Determination of succinic acid and lactic acid in pigs' serum, intestinal contents, and meat by ultrahigh-performance liquid chromatography-tandem mass spectrometry.

RATIONALE: Succinic acid and lactic acid have been associated with diarrhea in weaned piglets. The level of succinic acid and lactic acid in serum, meat, and intestinal contents is important to elucidate the mechanism of diarrhea in weaned piglets. METHODS: A facile method was developed for the quantification of succinic acid and lactic acid in pigs' serum, intestinal contents, and meat using ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC/MS/MS). The serum samples underwent protein precipitation with methanol. The meat and intestinal contents were freeze-dried and homogenized using a tissue grinding apparatus. Methanol-water mixture (80:20, v/v) was used for homogenizing the meat, while water was used for homogenizing the intestinal contents. An additional step of protein precipitation with acetonitrile was required for the intestinal contents. The resulting solution was diluted with water before being analyzed by UHPLC/MS/MS. Separation of succinic acid and lactic acid could be achieved within 3 min using a Kinetic XB-C18 column. RESULTS: The coefficients of variation for peak areas of succinic acid and lactic acid were less than 5.0%. The established method demonstrated good linearity as indicated by correlation coefficients exceeding 0.996. Additionally, satisfactory recoveries ranging from 88.58% to 108.8% were obtained. The detection limits (RS/N = 3) for succinic acid and lactic acid were determined to be 0.75 ng/mL and 0.02 µg/mL, respectively. CONCLUSION: This method exhibited high sensitivity, simplicity in operation, and small sample weight, making it suitable for quantitative determination of succinic acid and lactic acid in pigs' serum, intestinal contents, and meat. The method developed will provide valuable technical support in studying the metabolic mechanisms of succinic acid and lactic acid in pigs.

Characterization of chemical components of fresh and aged aerosol from vehicle exhaust emissions in Hong Kong.

The chemical properties of fresh and aged aerosol emitted during controlled vehicular exhaust emissions were characterized in the analysis. Pyrene (10417.1 ± 534.9 ng kg-1) is the most abundant of all analyzed compounds in total fresh emission and succinic acid (57359.8 ± 4000.3 ng kg-1) is for the total aged emission. The fresh emission factors (EFfresh) of all compounds in the n-alkanes group demonstrate higher average emissions for the two vehicles with EURO 3 standard compared to the other vehicles. The EFfresh for benzo [a]pyrene is in descending order: G1 (183.1 ± 144.7 ng kg-1) > G3 (103.4 ± 60.1 ng kg-1) > G4 (91.2 ± 80.1 ng kg-1) > G2 (88.6 ± 93.9 ng kg-1). Aged/fresh (A/F) emission ratios (>20) confirmed that these diacid compounds are generated by the photooxidation of primary pollutants that emitted from gasoline combustions. High A/F ratios (>200) in phthalic acid, isophthalic acid and terephthalic acid under idling mode imply relatively more intense photochemical reactions for their productions compared with other chemical groups. Strong positive correlations (r > 0.6) were observed between the degradation of toluene and formations of pinonic acid, succinic acid, adipic acid, terephthalic acid, glutaric acid and citramalic acid after the aging process, suggesting possible photooxidation of toluene that can lead to secondary organic aerosol (SOA) formation in the urban atmosphere. The findings demonstrate that vehicle emission standards for pollution in relation to the change of particulate matter chemical compositions and SOA formations. The results warrant a need for regulated reformulation for such vehicles.

Simultaneous determination of succinate-dehydrogenase-inhibitor fungicide traces in cereals by QuEChERS preparation and UPLC-MS/MS analysis.

A method for the simultaneous determination of 19 succinate dehydrogenase inhibitor (SDHI) fungicide residues in 8 kinds of cereals was established by combining UHPLC-MS/MS with the improved QuEChERS method. MgSO4 and octadecylsilane (C18) were used as the dispersive-solid phase extraction sorbent. The proposed method had good linearity in the range of 10-100 µg/L with correlation coefficients (R2 > 0.99). The limit of quantification of 19 fungicides was 10 µg/L, which is the minimum addition level of the method. The fortified recoveries of 19 SDHI fungicides at three levels were ranged from 79.57 % to 126.25 %. The developed method was utilized for the analysis of 45 real cereal samples, only 5 samples were detected with SDHI fungicides. The contents of the fungicides detected in the real samples are far lower than the MRL. The results indicated that the proposed method is reliable for detecting SDHI fungicides in cereals.

