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anaerobic fermentation, antibacterial, antioxidant, phenolics, tea
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Permata D, Nayohan S, Wahyuni S et al. Anaerobic fermentation of tea leaves and spent tea residues: Effects on phenolic and flavonoid contents, antioxidant capacity, and antibacterial activity [version 1; peer review: awaiting peer review]. F1000Research 2026, 15:935 (https://doi.org/10.12688/f1000research.179929.1)
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Brief Report
Anaerobic fermentation of tea leaves and spent tea residues: Effects on phenolic and flavonoid contents, antioxidant capacity, and antibacterial activity
[version 1; peer review: awaiting peer review]
PUBLISHED 15 Jun 2026
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This article is included in the Agriculture, Food and Nutrition gateway.
This article is included in the Plant Science gateway.
Abstract
Corresponding author:
Anuraga Jayanegara
Competing interests:
No competing interests were disclosed.
Grant information:
This research is funded by the Indonesian Endowment fund for Education (LPDP) on behalf of the Indonesian Ministry of Higher Education, Science and Technology and managed under the EQUITY Program (Contract No: 4297/B3/DT.03.08/2025 and No: 42011/IT3/HK.07.00-4/P/B/2025).  
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Copyright:
© 2026 Permata D et al.
This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
How to cite: Permata D, Nayohan S, Wahyuni S et al. Anaerobic fermentation of tea leaves and spent tea residues: Effects on phenolic and flavonoid contents, antioxidant capacity, and antibacterial activity [version 1; peer review: awaiting peer review]. F1000Research 2026, 15:935 (https://doi.org/10.12688/f1000research.179929.1)
First published: 15 Jun 2026, 15:935 (https://doi.org/10.12688/f1000research.179929.1)
Latest published: 15 Jun 2026, 15:935 (https://doi.org/10.12688/f1000research.179929.1)
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Introduction
Tea (Camellia sinensis) is widely consumed and is recognized as a rich dietary source of phenolic compounds and flavonoids, which contribute to antioxidant and antimicrobial properties and have been associated with various health-related benefits (Nibir et al., 2017; Wang and Kang, 2020). The antioxidant and antibacterial effects of tea are mainly attributed to catechins, including epigallocatechin gallate (EGCG), epicatechin (EC), and epicatechin gallate (ECG) (Zhao et al., 2014). These compounds work by neutralizing free radicals through metabolic processes in the body, thereby preventing oxidative stress associated with various degenerative diseases, including cancer, diabetes, and cardiovascular disorders (Wang and Kang, 2020). Beyond the brewed beverage, tea processing and consumption also generate substantial quantities of spent tea residues. Although these residues are often discarded, they may retain appreciable amounts of bioactive compounds and therefore represent potential resources for the development of functional ingredients or natural antioxidants for food applications (Çakmak et al., 2024).
Green, white, oolong, and black teas originate from the same plant, i.e., Camellia sinensis, but differ markedly in chemical composition because they undergo different degrees of enzymatic oxidation and processing (Alves et al., 2025). These differences influence the profile and extractability of phenolic compounds, which can translate into variation in antioxidant capacity and antibacterial activity across tea types (Tanaka & Matsuo, 2020; Esposito et al., 2023). Likewise, converting dried leaves into a spent residue by brewing can remove a portion of water-soluble phenolics, often lowering the functional potency of residue extracts compared with leaf extracts (Saklar et al., 2015). Furthermore, prolonged storage of these teas may reduce their bioactive compound content (Hazra et al., 2020), thereby decreasing their antimicrobial and antioxidant activities.
Fermentation is a food-relevant bioprocessing approach that can stabilize wet plant matrices and modulate phytochemical composition through microbial and enzymatic transformations. In sealed anaerobic systems, fermentation may alter the concentration, molecular forms, and extractability of tea phenolics; for example, changes in catechins and antioxidant activity during anaerobic storage have been reported (Nishino et al., 2007). From a food-processing perspective, a simple sealed-pouch anaerobic fermentation step may be particularly attractive for wet tea residues because it can serve as a low-input stabilization and valorization strategy. However, evidence remains limited regarding how anaerobic fermentation affects the bioactive potential of both tea leaves and spent residues across different processed tea types. Therefore, this study aimed to evaluate the effects of anaerobic fermentation, material fraction (leaf vs. spent residue), and processed tea type on total phenolic and flavonoid contents, antioxidant capacity, and antibacterial activity against Staphylococcus aureus and Escherichia coli.
