Study on the influence of microorganisms on the flavor of black tea

Abstract

In this study, microorganisms isolated from black tea (BT) processing samples were added to BT fermentation, and the volatile profile and chemical characteristics of BT fermented by adding single tea-derived microbe were investigated. Briefly, a total of 14 yeast and 33 bacteria were isolated, among which five were selected for BT fermentation. Adding tea-derived microorganisms during fermentation increased the contents of water extract, caffeine, and theabrownins while reduced soluble sugars, gallic acid, and ester catechins content in BT, with better quality observed in Hanseniaspora uvarum RL-B and Bacillus amyloliquefaciens N1 fermented samples. Additionally, tea-derived microbial fermentation altered the composition of volatile compounds, predominantly alcohols, esters, terpenoids, and hydrocarbons. It decreased volatiles content that presenting sweet and floral flavor whereas enhanced those exhibiting nutty and fruity-like green fragrance by modulating the monoterpenoid biosynthesis pathway. These findings provide new insights into the mechanisms by which microorganisms influence the quality formation of BT.

Highlights

• •14 yeast and 33 bacteria were isolated from black tea (BT) processing samples.
• •Tea-derived microbe fermentation (TDMF) altered the major quality components of BT.
• •Hanseniaspora uvarum RL-B and Bacillus amyloliquefaciens N1 were more effective.
• •TDMF reduced sweet and floral notes while enhanced nutty and green-fruity odor.
• •Adding TDMF affected BT aroma via the monoterpeniod biosynthesis pathway.

1. Introduction

As the most widely consumed tea globally, black tea is highly favored for its unique and attractive aroma. Aroma is a key sensory indicator for evaluating the quality of black tea, which is primarily characterized by floral (rose-like), honey-like, and sweet fragrance (Yao et al., 2023). Studies have revealed that the aroma profile of black tea comprises hundreds of volatile compounds, including alcohols, aldehydes, esters, ketones, acids, phenols, and sulfur compounds (Yang et al., 2024). As a fully fermented tea, the formation of distinctive quality and flavor characteristics of black tea occurs in the fermentation stage. In this stage, tea polyphenols were fully oxidized to form a large number of tea pigments like theaflavins and thearubigins (Chen et al., 2024), which attribute to the taste and color of black tea. Simultaneously, this period is critical for the transformation of scents substances, which dominates the formation of sweet and floral or fruity fragrance (Chen et al., 2024).

Microbes are decisive for quality of dark tea (Tian et al., 2024). Interestingly, recent researches have shown that numerous microorganisms are present in the processing of black tea, which predominated by Sphigomonas, Variovorax, Chryseobacterium, Pleosporales, Pantoea, Pseudomonas, Paucibacter, Cladosporium, Bacillus, and several yeast (Jia et al., 2022; Karunaratne et al., 2024; Liu et al., 2023; Wang et al., 2024). The composition of microbial community changes with the processing of black tea. In fresh leaves, the microbes were predominated by Proteobacteria, Bacteriodetes, Firmicutes, Actinobacteria, and Ascomycete, whereas Sphingomonas, Methylobacteria, and Didymellia during fermentation (Tong et al., 2021).

It has been reported that the quality of black tea is closely related to microbial communities during processing. Sterilization of fresh leaves decreased the content of total catechins and theanine in black tea, suggesting the involvement of leaf surface microbes in the formation of these compounds (Tong et al., 2021). Moreover, the dominant microbe in black tea were positively correlated with soluble sugars and tea pigments content, while negatively related to free amino acids and catechins concentration (Wang et al., 2024). In terms of aroma, Liao et al. (2024) found that Pedobacter, Acidovorax, Sphigomonas and Pantoea were the core functional microbe responsible for the characteristic aroma profile of Sichuan black tea. These findings demonstrated that microorganisms are indispensable in shaping the distinctive quality attributes of black tea. Furthermore, addition of microbes in the fermentation process is effective in improving the flavor of black tea, as evidenced by Aspergillus coronarius fermentation altered the content of quality components like catechins (Wang et al., 2023). However, current researches mainly focused on the overall changes of microbial communities during black tea processing based on Illumina Miseq, without isolation and purification of dominant strains. Meanwhile, systematic studies on the effect of fermentation with the addition of a single dominant strain on the quality of black tea are lacking.

Therefore, in this study, the composition of the main bacteria and yeasts in black tea processing was firstly clarified by isolating and purifying the microbes. Then the dominate microorganisms of black tea (tea-derived microbes) were screened out and the effect of adding single tea-derived microbial strains during fermentation on the quality and flavor of black tea was investigated. This finding provides theoretical support for the development of new tea products using microbial resources in the future.

2. Materials and methods

2.1. Microbial collection during black tea processing

2.1.1. Sample preparation

The fresh leaves (one bud with a leaf to two leaves) of ‘Fuding dabai’ (Camellia sinensis(L.) O. Ktze) were processed into black tea as the following procedures: withering (25 °C, 12 h), rolling (32 rpm for 1.5 h), fermentation (28 °C, 3 h, humidity: 96 %), and drying (initially at 100 °C for 15–20 min, followed by cooling for 5 h at 25 °C, and finally drying at 75 °C for 2 h until the moisture content fell below 4 %). During this process, fresh leaves, leaves after withering, rolling, and fermented for 1 h, 2 h, and 3 h were collected. All samples were immediately transferred to sterile polyethylene bags and stored at 4 °C until use.

2.1.2. Microbe isolation and morphological observation

Microbes (yeasts and bacteria) were isolated from black tea samples using direct inoculation, sterile water elution, and enrichment culture methods. For direct inoculation, black tea samples were aseptically spread on yeast extract peptone dextrose (YPD) agar (Haibo Biotechnology, Qingdao, China) and nutrient broth (NB) (Haibo Biotechnology) medium. In the elution method, samples were vortexed in sterile water for 5 min and then coated. Enrichment culture was performed by incubating samples in YPD and NB liquid media at 28 °C with shaking (120 rpm) for 36 h and then coated. YPD plates were incubated at 28 °C while NB plates at 37 °C. Single colonies were purified by streaking method.

Colony characteristics like size, shape, margin morphology, surface appearance, color, and transparency were observed. Colonies exhibiting undesirable odors (sour, putrid, or foul smells) were excluded. Then, yeast and bacteria were stained with Löffler's Methylene Blue Stain solution and Gram stain solution, respectively, followed by observation and imaging using a microscope (Carl Zeiss AG, Oberkochen, Germany).

2.1.3. Identification of isolated microbes

Genomic DNA of yeasts was extracted using the fungal DNA extraction kit (Solarbio Science & Technology, Beijing, China), while bacterial genomic DNA was purified with the Ezup column bacterial DNA purification kit (Sangon Biotech, Shanghai, China). Then the D1/D2 region of 26S rRNA, internal transcribed spacer (ITS), and RNA polymerase II subunit (RPB2) gene of yeast were amplified by PCR. Simultaneously, the 16S rRNA gene with its V4 hypervariable region of bacteria were amplified. The specific PCR procedure and primer sequence were list in Table S1 and Table S2, separately. Following this, the PCR products were sequenced by Sangon Biotech and were subsequently analyzed using BLASTn against the NCBI database. Sequences exhibiting ≥99 % identity with reference sequences were retained for subsequent phylogenetic analysis. Phylogenetic trees were constructed using the maximum likelihood method in MEGA-X software.

2.1.4. Scanning electron microscope (SEM) observation of screened strains

The selected strains (non-pathogenic, odorless, and previously reported exist in tea) were cultured in liquid medium for 24 h, then were harvested and washed twice with PBS (Servicebio, China). The pellets were fixed with electron microscopy fixative (Servicebio, China) at room temperature in the dark and stored at 4 °C. Dehydration was performed using a graded ethanol series (30 %, 50 %, 70 %, 80 %, 90 %, 95 %, and 100 %), with 15 min incubation at each concentration. Subsequently, the samples were treated with isoamyl acetate for 15 min. Finally, the processed samples were observed by using scanning electron microscopy (SEM), and high-resolution images were acquired for morphological analysis.

