Unraveling the formation mechanism of aroma compounds in pork during air frying using UHPLC-HRMS and Orbitrap Exploris GC-MS.

Lipids are the key matrix for the presence of odorants in meat products. The formation mechanism of odorants of air-fried (AF) pork at 230 °C was elucidated from the perspectives of lipids and heat transfer using physicochemical analyses and multidimensional statistics. Twenty-nine key aroma compounds were identified, with pyrazines predominantly contributing to the roasty aroma of air-fried roasted pork. Untargeted lipidomics revealed 1184 lipids in pork during roasting, with phosphatidylcholine (PC), phosphatidylethanolamine (PE), and triglyceride (TG) being the major lipids accounting for about 60 % of the total lipids. TG with C18 acyl groups, such as TG 16:1_18:1_18:2 and TG 18:0_18:0_20:3, were particularly significant in forming the aroma of AF pork. The OPLS-DA model identified seven potential biomarkers that differentiate five roasting times, including PC 16:0_18:3 and 2-ethyl-3,5-dimethylpyrazine. Notably, a lower specific heat capacity and water activity accelerated heat transfer, promoting the formation and retention of odorants in AF pork.

Metabolomics integrated with mass spectrometry imaging reveals novel action of tetramethylpyrazine in migraine.

Migraine as a common neurological disorder still lacks effective therapies. Tetramethylpyrazine (TMP) is the main bioactive component from Ligusticum chuanxiong hort., a traditional edible-medicinal herb. This study aimed to investigate the action of TMP on migraine by metabolomics with mass spectrometry imaging (MSI) analysis and molecular exploring, including random forest model analysis, KEGG enrichment analysis and metabolite-metabolite interaction network analysis. The results indicated that 26 key representative metabolic biomarkers were identified, especially γ-glu-cys, which were highly related to glutathione (GSH) metabolism. MSI found the abundance of eleven endogenous metabolites were modulated by TMP, particularly glucose, the most important energy metabolism molecule, and GSH were increased that maintains intracellular redox balance, which was consistent with activation of Nrf2 signals by TMP. These findings provide insights into the effectiveness of metabolomics integrated with MSI in explaining the metabolic mechanisms of TMP, and afford valuable information for healthy development of TMP in migraine.

An ultrasensitive green synchronous factorized spectrofluorimetric approach for the simultaneous determination of Favipiravir and Molnupiravir: Application to pharmaceutical, environmental, and biological matrices.

During the pandemic, Favipiravir (FVP) and Molnupiravir (MPV) have been widely used for COVID-19 treatment, leading to their presence in the environment. A green synchronous spectrofluorimetric method was developed to simultaneously detect them in environmental water, human plasma, and binary mixtures. Maximum fluorescence intensity was achieved at pH 8, with MPV exhibiting two peaks at 300 and 430 nm, and FVP showing one peak at 430 nm. A fluorescence subtraction method effectively removed interference, enabling direct determination of MPV at 300 nm and FVP at 430 nm. The method showed linearity within 2-13 ng/mL for FVP and 50-600 ng/mL for MPV, with recoveries of 100.35% and 100.12%, respectively. Limits of detection and quantification were 0.19 and 0.57 ng/mL for FVP and 10.52 and 31.88 ng/mL for MPV. Validation according to ICH and FDA guidelines yielded acceptable results. The method demonstrated good recoveries of FVP and MPV in pharmaceuticals, tap water and Nile water (99.62% ± 0.96% and 99.69% ± 0.64%) as per ICH guidelines and spiked human plasma (94.87% ± 2.111% and 94.79% ± 1.605%) following FDA guidelines, respectively. Its environmental friendliness was assessed using Green Analytical Procedure Index (GAPI) and the Analytical Greenness Metric (AGREE) tools.

Long term study on the fate and environmental risks of favipiravir in wastewater treatment plants and comparison with COVID-19 cases.

