Dynamic variation in the aroma characteristics of Rhus chinensis honey at different stages after capping.

The ripening characteristics after capping of honey are favourable for improving its quality. However, research on the variation and formation of aroma characteristics of honey in this process is lacking. Therefore, the present study was carried out with different stages of Rhus chinensis honeys (RCHs) after capping and identified 192 volatiles with varying levels of concentration. "Fruity" was the main aroma characteristic of RCHs at different stages after capping, mainly contributed by (E)-ß-damascenone. Methyl salicylate might be a potential indicator for differentiating RCHs at different stages after capping. The metabolic pathway analyses revealed that the aroma compounds in RCHs undergo transformation at different stages after capping, which subsequently affects its aroma characteristics formation. This work is the first to study the dynamic changes in honey aroma characteristics after capping from multiple perspectives, and the results are of great significance for understanding the aroma characteristics after capping and quality control of honey.

Characterization of Evodia rutaecarpa (Juss) Benth honey: volatile profile, odor-active compounds and odor properties.

BACKGROUND: Aroma is one of the most important quality criterion of different honeys and even defines their merchant value. The composition of volatile compounds, especially the characteristic odor-active compounds, contributes significantly to the aroma of honey. Evodia rutaecarpa (Juss) Benth honey (ERBH) is a special honey in China with unique flavor characteristics. However, no work in the literature has investigated the volatile compounds and characteristic odor-active compounds of ERBHs. Therefore, it is imperative to conduct systematic investigation into the volatile profile, odor-active compounds and odor properties of ERBHs. RESULTS: The characteristic fingerprint of ERBHs was successfully constructed with 12 characteristic peaks and a similarity range of 0.785-0.975. In total, 297 volatile compounds were identified and relatively quantified by headspace solid-phase microextraction coupled with gas chromatography quadrupole time-of-flight mass spectrometry, of which 61 and 31 were identified as odor-active compounds by relative odor activity values and GC-olfactometry analysis, respectively, especially the common 22 odor-active compounds (E)-ß-damascenone, phenethyl acetate, linalool, cis-linalool oxide (furanoid), octanal, hotrienol, trans-linalool oxide (furanoid), 4-oxoisophorone and eugenol, etc., contributed significantly to the aroma of ERBHs. The primary odor properties of ERBHs were floral, followed by fruity, herbaceous and woody aromas. The partial least-squares regression results showed that the odor-active compounds had good correlations with the odor properties. CONCLUSION: Identifying the aroma differences of different honeys is of great importance. The present study provides a reliable theoretical basis for the quality and authenticity of ERBHs. © 2023 Society of Chemical Industry.

Aromatic profiles and enantiomeric distributions of volatile compounds during the ripening of Dendropanax dentiger honey.

Dendropanax dentiger honey (DDH) is a specialty herbal honey from China. Previous research on DDH has mostly focused on its composition and potential chemical markers, no studies have been conducted on the changes in aroma characteristics and chiral odorants during its maturation. Therefore, the present study aims to address the missing parts. The proportions and total concentrations of 185 volatile compounds identified in different classes varied with DDHs ripening. Fourteen common odor-active compounds were identified by odor activity values (OAVs) and GC-olfactometry (GC-O) analysis. The aroma profiles of DDHs were observed to vary at different ripening stages, although the dominant aroma characteristic was "fruity" aroma, which became more pronounced with increasing maturity. The enantiomeric contents and distributions of 7 volatile enantiomers were related to specific physicochemical indicators and the maturity of DDHs, among which the enantiomers of linalool oxide A may be a potential indicator to identify its maturity. Furthermore, precise quantification and OAVs calculation showed that the enantiomer (2S, 5S)-linalool oxide A presented the highest concentration (8.83-27.39 ng/mL) and only the enantiomer R-linalool (OAVs: 5.56-6.14) was an important contributor to the aroma profiles of DDHs at different stages of maturity. These results provided a new research idea for quality control and identification of DDHs at different maturity stages.

