Modulatory Effects of Tea-Derived Bioactives on Oral Microbiota and their Virulence-Reducing Properties

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While their inhibitory effects on certain oral pathogens were well-documented, their impact on oral commensal bacteria remained unclear. This study investigated the effects of tea-derived bioactives on the composition of oral bacterial communities, with quantitative polymerase chain reaction (qPCR) utilized to assess changes in pathogenic and commensal bacteria. Epigallocatechin gallate (EGCG), green tea extract, and theaflavins were found to exert inhibitory effects on key oral pathogens, including Porphyromonas gingivalis , Fusobacterium nucleatum , and Prevotella intermedia . Concurrently, these polyphenols promoted the growth of beneficial oral commensal bacteria, such as Lactobacillus gasseri and Lactobacillus rhamnosus . At sub-inhibitory concentrations, tea polyphenols significantly suppressed the expression of virulence factors in P. gingivalis , suggesting that low concentrations were sufficient to mitigate pathogenicity without disrupting microbial homeostasis. Furthermore, tea polyphenols reduced oral VSCs by downregulating hydrogen sulfide/methyl mercaptan synthase genes. Hence, their dual capacity to inhibit pathogens and support commensal bacteria could make them promising candidates for oral health products. Health sciences/Health care/Dentistry/Oral microbiology Biological sciences/Molecular biology Tea-derived bioactives Oral microbiota Virulence factors Halitosis Figures Figure 1 Figure 2 Introduction The oral cavity represents a complex ecosystem, characterized by optimal temperatures, pH levels, and abundant nutrient sources that support diverse microbial communities 1 . Over 700 microbial species inhabit this environment, maintaining a dynamic equilibrium 2 . Disruptions of the balance due to environmental changes or host immune responses could lead to oral diseases, such as caries, halitosis, and gingivitis 3 . The oral cavity also served as a reservoir for various pathogens linked to localized and systemic diseases 4 . For instance, Porphyromonas gingivalis affected glucose and lipid metabolism 5 . Fusobacterium nucleatum had been implicated in promoting glycolysis in colon cancer cells 6 . Therefore, maintaining a balanced oral microbiota is essential for preventing oral diseases and mitigating systemic health conditions. Given the growing concerns over bacterial resistance stemming from antibiotic misuse, strategies focused on restoring oral microbial homeostasis offer promising avenues. Common oral conditions such as halitosis, caries, and gingivitis were closely associated with oral microbiota dysbiosis 7 . Halitosis arose from volatile sulfur compounds (VSCs), including hydrogen sulfide, methyl mercaptan, and dimethyl sulfide 8 . Key contributors to hydrogen sulfide production included Porphyromonas gingivalis , Treponema denticola , and Tannerella forsythia —collectively known as the “red complex” 9 . Methyl mercaptan was produced by anaerobic bacteria such as Prevotella , Veillonella , and Megasphaera 10 . Addressing halitosis requires a comprehensive understanding of the composition and functional dynamics of the oral microbiota. Gingivitis, characterized by inflammation of the gingival tissues, occurred when pathogens such as Porphyromonas gingivalis , Fusobacterium nucleatum , and Treponema denticola produced virulence factors that directly damaged periodontal tissues 11 , 12 . In recent years, probiotics and prebiotics had gained attention to combat oral diseases linked to microbial dysbiosis. Probiotics interact with the oral microbiota to promote health 13 . For instance, Lactobacillus species enhanced natural immunity and supported microbial balance 14 . Several common Lactobacillus strains could inhibit the growth of oral pathogens 15 , 16 . Similarly, Bifidobacterium species suppressed black-pigmented anaerobes by competing for essential growth factors like vitamin K 17 , reducing the presence of harmful bacteria the gingival of subjects 18 and improving gingival inflammation 19 . Moreover, prebiotics, defined as “substrates selectively utilized by host microorganisms that confer health benefits,” had also gained attention for promoting the proliferation of resident symbiotic bacteria 20 . Toothpaste containing arginine reduced the abundance of Streptococcus mutans in individuals with caries, thereby reducing the enamel demineralization potential 21 . Four novel potential prebiotic substrates, such as N-acetyl-D-glucosamine were identified, which downregulated the expression of virulence factors of oral pathogens 22 . Despite their promising potential, research on the applications of prebiotics for oral health were needed. Tea-derived bioactives, particularly polyphenolic components, had shown prebiotic potential. Studies suggested that tea polyphenols participated in regulating gut microbiota composition, similar to the effects of traditional prebiotics like oligosaccharides 23 . Tea bioactives, derived from water or alcohol-based extraction of tea leaves, including tea polyphenols, theanine, alkaloids, tea polysaccharides, saponins, vitamins, and minerals 24 . Tea polyphenols, the most characteristic metabolites in tea, included catechins, flavonoids, anthocyanins, and phenolic acids 25 . Catechins, especially epigallocatechin gallate (EGCG) were the most abundant and active catechin monomer, possessing anti-inflammatory and antioxidant properties 26 , 27 . Theaflavins, unique to black tea, were formed by the oxidation or polymerization of catechins under the action of enzymes (polyphenol oxidase and peroxidase) 28 . Theanine, chemically named glutamic acid γ-ethylamide, was a unique free amino acid in tea with functions of enhancing immunity 29 , 30 . Numerous studies had demonstrated that regular tea consumption could prevent dental caries, combat halitosis, and promote oral health 31 , 32 . For example, tea polyphenols exerted significant inhibitory effects on oral pathogens: they could prevent caries by inhibiting the metabolism of Streptococcus mutans 33 and reduce halitosis by suppressing the proliferation of Porphyromonas gingivalis and Fusobacterium nucleatum 34 . Additionally, tea polyphenols possessed prebiotic potential, by promoting the growth of some beneficial gut bacteria and altering the structure of the gut microbiota. In vitro anaerobic fermentation studies showed that EGCG promoted the growth of beneficial genera such as Bacteroides , Faecalibacterium , and Bifidobacterium , while inhibiting harmful bacteria 35 . Similarly, rodent studies indicated that phenolic compounds from tea extracts altered the proportions of various gut microorganisms 36 , 37 . However, most gut microbiota studies were focused primarily on genus-level changes, leaving species-level dynamics largely unexplored. Similarly, research on the effects of tea-derived bioactives on oral pathogens had been largely confined to single pathogens or single-species biofilms, with limited attention given to their interactions within complex microbial communities. To advance the application of tea-derived bioactives in oral health products, further in-depth studies were needed to explore their potential in balancing oral microbiota. In this study, we conducted a comprehensive investigation into the effects of tea polyphenols from various sources on specific oral bacterial species. We also examined the influence of tea polyphenols on the expression of virulence factors in oral pathogens. Finally, we demonstrated the inhibitory effects of tea polyphenols on hydrogen sulfide production in salivary bacteria and elucidated the underlying molecular mechanisms. Result Effects of Tea-Derived Bioactives on Oral Bacteria To comprehensively evaluate the regulatory effects of the four tea-derived bioactives, including epigallocatechin gallate (EGCG), green tea extract (GTE), theaflavins (TF), and L-theanine (L-T) on the structure of mixed oral bacteria, the relative abundances of six bacterial species, including P . gingivalis , F . nucleatum , P . intermedia , S . salivarius , L . gasseri , and L . rhamnosus were analyzed. Significant biological relevance was attributed to differences in the substrate-treated groups relative to untreated mixed oral bacteria, with either more than a 2-fold downregulation (2 − ΔΔCt 1.5). The addition of 0.1% EGCG led to a decrease in the relative abundance of F. nucleatum and P. intermedia , while significantly increasing L. gasseri and L. rhamnosus (Fig. 1a, 1b). Similarly, 0.1% GTE inhibited all three pathogenic bacteria and resulted in an 8.2-fold