In recent years especially during COVID-19, the increased usage of antiviral drugs has led to increased interest in monitoring their presence in wastewater worldwide. In this study, it was examined the occurrence, fate and environmental risks of favipiravir which is used for COVID-19 treatment in two wastewater treatment plants (WWTPs) with different treatment processes in Istanbul, Turkey. Favipiravir was measured in WWTPs influent samples, effluent samples and sludge samples with maximum concentrations of 97 µg/L, 64.11 µg/L and 182.47 µg/g, respectively. Favipiravir had removal efficiency below 55 % for both WWTPs. Mass balance analysis showed that favipiravir removal in WWTPs mainly attributed to biodegradation/biotransformation. Statistical analysis revealed a significant correlation between favipiravir concentration and COVID-19 incidence in Istanbul. The microbial distribution analysis indicated that comparison of collected COVID-19 pandemic sludge and post-pandemic period sludge samples, a noteworthy reduction in the Chloroflexi and Actinobacteriota phyla at the phylum level was observed. Environmental risk assessment using risk quotients ranged from 168 to 704, indicating that the presence of this antiviral drug posed significant ecological risks to aquatic organisms. The study concluded that WWTPs were releasing antiviral drugs into the environment, thereby posing risks to both the aquatic ecosystem and public health. The results of this study demonstrate the persistence of favipiravir in WWTPs and offer crucial supporting data for further research into the advancement of wastewater treatment technology. Also, this study shows wastewater based monitoring is supplementary and early warning system for determining the occurrence of antiviral drugs.

Characterization of Key Aroma Compounds and Main Contributing Amino Acids in Hot-Pressed Oil Prepared from Various Peanut Varieties.

The production of peanut oil in the industrial sector necessitates the utilization of diverse raw materials to generate consistent batches with stable flavor profiles, thereby leading to an increased focus on understanding the correlation between raw materials and flavor characteristics. In this study, sensory evaluations, headspace solid-phase micro-extraction gas chromatography mass spectrometry (HS-SPME-GC-MS), odor activity value (OAV) calculations, and correlation analysis were employed to investigate the flavors and main contributing amino acids of hot-pressed oils derived from different peanut varieties. The results confirmed that the levels of alcohols, aldehydes, and heterocyclic compounds in peanut oil varied among nine different peanut varieties under identical processing conditions. The OAVs of 25 key aroma compounds, such as methylthiol, 3-ethyl-2,5-dimethylpyrazine, and 2,3-glutarone, exceeded a value of 1. The sensory evaluations and flavor content analysis demonstrated that pyrazines significantly influenced the flavor profile of the peanut oil. The concentrations of 11 amino acids showed a strong correlation with the levels of pyrazines. Notably, phenylalanine, lysine, glutamic acid, arginine, and isoleucine demonstrated significant associations with both pyrazine and nut flavors. These findings will provide valuable insights for enhancing the sensory attributes of peanut oil and selecting optimal raw peanuts for its production.

The formation and conversion of characteristic aroma profiles and key harmful substances in different high-temperature processing of hairtail (Trichiurus Haumela).

The balance regulation between characteristic aroma and hazards in high-temperature processed fish is a hot spot. This study was aimed to explore the interactive relationship between the nutritional value, microstructures, aroma, and harmful substances of hairtail under different frying methods including traditional frying (TF), air frying (AF), and vacuum frying (VF) via chemical pattern recognition. The results indicated that VF-prepared hairtail could form a crunchy mouthfeel and retain the highest content of protein (645.53 mg/g) and the lowest content of fat (242.03 mg/g). Vacuum frying reduced lipid oxidation in hairtail, resulting in the POV reaching 0.02 mg/g, significantly lower than that of TF (0.05 mg/g) and AF (0.21 mg/g), and TBARS reached 0.83 mg/g, significantly lower than that of AF (1.96 mg/g) (P < 0.05), respectively. Notable variations were observedin the aroma profileof hairtail preparedfrom different frying methods. Vacuum frying of hairtail resulted in higher levels of pyrazines and alcohols, whereas traditional frying and air frying were associated with the formation of aldehydes and ketones, respectively. Air frying was not a healthy way to cook hairtail which produced the highest concentration of harmful substances (up to 190.63 ng/g), significantly higher than VF (5.72 ng/g) and TF (52.78 ng/g) (P < 0.05), especially norharman (122.57 ng/g), significantly higher than VF (4.50 ng/g) and TF (32.63 ng/g) (P < 0.05). Norharman and acrylamide were the key harmful substances in hairtail treated with traditional frying. The vacuum frying method was an excellent alternative for deep-fried hairtail as a snack food with fewer harmful substances and a fine aroma, providing a theoretic guidance for preparing healthy hairtail food with high nutrition and superior sensory attraction.