2.2. Microbial-fermented black tea processing

The selected bacteria (Bacillus amyloliquefaciens N1, Lysinibacillus fusiformis WL-B, and Enterobacter hormaechei P) and yeast (Hanseniaspora uvarum RL-B, Debaryomyces hansenii FLB-A) were individually cultured in 50 mL of corresponding medium at 28 °C for 36 h. The microbial suspensions were adjusted to 1 × 10⁶ CFU/mL, then were harvested and resuspended in sterile water, and stored at 4 °C for subsequent use.

The processing method and parameters of microbial-fermented black tea (MFBT) was in accordance with 2.1.1. After rolling, 25 mL of inoculum solution was mixed with 1 kg of rolled leaves for fermentation. Samples fermented with Hanseniaspora uvarum RL-B, Debaryomyces hansenii FLB-A, Bacillus amyloliquefaciens N1, Lysinibacillus fusiformis WL-B, and Enterobacter hormaechei P were named as RBT, FBT, NBT, WBT, and PBT, separately. Samples inoculated with equal volumes of sterile water was used as control (CBT). The specific producer was depicted in Fig. 1A. Samples were collected and stored at −80 °C for further analysis.

Fig. 1.

Morphological characteristics of the five tea-derived microbial strains used for black tea fermentation. (A) Schematic diagram of black tea fermented by tea-derived microbes. (B) Colony morphology and scanning electron microscopy (SEM) images of the five tea-derived microbial strains used for black tea fermentation.

2.3. Measurement of major biochemical components

The content of major biochemical components like water extract, tea polyphenols (TPs), amino acids (AA), soluble sugars, theaflavins (TFs), thearubigins (TRs), theabrownins (TBs), gallic acid (GA), catechin (C), epicatechin (EC), epigallocatechin (EGC), epigallocatechin gallate (EGCG), epicatechin gallate (ECG), gallocatechin gallate (GCG), catechin gallate (CG), and caffeine (CAF) were determined according to our previous study (Jia et al., 2022). Three replications were performed.

2.4. Extraction and identification of volatile compounds by HS-SPME-GC–MS

Volatile compounds in black tea were analyzed by HS-SPME-GC–MS. Specifically, 500 mg sample (powder), 20 μL (10 μg/mL) internal standard (3-hexanone-2,2,4,4-d4, Merk, Germany), and NaCl saturated solution were mixed in a 20 mL head-space vial (Agilent, USA). After equilibration (60 °C, 5 min), a 120 μm DVB/CAR/PDMS fiber (Agilent, USA) was inserted into the vial and kept in water bath (60 °C for 15 min) to extract volatile components. The fiber was then desorbed for 5 min at the GC–MS INJECTION PORT at 250 °C.

GC–MS analysis was performed using an Agilent 7890GC and 7000D mass spectrometer equipped with a DB-5MS capillary column (30 m × 0.25 μm × 0.25 μm, Agilent, USA). The specific procedure was strictly referenced to our previous study (Liao et al., 2024). An n-alkanes mixture (C6 ∼ C21) was injected to calculate the retention index (RI) of each peaks under the same GC–MS conditions. All volatile components were identified using the Metware database and confirmed by retention index (RI). Quantification was performed based on the peak areas of internal standard.

2.5. Analysis of relative odor activity value (rOAV) in volatile compounds

Relative odor activity value (rOAV) was calculated to evaluate the contribution of volatile compounds to black tea aroma. rOAV was determined as the ratio of the concentration (Ci) to its odor threshold (OTi) in water. Volatile compounds with rOAV>1were considered to contribute to the overall aroma profile. The thresholds of volatile substances were referred to a previous study (Huang et al., 2022).

2.6. Statistical analysis

Data are expressed as means ± SD. Statistical analysis were performed using SPSS (IBM Corp, v23, Armonk, NY, USA). For samples that pass the homogeneity of variance test, one-way ANOVA with Duncan multiple comparisons tests was used. For samples that did not pass the homogeneity of variance test, significance was calculated using the Tamhane T2 test. A value of p < 0.05 indicated statistical significance.

Principal component analysis (PCA), partial least squares-discriminant analysis (PLS-DA), and orthogonal projections to latent structures discriminant analysis (OPLS-DA) of volatiles were carried out by using R package (v4.1.2). Variable importance in projection (VIP) values was extracted from OPLS-DA results. Key volatile compounds between different treatments were screened by VIP > 1 and p < 0.05. KEGG analysis was using the KEGG Pathway database (http://www.kegg.jp/kegg/pathway.html).

3. Results

3.1. Strains identified in black tea samples

A total of 47 strains, including 14 yeast and 33 bacteria, were isolated from black tea processing samples using direct inoculation, sterile water elution, and enrichment culture methods. Following colony morphology observation (Table S3) and staining microscopy (Fig. S1, Fig. S2), strains producing undesirable odors (sour, putrid, or foul smells) were excluded. Ultimately, 4 yeast strains (FLB-A, FLB—C, FLC—B, and RL-B) and 11 bacterial strains (WL-B, AX1, JM WL-B, JM AE, L, JM M10, T1, P, J, N1, and C-1) were selected. Molecular identification showed that these strains were Debaryomyces hansenii, Hanseniaspora uvarum, Lysinibacillus fusiformis, Staphylococcus succinus, Weissella cibaria, Pantoea sp., Pantoea agglomerans, Mammaliicoccus sciuri, Enterobacter hormaechei, Staphylococcus saprophyticus, and Bacillus amyloliquefaciens, separately (Table S4, Fig. S3). Of which, the safety of Staphylococcus succinus, Staphylococcus saprophyticus and Mammaliicoccus sciuri are controversial, and may lead to several diseases like infective endocarditis (Ruffier d'Epenoux et al., 2024) and septicemia (Singh et al., 2024). Weissella cibaria is a common food spoilage microorganism that may cause adverse effects like diarrhea under certain circumstances (Abriouel et al., 2015). Both Pantoea sp. and Pantoea agglomerans have been identified as opportunistic human pathogens (Li et al., 2024). Therefore, Staphylococcus succinus, Weissella cibaria, Mammaliicoccus sciuri, Pantoea sp., and Pantoea agglomerans are unsuitable for black tea fermentation.

In contrast, Debaryomyces hansenii, a non-Saccharomyces yeast has been previously identified in dark tea (Zou et al., 2025). This strain contributes to the dark infusion color, sweet taste, and enhanced fruity aromas when used in dark tea pile-fermentation (Zou et al., 2025). Besides, it is non-pathogenic and participates in synthesizing volatiles like acids, aldehydes, hydrocarbons, and has been widely applied in the field of food fermentation (Zhang et al., 2021). Similarly, Hanseniaspora uvarum, another non-Saccharomyces yeast with high β-glucosidase activity, has been demonstrated to promote acetate esters content, thereby enhancing fruity flavors during wine fermentation (Zhang et al., 2023). Bacillus amyloliquefaciens exhibits glycosidase activity that promotes the formation of acids, sulfur compounds, and pyrazines (Haure et al., 2022; Lin et al., 2024), Besides, it can release monoterpenoids and benzenoids, both are important volatile constituents (Haure et al., 2022). Lysinibacillus fusiformis has been applied in post-harvest treatments of fruits and vegetables, demonstrating potential for degrading geraniol and fluorobenzoates (Shi et al., 2023). Enterobacter hormaechei possesses the capability to bio-transform lutein into novel volatile compounds (Zhong et al., 2017). Based on these studies, we infer that Debaryomyces hansenii FLB-A, Hanseniaspora uvarum RL-B, Bacillus amyloliquefaciens N1, Lysinibacillus fusiformis WL-B, and Enterobacter hormaechei P may be beneficial for the flavor of black tea and were thus selected for further investigation.