Effects of different floral periods and environmental factors on royal jelly identification by stable isotopes and machine learning analyses during non-migratory beekeeping.

It is crucial to monitor the authenticity of royal jelly (RJ) because the qualities of RJs produced by different floral periods vary substantially. In the context of non-migratory beekeeping, this study aims to identify rape RJ (RRJ), chaste RJ (CRJ), and sesame RJ (SRJ) based on δ13C, δ15N, δ2H, and δ18O combined with machine learning and to evaluate environmental effect factors. The results showed that δ13C (-27.62‰ ± 0.24‰), δ15N (2.88‰ ± 0.85‰), and δ18O (28.02‰ ± 1.30‰) of RRJ were significantly different from other RJs. The δ13C, δ2H, and δ18O in CRJ and SRJ were strongly correlated with temperature and precipitation, suggesting that these isotopes are influenced by environmental elements such as sunlight and rainfall. In addition, the artificial neural network (ANN) model was superior to the random forest (RF) model in terms of accuracy, sensitivity, and specificity. This study revealed that combining stable isotopes with ANN models and the unique correlation between stable isotopes and environmental factors could provide promising ideas for monitoring the authenticity of RJ.

C/H/O stable isotope of rape honey and its components combined with machine learning algorithms to identify geographical origin.

RATIONALE: The distribution of rape honey is among the largest and most diverse of all honeys available to humankind with respect to the geographical origin. Accurate isotopic reference values for rape honey are therefore important for precise verification of honey origin and its traceability. New combined rape honey δ13 C, δ2 H, and δ18 O values in combination with values on its compounds (protein and saccharides) were used to complement existing databases to better identify the geographical origin of Chinese rape honey. METHODS: Traceability methods based on elemental analyzer isotope ratio mass spectrometry and liquid chromatography isotope ratio mass spectrometry were established for geographical origin of rape honey. RESULTS: Rape honey harvested in the high-altitude region (QH; Qinghai) had significantly higher values (1.4 to 5.3‰ for δ13 C, 7.9 to 12.9‰ for δ2 Hprotein ) for the δ13 C of whole honey (-23.8‰), its protein (-24.4‰), fructose (-23.5‰), glucose (-23.6‰), and disaccharide (-24.7‰), and also δ2 H of the protein (103.5‰) than those in low-altitude regions (HB; Hubei, SC; Sicuan, and JS; Jingsu). The δ18 Orape honey was a useful index to differentiate whether rape honey from coastal (JS) or non-coastal (HB, SC, and QH) regions. The δ13 C, δ2 H, and δ18 O values in rape honey are affected by geographical factors, such as temperature and altitude. The δ13 Cprotein and δ13 Crape honey values were better to identify the geographical origin of rape honey than δ13 Csaccharides . The δ18 O and δ2 H values of rape honey protein were more suitable for traceability than those of rape honey. The combination of the δ13 C, δ2 H, and δ18 O values of rape honey and its extracted protein and saccharides improved the precision of three models (linear discriminant analysis, SVM, and random forest) used to discriminate rape honey from different regions in China. The SVM model obtained the best accuracy (93.2%). CONCLUSIONS: Stable isotopes could be significant predictors in determining the geographical origin of rape honey.

Study on stable carbon isotope fractionation of rape honey from rape flowers (Brassica napus L.) to its unifloral ripe honey.

A new idea and strategy for honey traceability and identification was provided by studying the carbon isotope fractionation of rape honey and its components in the different ripening process, as well as the fractionation from rape flowers, stamens, nectar to rape honey. The results showed the moisture content of rape honey continued to decrease, and the glucose and fructose content continued to increase during the ripening process. The δ13C of rape honey and its protein were less affected by honey ripeness, while the δ13C of sugars in rape honey were greatly affected by this. At the same time, the fractionation of carbon isotope from rape flowers to honey was significant. The δ13C of rape honey and its protein, disaccharide, fructose, and glucose had a strong correlation, and the δ13C of rape honey and its components were mainly related to rape flowers and its stamens.

