
==== Front
Food Chem X
Food Chem X
Food Chemistry: X
2590-1575
Elsevier

S2590-1575(24)00631-X
10.1016/j.fochx.2024.101743
101743
Research Article
Revealing novel insights into the enhancement of quality in black tea processing through microbial intervention
Wang Ailing ab
Lei Qingqing ab
Zhang Beibei c
Wu Junhai ab
Fu Zheyang ab
He Jiangfeng ab
Wang Yanbo ab
Wu Xinying ce.xywu@gzu.edu.cn
ab⁎
a College of Liquor and Food Engineering, Guizhou University, Guiyang, Guizhou 550025, China
b Key Laboratory of Fermentation Engineering and Biological Pharmacy of Guizhou Province, Guiyang, Guizhou 550025, China
c Guizhou Guitianxia Shengxing Tea Industry Co., LTD, Zunyi, Guizhou 563000, China
⁎ Corresponding author at: College of Liquor and Food Engineering, Guizhou University, Guiyang, Guizhou 550025, China. ce.xywu@gzu.edu.cn
15 8 2024
30 10 2024
15 8 2024
23 10174321 5 2024
11 8 2024
14 8 2024
© 2024 The Authors. Published by Elsevier Ltd.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Black tea is highly favored by consumers worldwide, with enzymatic reactions being recognized as a pivotal factor influencing tea quality. The role of microorganisms in shaping the composition of black tea has emerged as a focus of research due to their involvement in enzyme catalysis and metabolic processes. In this study, full-length amplicon sequencing combined with qPCR more accurately reflected microbial profile, and Pantoea, Pseudomonas, Paucibacter, and Cladosporium were identified as the main microbial genera. Moreover, by comprehensively analyzing color, aroma, and taste components over time in black tea samples, correlations were established between the dominant genus and various quality factors. Notably, peroxidase activity levels, total soluble sugar content, and tea pigments concentration exhibited significant associations with the dominant genus. Consequently, this microbiological perspective facilitated the exploration of driving factors for improving black tea quality while establishing a theoretical foundation for quality control in industrial production.

Highlights

• Pantoea & Cladosporium were the predominant genera by Full-length amplicon sequencing.

• By means of qPCR analysis, the fermentation was the main period of microbial growth.

• Microorganisms play a vital role in shaping the color, aroma, and flavor of black tea.

• Dominant genera were positively related to total soluble sugar, tea pigments.

• Microbial related peroxidase contributed significantly to the quality of black tea.

Keywords

Black tea
Microbial diversity
Full-length amplicon sequencing
qPCR
Enzymes
Quality-related components
Correlation analysis
Chemical compounds

Methyl salicylate (PubChem CID: 4133)
Leaf alcohol (PubChem CID: 5281167)
Linalool (PubChem CID: 6549)
(Z)-2-Hexen-1-ol (PubChem CID: 5324489)
(E)-2-Hexen-1-ol (PubChem CID: 5318042)
Hexanal (PubChem CID: 6184)
Benzyl alcohol (PubChem CID: 244)
Leaf acetate (PubChem CID: 5363388)
Leaf aldehyde (PubChem CID: 5281168)
Phenethyl alcohol (PubChem CID: 6054)
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pmc1 Introduction

Black tea, which belongs to the category of fully fermented tea, has gained popularity among consumers worldwide due to its attractive flavor and health benefits (Assad, Ashaolu, Khalifa, Baky, & Farag, 2023). Currently, black tea accounts for 70–80% of the total global tea consumption and has become Chinese second-largest category for tea exports. The primary processing of black tea involves withering, rolling, fermentation, and drying. The harvested tea leaves were allowed to wilt at room temperature for 2–5 h, leading to the evaporation of some water in the fresh tea. This process activated various enzymes in the leaves, particularly hydrolytic enzymes, thereby facilitating the degradation of complex components such as proteins and terpenes. Through mechanical rolling at ambient temperature for 0.5–1 h, gentle pressure was applied to disrupt surface tissues and release intracellular substances from the tea leaves. This process promoted optimal interaction between leaf surface microorganisms, and enhanced both aroma and flavor profiles during tea fermentation (Ren et al., 2024). Following rolling, controlled conditions including a temperature range of 25–30 °C, relative humidity between 70 and 80%, and interstitial ventilation were maintained in a fermentation chamber for 4–10 h. During this period, the tea components were oxidized, degraded, or transformed into special aromas and colors with the combined action of microorganisms, enzymes and oxygen (Wang et al., 2022). Finally, the fermented tea is dried with hot air at a temperature of 80–95 °C to remove excess water, inactivate the enzyme and terminate fermentation. During drying, Maillard reaction, caramelization, and other reactions occurred within the tea components further enhancing its flavor profile (Wan, 2008). In summary, the distinctive flavor of black tea is gradually formed through changes in color, aroma, and taste during processing. The quantification of quality changes was achieved by determining the chemical components associated with color, aroma, and taste (Alasalvar et al., 2012). The aroma of black tea is closely related to its volatile compound content and composition. Therefore, gas chromatography–mass spectrometry (GC–MS) was used to identify volatile components associated with aroma. Wu et al.'s research found that there was a significant increase in derivatives group of phenylpropanoid and carotenoids while a decrease in volatiles group of terpene- and fatty acid-derived during black tea processing (Wu et al., 2019). Benzaldehyde accumulation was identified as a crucial compound for almond flavor in almond-flavored black tea (Wang et al., 2020). The taste attributes of black tea can be described by fundamental chemical compositions such as polyphenols, amino acids, flavonoids, water extract, total soluble sugar, and catechins. Tea polyphenols contribute predominantly to the bitter and astringent taste; total soluble sugars provide sweetness; caffeine contributes bitterness; various free amino acids impart fresh sweet and bitter tastes to black tea (Yue, Wang, Peng, Li, & Yang, 2023). The reddish-brown coloration observed in black tea infusion was determined by the relative levels of theaflavins (TFs), thearubigins (TRs), and theabrownins (TBs).