Quantification of succinic acid levels, linked to succinate dehydrogenase (SDH) dysfunctions, by an automated and fully validated liquid chromatography tandem mass spectrometry method suitable for multi-matrix applications.

The hallmarks of cancer include metabolism with deregulating cellular energetics. Dysfunctions in succinate dehydrogenase (SDH) metabolic enzyme activity, leading to an abnormal accumulation of succinic acid has been described in solid tumors but also in inflammation and ischemia reperfusion injury. Succinic acid is a potential biomarker of SDH related pathologies for diagnostic, evaluation of treatment response and follow-up of the disease. We developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) method allowing a rapid, accurate and precise quantification of succinic acid levels in clinical (serum, urine) and preclinical (cellular pellets, supernatants) samples. 13C4 succinic acid disodium salt was used as internal standard and added to samples before a solid phase extraction (SPE) on Phenomenex STRATATM XL-A (200 mg - 3 mL) 33 µm cartridges. This method is automated by a Freedom EVO® platform from TECAN and succinic acid is separated on a C18 column combined to a Xevo® TQ-S micro Waters mass spectrometer with electrospray ionization (ESI) source. This biomedical analysis allows standard curves to be linear over the range 1.0-135.5 µM with r2 values > 0.999 and low matrix effects (<9.1 %). This method, which is validated according updated European Medicine Agency (EMA) guidelines, is accurate between-run (<11.0 %) and within-run (<7.8 %), precise between-run (<14.4 CV %) and within-run (<3.7 CV %), and is suitable for clinical and preclinical applications.

Abnormal composition of microbiota in the gut and skin of imiquimod-treated mice.

Psoriasis is a chronic, inflammatory skin disease. Although the precise etiology of psoriasis remains unclear, gut-microbiota axis might play a role in the pathogenesis of the disease. Here we investigated whether the composition of microbiota in the intestine and skin is altered in the imiquimod (IMQ)-treated mouse model of psoriasis. Topical application of IMQ to back skin caused significant changes in the composition of microbiota in the intestine and skin of IMQ-treated mice compared to control mice. The LEfSe algorithm identified the species Staphylococcus lentus as potential skin microbial marker for IMQ group. Furthermore, there were correlations for several microbes between the intestine and skin, suggesting a role of skin-gut-microbiota in IMQ-treated mice. Levels of succinic acid and lactic acid in feces from IMQ-treated mice were significantly higher than control mice. Moreover, the predictive functional analysis of the microbiota in the intestine and skin showed that IMQ caused alterations in several KEGG pathways. In conclusion, the current data indicated that topical application with IMQ to skin alters the composition of the microbiota in the gut and skin of host. It is likely that skin-gut microbiota axis plays a role in pathogenesis of psoriasis.

GHB related acids are useful in routine casework of suspected GHB intoxication cases.

GHB related acids (3,4-dihydroxy butyric acid, 2,4-dihydroxy butyric acid and glycolic acid) are produced through oxidative GHB metabolism. These analytes could be potential biomarkers to ensure the diagnosis of a GHB intoxication and even prolong the detection window. Within this study, forensic routine cases were measured to consider the potential of additional gas chromatographic mass spectrometric analysis on these acids. 17 GHB positive real cases (10 serum samples and 7 urine samples) and 40 cases with suspicion of drugging in DFC cases and negative GHB results (21 serum samples and 19 urine samples) were evaluated. Increased GHB related acid concentrations were detected in all serum and most urine samples positive on GHB. In some GHB negative cases, especially in serum samples, concentrations of GHB related acids gave hints that GHB actually was taken. We recommend to use the following cut-offs for a more reliable interpretation of potential GHB intoxication cases: 3,4-OH-BA:>3 mg/L in serum and>50 mg/L in urine; 2,4-OH-BA:>2 mg/L in serum and>25 mg/L in urine; GA:>5 mg/L in serum and>400 mg/L in urine.

Effect of Different Drying Methods on the Nutritional Value of Hibiscus sabdariffa Calyces as Revealed by NMR Metabolomics.

