Characterization of microbial community and yeast functional traits in Apocynum venetum honey from Xinjiang

TL;DR

This study explores the microbial community and yeast traits in Xinjiang Apocynum venetum honey, identifying unique yeast strains with potential for food fermentation.

Contribution

First characterization of microbial community in Xinjiang Apocynum venetum honey.

Findings

33 yeast strains (4 genera) isolated, enriching halophyte honey microbial resources.

Certain yeasts displayed favorable fermentation characteristics, albeit demonstrating lower ethanol tolerance compared to EC-1118.

Guides targeted strain selection for honey-derived functional foods.

Abstract

Apocynum venetum honey, a nutrient-rich product of natural fermentation, exhibits antimicrobial properties due to its low water activity and high sugar content. This study utilized four groups of Xinjiang A. venetum honey, with three biological replicates per group for amplicon sequencing and three technical replicates via gradient dilution for yeast isolation to isolate yeasts using various culture-dependent methods and to analyze microbial diversity and composition through culture-independent techniques. Yeast strains were identified using 26S rDNA sequencing, and their characteristics such as growth, production of ester, ethanol, and H₂S, as well as tolerance levels (ethanol, pH, glucose, SO₂), were compared to the commercial brewer's yeast Saccharomyces cerevisiae EC-1118. The culture-independent analysis revealed significant variations in bacterial richness and fungal diversity among different habitats. No bacterial or fungal species were found to be shared across the habitats with distinct group-specific compositions observed at phylum, class, and genus levels. The culture-dependent methods led to the isolation of 337 strains, of which 33 were yeasts belonging to four main species, representing 22.85% of the total isolates. Among these, EC-1118 demonstrated superior ethanol production and tolerance, while specific strains exhibited notable ester, H₂S and sugar fermentation capabilities. This integrated approach provides valuable insights into yeast characteristics and microbial community structure in A. venetum honey, offering a basis for targeted strain selection, fermentation optimization, and efficient utilization of microbial resource in the food industry.

•First characterization of microbial community in Xinjiang Apocynum venetum honey.•33 yeast strains (4 genera) isolated, enriching halophyte honey microbial resources.•Certain yeasts displayed favorable fermentation characteristics, albeit demonstrating lower ethanol tolerance compared to EC-1118.•Guides targeted strain selection for honey-derived functional foods.

First characterization of microbial community in Xinjiang Apocynum venetum honey.

33 yeast strains (4 genera) isolated, enriching halophyte honey microbial resources.

Certain yeasts displayed favorable fermentation characteristics, albeit demonstrating lower ethanol tolerance compared to EC-1118.

Guides targeted strain selection for honey-derived functional foods.

Figures

Investigate the microbial community structure and diversity in various sample groups of *A. venetum* honey from Xinjiang.Note: A–B. Species accumulation curves are provided for bacterial and fungal groups (AL, CL, LF, NL). C–D. Venn diagrams illustrate the species overlap within bacterial and fungal groups.Fig. 1

Composition characteristics and inter-sample heterogeneity of microbial communities in *A. venetum* honey.A–B. Stacked bar charts depict the microbial community composition across various sample groups, with C showing bacterial communities and D showing fungal communities.Fig. 3

Illustrates the diversity analysis of the isolation effect of culturable fungi using various culture media and samples.Note: A: Proportion of culturable fungi isolated from various culture media; B: Number of phyla, classes, orders, families, and genera of culturable fungi from diverse culture media; C: Proportion of culturable fungi isolated from different samples; D: Number of phyla, classes, orders, families, and genera of culturable fungi from varied samples.Fig. 4

Phylogenetic tree of dominant fungal taxa in *Apocynum venetum* honey samples.Fig. 5

Displays the colony morphology, phylogenetic relationships, and growth characteristics of yeast strains obtained from *A. venetum* honey.Note: A. The colony morphology of different yeast strains (labeled a–e corresponding to strains EC-1118, YLF8, RAL11, YNL4, and mNL1); B. The growth curves of each yeast strain in YPD liquid medium.Fig. 6

Color characteristics of *A. venetum* honey-derived yeast strains in different functional media.Note: A. Color development on TTC medium (reflecting ethanol-producing capacity); B. Color development on BIGGY medium (reflecting H₂S production capacity); C. Color reaction on ester-producing medium (reflecting ester-producing capacity); a–e correspond to strains EC-1118, YLF8, RAL11, YNL4, and mNL1, respectively.Fig. 7

Full text

Introduction

1

Apocynum venetum honey is a distinctive honey sourced from the nectar of the plant Apocynum venetum, primarily found in the arid regions of northwest China, particularly the Tarim River Basin in Xinjiang (Buttar et al., 2025). A. venetum, a perennial herb of the Apocynaceae family, exhibits remarkable tolerance to drought and salt-alkali conditions (Shoukat et al., 2026;Yang et al., 2017), typically thriving on the fringes of desert edges, saline-alkali soils, and sandy riverbank. At ambient temperature, A. venetum honey displays hues ranging from pale amber to dark amber, transforming into a delicate milky white upon crystallization (Buttar et al., 2025; Zhang et al., 2025). This honey possesses high viscosity, optimal fluidity, and a subtle, distinctive aroma reminiscent of A. venetum flowers.

In recent years, several studies have investigated honey-associated yeasts. Xue et al. (2025) examined yeast diversity in vitex honey from Apis mellifera ligustica and Apis cerana cerana in eastern China, revealing higher yeast diversity in A.mellifera honey. They also confirmed the osmotic/acid tolerance of isolated strains for potential fermentation applications; By generating progeny strains with enhanced inhibitor tolerance and adaptability to high-salt/high-temperature conditions through spore hybridization, they laid the groundwork for industrial fermentation strain development. Tiwari et al. (2021) isolated 48 yeast strains from tropical nectar in India, identifying osmotolerant/high-salt-tolerant strains with high xylitol productivity and highlighting tropical nectar as a potential reservoir of such yeasts; Li et al. (2025) compared the metabolic profiles of honey tea wine fermented by single vs. mixed yeast cultures, confirming that mixed fermentation enhances the content of active and volatile compounds, thereby supporting process optimization. These collective findings offer valuable theoretical and technical insights into functional honey food development, microbial resource utilization, and elucidation of metabolic mechanism.