increase in L. gasseri . TF at the same concentration inhibited F. nucleatum and promoted the growth of L. gasseri . No significant difference was obtained with an addition of 0.1% L-T. For S. salivarius , no substantial differences were observed under any of the substrate conditions (Fig. 1b). Among the tested tea-derived bioactives, EGCG and GTE proved the most effective in optimizing the oral bacterial structure, outperforming TF, while L-T exhibited no substantial impact. Effects of Tea Polyphenols on Virulence Factors The antibacterial activities of EGCG, GTE, TF and L-T against P. gingivalis, F. nucleatum and P. intermedia were assessed using a resazurin microplate assay. Among the tested tea-derived bioactives, EGCG, GTE, and TF all exhibited antibacterial effects against all three pathogenic bacteria (table 1), with EGCG exhibiting the strongest activity. The MIC value of cetylpyridinium chloride used as a reference compound, ranged from 3.91 to 7.81 µg/mL. Since L-T showed no inhibitory effects on these pathogens, subsequent studies on changes in virulence gene expression focused only on EGCG, GTE and TF. The expression of 16 virulence genes (for an overview of the associated functions of the corresponding virulence factors: see Supplementary Table S2) from the three periodontal pathogens was analyzed with the addition of low-concentration tea polyphenols. Gene expression of the three periodontal pathogens was relative to the expression of 16S rRNA. Significant changes in gene expression in the substrate-treated bacteria, relative to the untreated bacteria, were considered biologically relevant if they were more than 2-fold downregulated (2 − ΔΔCt 1.5). P. gingivalis , a primary pathogen responsible for gingivitis, relies heavily on gingipains as its main virulence factors. All three tea polyphenols significantly inhibited the expression of gingipain genes ( kgp and rgpA ). Regarding virulence factors involved in the colonization of subgingival sites, significant decreases of hagA , hagB , and fimA gene expression were observed for all polyphenols, with TF causing a particularly notable reduction in fimA expression, by over 29-fold. Similarly, the expression level of the hem gene, which encodes a hemolysin in P. gingivalis , was also repressed by EGCG, GTE, and TF. Additionally, the mgl gene, linked to halitosis, was downregulated across all treatments. Conversely, the stress protease gene htrA was upregulated, with EGCG increasing its expression by 4.8-fold and TF by 4.3-fold at higher concentrations (Table 2). Focal adhesion A ( fadA ) is one of the most important virulence genes of F. nucleatum , contributing to tissue destruction. Of the three tea polyphenols, only 125 ppm EGCG inhibited fadA expression. Additionally, the expression level of megl gene, linked to halitosis, was only downregulated by the addition of 125 ppm EGCG. Two genes ( aid1 and fomA ) associated with bacterial coaggregation were unaffected by any of the tea polyphenols. Interestingly, the ABC transporter permease gene (transp.), which encodes a type of resistance protein, was upregulated by all three tea polyphenols without a clear dose-response effect (Table 3). In P. intermedia , virulence genes ecf , inpA and groeS were significantly downregulated (2.1- to 9.9-fold) compared to the control after 8 hours of growth in the presence of the three tea polyphenols (Table 4). Effects of Tea-Derived Bioactives on Hydrogen Sulfide Production by Oral Pathogenic Bacteria Volatile sulfur compounds (VSCs), including hydrogen sulfide (H 2 S) and methyl mercaptan (CH 3 SH), were major contributors to halitosis. The inhibitory effects of the four tea-derived bioactives on hydrogen sulfide production by oral pathogenic bacteria were evaluated to assess their potential efficacy against halitosis-related oral bacterial species. At a substrate concentration of 0.1%, all three active polyphenols (EGCG, GTE, and TF) exhibited significant inhibitory effects on hydrogen sulfide production compared to the control (Fig. 1). Among them, TF demonstrated the highest inhibition rate (95%), followed by GTE (79%) and EGCG (44%). Conversely, L-T showed negligible effects on hydrogen sulfide reduction (Fig. 2). Discussion Our research indicated that EGCG (epigallocatechin gallate), GTE (green tea extracts), and TF (theaflavins) could regulate the structure of the salivary microbiome, and we analyzed the relative abundance changes of major oral bacterial species. Previous in vitro fermentation studies showed that EGCG regulated the structure of the gut microbiome and promoted the growth of Bifidobacterium .spp 38 . A clinical study also demonstrated the regulatory effect of theaflavin-containing toothpaste on the oral microbiome 39 . However, most existing studies lacked species-level analysis of the oral microbiome and comparisons of the effects of different tea-derived bioactives on balancing the microbiome. In this study, EGCG exhibited the most pronounced effect in inhibiting harmful bacteria while promoting the growth of beneficial bacteria. These effects could be influenced by factors such as the complexity of the in vitro model, the degree of oxidation, and the purity of the raw materials. Few studies reported the antibacterial effect of theanine, and this study did not observe any significant impact of theanine on salivary bacteria. Notably, all three tea-derived bioactives effectively inhibited Porphyromonas gingivalis , Fusobacterium nucleatum , and Prevotella intermedia . Furthermore, they increased the relative abundance of Lactobacillus gasseri and Lactobacillus rhamnosu . Several studies had suggested that tea polyphenols exerted their antibacterial effects by chelating essential bacterial nutrients, such as iron 40 , and inhibiting nucleic acid precursor synthesis 41 . Therefore, under normal circumstances, high doses of EGCG could also inhibit individual beneficial bacteria. Some research suggested that 2.5 mg/mL EGCG inhibits P. gingivalis , while 25 mg/mL EGCG was required to inhibit the growth of Lactobacillus salivarius WB21 42 , indicating that Lactobacilli possessed a relatively high tolerance to EGCG 43 . This implied that the effects of EGCG on oral bacteria were dose-dependent 44 . We hypothesized that within mixed bacterial communities, interactions among bacteria would allow low doses of tea polyphenols to selectively inhibit oral pathogens while promoting the growth of beneficial bacteria, ultimately contributing to oral microbiome balance and promoting oral health 45 . qPCR analysis revealed a marked reduction in Prevotella intermedia , a keystone pathogen in periodontitis pathogenesis 46 , along with a reduction in Fusobacterium nucleatum , a species implicated in periodontal disease and oral carcinogenesis. Conversely, the intervention significantly enhanced Lactobacillus abundances. The proliferation of Lactobacillus potentially inhibited pathogens through bacteriocin and organic acid production 15 , 16 . Notably, while Lactobacillus proliferation benefited microbial balance, its excessive growth could lower oral pH and increase enamel demineralization risk 47 , warranting longitudinal investigation. Collectively, these findings suggested tea polyphenols modulate oral health through suppressing periodontal pathogens while promoting symbiotic flora proliferation, thereby establishing a foundation for microbiome-targeted oral care strategies. Porphyromonas gingivalis was a key pathogen associated with periodontitis, with gingipains ( kgp and rgp ) being its primary virulence factors 48 . Consistent with previous studies 49 , we confirmed that EGCG and green tea extracts significantly inhibited the expression of these virulence factors. Notably, our findings revealed that theaflavins, at sub-MIC concentrations, demonstrated a stronger inhibitory effect on the expression of P. gingivalis virulence factors compared to EGCG and green tea extracts. This suggested that theaflavins had the potential to attenuate P. gingivalis virulence while maintaining the balance of the oral microbiome. Fusobacterium nucleatum utilized adhesins such as FadA to invade gingival epithelial cells and produces lipopolysaccharides (LPS) that damage periodontal tissue 50 . While a prior study using tea polyphenols found no effect on the expression of adhesin genes ( fap2 , fadA , aid1 ) 40 , our results indicated that 125 ppm EGCG significantly inhibited fadA gene expression. Since the original study did not provide specific data, differences in EGCG dosage or primer design could account for this discrepancy. Overall, EGCG not only reduced the virulence of F. nucleatum but also inhibited its