Methods
Sample preparation
Dried tea leaves representing four processed tea types (green, black, oolong, and white) were sourced as commercial products from the Indonesian Research Institute for Tea and Cinchona (PT Perkebunan Nusantara III, Bandung, West Java, Indonesia). The experiment followed a 2 × 2 × 4 factorial arrangement with three factors: fermentation status (unfermented vs. anaerobically fermented), material fraction (tea leaf vs. spent tea residue), and processed tea type (green, black, oolong, and white). For each processed tea type, two material fractions were prepared. The tea leaf fraction consisted of the original dried leaves. The spent tea residue fraction was produced by brewing the dried leaves with warm water, followed by oven-drying at 60 °C.
For the anaerobic fermentation treatment, 250 g of each material was packed in polyethylene bags and vacuum-sealed. Water was added to the leaf fraction at a 1:2 ratio for the fermented treatment, whereas unfermented leaves were prepared without added water. All bags were stored at −20 °C prior to fermentation. Samples were then subjected to anaerobic fermentation in the sealed pouches for 30 days. After fermentation, bags were opened and each sample was divided into two equal subsamples. All materials were freeze-dried and ground to pass a 1-mm sieve before analysis.
Extraction and analyses of bioactive compounds
Extraction followed ISO 14502-1 using 70% methanol (Merck, Sigma Aldrich, catalogue no. 109534). Briefly, 200 mg of ground sample was mixed with 5 mL of preheated 70% methanol and incubated in a water bath for 10 min with vortexing every 5 min. After cooling to room temperature, the mixture was centrifuged at 3,500 rpm for 10 min. The supernatant was collected and the pellet was re-extracted once using the same procedure. Combined extracts were adjusted to a final volume of 10 mL with cold 70% methanol.
Total phenolic content (TPC) was determined using the Folin–Ciocalteu method (Sari et al., 2023). A diluted extract (20 μL) was mixed with 20 μL Folin–Ciocalteu reagent (Merck, Sigma Aldrich, catalogue no. 1.09001) and vortexed for 1 min. After 5 min, 200 μL of 7% sodium carbonate (Merck, Sigma Aldrich, catalogue no. 1.06392) and 10 μL deionized water were added and mixed. The reaction was incubated in the dark at room temperature for 120 min and measured at 750 nm (Thermo Multiskan GO Microplate Reader, Massachusetts, USA). Gallic acid (10–60 mg L−1, Merck, Sigma Aldrich, catalogue no. 8.42649) was used for calibration. Results are reported as mg gallic acid equivalents (GAE) per g dry sample (mg GAE/g).
Total flavonoid content (TFC) was measured following Sari et al. (2023). In each well, 50 μL of extract or standard solution was mixed with 100 μL methanol (blank: 150 μL methanol). Then 20 μL of 10% AlCl3 (Merck, Sigma Aldrich, catalogue no. 8.01081) was added and incubated for 3 min, followed by 20 μL of 1 M sodium acetate (Merck, Sigma Aldrich, catalogue no. 1.06268) and 60 μL methanol. After incubation in the dark at room temperature for 40 min, absorbance was measured at 430 nm. Quercetin (Merck, Sigma Aldrich, catalogue no. Q4951) was used as the calibration standard, and results are reported as mg quercetin equivalents (QE) per g dry sample (mg QE/g).
Antioxidant and antibacterial assays
The radical-scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) was evaluated based on Cheng et al. (2006) with minor modifications. Extract (80 μL) was mixed with 20 μL of DPPH solution (Merck, Sigma Aldrich, catalogue no. D9132) in a 96-well microplate, and absorbance was read at 517 nm. Ascorbic acid (Merck, Sigma Aldrich, catalogue no. 1.00468) and quercetin were used as positive controls and analyses were performed in triplicate. Antioxidant activity was expressed as IC50 (ppm), defined as the extract concentration required to scavenge 50% of DPPH radicals.