Yeasts are widely present in honey, influencing its flavor, and shelf-life through fermentation traits (e.g., ethanol/ester/H₂S production) and adaptation to environmental conditions (e.g., high sugar/low pH). While yeast-derived esters enhance aroma, undesirable compounds like H₂S can lead to off-flavors. A. venetum honey, a nutrient-rich fermented food with high sugar content and low water activity, inhibits most microbes but favors osmotolerant yeasts. However, research on its yeast community is limited. This study isolated and identified yeasts from A. venetum honey, characterizing their growth, fermentation capacities (ethanol/ester/H₂S production), and stress tolerance. The aim is to identify superior strains, elucidate key functional traits, and support targeted strain selection and fermentation optimization in food production. This study makes academic contributions and improves A. venetum honey products by investigating yeast tolerance to high-sugar/low-pH conditions, thereby offering practical insights for enhancing honey storage stability in industrial applications.

The study seeks to characterize the microbial community structure of Xinjiang A. venetum honey through amplicon sequencing, isolate and identify its yeast strains, analyze their growth, fermentation, and stress tolerance characteristics sustematically, and establish a scientific basis for the utilization of honey microbial resources and fermentation process optimization.

Materials and methods

2

Materials

2.1

Four types of Xinjiang A. venetum honey, each from different sources and environments (not biological replicates), were chosen for this study. Sample 1 was collected from residual A. venetum honey in beehives of a Xinjiang honey brewery (NL). Sample 2 was obtained from A. venetum honey produced in Xinjiang (CL). Sample 3 was purchased from supermarkets as A. venetum honey (AL). Sample 4 was A. venetum honey from Shaya County, Xinjiang, provided by Teacher Li Yawen of the analysis and testing center (LF). The strain Saccharomyces cerevisiae EC-1118 was provided by Teacher Zhu Lixia from the College of Food Engineering, Tarim University.

Methods

2.2

Bioinformatics analysis of sample amplicon sequencing

2.2.1

A total of 0.5 g of A. venetum honey samples from each group (AL, CL, LF, NL) underwent amplicon sequencing on the Illumina MiSeq platform, provided by Nanjing Personalbio Technology Co., Ltd. The primer sequences utilized for amplicon sequencing and microbial community analysis in this study were as follows: The bacterial target gene amplification employed the 338F upstream primer (5′-ACTCCTACGGGAGGCAGCA-3′) and the 806R downstream primer(5′-GGACTACHVGGGTWTCTAAT-3′); for fungal target gene amplification, the ITS5 upstream primer (5′-GGAAGTAAAAGTCGTAACAAGG-3′) and the ITS2 downstream primer (sequence: 5′-GCTGCGTTCTTCATCGATGC-3′) were employed. All primer sequences were meticulously designed following molecular biology experimental standards to ensure the effective and specific amplification of target gene amplicons. Raw sequencing data were processed for paired-end sequence assembly using fastp software to generate operational taxonomic units (OTUs) (Chen, 2023; Chen et al., 2018). Subsequently, OTU clustering was conducted using vsearch software with simultaneous removal of chimeric sequence. Bacterial OTUs were taxonomically annotated against the RDP database (v18.0) (Paul et al., 2025), while fungal OTUs were annotated using the UNITE database (v8.0) to obtain species composition information at various taxonomic levels (phylum, class, genus, etc) (Abarenkov et al., 2024). The Richness index of bacterial and fungal communities in each group was calculated, and species richness line graphs were generated using R software (v4.2.0). Venn diagrams were constructed based on OTU-level data to analyze shared/unique species among the four groups. Furthermore, differences in community structure were assessed through Principal Coordinate Analysis (PCoA) using the Bray-Curtis distance matrix, leading to the generation of PCoA plots for bacterial and fungal communities (Islam et al., 2026). Genus-level abundance heatmaps were created to visualize the relationship between different groups and dominant genera. Stacked bar charts were plotted using the ggplot2 package to analyze genus-level relative abundance, identifying core dominant genera and their relative abundance characteristics in each group.

Isolation, identification, and growth characteristics determination of yeast strains

2.2.2

Two grams of each sample of A. venetum honey were accurately weighed and suspended in 18 mL of sterile water, then subjected to enrichment culture with shaking at 37 °C and 100 r/min for 2 h. The enriched suspension was evenly spread onto YPD medium, PDA medium, rose bengal agar medium, and malt extract solid medium using the gradient dilution plate spreading method （see Table 1）. Three biological replicates were prepared for each concentration, and the plates were incubated inverted at 28 °C in a constant-temperature incubator for 2 days. Single colonies exhibiting typical yeast morphological characteristics (e.g., circular shape, milky white color, smooth surface) were selected. Fungal genomic DNA was extracted using the enzymatic hydrolysis method, followed by PCR amplification with universal primers targeting the fungal ITS region: ITS1 (5’-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3′) (Bellemare et al., 2018). The PCR products underwent sequencing at Sangon Biotech (Shanghai) Co., Ltd. and the resulting sequences were analyzed for homology alignment using the NCBI database.Table 1. Formulations and sources of culture media.Table 1. MediumFormulaSource/ReferenceYPD MediumGlucose 2%, peptone 2%, yeast extract 1%; 2% agar added for solid mediumRefer to Olazabal et al. (2025)PDA MediumFor 1 L: Potato 200.0–250.0 g (peeled and sliced), boiled in 1 L distilled water until soft, then volumetrically adjusted; 1.6% agar added for solid mediumprepared in-houseMalt Extract Solid MediumMalt extract 130.0 g, chloramphenicol 0.1 g, agar 16.0 g, dissolved in distilled water and volumetrically adjusted to 1 Lprepared in-houseRose Bengal Agar MediumCommercial mediumBeijing Aoboxing Biotechnology Co., Ltd.TTC Medium (double-layer)Upper layer: TTC 0.5 g/L, glucose 5.0 g/L, agar 15.0 g/L (prepared fresh before use);Lower layer: Glucose 10.0 g/L, peptone 2.0 g/L, yeast extract 1.5 g/L, dipotassium hydrogen phosphate 1.0 g/L, magnesium sulfate 0.4 g/L, citric acid 0.3 g/L, agar 30.0 g/LRefer to Cen et al. (2015)BIGGY Agar Medium45.0 g commercial medium, dissolved in 1 L distilled water, boiled for no more than 1 minQingdao Haibo Biotechnology Co., Ltd. (Hi-Tech Industrial Park)Ester-Producing MediumBromocresol purple 0.04 g, tributyrin 15 mL/L, yeast extract 10.0 g/L, peptone 20.0 g/L, glucose 20.0 g/L, dissolved in distilled water and volumetrically adjusted to 1 LRefer to Liu et al. (2021)