As shown in Fig. 1B, the Hanseniaspora uvarum RL-B colonies appeared white and round, with moist surface and smooth rounded edges. The body was spherical, 2–5 μm long, 1–1.5 μm wide, and proliferated both budding and fission, with budding being the predominant mode, some of the cells retained bud scars and fission marks. Debaryomyces hansenii FLB-A formed white, circular colonies with convex, dry surfaces and smooth margins, displaying spherical cells (2–4 μm both in length and width) that reproduced by budding, with visible bud scars on cell surfaces. Bacillus amyloliquefaciens N1 developed white, circular colonies with dry surfaces and undulate margins on NB medium, showing Gram-positive, short rod-shaped cells (1–1.5 μm long and 0.3–0.5 μm wide) with rounded ends that reproduced through fission, with protein filaments observed between cells (Fig. 1B, Fig. S2, Table S3). Lysinibacillus fusiformis WL-B produced white, semi-transparent circular colonies with undulate margins, exhibiting Gram-negative rod-shaped cells (1–2 μm long and 0.3–0.5 μm wide) with rounded ends that reproduced by fission, displaying surface wrinkles and protein filament structures (Fig. 1B, Fig. S2, Table S3). Enterobacter hormaechei P formed white, semi-transparent circular colonies with smooth margins, showing Gram-negative ellipsoidal cells (0.5–2.5 μm long and 0.5–1 μm wide) that reproduced through fission, with numerous surface wrinkles and some cells displaying protein filaments (Fig. 1B, Fig. S2, Table S3).

3.2. Effect of adding tea-derived microbial strains fermentation on main quality components of black tea

As depicted in Fig. 2A, compared with the CBT group, both adding yeast and bacteria during fermentation increased the water extract content, with Debaryomyces hansenii FLB-A and Lysinibacillus fusiformis WL-B showing the most pronounced effect. The content of tea polyphenols was highest in black tea fermented with Lysinibacillus fusiformis WL-B while lowest in Hanseniaspora uvarum RL-B fermented tea, though neither differed significantly from the CBT group (Fig. 2B). The addition of Enterobacter hormaechei P during fermentation reduced the total free amino acid content compared to the CBT group (Fig. 2C). Except for Hanseniaspora uvarum RL-B, all other tea-derived microbial strains fermentation decreased soluble sugar level (Fig. 2D). Except for RBT, NBT and WBT, adding microbial during fermentation elevated caffeine level (Fig. 2E). The content of gallic acid (GA) was decreased in all adding tea-derived microorganism fermentation groups when compared to the CBT group (Fig. 2F). In contrast, no significant differences were observed in catechin (C) and gallocatechin gallate (GCG) contents among different groups (Fig. 2H). Simultaneously, inoculation of tea-derived microbial strains showed no significant effect on epicatechin (EC) content compared with the CBT group, while the RBT and NBT groups exhibited higher EC levels than those in the WBT and PBT groups (Fig. 2H). Meanwhile, epigallocatechin (EGC) and epicatechigallate (ECG) was reduced in RBT, FBT and PBT groups (Fig. 2H). Notably, except Bacillus amyloliquefaciens N1, all other tea-derived microorganisms significantly decreased epigallocatechin gallate (EGCG) level, with FBT group showing the lowest levels, followed by PBT, RBT, and WBT (Fig. 2H). Content of Theaflavins and thearubigins remained unchanged among these groups (Fig. 2G), whereas theabrownin was slightly increased in all treatments with the exception of RBT, with the Debaryomyces hansenii FLB-A and showing the most pronounced effect (Fig. 2G).

Fig. 2.

Effect of adding tea-derived microbial strains fermentation on main quality components of black tea. The content of water extract (A), tea polyphenols (B), free amino acid (C), soluble sugar (D), caffeine (E), gallic acid (F), theaflavins, thearubigins, and theabrownin (G), and catechins (H) in different groups. Data are means ± SD. Bars with different letters indicate a significant difference (p < 0.05).

3.3. Composition of volatile components in black tea fermented by adding tea-derived microorganisms

A total of 784 volatile components were identified in all groups, including terpenoids (181, 23.12 %), ester (133, 16.99 %), heterocyclic compound (110, 14.05 %), hydrocarbons (78, 9.96 %), ketone (70, 8.81 %), aldehyde (65, 8.3 %), alcohol (47, 6 %), aromatics (38, 4.85 %), amine (19, 2.43 %), acid (16, 2.04 %), phenol (13, 1.66 %), sulfur compound (4, 0.51 %), nitrogen compounds (3, 0.38 %), ether (3, 0.38 %), halogenated hydrocarbons (2, 0.26 %), and others (2, 0.26 %) (Table S5, Fig. 3A). PCA analysis revealed that the volatile profiles of black tea fermented with adding tea-derived microbial strains (including RBT, FBT, NBT, WB, and PBT) differed from the CBT group, whereas differences among microbial fermentation groups were minor (Fig. 3B). OPLS-DA further confirmed this discrimination (Fig. 3C), and permutation tests validated the robustness of the model (Fig. 3D). Further analysis showed that 211 differential volatiles (VIP > 1 and p < 0.05) were screened in all groups (Table S6), including acid (5), alcohol (13), aldehyde (18), amine (5), aromatics (5), ester (35), ether (1), heterocyclic compound (33), hydrocarbons (26), ketone (13), nitrogen compounds (1), phenol (7), sulfur compounds (2), and terpenoids (57) (Fig. 3E). Notably, most of these compounds were reduced in microbial-fermented black tea (MFBT) groups (Fig. 3F).

Fig. 3.

Composition of volatile components in black tea fermented by adding tea-derived microorganisms. (A) Types of volatile substances in black tea fermented with or without tea-derived microbes. (B) PCA scores of volatile components in black tea fermented with or without tea-derived microbes. (C) OPLS-DA plot of volatiles in black tea fermented under normal conditions (CBT) and by adding tea-derived microbes (MFBT). (D) Hypothesis testing of the OPLS-DA model in CBT_vs_MFBT group. (E) The number of different types of differential volatile components screened from CBT_vs_RBT_vs_FBT_vs_NBT_vs_WBT_vs_PBT. (F) Heatmap of differential volatile components (VIP top 50) screened from CBT_vs_RBT_vs_FBT_vs_NBT_vs_WBT_vs_PBT.

3.4. Effect of adding tea-derived yeast fermentation on the aroma of black tea

To investigate the effect of tea-derived yeast on black tea aroma, we analyzed key volatile components. The results showed that 214 and 246 differential volatile compounds were screened in the RBT_vs_CBT and FBT_vs_CBT groups, respectively (Table S6). These compounds mainly include terpenoids, esters, heterocyclic compounds, hydrocarbons, and aldehydes, most of which were reduced in the RBT and FBT groups (Fig. 4A). Among these, 95 compounds were solely affected by the Hanseniaspora uvarum RL-B, 127 by the Debaryomyces hansenii FLB-A, and 119 were affected by both tea-derived yeast strains (Fig. S4A). Of these substances, 60 showed a rOAV >1 (Table S6, Table 1), typically regarded as key aroma-active compounds. Except for a few terpenoids (o-Cymene), esters (Butanoic acid, 2-methyl-, hexyl ester), aldehydes (2,6-Nonadienal, (E,E)-, 2,6-Nonadienal, (E,Z)-, 3-Hexenal, (Z)- and Hexanal), and aromatics (Benzene, 1,4-diethyl- and p-Cymene), most key aroma compounds were more abundant in the CBT group (Fig. 4B). Compounds elevated in the tea-derived yeast fermentation groups showed green, citrus, and fruity flavor (Table S6, Table 1, Fig. 4C), whereas those with decreased levels exhibited sweet, green, waxy, fruity, and floral odor (Table S6, Table 1, Fig. 4D). This indicates that the addition of tea-derived yeast during fermentation weakened sweet and green fragrance of black tea slightly.

Fig. 4.

Effect of adding tea-derived yeast fermentation on the aroma of black tea. (A) Types of differential volatile components screened from RBT_vs_CBT and FBT_vs_CBT groups. (B) Heatmap of differential volatile components with rOAV>1 screened from RBT_vs_CBT and FBT_vs_CBT groups. (C) Flavor radarchart of volatiles (rOAV>1) with elevated levels in tea-derived yeast fermentation groups (RBT and FBT). (D) Flavor radarchart of volatiles (rOAV>1) with reduced contents in tea-derived yeast fermentation groups (RBT and FBT).

Table 1.

Key volatile components (rOAV>1) contribute to the aroma of black tea fermented by adding tea-derived yeast.