The alterations in composition contribute significantly to the quality of black tea, and previous studies have indicated that plant-derived enzymes played a pivotal role in catalyzing biochemical reactions that were primarily responsible for variations in composition (Wan, 2008). However, the microbial metabolism and its contribution to enzyme production have been overlooked. Oxidoreductases and hydrolases have been considered crucial enzymes involved in the transformation of substances during black tea processing, yet there is limited research on the impact of microorganisms and endophytes on the flavor constituents of tea. The application of high-throughput microbial sequencing technology has facilitated investigations into the diversity of bacteria and fungi participating in black tea processing, as well as their correlation with fundamental chemical composition. Nevertheless, further exploration is warranted to comprehend the interplay between microorganisms, functional enzymes, and components associated with color, aroma, and taste changes influencing black tea quality.

The advancement of microbial diversity detection and bioinformatics technology has created opportunities for further analysis of the role of microorganisms in black tea processing. Full-length amplicon sequencing, known for its superior accuracy and taxonomic resolution compared to other amplicon sequencing techniques, combined with real-time fluorescence quantitative PCR technology (qPCR), enables a comprehensive understanding of changes in microbial proliferation and composition within samples. Non-targeted based on GC–MS detection technology has been extensively employed in molecular basis analysis of food quality to uncover key flavor compounds. Enzyme activities serve as the manifestations of microbial activity, and is the key factor linking microbial metabolism and composition change. Therefore, our study dynamically assessed the absolute abundance of bacteria and fungi to identify the dominant genus using full-length amplicon sequencing technology combined with qPCR during black tea processing. By comparing functional differences in microbial flora between pre-processing samples and post-fermentation samples, we elucidated potential metabolic mechanisms underlying the impact of microorganisms on tea composition. Furthermore, by conducting dynamic assessments on changes in enzyme activity, basic chemical composition, and key volatile components closely related to quality formation during processing, associations were established between dominant bacteria and these factors to gain deeper insights into the connection between microorganisms and quality enhancement in black tea production. Building upon this microbiological perspective, the driving factors were explored for enhancing black tea quality while providing a theoretical foundation for quality control in industrial production.

2 Materials and methods

2.1 Collection of black tea samples

The sample came from the tea production area in Liupanshui City, Guizhou Province, China (26°08′N, 105°34′E). The tea samples were collected with a five-point sampling method at different processing stages: browish-green (BG); middle of withering (MW), end of withering (EW); middle of rolling (MR), end of rolling (ER); middle of fermentation (MF) and end of fermentation (EF). The sampling diagram was detailed in Supplementary material Fig. S1. During the withering stage, which lasted for 4 h, the temperature was maintained between 18 and 20 °C, while the relative humidity remained around 75%. The rolling stage took approximately 2 h, with the temperature ranging from 20 to 22 °C and a relative humidity of about 75%. The fermentation process extended over a period of 13 h, during which the temperature was kept between 20 °C and 22 °C, while maintaining a relative humidity of approximately 80%. Finally, these samples were placed in sterile sealed bags, stored in dry ice, transported to the laboratory and stored at −80 °C for testing.

2.2 Analysis of microbial diversity of black tea

2.2.1 DNA extraction of microorganisms

The sample was added to 100 mL of phosphate buffer solution (PBS) at pH 7.4, and then it was shaken, sonicated several times, and the eluate was collected by centrifugation. The precipitation obtained from multiple collections was used for DNA extraction. The microbial DNA from the precipitation was extracted using the soil genome DNA extraction kit (Tiangen Biochemical Technology Co., Ltd., Beijing).

2.2.2 Analysis of microbial diversity

The full-length region of bacteria, as determined by amplification of the extracted total DNA, was amplified using upstream primer 27F (5′-AGRGTTTGATYNTGGCTCAG-3′) and downstream primer 1492R (5′-TASGGHTACCTTGTTASGACTT-3′). Similarly, the full-length region of fungal ITS was amplified using upstream primer ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and downstream primer ITS4 (5′-TCCTCCGCTTATTGATATGC-3′). PCR reaction systems and procedures are described in Supplementary Table S1. The concentration and bands of the amplified PCR products were assessed using a NanoDrop 2000 microspectrophotometer (Thermo Fisher, Waltham, MA, USA) and agarose gel electrophoresis (Beijing Bomei Fuxin Technology Co., Ltd.), respectively. Following purification and quantification, the qualified PCR amplicons were subjected to sequencing on a PacBio sequel II platform. The resulting microbial sequences were analyzed using OIIME 2 software provided by Beijing Biomarker Biotechnology Co., Ltd. Effective sequences were clustered into operational taxonomic units (OTUs) based on a 97% similarity threshold. Representative OTU sequences were annotated against the Greengenes bacterial database and UNITE fungal database.

The alpha-diversity of microflora was analyzed using the Chao1 richness estimator, Ace richness estimator, Shannon-Wiener diversity index, Simpson diversity index, and Phylogenetic diversity index. The function of bacterial and fungal flora was predicted by PICRUST2 (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) and FUNGuild (Fungi Functional Guild), respectively. Raw sequencing reads from all samples can be accessed on the NCBI database under BioProject accession number PRJNA1081806.