For strain activation and seed liquid preparation, the isolated yeast strains were inoculated onto YPD solid medium and cultured invertedly at 28 °C for 48 h to activate them. A single activated colony was selected and inoculated into YPD liquid medium, then subjected to expanded culture with shaking at 28 °C and 150 r/min for 48 h to prepare seed liquid at a concentration of 1 × 10^6^ cells/mL. Subsequently, a 1 mL portion of the seed liquid was transferred into an Erlenmeyer flask containing 100 mL of YPD liquid medium and incubated with shaking at 28 °C and 150 r/min. Throughout the incubation, 200 μL of the bacterial suspension was sampled every 2 h. The OD₆₀₀ value of the bacterial suspension was measured at 600 nm using a microplate reader, with sterile YPD liquid medium serving as the blank control. 5 yeast strains (EC-1118, YLF8, RAL11, YNL4, mNL1) underwent quantitative growth curve analysis, with all strains being included for comprehensive profiling rather than selective representation. Each group underwent three biological replicates, and growth curves were constructed for all strains to assess their growth characteristics (Chen et al., 2025).

Fermentation capacity and stress tolerance assays

2.2.3

Basal mediumsupplemented with 0.6% yeast extract and 2% glucose, sucrose, or lactose as the sole carbon source was inoculated with a 1 mL aliquot of seed liquid (1 × 10^6^ cells/mL) using an inverted Durham tube. The cultures were then incubated at 28 °C for 48 h to assess sugar fermentation capacity through gas production (Liu et al., 2020). For metabolic product assays, activated strains were cultured on TTC lower medium for 48 h at 28 °C, followed by overlaying with freshly prepared TTC upper medium for further dark incubation at the same temperature to quantify ethanol production based on colony red color intensity. Additionally, strains were inoculated on BIGGY solid medium for 48 h at 28 °C to evaluate H₂S production by colony brown color depth and on ester-producing medium for 48 h at 28 °C to assess ester production by colony yellow color intensity, where darker color indicated stronger capacity in all assays (Zhao et al., 2020). Notably, the TTC/BIGGY and ester production colorimetric scoring methods are semi-quantitative and limited by subjective visual judgment, serving only for rapid phenotypic screening in this study. Stress tolerance was evaluated by modifying YPD liquid medium with gradient stress factors (ethanol: 3%, 6%, 9%, 12%, 15%, v/v; pH: 3.0, 3.5, 4.0, 4.5; glucose: 100, 200, 300, 400 g/L; SO₂: 150, 200, 250, 300, 350 mg/L, expressed as free SO₂). A 1 mL aliquot of seed liquid (1 × 10^6^ cells/mL) was then inoculated into each stressed medium using an inverted Durham tube and incubated with shaking (150 r/min) at 28 °C for 1–3 days. Growth was defined as both visible turbidity of the culture medium and gas accumulation in the Durham tube, rather than gas production alone. Stress tolerance was assessed by monitoring gas production in the Durham tube as an indicator of growth and metabolic activity (Ishmayana et al., 2017; Qiu & Jiang, 2017).

Statistical analysis

2.3

Microbial community β-diversity variances among groups were evaluated through PERMANOVA, utilizing the Bray-Curtis distance matrix with 999 permutations. This analysis aimed to determine significant difference in microbial community structure across various sources of A. venetum honey samples. Statistical computations were conducted using the vegan package within R software (v4.2.0).

Results

3

Analysis of microbial community diversity and structural characteristics in A. venetum honey samples

3.1

Microbial differential analysis

3.1.1

Microbial communities in four A. venetum Honey Samples (AL, CL, LF, NL) from Xinjiang were analyzed multidimensionally, the sequencing depth of the amplicon was 60,000 reads. Significant differences in bacterial species richness were observed among groups (P < 0.05): LF group and NL group were categorized as high-diversity, CL group as moderate, and AL group had the lowest richness (Fig. 1A). In the fungal community, NL group exhibited significantly higher richness compared to other groups (P < 0.05), while AL, CL, and LF groups had richness levels ≤10 with no significant differences between them (P > 0.05). The absence of a substantial distinction between AL, CL, and LF could be ascribed to their generally low fungal diversity and comparable community foundation. Therefore, the NL group demonstrated optimal microbial diversity (Fig. 1B).Fig. 1. Investigate the microbial community structure and diversity in various sample groups of A. venetum honey from Xinjiang.Note: A–B. Species accumulation curves are provided for bacterial and fungal groups (AL, CL, LF, NL). C–D. Venn diagrams illustrate the species overlap within bacterial and fungal groups.Fig. 1

The Venn diagram analysis demonstrated significant diversity in bacterial species among the four groups. The LF group had the highest number of unique bacterial species (Ma et al., 2018), followed by AL (Liu et al., 2021), NL (Liu et al., 2020), and CL (Li et al., 2025) groups. There were no common bacterial species among the groups, resulting in minimal overlap (Fig. 1C). Regarding fungal species, there were notable distribution differences. The NL group had 34 unique fungal species, AL group had 4, and CL/LF groups had 1 each. Similarly, no there were shared fungal species among the groups, indicating very little overlap (Fig. 1D).