Unravelling caramelization and Maillard reactions in glucose and glucose + leucine model cakes: Formation and degradation kinetics of volatile markers extracted during baking.

A large number of volatile compounds are formed during the baking of foods by reactions such as caramelization and Maillard reactions. Elucidating the reaction mechanisms may be useful to predict and control food quality. Ten reaction volatile markers were extracted during baking of solid model cakes implemented with known amounts of precursors (glucose with or without leucine) and then quantified by Thermal desorption-Gas chromatography-Mass spectrometry. The kinetic data showed that the level of air convection in the oven had no significant influence on the reaction rates. In contrast, increasing baking temperatures had a nonlinear accelerating impact on the generation of newly formed volatile compounds with a bell-shaped kinetic curve found for most of the markers at 200 °C. The presence of leucine triggered the activation of the Maillard and Strecker routes with a specific and very rapid formation of 3-Methylbutanal and pyrazines. A dynamic model was developed, combining evaporation flow rate and kinetic formation and consumption of reaction markers. It can be used to describe, for two furanic compounds of different volatilities, the vapor concentrations in the oven from the concentrations measured in the model cakes.

A novel LC-MS/MS method for the determination of favipiravir ribofuranosyl-5'-triphosphate (T-705-RTP) in human peripheral mononuclear cells.

Favipiravir is a broad-spectrum antiviral that is metabolised intracellularly into the active form, favipiravir ribofuranosyl-5'-triphosphate (F-RTP). Measurement of the intracellular concentration of F-RTP in mononuclear cells is a crucial step to characterising the pharmacokinetics of F-RTP and to enable more appropriate dose selection for the treatment of COVID-19 and emerging infectious diseases. The described method was validated over the range 24 - 2280 pmol/sample. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood and lysed using methanol-water (70:30, v/v) before cellular components were precipitated with acetonitrile and the supernatant further cleaned by weak anion exchange solid phase extraction. The method was found to be both precise and accurate and was successfully utilised to analyse F-RTP concentrations in patient samples collected as part of the AGILE CST-6 clinical trial.

Abscisic acid reduced methoxypyrazines concentration and its derived unpleasant odors in Cabernet Sauvignon grapes and wines.

BACKGROUND: The influences of abscisic acid (ABA) applications on precursors and gene expression in 3-alkyl-2-methoxypyrazines (MPs) biosynthetic pathway, MPs concentration and sensory evaluation of its derived peculiar odors in Cabernet Sauvignon grapes and wines were investigated. At the vineyard, ABA solution with 25, 100 and 400 mg L-1 (AT1, AT2 and AT3, respectively) and an aqueous solution (control) were sprayed three times from veraison to pre-harvest. RESULTS: Higher concentration ABA applications (AT2 and AT3) in grapes could significantly reduce MPs concentration and its derived peculiar odors in grapes and wines compared to a lower concentration ABA application (AT1) and control, with AT2 application having the strongest effect. The changes in MPs were mainly a result of the downregulated expression of VvOMTs genes at higher concentration ABA applications, independent of the levels of their potential precursors. CONCLUSION: The present study reveals that ABA application had the potential to decrease production of MPs in Cabernet Sauvignon grapes and wines, and this result provides reference values for the removal of unpleasant vegetable odors from Cabernet Sauvignon wines in production. © 2024 Society of Chemical Industry.

Effect of Dehulling Pretreatment on the Flavor of Sesame Paste.