2.2.3 Analysis of biomass

The total biomass of microorganisms in the samples was determined by qPCR (ABI7500, Applied Biosystems, Waltham, MA, USA). The total biomass of bacteria was analyzed by upstream primer 16SV34 (5’-ACTCCTACGGGGAGGCAGCAGCAG-3′) and downstream primer 16SV34 (5’-GGACTACHVGGGTWTCTAAT-3′). The total biomass of fungi was analyzed using upstream primer ITS1 (5’-CTTGGTCATTTAGAGGAAGTAA-3′) and downstream primer ITS1 (5’-TGCGTTCTTCATCGATGC-3′). Referring to Lou's method (Lou, Yang, Wang, Wu, & Xu, 2018), the absolute abundance of a microbial genus is the total microbial biomass (gene copy number) multiplied by the corresponding relative abundance obtained by amplicon sequencing.

2.3 Determination of enzyme activity

Firstly, the enzyme solution was prepared according to the method described by (Liu, Shi, Yuan, and Yue (2022) with certain modifications. A mixture of 1.5 g of tea powder, 0.9 g of Polyvinylpyrrolidone (PVP), a small quantity of quartz sand, and 15 mL of pre-cooled buffer was homogenized in a mortar. The extraction process was conducted for 24 h at a temperature of 4 °C and subsequently centrifuged at a speed of 4000 r/min for 10 min. Afterwards, the supernatant containing the crude enzyme solution was collected for determination of various enzyme activities. Finally, the enzyme activity could be converted into the amount of crude enzyme solution extracted per gram of dry sample.

2.3.1 Cellulase activity analysis

The cellulase activity was determined using the modified dinitrosalicylic acid (DNS) method, based on a previous study by (Saldana-Mendoza, Palacios-Ponce, Ruiz, Ascacio-Valdes, and Aguilar (2023). Cellulase activities were measured using 1.0% (w/v) carboxymethyl cellulose (CMC) as the substrate. After incubating the reaction mixture at 40 °C and pH 5.0 for 30 min, the amount of glucose produced was quantified at 540 nm by Multiskan Spectrum Microplate Spectrophotometer (Multiskan SkyHigh, Thermo Fisher, Waltham, MA, USA) with 3,5-dinitrosalicylic acid as the color indicator. One unit of cellulase activity was defined as producing l μg of glucose per minute from the substrate under these reaction conditions for every gram of sample used per minute. The standard curve equation for glucose was Y = 0.5973X-0.0404 (R2 = 0.991).

2.3.2 Pectinase activity analysis

The activity of pectinase was determined based on the work of Guimaraes et al. (2023) with some modifications. The activities of pectinases were measured by using 0.25% (w/v) pre-heated pectin solution as the substrate. The reaction mixture was incubated in a water bath at 40 °C, pH 5.6 for 30 min, the amount of generated galacturonic acid was measured at 540 nm using the 3,5-dinitrosalicylic acid agent as the color indicator. One unit of pectinase activity was defined by 1 g of sample per minute producing l μg of galacturonic acid from the substrate per minute under the above reaction conditions. The standard curve is the same as that of cellulase.

2.3.3 Amylase activity analysis

The determination of amylase activity was conducted following the method described by Liu, Shi, et al. (2022) with some modifications. Amylase activities were assessed using a 1% (w/v) soluble starch solution as the substrate. After incubating the reaction mixture in a water bath at 40 °C and pH 5.6 for 10 min, the generated amount of reducing sugars was measured at 540 nm using 3,5-dinitrosalicylic acid agent as color indicator. One unit of amylase activity was defined as producing l μg of reducing sugar liberated from soluble starch per minute. The standard curve is the same as that of cellulase.

2.3.4 β-Glucosidase activity analysis

The determination method of β-glucosidase was slightly enhanced based on the protocols established by Supriyadi, Nareswari, and Fitriani (2021). The enzymatic activity of β-glucosidase was assessed using 10 mmol/L p-NP-β-D-glucopyranoside as the substrate. Following a 30-min incubation in a water bath at pH 5.0 and 37 °C, the change in absorbance at 420 nm was measured using an enzyme marker. One unit of β-glucosidase activity was defined as an increase in absorbance of 0.001 per minute resulting from the hydrolysis of p-NP-β-D-glucopyranoside under the aforementioned reaction conditions.

2.3.5 Determination of the activity of peroxidase

The peroxidase activity was quantified according to a previously published method (Koohi et al., 2020). The crude enzyme was added into the reaction system containing 0.3% (w/v) mM guaiacol and H2O2. The dynamic changes in absorbance at 470 nm were monitored to measure the peroxidase-induced reaction at pH 5.6 and 35 °C. One unit of peroxidase activity was defined as an increase in absorbance of 0.001 per minute resulting from the hydrolysis of guaiacol under the aforementioned experimental conditions.

2.3.6 Polyphenol oxidase activity analysis

The determination of polyphenol oxidase activity was conducted by Liu, Shi, et al. (2022) and Liu, Wang, et al. (2022). Catechol was catalyzed by polyphenol oxidase for 10 min under the conditions of pH 5.6 and 37 °C. The dynamic measurement of absorbance change induced by polyphenol oxidase was performed, with one unit of activity defined as an increase in absorbance at 460 nm by 0.001 per minute.

2.3.7 Lipoxygenase activity analysis

The lipoxygenase activity assay method was conducted in accordance with our previously established protocol (Wu et al., 2023; Wu et al., 2023). In this procedure, the crude enzyme was added to a reaction solution containing linoleic acid, Tween 20, and sodium hydroxide (0.10 mol/L) at pH 6.5. Lipoxygenase activity was determined kinetically by measuring the absorbance change at 234 nm, with one unit defined as a change of 0.001 absorbance units per minute.

2.3.8 Glucoamylase activity analysis

The Glucoamylase activity was determined using the Dinitrosalicylic acid (DNS) method, as described in a previous study (Pasin et al., 2020). The reaction mixture, consisting of 2% (w/v) soluble starch, was incubated with crude enzyme at a temperature of 40 °C and pH value of 6.8 for 10 min. The resulting glucose generated from the reaction was quantified at an absorbance wavelength of 540 nm. One unit of glucoamylase activity was defined as l μg of glucose produced per minute under the aforementioned conditions. The standard curve for glucose was Y = 0.1732X + 0.0373 (R2 = 0.9901).

