Antibacterial activities of Miang extracts against selected pathogens and the potential of the tannin-free extracts in the growth inhibition of Streptococcus mutans

Aliyu Dantani Abdullahi; Kridsada Unban; Chalermpong Saenjum; Pratthana Kodchasee; Napapan Kangwan; Hathairat Thananchai; Kalidas Shetty; Chartchai Khanongnuch

doi:10.1371/journal.pone.0302717

. 2024 May 8;19(5):e0302717. doi: 10.1371/journal.pone.0302717

Antibacterial activities of Miang extracts against selected pathogens and the potential of the tannin-free extracts in the growth inhibition of Streptococcus mutans

Aliyu Dantani Abdullahi ¹, Kridsada Unban ², Chalermpong Saenjum ³, Pratthana Kodchasee ⁴, Napapan Kangwan ⁵, Hathairat Thananchai ⁶, Kalidas Shetty ⁷, Chartchai Khanongnuch ^4,^8,^9,^*

Editor: Mozaniel Santana de Oliveira¹⁰

PMCID: PMC11078415 PMID: 38718045

Abstract

Bacterial pathogens have remained a major public health concern for several decades. This study investigated the antibacterial activities of Miang extracts (at non-neutral and neutral pH) against Bacillus cereus TISTR 747, Escherichia coli ATCC 22595, Salmonella enterica serovar Typhimurium TISTR 292 and Streptococcus mutans DMST 18777. The potential of Polyvinylpolypyrrolidone (PVPP)-precipitated tannin-free Miang extracts in growth-inhibition of the cariogenic Streptococcus mutans DMST 18777 and its biofilms was also evaluated. The tannin-rich fermented extracts had the best bacterial growth inhibition against S. mutans DMST 18777 with an MIC of 0.29 and 0.72 mg/mL for nonfilamentous fungi (NFP) Miang and filamentous-fungi-processed (FFP) Miang respectively. This observed anti-streptococcal activity still remained after PVPP-mediated precipitation of bioactive tannins especially, in NFP and FFP Miang. Characterization of the PVPP-treated extracts using High performance liquid chromatography quadrupole-time of flight-mass spectrometry (HPLC-QToF-MS) analysis, also offered an insight into probable compound classes responsible for the activities. In addition, Crystal violet-staining also showed better IC₅₀ values for NFP Miang (4.30 ± 0.66 mg/mL) and FFP Miang (12.73 ± 0.11 mg/mL) against S. mutans DMST 18777 biofilms in vitro. Homology modeling and molecular docking analysis using HPLC-MS identified ligands in tannin-free Miang supernatants, was performed against modelled S. mutans DMST 18777 sortase A enzyme. The in silico analysis suggested that the inhibition by NFP and FFP Miang might be attributed to the presence of ellagic acid, flavonoid aglycones, and glycosides. Thus, these Miang extracts could be optimized and explored as natural active pharmaceutical ingredients (NAPIs) for applications in oral hygienic products.

Introduction

The United Nations’ drive to foster sustainable practices have encouraged the pursuit of good health and well-being as one of the sustainable development goals (SDGs) [1,2]. In this context, resilient efforts to combat infectious diseases and pathogenic bacteria (such as Bacillus cereus and Escherichia coli) that cause foodborne illnesses and/or pose serious problems to human health is a current research quest [3]. For a time, the discovery of antibiotics was a milestone that proved effective towards the eradication of these bacterial pathogens. However, this breakthrough has been significantly watered down due to the rapid spread of antimicrobial resistance thus necessitating a need to search for better alternatives including natural antimicrobial agents from food sources [4,5].

Miang is an ethnic fermented tea made from leaves of Camellia sinensis var. assamica and has been reported to possess strong antioxidant, anti-inflammatory and antimicrobial activities [6–8]. Kodchasee et al. [9] and Unban et al. [10] have extensively studied the two processes adopted in the production of fermented Miang products including nonfilamentous fungi growth based (NFP)-Miang and filamentous fungi growth based (FFP)-Miang respectively, where they attributed the bioactivities of these teas to tannins, phenolic acids, catechin and its derivatives. Therefore, Miang extracts could be explored as an eco-friendly bid to prolong the shelf-life of food products as well as inhibit the growth of pathogenic bacteria.

Second only to the gastrointestinal gut, the oral cavity is home to diverse and abundant microbial communities (> 700 bacterial species) some of which are major players in disease pathogenesis especially, in events of oral Dysbiosis [11,12]. The translocation of oral microbes to the gastrointestinal tract (GIT) via enteral or hematogenous routes has been linked to colorectal cancer, irritable and inflammatory bowel diseases [11]. Streptococcus mutans is an aciduric and acidogenic dental pathogen implicated in dental caries and has closely evolved in association with the human host making it a model Gram-positive organism [13]. Together with other anaerobes and oral Streptococci, these pathogens organize into biofilms on dental surfaces and metabolize food remnants (fermentable sugars). The biofilm formation is mediated through sortase A enzyme which anchors surface proteins on bacterial cell walls to allow host attachment by S. mutans biofilms. Consequently, these events lead to the formation of dental plaque, demineralization of enamel, and leaching of teeth components [14,15]. Even though antimicrobials such as Chlorhexidine (CHX) are usually incorporated in mouth rinses as chemical anti-plaque agents that serve as adjuncts to the conventional mechanical plaque control strategies such as tooth-brushing and dental flossing [16,17], long term usage leads to bacterial resistance, teeth stains, erosion of the oral mucosa and taste alteration [18,19]. Moreover, tannins found in tea and numerous other plants have been reported to exert potent bacterial growth inhibition against Gram-positive and Gram-negative bacteria [20]. However, these compounds are known to be bitter and astringent, cause tooth stains/discolorations as well as bind histidine-rich proteins in the saliva to cause a moisture void that leads to dry mouth [21,22]. These drawbacks necessitate the need to explore other effective food products-derived antimicrobial alternatives that are preferably devoid of tannins, with an acceptable sensory, organoleptic and consumer-friendly properties that could find application as constituents of mouthwash.

In this study, the antimicrobial activities of acetone extracts including NFP Miang, FFP Miang, young (YTL) and mature (MTL) tea leaves (raw materials for NFP and FFP Miang respectively) were evaluated against two Gram-positive (Bacillus cereus TISTR 747 and Streptococcus mutans DMST 18777) and Gram-negative (Escherichia coli ATCC 22595 and Salmonella enterica serovar Typhimurium TISTR 292) bacteria respectively. In addition, the tea samples were chemically treated to produce tannin-free extracts after which their antimicrobial activities were further investigated against S. mutans DMST 18777.

Materials and methods

Sample collection

FFP and NFP Miang samples were purchased from local producers in Pa-dang sub-district, Muang district, Phrae Province and Papae sub-district, Mae-Tang district, Chiang Mai Province Thailand, respectively. The starting materials for FFP and NFP Miang fermentation were mature tea leaves MTL (begins at the sixth leaf from the tea shoot) and fresh young tea leaves YTL (covers the second to the sixth leaf from the tea shoot) were also obtained from the above-mentioned locations.

Preparation of Miang extracts

FFP Miang, NFP Miang, MTL and YTL were dried in a vacuum dryer (VD53 Binder Oven, Germany) for 24 h at 50 °C then ground into a powder with a blender respectively. 80% acetone (100 mL) was added to each sample (5g) and the extraction was carried out in a shaking incubator (Daihan Labtech Co., Ltd., Namyangju-City, Korea) set at 30 °C, 150 rpm for 1 h. The solution was filtered using a 125 mm Whatman filter paper and concentrated at 40 °C using rotary evaporator (N-1000 Eyela Rotary Evaporator; Tokyo, Japan). The crude extract was dissolved in 20 mL of deionized (DI) water (mg extract/mL), and then stored at -20 °C freezer until further use. For the antimicrobial tests, an aliquot of each extract was prepared and the pH was adjusted to neutral using 0.1 M NaOH.

Bacterial strains and growth conditions

Bacterial strains were collected from Thailand Institute of Scientific and Technological Research (TISTR), Department of Medical Sciences Thailand (DMST) and American Type Culture Collection (ATCC) including Bacillus cereus TISTR 747, Salmonella enterica ser. Typhimurium TISTR 292, Streptococcus mutans DMST 18777 and Escherichia coli ATCC 22595. Three pathogens were cultivated in Mueller Hinton agar (HiMedia, India) at 37 °C while S. mutans DMST 18777 was cultivated in Trypticase soy medium (HiMedia, India) supplemented with 1% sucrose at 37 °C under microaerophilic condition.