Gas chromatography-olfactory-mass spectrometry (GC-O-MS) combined with Aroma Extract Dilution Analysis (AEDA) were employed to characterize the key odor-active compounds in sesame paste (SP) and dehulled sesame paste (DSP). The AEDA results revealed the presence of 32 and 22 odor-active compounds in SP and DSP, respectively. Furthermore, 13 aroma compounds with FD ≥ 2, OAV ≥ 1, and VIP ≥ 1 were identified as key differential aroma compounds between SP and DSP. Specifically, compounds such as 3-methylbutyraldehyde (OAV = 100.70-442.57; fruity), 2-methylbutyraldehyde (OAV = 106.89-170.31; almond), m-xylene (FD = 16; salty pastry), and 2,5-dimethylpyrazine (FD = 8-16; roasted, salty pastry) played an important role in this differentiation. Additionally, the dehulling process led to increased fermented, sweet, green, and nutty aroma notes in DSP compared to the more pronounced burnt and roasted sesame aroma notes in SP. Our findings offer a theoretical foundation for the regulation of sesame paste aroma profiles.

QbD-Based Stability-Indicating RP-HPLC Method Development and Validation for the Estimation of Favipiravir-An Eco-Friendly Approach.

BACKGROUND: Analytical quality by design (AQbD) affords a systematic scaffolding to triumph a continuously validated, robust assay as well as life cycle management. The resuscitative repurposed drug favipiravir, an oral drug approved for reemerging pandemic influenza in Japan in 2014, is used for the treatment of life-threatening pathogens such as Ebola, Lassa virus, and currently COVID-19. Favipiravir is gaining a great deal of medical importance due to its pharmaceutical applications. OBJECTIVE: To develop and validate a risk-based stability-indicating RP-HPLC method employing an AQbD approach using Central Composite Design (Design Expert Software 13.0) for the estimation of favipiravir. METHOD: The Quality Target Product Profile optimized were flow rate and mobile phase composition, thus assessing the critical analytical attributes (retention time, tailing factor, and number of theoretical plates) as the constraints of method robustness. The proposed technique was optimized with a C18 (150 × 4.6 mm, 5 µm) column and 0.1% orthophosphoric acid buffer-acetonitrile (50:50, v/v) as the mobile phase at a flow rate of 1 mL/min using diode-array detector (230 nm) eluted favipiravir at 2.3 min. RESULTS: The optimized method validated as per ICH guideline Q2 (R1) was found to be eco-friendly, simple, precise (RSD 0.0051-1.2%), accurate (99.86-100.22%), linear (25-150 µg/mL), rugged (RSD 0.70%), and robust (RSD 0.6-1.6%) with a limit of detection and limit of quantitation of 1.140 µg/mL and 4.424 µg/mL, respectively. CONCLUSION: Forced degradation studies (acidic, alkaline, thermal, photolytic, and oxidative conditions) revealed the suitability of the AQbD method for the analysis of favipiravir in tablet formulation.The developed and validated AQbD method is less time consuming and can be used in the industry for routine quality control/analysis of bulk drug and marketed Favipiravir products. HIGHLIGHTS: A robust Design of Experiment enhanced stability-indicating analytical method was developed and validated for the estimation of favipiravir. Furthermore, the contemporary method would aid in extending the analysis of favipiravir in other formulations.

An Eco-Friendly RP-HPLC Method Development and Validation for Quantification of Favipiravir in Bulk and Tablet Dosage Form Followed by Forced Degradation Study.