Analysis of microbial community structure using CPCoA and PCoA based on Bray-Curtis distance revealed significant inter-group differentiation. In the bacterial community, all four groups exhibited independent clustering without overlap in both CPCoA (Fig. 2A, explaining 47.88% of variation) and PCoA (Fig. 2C, explaining 63.55% of variation). PCoA and PERMANOVA analysis further confirmed that the microbial community structure differed significantly among groups (P < 0.001). The inter-group separation pattern of the fungal community mirrored that of bacteria, with distinct clusters formed by the four groups in both CPCoA (Fig. 2B, explaining 33.41% of variation) and PCoA (Fig. 2D, explaining 33.49% of variation). Overall, both bacterial and fungal community structures displayed high inter-group specificity.Fig. 2β-Diversity and community structure patterns of microorganisms in A. venetum honey samples.Note: A–D represent the principal coordinate analysis (PCoA) based on Bray-Curtis distance, visualizing the clustering and separation characteristics of microbial communities across four sample groups (AL, CL, LF, NL). Each PCoA plot explained 100% of the total variance, and permutation tests showed P < 0.001, suggesting extremely significant differences in the community composition among different groups.Fig. 2

Microbial community composition analysis

3.1.2

Species-sample association heatmaps illustrated the bacterial community, reflecting rich dominant species types and significant compositional heterogeneity, with scattered color blocks representing species abundance across samples (Fig. 3A). In contrast, the fungal community exhibited high compositional similarity in NL, AL, and LF samples, while the CL sample showed prominent community composition specificity, indicating the complexity and heterogeneity of microbial community composition among samples (Fig. 3B). Genus-level analysis of bacteria identified distinct dominant genera within groups: AL was dominated by Pseudomonas and Methylobacterium; CL by Pseudomonas, LF by Rosenbergiella, and NL by Rosenbergiella and Aplastobacter, reflecting habitat specificity dependence of dominant bacterial groups (Fig. 3C). For fungi, genus-level analysis revealed Zygosaccharomyces and Schizosaccharomyces as dominant in AL, Meyerozyma and Cladosporium in CL, and unannotated taxa in LF and NL (with a small amount of Exidia in NL), highlighting significant divergence in fungal community composition (Fig. 3D).Fig. 3. Composition characteristics and inter-sample heterogeneity of microbial communities in A. venetum honey.A–B. Stacked bar charts depict the microbial community composition across various sample groups, with C showing bacterial communities and D showing fungal communities.Fig. 3

Isolation, identification and growth profiling of yeasts from A. venetum honey

3.2

Four types of A. venetum honey from Xinjiang were used as samples in this study. These included. Residual honey from beehives of an Xinjiang honey brewery (NL), honey produced in Xinjiang (CL), commercially available honey from supermarkets (AL), and honey from Shaya County, Xinjiang (LF). Culturable fungi were isolated using YPD, PDA, malt extract, and rose bengal media, resulting in a total of 77 yeast strains. By applying the principle of consistent colony morphology of strains from the same sample and medium, 33 strains were chosen for 26S rDNA gene sequencing identification. The study revealed that the fungi belonged to 2 phyla, 4 classes, 4 orders, 4 families, and 4 genera. The type of medium and the sample source were found to have a significant impact on the diversity of culturable fungi. In terms of medium diversity, YPD medium had the highest isolation efficiency with 37 strains (48.1%), followed by malt extract medium with 17 strains (22.1%), rose bengal medium with14 strains (18.2%), and PDA medium with 9 strains (11.7%) having the lowest number (Fig. 4A,B). Ascomycota was the dominant phylum across all four media, reaching its highest proportion of 44.4% in YPD medium, while Basidiomycota was not isolated from PDA or rose bengal media. Malt extract medium exhibited the highest taxonomic diversity with 4 classes, 3 orders, 3 families and 4 genera followed by rose bengal and YPD media, both showing 3 classes, 2 orders, 2 families and 3 genera. PDA medium displayed the lowest diversity with 1 class, 1 order, 1 family, 1 genus. In terms of sample diversity, the NL sample had the highest number of strains (29 strains, 37.7%), followed by CL sample (18 strains, 23.4%), the AL sample (16 strains, 20.8%), and the LF sample had the fewest strains (14 strains, 18.2%). This indicates a higherrichness of strains in NL and AL samples compared to LF and CL samples. The AL and NL samples showed greater diversity across all taxonomic levels, with 2 phyla, 3 classes, 2 orders, 2 families and 3 genera each, while the LF and CL samples were relatively homogeneous, with 1 phylum, 1 class, 1 order, 1 family, and 1 genus each(Fig. 4C,D).Fig. 4. Illustrates the diversity analysis of the isolation effect of culturable fungi using various culture media and samples.Note: A: Proportion of culturable fungi isolated from various culture media; B: Number of phyla, classes, orders, families, and genera of culturable fungi from diverse culture media; C: Proportion of culturable fungi isolated from different samples; D: Number of phyla, classes, orders, families, and genera of culturable fungi from varied samples.Fig. 4

This study investigated the fermentation characteristics of yeasts isolated from A. venetum honey in Xinjiang. The morphological features of the isolated yeast strains were examined using honey as the substrate, with the industrial strain EC-1118 employed as a reference. Strain EC-1118 exhibited circular colonies with a diameter of 2.0–4.0 mm, well-defined edges, a convex dome shape, milky white pigmentation, and a smooth, glossy surface (Fig. 6A-a). Strain YLF8 produced circular colonies (2.0–3.5 mm) with clear margins, a smooth, glossy surface, some roughness in densely populated areas, and milky white coloration (Fig. 6A-b). Strain RAL11 formed circular colonies with distinct edges, a pale pink to light reddish pigment, a smooth, glossy surface, and a gelatinous, semi-transparent texture with moderate protrusion (Fig. 6A-c). Strain YNL4 developed circular colonies (2.5–4.0 mm) with well-defined edges, pale pink coloration, a smooth, glossy surface, and a gelatinous, viscous, semi-transparent texture (Fig. 6A-d). Strain mNL1 exhibited nearly circular colonies (1.5–3.0 mm) that were milky white, semi-transparent, with sharp, well-defined edges and moderate central protrusion (Fig. 6A-e). Lastly, Strain RAL11 developed circular colonies with well-defined edges, white and opaque color, and a smooth surface.