Volatiles	CAS	RI	Threshold	rOAV			Odor
				RBT	FBT	CBT
Acid
Benzeneacetic acid	103–82-2	1262	0.135	1.46 ± 0.48b	2.42 ± 0.86b	4.36 ± 0.71a	sweet, honey, floral, honeysuckle, sour, waxy, civet
Alcohol
2-Nonanol	628–99-9	1099.20	0.058	1.98 ± 0.09ab	1.81 ± 0.05b	2.2 ± 0.17a	rose
2-Octen-1-ol, (E)-	18,409–17-1	1067.62	0.02	9.81 ± 0.32b	9.2 ± 0.38b	11.23 ± 0.9a	green, citrus, vegetable, fatty
Aldehyde
(E)-2-Decenal	3913-81-3	1263	0.005	118.96 ± 7.18b	132.95 ± 11.84b	150.55 ± 2.45a	waxy, fatty, earthy, green, cilantro, mushroom, aldehydic, fried, chicken, fatty, tallow
2,6-Nonadienal, (E,E)-	17,587–33-6	1153	0.0005	25.02 ± 5.36b	50.29 ± 7.07a	36.09 ± 4.46b	fresh, citrus, green, cucumber, melon
2,6-Nonadienal, (E,Z)-	557–48-2	1154.75	0.00001	1250.87 ± 268.18b	2514.7 ± 353.61a	1804.46 ± 223.15b	cucumber, green
4-Nonenal, (E)-	2277-16-9	1105	0.0022	91.65 ± 4.47b	89.39 ± 1.08b	119.56 ± 15.75a	fruity
6-Nonenal, (Z)-	2277-19-2	1103.52	0.00014	20,525.41 ± 928.47b	18,306.7 ± 1568.62b	24,273.62 ± 1280.66a	green, cucumber, melon, cantaloupe, honeydew, waxy, vegetable, orris, violet, leafy
2-Nonenal	2463-53-8	1161	0.0001	486.13 ± 59.07b	524.2 ± 23.66b	671.12 ± 55.06a	fatty, green, waxy, cucumber, melon
2-Nonenal, (E)-	18,829–56-6	1161.21	0.00008	2687.69 ± 41.95b	2489.43 ± 71.83b	3633.95 ± 342.31a	fatty, green, cucumber, aldehydic, citrus
2-Nonenal, (Z)-	60,784–31-8	1148	0.0045	168.97 ± 1.16b	175.87 ± 5.56b	267.6 ± 26.18a	orris, fatty, waxy, cucumber
3-Hexenal, (Z)-	6789-80-6	800	0.004	11.25 ± 0.92a	12.47 ± 1.24a	8.46 ± 0.8b	green, fatty, grassy, weedy, fruity, apple
Hexanal	66–25-1	799.79	0.005	47.1 ± 4.46b	60.84 ± 9.22a	32.02 ± 2.99c	aldehyde, grassy, green, leafy, vinegar
cis-7-Decen-1-al	21,661–97-2	1212	0.0022	106.63 ± 1.29b	109.22 ± 0.2b	123.98 ± 2.91a	citrus, aldehydic, cucumber
Aromatics
Benzene, nitro-	98–95-3	1080	0.15	1.64 ± 0.07ab	1.52 ± 0.05b	1.71 ± 0.1a	–
Benzene, 1,4-diethyl-	105–05-5	1041	0.0021	12.21 ± 0.08b	14.3 ± 1.51a	10.02 ± 0.83c	–
p-Cymene	99–87-6	1025.99	0.0114	2.25 ± 0.01b	2.63 ± 0.28a	1.85 ± 0.15c	woody, citrus
Amine
BenzenAmine, N,N-dimethyl-	121–69-7	1101	0.012	5.39 ± 0.26b	5.24 ± 0.18b	6.51 ± 0.51a	–
Ester
2,4-Decadienoic acid, ethyl ester, (E,Z)-	3025-30-7	1479	0.1	0.96 ± 0.12b	1.01 ± 0.09b	1.27 ± 0.08a	green, waxy, pear, apple, sweet, fruity, tropical
Butanoic acid, 2-methyl-, hexyl ester	10,032–15-2	1236	0.022	2.25 ± 0.08a	1.04 ± 0.23c	1.44 ± 0.05b	green, waxy, fruity, apple, spicy, tropical
Butanoic acid, 3-methyl-, 2-phenylethyl ester	140–26-1	1491	0.00001	5231.47 ± 437.97a	4785.3 ± 2590.9a	6486.21 ± 156.42a	floral, fruity, sweet, rose, peach, apricot
Propanoic acid, 2-methyl-, propyl ester	644–49-5	859.34	0.000086	29.12 ± 0.74ab	21.12 ± 9.7b	36.36 ± 2.69a	sweet, ripe fruit, tropical, melon, berry
2(3H)-Furanone, 5-butyldihydro-	104–50-7	1261	0.0179	16.88 ± 1.83b	19.46 ± 0.47b	24.85 ± 1.41a	sweet, coconut, waxy, creamy, tonka, dairy, fatty
2(5H)-Furanone, 3-hydroxy-4,5-dimethyl-	28,664–35-9	1110	0.011	128.05 ± 5.32b	130.81 ± 10.81b	165.9 ± 16.7a	extremely sweet, strong caramel, maple, burnt, sugar, coffee
Acetic acid, hexyl ester	142–92-7	1013.054	0.115	0.11 ± 0.02c	2.09 ± 0.01b	2.37 ± 0.06a	fruity, green, apple, banana, sweet
Methyl salicylate	119–36-8	1200	0.04	74.02 ± 4.13c	89.81 ± 2.84b	111.69 ± 8.57a	caramel, pepperminty
Heterocyclic compound
2-Thiophenemethanethiol	6258-63-5	1105	0.00004	5416.94 ± 181.87b	5566.3 ± 90.4b	6694.32 ± 623.33a	roasted, coffee, fishy
Pyrazine, 2-ethyl-3,5-dimethyl-	13,925–07-0	1084	0.00004	476.05 ± 23.48a	678.76 ± 320.28a	744.33 ± 133.95a	burnt, almond, roasted, nutty, coffee
Pyrazine, 3-ethyl-2,5-dimethyl-	13,360–65-1	1081	0.0086	2.21 ± 0.11a	3.16 ± 1.49a	3.46 ± 0.62a	potato, cocoa, roasted, nutty
2-Ethoxy-3-methylpyrazine	32,737–14-7	1065	0.0008	40.85 ± 1.8b	36.83 ± 1.77b	54.58 ± 6.23a	hazelnut, roasted, almond, pineapple, earthy
2H-Pyran-2-one, tetrahydro-6-methyl-	823–22-3	1095.14	0.02683	5.43 ± 0.87b	5.52 ± 0.24b	7.48 ± 0.13a	creamy, fruity, coconut
Pyrazine, 2-methoxy-3-(1-methylethyl)-	25,773–40-4	1097	0.000002	638,459.27 ± 24,584.45ab	585,253.33 ± 16,572.14b	701,907.93 ± 51,880.13a	beany, pea, earthy, chocolate, nutty
Ethanone, 1-(2-pyridinyl)-	1122-62-9	1034	0.1	5.76 ± 0.71b	6.81 ± 0.11a	5.83 ± 0.12b	popcorn, heavy, corn, chip, fatty, tobacco
Ethanone, 1-(2-thienyl)-	88–15-3	1092	0.001	5657.21 ± 195.35b	5227.77 ± 147.64b	6528.65 ± 620.74a	sulfury, nutty, hazelnut, walnut
Pyrazine, 2-ethyl-5-methyl-	13,360–64-0	1005	0.016	2.24 ± 0.09b	2.99 ± 0.19a	1.81 ± 0.16c	coffee, beany, nutty, grassy, roasted
2-Acetyl-3-methylpyrazine	23,787–80-6	1082	0.02	2.81 ± 0.18b	3.51 ± 0.1a	2 ± 0.22c	nutty, flesh, roasted hazelnut, toasted grain, corn, chip, vegetable, nut skin, caramel
5-Thiazoleethanol, 4-methyl-	137–00-8	1278	0.1	2.35 ± 0.03b	2.4 ± 0.01b	2.73 ± 0.06a	fatty, cooked, beefy, juice
Hydrocarbons
Tridecane	629–50-5	1300	0.042	1.97 ± 0.03b	2.23 ± 0.08b	2.86 ± 0.22a	alkane
Ketone
3,5-Octadien-2-one, (E,E)-	30,086–02-3	1073	0.0005	2691.75 ± 330.6ab	2367.3 ± 76.27b	2967.23 ± 319.63a	fruity, green, grassy
5,9-Undecadien-2-one, 6,10-dimethyl-, (E)-	3796-70-1	1453	0.01	6.44 ± 0.33ab	7.13 ± 0.34a	5.66 ± 0.51a	fresh, green, fruity, waxy, rose, woody, magnolia, tropical
Cyclohexanone, 2,2,6-trimethyl-	2408-37-9	1036	0.0001	1554.86 ± 158.82a	1435.76 ± 34.02a	1584.99 ± 30.26a	pungent, thujone, labdanum, honey, cistus
4-Undecanone	14,476–37-0	1208	0.041	0.94 ± 0.04b	0.8 ± 0.06c	1.23 ± 0.1a	fruity
Nitrogen compounds
Dodecanenitrile	2437-25-4	1490	0.00009	101.51 ± 4.64a	89.2 ± 6.82ab	81.83 ± 6.8b	citrus, orange, peel, metallic, spicy
Phenol
Phenol, 2,4-dichloro-	120–83-2	1171	0.0014	20.22 ± 3.41b	40.84 ± 2.89a	41.53 ± 6.87a	–
p-Cresol	106–44-5	1073.39	0.00024	448.54 ± 50.79b	475.63 ± 16.12ab	558.21 ± 50.41a	phenol, narcissus, animalic, mimosa
Sulfur compounds
Propanoic acid, 3-(methylthio)-	646–01-5	1092.92	0.05	1.09 ± 0.04b	0.9 ± 0.06c	1.23 ± 0.06a	sweet, sulfury
Terpenoids
D-Carvone	2244-16-8	1246	0.01	11.15 ± 0.04ab	10.56 ± 1.71b	13.34 ± 1.55a	spice, minty, bread, caraway
o-Cymene	527–84-4	1022	0.01144	6.05 ± 0.22a	4.53 ± 0.94b	5.09 ± 0.17ab	gasoline
Copaene	3856-25-5	1376	0.006	4.3 ± 0.85a	2.74 ± 0.12b	4.85 ± 0.12a	woody, spicy, honey
trans-.beta.-Ocimene	3779–61–1	1049	0.034	16.46 ± 0.27b	20.34 ± 0.56a	15.36 ± 0.65c	sweet, herbal
.beta.-Pinene	127–91-3	979.72	0.14	5.33 ± 0.34b	7.36 ± 0.3a	3.98 ± 0.35c	dry, woody, resinous, pine, hay, green
(2S,4R)-4-Methyl-2-(2-methylprop-1-en-1-yl)tetrahydro-2H-pyran	3033-23-6	1110	0.0002	1477.66 ± 51.79b	1457.49 ± 87.84b	1827.55 ± 119.77a	rose, cortex, green, floral, geranium, powdery, metallic
2,4,6-Octatriene, 2,6-dimethyl-	673–84-7	1131	0.034	7.19 ± 0.37b	8.19 ± 0.89b	9.91 ± 0.19a	sweet, floral, nut skin, peppery, herbal, tropical
2,4,6-Octatriene, 2,6-dimethyl-, (E,Z)-	7216-56-0	1131	0.034	7.19 ± 0.37b	8.19 ± 0.89b	9.91 ± 0.19a	–
2H-Pyran, tetrahydro-4-methyl-2-(2-methyl-1-propenyl)-	16,409–43-1	1112.96	0.0002	1477.66 ± 51.79b	1457.49 ± 87.84b	1827.55 ± 119.77a	sweet, floral, aromatic, rose, fresh, bay, leafy
Bicyclo[2.2.1]heptan-2-ol, 1,7,7-trimethyl-, (1S-endo)-	464–45-9	1170	0.048	11.12 ± 0.08b	11.43 ± 0.38b	17.32 ± 1.59a	pine, woody, camphor
Citronellal	106–23-0	1154.55	0.06	0.69 ± 0.01b	0.71 ± 0.01b	1.08 ± 0.10a	sweet, dry, floral, herbal, waxy, aldehydic, citrus
Geranyl acetate	105–87-3	1384.01	0.1	0.93 ± 0.04b	0.98 ± 0.03b	1.25 ± 0.07a	lemon
Linalool	78–70-6	1100.59	0.006	348.95 ± 14.23b	341.17 ± 3.81b	460.01 ± 55.62a	floral, green
endo-Borneol	507–70-0	1170.42	0.18	2.97 ± 0.02b	3.05 ± 0.1b	4.62 ± 0.42a	pine, woody, camphor, balsamic