Red mature calyces of Hibiscus sabdariffa were collected from 16 different locations in Meghalaya, India. Samples were processed using shade drying (SD) and tray drying (TD). NMR spectroscopy was used to assess the metabolic composition of the calyces. In this study, 18 polar metabolites were assigned using 1D and 2D NMR spectra, and 10 of them were quantified. Proximate analysis showed that the TD method is more efficient at reducing moisture and maintaining the ash content of the Hibiscus biomass. NMR metabolomics indicates that the metabolite composition significantly differs between SD and TD samples and is more stable in TD plant processing. The differences in post-harvest drying has a greater impact on the metabolite composition of Hibiscus than the plant location.

Electrophysiological Activity and Survival Rate of Rats Nervous Tissue Cells Depends on D/H Isotopic Composition of Medium.

The deuterium content modification in an organism has a neuroprotective effect during the hypoxia model, affecting anxiety, memory and stress resistance. The aim of this work was to elucidate the possible mechanisms of the medium D/H composition modification on nerve cells. We studied the effect of an incubation medium with a 50 ppm deuterium content compared to a medium with 150 ppm on: (1) the activity of Wistar rats' hippocampus CA1 field neurons, (2) the level of cultured cerebellar neuron death during glucose deprivation and temperature stress, (3) mitochondrial membrane potential (MMP) and the generation of reactive oxygen species in cultures of cerebellar neurons. The results of the analysis showed that the incubation of hippocampal sections in a medium with a 50 ppm deuterium reduced the amplitude of the pop-spike. The restoration of neuron activity was observed when sections were returned to the incubation medium with a 150 ppm deuterium content. An environment with a 50 ppm deuterium did not significantly affect the level of reactive oxygen species in neuron cultures, while MMP decreased by 16-20%. In experiments with glucose deprivation and temperature stress, the medium with 50 ppm increased the death of neurons. Thus, a short exposure of nerve cells in the medium with 50 ppm deuterium acts as an additional stressful factor, which is possibly associated with the violation of the cell energy balance. The decrease in the mitochondrial membrane potential, which is known to be associated with ATP synthesis, indicates that this effect may be associated with the cell energy imbalance. The decrease in the activity of the CA1 field hippocampal neurons may reflect reversible adaptive changes in the operation of fast-reacting ion channels.

Faecal microbial metabolites of proteolytic and saccharolytic fermentation in relation to degree of insulin resistance in adult individuals.

The gut microbiota may affect host metabolic health through microbial metabolites. The balance between the production of microbial metabolites by saccharolytic and proteolytic fermentation may be an important determinant of metabolic health. Amongst the best-studied saccharolytic microbial metabolites are the short-chain fatty acids acetate, propionate and butyrate. However, human data on the role of other microbial fermentation by-products in metabolic health are greatly lacking. Therefore, we compared in a cross-sectional study the faecal microbial metabolites (caproate, lactate, valerate, succinate, and the branched-chain fatty acids (BCFA) (isobutyrate, isovalerate)) between insulin sensitive (homeostatic model assessment of insulin resistance (HOMA-IR), HOMA-IR<1.85, IS) and insulin resistant (HOMA-IR>1.85, IR) individuals. Additionally, we assessed the relationships between faecal metabolites and markers of metabolic health including fasting glucose, insulin, free fatty acids, insulin resistance (HOMA-IR) and fasting substrate oxidation in 86 individuals with a wide range of body mass index. Faecal metabolite concentrations did not significantly differ between IS and IR. Furthermore, there were no associations between microbial metabolites and metabolic health markers, except for a slight positive association of isovalerate with carbohydrate oxidation (E%, std ß 0.194, P=0.011) and fat oxidation (E%, std ß -0.075, P=0.047), also after adjustment for age, sex and BMI. In summary, faecal caproate, lactate, valerate, succinate, and BCFA (isobutyrate, isovalerate) were not different between IR and IS individuals, nor was there any association between these faecal metabolites and parameters of metabolic health. Further human intervention studies are warranted to investigate the role of these microbially-derived fermentation products and their kinetics in metabolic health and insulin sensitivity.

A rapid liquid chromatography-electrospray ionization-ion mobility spectrometry method for monitoring nine representative metabolites in the seedlings of cucumber and wheat.