2.3.9 Alcohol dehydrogenase activity analysis

The determination of alcohol dehydrogenase activity is based on the method developed by Guo, Deng, and Lu (2019) with certain modifications. Alcohol dehydrogenase activities were assessed using a mixture consisting of 0.1 mol/L Glycine-NaOH buffer (pH 9.0), 5 mg/mL NAD+, and 1.7 mol/L ethanol as the substrate. One unit of alcohol dehydrogenase activity was defined as a change in absorbance of 0.001 per minute at 340 nm under the aforementioned reaction conditions.

2.4 Determination of fundamental chemical components

The black tea samples were ground into powder using a grinder (JXFSTPRP-64, Shanghai Jingxin Industrial Development Co.).

2.4.1 Water extract content

The water extract content in tea was measured according to the nationally recommended standards (GB/T 8305 2013). Two grams of tea powder were added to 100 mL of distilled water and incubated for 45 min in a boiling water bath. Subsequently, the filtered extract was concentrated and dried under vacuum conditions, and finally, the water extract content was determined based on the weight of the dried sample.

2.4.2 Total soluble sugar

The total soluble sugar content was determined using the sulfuric acid-anthrone method as described by Wei et al. (2023). The extraction followed the water extract method, and the absorbance at 620 nm was measured using a Multiskan Spectrum Microplate Spectrophotometer. Finally, the quantification of total soluble sugar in tea samples was performed using glucose as a standard with a linear regression equation: Y = 2.9606X + 0.0851, R2 = 0.998.

2.4.3 Free amino acids

The determination of free amino acids was conducted in accordance with the nationally recommended standards (GB/T 8314–2013). Similarly, two grams of tea samples were added to 100 mL distilled water and subjected to boiling conditions for 45 min. Subsequently, the extraction was filtered under vacuum conditions. The absorbance value was measured at 570 nm, and the content of free amino acids was quantified using glutamic acid as a reference compound. A standard curve eq. Y = 0.861X + 0.024 (R2 = 0.9905) was employed.

2.4.4 Tea polyphenols and catechins

The determination of tea polyphenols and catechin content followed the Folin-phenol method specified by nationally recommended standards (GB/T8313–2018). Initially, two grams of powdered samples underwent extraction with 70% methanol under a water bath temperature of 70 °C. The concentration of tea polyphenols was quantified using a Multiskan Spectrum Microplate Spectrophotometer at a wavelength of 765 nm. A standard curve (Y = 0.0056X + 0.0544, R2 = 0.9961) was established with gallic acid as the reference material.

The content of catechins in extracts was determined by High Performance Liquid Chromatography (HPLC) equipped with a ZORBAX SB-Aq chromatographic column (4.6 × 250 mm, 5 μm). The mobile phase A was composed of 9% acetonitrile, 2% acetic acid, and a solution of EDTA-2Na (10 mg/mL), while the mobile phase B consisted of 80% acetonitrile, 2% acetic acid, and a solution of EDTA-2Na (10 mg/mL). The chromatographic conditions comprised a flow rate of 1 mL/min, column temperature maintained at 35 °C, injection volume of 10 μL, and detection wavelength set at λ = 278 nm using an ultraviolet detector. Gradient elution involved maintaining phase A for 10 min followed by transitioning to a mixture containing 68% A phase and 32% B phase within 15 min which was then sustained for another 10 min before returning to 100% A phase. There is a linear relationship between the concentration of gallic acid (GA) and the peak area, the equation is Y = 55.04X-12.4, the coefficient of determination R2 = 0.9993; the concentration of epigallocatechin (EGC) can be described by the equation of Y = 2.9358X-38.646 with a high determination coefficient of R2 = 0.9999; catechin (C): Y = 7.7021X-56.5, R2 = 0.9976; caffeine (CAF): Y = 28.259X + 17.198, R2 = 0.999; epicatechin (EC): Y = 9.7606X-44.861, R2 = 0.9999; epigallocatechin gallate (EGCG): Y = 14.003X-968.83, R2 = 0.993; and epicatechin gallate (ECG): Y = 16.98X-627.77, R2 = 0.9922.

2.4.5 Total flavonoids

Total flavonoids were analyzed with reference to Wang's method (Wang et al., 2023) with some modifications. At 510 nm, the content of total flavonoids in tea samples was quantized by rutin, and the standard curve was Y = 0.6301X + 0.0399, R2 = 0.9969.

2.4.6 Tea pigments

The content of tea pigments was determined by spectrophotometer at 380 nm with reference to the method of Wang, Peng, and Gong (2011).

2.5 Volatile components

Volatile compounds in the samples were detected using untargeted metabolomics based on headspace solid-phase microextraction gas chromatography–mass spectrometry (HS-SPME GC/MS, 7890 A-5975C, Agilent Technology (China) Co., LTD). A total of 2.00 ± 0.01 g of sample powder and 5 mL of distilled water were placed into a 20 mL headspace bottle along with 10 μL of internal standard (0.25 mg/mL 4-methyl-2-Pentanol as an internal standard, Tokyo Chemical Industry CoLtd, Japan). After oscillating the samples at a temperature of 60 °C for 15 min, volatiles were adsorbed for 60 min using an SPME fiber assembled with DVB/CAR/PDMS (50/30 μm, Divinylbenzene/Carboxen/Polydimethylsiloxane), and then desorbed at a temperature of 250 °C for 5 min. The volatile components of the samples were detected using a GC–MS system coupled with a DB-WAX column (30 mm × 0.25 mm × 0.25 μm, Agilent Technologies Ltd., Santa Clara, CA, USA). The chromatographic conditions included an inlet temperature set to be at 260 °C and maintaining the heating temperature at 40 °C for 3 min before increasing it to 180 °C at a rate of 2 °C/min for 10 min, and finally raising it to 220 °C at 10 °C/min. The mass spectrometer was operated in the forward mode of EI (Electron impact) ion source with electron impact ionization set at 70 eV. Ion source temperature was maintained at 230 °C and quadrupole temperature was kept 150 °C. The mass scanning range was 35–350 m/z. The carrier gas used was helium (99.99%) at a constant flow rate of 1 mL/min. Qualitative analysis was carried out by NIST11 library and quantitative analysis was carried out by internal standard method. Calculating by the following formula:(1) Ci=Vs×AiAs×m×Ws