In this work, an eco-friendly simple, precise reverse phase high-performance liquid chromatography (HPLC) method has been developed and validated for Favipiravir in bulk and tablet dosage form followed by its force degradation study. The proposed method was validated to obtain official requirements including stability, accuracy, precision, linearity, robustness and selectivity as per International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Guidelines. The estimation was developed on C (18) column reversed-phase using the mobile phase composition as methanol:water (10:90 v/v). The flow rate was set as 1 ml/min, and the maximum absorption was observed at 323 nm using Shimadzu Photo Diode Array detector. The Favipiravir, drug showed a precise and good linearity at the concentration ranges of 10-50 µg/mL. The Revearse Phase High Perforance Liquid Chromatography assay showed the highest purity ranging from 99.90 to 100.02% for Favipiravir, tablet dosage form, and 100.15% was the mean percentage purity. The percent recovery was found within the acceptance limit of (98.6-100.0%). Intra- and inter-day precision studies of the method were less than the maximum allowable limit percentage of relative standard deviation ≤ 2.0. The Favipiravir retention time was found to be 5.00 min. To examine the stability of the drug, various forced degradation studies were conducted on Favipiravir Active Pharmaceutical Ingredient. The developed method was validated according to the ICH guidelines. A very quick, cost-effective, precise and accurate HPLC method for the determination of Favipiravir has been developed and validated in compliance with ICH guidance Q2.

Effects of different over-fired drying methods on the aroma of Lu'an Guapian tea.

Over-fired drying, a crucial process in the production of Lu'an Guapian (LAGP) tea, greatly enriches the tea's aroma. In this study, the aroma compounds of LAGP tea processed through pulley charcoal drying (PCD), roller drying (RD), roller-conveyor drying (RCD), and hot air drying (HD) were analyzed using gas chromatography-mass spectrometry. A subsequent analysis of aroma extraction dilution analysis and odor activity values revealed that (E)-ß-ionone, dimethyl sulfide, (E,E)-2,4-heptadienal, geraniol, linalool, benzeneacetaldehyde, coumarin, 2-ethyl-3,5-dimethyl-pyrazine, indole, hexanal, (Z)-jasmone, and (Z)-3-hexen-1-ol were the key contributors to the samples' aroma variation. Moreover, a quantitative descriptive analysis and aroma recombination and omission experiments analysis revealed that (E)-ß-ionone is the most critical contributor to the formation of floral aroma in tea processed using PCD, whereas (E,E)-2,4-heptadienal is responsible for the more pronounced fresh aroma in tea processed using HD. In addition, 2-ethyl-3,5-dimethyl-pyrazine contributes to the formation of a roasted aroma in tea processed using RD and RCD. The study results provide a theoretical basis for choosing the processing method, especially for drying, to obtain high-quality LAGP tea.

Contribution of theanine to the temperature-induced changes in aroma profile of Wuyi rock tea.

Theanine is a distinctive amino acid in tea that plays a vital role in tea flavor during the roasting process. Model thermal reactions of total amino acids and sugars with different roasting conditions (low-fire, middle-fire, and high-fire) showed theanine competitively inhibited the formation of indole, skatole, 4-hydroxy-2,5-dimethyl-3(2H)-furanone, and Strecker aldehydes, while greatly stimulated the production of roasty pyrazines. In addition, highest amounts of pyrazines were obtained under high-fire degree. Quantification of these reaction products in Wuyi rock tea (WRT) was realized in different roasted Dahongpao teas by means of sensomics approach. The quantitative data revealed the biggest influence of roasting temperatures on the formation of reaction products among indole, lipid oxidation products, and pyrazines, while other reaction products were only slightly affected. The findings of this study provide a fresh perspective on the impact of theanine on aroma formation during the roasting process, which will help to explore the formation of key odorants during tea production.

Isolation and Identification of Lactic Acid Bacteria and Their Effects on the Off-odor of Burdocks.

Burdocks have diverse nutritional and pharmacological functions, but their unique odor is unwelcome. Here, the effect and mechanism of lactic acid bacteria fermentation on the off-odor of burdocks were investigated. The sensory evaluation showed that burdocks had earthy, musty, grassy, and pepper odors. 2-Isobutyl-3-methoxypyrazine (IBMP) and 2-secbutyl-3-methoxypyrazine (IPMP) mainly contributed to burdock's unique off-odor and were identified by gas chromatography-mass spectrometry combined with headspace-solid phase microextraction (HS-SPME-GC-MS) and relative odor activity value (ROAV) analysis. Weissella cibaria ZJ-5 from screened strains performed with the strongest ability to remove the off-odor and generate a fragrant odor, as determined by sensory evaluation. When incubated aerobically together with IBMP during fermentation, ZJ-5 degraded IBMP directly from 149.56 ± 0.72 to 71.55 ± 1.81 ng/mL. Additionally, linoleic acid content in fermented burdocks was significantly decreased compared with unfermented burdocks. (E,Z)-2,6-Nonadienal, which mainly contributed to fermented burdock's odor, may have been generated from linoleic acid during ZJ-5 fermentation, through the acid catalysis pathway. It indicated that LAB fermentation could improve burdock odor by degrading off-odor compounds and precursors and by generating new aldehydes.