After sequencing the PCR products to obtain the 26S rDNA D1/D2 region sequences, they were compared with entries in the GenBank database using the NCBI BLAST tool. A phylogenetic tree of the isolated strains and type strains was generated using the neighbor-joining method in MEGA 11.0 software, with bootstrap values computed from 1000 replicates. Strain YLF8 showed the closest relation to Meyerozyma guilliermondii, Strain RAL11 to Torulaspora delbrueckii, Strain mNL1 to Naganishia albida, and Strain YNL4 was relatively closely related to Rhodotorula frigidialcoholis. Based on morphological characteristics and molecular identification, Strain YLF8 was identified as Meyerozyma guilliermondii, Strain RAL11 as Torulaspora delbrueckii, Strain YNL4 as Rhodotorula mucilaginosa, and Strain mNL1 as Naganishia albida(Fig. 5).Fig. 5. Phylogenetic tree of dominant fungal taxa in Apocynum venetum honey samples.Fig. 5

This study examined the fermentation characteristics of yeasts in A. venetum honey from Xinjiang by analyzing yeast growth patterns isolated from the honey, using the industrial strain EC-1118 as a reference (Fig. 6B). Statistical comparisons were made on the maximum OD₆₀₀ (ODmax) and maximum specific growth rate (μmax) to assess growth variations among all tested strains. Results showed that all strains exhibited a lag phase lasting from 0 to 8 h, with a gradual increase in OD₆₀₀ values and minor differences in initial values. Subsequently, they entered the logarithmic growth phase from 8 to 20 h, where EC-1118 displayed the highest growth rate and μmax, indicating robust proliferative activity. YLF8 and RAL11 showed growth rates and μmax values during this phase that were relatively similar to EC-1118, with no significant difference (P > 0.05), suggesting advantages in nutrient utilization and metabolism. In contrast, YNL4 and mNL1 entered the logarithmic growth phase later with slower growth rates, and their μmax values were significantly lower than EC-1118 (P < 0.05), indicating weak proliferative capacity. All strains reached the stationary phase after 20 h. YLF8 and RAL11 maintained ODmax levels comparable to EC-1118 (P > 0.05), while YNL4 and mNL1 exhibited significantly lower ODmax values (P < 0.05) than the control strain, indicating inferior growth ability or tolerance to culture conditions. In conclusion, notable differences were observed between the isolated strains and the industrial strain EC-1118 in terms of growth rate, ODmax, μmax, nutrient utilization, and environmental tolerance.Fig. 6. Displays the colony morphology, phylogenetic relationships, and growth characteristics of yeast strains obtained from A. venetum honey.Note: A. The colony morphology of different yeast strains (labeled a–e corresponding to strains EC-1118, YLF8, RAL11, YNL4, and mNL1); B. The growth curves of each yeast strain in YPD liquid medium.Fig. 6

Yeast strains from A. venetum honey: fermentation capabilities and multi-stress tolerance

3.3

The fermentation characteristics of yeasts in Xinjiang A. venetum honey were investigated in this study. The ethanol tolerance of indigenous yeasts from the honey was analyzed, with the industrial strain EC-1118 serving as the control. High ethanol concentrations can disrupt yeast cell membranes and walls, inhibit glucose transport and microbial growth, and exert stronger the toxic effect as concentrations increase, ultimately leading to slow or halted fermentation. Ethanol tolerance is essential for yeasts to effectively ferment substrates. Table 2 results revealed that at a 3% ethanol concentration, EC-1118 and YLF8 displayed high tolerance, RAL11 exhibited moderate tolerance, while YNL4 and mNL1 showed minimal tolerance. In ethanol concentrations ranging from 6% to 15%, all strains tested did not grow or produce gas, indicating very low ethanol tolerance under high stress levels. Notably, all isolated yeast strains demonstrated poor ethanol tolerance, with no growth observed beyond 3% ethanol, except for the industrial strain EC-1118 which survived higher ethanol levels. This lack of ethanol tolerance significantly hinders the direct industrial application of these indigenous strains in high-strength alcoholic fermentation, representing a key limitation of this study. Despite EC-1118 and YLF8 showing relatively better tolerance at 3% ethanol, all indigenous strains were notably less tolerant than the industrial strain EC-1118, making them challenging to use directly in typical industrial fermentation settings.Table 2. Tolerance of four yeast strains to ethanol.Table 2. StrainEthanol volume fraction (%)3691215EC-1118+++−−−−YLF8+++−−−−YNL4−−−−−mNL1−−−−−RAL11+−−−−Note: “+”: Slight gas production (<1/4 of small test tube volume); “++”: Moderate gas production (1/3 of small test tube volume); “+++”: Abundant gas production (>1/2 of small test tube volume).

The fermentation characteristics of yeasts in Xinjiang A. venetum honey were investigated by analyzing the pH tolerance of its indigenous yeasts in the honey, with the industrial strain EC-1118 serving as the control. No buffered medium was employed in the pH tolerance assay; solely the initial pH was adjusted, devoid of supplementary buffering agents to ensure pH stability throughout cultivation. The optimal pH range for yeast growth is 4–5, and pH < 4 inhibiting growth and metabolism. Acidic byproducts of fermentation further lower the substrate pH, hindering yeast metabolism and growth. The study's results (Table 3) revealed that under strongly acidic conditions (pH 3–3.5), EC-1118 exhibited the highest tolerance, followed by YLF8 and RAL11, while YNL4 and mNL1 showed poor tolerance. Under neutral-to-slightly acidic conditions (pH 4–4.5), EC-1118, YLF8, and RAL11 demonstrated good tolerance, whereas YNL4 and mNL4 exhibited weak tolerance. Overall, YNL4 and mNL1 displayed significantly lower pH tolerance compared to EC-1118, with YLF8 and RAL11 showing inferior performance overall despite some tolerance under certain pH conditions.Table 3. Tolerance of four yeast strains to pH.Table 3. StrainpH33.544.5EC-1118++++++++++++YLF8++++++++++YNL4−−−−mNL1−−−−RAL11++++++++++Note: “+” indicates a small amount of gas production, with the gas volume accounting for less than 1/4 of the small test tube volume; “++” indicates a moderate amount of gas production, with the gas volume accounting for 1/3 of the small test tube volume; “+++” indicates a large amount of gas production, with the gas volume exceeding 1/2 of the small test tube volume.