The concentration of the components (μg/g) is denoted as Ci. The volume of the standard is represented by Vs, and its concentration in sampls (μg/L) is denoted as WS. Ai is the peak area of the component to be measured, and As is the peak area of the internal standard. Finally, m denotes the mass of the sample to be measured (g).

2.6 Statistical analysis

The determination of each result was based on a minimum of three parallel samples, taking into account the influence of moisture. Univariate analysis of variance (ANOVA) was employed to assess differences among the samples, followed by Duncan's test using SPSS 20.0 software (SPSS Inc., Chicago, IL, USA). The results were presented as mean ± standard deviation. The Spearman's correlation coefficients were computed using the R software package. A robust correlation was considered when |ρ| > 0.5. The range of Spearman correlation coefficient (ρ) lies between −1 and 1. A positive correlation exists when ρ ranges from 0 to 1; conversely, a negative correlation occurs when ρ ranges from −1 to 0. Furthermore, larger absolute values of ρ indicate stronger correlations. PLS-DA (a partial least squares-discriminant analysis) model was constructed and VIP (Variable Importance Projection) values were determined using SIMCA software (v13.0), Umetrics Inc., Sweden). Data visualization was performed with OriginPro software (v9.0, MicroCal Inc., MA, United States), R software (v4.2.1, University of Auckland, Auckland New Zealand), and Gephi software (v0.9.3; Web Atlas Paris France).

3 Results and discussion

3.1 Analysis of microbial diversity during black tea processing

In this study, the full-length amplicon sequencing technology was used to dynamically detect microbial diversity. During the processing, a total of 7 phyla, 8 classes, 13 orders, 16 families, 23 genus, and 31 species of bacteria were identified, as well as 9 phyla, 37 classes, 97 orders, 225 families, 431 genera, and 647 species of fungi. The α-diversity index of the samples was shown in supplementary Table S2. Good's coverage index of all sequences was above 99.00%, indicating that the sequencing depth could meet the needs of microbial diversity analysis. Shannon and Simpson indices reflected the diversity, while ACE and Chao1 indices reflected the richness of microorganisms. Therefore, the microbial composition in the MR and MF stages had the highest bacterial diversity and richness, respectively, however the BG and MR stages had the highest fungal diversity and richness. Fig. 1 shows the top 10 microbial genera in relative abundance, while “ Others “ represent microbes outside the top 10. As shown in Fig. 1A and B, the dominant genera of the samples before the withering stage were mainly Pantoea (23.25% - 83.68%) and Pseudomonas (9.93% - 74.79%). After withering stage, the dominant genera became Pantoea (33.35% - 91.09%) and Paucibacter (7.32% - 47.88%). Cladosporium (35.66% - 54.19%) was the fungus genus with the highest relative abundance throughout the processing of black tea. The biomass was measured by qPCR technology, and it was found that the microorganisms increased greatly through fermentation, which made the biomass of bacteria reach 9.03 × 1010 copies/g at EF, and the biomass of fungi reach 7.36 × 107 copies/g at MF. It is worth noting that bacterial biomass is much greater than that of the fungi. Fig. 1C and D respectively show the biomass composition of dominant bacteria and fungi in the processing after considering the total biomass.Fig. 1 Relative abundance of bacteria (A) and fungi (B), as well as absolute abundance of bacteria (C) and fungi (D) at the genus level in black tea samples during processing. BG: Browish-green, MW: Middle of withering, EW: End of withering, MR: Middle of rolling; ER: End of rolling, MF: Middle of fermentation, EF: End of fermentation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 1

During the processing of black tea, fermentation created an optimal environment for microorganism proliferation. As a result, microbial biomass reached its peak during this stage, highlighting the indispensable role of fermentation in facilitating microorganism activity throughout the process. Previous studies on black tea have identified Pantoea, Pseudomonas, Cladosporium, Thermoascus and Thermoomyces as functional bacteria that plaied important roles in determining black tea quality. For example, Pantoea has been found to be closely associated with catechins and tea polyphenols (Li et al., 2021), while Pseudomonas was not only a dominant bacterial genus in black tea but also plays a key role in kombucha fermentation (Meng, Wang, Li, Chen, & Chen, 2024). Cladosporium has been identified as the predominant functional microorganism in Liupao tea and was significantly positively correlated with catechin content, water extract content and total polyphenol content (Wang et al., 2021). Both Thermoascus and Thermoomyces were highly associated with fragrance-related aldehyde formation in black tea (Hu et al., 2021).