Determination of antibacterial activities of neutralized (pH 7) and non-neutralized (pH 4.0–4.6) Miang extracts

The standard agar well diffusion method as described by Hossain et al. [23] with slight modification, was used to measure the antibacterial activities of Miang extracts. Briefly, 4–5 isolated colonies were taken from each 18–24 h pure culture, for subsequent subculture overnight in 5 mL normal saline. Afterward, the culture was adjusted to 0.5 McFarland turbidity standard (~1.0 × 10⁸) and 200 μL of each bacterial culture was spread on agar plate surfaces. A sterile borer (6mm diameter) was used to make wells on the agar and 30 μL of the Miang extracts each or control (DI water) were dispensed into the well and the plates were incubated for 24 h at 37 °C. The zone of inhibition around each well was measured as diameters and the units were represented in millimeters (mm). Inhibition by neutralized extract and organic acids is calculated according to the following equation: inhibition by neutralized extract (%) = (zone of inhibition for neutralized extract/zone of inhibition for non-neutralized extract) × 100, and inhibition by organic acids (%) = 100 ˗ inhibition by neutralized extract.

Determination of minimum inhibitory (MIC) and minimum bactericidal concentrations (MBC) of neutralized (pH 7) and non-neutralized (pH 4.0–4.6) Miang extracts

The broth dilution method was used to determine MIC and MBC according to the method of Nibir et al. [24] with little modifications. Briefly, overnight cultures were adjusted to 0.5 McFarland standards (~1.0 × 10⁸ CFU/mL) and a 1:1 dilution of each bacterial culture was inoculated into two-fold serially diluted Miang extracts respectively. After incubation for 18 h, 5 μL of the serially diluted cultures were plated onto Mueller Hinton agar and Trypticase soy agar (Streptococcus mutans) as drop plates and incubated for 24 h at 37 °C. The bacteriostatic (MIC) and bactericidal (MBC) effects for this study were considered as <3-log and ≥3-log decrease in CFU/mL, respectively, compared to the control inoculum [25,26].

Total tannin contents of polyvinylpolypyrrolidone (PVPP)–Miang extract supernatants

The method of Makkar et al. [27] was adopted where the Miang extracts were chemically treated with Polyvinylpolypyrrolidone (PVPP) to separate tannins from other phenols. Briefly, 50 mg/mL PVPP was mixed with Miang extracts each, the mixtures were stirred for 15 mins and centrifuged afterward. The antibacterial activity of the PVPP-treated extract adjusted to pH 5 [28], was evaluated using the agar well diffusion method as described in 2.4 above. The total tannin (TT) content in the PVPP-treated extracts and original extracts respectively, were measured using Folin–Ciocalteu reagent [29] and the absorbance was recorded at 750 nm for all samples. All results were expressed as mg tannic acid equivalent per gram of sample (TAE/g).

HPLC-QToF-MS analysis Polyvinylpolypyrrolidone (PVPP)–treated Miang extracts

The supernatants were analyzed using a Hitaci Chromaster HPLC system (Hitaci HighTech, Chiyoda-ku, Tokyo, Japan) coupled to a time-of flight mass spectrometer (Chromaster 5610 Q-TOF, Tokyo, Japan). The separation of the chemical compounds was conducted using a Pinnacle-II C18 column (250 × 4.6 mm, 5 μm) at a flow rate of 0.8 mL/min. The column temperature and detection wavelengths were set at 40 °C, 210 and 270 nm, respectively. 10.0 μL of each sample was injected. The mobile phase was composed of 0.1% acetic acid in water (v/v, A) and acetonitrile (B), with an elution of 95% A and 5% B. The mass spectrometer was operated in the positive ionization mode over a scan range of m/z 50–1000 with the following settings: capillary voltage, 3.5 kV; counter gas flow, 0.6 L/min; nebulizer, 72.5189 psi; ionizing temperature, 2500 V.

Inhibition of biofilm formation

Streptococcus mutans DMST 18777 was cultured overnight at 37 °C in Trypticase soy broth to attain an optical density at 600 nm (OD600) of ~1 [30]. The overnight culture was then diluted (1:10) in TSB supplemented with 1% sucrose and extracts’ PVPP-treated extracts were added to desired concentrations respectively. 200 μL of the bacterial inoculum were aliquoted into wells of a 96-well microplate (flat-bottom) in the absence (untreated control) or presence of the supernatants and incubated at 37 °C for 24 h. After incubation, the medium was removed and biofilms were washed with phosphate-buffered saline (PBS) twice. The plates were stained with 200 μL 0.1% aqueous crystal violet solution (CV) and incubated for 15 mins at room temperature. The plates were washed twice with water and the remaining CV was solubilized using 33% acetic acid (150 μL) and incubated under constant shaking for 5 mins. The absorbance was recorded at 595 nm and biofilm inhibition was estimated by calculating the absorbance at 595 nm of supernatant-treated wells as a percentage of the untreated control wells while the concentration for each supernatant at which 50% reduction in biofilm formation was observed when compared to the untreated wells was considered as the IC₅₀.

Homology modelling

The target amino acid sequence of S. mutans sortase (WP_002269270.1) was retrieved form the National Center of Biotechnology Information (NCBI) database. The three dimensional (3D) structure of S. mutans sortase is available in the protein data bank (4tqX.pdb, www.rcsb.org). However, this template had 40 amino acid residues (predicted transmembrane domain) truncated from its N-terminal, residues 49–53 were omitted and active site amino acid (His139) was mutated to stabilize the template crystal structure [31]. The academic version of MODELLER10.4 was used to generate the 3D structure based on target-template alignment information (99% identity) using slightly modified python codes [32,33]. Multiple 3D models were generated but the best model with the lowest discrete optimized protein energy (DOPE) score was selected. The accuracy and stereochemical quality of the model (.pdb file) was evaluated by PROCHECK, ERRAT, Verify3D and WHATCHECK [34]. The model satisfied all the evaluation criteria thus was employed for molecular docking study.

Molecular docking

The 3D structures of the identified compounds in the PVPP-treated extracts, were retrieved from PubChem database (https://pubchem.ncbi.nlm.nih.gov/) as 3D SDF conformer (accessed on July 4^th, 2023). To understand binding interactions between the compounds and sortase A (.pdb model), docking analyses were performed using Autodock Vina [35]. The.pdb file was prepared for docking by removal of all fluids and ions in the crystal structure, followed by the addition of hydrogen atoms, Gasteiger charges and protonation states to ionizable amino acids at physiologic conditions (pH 7.0). The 3D geometry of the ligands was converted from SDF format to PDBQT format and optimized via the Merck Molecular Force Field (MMFF) in open BaBel software. Using Autodock 4.2 [36]. The catalytic triad (His139, Arg213, and Cys205) were used to set up a grid box in order to cover the active site region whereas the center (12.158, 26.714, -14.504) and grid dimension (38, 52, 68) were generated in Autogrid. Molecular docking was carried out using the optimized ligands in Autodock Vina while trans-chalcone (co-crystalized with the template 4tqX), doxercalciferol, and biapenem were included in the docking analyses as reference sortase inhibitors for validation [31,37]. Results were clustered by positional root mean square deviation (RMSD) and represented by the lowest binding free energy (kcal/mol) followed by examination of binding interaction in Biovia discovery studio software [38].

Statistical analysis

All experiments were carried out in triplicate and data are expressed as mean ± SD whereas one-way analysis of variance (ANOVA) was used to determine significant differences for multiple comparisons via SPSS software for Windows version 20 (SPSS Inc., Chicago, IL, USA). p<0.05 was considered statistically significant.

Results

In this study, the antibacterial activities of original (pH 4.0–4.6) and neutralized (pH 7) Miang extracts were investigated against Gram-positive Bacillus cereus TISTR 747; Streptococcus mutans DMST 18777, and Gram-negative Escherichia coli ATCC 22595; Salmonella enterica ser. Typhimurium TISTR 292, respectively. Using the agar-well diffusion technique, NFP and FFP Miang extracts showed better antibacterial activities compared to young (YTL) and mature tea leaves (MTL). At neutral pH, NFP and FFP Miang still inhibited the growth of B. cereus TISTR 747 and S. mutans DMST 18777 with organic acids in NFP Miang contributing ~11.4% and 34.54% of the inhibition respectively (Table 1).

Table 1. The inhibition zone of Miang extracts tested against Bacillus cereus TISTR 747, Escherichia coli ATCC 22595, Salmonella Typhimurium TISTR 292 and Streptococcus mutans DMST 18777.

Sample	Zone of Inhibition
	Non-neutralized extracts				Neutralized extracts
	YTL	MTL	NFP	FFP	YTL	MTL	NFP	FFP
pH	4.3	4.6	4.3	4.0	7.0
Bacillus cereus TISTR 747 (mm)	5.88 ± 0.63^c	3.00 ± 1.00^d	8.75 ± 0.25^a	7.00 ± 0.00^b	-	-	7.75 ± 0.75^b	5.25 ± 1.25^c
Escherichia coli ATCC 22595 (mm)	-	-	4.25 ± 0.75^b	5.50 ± 0.50^a	-	-	4.75 ± 0.25^b	2.75 ± 0.25^c
Salmonella Typhimurium TISTR 292(mm)	-	-	6.50 ± 0.50^c	6.00 ± 1.00^c	-	-	8.25 ± 0.75^a	7.75 ± 0.25^b
Streptococcus mutans DMST 18777 (mm)	4.00 ± 1.00^e	3.00 ± 0.50^e	13.75 ± 1.25^a	12.00 ± 0.50^b	4.50 ± 0.50^e	4.00 ± 0.00^e	9.00 ± 0.00^c	6.75 ± 0.25^d

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YTL: Young tea leaves (109.5 mg/mL), MTL: Mature tea leaves (100.5 mg/mL), NFP: Non-filamentous fungi growth based process fermented Miang (74.5 mg/mL), FFP: Filamentous fungi-growth based process fermented Miang (91.5 mg/mL). Different alphabets represent significant differences (p<0.05, Tukey’s test).