3.5. Influence of adding tea-derived bacteria during fermentation on black tea fragrance

In the NBT_vs_CBT, WBT_vs_CBT and PBT_vs_CBT groups, 190, 199 and 229 differential volatile compounds were screened, respectively (Table S6), which were mainly terpenoids, hydrocarbons, heterocyclic compounds, esters, aldehydes and alcohols (Fig. 5A). The majority of these compounds were decreased in black tea fermented by adding tea-derived bacterias (Fig. 5A). Among these substances, 51 were affected by Bacillus amyloliquefaciens N1, 40 by Lysinibacillus fusiformis WL-B, and 90 by Enterobacter hormaechei P (Fig. S4B). Among the 66 compounds with rOAV >1 (Table S6, Table 2), except Styrene, Benzene, 1,4-diethyl-, p-Cymene, Propanoic acid, 2-methyl-, 2-methylbutyl ester, 2-Acetyl-3-methylpyrazine, Pyrazine, 2-ethyl-5-methyl-, trans-.beta.-Ocimene, and beta.-Pinene, all others exhibited higher concentrations in the CBT group (Fig. 5B). Compounds elevated in adding tea-derived bacteria fermentation groups exhibited nutty and woody attributes (Tables S6, Table 2, Fig. 5C), whereas those decreased in adding tea-derived bacteria fermentation groups showed green, sweet, floral, waxy, fruity, and fatty fragrance (Tables S6, Table 2, Fig. 5D). These results suggest that adding tea-derived bacteria during fermentation attenuated green and sweet aroma of black tea.

Fig. 5.

Influence of adding tea-derived bacteria fermentation on black tea fragrance. (A) Types of differential volatile components screened from NBT_vs_CBT, WBT_vs_CBT, and PBT_vs_CBT groups. (B) Heatmap of differential volatile components with rOAV>1 screened from NBT_vs_CBT, WBT_vs_CBT, and PBT_vs_CBT groups. (C) Flavor radarchart of volatiles (rOAV>1) with enhanced levels in tea-derived bacteria fermentation groups (RBT and FBT). (D) Flavor radarchart of volatiles (rOAV>1) with decreased contents in tea-derived bacteria fermentation groups (RBT and FBT).

Table 2.

Key volatile components (rOAV>1) contribute to the aroma of black tea fermented by adding tea-derived bacteria.