growth. For Prevotella intermedia , the RNA polymerase ECF-type sigma factor ( ecf ) played a role in polysaccharide production, enhancing its resistance to host defenses 51 . Additionally, Interpain A ( InpA ) was associated with the bacterium’s serum resistance 52 . Our findings revealed that all three tea polyphenols significantly inhibited the expression of virulence factors such as ecf and InpA . At sub-MIC concentrations, EGCG and green tea extracts demonstrated stronger inhibitory effects than theaflavins, consistent with the slight effect of theaflavins on the growth of P. intermedia . Few studies had reported the effects of tea polyphenols on P. intermedia 's antimicrobial mechanisms and virulence factors, but our results suggested that the inhibition of virulence factor expression could play a crucial role in inhibiting its growth. Moreover, the three tea polyphenols inhibited the expression of genes related to halitosis, such as mgl and megl , offering insight into their ability to decrease the production of hydrogen sulfide and other odor-causing gases. Oral pathogenic bacteria, such as P. gingivalis and F. nucleatum , produce volatile sulfur compounds (VSCs) like hydrogen sulfide (H₂S), which were primary contributors to halitosis 53 . Beyond causing bad breath, these sulfur compounds were toxic to oral mucosal cells and trigger inflammatory responses, potentially leading to gingivitis 54 . Therefore, identifying natural substances to control VSC production was critical for the prevention and management of both halitosis and gingivitis. In our study, we found that, except theanine, EGCG, green tea extracts, and theaflavins all demonstrated effects in inhibiting hydrogen sulfide production in vitro, which aligned with the results that theanine had minimal impact on oral microbiota structure. We speculated that the reduction in hydrogen sulfide might be related to the role of the three tea polyphenols in promoting a balanced oral microbiome. Previous clinical studies showed that green tea mouthwash was more effective in eliminating halitosis than black tea 55 . However, our results suggested that theaflavins were more effective than green tea polyphenols in inhibiting hydrogen sulfide production. This discrepancy could stem from the fact that halitosis gases include methanethiol, hydrogen sulfide, and dimethyl sulfide 56 , whereas our study exclusively measured hydrogen sulfide levels. As a result, direct comparisons among the three tea-derived bioactives for overall halitosis gas elimination remain inconclusive. In summary, this study demonstrated that EGCG, green tea extracts, and theaflavins effectively inhibited the production of volatile sulfur compounds, thereby reducing halitosis gases. More specifically, these compounds promoted the growth of beneficial bacteria and inhibited the proliferation of harmful bacteria, contributing to the regulation of the salivary microbiome. Additionally, tea polyphenols reduced the expression of virulence factors and volatile sulfur compound-producing genes in P. gingivalis , F. nucleatum , and P. intermedia under sub-MIC conditions. Overall, our findings suggested that polyphenols from green and black tea held promise as adjunct therapies for the treatment of halitosis and gingivitis. Materials and methods Tea-Derived Bioactives Theaflavins (TF) with a purity of 40%, was purchased from Yaizu Suisankagaku (Japan). Epigallocatechin gallate (EGCG) with a purity of 94%, green tea extract (GTE) with a purity of at least 70%, and L-theanine (L-T) with a purity of at least 30%, were all naturally extracted from the plant Camellia sinensis and purchased from Novanat (Shanghai, China). Bacteria and Growth Conditions The following bacterial strains were used as representative oral pathogens: Porphyromonas gingivalis ATCC 33277, Fusobacterium nucleatum ATCC 10953, Prevotella intermedia ATCC 25611. Conversely, Streptococcus salivarius BAA1024, Lactobacillus gasseri ATCC 33323, Lactobacillus rhamnosus ATCC 7469 were used as representative commensal/beneficial oral bacterial strains. All six bacteria were grown in Brain-Heart Infusion broth (BHI; Difco, Sparks, MD), which contained no salivary proteins. All bacteria cultures were incubated at 37 ℃ in an anaerobic chamber. In Vitro Co-culture of Tea-Derived Bioactives with Oral Bacteria Six oral bacteria were co-cultured using BHI culture medium, including P. gingivalis , F. nucleatum , P. intermedia , S. salivarius, L. gasseri , and L. rhamnosus . Four tea-derived bioactives were added to the cultures at a final concentration of 0.1%. After 24 hus of anaerobic incubation, 1 mL of each culture was collected and immediately frozen at -80°C for subsequent bacterial DNA extraction. DNA Extraction and Real-time Fluorescence Quantitative PCR (qPCR) Bacterial genomic DNA was extracted using a TIANamp Bacteria DNA Kit (TIANGEN, Beijing, China) according to the manufacturer’s instructions. DNA concentrations and purity were measured using UV-spectrophotometry (Implen NanoPhotometer N50, Germany). qPCR tests were performed in a total reaction volume of 20 µl, containing 10 µl SYBR Green qPCR SuperMix (Transgen Biotech, Beijing, China), 1 µl template DNA, 1 µl primers (Sangon Biotech, Beijing, China), and 8 µl DNase/RNase-free water (Transgen Biotech, Beijing, China). The qPCR reaction was conducted on a MiniOpticon System (Bio-Rad, USA) using the following assay conditions: an initial 3 min denaturation step at 94°C, followed by 40 cycles of 5 s at 94°C, 15 s at 60°C and 10 s at 72°C. Data from qPCR experiments were analyzed using the 2 (−ΔΔCT) method. Primer sequences were listed in Table 1 . Quantification of Virulence Genes Expression in P. gingivalis , F. ucleatum and P. intermedia The minimal inhibitory concentration (MIC) values of EGCG, GTE, TF and L-T for P. gingivalis, F. ucleatum and P. intermedia were determined by a resazurin microplate assay. MIC values were determined as the lowest concentrations at which no bacterial growth occurred. Cetylpyridinium chloride were used as a reference bacteriostatic agent. Porphyromonas gingivalis ATCC 33277, Fusobacterium nucleatum ATCC 10953, and Prevotella intermedia ATCC 25611 were grown to mid-log phase respectively and then three tea-derived bioactives were added at sub-MIC values. The bacterial pellets were collected at 8 h for RNA extraction. RNA was extracted using an Eastep Super Total RNA Extraction Kit (Promega USA) according to the manufacturer’s instructions. RNA concentrations and purities were determined by UV-spectrophotometry (Implen NanoPhotometer N50, Germany), and agarose gel electrophoresis was used to detect RNA integrity. cDNA synthesis was conducted using a PrimeScript RT Master Mix Kit (Takara Japan) in a 20 µl reaction mixture. Reverse transcription qPCR (RT-qPCR) was conducted on a MiniOption System (Bio-Rad, USA). Primer sequences were listed in Table 1 . In Vitro Detection of Hydrogen Sulfide Inhibition Rate To evaluate hydrogen sulfide inhibition, four tea-derived bioactives were added to oral pathogenic bacteria samples (PBS served as the control). Hydrogen sulfide absorption tubes were then inserted into the headspace bottles. The headspace bottles were incubated anaerobically at 37°C for 24 hours. Hydrogen sulfide entered the absorption tubes, reacted chemically with the substances inside, produced color, and reached the corresponding scale. The content and inhibition rate of hydrogen sulfide were calculated based on the color scale. Statistics Means and standard deviations were calculated and analyzed for statistical significance using the Student’s t-test, with a p -value < 0.05 considered statistically significant. Declarations Competing Interests: The authors declare that they have no competing interests. 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Agents Chemother. 49 , 4339–4343 (2005). Yussof, A., Cammalleri, B., Fayemiwo, O., Lopez, S. & Chu, T. Antibacterial and Sporicidal Activity Evaluation of Theaflavin-3,3′-digallate. International J. Mol. Sciences 23 (2022). Könönen, E., Fteita, D., Gursoy, U. K. & Gursoy, M. Prevotella species as oral residents and infectious agents with potential impact on systemic conditions. J. Oral Microbiol. 14 , 2079814 (2022). Han, Y. W. et al. Interactions between periodontal bacteria and human oral epithelial cells: Fusobacterium nucleatum adheres to and invades epithelial cells. Infect. Immun. 68 , 3140–3146 (2000). Marsh, P. D. Dental plaque as a biofilm and a microbial community - implications for health and disease. BMC Oral Health . 6 (Suppl 1), S14 (2006). Stathopoulou, P. G. et al. Porphyromonas gingivalis induce apoptosis in human gingival epithelial cells through a gingipain-dependent mechanism. BMC Microbiol. 