Analytical Strategies for LC-MS-Based Untargeted and Targeted Metabolomics Approaches Reveal the Entomological Origins of Honey.

A comprehensive liquid chromatography-mass spectrometry (LC-MS)-based metabolomics approach was developed to discriminate honey harvested from Apis mellifera ligustica Spinola (A. mellifera) and Apis cerana cerana Fabricius (A. cerana). Based on an untargeted strategy, ultrahigh-performance liquid chromatography electrospray ionization quadrupole orbitrap high-resolution mass spectrometry (UPLC Q-Orbitrap) was combined with chemometrics techniques to screen and identify tentative markers from A. mellifera and A. cerana honey. In targeted metabolomics analysis, a sensitive method of solid-phase extraction followed by ultrahigh-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UPLC-MS/MS) was established for quantifying three markers, and the results showed that 3-amino-2-naphthoic acid and methyl indole-3-acetate could be considered markers of A. cerana honey, as they were present in higher amounts in A. cerana honey than in A. mellifera honey, whereas kynurenic acid was determined to be a marker of A. mellifera honey. This work highlights critical information for the authentication of A. cerana and A. mellifera honey.

The landscape of COVID-19 vaccination among healthcare workers at the first round of COVID-19 vaccination in China: willingness, acceptance and self-reported adverse effects.

The COVID-19 vaccines have been developed in a wide range of countries. This study aimed to examine factors that related to vaccination rates and willingness to be vaccinated against COVID-19 among Chinese healthcare workers (HCWs). From 3rd February to 18th February, 2021, an online cross-sectional survey was conducted among HCWs to investigate factors associated with the acceptance and willingness of COVID-19 vaccination. Sociodemographic characteristics and the acceptance of COVID-19 vaccination among Chinese HCWs were evaluated. A total of 2156 HCWs from 21 provinces in China responded to this survey (effective rate: 98.99%)), among whom 1433 (66.5%) were vaccinated with at least one dose. Higher vaccination rates were associated with older age, working as a clinician, having no personal religion, working in a fever clinic or higher hospital grade, and having received vaccine education, family history for influenza vaccination and strong familiarity with the vaccine. Willingness for vaccination was related to working in midwestern China, considerable knowledge of the vaccine, received vaccine education, and strong confidence in the vaccine. Results of this study can provide evidence for the government to improve vaccine coverage by addressing vaccine hesitancy in the COVID-19 pandemic and future public health emergencies.

Occurrence of Neonicotinoids in Chinese Apiculture and a Corresponding Risk Exposure Assessment.

Neonicotinoids are the most widely used insecticides worldwide, but there is mounting evidence demonstrating that they have adverse effects on nontarget organisms. However, little is known about the extent of environmental neonicotinoids contamination in China. In this study, a total of 693 honey samples from across China, from both Apis melifera and Apis cerana, were analyzed to examine neonicotinoid concentrations and their geographical distribution, and correlation with the primary plant species from which the honey was obtained. Furthermore, chronic and acute exposure risk and risk ranking for humans eating honey were investigated, and risks to bees were also considered. The results revealed that 40.8% of the samples contained at least one of the five neonicotinoids tested. Honeys from commercial crops were found to be more frequently contaminated with neonicotinoids than those from noncommercial crops. Honey samples from Apis mellifera were more frequently contaminated than those from Apis cerana. The concentrations of neonicotinoids found in honey overlapped with those that have been found to have significant adverse effects on honeybee health. The dietary risk assessments indicated that the levels of neonicotinoids detected in honey were likely to be safe for human consumption.

GABA negatively regulates adventitious root development in poplar.