3.2 Prediction of flora function

During the processing of black tea, functional differences were observed in the changes of microflora structure. In this study, PICRUSt 2 and FUNGuild were utilized to predict bacterial and fungal flora functions, respectively. Based on the analysis of KEGG metabolic pathways, a total of 36 significantly different functions (p < 0.05) were identified in the bacterial flora between samples after fermentation and those at the BG stage, as illustrated in Fig. 2A. The up-regulated functions included carbohydrate metabolism, membrane transport, energy metabolism, replication and repair, nucleotide metabolism, biosynthesis of other secondary metabolites, glycan biosynthesis and metabolism among others; while down-regulated functions included amino acid metabolism, lipid metabolism, metabolism terpenoids and polyketides among others. The fungal community exhibited over 30 predominant functions as depicted in Fig. 2B. Notably during the BG stage Fungal Parasite-Plant Pathogen-Plant Saprotroph accounted for 30.15 ± 0.02% of relative abundance; however, after fermentation there was an increase in relative abundance to 37.26 ± 0.06% for Plant pathogen function. Undefined Fungal parasite-Plant Pathogen-Plant Saprotroph decreased significantly.Fig. 2 Prediction of functional profiles for bacterial (A) and fungal (B) communities. BG: Browish-green; EF: End of fermentation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Based on the predicted function, proliferation of bacteria and fungi, particularly endophytic fungi, was observed during the fermentation stage. The up-regulated functions primarily involved microbial proliferation and metabolism, including carbohydrate metabolism, membrane transport, replication and repair, energy metabolism, and nucleotide metabolism. The active carbohydrate metabolism with the highest relative abundance provided both necessary carbon source and energy for microbial growth while also accumulating total soluble sugars that contribute to the mellow-sweet taste of black tea. Most of the down-regulated metabolic pathways were associated with degradation or transformation of flavor precursors. Amino acid metabolism facilitates the production of aromatic substances such as aldehydes (e.g., phenylacetaldehyde) and alcohols (e.g., phenylethanol). Additionally, lipid metabolism along with terpenoid and polyketide metabolisms play crucial roles in flavor formation. For example, the formation of hexanal, (Z)-3-Hexanal and their derivatives and linalool, geraniol, etc., contributed to the grassy, fruity, and floral aromas of black tea (Liu et al., 2023). According to FUNGuild predictions, the majority of fungal flora functions were closely associated with endophytic microorganisms, indicating that both surface microorganisms and endophytes plaied a significant role in component change during black tea production. As the database and methods for fungal function continue to improve, prediction results based on amplified sequencing technology will become more comprehensive.

3.3 Analysis of enzyme activity

Enzymes are recognized as critical drivers of black tea quality during black tea processing, especially hydrolase and oxidoreductase. As illustrated in Fig. 3, dynamic analysis was conducted to investigate changes in enzyme activity during processing. Peroxidase, polyphenol oxidase, and lipoxygenase activities exhibited an increasing trend, while amylase, glucoamylase, pectinase, and β-glucosidase activities showed a decreasing trend. Amylase, glycosylase, pectinase, and β-glucosidases reached peak values at the withering stage. Lipoxygenases cellulases and β-glucosidases displayed the highest activity levels at the rolling stage. Conversely, peroxidase, polyphenol oxidase, and alcohol dehydrogenase revealed maximum activity levels at the fermentation stage.Fig. 3 Dynamic changes of the activities of alcohol dehydrogenase (A), cellulase (B), glucoamylase (C), peroxidase (D), lipoxygenase (E), pectinase (F), polyphenol oxidase (G), amylase (H) and β-glucosidase (I) in black tea samples during processing. BG: Browish-green, MW: Middle of withering, EW: End of withering, MR: Middle of rolling, ER: End of rolling, MF: Middle of fermentation, EF: End of fermentation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Investigating the variations in enzyme activities throughout each stage of processing can provide valuable insights into the changes occurring in flavor-related compounds. Amylase, pectinase, glucoamylase, and cellulase facilitate the degradation of their respective substrates into soluble sugars. These enzymes contribute to the development of sweetness in black tea and serve as a vital carbon source for microorganism proliferation during fermentation, which explains the observed increase in soluble total sugars depicted in Fig. 3. Simultaneously, hydrolases promote the efficient release of aroma precursors. β-glucosidase hydrolyzes the flavor substances in tea that bind to glucosides, so its high activity during the withering and rolling stages helped accumulate volatile flavor substances during the fermentation stage. Oxidoreductases play a significant role in degrading and converting polyphenols, flavonoids, and higher fatty acids leading to diverse volatile flavors such as alcohols, aldehydes, and acids (Wan, 2008). Polyphenol oxidase can oxidize flavanols in tea to quinone and further form complex compounds that contribute to tea pigment formation. Additionally, peroxidase also promotes pigment formation. Alcohol dehydrogenases catalyze REDOX reactions of aliphatic or aromatic alcohols and aldehydes while lipoxygenases catalyze oxygenation of unsaturated fatty acids containing cis, cis-1,-4-glutaryl resulting peroxide formation which then degrades into volatile aldehydes, alcohols, and other flavor compounds. During the fermentation stage, the high activity exhibited by these enzymes is found consistent with microbial biomass proliferation trend thereby indicating significant correlation between them and microorganisms.

3.4 Analysis of fundamental chemical components

The content of water extracts, total soluble sugars, free amino acids, tea polyphenols, total flavonoids, and caffeine in black tea contributed to the formation of its taste in the manufacturing process. Compared with the tea in the BG stage, each component played a different dynamic change. As shown in Fig. 4A, the content of water extract in tea increased significantly in the MW stage (p < 0.05) reached 71.11 ± 0.22%, and then gradually decreased. The content of total soluble sugars reached a high level at the MR and EF stages (Fig. 4B). The levels of free amino acids and tea polyphenols peaked at the EW stage, before gradually declining (Fig. 4C and D). The total flavonoid content significantly increased to a peak value of 2.44 ± 0.04% at the MR stage and then declined rapidly (Fig. 4E). The caffeine content exhibited fluctuations throughout processing, peaking at 0.83 ± 0.01% at the ER stage (Fig. 4F). Fig. 4G illustrated the dynamic variations of total catechins including GA, C, EC, EGC, and EGCG. The GA content ranged from approximately 0.0129% to 0.0167%. Compared to fresh tea, the levels of C and EGC in processed samples exhibited a significant decrease. The content of EGCG increased after the withering stage, and there was almost no change in the subsequent stage. However, there was little change in ECC content. At last, Fig. 4H showed, TBs increased rapidly, while TRs decreased, but TFs increased little during the fermentation stage.Fig. 4 Dynamic changes of the content of water extract (A), total soluble sugar (B), free amino acid (C), tea polyphenols (D), total flavonoid (E), caffeine (F), catechins (G) and tea pigments (H) in black tea samples during processing. Catechins consisting of epicatechin gallate (ECG), epigallocatechin gallate (EGCG), epigallocatechin (EGC), catechin (C), gallic acid (GA), and tea pigments including theaflavins (TFs), thearubigins (TRs) and theabrownins (TBs).