Similarly, ~25% and 43.75% of the antibacterial activity by FFP Miang against the Gram-positive pathogens was due to organic acids. On the contrary, neutralizing the extracts did not significantly (p>0.05) alter the antibacterial activity of NFP Miang against E. coli ATCC 22595 while FFP Miang showed ~50% antibacterial activity of the extract was as a result of tea polyphenols indicating bioactive acids constituents contributed the remaining 50% inhibition (Figs 1 and 2).

Fig 1 — Antibacterial activities of non-neutralized tea leaf extracts and non-neutralized Miang extracts against *Bacillus cereus* TISTR 747 (A), *Escherichia coli* ATCC 22595 (B), *Salmonella* Typhimurium TISTR 292 (C), *Streptococcus mutans* DMST 18777 (D). Sterile water was used as the control. YTL: Young tea leaves, MTL: Mature tea leaves, NFP: Non-filamentous fungi growth based process fermented Miang, FFP: Filamentous fungi-growth based process fermented Miang (pH 4.0–4.9).

Fig 2 — Antibacterial activities of neutralized tea leaf extracts and neutralized Miang extracts against *Bacillus cereus* TISTR 747 (A), *Escherichia coli* ATCC 22595 (B), *Salmonella* Typhimurium TISTR 292 (C), *Streptococcus mutans* DMST 18777 (D). Sterile water was used as the control. YTL: Young tea leaves, MTL: Mature tea leaves, NFP: Non-filamentous fungi growth based process fermented Miang, FFP: Filamentous fungi-growth based process fermented Miang (pH 7.0).

NFP and FFP Miang (pH 7.0) showed better growth inhibition against Sm. Typhimurium TISTR 292 compared to the original (non-neutralized) Miang extracts (Table 1). Antibacterial quantitative assays showed an overall better MIC (< 3 log decrease in CFU/mL) and MBC (> 3 log decrease in CFU/mL) for NFP and FFP Miang compared to the young (YTL) and mature tea leaves (MTL) against all the pathogens at non-neutralized and neutralized pH of the extracts respectively. Against B. cereus TISTR 747, an MBC of 109.5 mg/mL and 100.5 mg/mL was observed at non-neutral pH for YTL and MTL. On the contrary, the MBC observed at both pH of the extracts for NFP Miang (37.25 mg/mL) and FFP Miang (45.75 mg/mL) was significantly (p<0.05) lower when tested against the same pathogen (Fig 3A). YTL, MTL and FFP Miang showed significantly (p<0.05) better MICs (54.75 mg/mL, 25.13 mg/mL, 5.72 mg/mL) and MBCs (109.5 mg/mL, 50.25 mg/mL, 22.88 mg/mL) at non-neutralized pH against E. coli ATCC 22595 compared to the neutralized extracts. However, the MIC (9.31 mg/mL) and MBC (18.63 mg/mL) of NFP Miang when tested against the Gram-negative E. coli was the same irrespective of extracts’ pH (Fig 3B). Moreover, YTL (non-neutralized) inhibited Sm. Typhimurium TISTR 292 with an observed MIC and MBC of 54.75 mg/mL and 109.5 mg/mL respectively, but no growth inhibition was observed for YTL and MTL at pH 7 nor for the normal (non-neutralized) extract of MTL (Fig 3C). On the other hand, NFP Miang had a similar MIC (9.31 mg/mL) and MBC (18.63 mg/mL) at both tested pH values whereas FFP Miang (at pH 7) showed a lower MIC (11.44 mg/mL) compared to the non-neutralized FFP Miang extract (22.88 mg/mL). The extracts had the best bacterial growth inhibition against S. mutans DMST 18777 with an MBC range of 3.42–6.84 mg/mL in YTL and MTL; while NFP and FFP Miang showed an MIC of 0.29 and 0.72 mg/mL respectively, albeit the MIC of NFP Miang (pH 7) was significantly (p<0.05) lower (0.15 mg/mL) compared to the non-neutralized extract (Fig 3D).

Fig 3 — Antibacterial activities of non-neutralized and neutralized extracts of Miang and tea leaves against *Bacillus cereus* TISTR 747 (A), *Escherichia coli* ATCC 22595 (B), *Salmonella* Typhimurium TISTR 292 (C), *Streptococcus mutans* DMST 18777 (D). Young tea leaves (YTL), mature tea leaves (MTL), non-filamentous fungi-process based fermented Miang (NFP) and filamentous fungi-process based fermented Miang (FFP). “Different asterisk * and ** represent significant differences of MIC and MBC, respectively (p<0.05)”.

In this regard, tannin-free extracts were further prepared and tested against S. mutans DMST 1877 and the result showed a significant (p<0.05) difference in the zone of inhibition compared to the original (tannin-rich) extracts (Table 2).

Table 2. The inhibition zone of tannin-free extracts of Miang and tea leaves against Streptococcus mutans DMST 18777.

SAMPLE
	YTL		MTL		NFP		FFP
	Tannin-rich extract	Tannin-free extract	Tannin-rich extract	Tannin-free extract	Tannin-rich extract	Tannin-free extract	Tannin-rich extract	Tannin-free extract
Streptococcus mutans DMST 18777 (mm)	15.00 ± 1.00^a	8.00 ± 0.00^b	13.00 ± 0.50^a	7.00 ± 0.50^b	18.50 ± 0.25^a	12.50 ± 1.00^b	22.50 ± 0.50^a	10.50 ± 0.25^b

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However, tannin-free extracts still maintained potent inhibitions against S. mutans DMST 18777 (Fig 4). It was important to test the antibacterial activities of the extracts at non-neutralized and neutralized pH because it showed the observed activities was not exclusively due to low extract pH. Moreover, the pH of the tannin-free extract was adjusted to 5 which is still within the acceptable criteria for mouthwash (4.0–6.5) [28].

Considering the residual tannin contents after precipitation was significantly (p<0.05) low, this suggests that antimicrobial activities of the PVPP-treated extracts may be due to other compound classes (Fig 5).

Fig 5 — Young tea leaves, mature tea leaves, non-filamentous fungi-process based fermented Miang (NFP) and filamentous fungi-process based fermented Miang (FFP). Different alphabets represent significant differences (p<0.05, Tukey’s test).

Compound annotations obtained from LCMS analysis (S1 Table) shows the presence of gallic acid, ellagic acid, quinic acid, lactic acid and butyric acid, flavonoid aglycones and flavonoid glycosides in the fermented PVPP-treated extracts (S1 Fig). The inhibitory activity of PVPP-treated extracts against the formation of S. mutans DMST 18777 biofilms was also investigated. CV-staining showed better IC₅₀ values for NFP Miang (4.30 ± 0.66 mg/mL) and FFP Miang (12.73 ± 0.11 mg/mL) compared to the young and mature tea leaves respectively (Fig 6).

Fig 6 — Young tea leaves, mature leaves, non-filamentous fungi-process based fermented Miang (NFP) and filamentous fungi-process based fermented Miang (FFP). Different alphabets represent significant differences (p<0.05, Tukey’s test).

Molecular docking studies using the identified compounds against S. mutans sortase A showed stronger inhibition for ellagic acid, flavonoid aglycones and glycosides (Fig 7) which have been previously detected in NFP Miang and FFP Miang.

Fig 7 — (A) Discrete optimized protein energy (DOPE) score profiles of the 4tqX template (-20008.758) and Model (-20812.996). The overlay shows the missing N-terminal residues in 4tqX template as well as the omitted residues (49–53). (B) Ramachandran plot generated via RamachanDraw for the best *Streptococcus mutans* sortase model (.pdb output file) from MODELLER10.4. (C) Binding free energies in kcal/mol for Compound-Sortase complex grouped in a descending order from left—right. ^F: Detected in fermented teas, ^uF: Detected in young and mature teas, ^Ref.: Reference compound.

Like the reference compounds (Fig 8), epicatechin, gallocatechin, chlorogenic acid, and 5-feruloylquinic acid interacted with the active site catalytic triad (Cys205, Arg213 and His139) suggesting a probable competitive-like inhibition (Table 3).

Table 3. Binding energies and bonding interactions of docked compounds.