γ-Aminobutyric acid (GABA) influences plant growth, but little is known about how this metabolite regulates adventitious root (AR) development. Here, we investigate the effects of GABA on ARs using poplar lines overexpressing glutamate decarboxilase 2 (GAD2) and by treating poplar stem cuttings with exogenous GABA or vigabatrin (VGB; a specific GABA transaminase inhibitor). Endogenous GABA accumulation not only inhibited AR growth, but it also suppressed or delayed AR formation. Anatomical observations revealed that the GABA and VGB treatments resulted in a 1 d delay in the formation of AR primordia and the appearance of ARs. This delay coincided with changes in primary metabolism, including transient increases in hexose and amino acid levels. GABA-dependent changes in the expression of genes related to hormone synthesis and signalling, as well as analysis of hormone levels revealed that ethylene-dependent pathways were decreased at the earliest stage of AR formation. In contrast, auxin and abscisic acid were increased at 1-5 d as well as GA4 over a 5 d period of AR formation. These results demonstrate that GABA plays a crucial role in AR development. Evidence is presented demonstrating that GABA can interact with hormone-related pathways as well as carbon/nitrogen metabolism. These findings also elucidate the functions of GABA in plant development.

Untargeted and Targeted Discrimination of Honey Collected by Apis cerana and Apis mellifera Based on Volatiles Using HS-GC-IMS and HS-SPME-GC-MS.

Fraudulent acts regarding honey authenticity that use Apis mellifera honey as a substitute for Apis cerana honey have garnered considerable concern in China and triggered a trust crisis from consumers. In this study, untargeted metabolomics analysis was carried out based on volatile fractions in honey from A. cerana and A. mellifera using headspace gas chromatography-ion mobility spectrometry (HS-GC-IMS). Honey from A. cerana and A. mellifera was discriminated by HS-GC-IMS profiling, principal component analysis, and orthogonal partial least-squares discrimination analysis. Tentative markers were identified from p-values and the variable importance in projection analysis and confirmed using the retention index, mass fragments, and reference standards by gas chromatography-mass spectrometry (GC-MS). A targeted method was established using the headspace solid phase coupled with microextraction GC-MS (HS-SPME-GC-MS) to quantitate the markers. The results demonstrated that the developed untargeted and targeted metabolomics approach performed well when discriminating honey from A. cerana and A. mellifera.

γ-Aminobutyric acid is closely associated with accumulation of flavonoids.

γ-Aminobutyric acid (GABA) is an important neurotransmitter in mammals whose receptor is reported to be regulated by flavonoids. In plants, it is considered to be at the intersection of carbon and nitrogen metabolism, but its relationship with flavonoid metabolism remains unclear. Our recent RNA-seq analysis showed that expression of flavonoid biosynthetic genes was influenced in poplar by the blockage of α-ketoglutarate dehydrogenase (α-KGDH) activity and the application of GABA under NaCl stress, accompanied by the changes in GABA shunt activity. Here, we further found that the flavonoid accumulation was significantly affected by blocking the activities of α-KGDH and GABA transaminase as well as applying exogenous GABA, coupled with the changes of endogenous GABA contents. Key genes involved in the flavonoid biosynthetic pathway were also significantly influenced, including two PALs, 4CL, and two CHSs. Our results suggest that the GABA shunt is closely associated with the metabolism of flavonoids, which would benefit future understanding of GABA's roles in carbon allocation by regulating the pathway of flavonoid biosynthesis under normal or stress conditions.

A green triple-locked strategy based on volatile-compound imaging, chemometrics, and markers to discriminate winter honey and sapium honey using headspace gas chromatography-ion mobility spectrometry.

A simple and environmentally approach using untargeted imaging of volatile substances combined with chemometrics and markers response was proposed for discriminating different species of honey with headspace gas-chromatography-ion-mobility (HS-GC-IMS). The 3D HS-GC-IMS imaging and their response differences enabled the clear discrimination between winter honey and sapium honey. Principal component analysis (PCA) and partial least-squares discrimination analysis (PLS-DA) were employed to discriminate different honey. Markers of two kinds of honey were identified and confirmed with a user-built imaging database combined with multivariate analysis. Benzaldehyde dimer and phenylacetaldehyde dimer were found to be reliable markers of winter honey, and phenylethyl acetate dimer was of sapium honey. Adulteration identification of the honey samples with different adulteration ratios were subjected to this triple-locked strategy analysis. The results demonstrate that HS-GC-IMS imaging coupled with chemometrics and marker identification is a useful triple-locked strategy to discriminate honey from different floral origins and adulterated honey.