Fig. 4

Typically, non-volatile components in tea were important for taste, color, and health benefits, The presence of total soluble sugars and amino acids in tea contributesd to the pleasant sweetness and refreshing flavor. Caffeine, polyphenols, and flavonoids contributed to both its taste and health benefits. Catechins or tea tannins were a group of flavanols found in tea with antioxidant activity. Total catechins accounted for approximately 70% of the weight of tea polyphenols characterized by a robust astringent and bitter taste (Wan, 2008). In addition, caffeine was also one of the abundant alkaloids in tea and contributes to the bitter taste of tea. Consistent with previous research findings (Fu et al., 2024; Liu, Wang, et al., 2022), the levels of tea polyphenols, total flavonoids, and free amino acids exhibited a declining pattern throughout the tea process due to the synergistic effects of enzyme catalysis and microbial metabolism that generated volatiles. The fluctuation of total soluble sugar content was related to the consumption of microorganisms. The total catechin content were a result of both the liberation of free catechin and their subsequent oxidation into polymers, leading to the formation of tea pigments. The caffeine content increased significantly during the withering stage may stemmed from the production of the nucleosides and nucleotides, and the caffeine content decreased again during the fermentation probably due to the disintegration of the cell membrane (Chen et al., 2020). Tea pigments were plant phenolic pigments that were oxidized to form orthoquinone through oxidative polymerization mediated by endogenous or microbial polyphenol oxidase and peroxidase, which then polymerized into TFs, TRs, and TBs (Zhao, Yan, Li, Qu, & Xu, 2023).

3.5 Analysis of volatile compounds

The non-targeted detection technology based on GC–MS was employed to dynamically analyze the compositional changes of 28 samples. A total of 42 components, including 12 alcohols, 9 aldehydes, 5 esters, 4 aromatics, 4 terpenes, 4 ketones, 2 alkanes, 1 heterocyclic compound and 1 acid, were shown in Supplementary Table S3. To investigate the dynamic changes of volatile compounds during black tea processing, The reliability of established PLS-DA model was assessed using a permutation test (n = 200) with R2 > 0 and Q2 < 0.05 (R2 = 0.0878, Q2 = −0.624), indicating that the PLS-DA model does not overfit (Supplementary Fig. S2A and B). Based on VIP value ≥1 as described by Wu, Cai, et al. (2023) and Wu, Chen, et al. (2023), a total of fourteen differential compounds were identified (Supplementary Table S4) and visualized in Fig. 5A. The levels of methyl salicylate and phenylethylene were found to be high during the BG stage, exhibiting a significant decrease in the withering stage, followed by an increase again during the rolling stage. During the withering stage, the content of hexanol reached the peak. In the rolling stage, the contents of methyl salicylate, linalool and benzyl alcohol were 44.70 ± 4.82 μg/g, 132.68 ± 4.58 μg/g and 44.47 ± 1.84 μg/g, respectively. In the fermentation stage, (Z)-2-hexen-1-ol peaked at 8.03 ± 0.89 μg/g.Fig. 5 Ring heat maps about changes of the content of 14 kinds of differential compounds (A) and possible metabolic pathways of some differential volatile compounds (B) in black tea samples during processing. BG: Browish-green, MW: Middle of withering, EW: End of withering, MR: Middle of rolling; ER: End of rolling, MF: Middle of fermentation; EF: End of fermentation. LOX: lipoxygenase; HPL: hydroperoxide; ADH: alcohol dehydrogenase; AAT: alcohol acyl CoA transferase; NDO:naphthalene 1,2-dioxygenase; SMO: styrene monooxygenase;TPS: terpene synthase. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

During the processing of black tea, a series of reactions including oxidation, condensation, degradation, glycosylation, methylation and others have been observed to induce changes in chemical components (Liu et al., 2023). In the rolling stage, mechanical action facilitated the release of various enzymes and components from leaf cells, thereby promoting biochemical reactions between them, particularly oxidation-reduction reactions. Subsequently, fermentation significantly enhanced microbial metabolism for the transformation and degradation of components under optimal temperature and humidity conditions. Consequently, certain flavor-related volatiles accumulated. As illustrated in Fig. 5B, hexanal and (Z)-3-hexenal may originate from the breakdown of fatty acids and their derivatives by lipoxygenase and hydroperoxide lyase (Liu et al., 2023). Hexanal in a moderate concentration has a pleasant grassy and chestnut aroma. Meanwhile, (Z)-3-hexenal is converted into leaf alcohol by alcohol dehydrogenase followed by conversion into leaf acetate by alcohol acyl CoA transferase, and they have a positive contribution to the grassy or green fruit aroma in black tea (Zheng et al., 2023). The anaerobic isomerization of (Z)-3-hexenal yields fresh sweet leaf aldehydes, while alcohol dehydrogenase catalyzes the production of lemon-flavored (E)-2-hexene-1-ol and floral-flavored (E)-3-hexene-1-ol (Yang et al., 2023). Hexanal can also be converted into l-hexanol via alcohol dehydrogenase and then isomerized into l-pentanol which gives black tea its sweet fruity flavor.