A fast and convenient high-performance liquid chromatography-electrospray ionization-ion mobility spectrometry method was developed to determine nine representative metabolites in the seedlings of cucumber and wheat. The analytical conditions were obtained by optimizing the parameters of high-performance liquid chromatography and ion mobility spectrometry. Briefly, acetonitrile-0.1% formic acid solution was selected as the mobile phase for gradient elution at a flow velocity of 0.4 mL/min. Under negative electrospray ionization mode, spray voltage of ion mobility spectrometry was 4.5 kV, and drift tube temperature was set at 90°C. The metabolites from seedling leaves were extracted using 80% acetonitrile as the solvent at 4°C for 12 h. Results showed that under soilless culture conditions, the contents of maltose, citric acid, and p-hydroxybenzoic acid in the seedlings of cucumber and wheat were reduced by low concentration of itaconic acid, succinic acid, and citric acid. Importantly, this analytical approach demonstrated high sensitivity, good linear response, and high selectivity. The lowest limit of detection was 0.004 µg for p-hydroxybenzoic acid. Overall, this high-performance liquid chromatography-electrospray ionization-ion mobility spectrometry method is sensitive and efficient for rapid separation and identification of plant metabolites.

Carbon isotope ratio of organic acids in sake and wine by solid-phase extraction combined with LC/IRMS.

We developed an analytical procedure for determining the δ13C values of organic acids in sake and wine using solid-phase extraction combined with liquid chromatography/isotope ratio mass spectrometry (LC/IRMS). First, the solid-phase extraction (SPE) procedure was performed and various tests were conducted to extract organic acids from alcoholic beverages using the simulated sake sample. Under the optimal SPE procedure, high recovery rates (96-118%) and good accuracies (≤ 0.7‰) were thus achieved for the simulated sake and wine samples. Next, we determined the δ13C of organic acid (tartaric acid, malic acid, lactic acid, succinic acid) in 9 sake and 11 wine samples. Finally, the δ13C values of lactic acid in nine sake samples suggested that lactic acid had been added during the brewing process. The high correlation between the δ13C values of tartaric acid and malic acid in 11 wine samples was consistent with their common source, grapes. This analytical method may help to identify when organic acids have been added to sake and wine and to elucidate the process of organic acid production therein. Graphical abstract.

GHB related acids (dihydroxy butyric acids, glycolic acid) can help in the interpretation of post mortem GHB results.

Post mortem gamma hydroxy butyric acid (GHB) concentrations should be interpreted with caution since GHB concentrations can increase after death. Post mortem concentrations after the intake of GHB ante mortem do overlap with concentration ranges in cases without known exposure to GHB and make an interpretation challenging. GHB is known to undergo intensive metabolism to related acids (glycolic acid (GA), succinic acid (SA), 2,4- and 3,4-dihydroxy butyric acid (2,4-OH-BA and 3,4-OH-BA)). GHB and these related acids were analyzed using a validated gas chromatographic mass spectrometric (GC-MS) method after liquid liquid extraction and trimethylsilylation. SA concentrations were not usable post mortem due to instability. Concentrations in cases without known exposure to GHB (urine: n = 80; femoral blood: n = 103) were: for GA 4.6-121 mg/L in urine and 1.6-11.2 mg/L in blood, for 2,4-OH-BA < LoD-25,3 mg/L in urine and < LoD-3.7 mg/L in blood and for 3,4-OH-BA < LoD-54,3 mg/L in urine and < LoD-5.3 mg/L in blood. In death cases involving GHB (n = 11) concentrations of GHB related acids were increased compared to these levels (for GA in 7/10 cases and up to 391 mg/L in urine, in 6/11 cases and up to 34 mg/L in blood; for 2,4-OH-BA in 9/10 cases and up to 144 mg/L in urine, in 11/11 cases and up to 9.1 mg/L in blood; for 3,4-OH-BA in 7/10 cases and up to 665 mg/L in urine, in 11/11 cases and up to 19 mg/L in blood). Therefore, the concentrations of these GHB related acids can aid in a more reliable differentiation of GHB exposure in post mortem toxicology. We recommend to add the analysis of 2,4-OH-BA, 3,4-OH-BA and GA in femoral blood for the diagnosis of a GHB intake post mortem. Post mortem femoral blood concentrations > 4 mg/L for 2,4-OH-BA, > 5 mg/L for 3,4-OH-BA and > 12 mg/L for GA give hints for a GHB intake.

Quantitative analyses of the umami characteristics of disodium succinate in aqueous solution.

