfully quantified saline samples with an excellent quantitative ability (R2 ≥ 0.998). We also achieved some analytical samples in the buffer solution at a very high concentration and even in a saturated salt solution. Overall, AEAI-MS has protonated ions for most target analytes. In addition, the relationship between auxiliary temperature and the distance from the sample to the arc was investigated, and the results indicated that thermal desorption plays an important role in AEAI source.

Poly-l-lysine-functionalized magnetic beads combined with polymerase chain reaction for the detection of Staphylococcus aureus and Escherichia coli O157:H7 in milk

Rapid and credible detection of pathogens is essential to prevent and control outbreaks of foodborne diseases. In this study, a poly-l-lysine-functionalized magnetic beads (PLL-MB) strategy combined with a PCR assay was established to detect Staphylococcus aureus. We also detected Escherichia coli O157:H7 to further verify the strategy for gram-negative bacteria detection. Poly-l-lysine has strong positive charges because of its amino groups, which can conjugate with the carboxyl of carboxyl magnetic beads. Furthermore, it can be used to combine with bacteria through electrostatic adsorption. Under optimum conditions, the developed PLL-MB complexes showed 90% capture efficiency in phosphate-buffered saline and 85% capture efficiency in milk for S. aureus detection. The limit of detection of the PLL-MB-PCR assay was 102 cfu/mL (1.8 × 102 cfu/mL for S. aureus and 7 × 102 cfu/mL for E. coli O157:H7) in phosphate-buffered saline and milk samples. The whole assay can be performed within 4 h. The proposed strategy showed a lower limit of detection when compared with the conventional PCR assay without enrichment. In addition, this method exhibited the advantages of a high-efficient, cost-efficient, and simple operation, indicating its potential applications in foodborne pathogen detection.

Double-Antibody Sandwich Immunoassay and Plasmonic Coupling Synergistically Improved Long-Range SPR Biosensor with Low Detection Limit.

A long-range surface plasmonic resonance (LR-SPR) biosensor modified with double-antibody sandwich immunoassay and plasmonic coupling is demonstrated for human-immunoglobulin G detection with a low limit of detection (LOD). The double-antibody sandwich immunoassay dramatically changes the average refractive index of the medium layer on the sensor surface. The near-field electron coupling between the localized surface plasmon and the long-range surface plasmon leads to a significant perturbation of the evanescent field. The large penetration depth and the long propagation distance of the long-range surface plasmonic waves facilitate the LR-SPR sensor in the detection of biological macromolecules. The unique light absorption characteristic of the nanocomposite material in the sensor provides the in situ self-compensation for the disturbance. Therefore, besides the inherent advantages of optical fiber sensors, the developed biosensor can realize the detection of biomolecules with high sensitivity, low LOD and high accuracy and reliability. Experimental results demonstrate that the LOD of the biosensor is as low as 0.11 μg/mL in the detection of the phosphate-buffered saline sample, and the spike-and-repetition rate is 105.56% in the detection of the real serum sample, which partly shows the practicability of the biosensor. This indicates that the LR-SPR biosensor provides better response compared with existing similar sensors and can be regarded as a valuable method for biochemical analysis and disease detection.

Some physicochemical, textural properties and acrylamide contents of chips produced from the teleme of white cheese

The aim of this study is to evaluate some properties (acrylamide content, textural, and sensory) of cheese chips produced from the teleme of white cheese. Cheese chips were flavored with sucrose and stevia, salt, and evaluated against the control group (without salt, stevia, and sucrose). Cheese dough formulations were thinned to the thickness of chips and turned into round-shaped chips and fried in the oven at the specified temperature and time (180 °C, 6 min.). The produced chips were packed and stored at + 4 °C for 30 days. The difference between chips and storage time of pH, lactic acid (%), and water activity analysis was found to be statistically significant (p<0.05). The sucrose (CSU) added chips were determined to be harder but less brittle than the stevia added chips (CST). The acrylamide value was found between 0.218 ppm and 0.573 ppm (with sucrose sample) in cheese chips. The acrylamide content of cheese chips was determined below the legal limit. All chips produced were found to be generally acceptable. Starting from the most liked according to the total sensory score, the ranking is as follows; saline (CSA), control (CNA), stevia (CST), and sucrose (CSU) sample.