#	Compound	Binding energy (kcal/mol)	Hydrogen bonding	Van der Waals^a, Hydrophobic & Electrostatic^b interactions
Ref.	Trans-chalcone	-6.3	-	Cys205, Ala137, Val203, Val190, Arg213.
Ref.	Biapenem	-6.0	Cys205, His139	His139^b, Ala210, Leu116
Ref.	Doxercalciferol	-6.8	Cys205	His139, Leu116, Leu111, Arg213, Val203, Ile215, Val190, Ile191, Val188, Met123.
1	Ellagic acid	-9.5	Thr204, Ser138, Val203.	Arg213^*
2	Quercetin dihexoside	-7.3	Gln239, Ser240, Ser245, Gln243, Phe246.	Lys102.
3	Apigenin-6-C-glucosyl-8-C-arabinoside	-7.2	Ser240, Phe246, Gln243, Lys102, Asn242.	Tyr241^a, Lys102, Pro99.
4	Quercetin	-7.1	Ile103, Tyr241.	Lys102.
5	Quercetin-3-glucoside	-7.0	Ser245, Tyr241, Gly146.	Lys102, Gln149.
6	Epigallocatechin gallate	-6.9	Tyr241, Gln243, Phe246, Gln239.	Lys102.
7	Myricetin-3-galactoside	-6.7	Asn193, Asp192, Val190, Asn133, Gu199, Glu189, Lys181.	His187, His217, Arg194.
8	Chlorogenic acid	-6.6	Cys205, Ser138, His187, Pro185.	Arg213^a, Val203^a, His139^a, Ala137, Leu116.
9	Gallocatechin	-6.6	Cys205, Arg213, His187, Val203, Ser138.	Ala137, Met123.
10	Epicatechin	-6.5	His187, Arg213, Cys205.	Leu111^a, Ile119^a, Val188^a, Met123, Val203, Ala137.
11	5-Feruloylquinic acid	-6.4	Cys205, Pro185, Thr184.	Ala137, Met123^a, Val203^a.
12	3-p-Coumaroylquinic acid	-6.4	Tyr241, Gln243, Ser245.	Lys102.
13	Dicaffeoylquinic acid 1	-5.7	Gln239.	Phe246.
14	Dicaffeoylquinic acid 3	-5.6	Glu156, Ser148, Gly146, Gly143, Met144, Ser147.	Thr206^a, Glu156, Phe142.
15	Gallic acid	-5.6	Arg194, Val190, Glu189.	His217.
16	Uronic acid	-5.6	Phe246, Ser245, Gln243.	Lys102
17	Pyrogallol	-5.0	Asp192, Val190.	Arg194, His217.
18	Quinic acid	-5.0	Lys181, Glu189, Glu179.	-
19	Pyroglutamic acid	-4.6	Thr184, Pro185, Val188.	-
20	Lactic acid	-4.1	Asn132, Asn193, Lys124.	-
21	Butyric acid	-3.7	Ile103.	-

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*represents an unfavorable positive-positive interaction.

Discussion

A cheaper, reliable, and sustainable bioprospection of plant extracts for possible application in innovative functional foods, natural active pharmaceutical ingredients (NAPIs) and/or therapeutics is a current research trend [39]. In this study, fermented Miang extracts (NFP and FFP Miang) showed better growth inhibition against both Gram positive (B. cereus TISTR 747 and S. mutans DMST 18777) and Gram-negative (E. coli ATCC 22595 and Sm. Typhimurium TISTR 292) pathogens compared to young (YTL) and mature (MTL) tea leaves respectively. The contents of tannins and organic acids including ellagic acid, galacturonic acid, tartaric acid, lactic acid, succinic acid, and acetic acid; in NFP and FFP Miang have been reported to significantly increase after fermentation [6] thus may have translated into the observed antimicrobial activities. The contribution of the compound classes to the antimicrobial activities was investigated by neutralizing the pH of Miang extracts (Table 1) and the results suggested that organic acids and/or essential oils may actually confer significant antimicrobial activities in FFP Miang compared to NFP Miang especially, against S. mutans DMST 18777, B. cereus TISTR 747, and E. coli ATCC 22595. This observation for FFP Miang might be due the simultaneous stepwise fermentation process applied to produce the fermented product which uses filamentous fungi, yeast, and bacteria subsequently leading to microbial-mediated synthesis of acids, terpenes, esters, and other volatile aromatic compounds (VOCs) as these compounds have been previously characterized in FFP Miang using GC/MS analysis [9,40]. Some of these compounds including terpenes and alkaloids are known to permeate membranes and intercalate DNA in bacterial pathogens [41]. There are confirmed reports that show essential oils such as eugenol, in plant extracts contributed to potent growth inhibition of Gram-positive bacteria such as S. mutans. [42]. The zones of inhibition were larger for the fermented extracts tested under non-neutral pH (4.0–4.9) conditions against S. mutans DMST 18777 and B. cereus TISTR 747 (Figs 1 and 2) which suggests organic acids in NFP Miang and more so FFP Miang (S1 Fig), diffuse easier into the more permeable Gram-positive cell walls compared to the outer phospholipid membrane of Gram-negative bacteria which precludes cell invasion by lipophilic compounds [43,44]. Interestingly, FFP Miang (pH 7) showed a smaller zone of inhibition against E. coli ATCC 22595 while the same extract as well as NFP Miang (pH 7) extracts showed a better growth inhibition of Sm. Typhimurium TISTR 292 compared to their non-neutral counterparts (Table 1). The tannins and/or other non-acidic compounds alone are thought to be responsible for this inhibition against Sm. Typhimurium TISTR 292 at pH 7 which suggest the presence of potent compounds with salient pharmacophores capable of probable penetration and targeting of cellular machineries in this pathogen. In addition, catechins have been previously implicated in the disruption of bacterial membranes by interfering with the normal function of proteins on their cell surface [29,30]. However, the discrepancy observed for FFP Miang (at pH 7) against E. coli in this study could be further investigated in the future so as to shed more light into how the non-neutral and/or volatile compounds contained in FFP Miang exerted their growth inhibition against this Gram-negative pathogen.

Moreover, the inhibition pattern reported using agar well diffusion technique agreed with that observed for the quantitative experiments albeit the latter was able to give an idea as to the strength of extract inhibition through the MIC and MBC estimations. Overall, the fermented teas showed better MIC/MBCs compared to the young and mature tea extracts (NFP Miang > FFP Miang > YTL ~ MTL) against the bacterial pathogens with S. mutans DMST 18777 showing the best activity especially, for extracts tested at pH 7 (Fig 3D). On this note, the effect of PVPP-treated Miang extracts (tannin-free) were investigated against cariogenic S. mutans DMST 18777 since this pathogen is known to thrive on food/sugar remnants (such as sucrose & fructose) thereby causing infection by attaching to hosts’ oral cavities using bacterial adhesins, exopolysaccharides amongst others, thus aggregating into effective and infectious biofilms that are less susceptible to antibiotics compared to planktonic pathogens [45–48]. Streptococcus mutans has been reported to recruit sucrose-dependent mechanisms where glucosyltransferase (GTF) mediates a reactions with glucan-binding proteins (GBP) to cause the formation of dental plaques [49–51]. Interestingly, tannins have been reported to inhibit GTF-mediated synthesis of insoluble glucans thus suppressed S. mutans proliferation [52]. Therefore, targeting these biofilms will require 2–4 folds the original MICs calculated for the test fermented extracts to be able to arrive at experimentally-relevant concentrations that could effectively inhibit the bacterial biofilms [53]. The lower MIC and MBC observed for the extracts at pH 7 (Fig 3D) also suggests a retained antimicrobial activity independent of organic acids which could make the extracts choice selections to be applied in therapeutic formulations targeted against aciduric and acidogenic S. mutans strains. However, 2–4 folds the original MICs might increase tannin contents to thresholds that might be toxic to the host system in addition to the sensory drawbacks of tannin deposition which causes tooth stains and bitter sensation thus will perhaps guarantee consumers’ dissatisfaction with such therapeutic formulations [21].