9 , 107 (2009). Fournier-Larente, J., Morin, M. P. & Grenier, D. Green tea catechins potentiate the effect of antibiotics and modulate adherence and gene expression in Porphyromonas gingivalis . Arch. Oral Biol. 65 , 35–43 (2016). Fardini, Y. et al. Fusobacterium nucleatum adhesin FadA binds vascular endothelial cadherin and alters endothelial integrity. Mol. Microbiol. 82 , 1468–1480 (2011). Herrero, E. R. et al. Dysbiotic Biofilms Deregulate the Periodontal Inflammatory Response. J. Dent. Res. 97 , 547–555 (2018). Potempa, M. et al. Interpain A, a cysteine proteinase from Prevotella intermedia , inhibits complement by degrading complement factor C3. PLoS Pathog . 5 , e1000316 (2009). Ye, W., Zhang, Y., He, M., Zhu, C. & Feng, X. P. Relationship of tongue coating microbiome on volatile sulfur compounds in healthy and halitosis adults. J. Breath. Res. 14 , 016005 (2019). Chen, W. et al. Bacteria-derived hydrogen sulfide promotes IL-8 production from epithelial cells. Biochem. Biophys. Res. Commun. 391 , 645–650 (2010). Sari, N. N. G., Dermawan, I. G. N. P. & Dewi, A. R. K. A comparison of the effectiveness of gargling green tea and black tea on halitosis reduction. Makassar Dent. J. 12 , 149–152 (2023). Van den Velde, S., van Steenberghe, D., Van Hee, P. & Quirynen, M. Detection of odorous compounds in breath. J. Dent. Res. 88 , 285–289 (2009). Tables Table 1 to 4 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Tables.docx SupplementaryInformation.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6468245","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":498277977,"identity":"f3e52bdd-f46b-469a-a740-2346f43a6bf1","order_by":0,"name":"Meijing Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYBACNgSZwPggoaKGNC3MBg/OHCPJrgQ2yYctzIQV87EfPvi5oMxO3pw9x6wisYGNgb+9OwG/+TxpydIzziUb7ux5Y3YjcYcMg8SZsxvwa5HgMZDmbTvAuOFGDlDLGTYGA4lcQlr4P/8GarEHaSlIbGMmRgsPG8iWRJAWBuK08KSZWfOcS07ecOZZsUTCmWM8BP0i33748W2eMjvbDceTN378UVEjx9/ei18LEuAwAJE8xCoHAfYHpKgeBaNgFIyCEQQAc49F4mtwSO4AAAAASUVORK5CYII=","orcid":"","institution":"Hawley \u0026 Hazel Chemical Co. (ZS) Ltd","correspondingAuthor":true,"prefix":"","firstName":"Meijing","middleName":"","lastName":"Liu","suffix":""},{"id":498277978,"identity":"430a1eca-b5aa-479f-838e-3fc25ca21ba4","order_by":1,"name":"Qian Luo","email":"","orcid":"","institution":"Hawley \u0026 Hazel Chemical Co. (ZS) Ltd","correspondingAuthor":false,"prefix":"","firstName":"Qian","middleName":"","lastName":"Luo","suffix":""},{"id":498277979,"identity":"866574cf-5c5b-4fb7-9f81-5312da542da0","order_by":2,"name":"Xuefen Ding","email":"","orcid":"","institution":"Hawley \u0026 Hazel Chemical Co. (ZS) Ltd","correspondingAuthor":false,"prefix":"","firstName":"Xuefen","middleName":"","lastName":"Ding","suffix":""},{"id":498277980,"identity":"92584680-712e-463d-8ebf-96937d665b99","order_by":3,"name":"Lin Gao","email":"","orcid":"","institution":"Hawley \u0026 Hazel Chemical Co. (ZS) Ltd","correspondingAuthor":false,"prefix":"","firstName":"Lin","middleName":"","lastName":"Gao","suffix":""},{"id":498277981,"identity":"23f469b3-3291-4013-a3a8-eb0f19a51265","order_by":4,"name":"Yi Zhou","email":"","orcid":"","institution":"Hawley \u0026 Hazel Chemical Co. (ZS) Ltd","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Zhou","suffix":""},{"id":498277982,"identity":"d2ed4a44-cc25-4c73-a06f-02a9da2c1731","order_by":5,"name":"Xiaobin Chen","email":"","orcid":"","institution":"Hawley \u0026 Hazel Chemical Co. (ZS) Ltd","correspondingAuthor":false,"prefix":"","firstName":"Xiaobin","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2025-04-17 05:38:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6468245/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6468245/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88793027,"identity":"3ed13363-e701-48a5-bef8-fcceed40ae78","added_by":"auto","created_at":"2025-08-11 13:09:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":23540,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the effects of EGCG, GTE, TF, and L-T on six-species bacterial mixture composition. The relative abundances of pathogenic oral species (Fig. 1a) and beneficial/commensal oral species (Fig. 1b) are shown. All substrates were dissolved in PBS at a concentration of 0.1%. Statistically significant differences compared to the control (PBS) are marked with ‘*’ (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) or ‘**’ (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01). \u003cem\u003ePg\u003c/em\u003e: \u003cem\u003eP. gingivalis\u003c/em\u003e; \u003cem\u003eFn\u003c/em\u003e: \u003cem\u003eF. nucleatum\u003c/em\u003e; \u003cem\u003ePi\u003c/em\u003e: \u003cem\u003eP. intermedia\u003c/em\u003e; \u003cem\u003eLg\u003c/em\u003e: \u003cem\u003eL. gasseri\u003c/em\u003e; \u003cem\u003eLr\u003c/em\u003e: \u003cem\u003eL. rhamnosus\u003c/em\u003e; \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esal\u003c/em\u003e.: \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esalivarius\u003c/em\u003e; EGCG: Epigallocatechin gallate; GTE: Green tea extract; TF: Theaflavin; L-T: L-theanine.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6468245/v1/55061ae718dab9a2ebc21a87.png"},{"id":88794346,"identity":"167afcf4-e7c0-4ac6-abd8-1969af197dd6","added_by":"auto","created_at":"2025-08-11 13:17:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":19483,"visible":true,"origin":"","legend":"\u003cp\u003eThe inhibition rate of hydrogen sulfide of EGCG, GTE, TF, and L-T. All substrates were dissolved in PBS at a concentration of 0.1%. Statistically significant differences compared to the control (PBS) are marked with ‘*’ (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05) or ‘**’ (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). Zinc citrate served as a positive control. EGCG: Epigallocatechin gallate; GTE: Green tea extract; TF: Theaflavin; L-T: L-theanine; H\u003csub\u003e2\u003c/sub\u003eS: Hydrogen sulfide.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6468245/v1/0e64c790601129879fbf13fb.png"},{"id":89531221,"identity":"4f671d4f-d334-4b36-bfb5-a7bf437afab5","added_by":"auto","created_at":"2025-08-21 04:01:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":810923,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6468245/v1/51e5b4dc-1863-400f-9152-bfc69d6c6da5.pdf"},{"id":88793025,"identity":"c2d3919a-67e4-4452-8ed5-ad78cad3bf28","added_by":"auto","created_at":"2025-08-11 13:09:06","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":26745,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-6468245/v1/5cf11ab1e478d6d538a78a43.docx"},{"id":88793021,"identity":"67434b79-6118-4814-bdd8-0c73debf222c","added_by":"auto","created_at":"2025-08-11 13:09:06","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":31903,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-6468245/v1/0074ff757d0f918892d137c7.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Modulatory Effects of Tea-Derived Bioactives on Oral Microbiota and their Virulence-Reducing Properties","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe oral cavity represents a complex ecosystem, characterized by optimal temperatures, pH levels, and abundant nutrient sources that support diverse microbial communities\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Over 700 microbial species inhabit this environment, maintaining a dynamic equilibrium\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Disruptions of the balance due to environmental changes or host immune responses could lead to oral diseases, such as caries, halitosis, and gingivitis\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The oral cavity also served as a reservoir for various pathogens linked to localized and systemic diseases\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. For instance, \u003cem\u003ePorphyromonas gingivalis\u003c/em\u003e affected glucose and lipid metabolism\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eFusobacterium nucleatum\u003c/em\u003e had been implicated in promoting glycolysis in colon cancer cells\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Therefore, maintaining a balanced oral microbiota is essential for preventing oral diseases and mitigating systemic health conditions. Given the growing concerns over bacterial resistance stemming from antibiotic misuse, strategies focused on restoring oral microbial homeostasis offer promising avenues.