Discrimination of geographical origins of Chinese acacia honey using complex 13C/12C, oligosaccharides and polyphenols.

The aim of this study was to predict the geographical origin of acacia honey of China through analysis of physicochemical parameters combination with chemometrics. Samples from six different origins were investigated on parameters of δ13C value, oligosaccharides and polyphenols, using EA-IRMS/LC-IRMS, GC-MS and HPLC-MS, respectively. The results indicated that the δ13C value of honey from Gansu region were lower than those of other regions. Oligosaccharides of honey from Shanxi and Shaanxi regions were both higher than other four regions. Polyphenols of honey from Shandong region was the highest and were better parameters than both δ13C and oligosaccharides in discrimination of geographical origins. Partial Least Square Discriminant Analysis (PLS-DA) showed that when all 31 different parameters were combined, a correct classification rate of 94.12% could be achieved using external cross validation method. In conclusion, the method in discrimination of geographical can be used to provide reliable and useful reference information.

Inhibition of α-ketoglutarate dehydrogenase activity affects adventitious root growth in poplar via changes in GABA shunt.

MAIN CONCLUSION: Blocking α-ketoglutarate dehydrogenase results in up-regulation of γ-aminobutyric acid (GABA) shunt activity, and inhibits the growth of poplar adventitious roots (ARs), indicating that AR growth is closely associated with GABA shunt. γ-Aminobutyric acid (GABA) shunt starts from α-ketoglutarate in the tricarboxylic acid cycle, which is thought to represent the cross road between carbon and nitrogen metabolism. Previous studies (Araújo et al. 2012b, Plant Cell 24: 2328-2351) have shown that blocking α-ketoglutarate dehydrogenase (α-KGDH) affects the GABA shunt activity, and inhibits growth. However, its effects on the growth of adventitious roots (ARs) are unclear. In this study, the growth of ARs in tissue-cultured 84K poplar (Populus alba × Populus glandulosa cv. '84K') was significantly inhibited when succinyl phosphate (SP), a specific inhibitor of α-KGDH, was supplied. The inhibition of ARs was associated with significant changes in the levels of soluble sugars, organic acids, and amino acids, and was coupled with the up-regulation of the GABA shunt activity at the transcriptional and translational levels. Exogenous GABA also inhibited AR growth following the increase of the endogenous GABA level. Transcriptomic analyses further showed that genes related to cell wall carbon metabolism and phytohormone (indoleacetic acid, ABA, and ethylene) signaling were affected by the changes of GABA shunt activity, resulting from the α-KGDH inhibition. Thus, our study indicates that the inhibition of poplar AR growth by blocking α-KGDH is closely associated with GABA shunt, which would benefit a better understanding of GABA's roles in plant development and stress response.

Roles of γ-aminobutyric acid on salinity-responsive genes at transcriptomic level in poplar: involving in abscisic acid and ethylene-signalling pathways.