Disodium succinate (WSA) contributes to umami taste in seafoods and it is abundantly found in scallops. However, the actual application of WSA in foods is limited due to a lack of understanding of its taste characteristics and stability. In this study, two-alternative forced choice method was used to determine the relative umami intensity of WSA compared to monosodium glutamate, as well as the relative umami intensity under different conditions (pH and temperature). WSA concentration-taste intensity curve was established, which fitted well with a logarithmic-linear regression (R2 = 0.96). WSA exhibited the strongest umami intensity at 25 °C, 0.1% Na+ addition, and pH 7.5. It also had a good thermal stability, which met the needs of high temperature heating during food processing. In conclusion, this research provided useful information on umami characteristics of WSA and the results widen the application of WSA in the food industry.

The Use of a Biostimulant Composition to Increase Turkey Viability during the Key Critical Periods of Development.

The paper reports that the treatment of hatching turkey eggs with a mixture composed of colamine, succinic acid, serine, and pyridoxine hydrochloride increased the viability of embryos and reduced incubation wastes. This effect allowed increasing the hatching of turkey poults by 6.73% and the hatchability of eggs, by 4.43%. At the same time, a statistically significant decrease in the key lipid peroxidation products in one-day-old turkey poults was observed. In particular, the content of isolated double bonds decreased 1.47-fold (p < 0.01); diene conjugates, 1.67-fold (p < 0.01); triene conjugates, 1.46-fold (p < 0.05); oxidiene conjugates, 1.48-fold (p < 0.01); and Schiff bases, 1.3-fold compared to the control. All the above-mentioned positively affected survivability in the experimental group, which appeared to be increased by 1% compared to the control.

HPLC and NMR quantification of bioactive compounds in flowers and leaves of Brassica rapa: the influence of aging.

Several members of the Brassicaeae family are known to possess beneficial properties which positively impact human diet, thanks to the presence of antioxidants, bioactive polyphenols and amino acids. B. rapa, one of the most widespread and economically relevant species, represents an outstanding example. The aim of this study is to investigate, at the molecular level, the effect of plant aging on the concentration of some biologically relevant compounds in different parts of the plant. Using HPLC and NMR techniques, the quantification of polyphenolic species (caffeic acid, quercetin and rutin), succinic acid and alanine was performed in flowers and leaves of young and mature B. rapa plants.

Improved succinate production from galactose-rich feedstocks by engineered Escherichia coli under anaerobic conditions.

It is of great economic interest to produce succinate from low-grade carbon sources, which can make it more economically competitive against petrochemical-based succinate. Galactose sugars constitute a significant fraction of the soluble carbohydrate in a meal from agricultural sources which is considered a low value or waste byproduct of oilseed processing. To improve the galactose utilization, the effect of galR and glk on sugars uptake was investigated by deactivation of each gene in three previously engineered host strains. As expected, glk plays an important role in glucose uptake, while, the effect of deactivation of galR is highly dependent on the strength of the downstream module (succinate production module). A new succinate producer FZ661T was constructed by enhancement of the succinate producing module and manipulation of the gal operon. The succinate productivity reached 4.57 g/L/hr when a mixed sugar feedstock was used as a carbon source in shake-flask fermentation, up to 812 mM succinate was accumulated in 80 hr in fed-batch fermentation. When SoyMolaGal hydrolysate was used as a carbon source, 628 mM (74 g/L) succinate was produced within 72 hr. In this study, we demonstrate that FZ661T can produce succinate quickly with relatively high yield, giving it the potential for industrial application.

Succinylome Analysis Reveals the Involvement of Lysine Succinylation in the Extreme Resistance of Deinococcus radiodurans.

Increasing evidence shows that the succinylation of lysine residues mainly regulates enzymes involved in the carbon metabolism pathway, in both prokaryotic and eukaryotic cells. Deinococcus radiodurans is one of the most radioresistant organisms on earth and is famous for its robust resistance. A major goal in the current study of protein succinylation is to explore its function in D. radiodurans. High-resolution LC-MS/MS is used for qualitative proteomics to perform a global succinylation analysis of D. radiodurans and 492 succinylation sites in 270 proteins are identified. These proteins are involved in a variety of biological processes and pathways. It is found that the enzymes involved in nucleic acid binding/processing are enriched in D. radiodurans compared with their previously reported levels in other bacteria. The mutagenesis studies confirm that succinylation regulates the enzymatic activities of species-specific proteins PprI and DdrB, which belong to the radiation-desiccation response regulon. Together, these results provide insight into the role of lysine succinylation in the extreme resistance of D. radiodurans.