\u003c/p\u003e\u003cp\u003eCommon oral conditions such as halitosis, caries, and gingivitis were closely associated with oral microbiota dysbiosis\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Halitosis arose from volatile sulfur compounds (VSCs), including hydrogen sulfide, methyl mercaptan, and dimethyl sulfide\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Key contributors to hydrogen sulfide production included \u003cem\u003ePorphyromonas gingivalis\u003c/em\u003e, \u003cem\u003eTreponema denticola\u003c/em\u003e, and \u003cem\u003eTannerella forsythia\u003c/em\u003e—collectively known as the “red complex”\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Methyl mercaptan was produced by anaerobic bacteria such as \u003cem\u003ePrevotella\u003c/em\u003e, \u003cem\u003eVeillonella\u003c/em\u003e, and \u003cem\u003eMegasphaera\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Addressing halitosis requires a comprehensive understanding of the composition and functional dynamics of the oral microbiota. Gingivitis, characterized by inflammation of the gingival tissues, occurred when pathogens such as \u003cem\u003ePorphyromonas gingivalis\u003c/em\u003e, \u003cem\u003eFusobacterium nucleatum\u003c/em\u003e, and \u003cem\u003eTreponema denticola\u003c/em\u003e produced virulence factors that directly damaged periodontal tissues\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn recent years, probiotics and prebiotics had gained attention to combat oral diseases linked to microbial dysbiosis. Probiotics interact with the oral microbiota to promote health\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. For instance, \u003cem\u003eLactobacillus\u003c/em\u003e species enhanced natural immunity and supported microbial balance\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Several common \u003cem\u003eLactobacillus\u003c/em\u003e strains could inhibit the growth of oral pathogens\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Similarly, \u003cem\u003eBifidobacterium\u003c/em\u003e species suppressed black-pigmented anaerobes by competing for essential growth factors like vitamin K\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, reducing the presence of harmful bacteria the gingival of subjects\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e and improving gingival inflammation\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Moreover, prebiotics, defined as “substrates selectively utilized by host microorganisms that confer health benefits,” had also gained attention for promoting the proliferation of resident symbiotic bacteria\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Toothpaste containing arginine reduced the abundance of \u003cem\u003eStreptococcus mutans\u003c/em\u003e in individuals with caries, thereby reducing the enamel demineralization potential\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Four novel potential prebiotic substrates, such as N-acetyl-D-glucosamine were identified, which downregulated the expression of virulence factors of oral pathogens\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Despite their promising potential, research on the applications of prebiotics for oral health were needed.\u003c/p\u003e\u003cp\u003eTea-derived bioactives, particularly polyphenolic components, had shown prebiotic potential. Studies suggested that tea polyphenols participated in regulating gut microbiota composition, similar to the effects of traditional prebiotics like oligosaccharides\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Tea bioactives, derived from water or alcohol-based extraction of tea leaves, including tea polyphenols, theanine, alkaloids, tea polysaccharides, saponins, vitamins, and minerals\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Tea polyphenols, the most characteristic metabolites in tea, included catechins, flavonoids, anthocyanins, and phenolic acids\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Catechins, especially epigallocatechin gallate (EGCG) were the most abundant and active catechin monomer, possessing anti-inflammatory and antioxidant properties\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Theaflavins, unique to black tea, were formed by the oxidation or polymerization of catechins under the action of enzymes (polyphenol oxidase and peroxidase)\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Theanine, chemically named glutamic acid γ-ethylamide, was a unique free amino acid in tea with functions of enhancing immunity\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Numerous studies had demonstrated that regular tea consumption could prevent dental caries, combat halitosis, and promote oral health\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. For example, tea polyphenols exerted significant inhibitory effects on oral pathogens: they could prevent caries by inhibiting the metabolism of \u003cem\u003eStreptococcus mutans\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e and reduce halitosis by suppressing the proliferation of \u003cem\u003ePorphyromonas gingivalis\u003c/em\u003e and \u003cem\u003eFusobacterium nucleatum\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Additionally, tea polyphenols possessed prebiotic potential, by promoting the growth of some beneficial gut bacteria and altering the structure of the gut microbiota. In vitro anaerobic fermentation studies showed that EGCG promoted the growth of beneficial genera such as \u003cem\u003eBacteroides\u003c/em\u003e, \u003cem\u003eFaecalibacterium\u003c/em\u003e, and \u003cem\u003eBifidobacterium\u003c/em\u003e, while inhibiting harmful bacteria\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Similarly, rodent studies indicated that phenolic compounds from tea extracts altered the proportions of various gut microorganisms\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. However, most gut microbiota studies were focused primarily on genus-level changes, leaving species-level dynamics largely unexplored. Similarly, research on the effects of tea-derived bioactives on oral pathogens had been largely confined to single pathogens or single-species biofilms, with limited attention given to their interactions within complex microbial communities.\u003c/p\u003e\u003cp\u003eTo advance the application of tea-derived bioactives in oral health products, further in-depth studies were needed to explore their potential in balancing oral microbiota. In this study, we conducted a comprehensive investigation into the effects of tea polyphenols from various sources on specific oral bacterial species. We also examined the influence of tea polyphenols on the expression of virulence factors in oral pathogens. Finally, we demonstrated the inhibitory effects of tea polyphenols on hydrogen sulfide production in salivary bacteria and elucidated the underlying molecular mechanisms.\u003c/p\u003e"},{"header":"Result","content":"\u003cp\u003e\u003cstrong\u003eEffects of Tea-Derived Bioactives on Oral Bacteria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo comprehensively evaluate the regulatory effects of the four tea-derived bioactives, including epigallocatechin gallate (EGCG), green tea extract (GTE), theaflavins (TF), and L-theanine (L-T) on the structure of mixed oral bacteria, the relative abundances of six bacterial species, including \u003cem\u003eP\u003c/em\u003e. \u003cem\u003egingivalis\u003c/em\u003e, \u003cem\u003eF\u003c/em\u003e. \u003cem\u003enucleatum\u003c/em\u003e, \u003cem\u003eP\u003c/em\u003e. \u003cem\u003eintermedia\u003c/em\u003e, \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esalivarius\u003c/em\u003e, \u003cem\u003eL\u003c/em\u003e. \u003cem\u003egasseri\u003c/em\u003e, and \u003cem\u003eL\u003c/em\u003e. \u003cem\u003erhamnosus\u0026nbsp;\u003c/em\u003ewere analyzed. Significant biological relevance was attributed to differences in the substrate-treated groups relative to untreated mixed oral bacteria, with either more than a 2-fold downregulation (2\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003csup\u003e\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.5) or more than a 1.5-fold upregulation (2\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003csup\u003e\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e\u0026thinsp;\u0026gt;\u0026thinsp;1.5).