The fermentation characteristics of yeasts in Xinjiang A. venetum honey were investigated by analyzing the tolerance of indigenous yeasts to high glucose concentrations using the honey as the research material. The industrial strain EC-1118 served as the control. Yeast fermentation primarily depends on glycolysis of glucose to produce ethanol. The glucose content impacts yeast growth, reproduction, and metabolic capacity. However, high osmotic pressure resulting from high glucose concentrations leads to water loss and rupture in yeast cells, hindering their growth and reproduction. Results （Table 4）indicated that at a low glucose concentration of 100 g/L, EC-1118, YLF8 and RAL11 exhibited good tolerance, producING gas equivalent to half of the small test tube volume and displaying vigorous growth. In contrast, YNL4 and mNL1 showed poor tolerance, producing no gas and failing to grow. At medium to high glucose concentrations of 200–400 g/L, the gas production of EC-1118, YLF8 and RAL11 decreased to one-third to one-fourth of the test tube volume with increasing glucose concentration, yet they still sustained growth indicating moderate tolerance. Notably, EC-1118 and YLF8 maintained gas production equivalent to one-third of the small test tube volume at a high glucose concentration of 300 g/L, with EC-1118 demonstrating slightly superior tolerance compared to RAL11. Conversely, YNL4 and mNL1 showed no gas production and failed to grow throughout the experiment, displaying minimal glucose tolerance.Table 4. Tolerance of four yeast strains to glucose.Table 4. StrainGlucose mass concentration (g/L)100200300400EC-1118++++++++YLF8++++++++YNL4−−−−mNL1−−−−RAL11+++++++Note: “+”: Slight gas production (<1/4 of small test tube volume); “++”: Moderate gas production (1/3 of small test tube volume); “+++”: Abundant gas production (>1/2 of small test tube volume).

The fermentation characteristics of yeasts in Xinjiang A. venetum honey were investigated in this study. The indigenous yeasts' tolerance to SO₂ was analyzed using the honey as the research material, with the industrial strain EC-1118 serving as the control. Results from Table 5 indicated that at low SO₂ concentrations of 150–250 mg/L, EC-1118 exhibited vigorous growth, producing gas equivalent to 1/2 of the small test tube volume. In comparison, RAL11 produced gas equal to 1/2 of the test tube volume at 150–200 mg/L, decreasing to 1/3 at 250 mg/L; YLF8 maintained stable gas production at 1/3 of the test tube volume within this range, while YNL4 and mNL1 showed no gas production or growth. At high SO₂ concentrations of 300–350 mg/L, EC-1118 continued to grow, producing gas equivalent to 1/3 of the test tube volume at 300 mg/L and 1/4 at 350 mg/L. In contrast, RAL11 and YLF8 ceased gas production at 350 mg/L and 300 mg/L, respectively, while YNL4 and mNL1 did not produce any gas. Overall, EC-1118 demonstrated superior tolerance to glucose and SO₂ compared to YNL4 and mNL1, with YNL4 and mNL1 showing moderate tolerance. Particularly, EC-1118 exhibited the highest tolerance under high-concentration conditions.Table 5. Tolerance of four yeast strains to SO_2_.Table 5. StrainSO_2_ mass concentration (mg/L)150200250300350EC-1118++++++++++++YLF8+++++++−YNL4−−−−−mNL1−−−−−RAL11+++++++++−Note: “+”: Slight gas production (<1/4 of small test tube volume); “++”: Moderate gas production (1/3 of small test tube volume); “+++”: Abundant gas production (> 1/2 of small test tube volume).

The fermentation characteristics of yeasts in Xinjiang A. venetum honey were investigated by analyzing the fermentation capacity of indigenous yeasts in the honey for different sugars, using the industrial strain EC-1118 as a control. Results (Table 6) demonstrated that in glucose fermentation, RAL11 produced gas equivalent to 1/2 of the small test tube volume, exhibiting superior fermentation performance compared to EC-1118. In sucrose fermentation, RAL11 produced gas equivalent to 1/3 of the small test tube volume, slightly surpassing EC-1118. EC-1118 displayed a certain fermentation capacity for both glucose and sucrose. By utilizing EC-1118 as a reference control strain, this study systematically compared the fermentation characteristics of yeasts from Xinjiang A. venetum honey with other strains, enabling an analysis of their advantages and limitations. This comparison provides a theoretical foundation for further exploration of the fermentation potential. Gas production volume, which only indicates CO₂ release during sugar fermentatio, should not be directly correlated with actual ethanol yield. To achieve a more precise quantification of ethanol production, it is advisable to employ chemical determinationmethods like HPLC or GC in subsequent investigations.Table 6. Sugar fermentation capacity of four yeast strains.Table 6. StrainGlucoseSucroseLactoseEC-1118+++−YLF8+++−YNL4+−−mNL1+−−RAL11+++++−Note: “+”: Slight gas production (<1/4 of small test tube volume); “++”: Moderate gas production (1/3 of small test tube volume); “+++”: Abundant gas production (> 1/2 of small test tube volume).

The fermentation characteristics of yeasts in Xinjiang A. venetum honey were assessed by comparing the ethanol-producing capacity of indigenous yeasts with the industrial strain EC-1118. Ethanol production was quantified using the TTC colorimetric method, with a darker red color indicating higher ethanol-producing capacity. It is important to note that TTC color development is an indirect and semi-quantitative measure of ethanol production. Results (Fig. 7) showed that the ranking of ethanol-producing capacity was EC-1118 > YLF8 > YNL4 > RAL11 > mNL1. EC-1118 exhibited the highest ethanol-producing capacity, followed by YLF8, while YNL4, RAL11, and mNL1 showed decreasing capacities. Possible reasons for this decline include substrate uptake limitations, metabolic pathway blockages, or reduced enzyme activity. For precise quantification of ethanol yield, it is recommended to confirm the results using analytical techniques like gas chromatography (GC) or high-performance liquid chromatography (HPLC). In practical fermentation scenarios, EC-1118 and YLF8 are suggested as primary fermentation strains. Co-fermentation of strains with diverse metabolic characteristics, such as combining high ethanol-producing strains with those adept at utilizing other honey components, could improve ethanol yield, fermentation efficiency, and flavor equilibrium.Fig. 7. Color characteristics of A. venetum honey-derived yeast strains in different functional media.Note: A. Color development on TTC medium (reflecting ethanol-producing capacity); B. Color development on BIGGY medium (reflecting H₂S production capacity); C. Color reaction on ester-producing medium (reflecting ester-producing capacity); a–e correspond to strains EC-1118, YLF8, RAL11, YNL4, and mNL1, respectively.Fig. 7

Conclusion

4

This study systematically examined the diversity and functional characteristics of culturable yeasts in A. venetum honey from Xinjiang, utilizing four types of honey samples (NL, CL, AL, LF) and four culture media (YPD, PDA, malt extract, rose bengal media). A total of 77 yeast strains were isolated, after excluding redundant and duplicate isolates, 33 representative strains were identified through sequencing of the 26S rDNA D1/D2 regiong. The findings indicated that these yeasts were classified into 2 phyla, 4 classes, 4 orders, 4 families, and 4 genera, with both medium type and sample source significantly impacting the diversity of culturable fungi. YPD medium demonstrated the highest isolation efficiency (48.1%), while malt extract medium displayed the most abundant taxonomic diversity (4 classes, 3 orders, 3 families, 4 genera). Regarding the samples, NL and AL exhibited greater strain richness and taxonomic diversity compared to LF and CL.