To address this, PVPP-treated extracts tested against S. mutans DMST 18777 using agar well diffusion technique showed residual antimicrobial activity after precipitating out tannins (Table 2) more so for NFP Miang compared to the other extracts (NFP Miang > FFP Miang > YTL ~ MTL) (Fig 4). The efficiency of tannin precipitation was evaluated by quantitative estimation of tannin contents in the original Miang extracts and their corresponding PVPP-treated extracts where the result showed a > 90% precipitation efficiency overall (Fig 5) which mildly suggest other compound classes and/or the residual tannins (in young and mature teas) might be responsible for the growth inhibition of this supernatant against S. mutans DMST 18777. Interestingly NFP Miang (4.30 mg/mL; IC₅₀) and FFP Miang (12.73 mg/mL; IC₅₀) supernatants showed appreciable inhibitory activities against S. mutans DMST 18777 biofilm formation which indicates that the antibacterial activity of the tannin-free supernatants from these fermented samples were potent enough to exert anti-biofilm activities. A computational docking study was considered given the effectiveness of the PVPP-treated extracts in the inhibition of the Gram-positive S. mutans DMST 18777. Sortase A is a highly conserved membrane-associated enzyme in Gram-positive bacteria that facilitate the covalent attachment of bacterial virulent-surface proteins on bacterial cell walls to mediate host cell attachment, biofilm formation, iron acquisition, signaling, invasion and pili formation [31,54,55]. Sortase A recognizes the conserved LPXTG (Leucine, proline, X = any amino acid, threonine, glycine) motif at the C-terminus of S. mutans surface proteins and hydrolyzes the amide bond between threonine and glycine residues using a conserved active site cysteine (Cys205) residue. The thioacyl intermediate formed attaches to the cell wall precursor lipid II pentaglycine side chain. On the other hand, side chains of His139 and Arg213 in the active site serve as a general base as well as stabilizer of ionic intermediates in the sortase catalyzed hydrolysis thus making sortase A an attractive chemotherapeutic target [31,56–58]. Medicinal plants rich in flavonoids (including kempferol, apigenin and quercetin) and tannins (such as ellagitannins and proanthocyanidins) have been shown to contribute to the maintenance of oral health and are therefore applied as additives or major ingredients in toothpastes or mouthwashes [59]. Our results suggested ellagic acid, flavonoid aglycones, flavonoid glycosides and organic acid derivatives might be responsible for the antimicrobial activities of NFP and FFP Miang (Table 3, Fig 7C) from the observed docking binding energies. Further experiments could explore possibilities of enriching and/or optimizing tannin-free Miang extracts as potential candidates to be applied as natural active pharmaceutical ingredients (NAPIs).

Conclusion

This study confirmed that microbial fermentation increased the antimicrobial activities of neutralized and non-neutralized Miang extracts against both Gram-positive (B. cereus TISTR 747 and S. mutans DMST 18777) and Gram-negative (E. coli ATCC 22595 and Sm. Typhimurium TISTR 292) bacteria. The quantitative estimation of antibacterial activities showed a lower MIC and MBC for all especially, the fermented extracts tested against S. mutans DMST 18777. The extracts were precipitated using PVPP and the tannin-free extracts still retained appreciable growth inhibition against S. mutans DMST 18777 while NFP and FFP Miang showed appreciable IC50 values against S. mutans DMST 18777 biofilms. Overall, the result showed the potential of these extracts to be enriched, optimized and possibly find future applications as a mainstay in combating bacterial pathogens and as potential ingredients in mouthwashes.

Supporting information

S1 Fig. HPLC chromatogram of polyvinylpyrrolidone (PVPP)–treated Miang extracts.

YTL: Young tea leaves, MTL: Mature tea leaves, NFP: Non-filamentous fungi growth based process fermented Miang, FFP: Filamentous fungi-growth based process fermented Miang.

(PDF)

pone.0302717.s001.pdf^{(385.6KB, pdf)}

S1 Table. Identification of compounds in polyvinylpyrrolidone (PVPP)–treated Miang extracts.

^aAbbreviations are listed as follows: OA; organic acid; F3, flavan-3-ol; FL, flavonol/flavone; PA, phenolic acid; PP, Polyphenol; AA, amino acid. ^bRT: Retention time All acquisitions were carried out in the positive mode (m/z, [M + H] ⁺) using LC-MS (at 210 and 270 nm wavelengths).

(PDF)

pone.0302717.s002.pdf^{(207.1KB, pdf)}

Acknowledgments

The authors would like to acknowledge the Chiang Mai University’s Graduate school and Presidential scholarship program, respectively. We also acknowledge Faculty of Agro-Industry, Chiang Mai University, for providing the research facilities.

Data Availability

All relevant data are within the paper and its Supporting information files.

Funding Statement

The author(s) received no specific funding for this work.

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Antibacterial activities of Miang extracts against selected pathogens and the potential of the tannin-free extracts in the growth inhibition of Streptococcus mutans

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Additional Editor Comments:

Dear Dr. Khanongnuch, I have made a decision about your manuscript, please review each reviewer's comment point by point, changes should be highlighted so we can track.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

Reviewer #4: Partly

Reviewer #5: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

Reviewer #5: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript PONE-D-23-35234 entitled “Antibacterial activities of Miang extracts against selected pathogens and the potential of the tannin-free extracts in the growth inhibition of Streptococcus mutans” has described solvent extraction of Miang (fermented tea leaves) and tea leaves, raw material for Miang production and determination of antibacterial activities of the obtained extracts, and those treated with tannin-free against selected bacterial pathogens, examination of compounds. The results have found that tannin-free Miang extracts showed more potential to inhibit growth of pathogens, specifically Streptococcus mutans than tannin-free tea leaf extracts. In addition, the manuscript has described analysis of the extracts by HPLC-MS in order to examine the chemical compounds that are relevant to growth inhibition of Streptococcus mutans. The identified compounds were used to determine interactions between each identified compound and sortase A enzyme, the key enzyme associated with biofilm formation of S. mutans. The authors conclude that the tannin-free Miang extracts could be used as a natural active pharmaceutical ingredient for applications in oral hygiene products.

After the evaluation, this manuscript presents sufficient experimental designs and statistical analysis. However, there is unclear information and errors. To meet the standard manuscript for publication, the current form of the manuscript must be substantially revised. The following comments and suggestions would help increase the completement of the manuscript.

General comments and suggestions

Please check the writing format of the scientific name of all microorganisms. There are “Streptococcus” and “Salmonella” which can be abbreviated as “S.”. This would make readers confused. It is suggested to present “S.” for Streptococcus mutans and “Sm”. for Salmonella.

To describe Salmonella enterica serovar Typhimurium, it is recommended to state it as “Salmonella var. Typhimurium” or “Salmonella Typhimurium” or “Sm. Typhimurium”. Please correct it in the whole manuscript.

Please indicate strain to all microorganisms used in this study.

Full name of a microorganism must be present for the first time of the main section and then it should be abbreviated. However, when the authors state it as a subject of the sentence, it is recommended to state its full name.

It is confusing when the authors state NFP and FFP as Miang because either NFP or FFP is a process of Miang fermentation. It is recommended to state it as “NFP Miang” or “FFP Miang”. Please also correct these words throughout the manuscript.

Comma symbol (,) must be added after a specific word such as respectively, especially, etc.,

Considering the type of extracts used in this study and their consistency, please reconsider these words: non-neutral, neutral, acidic, and pH 7. If tea leaf extracts (YTL and MTL) and NFP and FFP Miang extracts are generally acidic, it could state them as “non-neutralized extracts”. After the authors have neutralized the acidic extracts, it could state them as “neutralized extracts”.

Due to Miang is made from tea leaves, it is relevant to express only young tea leaves or mature tea leaves rather than unfermented young tea leaves and unfermented mature tea leaves, respectively (Line 228 and others in discussion section).

Please italicize p of p-value and t of t-value in whole manuscript.

It is more relevant to replace “PVPP-treated extract’ supernatant” by “PVPP-treated extract”. (lines 148, 398, 408, …)

Major and minor comments

Introduction section, add more information about role of sortase A enzyme in dental plaque formation. This information would at least let readers primarily understand why the authors chose molecular docking to investigate interactions between active compounds in Miang extracts and the sortase A enzyme.

Line 109-107 how did the authors express the unit of all extract? Is it mg/mL total polyphenol content or mg/mL total tannin content, or mg extract/mL, etc., Please add more information.

Line 127, is it non-neutralized Miang extracts?

Line 139, is it neutralized Miang extracts? Please also consider as above mentioned.

Line 133, indicate diameter of borer.

Line 134, indicate temperature.

Line 163, remove “be”

Line 172, edit to “Streptococcus mutans” when it was first used as the subject.

Line 173, edit to “an optical density at 600 nm (OD600)”

Liness 223, 247, and 253 add the strain of S. mutans.

Lines 230 and 239 how did the authors calculate percentages of inhibition? Please clarify.

Table 1, the authors can categorize the extracts into two groups including non-neutralized (acidic) and neutralized extracts. Each group consists of YTL, MTL, NFP, and FFP.

Line 236-237, different alphabets represent significant differences vs control. The control was just water. If the authors compared the experimental values to the control, extract samples exhibited the clear zone will have significant differences. From the point of view, multiple comparisons were made after ANOVA results showed significant differences (p < 0.05). It is supposed that all pairwise comparisons were used to analyze the data within the same row, not just compared with the control. It is noted that data with positive results are always significant when they are compared with the control. Please consider this comment for Fig 3., Table 2, Fig 5, and Fig 6.

Lines 245 and 251, it is recommended to revise the figure legend as follows:

Fig 1. Antibacterial activities of non-neutralized tea leaf extracts and non-neutralized Miang extracts against Bacillus cereus TISTR 747 (A), Escherichia coli ATCC 22595 (B), Salmonella Typhimurium TISTR 292 (C), Streptococcus mutans DMST 18777 (D). Sterile water was used as the control.

Fig 2. Antibacterial activities of neutralized tea leaf extracts and neutralized Miang extracts against Bacillus cereus TISTR 747 (A), Escherichia coli ATCC 22595 (B), Salmonella Typhimurium TISTR 292 (C), Streptococcus mutans DMST 18777 (D). Sterile water was used as the control.