Volatiles	CAS	RI	Threshold	rOAV				Odor
				NBT	WBT	PBT	CBT
Acid
Benzeneacetic acid	103–82-2	1262	0.135	1.59 ± 0.07b	2.28 ± 0.86b	1.23 ± 0.35b	4.36 ± 0.71a	sweet, honey, floral, honeysuckle, sour, waxy, civet
Alcohol
2-Octen-1-ol, (E)-	18,409–17-1	1067.62	0.02	9.91 ± 0.34b	10.08 ± 0.62ab	8.86 ± 0.68b	11.23 ± 0.9a	green, citrus, vegetable, fatty
2-Nonanol	628–99-9	1099.20	0.058	1.66 ± 0.06c	1.94 ± 0.03b	1.82 ± 0.15bc	2.2 ± 0.17a	rose
Aldehyde
2-FurancarboxAldehyde, 5-methyl-	620–02-0	964.60	0.5	1.76 ± 0.1ab	1.64 ± 0.14bc	1.49 ± 0.09c	1.94 ± 0.15a	spice, caramel, maple
4-Nonenal, (E)-	2277-16-9	1105	0.0022	107.63 ± 5.8ab	100.5 ± 2.61bc	82.94 ± 7.72c	119.56 ± 15.75a	fruity
6-Nonenal, (Z)-	2277-19-2	1103.52	0.00014	20,080.5 ± 908.79b	20,450.35 ± 1560.88b	15,886.63 ± 1758.31c	24,273.62 ± 1280.66a	green, cucumber, melon, cantaloupe, honeydew, waxy, vegetable, orris, violet, leafy
cis-7-Decen-1-al	21,661–97-2	1212	0.0022	107.17 ± 2.28a	36.16 ± 57.73b	93.28 ± 7.56b	123.98 ± 2.91a	citrus, aldehydic, cucumber
2-Nonenal	2463-53-8	1161	0.0001	588.22 ± 38.97b	489.44 ± 11.73c	496.42 ± 15.29c	671.12 ± 55.06a	fatty, green, waxy, cucumber, melon
(E)-2-Decenal	3913-81-3	1263	0.005	90.62 ± 21.52c	124.35 ± 9.31ab	113.58 ± 15.28bc	150.55 ± 2.45a	waxy, fatty, earthy, green, cilantro, mushroom, aldehydic, fried, chicken, fatty, tallow
2-Nonenal, (E)-	18,829–56-6	1161.21	0.00008	2710.23 ± 205.06b	2509.19 ± 340.15b	2469.43 ± 150.42b	3633.95 ± 342.31a	fatty, green, cucumber, aldehydic, citrus
2-Nonenal, (Z)-	60,784–31-8	1148	0.0045	192.83 ± 16.9b	177.67 ± 10.76b	181.9 ± 11.18b	267.6 ± 26.18a	orris, fatty, waxy, cucumber
Amine
BenzenAmine, N,N-dimethyl-	121–69-7	1101	0.012	6.15 ± 0.33a	5.87 ± 0.11a	4.83 ± 0.59b	6.51 ± 0.51a	–
Aromatics
Benzene, nitro-	98–95-3	1080	0.15	1.4 ± 0.13c	1.61 ± 0.02ab	1.5 ± 0.11bc	1.71 ± 0.1a	–
Styrene	100–42-5	893	0.0036	8.08 ± 0.77ab	10.12 ± 2.99ab	10.73 ± 1.54a	7.1 ± 0.82b	penetrating, balsamic, gasoline
Benzene, 1,4-diethyl-	105–05-5	1041	0.0021	11.68 ± 1.05a	12.63 ± 1.09a	10.9 ± 2.4a	10.02 ± 0.83a	–
p-Cymene	99–87-6	1025.99	0.0114	2.15 ± 0.19a	2.33 ± 0.2a	2.01 ± 0.44a	1.85 ± 0.15a	woody, citrus
Ester
Butanoic acid, 3-methyl-, 2-phenylethyl ester	140–26-1	1491	0.00001	5062.62 ± 223.37a	4548.14 ± 2588.85a	5717.35 ± 554.59a	6486.21 ± 156.42a	floral, fruity, sweet, rose, peach, apricot
Propanoic acid, 2-methyl-, 2-methylbutyl ester	2445-69-4	1016	0.014	8.78 ± 0.31b	10.76 ± 1.34a	9.74 ± 1.02ab	8.64 ± 0.37b	fruity, ethereal, tropical, banana
2(5H)-Furanone, 3-hydroxy-4,5-dimethyl-	28,664–35-9	1110	0.011	147.57 ± 6.91a	143.12 ± 10.01a	113.32 ± 10.62b	165.9 ± 16.7a	extremely sweet, strong caramel, maple, burnt, sugar, coffee
Heptanoic acid, ethyl ester	106–30-9	1097.80	0.002	2.37 ± 0.16ab	2.25 ± 0.34ab	1.97 ± 0.15b	2.59 ± 0.26a	fruity, pineapple, cognac, rummy, wine
Methyl salicylate	119–36-8	1200	0.04	79.44 ± 3.65bc	84.1 ± 4.9b	70.03 ± 8.37c	111.69 ± 8.57a	caramel, pepperminty
Acetic acid, hexyl ester	142–92-7	1013.05	0.115	2.05 ± 0.04b	2.05 ± 0.08b	1.78 ± 0.14c	2.37 ± 0.06a	fruity, green, apple, banana, sweet
2,4-Decadienoic acid, ethyl ester, (E,Z)-	3025-30-7	1479	0.1	1.01 ± 0.08b	0.93 ± 0.04b	0.95 ± 0.15b	1.27 ± 0.08a	green, waxy, pear, apple, sweet, fruity, tropical
Propanoic acid, 2-methyl-, propyl ester	644–49-5	859.34	0.000086	17.69 ± 17.15a	23.73 ± 4.78a	18.28 ± 7.28a	36.36 ± 2.69a	sweet, ripe fruit, tropical, melon, berry
2(3H)-Furanone, 5-butyldihydro-	104–50-7	1261	0.0179	18.07 ± 0.16b	17.61 ± 1.06b	16.14 ± 1.71b	24.85 ± 1.41a	sweet, coconut, waxy, creamy, tonka, dairy, fatty
Heterocyclic compound
2(5H)-Furanone, 5-ethyl-	2407-43-4	966	0.0097	88.67 ± 5.13a	82.75 ± 6.56ab	75.91 ± 4.12b	92.03 ± 2.5a	spice
2-Furanmethanol, 5-ethenyltetrahydro-.alpha.,.alpha.,5-trimethyl-, cis-	5989-33-3	1074	0.32	13.02 ± 0.48ab	13.6 ± 1.11a	11.64 ± 0.93b	14.58 ± 1.17a	earthy, floral, sweet, woody
2-Thiophenemethanethiol	6258-63-5	1105	0.00004	6122.53 ± 218.58a	5887.4 ± 406.93a	4872.61 ± 604.76b	6694.32 ± 623.33a	roasted, coffee, fishy
5-Thiazoleethanol, 4-methyl-	137–00-8	1278	0.1	2.36 ± 0.05a	1.14 ± 0.97b	2.05 ± 0.17ab	2.73 ± 0.06a	fatty, cooked, beefy, juice
Ethanone, 1-(2-thienyl)-	88–15-3	1092	0.001	5670.4 ± 181.42b	5736.17 ± 237.69b	5199.57 ± 439.61b	6528.65 ± 620.74a	sulfury, nutty, hazelnut, walnut
Pyrazine, (2-methylpropyl)-	29,460–92-2	1074	0.4	8.15 ± 0.29ab	8.47 ± 0.23a	7.47 ± 0.59b	8.66 ± 0.43a	green, vegetable, fruity
Pyrazine, 2-ethyl-3,5-dimethyl-	13,925–07-0	1084	0.00004	607.94 ± 35.43ab	543.53 ± 26.93bc	436.36 ± 36.57c	744.33 ± 133.95a	burnt, almond, roasted, nutty, coffee