Morphological observation and molecular identification confirmed four key strains: Meyerozyma guilliermondii YLF8, Torulaspora delbrueckii RAL11, Rhodotorula mucilaginosa YNL4, and Naganishia albida mNL1. Growth characteristic analysis revealed that YLF8 and RAL11 exhibited growth rates and biomass accumulation similar to the industrial strain EC-1118, indicating advantages in nutrient utilization and environmental tolerance. Conversely, YNL4 and mNL1 displayed slower growth and lower biomass, suggesting relatively inferior adaptability to the culture conditions.

A major limitation of this study is the lack of direct measurements of antioxidant activities (DPPH radical scavenging activity, ABTS^+^ radical cation decolorization activity, FRAP ferric reducing antioxidant power) and biochemical components (total phenolic content, TPC; total flavonoid content, TFC) in Apocynum venetum honey. This gap prevents us from establishing correlations between these functional quality indicators and microbial community structure, especially the four core strains identified in this study: Meyerozyma guilliermondii YLF8, Torulaspora delbrueckii RAL11, Rhodotorula mucilaginosa YNL4, and Naganishia albida mNL1. However, existing studies on these strains have provided sufficient evidence for their antioxidant potential and ability to synthesize phenolic/flavonoid substances, which highlights the research value of these strains in the honey samples of this study and lays a foundation for subsequent functional verification.

Rhodotorula mucilaginosa

Numerous studies have confirmed that Rhodotorula mucilaginosa and its metabolites exhibit excellent antioxidant properties. Kheyrandish et al. (2022) isolated an exopolysaccharide (EPS) from the cold-adapted yeast Rhodotorula mucilaginosa strain GUMS16 and found that the DPPH radical scavenging activity of this EPS was significantly higher than that of the control group (p < 0.0001), which was attributed to its ability to neutralize reactive oxygen species. Ma et al. (2018) further reported that REPS2-A, an exopolysaccharide extracted from Rhodotorula mucilaginosa strain CICC 33013, showed excellent DPPH and ABTS radical scavenging activities as well as strong reducing power (a key indicator related to FRAP), and its antioxidant capacity was closely associated with its branched structural characteristics and molecular weight. In addition, Santander et al. (2024) found that the TEAC (related to ABTS), CUPRAC (related to FRAP), and DPPH activities in the leaves of lettuce plants inoculated with Rhodotorula mucilaginosa were significantly increased, along with a slight increase in total phenolic content. This indirectly indicates that this strain may enhance antioxidant capacity by regulating plant phenolic metabolism, suggesting that Rhodotorula mucilaginosa strain YNL4 in the honey samples of this study may play a role in the antioxidant function of honey by producing phenolic substances, flavonoids, or antioxidant-active polysaccharides.

Naganishia albida

Naganishia albida is a typical extremophile-tolerant yeast, and its antioxidant potential has been reflected in studies related to extreme environments. Wei et al. (2022) identified Naganishia albida as one of the dominant yeast species in the Qaidam Basin Desert (a Mars analog site in China) and emphasized that microorganisms adapting to hyper-arid, high-salt, and high-UV environments usually evolve efficient antioxidant systems to cope with oxidative stress. In addition, González et al. (2025) showed that the total phenolic content (TPC) and DPPH, TEAC, and CUPRAC antioxidant activities of lettuce plants inoculated with Naganishia albida changed significantly, indicating that this strain can affect plant phenolic metabolism and antioxidant capacity. This indirectly suggests that Naganishia albida strain mNL1 in the honey samples of this study may have the potential to produce antioxidant substances and accumulate phenolic/flavonoid substances, and its presence in honey may be closely related to the antioxidant function of honey.

Torulaspora delbrueckii

As an unconventional yeast widely used in food fermentation, Torulaspora delbrueckii has been reported to be involved in the regulation of antioxidant components. Alfonzo et al. (2021) pointed out that Torulaspora delbrueckii can regulate metabolic pathways during dough fermentation to increase the content of bioactive substances, including phenolic substances with antioxidant activity. Aslankoohi et al. (2016) further confirmed that when Torulaspora delbrueckii is co-cultured with Saccharomyces cerevisiae, it can promote the release and transformation of phenolic substances in fermented products, thereby enhancing the antioxidant capacity of the final product. Although this study did not determine the TPC, TFC, and antioxidant activities related to Torulaspora delbrueckii strain RAL11 in honey, existing studies have shown that this strain can regulate phenolic metabolism, suggesting that it may enhance the antioxidant function of honey by participating in the transformation of phenolic and flavonoid substances in honey.

Meyerozyma guilliermondii

The antioxidant potential and phenolic metabolism ability of Meyerozyma guilliermondii (formerly Candida guilliermondii) have been verified in multiple studies. Santander et al. (2024) found that the total phenolic content of lettuce plants inoculated with Meyerozyma guilliermondii was significantly increased, among which the concentration of phenolic compounds such as chicoric acid was greatly improved, and the TEAC, CUPRAC, and DPPH antioxidant activities were also significantly enhanced. González et al. (2025) further showed that under drought stress, the antioxidant activity and phenolic content of lettuce plants inoculated with Meyerozyma guilliermondii were significantly higher than those of the control group, indicating that this strain can enhance antioxidant capacity by regulating plant phenolic metabolism. These studies suggest that Meyerozyma guilliermondii strain YLF8 in this study may have the ability to produce antioxidant metabolites and regulate the accumulation of phenolic/flavonoid substances, making it a potential functional strain involved in the formation of the antioxidant function of honey.