\u003c/p\u003e\n\u003cp\u003eThe addition of 0.1% EGCG led to a decrease in the relative abundance of \u003cem\u003eF. nucleatum\u003c/em\u003e and \u003cem\u003eP. intermedia\u003c/em\u003e, while significantly increasing \u003cem\u003eL. gasseri\u003c/em\u003e and \u003cem\u003eL. rhamnosus\u003c/em\u003e (Fig. 1a, 1b). Similarly, 0.1% GTE inhibited all three pathogenic bacteria and resulted in an 8.2-fold increase in \u003cem\u003eL. gasseri\u003c/em\u003e. TF at the same concentration inhibited \u003cem\u003eF. nucleatum\u003c/em\u003e and promoted the growth of \u003cem\u003eL. gasseri\u003c/em\u003e. No significant difference was obtained with an addition of 0.1% L-T. For \u003cem\u003eS. salivarius\u003c/em\u003e, no substantial differences were observed under any of the substrate conditions (Fig. 1b). Among the tested tea-derived bioactives, EGCG and GTE proved the most effective in optimizing the oral bacterial structure, outperforming TF, while L-T exhibited no substantial impact.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of Tea Polyphenols on Virulence Factors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe antibacterial activities of EGCG, GTE, TF and L-T against\u003cem\u003e\u0026nbsp;P. gingivalis, F. nucleatum\u003c/em\u003e and \u003cem\u003eP. intermedia\u0026nbsp;\u003c/em\u003ewere assessed using a resazurin microplate assay. Among the tested tea-derived bioactives, EGCG, GTE, and TF all exhibited antibacterial effects against all three pathogenic bacteria (table 1), with EGCG exhibiting the strongest activity. The MIC value of cetylpyridinium chloride used as a reference compound, ranged from 3.91 to 7.81 \u0026micro;g/mL. Since L-T showed no inhibitory effects on\u0026nbsp;these pathogens, subsequent studies on changes in virulence gene expression focused only on EGCG, GTE and TF.\u003c/p\u003e\n\u003cp\u003eThe expression of 16 virulence genes (for an overview of the associated functions of the corresponding virulence factors: see Supplementary Table S2) from the three periodontal pathogens was analyzed with the addition of low-concentration tea polyphenols. Gene expression of the three periodontal pathogens was relative to the expression of 16S rRNA. Significant changes in gene expression in the substrate-treated bacteria, relative to the untreated bacteria, were considered biologically relevant if they were more than 2-fold downregulated (2\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003csup\u003e\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.5) or more than 1.5-fold upregulated (2\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003csup\u003e\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e\u0026thinsp;\u0026gt;\u0026thinsp;1.5).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eP. gingivalis\u003c/em\u003e, a primary pathogen responsible for gingivitis, relies heavily on gingipains as its main virulence factors. All three tea polyphenols significantly inhibited the expression of gingipain genes (\u003cem\u003ekgp\u003c/em\u003e and \u003cem\u003ergpA\u003c/em\u003e). Regarding virulence factors involved in the colonization of subgingival sites, significant decreases of \u003cem\u003ehagA\u003c/em\u003e, \u003cem\u003ehagB\u003c/em\u003e, and \u003cem\u003efimA\u003c/em\u003e gene expression were observed for all polyphenols, with TF causing a particularly notable reduction in \u003cem\u003efimA\u003c/em\u003e expression, by over 29-fold. Similarly, the expression level of the \u003cem\u003ehem\u003c/em\u003e gene, which encodes a hemolysin in \u003cem\u003eP. gingivalis\u003c/em\u003e, was also repressed by EGCG, GTE, and TF. Additionally, the \u003cem\u003emgl\u003c/em\u003e gene, linked to halitosis, was downregulated across all treatments. Conversely, the stress protease gene\u003cem\u003e\u0026nbsp;htrA\u003c/em\u003e was upregulated, with EGCG increasing its expression by 4.8-fold and TF by 4.3-fold at higher concentrations (Table 2).\u003c/p\u003e\n\u003cp\u003eFocal adhesion A (\u003cem\u003efadA\u003c/em\u003e) is one of the most important virulence genes of \u003cem\u003eF. nucleatum\u003c/em\u003e, contributing to tissue destruction. Of the three tea polyphenols, only 125 ppm EGCG inhibited\u003cem\u003e\u0026nbsp;fadA\u0026nbsp;\u003c/em\u003eexpression. Additionally, the expression level of \u003cem\u003emegl\u003c/em\u003e gene, linked to halitosis, was only downregulated by the addition of 125 ppm EGCG. Two genes (\u003cem\u003eaid1\u003c/em\u003e and \u003cem\u003efomA\u003c/em\u003e) associated with bacterial coaggregation were unaffected by any of the tea polyphenols. Interestingly, the ABC transporter permease gene (transp.), which encodes a type of resistance protein, was upregulated by all three tea polyphenols without a clear dose-response effect (Table 3). In \u003cem\u003eP. intermedia\u003c/em\u003e, virulence genes \u003cem\u003eecf\u003c/em\u003e, \u003cem\u003einpA\u003c/em\u003e and \u003cem\u003egroeS\u003c/em\u003e were significantly downregulated (2.1- to 9.9-fold) compared to the control after 8 hours of growth in the presence of the three tea polyphenols (Table 4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of Tea-Derived Bioactives on Hydrogen Sulfide Production by Oral Pathogenic Bacteria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVolatile sulfur compounds (VSCs), including hydrogen sulfide (H\u003csub\u003e2\u003c/sub\u003eS) and methyl mercaptan (CH\u003csub\u003e3\u003c/sub\u003eSH), were major contributors to halitosis. The inhibitory effects of the four tea-derived bioactives on hydrogen sulfide production by oral pathogenic bacteria were evaluated to assess their potential efficacy against halitosis-related oral bacterial species. At a substrate concentration of 0.1%, all three active polyphenols (EGCG, GTE, and TF) exhibited significant inhibitory effects on hydrogen sulfide production compared to the control (Fig. 1). Among them, TF demonstrated the highest inhibition rate (95%), followed by GTE (79%) and EGCG (44%). Conversely, L-T showed negligible effects on hydrogen sulfide reduction (Fig. 2).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur research indicated that EGCG (epigallocatechin gallate), GTE (green tea extracts), and TF (theaflavins) could regulate the structure of the salivary microbiome, and we analyzed the relative abundance changes of major oral bacterial species. Previous in vitro fermentation studies showed that EGCG regulated the structure of the gut microbiome and promoted the growth of \u003cem\u003eBifidobacterium\u003c/em\u003e.spp\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. A clinical study also demonstrated the regulatory effect of theaflavin-containing toothpaste on the oral microbiome\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. However, most existing studies lacked species-level analysis of the oral microbiome and comparisons of the effects of different tea-derived bioactives on balancing the microbiome.\u003c/p\u003e\u003cp\u003eIn this study, EGCG exhibited the most pronounced effect in inhibiting harmful bacteria while promoting the growth of beneficial bacteria. These effects could be influenced by factors such as the complexity of the in vitro model, the degree of oxidation, and the purity of the raw materials. Few studies reported the antibacterial effect of theanine, and this study did not observe any significant impact of theanine on salivary bacteria. Notably, all three tea-derived bioactives effectively inhibited \u003cem\u003ePorphyromonas gingivalis\u003c/em\u003e, \u003cem\u003eFusobacterium nucleatum\u003c/em\u003e, and \u003cem\u003ePrevotella intermedia\u003c/em\u003e. Furthermore, they increased the relative abundance of \u003cem\u003eLactobacillus gasseri and Lactobacillus rhamnosu\u003c/em\u003e. Several studies had suggested that tea polyphenols exerted their antibacterial effects by chelating essential bacterial nutrients, such as iron\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, and inhibiting nucleic acid precursor synthesis\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Therefore, under normal circumstances, high doses of EGCG could also inhibit individual beneficial bacteria. Some research suggested that 2.5 mg/mL EGCG inhibits \u003cem\u003eP. gingivalis\u003c/em\u003e, while 25 mg/mL EGCG was required to inhibit the growth of \u003cem\u003eLactobacillus salivarius\u003c/em\u003e WB21\u003csup\u003e42\u003c/sup\u003e, indicating that \u003cem\u003eLactobacilli\u003c/em\u003e possessed a relatively high tolerance to EGCG\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. This implied that the effects of EGCG on oral bacteria were dose-dependent\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. We hypothesized that within mixed bacterial communities, interactions among bacteria would allow low doses of tea polyphenols to selectively inhibit oral pathogens while promoting the growth of beneficial bacteria, ultimately contributing to oral microbiome balance and promoting oral health\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eqPCR analysis revealed a marked reduction in \u003cem\u003ePrevotella intermedia\u003c/em\u003e, a keystone pathogen in periodontitis pathogenesis\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, along with a reduction in \u003cem\u003eFusobacterium nucleatum\u003c/em\u003e, a species implicated in periodontal disease and oral carcinogenesis. Conversely, the intervention significantly enhanced \u003cem\u003eLactobacillus\u003c/em\u003e abundances. The proliferation of \u003cem\u003eLactobacillus\u003c/em\u003e potentially inhibited pathogens through bacteriocin and organic acid production\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Notably, while \u003cem\u003eLactobacillus\u003c/em\u003e proliferation benefited microbial balance, its excessive growth could lower oral pH and increase enamel demineralization risk\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, warranting longitudinal investigation. Collectively, these findings suggested tea polyphenols modulate oral health through suppressing periodontal pathogens while promoting symbiotic flora proliferation, thereby establishing a foundation for microbiome-targeted oral care strategies.