Lines 256-277, it is recommended to initially describe line 257-260 (Antibacterial quantitative assays showed …….at acidic and neutral extracts’ pH respectively). However, the phrase “at acidic and neutral extracts’ pH respectively” should be revised to proper format as previously suggested. For example, Antibacterial quantitative assays showed an overall better MIC and MBC for NFP and FFP Miang extracts compared to the YTL and MTL when they were tested against all the pathogens, respectively.

Line 278, it is recommended to revise the figure legend as follows:

Fig 3. Antibacterial activities of non-neutralized and neutralized extracts of Miang and tea leaves against Bacillus cereus TISTR 747 (A), Escherichia coli ATCC 22595 (B), Salmonella Typhimurium TISTR 292 (C), Streptococcus mutans DMST 18777 (D).

Line 282, Different asterisk (*) represent significant differences vs control. What was the control of this experiment? Is it the initial cell concentration or just sterile water? This phrase could be modified to “Different asterisk symbols * and ** represent significant differences of MIC and MBC, respectively (p < 0.05)”.

Line 289, please consider modifying description of table 2 as follows:

Table 2. The inhibition zone of tannin-free extracts of Miang and tea leaves against Streptococcus mutans DMST 18777. Avoid extension of figure description.

Line 293 the comment is similar to that is given for Line 236-237

Line 296, it is unnecessary to add Fig 4 because the authors have already displayed the results in Table 2.

Lines 309-314 it is necessary to compare effect of tannin-free extracts and their original extract (in the presence of tannin)

Lines 384-386 Streptococcus mutans has been reported to recruit sucrose-dependent mechanisms where glucosyltransferase (GTF) reacts with sucrose and convert into dextran polymer or glucan that can be further bound with glucan-binding proteins (GBP) which leads to the formation of dental plaque. Please consider modifying the sentence.

Lines 411-425 please shorten principal discussion about role of sortase A in plaque formation but discuss more about finding of this study. How could active compounds from PVPP-treated Miang extracts inhibit sortase A? Are there any literatures reported active compounds that have potential to inhibit the enzyme?

Line 438-440 the sentence in the conclusion is not relevant to what the author proposes in introduction section (Lines 51-53, and 90-93).

Reviewer #2: It was advantageous to discover a plant extract that inhibited pathogen growth. Several concerns, however, needed to be elucidated in order to have a better knowledge of Miang extracts' antibacterial activity.

It was unclear how the authors obtained the quantities of Miang extracts utilized in Table 1 to establish zones of inhibition against bacteria.

Please double-check whether it was Turkey's or Tukey's test (line 237).

Only triplicate samples used to run a t-test comparing tannin rich extract to tannin-free extract were sufficient to accept the results (Table 2). Please specify the concentration of Miang extracts that were used.

Please modify the titles of all figures to improve comprehension.

In the conclusion section, the authors should summarize the optimal concentration of each Miang extract that can be employed to inhibit bacterial growth based on the MIC:MBC ratio of Miang extracts.

Reviewer #3: Materials and Methods

1. Line 126 to 136 talks about ‘Determination of antibacterial activities of neutralized (pH 7) and non-neutral (pH 4.0 – 4.6) Miang extracts’. The description, however, misses out on how these extracts were obtained before using them for the microbial tests. The ‘Preparation of Miang extracts’ is also silent on how the neutral and non-neutral extracts were obtained. Authors should kindly take a look at that.

2. What was the rationale for testing neutralized and non-neutralized extracts in the study? Could the justification be clearly demonstrated in the write-up?

3. Line 127 – I guess ‘non-neural’ should be ‘non-neutral’.

4. Kindly take a second look at the format for writing your units. At one point, I see CFU/mL, then at other places, I see CFU mL-1.

5. Line 163-164 : authors should kindly the sentence ‘The separation of the chemical compounds was be conducted……..’

Results

1. From Table 2, the zone of inhibition for tannin-rich and tannin-free extracts against Streptococcus mutans were significantly different for each type of sample. How do you authors then arrive at the observation that ‘tannin-free NFP and FFP extracts had significantly (p> 0.05) comparable zones of inhibition with the tannin-rich extracts’ and refer to figure 4, which I suppose is the primary source of data for Table 2?

2. Based on the observations in Table 2, it would be appreciated if authors could reconsider the point on tannin-free producing similar effects as tannin-rich extracts. That obviously also affect the reasoning that

Reviewer #4: This article explain on the antibacterial activity of Miang extract againts selected foodborne pathogens and the finding showed that the extract had the ability to inhibit the growth of pathogens. However, there is no problem statement regarding foodborne pathogens eventhough at the the author mention about foodborne problem at the beginning. Author is suggested to do proof read on their article content. This research give new info on the antibacteria activity of miang extract

Reviewer #5: I extend my appreciation to the authors for their dedication and commendable efforts in completing this excellent piece of research. This work is of considerable importance within the context of oral health, addressing a notable gap in the current literature and searching for novel antimicrobial agents is of great interest. I recommend its publication in PLOS for the following reasons:

• The title effectively reflects the study, and the abstract is clear and concise for readers.

• The comprehensive literature review demonstrates a good understanding of existing research, particularly in the quest for alternative antimicrobial agents.

• The methodology is well-structured, offering a clear and systematic approach to the research, which can serve as a valuable guide for future researchers. I just suggest standardizing the writing style concerning bacteria.

• The data analysis is well-performed, and the presentation is clear.

• The discussion is thoughtful, providing significant interpretations of the results.

• The author displays a high level of proficiency in English.

• The use of references is appropriate.

Overall, this work makes a noteworthy contribution to the field in the search for a new antimicrobial agent and aligns well with the standards of PLOS. I strongly recommend its publication.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

Reviewer #5: No

**********

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Attachment

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PLoS One. 2024 May 8;19(5):e0302717. doi: 10.1371/journal.pone.0302717.r002

Author response to Decision Letter 0

4 Mar 2024

POINT BY POINT RESPONSE TO THE EDITOR AND REVIEWERS’ COMMENTS ON THE MANUSCRIPT (PONE-D-23-35234).

Thank you for giving us the opportunity to revise the manuscript and the willingness to accept the article after the major revision. We have considered each of the comments given by both editor and reviewers and have provided revisions/responses to each comment in a point-by-point pattern. Please note that all corrections by the authors are made using Tracked changes and saved as “Revised document with Track changes”. Line numbers (LNs) are also included in the point by point responses to reviewers for efficient tracking of corrections.

Editor

General Response: We are grateful to the editor for the kind comments and suggestions meant to improve the manuscript.

Comment 1: Please include a rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labelled 'Response to Reviewers'.

Response 1: A rebuttal letter labelled 'Response to Reviewers' has been included per directive of the editor

Comment 2: Please include a marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labelled 'Revised Manuscript with Track Changes'.

Response 2: “Track changes” in MS word has been used to highlight the revisions and the file has been saved as 'Revised Manuscript with Track Changes'.

Comment 3: Please an unmarked version of your revised paper without tracked changes. You should upload this as a separate file labelled 'Manuscript'.

Response 3: A clean copy of the Manuscript devoid of highlights and tracked changes has been labelled and saved as 'Manuscript'.

Reviewer 1

General Response: We are grateful to the reviewer for the kind comments and efforts to make the manuscript better. All the corrections have been implemented and can be confirmed easily in the file saved as “Revised manuscript with track changes”. Line numbers (LNs) are also included in the point by point responses to reviewers for efficient tracking of corrections.

Comment 1: Please check the writing format of the scientific name of all microorganisms. There are “Streptococcus” and “Salmonella” which can be abbreviated as “S.”. This would make readers confused. It is suggested to present “S.” for Streptococcus mutans and “Sm”. for Salmonella. To describe Salmonella enterica serovar Typhimurium, it is recommended to state it as “Salmonella var. Typhimurium” or “Salmonella Typhimurium” or “Sm. Typhimurium”. Please correct it in the whole manuscript. Please indicate strain to all microorganisms used in this study. Full name of a microorganism must be present for the first time of the main section and then it should be abbreviated. However, when the authors state it as a subject of the sentence, it is recommended to state its full name.

Response 1: Thank you for the comment. The writing format for the scientific names have been modified as suggested and the corrections was applied throughout the manuscript.

Comment 2: It is confusing when the authors state NFP and FFP as Miang because either NFP or FFP is a process of Miang fermentation. It is recommended to state it as “NFP Miang” or “FFP Miang”. Please also correct these words throughout the manuscript.

Response 2: “NFP” and “FFP” has been clarified and changed throughout the manuscript to “NFP Miang” or “FFP Miang” (LNs 43, 47, 52, 60, 98, 99, 107, 109, 114, 233, 234, 236, 249, 270,273, 277, 283, 287, 291-293, 342, 352, 371, 376, 380, 392, 396, 412, 435-436, 441-442 and 471) as recommended.

Comment 3: Comma symbol (,) must be added after a specific word such as respectively, especially, (LNs 70, 152, 161, 286, 292, 303, 381, 413 and 480) etc.