MAIN CONCLUSION: γ-Aminobutyric acid (GABA) affected ABA and ethylene metabolic genes and signal components in salt-treated poplar, indicating its potential role in signal pathways of ABA and ethylene during salt stress. GABA is a small signalling molecule that accumulates rapidly in plants exposed to various stresses. However, the relationship between GABA and other signalling molecules, such as hormones, remains unclear. Here, in the poplar woody plant under 200-mM NaCl conditions, the application of low (0.25 mM) and high (10 mM) exogenous GABA, compared to 0 mM, affected the accumulation of hydrogen peroxide and hormones, including ABA and ethylene, in different manners. Transcriptomic analysis demonstrated that 1025 differentially expressed genes (DEGs; |log2Ratio| ≥ 1.5) were widely affected by exogenous GABA under salt stress. A clustering analysis revealed that GABA could rescue or promote the effects of salt stress on gene expression. Among them, 146 genes involved in six hormone-signalling pathways were enriched, including 22 ABA- and 50 ethylene-related genes. Quantitative expression of selected genes involved in hormone-related pathways showed that ABA metabolic genes (ABAG, ABAH2, and ABAH4), ethylene biosynthetic genes (ACO1, ACO2, ACO5, ACOH1, ACS1, and ACS7) and receptor genes (PYL1, PYL2, PYL4, and PYL6) were regulated by exogenous GABA, even at a 0.1 mM level. The production of ABA was negatively correlated with ABAH expression levels at different GABA concentrations. The increase of endogenous GABA, resulting from inhibitor (succinyl phosphonate) of α-ketoglutarate dehydrogenase, affected the PYLs levels. Thus, GABA may be involved in ABA- and ethylene-signalling pathways. Our data provide a better understanding of GABA's roles in the plant responses to environmental stresses.

Proteome-Level Analysis of Metabolism- and Stress-Related Proteins during Seed Dormancy and Germination in Gnetum parvifolium.

Gnetum parvifolium is a rich source of materials for traditional medicines, food, and oil, but little is known about the mechanism underlying its seed dormancy and germination. In this study, we analyzed the proteome-level changes in its seeds during germination using isobaric tags for relative and absolute quantitation. In total, 1,040 differentially expressed proteins were identified, and cluster analysis revealed the distinct time points during which signal transduction and oxidation-reduction activity changed. Gene Ontology analysis showed that "carbohydrate metabolic process" and "response to oxidative stress" were the main enriched terms. Proteins associated with starch degradation and antioxidant enzymes were important for dormancy-release, while proteins associated with energy metabolism and protein synthesis were up-regulated during germination. Moreover, protein-interaction networks were mainly associated with heat-shock proteins. Furthermore, in accord with changes in the energy metabolism- and antioxidant-related proteins, indole-3-acetic acid, Peroxidase, and soluble sugar content increased, and the starch content decreased in almost all six stages of dormancy and germination analyzed (S1-S6). The activity of superoxide dismutase, abscisic acid, and malondialdehyde content increased in the dormancy stages (S1-S3) and then decreased in the germination stages (S4-S6). Our results provide new insights into G. parvifolium seed dormancy and germination at the proteome and physiological levels, with implications for improving seed propagation.

Identification of two CiGADs from Caragana intermedia and their transcriptional responses to abiotic stresses and exogenous abscisic acid.

BACKGROUND: Glutamate decarboxylase (GAD), as a key enzyme in the γ -aminobutyric acid (GABA) shunt, catalyzes the decarboxylation of L-glutamate to form GABA. This pathway has attracted much interest because of its roles in carbon and nitrogen metabolism, stress responses, and signaling in higher plants. The aim of this study was to isolate and characterize genes encoding GADs from Caragana intermedia, an important nitrogen-fixing leguminous shrub. METHODS: Two full-length cDNAs encoding GADs (designated as CiGAD1 and CiGAD2) were isolated and characterized. Multiple alignment and phylogenetic analyses were conducted to evaluate their structures and identities to each other and to homologs in other plants. Tissue expression analyses were conducted to evaluate their transcriptional responses to stress (NaCl, ZnSO4, CdCl2, high/low temperature, and dehydration) and exogenous abscisic acid. RESULTS: The CiGADs contained the conserved PLP domain and calmodulin (CaM)-binding domain in the C-terminal region. The phylogenetic analysis showed that they were more closely related to the GADs of soybean, another legume, than to GADs of other model plants. According to Southern blotting analysis, CiGAD1 had one copy and CiGAD2-related genes were present as two copies in C. intermedia. In the tissue expression analyses, there were much higher transcript levels of CiGAD2 than CiGAD1 in bark, suggesting that CiGAD2 might play a role in secondary growth of woody plants. Several stress treatments (NaCl, ZnSO4, CdCl2, high/low temperature, and dehydration) significantly increased the transcript levels of both CiGADs, except for CiGAD2 under Cd stress. The CiGAD1 transcript levels strongly increased in response to Zn stress (74.3-fold increase in roots) and heat stress (218.1-fold increase in leaves). The transcript levels of both CiGADs significantly increased as GABA accumulated during a 24-h salt treatment. Abscisic acid was involved in regulating the expression of these two CiGADs under salt stress. DISCUSSION: This study showed that two CiGADs cloned from C. intermedia are closely related to homologs in another legume, soybean. CiGAD2 expression was much higher than that of CiGAD1 in bark, indicating that CiGAD2 might participate in the process of secondary growth in woody plants. Multiple stresses, interestingly, showed that Zn and heat stresses had the strongest effects on CiGAD1 expression, suggesting that CiGAD1 plays important roles in the responses to Zn and heat stresses. Additionally, these two genes might be involved in ABA dependent pathway during stress. This result provides important information about the role of GADs in woody plants' responses to environmental stresses.