Monitoring alkyl pyrazines in roasted hazelnuts by SHS-GC-IMS: IMS response assessment and standardization.

Gas chromatography coupled with ion mobility spectrometry (IMS) is an analytical tool which is rapidly becoming widespread in the analysis of food volatiles. Despite this increasing popularity, an assessment of the IMS response for several flavor compound classes is not yet available. This study focuses on alkyl pyrazines and their determination in roasted hazelnut pastes. These Maillard reaction products are crucial to determine the aromatic profile of roasted foods and are suitable markers for industrial roasting monitoring. The instrumental response of 8 alkyl pyrazines was studied using a model matrix and a matrix matching approach. The results showed a relevant effect of the pyrazine ring substitution pattern on the concentration-response curve trends, highlighting that an external standardization of the IMS response is required to make possible relative abundance comparisons between analytes. A response standardization was therefore developed and applied to determine alkyl pyrazines in samples with different roasting intensity and geographical and botanical origin.

Insights into the Uptake, Distribution, and Metabolism of 3-Isobutyl-2-hydroxypyrazine in Grapevine Using a Stable Isotope Tracer.

Methoxypyrazines (MPs) are potent aroma compounds that have been predominately studied in grape berries but can also be detected in other vine tissues. The synthesis of MPs in berries from hydroxypyrazines by VvOMT3 is well established, but the origin of MPs in vine tissues that have negligible VvOMT3 gene expression is unknown. This research gap was addressed through the application of stable isotope tracer 3-isobutyl-2-hydroxy-[2H2]-pyrazine (d2-IBHP) to the roots of Pinot Meunier L1 microvines and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) quantification of HPs from grapevine tissues following a novel solid-phase extraction method. Four weeks post-application, d2-IBHP and its O-methylated product 3-isobutyl-2-methoxy-[2H2]-pyrazine (d2-IBMP) were present in excised cane, berry, leaf, root, and rachis material. Translocation of d2-IBHP and d2-IBMP was investigated, but results were inconclusive. Nonetheless, knowledge that d2-IBHP, and potentially d2-IBMP, are translocated from roots to other vine organs, including the berries, could provide opportunities for controlling MP accumulation in grapevine tissues pertinent to winemaking.

Optimization of Jinhua Ham Classification Method Based on Volatile Flavor Substances and Determination of Key Odor Biomarkers.

Jinhua ham is a traditional cured meat food in China. For a long time, its grade has mainly been evaluated by the human nose through the three-sticks method, which is highly subjective and is not conducive to establishing evaluation standards through odor markers. In this paper, we analyzed the well-graded Grade I-III hams provided by Jinzi Ham Co., Ltd. (Jinhua, China). Firstly, we used different extraction fibers, extraction temperatures, and extraction time to determine the optimal conditions for headspace solid-phase microextraction (HS-SPME). Then, the aroma components of Jinhua ham were analyzed by headspace solid-phase microextraction combined with gas chromatography-mass spectrometry (GC-MS), and OAV was calculated to screen the key aroma volatiles of three kinds of Jinhua ham. It was found that a total of 56 components were detected in the three types of ham. Among them, there are 21 kinds of key aroma volatiles. Aldehydes, alcohols, and acids are the three main components of Jinhua ham, and the content of aldehydes gradually decreases from Grade I to Grade III ham. The content of acids gradually increased, and we speculated that the increase in acid content was caused by the proliferation of microorganisms in Grade III ham. The key flavor volatiles in Grade I hams was hexanal and 2-methylbutanal. Grade I hams had a strong meat aroma, pleasant fatty, and roasted aroma without any off-flavors. In Grade II ham, the characteristic volatiles (E,E)-2,4-decadienal and ethyl isovalerate were detected. These two volatiles contribute greatly to the flavor of Grade II ham, which makes the flavor of Grade II ham have a special fruity aroma. They also may be prone to sourness and affect the flavor of the ham. Volatiles with low threshold values, such as pyrazines, furans, and sulfur-containing compounds, were relatively high in Grade III hams. This may also contribute to the poorer flavor quality of Grade III hams. This experiment provided a reliable test method and evaluation basis for the rating of Jinhua ham. These results have positive implications for the establishment of odor markers-based grading criteria.