Response 3: Comma symbols have been added as suggested for specific words.

Comment 4: Considering the type of extracts used in this study and their consistency, please reconsider these words: non-neutral, neutral, acidic, and pH 7. If tea leaf extracts (YTL and MTL) and NFP and FFP Miang extracts are generally acidic, it could state them as “non-neutralized extracts”. After the authors have neutralized the acidic extracts, it could state them as “neutralized extracts”.

Response 4: Thank you for the comment. “Non-neutral extract” and “neutral extract” were replaced with “non-neutralized extracts” and “neutralized extracts” respectively (LNs 131-132, 144, 253, 262, 271, 274, 284, 287, 289, 295, 322 and 477). However, words such as non-neutral pH, and neutral pH were not altered.

Comment 5: Due to Miang is made from tea leaves, it is relevant to express only young tea leaves or mature tea leaves rather than unfermented young tea leaves and unfermented mature tea leaves, respectively (Line 228 and others in discussion section).

Response 5: The word “unfermented” in LNs 234, 359, 373, 411 and 440, has been erased due to its redundancy. Thank you for the suggestion.

Comment 6: Please italicize p of p-value and t of t-value in whole manuscript.

Response 6: All p-values found in LNs 227, 244-245, 249, 278, 280, 293, 303, 308, 316, 329, 335 and 348, have been italicized.

Comment 7: It is more relevant to replace “PVPP-treated extract’ supernatant” by “PVPP-treated extract”. (Lines 148, 398, 408 …).

Response 7: Thank you for the comment. “PVPP-treated extract’ supernatant” has been changed to “PVPP-treated extract” (LNs 160, 180, 206, 330, 339-340, 433, 438 and 446) throughout the manuscript.

Comment 8: Introduction section, add more information about role of sortase A enzyme in dental plaque formation. This information would at least let readers primarily understand why the authors chose molecular docking to investigate interactions between active compounds in Miang extracts and the sortase A enzyme.

Response 8: Additional information has been included in the Introductory part as recommended: LNs 80-82, “The biofilm formation is mediated through sortase A enzyme which anchors surface proteins on bacterial cell walls to allow host attachment by S. mutans biofilms. Consequently, these events lead to the formation of dental plaque, demineralization of enamel and leaching of teeth components [14,15]”.

Comment 9: Line 109-107 how did the authors express the unit of all extract? Is it mg/mL total polyphenol content or mg/mL total tannin content, or mg extract/mL, etc., Please add more information.

Response 9: The unit of the extract was expressed as mg extract/mL as indicated now in LN 121 as suggested.

Comment 10: Line 127, is it non-neutralized Miang extracts? Line 139, is it neutralized Miang extracts? Please also consider as above mentioned.

Response 10: Thank you for the comment, the previous recommendation (comment 4) also relates to this and has been judiciously implemented throughout the manuscript.

Comment 11: Line 133, indicate diameter of borer. Line 134, indicate temperature. Line 163, remove “be” Line 172, edit to “Streptococcus mutans” when it was first used as the subject. Line 173, edit to “an optical density at 600 nm (OD600)” Lines 223, 247, and 253 add the strain of S. mutans.

Response 11: The diameter of the borer: “6mm diameter”, has been indicated (LN 138). Temperature has been indicated on LN 140 “incubated for 24 h at 37 oC”. “be” has been removed from LN 168. Line 177, has been edited to “Streptococcus mutans DMST 18777” due to its use here as the subject. LN 178 has been edited to “to attain an optical density at 600 nm (OD600) of ~1”. The strain of S. mutans has been included in LNs 235, 291 and 308. All the corrections have been implemented and can be confirmed easily in the file saved as “Revised manuscript with track changes”.

Comment 12: LNs 230 and 239 how did the authors calculate percentages of inhibition? Please clarify.

Response 12: Now LNs 236 and 247, % Contribution of neutralized compounds (Table 1) was expressed as (Zone of inhibition for neutralized extract/ Zone of inhibition for non-neutralized extract) x 100%. Then, contribution of organic acids = 100 % ˗ % Contribution of neutralized compounds.

Comment 13: Table 1, the authors can categorize the extracts into two groups including non-neutralized (acidic) and neutralized extracts. Each group consists of YTL, MTL, NFP, and FFP.

Response 13: Thank you for the comment, the change was duly noted (LNs 238-245) and incorporated in the table.

Comment 14: Line 236-237, different alphabets represent significant differences vs control. The control was just water. If the authors compared the experimental values to the control, extract samples exhibited the clear zone will have significant differences. From the point of view, multiple comparisons were made after ANOVA results showed significant differences (p < 0.05). It is supposed that all pairwise comparisons were used to analyze the data within the same row, not just compared with the control. It is noted that data with positive results are always significant when they are compared with the control. Please consider this comment for Fig 3., Table 2, Fig 5, and Fig 6.

Response 14: Thank you for the comment, the comment was considered and a modification (“Different alphabets represent significant differences (p<0.05, Turkey’s test).”) was applied accordingly to the affected figures and tables mentioned above (LNs 244, 302, 316-317, 335 and 347).

Comment 15: Lines 245 and 251, it is recommended to revise the figure legend as follows:

Response 15: Thank you for the comment. The Figure title has been revised as recommended (LNs 253-269).

Comment 16: Lines 256-277, it is recommended to initially describe line 257-260 (Antibacterial quantitative assays showed …….at acidic and neutral extracts’ pH respectively). However, the phrase “at acidic and neutral extracts’ pH respectively” should be revised to proper format as previously suggested. For example, Antibacterial quantitative assays showed an overall better MIC and MBC for NFP and FFP Miang extracts compared to the YTL and MTL when they were tested against all the pathogens, respectively.

Response 16: Now LNs 272 – 275, the MIC and MBC was initially defined in Lines 151 and 152. However, it has been modified to “Antibacterial quantitative assays showed an overall better MIC (< 3 log decrease in CFU/mL) and MBC (> 3 log decrease in CFU/mL) for NFP and FFP Miang compared to the young (YTL) and mature tea leaves (MTL) against all the pathogens at non-neutralized and neutralized pH of the extracts respectively”.

Comment 17: Line 278, it is recommended to revise the figure legend as follows:

Response 17: The figure title has been revised as recommended (LN 295).

Comment 18: Line 282, Different asterisk (*) represent significant differences vs control. What was the control of this experiment? Is it the initial cell concentration or just sterile water? This phrase could be modified to “Different asterisk * and ** represent significant differences of MIC and MBC, respectively (p < 0.05)”.

Response 18: The initial cell concentration and sterile water were both incorporated in the experimental design. However, “Different asterisk * and ** represent significant differences of MIC and MBC, respectively (p<0.05)” better captures the multiple comparison from Turkey’s post hoc test. Thank you very much for this comment (LN 302).

Comment 19: Line 289, please consider modifying description of table 2 as follows:

Table 2. The inhibition zone of tannin-free extracts of Miang and tea leaves against Streptococcus mutans DMST 18777. Avoid extension of figure description.

Response 19: Thank you for the comment. The simpler description has been implemented (LN 310) as suggested.

Comment 20: Line 293 the comment is similar to that is given for Line 236-237. Line 296, it is unnecessary to add Fig 4 because the authors have already displayed the results in Table 2.

Response 20: “Different alphabets…” indicating the multiple comparisons from Turkey’s test have been modified throughout the manuscript as recommended. However, we feel some readers resonate more with figures so we showed the fig 2 in addition to the table (which shows the statistical difference) in a bid to accelerate readers’ understanding of the result.

Comment 21: Lines 309-314 it is necessary to compare effect of tannin-free extracts and their original extract (in the presence of tannin)

Response 21: Thank you for the comment, the effect of tannin-free extracts and their original extracts were compared in table 2 where independent sample T-test was used to explain the difference. Against the S. mutans DMST 18777 biofilms, the point of this publication was to explore the potential of tannin free extracts in the growth inhibition of the biofilms. The justification for this was mentioned in the introduction (Lines 90 – 97).

Comment 22: Lines 384-386 Streptococcus mutans has been reported to recruit sucrose-dependent mechanisms where glucosyltransferase (GTF) reacts with sucrose and convert into dextran polymer or glucan that can be further bound with glucan-binding proteins (GBP) which leads to the formation of dental plaque. Please consider modifying the sentence.

Response 22: Lines 394 – 397), the sentence has been modified to “Streptococcus mutans has been reported to recruit sucrose-dependent mechanisms where glucosyltransferase (GTF) mediates a reaction with glucan-binding proteins (GBP) to cause the formation of dental plaques [49–51]. Interestingly, tannins have been reported to inhibit GTF-mediated synthesis of insoluble glucans thus suppressed S. mutans proliferation”.

Comment 23: Lines 411-425 please shorten principal discussion about role of sortase A in plaque formation but discuss more about finding of this study. How could active compounds from PVPP-treated Miang extracts inhibit sortase A? Are there any literatures reported active compounds that have potential to inhibit the enzyme?