Characterization of Chinese Unifloral Honeys Based on Proline and Phenolic Content as Markers of Botanical Origin, Using Multivariate Analysis.

The phenolic and proline content were determined in honey samples of different floral origins (rapeseed, sunflower, buckwheat and Codonopsis) from five different regions of China. The phenolic and proline profile of these samples were used to construct a statistical model to distinguish honeys from different floral origins. Significant differences were identified among the studied honey samples from multivariate chemometric methods. The proline content varied among the four types of honeys, with the values decreasing in the order: buckwheat > Codonopsis > sunflower > rapeseed. Rapeseed honeys contained a high level of benzoic acid, while rutin, p-coumaric acid, p-hydroxybenzoic acid were present at relatively high levels in buckwheat honeys. Principal component analysis (PCA) revealed that rapeseed honey could be distinguished from the other three unifloral honeys, and benzoic acid, proline and kaempferol could serve as potential floral markers. Using 18 phenolic compounds and proline the honey samples were satisfactorily classified according to floral origin at 94% correct prediction by linear discriminant analysis (LDA). The results indicated that phenolic compounds and proline were useful for the identification of the floral origin of the four type honeys.

Strategy for comparative untargeted metabolomics reveals honey markers of different floral and geographic origins using ultrahigh-performance liquid chromatography-hybrid quadrupole-orbitrap mass spectrometry.

Honey discrimination based on floral and geographic origins is limited by the ability to determine reliable markers because developing hypothetical substances in advance considerably limits the throughput of metabolomics studies. Here, we present a novel approach to screen and elucidate honey markers based on comparative untargeted metabolomics using ultrahigh-performance liquid chromatography-hybrid quadrupole-orbitrap mass spectrometry (UHPLC-Q-Orbitrap). To reduce metabolite information losses during sample preparation, the honey samples were dissolved in water and centrifuged to remove insoluble particles prior to UHPLC-Q-Orbitrap analysis in positive and negative electrospray ionization modes. The data were pretreated using background subtraction, chromatographic peak extraction, normalization, transformation and scaling to remove interferences from unwanted biases and variance in the experimental data. The pretreated data were further processed using principal component analysis (PCA) and a three-stage approach (t-test, volcano plot and variable importance in projection (VIP) plot) to ensure marker authenticity. A correlation between the molecular and fragment ions with a mass accuracy of less than 1.0ppm was used to annotate and elucidate the marker structures, and the marker responses in real samples were used to confirm the effectiveness of the honey discrimination. Moreover, we evaluated the data quality using blank and quality control (QC) samples based on PCA clustering, retention times, normalized levels and peak areas. This strategy will help guide standardized, comparative untargeted metabolomics studies of honey and other agro-products from different floral and geographic origins.