\u003c/p\u003e\u003cp\u003e\u003cem\u003ePorphyromonas gingivalis\u003c/em\u003e was a key pathogen associated with periodontitis, with gingipains (\u003cem\u003ekgp\u003c/em\u003e and \u003cem\u003ergp\u003c/em\u003e) being its primary virulence factors\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Consistent with previous studies\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, we confirmed that EGCG and green tea extracts significantly inhibited the expression of these virulence factors. Notably, our findings revealed that theaflavins, at sub-MIC concentrations, demonstrated a stronger inhibitory effect on the expression of \u003cem\u003eP. gingivalis\u003c/em\u003e virulence factors compared to EGCG and green tea extracts. This suggested that theaflavins had the potential to attenuate \u003cem\u003eP. gingivalis\u003c/em\u003e virulence while maintaining the balance of the oral microbiome.\u003c/p\u003e\u003cp\u003e\u003cem\u003eFusobacterium nucleatum\u003c/em\u003e utilized adhesins such as \u003cem\u003eFadA\u003c/em\u003e to invade gingival epithelial cells and produces lipopolysaccharides (LPS) that damage periodontal tissue\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. While a prior study using tea polyphenols found no effect on the expression of adhesin genes (\u003cem\u003efap2\u003c/em\u003e, \u003cem\u003efadA\u003c/em\u003e, \u003cem\u003eaid1\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, our results indicated that 125 ppm EGCG significantly inhibited \u003cem\u003efadA\u003c/em\u003e gene expression. Since the original study did not provide specific data, differences in EGCG dosage or primer design could account for this discrepancy. Overall, EGCG not only reduced the virulence of \u003cem\u003eF. nucleatum\u003c/em\u003e but also inhibited its growth.\u003c/p\u003e\u003cp\u003eFor \u003cem\u003ePrevotella intermedia\u003c/em\u003e, the RNA polymerase ECF-type sigma factor (\u003cem\u003eecf\u003c/em\u003e) played a role in polysaccharide production, enhancing its resistance to host defenses\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Additionally, Interpain A (\u003cem\u003eInpA\u003c/em\u003e) was associated with the bacterium\u0026rsquo;s serum resistance\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Our findings revealed that all three tea polyphenols significantly inhibited the expression of virulence factors such as \u003cem\u003eecf\u003c/em\u003e and \u003cem\u003eInpA\u003c/em\u003e. At sub-MIC concentrations, EGCG and green tea extracts demonstrated stronger inhibitory effects than theaflavins, consistent with the slight effect of theaflavins on the growth of \u003cem\u003eP. intermedia\u003c/em\u003e. Few studies had reported the effects of tea polyphenols on \u003cem\u003eP. intermedia\u003c/em\u003e's antimicrobial mechanisms and virulence factors, but our results suggested that the inhibition of virulence factor expression could play a crucial role in inhibiting its growth. Moreover, the three tea polyphenols inhibited the expression of genes related to halitosis, such as \u003cem\u003emgl\u003c/em\u003e and \u003cem\u003emegl\u003c/em\u003e, offering insight into their ability to decrease the production of hydrogen sulfide and other odor-causing gases.\u003c/p\u003e\u003cp\u003eOral pathogenic bacteria, such as \u003cem\u003eP. gingivalis\u003c/em\u003e and \u003cem\u003eF. nucleatum\u003c/em\u003e, produce volatile sulfur compounds (VSCs) like hydrogen sulfide (H₂S), which were primary contributors to halitosis\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Beyond causing bad breath, these sulfur compounds were toxic to oral mucosal cells and trigger inflammatory responses, potentially leading to gingivitis\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Therefore, identifying natural substances to control VSC production was critical for the prevention and management of both halitosis and gingivitis. In our study, we found that, except theanine, EGCG, green tea extracts, and theaflavins all demonstrated effects in inhibiting hydrogen sulfide production in vitro, which aligned with the results that theanine had minimal impact on oral microbiota structure. We speculated that the reduction in hydrogen sulfide might be related to the role of the three tea polyphenols in promoting a balanced oral microbiome. Previous clinical studies showed that green tea mouthwash was more effective in eliminating halitosis than black tea\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. However, our results suggested that theaflavins were more effective than green tea polyphenols in inhibiting hydrogen sulfide production. This discrepancy could stem from the fact that halitosis gases include methanethiol, hydrogen sulfide, and dimethyl sulfide\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e, whereas our study exclusively measured hydrogen sulfide levels. As a result, direct comparisons among the three tea-derived bioactives for overall halitosis gas elimination remain inconclusive.\u003c/p\u003e\u003cp\u003eIn summary, this study demonstrated that EGCG, green tea extracts, and theaflavins effectively inhibited the production of volatile sulfur compounds, thereby reducing halitosis gases. More specifically, these compounds promoted the growth of beneficial bacteria and inhibited the proliferation of harmful bacteria, contributing to the regulation of the salivary microbiome. Additionally, tea polyphenols reduced the expression of virulence factors and volatile sulfur compound-producing genes in \u003cem\u003eP. gingivalis\u003c/em\u003e, \u003cem\u003eF. nucleatum\u003c/em\u003e, and \u003cem\u003eP. intermedia\u003c/em\u003e under sub-MIC conditions. Overall, our findings suggested that polyphenols from green and black tea held promise as adjunct therapies for the treatment of halitosis and gingivitis.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cb\u003eTea-Derived Bioactives\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTheaflavins (TF) with a purity of 40%, was purchased from Yaizu Suisankagaku (Japan). Epigallocatechin gallate (EGCG) with a purity of 94%, green tea extract (GTE) with a purity of at least 70%, and L-theanine (L-T) with a purity of at least 30%, were all naturally extracted from the plant \u003cem\u003eCamellia sinensis\u003c/em\u003e and purchased from Novanat (Shanghai, China).\u003c/p\u003e\u003cp\u003e\u003cb\u003eBacteria and Growth Conditions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe following bacterial strains were used as representative oral pathogens: \u003cem\u003ePorphyromonas gingivalis\u003c/em\u003e ATCC 33277, \u003cem\u003eFusobacterium nucleatum\u003c/em\u003e ATCC 10953, \u003cem\u003ePrevotella intermedia\u003c/em\u003e ATCC 25611. Conversely, \u003cem\u003eStreptococcus salivarius\u003c/em\u003e BAA1024, \u003cem\u003eLactobacillus gasseri\u003c/em\u003e ATCC 33323, \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e ATCC 7469 were used as representative commensal/beneficial oral bacterial strains.\u003c/p\u003e\u003cp\u003eAll six bacteria were grown in Brain-Heart Infusion broth (BHI; Difco, Sparks, MD), which contained no salivary proteins. All bacteria cultures were incubated at 37 ℃ in an anaerobic chamber.