Antibacterial activity was evaluated against Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) using a microdilution assay adapted from Nabila et al. (2025). Mueller-Hinton broth (MHB, Merck, Sigma-Aldrich, Catalogue No. 1.10293) was used as the growth medium and inocula from 24-h cultures were adjusted to 1 × 106 McFarland. In a 96-well microplate, 100 μL sterile MHB was added per well, followed by two-fold serial dilutions of the extracts from 400 to 25 ppm. Each well received 10 μL bacterial suspension (except the media control). Optical density (OD) was recorded at 540 nm at 0 and 24 h (Thermo Multiskan GO, Massachusetts, USA). Growth inhibition was calculated from OD changes relative to the growth control, and MIC50 (ppm) was defined as the extract concentration producing 50% growth inhibition.
Statistical analysis
Data were analyzed using three-way analysis of variance (ANOVA) in CoStat software (version 6.400, CoHort software, Monterey, CA, USA), including main effects of fermentation status, material fraction, processed tea type, and their interactions. Least Significant Difference (LSD) tests were used for post hoc mean comparisons. Differences were considered significant at p < 0.05.
Results
Across tea types, oolong and white teas showed higher TPC than black tea (p < 0.01), whereas black tea had the highest mean TFC (p < 0.01; Tables 1–2). Tea leaves contained substantially higher TPC than spent residues. The effects of anaerobic fermentation on TPC and TFC depended on both material fraction and tea type. Fermented leaves showed a slight increase in TPC across all tea types (p < 0.01), whereas fermentation increased TFC only in green tea and black tea leaves (p < 0.01). In the residue fraction, fermentation consistently increased TPC and TFC in black tea residues, whereas the same treatment led to a consistent decline in green tea residues (p < 0.01).
Table 1. Effects of fermentation, tea material, and tea types on total phenolic content.
| Significance of treatment factors | Fermentation | Material | Tea | F × M | F × T | M × T | F × M × T |
|---|---|---|---|---|---|---|---|
| (p value) | <0.01 | <0.01 | <0.01 | <0.01 | <0.01 | <0.01 | <0,01 |
Table 2. Effects of fermentation, tea material, and tea types on total flavonoid content.
| Significance of treatment factors | Fermentation | Material | Tea | F × M | F × T | M × T | F × M × T |
|---|---|---|---|---|---|---|---|
| (p value) | <0.01 | <0.01 | <0.01 | <0.01 | <0.01 | <0.01 | <0.01 |
The DPPH IC50 values indicated stronger antioxidant activity, reflected by lower IC50 values, in oolong and white teas than in black tea ( Table 3). Tea leaves exhibited substantially lower IC50 values than spent residues across all tea types and fermentation statuses (p < 0.01). Fermentation increased IC50 values in the leaf fraction in almost all tea types (p < 0.01), with white tea leaf being the only exception. In contrast, the residue fraction showed a consistent reduction in IC50 after fermentation across all tea types (p < 0.01), indicating improved antioxidant capacity following fermentation.
Table 3. Effects of fermentation, tea material, and tea types on inhibitory concentration (IC50).
| Significance of treatment factors | Fermentation | Material | Tea | F × M | F × T | M × T | F × M × T |
|---|---|---|---|---|---|---|---|
| (p value) | <0.01 | <0.01 | <0.01 | <0.01 | <0.01 | <0.01 | <0.01 |
Regarding antibacterial activity, white tea showed the lowest MIC50 values against S. aureus and E. coli (p < 0.01), while oolong tea showed a lower MIC50 only against E. coli (p < 0.01; Tables 4–5). Fermentation increased the MIC50 against S. aureus in the leaf fraction across all tea types (p < 0.01). A similar trend was observed in the residue fraction, in which fermentation increased the MIC50 of green tea, white tea, and oolong tea (p < 0.01). For antimicrobial activity against E. coli, green tea and oolong tea leaves showed higher MIC50 values after fermentation (p < 0.01). In the residue fraction, fermentation consistently increased MIC50 values across all tea types (p < 0.01), indicating reduced antibacterial effectiveness.