19 Interaction of kisspeptin and the somatotropic and lactotropic axes in mares and geldings

Several experiments were conducted to investigate the interaction between kisspeptin and the somatotropic and lactotropic axes in mares and geldings. Light horse, diestrous mares and geldings, weighing approximately 500 kg and aged 6 to 24 years, were randomly assigned to treatment with 1 mg equine kisspeptin-10 (Kp10; n = 10) or 1 mL saline (n = 9), i.v. Blood samples were collected via jugular venipuncture −10, 0, 5, 10, 15, 20 and 30 min relative to treatment. Plasma concentrations of growth hormone (GH) and prolactin (PRL) were determined by RIA and means compared using ANOVA with repeated sampling. Neither GH nor PRL were stimulated by Kp10. In a second experiment, 30 mares and geldings were used to determine if pre-treatment with estradiol or progesterone would elicit a GH or PRL response to Kp10. Five mares and 5 geldings each were assigned to pre-treatment with 50 mg estradiol cypionate in oil (ECP), 600 mg long-acting progesterone, or oil only, i.m. Two days later, all horses received 1 mg Kp10 in saline, i.v., and blood sampling occurred 0, 5, 10, 15 and 20 min relative to Kp10 administration. Mean GH and PRL concentrations and area under the curve were compared by ANOVA. No stimulation of GH or PRL was observed. In a third experiment, 12 diestrous mares, confirmed by plasma progesterone and ultrasound findings, were randomly assigned to treatment with 1 mg Kp10 (n = 6) or 1 mL saline (n = 6) followed by the dopamine antagonist, sulpiride (0.01 mg/kg body weight), 5 min later. Blood sampling occurred 0, 5, 10 and 15 min relative to sulpiride administration. Sulpiride stimulated PRL ( P < 0.0001) in both groups of mares; however, PRL concentrations were lower ( P ≤ 0.01) in Kp10-treated mares compared with saline-treated mares. To determine if estrogen altered the PRL response to sulpiride, 12 seasonally anovulatory mares, confirmed by plasma progesterone and ultrasound findings, were used in a fourth experiment and randomly assigned to receive 50 mg ECP (n = 6) or oil (n = 6), i.m. The next day, mares were challenged with sulpiride. Treatment and blood sampling occurred as described in experiment 3. Plasma PRL was stimulated ( P < 0.001) in both groups of mares and responses were similar between treatments. In the described experiments, 1 mg Kp10 given as a bolus failed to stimulate circulating GH or PRL within 30 min in mares or geldings regardless of steroid milieu. This is in contrast to reports of GH stimulation in ruminants. Kp10 did alter the PRL response to sulpiride in diestrous mares, perhaps suggesting an interaction between Kp10 and dopaminergic input to the pituitary in a progesterone dominant environment.

Programmable flow injection in batch mode: Determination of nutrients in sea water by using a single, salinity independent calibration line, obtained with standards prepared in distilled water

Interference of the Schlieren effect on sea water analysis by spectrophotometry is caused by the flow of solutions of different ionic strengths through a flow cell. A flow injection assay protocol programmed in a flow-batch format removes this interference and allows the use of a calibration line, obtained in deionized water, for determination of analytes in sea water samples of different salinity. This Single Line Calibration (SLC) technique is validated on the most frequently performed nutrient assays. Automated determinations, performed at rates ranging from 20 to 60 samples/hr, covered seawater sample ranges from nM to mM with limits of detection: 12 nM for nitrite, 94 nM for nitrate, 47 nM for phosphate, and 240 nM for silicate. Reproducibility of the determinations was equal to or better than, 3% r.s.d. and day to day calibration was within 10%. The programmable FI, uses about 1/5th volume of reagents compared to continuous flow techniques.

Quantification of Amine- and Alcohol-Containing Metabolites in Saline Samples Using Pre-extraction Benzoyl Chloride Derivatization and Ultrahigh Performance Liquid Chromatography Tandem Mass Spectrometry (UHPLC MS/MS).

Dissolved metabolites serve as nutrition, energy, and chemical signals for microbial systems. However, the full scope and magnitude of these processes in marine systems are unknown, largely due to insufficient methods, including poor extraction of small, polar compounds using common solid-phase extraction resins. Here, we utilized pre-extraction derivatization and ultrahigh performance liquid chromatography electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) to detect and quantify targeted dissolved metabolites in seawater and saline culture media. Metabolites were derivatized with benzoyl chloride by their primary and secondary amine and alcohol functionalities and quantified using stable isotope-labeled internal standards (SIL-ISs) produced from 13C6-labeled benzoyl chloride. We optimized derivatization, extraction, and sample preparation for field and culture samples and evaluated matrix-derived biases. We have optimized this quantitative method for 73 common metabolites, of which 50 cannot be quantified without derivatization due to low extraction efficiencies. Of the 73 metabolites, 66 were identified in either culture media or seawater and 45 of those were quantified. This derivatization method is sensitive (detection limits = pM to nM), rapid (∼5 min per sample), and high throughput.

Illumina MiSeq sequencing reveals microbial community succession in salted peppers with different salinity during preservation

Chopped pepper is one of the traditional fermented pepper products in China. At present, the industrial production method is mainly to preserve the peppers with high salt about 1 year, and then make the product after desalination and seasoning when it is processed. However, the composition and succession of the bacterial community involved in the long-term preservation of salted pepper was complex. In this study, Illumina Miseq sequencing technology was used to reveal the succession in the bacterial community structure of different salted pepper within 10 months of preservation. The results showed that Firmicutes and Proteobacteria were dominant bacteria in all samples at the Phylum level. At the Genus level, among fresh unsalted capsicum, Fructobacillus (44.66%), Enterobacteriaceae unclassified (26.78%), Leuconostoc (12.04%) and Lactococcus (8.45%) had relatively high abundance. Enterobacteriaceae unclassified, Lactobacillus, Marinospirillum and Halomonas were identified as the main dominant bacteria in the samples with 6%-12% (w/w) salinity, and Enterobacteriaceae unclassified mainly appeared in the early stage of preservation. In 15% and 18%(w/w) salinity samples, with the increase of preservation time, the dominant genus was changed from Enterobacteriaceae unclassified to Chromohalobacterter, Tetragenococcus, Halomonas, Halovibrio, etc., while the relative abundance of Lactobacillus remained at an extremely low level. The bacterial structure of 6% (w/w) salinity samples changed significantly during preservation, while the distribution in PCoA analysis of salinity samples of 9% was similar to that of 12%. In the high-salinity samples (15%-18%), the composition of the community was highly similar in 0–6 months, but the composition changed significantly with the increase of the preservation time and the growth of halophilic bacteria (p < 0.01). Pearson correlation analysis was used to investigate that Lactobacillus exhibited a negative correlation with salinity (p < 0.01). And the salinity had a positive correlation with both the species richness and evenness in the samples, which might be the key factor for the change of the microbial community.