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn Vitro Co-culture of Tea-Derived Bioactives with Oral Bacteria\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSix oral bacteria were co-cultured using BHI culture medium, including \u003cem\u003eP. gingivalis\u003c/em\u003e, \u003cem\u003eF. nucleatum\u003c/em\u003e, \u003cem\u003eP. intermedia\u003c/em\u003e, \u003cem\u003eS. salivarius, L. gasseri\u003c/em\u003e, and \u003cem\u003eL. rhamnosus\u003c/em\u003e. Four tea-derived bioactives were added to the cultures at a final concentration of 0.1%. After 24 hus of anaerobic incubation, 1 mL of each culture was collected and immediately frozen at -80\u0026deg;C for subsequent bacterial DNA extraction.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDNA Extraction and Real-time Fluorescence Quantitative PCR (qPCR)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBacterial genomic DNA was extracted using a TIANamp Bacteria DNA Kit (TIANGEN, Beijing, China) according to the manufacturer\u0026rsquo;s instructions. DNA concentrations and purity were measured using UV-spectrophotometry (Implen NanoPhotometer N50, Germany).\u003c/p\u003e\u003cp\u003eqPCR tests were performed in a total reaction volume of 20 \u0026micro;l, containing 10 \u0026micro;l SYBR Green qPCR SuperMix (Transgen Biotech, Beijing, China), 1 \u0026micro;l template DNA, 1 \u0026micro;l primers (Sangon Biotech, Beijing, China), and 8 \u0026micro;l DNase/RNase-free water (Transgen Biotech, Beijing, China). The qPCR reaction was conducted on a MiniOpticon System (Bio-Rad, USA) using the following assay conditions: an initial 3 min denaturation step at 94\u0026deg;C, followed by 40 cycles of 5 s at 94\u0026deg;C, 15 s at 60\u0026deg;C and 10 s at 72\u0026deg;C. Data from qPCR experiments were analyzed using the 2\u003csup\u003e(\u0026minus;ΔΔCT)\u003c/sup\u003e method. Primer sequences were listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eQuantification of Virulence Genes Expression in\u003c/b\u003e \u003cb\u003eP. gingivalis\u003c/b\u003e, \u003cb\u003eF. ucleatum\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eP. intermedia\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe minimal inhibitory concentration (MIC) values of EGCG, GTE, TF and L-T for \u003cem\u003eP. gingivalis, F. ucleatum\u003c/em\u003e and \u003cem\u003eP. intermedia\u003c/em\u003e were determined by a resazurin microplate assay. MIC values were determined as the lowest concentrations at which no bacterial growth occurred. Cetylpyridinium chloride were used as a reference bacteriostatic agent. \u003cem\u003ePorphyromonas gingivalis\u003c/em\u003e ATCC 33277, \u003cem\u003eFusobacterium nucleatum\u003c/em\u003e ATCC 10953, and \u003cem\u003ePrevotella intermedia\u003c/em\u003e ATCC 25611 were grown to mid-log phase respectively and then three tea-derived bioactives were added at sub-MIC values. The bacterial pellets were collected at 8 h for RNA extraction. RNA was extracted using an Eastep Super Total RNA Extraction Kit (Promega USA) according to the manufacturer\u0026rsquo;s instructions. RNA concentrations and purities were determined by UV-spectrophotometry (Implen NanoPhotometer N50, Germany), and agarose gel electrophoresis was used to detect RNA integrity. cDNA synthesis was conducted using a PrimeScript RT Master Mix Kit (Takara Japan) in a 20 \u0026micro;l reaction mixture. Reverse transcription qPCR (RT-qPCR) was conducted on a MiniOption System (Bio-Rad, USA). Primer sequences were listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn Vitro Detection of Hydrogen Sulfide Inhibition Rate\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo evaluate hydrogen sulfide inhibition, four tea-derived bioactives were added to oral pathogenic bacteria samples (PBS served as the control). Hydrogen sulfide absorption tubes were then inserted into the headspace bottles. The headspace bottles were incubated anaerobically at 37\u0026deg;C for 24 hours. Hydrogen sulfide entered the absorption tubes, reacted chemically with the substances inside, produced color, and reached the corresponding scale. The content and inhibition rate of hydrogen sulfide were calculated based on the color scale.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistics\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMeans and standard deviations were calculated and analyzed for statistical significance using the Student\u0026rsquo;s t-test, with a \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered statistically significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting Interests:\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM.L. contributed to conception, design, data acquisition and analysis, interpretation, drafted and revised the manuscript. M.L. Q.L. and X.D. conducted the majority of the experiments. L.G. and Y.Z. provided suggestions for the project. X.C. supervised the project and critically revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe authors declared that all data supporting the findings of this study were available within the paper and its supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFaran Ali, S. M. \u0026amp; Tanwir, F. Oral microbial habitat a dynamic entity. \u003cem\u003eJ. Oral Biology Craniofac. Res.\u003c/em\u003e \u003cb\u003e2\u003c/b\u003e, 181\u0026ndash;187 (2012).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKuramitsu Howard, K., He, X., Lux, R., Anderson Maxwell, H. \u0026amp; Shi, W. Interspecies Interactions within Oral Microbial Communities. \u003cem\u003eMicrobiol. Mol. Biol. Rev.\u003c/em\u003e \u003cb\u003e71\u003c/b\u003e, 653\u0026ndash;670 (2007).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKozak, M. \u0026amp; Pawlik, A. 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A comparison of the effectiveness of gargling green tea and black tea on halitosis reduction. \u003cem\u003eMakassar Dent. J.\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 149\u0026ndash;152 (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVan den Velde, S., van Steenberghe, D., Van Hee, P. \u0026amp; Quirynen, M. Detection of odorous compounds in breath. \u003cem\u003eJ. Dent. Res.\u003c/em\u003e \u003cb\u003e88\u003c/b\u003e, 285\u0026ndash;289 (2009).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 to 4 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Tea-derived bioactives, Oral microbiota, Virulence factors, Halitosis","lastPublishedDoi":"10.21203/rs.3.rs-6468245/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6468245/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTea polyphenols exhibited potent biological activity and were increasingly recognized as potential prebiotics. While their inhibitory effects on certain oral pathogens were well-documented, their impact on oral commensal bacteria remained unclear. This study investigated the effects of tea-derived bioactives on the composition of oral bacterial communities, with quantitative polymerase chain reaction (qPCR) utilized to assess changes in pathogenic and commensal bacteria. Epigallocatechin gallate (EGCG), green tea extract, and theaflavins were found to exert inhibitory effects on key oral pathogens, including \u003cem\u003ePorphyromonas gingivalis\u003c/em\u003e, \u003cem\u003eFusobacterium nucleatum\u003c/em\u003e, and \u003cem\u003ePrevotella intermedia\u003c/em\u003e. Concurrently, these polyphenols promoted the growth of beneficial oral commensal bacteria, such as \u003cem\u003eLactobacillus gasseri\u003c/em\u003e and \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e. At sub-inhibitory concentrations, tea polyphenols significantly suppressed the expression of virulence factors in \u003cem\u003eP. gingivalis\u003c/em\u003e, suggesting that low concentrations were sufficient to mitigate pathogenicity without disrupting microbial homeostasis. Furthermore, tea polyphenols reduced oral VSCs by downregulating hydrogen sulfide/methyl mercaptan synthase genes. Hence, their dual capacity to inhibit pathogens and support commensal bacteria could make them promising candidates for oral health products.\u003c/p\u003e","manuscriptTitle":"Modulatory Effects of Tea-Derived Bioactives on Oral Microbiota and their Virulence-Reducing Properties","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-11 13:08:44","doi":"10.21203/rs.3.rs-6468245/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cc3718c0-883e-4efa-8795-6e8db365943c","owner":[],"postedDate":"August 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":52926204,"name":"Health sciences/Health care/Dentistry/Oral microbiology"},{"id":52926205,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2025-08-21T03:53:39+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-11 13:08:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6468245","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6468245","identity":"rs-6468245","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}