Comprehensive analysis of the lysine acetylome in Aeromonas hydrophila reveals cross-talk between lysine acetylation and succinylation in LuxS.

Lysine acetylation and succinylation are both prevalent protein post-translational modifications (PTMs) in bacteria species, whereas the effect of the cross-talk between both PTMs on bacterial biological function remains largely unknown. Our previously study found lysine succinylated sites on proteins play important role on metabolic pathways in fish pathogenic Aeromonas hydrophila. A total of 3189 lysine-acetylation sites were further identified on 1013 proteins of this pathogen using LC-MS/MS in this study. Functional examination of these PTMs peptides showed associations with basal biological processes, especially metabolic pathways. Additionally, when comparing the obtained lysine acetylome to a previously obtained lysine succinylome, 1198 sites in a total of 547 proteins were found to be in common and associated with various metabolic pathways. As the autoinducer-2 (AI-2) synthase involved in quorum sensing of bacteria, the site-directed mutagenesis of LuxS at the K165 site was performed and revealed that the cross-talk between lysine acetylation and succinylation exerts an inverse influence on bacterial quorum sensing and on LuxS enzymatic activity. In summary, this study provides an in-depth A. hydrophila lysine acetylome profile and for the first time reveals the role of cross-talk between lysine acetylation and succinylation, and its potential impact on bacterial physiological functions.

Baltic amber teething necklaces: could succinic acid leaching from beads provide anti-inflammatory effects?

BACKGROUND: Baltic amber teething necklaces have been popularized as a safe and natural alternative to conventional or pharmacological medicines for the management of teething pain. However, claims made by retailers regarding the efficacy and mechanism of action of these necklaces lack scientific or clinical basis. The claim most closely resembling science is the assertion that succinic acid will leach out of the beads and through the skin of the wearer and carry out anti-inflammatory and analgesic effects. The objective of the current research is to scientifically assess this claim. METHODS: Beads from necklaces were powdered for identification by infrared spectroscopy, and dissolved in sulfuric acid for quantification of succinic acid using HPLC. Succinic acid release from beads was assessed by long-term submersion of amber beads (separated according to light, medium and dark brown colour) in solvents relevant to human skin conditions. The potential for succinic acid to have anti-inflammatory effects was assessed by measuring the release of inflammatory cytokines IL-1α, IL-1ß, IL-8 and TNFα, and the inflammatory messenger PGE2, from THP-1 human macrophages after treatment with succinic acid and LPS. RESULTS: Amber teething necklaces were positively identified as Baltic amber, by comparison of the beads' infrared spectrum to the literature, and by their succinic acid content (1.5 mg per bead; 1.44% w/w). However, whole amber beads submerged in octanol or pH 5.5 phosphate buffered saline did not release any measurable succinic acid, except for the light-coloured beads in octanol which broke into tiny fragments. Additionally, treatment of macrophages with succinic acid did not reduce the release of any inflammatory cytokines measured, and displayed toxicity to the cells at high concentrations. CONCLUSIONS: While amber teething necklaces are genuine Baltic amber, we have found no evidence to suggest that the purported active ingredient succinic acid could be released from the beads into human skin. Additionally, we found no evidence to suggest that succinic acid has anti-inflammatory properties.

Enhancing wine ester biosynthesis in mixed Hanseniaspora uvarum/Saccharomyces cerevisiae fermentation by nitrogen nutrient addition.

The dynamic changes of wine ester production during mixed fermentation with Hanseniaspora uvarum Yun268 and Saccharomyces cerevisiae F5 was investigated at different levels and timings of nitrogen nutrient addition. Nitrogen additions were performed by supplementing yeast assimilable nitrogen (YAN) into a synthetic grape must with defined composition. Ester precursors and extracellular metabolites involved in ester synthesis were analyzed throughout the fermentation. Results showed that nitrogen additions covering 50-200 mg/L YAN at the point of yeast inoculation slightly affected yeast competition and ester profiles. Interestingly, when YAN was supplemented in the mid-stage, the survival of H. uvarum Yun268 was enhanced, resulting in more than a 2-fold increase in the levels of higher alcohol acetates compared to that at the initial stage. Furthermore, carbon fluxes may be redistributed in the central pathway, which contributed to the production of medium-chain fatty acids and eventually triggered a 1.2-fold elevation in corresponding ethyl ester levels.