Pyrazine, 2-methoxy-3-(1-methylethyl)-	25,773–40-4	1097	0.000002	596,324.43 ± 55,314.76b	635,632.1 ± 11,215.83ab	586,013.33 ± 46,433.9b	701,907.93 ± 51,880.13a	beany, pea, earthy, chocolate, nutty
Pyridine, 2-pentyl-	2294-76-0	1202	0.0006	16,868.58 ± 799.02b	15,065.64 ± 900.56c	7314.23 ± 527.29d	19,549.46 ± 1314.82a	fatty, tallow, green, pepper, mushroom, herbal
Pyrazine, 3-ethyl-2,5-dimethyl-	13,360–65-1	1081	0.0086	2.83 ± 0.16ab	2.53 ± 0.13bc	2.03 ± 0.17c	3.46 ± 0.62a	potato, cocoa, roasted, nutty
trans-Linalool oxide (furanoid)	34,995–77-2	1075.24	0.19	21.92 ± 0.81ab	22.9 ± 1.87a	19.61 ± 1.56b	24.56 ± 1.98a	floral
2-Acetyl-3-methylpyrazine	23,787–80-6	1082	0.02	2.57 ± 0.14bc	3.51 ± 0.12a	2.83 ± 0.78ab	2 ± 0.22c	nutty, flesh, roasted hazelnut, toasted grain, corn, chip, vegetable, nut skin, caramel
Pyrazine, 2-ethyl-5-methyl-	13,360–64-0	1005	0.016	2.25 ± 0.04b	3.1 ± 0.32a	2.53 ± 0.67ab	1.81 ± 0.16a	coffee, beany, nutty, grassy, roasted
Benzothiazole	95–16-9	1228	0.08	1.05 ± 0.11b	1.06 ± 0.03b	0.87 ± 0.08c	1.3 ± 0.06a	meaty, vegetable, brown, cooked, beefy, coffee
2-Ethoxy-3-methylpyrazine	32,737–14-7	1065	0.0008	33.75 ± 2.01b	34.96 ± 2.41b	31.14 ± 3.79b	54.58 ± 6.23a	hazelnut, roasted, almond, pineapple, earthy
Hydrocarbons
Tridecane	629–50-5	1300	0.042	2.28 ± 0.14b	2.37 ± 0.39ab	2.34 ± 0.33ab	2.86 ± 0.22a	alkane
Ketone
1-(4-methylphenyl)-Ethanone	122–00-9	1183	0.021	14.49 ± 0.62b	13 ± 0.84bc	11.46 ± 0.8c	18.35 ± 1.76a	green, pea, bell pepper, galbanum
2-Butanone, 4-(2,6,6-trimethyl-1-cyclohexen-1-yl)-	17,283–81-7	1433	0.0036	0.78 ± 0.12b	0.98 ± 0.05a	0.92 ± 0.15ab	1.11 ± 0.04a	earthy, woody, mahogany, orris, dry, amber
1-Octen-3-one	4312-99-6	976	0.000005	1423.84 ± 123.83bc	1694.31 ± 119.39ab	1012.42 ± 155.48c	2005.68 ± 397.52a	mushroom
2-Cyclopenten-1-one, 2-hydroxy-3,4-dimethyl-	21,835–00-7	1075	0.02	108.31 ± 4.43b	130.76 ± 1.52a	117.1 ± 9.11b	136.04 ± 7.19a	strong, caramel
4-Undecanone	14,476–37-0	1208	0.041	0.88 ± 0.07b	1.1 ± 0.18a	0.83 ± 0.04b	1.23 ± 0.1a	fruity
2-Butanone, 4-(2,6,6-trimethyl-2-cyclohexen-1-yl)-	31,499–72-6	1406	0.0017	6.97 ± 0.35b	7.38 ± 0.58b	8.22 ± 1.38b	11.18 ± 0.5a	woody, floral, berry, orris, powdery, violet, raspberry, fruity
Phenol
Phenol, 2,4-dichloro-	120–83-2	1171	0.0014	19.2 ± 2.66c	23.99 ± 7.95bc	30.96 ± 3.5ab	41.53 ± 6.87a	–
p-Cresol	106–44-5	1073.39	0.00024	460.67 ± 7.39b	472.45 ± 17.67b	458.17 ± 42.75b	558.21 ± 50.41a	phenol, narcissus, animalic, mimosa
Sulfur compounds
Propanoic acid, 3-(methylthio)-	646–01-5	1092.92	0.05	0.93 ± 0.04c	1.07 ± 0.02b	1 ± 0.07bc	1.23 ± 0.06a	sweet, sulfury
Terpenoids
Geranyl acetate	105–87-3	1384.019	0.1	0.96 ± 0.03ab	0.71 ± 0.34b	1.15 ± 0.2a	1.25 ± 0.07a	lemon
trans-.beta.-Ocimene	3779–61–1	1049	0.034	18.36 ± 0.47a	18.72 ± 2.57a	17.38 ± 3.55a	15.36 ± 0.65a	sweet, herbal
o-Cymene	527–84-4	1022	0.01144	4.53 ± 1.77a	3.55 ± 0.55a	3.9 ± 0.61a	5.09 ± 0.17a	gasoline
Cyclohexene, 1-methyl-4-(1-methylethylidene)-	586–62-9	1090.669	0.2	31.42 ± 1.55a	29.28 ± 1.05a	23.92 ± 2.5b	31.48 ± 2.3a	citrus, pine
Linalool	78–70-6	1100.5889	0.006	410.98 ± 21.3ab	381.81 ± 13.02b	310.93 ± 32.44c	460.01 ± 55.62a	floral, green
.beta.-Pinene	127–91-3	979.72	0.14	7.29 ± 0.3a	6.95 ± 1.15a	6.02 ± 1.49a	3.98 ± 0.35b	dry, woody, resinous, pine, hay, green
Copaene	3856-25-5	1376	0.006	2.88 ± 0.1b	3.07 ± 0.53b	3.67 ± 0.8b	4.85 ± 0.12a	woody, spicy, honey
(2S,4R)-4-Methyl-2-(2-methylprop-1-en-1-yl)tetrahydro-2H-pyran	3033-23-6	1110	0.0002	1560.85 ± 64.23b	1515.84 ± 61.55b	1305.27 ± 77.25c	1827.55 ± 119.77a	rose, cortex, green, floral, geranium, powdery, metallic
2,4,6-Octatriene, 2,6-dimethyl-	673–84-7	1131	0.034	4.76 ± 3b	5.82 ± 0.72b	6.38 ± 1.01b	9.91 ± 0.19a	sweet, floral, nut skin, peppery, herbal, tropical
2,4,6-Octatriene, 2,6-dimethyl-, (E,Z)-	7216-56-0	1131	0.034	4.76 ± 3b	5.82 ± 0.72b	6.38 ± 1.01b	9.91 ± 0.19a	–
2H-Pyran, tetrahydro-4-methyl-2-(2-methyl-1-propenyl)-	16,409–43-1	1112.96	0.0002	1560.85 ± 64.23b	1515.84 ± 61.55b	1305.27 ± 77.25c	1827.55 ± 119.77a	sweet, floral, aromatic, rose, fresh, bay, leafy
2,6-Octadien-1-ol, 3,7-dimethyl-, (Z)-	106–25-2	1230.47	0.049	4.19 ± 0.26c	4.56 ± 0.16b	3.82 ± 0.18d	6 ± 0.05a	lemon, fresh
D-Carvone	2244-16-8	1246	0.01	7.71 ± 0.19c	10.41 ± 0.63b	9.75 ± 0.83b	13.34 ± 1.55a	spice, minty, bread, caraway
Bicyclo[2.2.1]heptan-2-ol, 1,7,7-trimethyl-, (1S-endo)-	464–45-9	1170	0.048	12.55 ± 1.14b	11.6 ± 0.71b	11.97 ± 0.8b	17.32 ± 1.59a	pine, woody, camphor
Citronellal	106–23-0	1154.55	0.06	0.77 ± 0.06b	0.72 ± 0.04b	0.74 ± 0.05b	1.08 ± 0.1a	sweet, dry, floral, herbal, waxy, aldehydic, citrus
endo-Borneol	507–70-0	1170.42	0.18	3.35 ± 0.3b	3.09 ± 0.19b	3.19 ± 0.21b	4.62 ± 0.42a	pine, woody, camphor, balsamic