Table 4. Effects of fermentation, tea material, and tea types on antimicrobial activity against Staphylococcus aureus (MIC50).
| Significance of treatment factors | Fermentation | Material | Tea | F × M | F × T | M × T | F × M × T |
|---|---|---|---|---|---|---|---|
| (p value) | <0.01 | <0.01 | <0.01 | <0.01 | <0.01 | <0.01 | <0.01 |
Table 5. Effects of fermentation, tea material, and tea types on antimicrobial activity against Escherichia coli (MIC50).
| Significance of treatment factors | Fermentation | Material | Tea | F × M | F × T | M × T | F × M × T |
|---|---|---|---|---|---|---|---|
| (p value) | <0.01 | <0.01 | <0.01 | <0.01 | <0.01 | <0.01 | <0.01 |
Discussion
The higher TPC observed in oolong and white teas than in black tea may reflect differences in leaf maturity and processing intensity. In particular, the high TPC in white tea may be attributed to the use of young leaves, which are naturally rich in epigallocatechin gallate (EGCG) and epicatechin gallate (ECG) (Esposito et al., 2023). By contrast, the substantially higher TPC in tea leaves than in spent residues is consistent with the removal of water-soluble phenolics during brewing (Saklar et al., 2015). The slight increase in TPC after fermentation, especially in the leaf fraction, may reflect degradation of native phenolic compounds such as catechins into smaller molecules that still retain phenolic properties rather than being converted into non-phenolic acids (Nishino et al., 2007). These breakdown products may continue to react with the Folin–Ciocalteu reagent, thereby contributing to a slight apparent increase in measured total phenols.
The stronger antioxidant activity of oolong and white teas, as indicated by lower IC50 values, is in agreement with their higher phenolic concentrations. Dudonné et al. (2009) reported a strong correlation between total phenolic content and radical-scavenging activity (R = 0.939 for DPPH), and this activity is mediated through mechanisms such as hydrogen atom transfer, single-electron transfer, sequential proton loss electron transfer, and transition metal chelation (Zeb, 2020). Although black tea contained more flavonoids than the other tea types, its antioxidant activity was weaker, as shown by its higher IC50 values. This may be explained by the transformation of catechins into theaflavins and other polymeric compounds during full oxidation in black tea processing (Tanaka & Matsuo, 2020).
The lower IC50 values in the leaf fraction than in the residue fraction are also consistent with the substantially higher TPC and TFC of the leaves. Conversely, the higher IC50 values in tea residues likely resulted from the brewing process, which extracted most of the water-soluble phenolic compounds (Saklar et al., 2015). Interestingly, fermentation had contrasting effects on antioxidant capacity depending on the material fraction. In leaves, fermentation generally increased IC50 values and thus reduced antioxidant capacity. In residues, however, fermentation consistently reduced IC50 values across tea types, indicating improved antioxidant activity. This contrasting response may be related to the presence of bioactive compounds that remain bound to the fibrous matrix of tea residues (Çakmak et al., 2024). Fermentation may facilitate the release of these bound phenolics, thereby increasing the availability of antioxidant compounds in the residue fraction.
With respect to antibacterial activity, the lower MIC50 values of white tea against both S. aureus and E. coli, and of oolong tea against E. coli, indicate a stronger antibacterial potential of these tea types. This finding is consistent with their higher phenolic content, as phenolic compounds can disrupt microbial cell membranes, inhibit metabolic enzymes, and interfere with biofilm formation in pathogenic bacteria such as E. coli and S. aureus (Huang et al., 2022). Nevertheless, fermentation generally increased MIC50 values in both leaf and residue fractions, indicating reduced antibacterial effectiveness after fermentation. Although TPC appeared to increase after fermentation, antimicrobial activity against both S. aureus and E. coli declined. This reduction may be attributed to polymerization of phenolic compounds during fermentation or to their degradation by microbial activity, producing derivatives with weaker antibacterial effects (Romero et al., 2025).
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how to cite this article
Permata D, Nayohan S, Wahyuni S et al. Anaerobic fermentation of tea leaves and spent tea residues: Effects on phenolic and flavonoid contents, antioxidant capacity, and antibacterial activity [version 1; peer review: awaiting peer review]. F1000Research 2026, 15:935 (https://doi.org/10.12688/f1000research.179929.1)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
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