Microbial characteristics and metabolite profiles of high-temperature Daqu in different maturation stages.

The maturation period of high-temperature Daqu (HTD) is usually 3-6 months, and the characteristics of HTD at different maturation stages were different. In this study, the microbial characteristics and metabolite profiles of HTD at different maturation stages were revealed with the combination of physicochemical detection, the third generation Pacific Biosciences (PacBio) single-molecule, real-time (SMRT) sequencing technology, gas chromatography-mass spectrometry (GC-MS), and gas chromatography-ion mobility spectrometry (GC-IMS). Results showed that HTD matured for 6 months (Mix_m6) had higher saccharification power but less culturable thermotolerant bacteria and fungi than HTD matured for 3 months (Mix_m3). The average relative abundances of Thermoactinomyces, Paenibacillus, and Rasamsonia in Mix_m3 were higher than that in Mix_m6, while the average relative abundances of Bacillus, Pseudomonas, Thermoascus increased obviously with the prolongation of the maturation period. Streptomyces and Thermoactinomyces were biomarkers in Mix_m3, while Burkholderia and Pseudomonas were regarded as biomarkers in Mix_m6. Differences in microbiota structure led to different enrichment of metabolic pathways in HTD at different maturation stages, resulting in different flavor profiles, especially in ethyl acetate, 1-octen-3-one, (E)-3-Hexen-1, 2,3,5-trimethy-6-ethylpyrazine, pyrazine, tetramethyl content. The microbiota and metabolite characteristics of HTD comprehensively reflected the HTD quality in different maturation stages, which provided a reference for determining the optimal maturation time.

Optimized Solid-Phase Mesh-Enhanced Sorption from Headspace (SPMESH) for Rapid Sub-ng/kg Measurements of 3-Isobutyl-2-methoxypyrazine (IBMP) in Grapes.

Parallel extraction of headspace volatiles from multiwell plates using sorbent sheets (HS-SPMESH) followed by direct analysis in real-time high-resolution mass spectrometry (DART-HRMS) can be used as a rapid alternative to solid-phase micro-extraction (SPME) gas-chromatography mass-spectrometry (GC-MS) for trace level volatile analyses. However, an earlier validation study of SPMESH-DART-MS using 3-isobutyl-2-methoxypyrazine (IBMP) in grape juice showed poor correlation between SPMESH-DART-MS and a gold standard SPME-GC-MS around the compound's odor detection threshold (<10 ng/kg) in grape juice, and lacked sufficient sensitivity to detect IBMP at this concentration in grape homogenate. In this work, we report on the development and validation of an improved SPMESH extraction approach that lowers the limit of detection (LOD < 0.5 ng/kg), and regulates crosstalk between wells (<0.5%) over a calibration range of 0.5−100 ng/kg. The optimized SPMESH-DART-MS method was validated using Cabernet Sauvignon and Merlot grape samples harvested from commercial vineyards in the central valley of California (n = 302) and achieved good correlation and agreement with SPME-GC-MS (R2 = 0.84) over the native range of IBMP (<0.5−20 ng/kg). Coupling of SPMESH to a lower resolution triple quadrupole (QqQ)-MS via a new JumpShot-HTS DART source also achieved low ng/kg detection limits, and throughput was improved through positioning stage optimizations which reduced time spent on intra-well SPMESH areas.