Response 23: The discussion was made to be as concise as possible (Lines 448 – 465). And a Paper which was recently published (December, 2022) was cited which mentioned related active compounds that have found use in maintaining oral health. Line 466 – 469, “Medicinal plants rich in flavonoids (including kempferol, apigenin and quercetin) and tannins (such as ellagitannins and proanthocyanidins) have been shown to contribute to the maintenance of oral health and are therefore applied as additives or main ingredients in mouthwashes or toothpastes [59]”.

Comment 24: Line 438-440 the sentence in the conclusion is not relevant to what the author proposes in introduction section (Lines 51-53, and 90-93).

Response 24: The sentence has been modified to “Overall, the result showed the potential of these extracts to be enriched, optimized and possibly find future applications as a mainstay in combating bacterial pathogens and as potential ingredients in mouthwashes” (484 – 486).

Reviewer 2

General Comment: It was advantageous to discover a plant extract that inhibited pathogen growth. Several concerns, however, needed to be elucidated in order to have a better knowledge of Miang extracts' antibacterial activity.

General Response: Thank you very much for the comment. Your concerns will be given due attention and addressed appropriately. All corrections by the authors are made using Tracked changes and saved as “Revised document with Track changes”. Line numbers (LNs) are also included in the point by point responses to reviewers for effic

Attachment

Submitted filename: Response to Reviewers.doc

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PLoS One. doi: 10.1371/journal.pone.0302717.r003

Decision Letter 1

Mozaniel Santana de Oliveira

20 Mar 2024

PONE-D-23-35234R1Antibacterial activities of Miang extracts against selected pathogens and the potential of the tannin-free extracts in the growth inhibition of Streptococcus mutansPLOS ONE

Dear Dr. Khanongnuch,

==============================

ACADEMIC EDITOR:

Dear author, I received the opinion on your manuscript. Review reviewers' recommendations point by point

==============================

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Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

Reviewer #3: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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6. Review Comments to the Author

Reviewer #1: Dear Authors,

Thank you for your revised manuscript. All of my comments and suggestions have been almost completed. It would be great if the authors could include response 12 in the manuscript.

Reviewer #2: Although the authors responded to the comments, some points needed to be elucidated.

The authors should determine which active ingredients in Miang extract prevent antibacterial growth.

To describe the study's findings, the authors should include the concentration of Miang extract that was observed to limit bacterial growth.

Reviewer #3: All the comments that I raised have been addressed adequately. I comend the authors for the efforts put into addressing all the comments posed by the reviewers.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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PLoS One. 2024 May 8;19(5):e0302717. doi: 10.1371/journal.pone.0302717.r004

Author response to Decision Letter 1

23 Mar 2024

Reviewer 1

Comment: Dear Authors, thank you for your revised manuscript. All of my comments and suggestions have been almost completed. It would be great if the authors could include response 12 in the manuscript.

Response: Thank you very much for the comment. Response 12 from the major revision responded the question on how percentage inhibition was calculated. The response has been included in materials and methods section on LNs 140 – 142, “Inhibition by neutralized extract and organic acids is calculated according to the following equation: inhibition by neutralized extract (%) = (zone of inhibition for neutralized extract/zone of inhibition for non-neutralized extract) × 100, and inhibition by organic acids (%) = 100 ˗ inhibition by neutralized extract”.

Reviewer 2

General Response: Thank you very much for the comment. All corrections by the authors are made using Tracked changes and saved as “Revised document with Track changes”. Line numbers (LNs) are also included in the point by point responses to reviewers for efficient tracking of corrections. Responses to R2 are highlighted in green in the “Revised manuscript with track changes”.

Comment: Although the authors responded to the comments, some points needed to be elucidated. The authors should determine which active ingredients in Miang extract prevent antibacterial growth. To describe the study's findings, the authors should include the concentration of Miang extract that was observed to limit bacterial growth.

Response 1: We already established previously (Reference 7, LN 502) that tannins/proanthocyanidins are major bioactive compounds in Miang and play important roles as antioxidant and antimicrobial agents. In the context of this study, we suggested that ellagic acid, flavonoid aglycones, flavonoid glycosides and organic acid derivatives in tannin-free extracts (See supplementary information) as probably responsible for the observed antibacterial activities against S. mutans DMST 18777. LN 441 – 444, “Our results suggested ellagic acid, flavonoid aglycones, flavonoid glycosides and organic acid derivatives might be responsible for the antimicrobial activities of NFP and FFP Miang (Table 3, Fig 7C) from the observed docking binding energies”.

In addition, the concentrations of Miang extracts that inhibited bacterial growth were represented as minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) as seen in LNs 264 – 287 (Result section) and illustrated in Fig 3. Future studies will try evaluating the potential activities of these suggested compounds against the bacterial pathogen.

Attachment

Submitted filename: Response to Academic editor_R2.doc

pone.0302717.s006.doc^{(30KB, doc)}

PLoS One. doi: 10.1371/journal.pone.0302717.r005

Decision Letter 2

Mozaniel Santana de Oliveira

9 Apr 2024

Antibacterial activities of Miang extracts against selected pathogens and the potential of the tannin-free extracts in the growth inhibition of Streptococcus mutans

PONE-D-23-35234R2

Dear Dr. Khanongnuch,

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PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: Yes

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6. Review Comments to the Author

Reviewer #2: The authors had responded to all comments. Please remove value 3.76 from Table 3, as it did not reflect any parameter.

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PLoS One. doi: 10.1371/journal.pone.0302717.r006

Acceptance letter

Mozaniel Santana de Oliveira

26 Apr 2024

PONE-D-23-35234R2

PLOS ONE

Dear Dr. Khanongnuch,

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. HPLC chromatogram of polyvinylpyrrolidone (PVPP)–treated Miang extracts.

YTL: Young tea leaves, MTL: Mature tea leaves, NFP: Non-filamentous fungi growth based process fermented Miang, FFP: Filamentous fungi-growth based process fermented Miang.

(PDF)

pone.0302717.s001.pdf^{(385.6KB, pdf)}

S1 Table. Identification of compounds in polyvinylpyrrolidone (PVPP)–treated Miang extracts.

(PDF)

pone.0302717.s002.pdf^{(207.1KB, pdf)}

Attachment

Submitted filename: PONE-D-23-35234_Comments.docx

pone.0302717.s003.docx^{(17.7KB, docx)}

Attachment

Submitted filename: Comments PLos one.pdf

pone.0302717.s004.pdf^{(71.6KB, pdf)}

Attachment

Submitted filename: Response to Reviewers.doc

pone.0302717.s005.doc^{(110.5KB, doc)}

Attachment

Submitted filename: Response to Academic editor_R2.doc

pone.0302717.s006.doc^{(30KB, doc)}

Data Availability Statement

All relevant data are within the paper and its Supporting information files.

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Antibacterial activities of Miang extracts against selected pathogens and the potential of the tannin-free extracts in the growth inhibition of Streptococcus mutans

Aliyu Dantani Abdullahi

Kridsada Unban

Chalermpong Saenjum

Pratthana Kodchasee

Napapan Kangwan

Hathairat Thananchai

Kalidas Shetty

Chartchai Khanongnuch

Roles

Abstract

Introduction

Materials and methods

Sample collection

Preparation of Miang extracts

Bacterial strains and growth conditions

Determination of antibacterial activities of neutralized (pH 7) and non-neutralized (pH 4.0–4.6) Miang extracts

Determination of minimum inhibitory (MIC) and minimum bactericidal concentrations (MBC) of neutralized (pH 7) and non-neutralized (pH 4.0–4.6) Miang extracts

Total tannin contents of polyvinylpolypyrrolidone (PVPP)–Miang extract supernatants

HPLC-QToF-MS analysis Polyvinylpolypyrrolidone (PVPP)–treated Miang extracts

Inhibition of biofilm formation

Homology modelling

Molecular docking

Statistical analysis

Results

Table 1. The inhibition zone of Miang extracts tested against Bacillus cereus TISTR 747, Escherichia coli ATCC 22595, Salmonella Typhimurium TISTR 292 and Streptococcus mutans DMST 18777.

Fig 1.

Fig 2.

Fig 3.

Table 2. The inhibition zone of tannin-free extracts of Miang and tea leaves against Streptococcus mutans DMST 18777.

Fig 4.

Fig 5. Total tannin contents in Miang extracts before and after polyvinylpyrrolidone (PVPP)–precipitation.

Fig 6. Anti-biofilm activity of polyvinylpyrrolidone (PVPP)-treated Miang extracts on S. mutans DMST 18777.

Fig 7.

Fig 8. 2D and 3D Ligand-receptor interactions for reference compounds and ellagic acid (bottom right).

Table 3. Binding energies and bonding interactions of docked compounds.

Discussion

Conclusion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Mozaniel Santana de Oliveira

Roles

Author response to Decision Letter 0

Decision Letter 1

Mozaniel Santana de Oliveira

Roles

Author response to Decision Letter 1

Decision Letter 2

Mozaniel Santana de Oliveira

Roles

Acceptance letter

Mozaniel Santana de Oliveira

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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RESOURCES

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