Aminotransferase, alcohol dehydrogenases, and alcohol acyl CoA transferase played pivotal roles in the accumulation of benzaldehyde, phenylacetaldehyde, benzyl alcohol, and phenethyl alcohol during amino acid degradation (Liu et al., 2023). Phenylacetaldehyde and benzyl alcohol exhibit distinct berry, honey, nutty, and spicy flavors at low concentrations (Jin et al., 2024). Benzyl alcohol and phenethyl alcohol can be enzymatically converted into methyl salicylate with a holly-like aroma by alcohol acyl CoA transferase (Su, He, Zhou, Li, & Zhou, 2022). Linalool and geraniol are significant monoterpenes characterized by their typical floral attributes produced via the terpenoid metabolic pathway from geranyl diphosphate (Jiang et al., 2023), contributing significantly to the aromatic profile of tea.

3.6 Correlation analysis

The correlation analysis was performed based on the dominant genus and basic chemical composition, enzyme activities, and differential compounds. A significant correlation threshold of |ρ| > 0.5 was identified. As shown in Fig. 6A, a total of 18 dominant genera exhibited significant correlations with 9 enzymes. Erwinia displayed the highest number of positively correlated enzymes, including peroxidase (ρ = 0.8657) and cellulase (ρ = 0.7529). Moreover, Pedobacter and Paucibacter also showed positive correlations with peroxidase activity. Regarding fungi, Pseudopithomyces, Leiothecium, and Thermomyces demonstrated the strongest correlation with many enzymes, encompassing peroxidases and amylases. Fig. 6B found significant correlations between 20 dominant microbial genera and 10 basic components of tea. Among the dominant bacteria genera, Erwinia was positively correlated with tea pigments (TFs: ρ = 0.7646; TRs: ρ = 0.6190; TBs: ρ = 0.7178) and total soluble sugar (ρ = 0.8804), but it was negatively correlated with catechin (ρ = −0.8075). Pedobacter and Paucibacter also exhibited positive correlations with tea pigments. Among the dominant fungi, Thermobacterium exhibited a remarkably strong positive correlation with tea pigment (ρ > 0.9000), while displaying negative correlations with free amino acids (ρ = −0.6130), total flavonoids (ρ = −0.6762), and catechins (ρ = −0.8273). A total of 20 microorganisms significantly correlated to 14 different compounds were presented in Fig. 6C. Pantoea was strongly related to compounds derived from C6-C9 aldehydes, alcohols, and fatty acids esters such as (Z)-2-Hexen-1-ol, (E)-2-Hexen-1-ol, hexanal, leaf acetate, and leaf aldehyde. Cladosporium exhibited strong correlations with seven different compounds, notably its ρ for hexanal was the highest at 0.8766.Fig. 6 Correlation analysis of dominant microbial genus with enzymes (A), basic components of tea (B), and differential compounds (C), respectively.

Fig. 6

To ensure survival, microorganisms in tea employ enzymatic secretion to degrade compounds for energy acquisition and the fulfillment of bacterial growth requirements, thereby exerting an influence on the chemical composition and quality of tea (Assad et al., 2023). Erwinia had the capability to secrete various hydrolytic enzymes including cellulases and so on (Cooper & Salmond, 1993), which was consistent with our conclusion that there was a significant correlation between Erwinia and cellulase and total soluble sugar. Similarly, pedobacter could secrete peroxidase to promote lignin degradation. Similarly, pedobacter could secrete peroxidase to promote lignin degradation (Pan et al., 2023). Peroxidase serves as a crucial enzyme involved in the conversion of catechins into tea pigments (Chiang, Yang, Wang, & Chen, 2022). Pantoea was found as a genus with the potential to produce fatty acids (Tanaka et al., 2015), which explained its close relationship with (Z)-2-Hexen-1-ol, (E)-2-Hexen-1-ol, hexanal, leaf acetate, leaf aldehyde, among others. Additionally, Cladosporium has been reported to have potential capabilities in lipid, fatty acid and protein biosynthesis (De & Verma, 2011), and might be closely related to hexanal via secreting lipoxygenase (Yang et al., 2023).

In summary, this study comprehensively investigated the dynamic changes in microorganisms, key enzyme activities, basic chemical components, and volatile components of black tea. The correlation analysis revealed that microorganisms significantly influenced compositional changes and contributed to the formation of black tea quality through metabolic processes and enzyme secretion during tea processing. Our study's findings provide a strong foundation for further exploration into functional microorganisms present in black tea and optimization of processing techniques to enhance its quality. Additionally, screening for functional microorganisms can be conducted to investigate optimal fermentation process parameters. By controlling parameters such as temperature, humidity, time and ventilation during fermentation, the growth of functional microorganisms and their enzyme activity and metabolism can be promoted to improve the quality of black tea.

Ethical approval

This study does not involve any human or animal testing.

CRediT authorship contribution statement

Ailing Wang: Writing – original draft, Methodology, Investigation, Data curation, Conceptualization. Qingqing Lei: Methodology, Investigation. Beibei Zhang: Resources. Junhai Wu: Writing – review & editing, Software. Zheyang Fu: Investigation. Jiangfeng He: Investigation. Yanbo Wang: Investigation. Xinying Wu: Writing – review & editing, Resources, Methodology, Funding acquisition, 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

Image 1

Data availability

Data will be made available on request.

Acknowledgment

The authors would like to thank Guizhou University for providing the necessary Talent Fund Project for financial support to execute this project.

Funding

Study on the contribution of microorganisms to the formation of Guizhou black tea flavor (Guizhou University Doctoral Fund [GDRJHZ [2022]02] ).

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.fochx.2024.101743.
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