In summary, the absence of measurements of antioxidant activities, TPC, and TFC limits our comprehensive understanding of the relationship between the microbial community and functional quality of A. venetum honey. However, existing literature clearly indicates that Meyerozyma guilliermondii YLF8, Torulaspora delbrueckii RAL11, Rhodotorula mucilaginosa YNL4, and Naganishia albida mNL1 all have significant potential to produce antioxidant metabolites and synthesize phenolic/flavonoid substances. Future studies should systematically determine the DPPH, ABTS, and FRAP activities, as well as TPC and TFC contents, of A. venetum honey, and combine pure culture experiments of these strains to analyze their ability to produce functional substances. This will help clarify the molecular mechanism by which these strains regulate the antioxidant quality of honey and provide theoretical support for the development of functional honey products and microbial inoculants.

This study advances the understanding of yeast resources found in A. venetum honey from Xinjiang, highlighting strains like YLF8 and RAL11 with beneficial fermentation properties. These strains offer valuable microbial resources for creating and utilizing fermented products derived from honey. Additionally, the results lay the groundwork for refining yeast isolation techniques and prolonging the shelf life of honey.

Discussion

5

The microbial community structure and functional properties of yeast strains isolated from A. venetum honey in Xinjiang were systematically characterized in this study. By integrating the research findings of Buttar et al. (2025) and Jiang et al. (2016)., the formation mechanisms and application potential of the functional traits of A. venetum honey yeasts were elucidated. A. venetum honey, a monofloral honey from halophytes, exhibits a metabolic profile influenced by the extreme arid and saline environments of Xinjiang, with a total soluble sugar content of 75.6 ± 1.3%, moisture content of 15.3 ± 0.2%, and abundant secondary metabolites such as flavonoids and phenolic acids (Buttar et al., 2025; Jiang et al., 2016; Zhang et al., 2025). The osmotolerant yeast strains identified in this study (Meyerozyma guilliermondii YLF8 and Torulaspora delbrueckii RAL11) were directly selected due to the high-sugar, low-water-activity environment. Strain YLF8 demonstrated a maximum glucose tolerance concentration of 300 g/L, aligning with the high-sugar stress adaptation mechanism of halophyte honey microorganisms as reported by Buttar et al. (2025). The peroxidase (POD) activity and adenine content in A. venetum honey were measured at 7.5 ± 0.3 U/g and 6.2 ± 0.1 mg/kg, respectively. The weak tolerance of strains YNL4 and mNL1 to ethanol and SO₂ in this study was attributed to insufficient energy metabolism substrates and low antioxidant enzyme levels, supporting Buttar et al.'s (2025) proposition that adenine content correlates positively with the stress resistance of honey microorganisms.

The industrial strain EC-1118 demonstrated superior ethanol production capacity and low hydrogen sulfide yield. As reported by Jiang et al., flavonoids in A. venetum honey can inhibit CYP2E1 activity, which may be related to the improved safety and reduced off-flavors associated with low hydrogen sulfide production in fermentation.

In addition, Jiang et al. (2016) reported that metabolites of flavonoids may contribute to the bioactivity of A. venetum extracts, suggesting that yeast-fermented A. venetum honey products have potential for development as value-added food materials.

In terms of microbial community diversity, this study revealed that fungal richness in NL samples (residual honey in beehives) exceeded 40 operational taxonomic units (OTUs), significantly higher than that in AL, CL, and LF samples (≤ 10 OTUs). No shared fungal species was observed among groups, indicating distinct habitat preferences. This finding is consistent with that of Buttar et al. (2025), who reported that mineral elements (K, Mg, Zn) in A. venetum honey influence microbial community structure. The relatively high mineral content in residual honey may improve nutrient availability for yeast growth, thus supporting higher microbial diversity in NL samples.

The high glucose-tolerant strain YLF8, strong sugar-fermenting strain RAL11, and high ester-producing strain YNL4 identified in this study showed lower tolerance to ≥6% ethanol and ≥ 300 mg/L SO₂ compared with the industrial strain EC-1118. This observation agrees with the metabolic characteristics of A. venetum honey reported by Buttar et al., who noted that the relatively simple nutritional matrix may lead to weaker stress resistance in indigenous yeasts than in commercial industrial strains.

Furthermore, Buttar et al. reported lower contents of certain fatty acids (e.g., palmitoleic acid, heptadecenoic acid) in A. venetum honey than in A. pictum honey; these fatty acids are thought to promote protein binding affinity. Such differences may partially explain the relatively lower protein synthesis efficiency observed in A. venetum honey-derived yeasts in the present study.

As documented in previous studies (Jiang et al., 2016), A. venetum extracts have been reported to alleviate liver-related injury induced by alcohol or APAP. Combined with the low toxicity of fermentation products observed in this study, these background observations support the potential value of A. venetum honey and its associated microbes for developing high-value fermented food products with enhanced functional attributes. These findings also provide new insights for the high-value utilization of microbial resources from this unique nectar source (Li et al., 2025).

The functional traits of A. venetum honey yeasts in Xinjiang are a result of their synergistic adaptation to the unique nutritional environment and extreme habitat of A. venetum honey. The habitat specificity of the microbial community in A. venetum honey provides a basis for the targeted screening of functional strains. Future research can focus on the metabolic engineering modification of yeasts to improve their stress resistance and metabolic efficiency, further exploring the flavor and functional value of A. venetum honey fermented products.

CRediT authorship contribution statement

Yi Huang Chen: Writing – original draft, Data curation. Ya Wen Li: Formal analysis, Data curation. Xi Rui Wang: Investigation, Formal analysis, Data curation. Li Jun Wang: Methodology. Shu Han Yu: Visualization, Validation. Xiao Xia Luo: Project administration, Formal analysis. Rui Li Zhang: Funding acquisition, Formal analysis. Hua Ying Liu: Writing – review & editing, Funding acquisition, Formal analysis.

Ethics approval

No studies with human participants or animals were performed.

Funding

This research was funded by “Study on Polyphenols and Fingerprint Construction in Apocynum venetum Nectar (Southern Xinjiang)” (TDZKYB202405).

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The research was funded by the project “Study on Polyphenols and Fingerprint Construction in Apocynum venetum Nectar (Southern Xinjiang)” (Grant No.: TDZKYB202405). The funder had no role in the design of the study, collection, analysis, or interpretation of data, writing of the manuscript, or the decision to submit the manuscript for publication. All authors confirm that there are no conflicts of interest to disclose.