3.6. Pathways involved in the effect of adding tea-derived microorganism fermentation on black tea aroma

To elucidate the potential mechanism by which adding tea-derived microorganism fermentation on the aroma of black tea, KEGG enrichment analysis of differential volatile compounds screen from MFBT_vs_ CBT was performed. As shown in Fig. 6, the addition of tea-derived microorganism during fermentation mainly affected the monoterpenoid biosynthesis. Apart from (−)-beta-Pinene，the content of compounds enriched in this pathway were reduced by tea-derived microorganism. This indicates that exogenously adding tea-derived microbe during fermentation primarily affects black tea aroma by suppressing monoterpenoid biosynthesis, thereby reducing sweet and green fragrance while enhancing nutty and fruity-like green odor.

Fig. 6.

Pathways involved in the effect of adding tea-derived microorganism fermentation on black tea aroma.

4. Discussion

Currently, research focused on the microorganisms in black tea remains limited. A recent study identified bacteria, fungi and yeast during black tea processing samples (Karunaratne et al., 2024). Similarly, in the present study, bacteria and yeast were isolated from samples at different processing stages of black tea, whereas no fungi were found. This may be attributed to the variations in processing environments like air humidity and microbial distribution (Agarwal et al., 2025). Among the 15 initially identified strains, Debaryomyces hansenii, Pantoea sp, and Mammaliicoccus sciuri were more abundant. This is aligning with previous studies in which Pantoea, Pseudomonas, Paucibacter, and Cladosporium were identified as the main microbial genera (Wang et al., 2024) and Debaryomyces, Candida, and Wickerhamomyces were the major yeast genera (Karunaratne et al., 2024) in black tea. This suggests that the formation of black tea quality not only relies on enzymatic oxidation, glycosidic bond cleavage and recombination, and the Maillard reaction (Bokuchava & Skobeleva, 1980), the microbial community present during processing also plays crucial roles.

It has been demonstrated that microbial fermentation can effectively improve black tea quality (Wang et al., 2023), primarily through metabolic activities and enzyme secretion (Wang et al., 2024). In this study, the addition of tea-derived microbe during fermentation increased water extract content while reduced free amino acid levels to some extent, which is consistent with the result of Wang et al. (2024). This may because amino acids were converted into volatile organic compounds during fermentation through deamination, decarboxylation, oxidation reactions, and Maillard reactions (Hu et al., 2021). Simultaneously, amino acids may be served as carbon and nitrogen sources during fermentation (Zhang et al., 2025). However, the gallic acid content was decreased in MFBT, which is contrary to Shi et al. (2021), and this may due to differences in microbial species. Notably, unlike the pile-fermentation of dark tea, the addition of microbe during black tea fermentation did not increase soluble sugar content, probably attribute to the differences of strains in dark tea and black tea. Interestingly, the content of theabrownins increased in MFBT, suggesting that microorganisms may involving in the enzymatic oxidation of catechins (Cheng et al., 2025). However, the precise mechanisms require further investigation. Consistent with Wang et al. (2023), adding microbes during fermentation decreased the levels of ester catechins, indicating that adding tea-derived microbe can reduce astringency and improve sensory quality of black tea. Notably, caffeine was elevated in microbial-fermented groups, presumably due to the promotional effect of these microorganisms on the synthesis of caffeine. And this result is aligning with previous observations of increased caffeine levels in fungal and yeast fermentation systems (Wang et al., 2005). Collectively, among the five tea-derived microbes, the quality of black tea fermented with the addition of Hanseniaspora uvarum RL-B and Bacillus amyloliquefaciens N1 were closer to that of the normal fermented group.

Results showed that adding tea-derived microbe fermentation altered the composition of black tea volatile compounds, including terpenoids, hydrocarbons, esters, and aromatics, which are the main substances contributing to the aroma of black tea. However, unlike dark tea (Chen et al., 2021), addition of microbial decreased the content of overall volatile compounds, which aligns partially with prior findings where fermentation with Bacillus spp. decreased ketones and aldehydes content in dark tea (Yang et al., 2025). Nevertheless, the levels of alcohols, esters, and terpenoids were elevated in the previous study (Yang et al., 2025). This discrepancy arises from differences in processing conditions like temperature and fermentation time, as well as microbial strains used. Compounds (rOAV >1) reduced in MFBT groups exhibited sweet and floral fragrance, such as linalool and phenylacetaldehyde. This is inconsistent with previous studies using single-microorganism fermentation of dark tea (Xiao et al., 2024; Zhang et al., 2025), potentially resulted from distinct effects on the synthesis and hydrolysis of volatile compounds of different microorganisms. As it has been documented that certain microorganisms facilitate the hydrolysis of linalool into geraniol and geranial during fermentation, thus leading to its reduced content in tea (Marmulla et al., 2016).

Conversely, tea-derived microbe fermentation slightly increased the content of compounds (rOAV >1) presenting nutty and woody notes, in which substances exhibiting nutty aroma were primarily pyrazines like 2-Acetyl-3-methylpyrazine and Pyrazine, 2-ethyl-5-methyl-. The elevated pyrazine levels can be attributed to microbial degradation of proteins and amino acids in tea leaves, which supplied essential amino source for pyrazine synthesis (Yu et al., 2021). Besides, pyrazines may undergo further degradation into volatile aldehydes, ketones, and alcohols (Liu & Quan, 2024), thereby enriching the overall flavor profile. Interestingly, both the elevated and decreased volatile substances presented green odor, a fragrance compromise both fresh fruits and leaves. Adding tea-derived microbe fermentation increased aldehydes associated with green-fruity odor (including 2,6-nonadienal, predominantly found in melon, and 3-hexenal), a result may be attributed to the promotion of fatty acid oxidation by microorganisms (Gong et al., 2024), which endow black tea with fruity green aroma. In contrast, substances exhibiting green odor that decreased in the tea-derived microorganism-fermented groups included 2-Nonenal and 6-Nonenal, which are typically present in fresh grass. This indicates that the addition of tea-derived microbial fermentation enhanced the green odor of fruits like citrus, while reduced the green odor of fresh leaves. Overall, the volatile compound results indicate that adding tea-derived microbial fermentation imparts fruity and nutty flavors to black tea. This is consistent with the result observed in wine fermented by using Hanseniaspora uvarum (Zhang et al., 2023). However, the findings differ from previous studies in which solid-state fermentation of dark tea with single strain enriched floral characteristics (Yang et al., 2025; Zhang et al., 2025). This discrepancy primarily stems from the extended fermentation duration of dark tea, which result in different flavor profiles.

Microorganisms can synthesize precursor substances for monoterpene synthesis like geranyl diphosphate via their own enzyme systems. And several microbes (streptomyces, Escherichia coli, and yeasts) have been widely employed in monoterpenoids biosynthesis currently (Helfrich et al., 2019; Kant et al., 2024). Nevertheless, the addition of tea-derived microbes during fermentation reduces the content of compounds (like (L)-alpha-Terpineol and Geraniol) derived from geranyl diphosphate in the present study. This reduction may be attributed to the inoculated bacteria or yeasts may utilize oxidases to oxidize monoterpenes, yielding derivatives such as alcohols, aldehydes, ketones, or carboxylic acids. Additionally, microbial secretion of reductases could lead to the reduction of monoterpenes (Gozari et al., 2021).

5. Conclusion

In conclusion, this study isolated 14 yeast and 33 bacteria from the processing samples of black tea, from which five tea-derived microbial strains were selected, namely, Debaryomyces hansenii FLB-A, Hanseniaspora uvarum RL-B, Bacillus amyloliquefaciens N1, Lysinibacillus fusiformis WL-B, and Enterobacter hormaechei P. Adding tea-derived microbes during fermentation increased the content of water extract, caffeine, and theabrownins, while decreased the level of soluble sugars, gallic acid, and ester catechins of black tea to a certain extent, with Hanseniaspora uvarum RL-B and Bacillus amyloliquefaciens N1 exerting superior effect. In terms of aroma, the addition of microbes during fermentation altered the types of black tea volatile compounds, primarily alcohols, esters, terpenoids, and hydrocarbons. Tea-derived microbes decreased the content of the substances that presenting sweet or floral fragrance, whereas increased those exhibiting nutty and fruity-like green odor of black tea, and it mainly achieved through affecting the monoterpeniod biosynthesis pathway.

CRediT authorship contribution statement

Siyu Liao: Writing – original draft, Methodology, Investigation, Data curation, Conceptualization. Hongting Mo: Resources, Investigation, Data curation. Yixuan Gao: Validation, Software, Resources, Investigation. Tunyaluk Bouphun: Software, Resources, Investigation. Wei Xu: Writing – review & editing, Funding acquisition, Conceptualization. Ling Lin: Writing – review & editing, Writing – original draft, Supervision, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

Supplementary material 1

Supplementary material 2

Supplementary material 3

Supplementary material 4

Data availability

Data will be made available on request.