Evolution of Kombucha Tea from Isolated Acetic Acid Bacteria, Lactic Acid Bacteria and Yeast in Single- and Mixed-Cultures: Characteristics, Bioactivities, Fermentation Performance and Kinetics

ABSTRACT In this study, three microbial isolates from kombucha (Saccharomyces cerevisiae, Komagataeibacter saccharivorans, and Levilactobacillus brevis) were used as single- and mixed-inoculum to produce kombucha. During 18-day fermentation, phenolic content was shown to rise from 621.4–633.1 to 817.8–937.7 mg/L while DPPH radical scavenging and ferric reducing activities reached their peaks (1191.3–1343.3 and 742.9–837.9 mg/L, respectively) at day 2–8 and constantly declined throughout the remaining days. Higher sugar concentration and longer fermentation time also resulted in greater antibacterial activity, particularly against Gram-positive bacteria. Regarding bacterial cellulose productivity, 50 g/L glucose was proven to be the effective concentration regardless of microbial combinations with the maximum yield of 193.3–263.9 g/L. A close interaction was seen between S. cerevisiae and K. saccharivorans, while L. brevis exhibited limited interaction with others. Therefore, the application of single culture of K. saccharivorans, or its mixed-culture with S. cerevisiae is considered a feasible approach to control Kombucha quality.


Introduction
Fermentation technology is a prevalent approach utilized in the preservation and development of fermented foods based on the action of microorganisms, most commonly lactic acid bacteria (LAB).It operates through the mechanism by which complex compounds, predominantly carbohydrates, are degraded into simple molecules along with the biosynthesis of other metabolites (Skowron et al. 2022).Traditional fermented foods including black rice vinegar, soy sauce, and Kombucha are employed as a source of potential functional foods due to their ability to enhance product quality, diversify product lines, and aid in the prevention and management of a variety of health issues (Murooka and Yamshita 2008;Sharma et al. 2020).Kombucha is CONTACT Quoc-Duy Nguyen nqduy@ntt.edu.vnInstitute of Applied Technology and Sustainable Development, Nguyen Tat Thanh University, 300A Nguyen Tat Thanh Street, Ward 13, District 4, Ho Chi Minh City 700000, Vietnam Supplemental data for this article can be accessed online at https://doi.org/10.1080/08905436.2024.2306505 2019) while still maintaining the characteristic features of Kombucha tea, such as its chemical composition and biological benefits.The microbial isolates from Kombucha were also applied in other food commodities, such as dairy products (Sarkaya, Akan, and Kinik 2021) or cocoa beans (Díaz-Muñoz et al. 2021).
Natural fermentation based on backsloping methods, which involves inoculating a new batch with previously fermented products, is still used at household scale with the disadvantage of inconsistent quality.Therefore, the fermentation process using well-defined microorganisms as starter culture under controlled conditions shows potential application in the food industry by limiting the growth of other unwanted microbiota (Skowron et al. 2022).Despite the fact that acetic acid bacteria are the predominant microorganisms responsible for imparting the distinctive flavor to Kombucha, a symbiotic connection with lactic acid bacteria and yeast can also influence product quality.Therefore, to evaluate the applicability of pure starter culture isolated from Kombucha in Kombucha production, this study aimed to isolate some microbial strains from wild Kombucha for application in the fermentation of black tea Kombucha by using single-and mixedcultures at different sugar concentrations.Apart from some physicochemical properties of Kombucha tea including phenolic content, antioxidant, antibacterial activities, and bacterial cellulose (BC) biosynthesis, the symbiotic relationship between these microorganisms was also considered based on some kinetic data.

Materials and chemicals
Black tea was purchased from Wonderful Foods Co. Ltd. (Vietnam) in a sealed plastic bag and stored at room temperature.

Isolation and identification of microbial strains
The Kombucha was naturally fermented from a mixture of black tea (8 g/L), glucose (20 g/L) and wild SCOBY for 7 days at ambient temperature.After being brewed for 15 min in boiling water, black tea was filtered, followed by sugar addition, cooling to 25°C and SCOBY inoculation.After that, Kombucha was collected and the serial dilution with 0.9% saline was conducted to yield a series of dilutions from 10 −1 to 10 −6 .Subsequently, 0.1 mL of each diluent was spread separately on the surface of YDPG agar (yeast extract 5.0 g/L, peptone 5.0 g/L, glucose 10.0 g/L, agar 20.0 g/L with added 0.5% CaCO 3 ), YPDA agar (glucose 50.0 g/L, KH 2 PO 4 3.0 g/L, MgSO 4 .7 H 2 O 3.0 g/L, peptone 10.0 g/L, agar 20.0 g/L with 1.5 mg/L chloramphenicol), and MRS agar.After incubation for 48 h at 30°C, colonies of different shapes were selected and purified by repeatedly streaking on the YDPG, YPDA, and MRS agar for obtaining homogenized colonies.After purification, the bacterial strains were subjected to Gram staining for cell morphology, biochemical tests such as sugar fermentation, film-forming ability, catalase test, oxidase test, the ability to oxidize ethanol to acetic acid, and microbial identification based on 16S ribosomal RNA (16S rRNA) gene sequencing.

Biochemical tests
Colonies from purified bacterial strains on YDPG and MRS agar plates were collected and Gram-stained to determine the bacterial morphology in which purple-and pink-stained bacteria were identified as Gram-positive and Gramnegative.Subsequently, biochemical tests were carried out, such as the ability to assimilate sugars (glucose, lactose, sucrose), catalase test (based on the decomposition of hydrogen peroxide to produce oxygen gas), oxidase test (cytochrome oxidase-producing bacteria oxidize the TMPD reagent to purple indophenol), and the ability to form exopolysaccharides on Hestrin -Schramm medium (glucose 50.0 g/L, peptone 5.0 g/L, yeast extract 5.0 g/L, Na 2 HPO 4 2.7 g/L, and citric acid 1.15 g/L at pH 5.0 with addition of 2% (v/v) ethanol).

16S rRNA sequencing
For sequencing, the purified colonies were collected and extracted for genomic DNA segments to amplify the 16S rRNA gene sequence by PCR amplification method using a primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and a 1492 R (5′-TACGGCTACCTTGTTACGACTT-3′) for bacteria and primers NL1 5′-GCATATCAATAAGCGGAGGAAAAG-3′, NL4 5′-GGTCCGTGTT TCAAGACGG-3′ for fungi.The purified PCR product was then subject to electrophoresis in 1% agarose gel with 1X TBE buffer (Tris-HCl/Boric Acid/ EDTA) in ethidium bromide, sequencing and compared for the similarity with the gene sequences on the NCBI database system.Phylogenetic tree was built based on the Neighbor-Joining method using Molecular Evolutionary Genetics Analysis (MEGA) software version 11 to determine the genetic distance between microbial isolates and strains in the gene bank.

Determination of probiotic activity
Preparation of microbial biomass: The isolates were cultured in the liquid medium at 37°C overnight.Then, the biomass was collected by centrifugation at 8000 rpm for 20 min and resuspended in PBS buffer at pH 2 for acid-bile tolerance test and pH 6.5 (OD 600 = 1) for auto-aggregation and co-aggregation ability test.
Acid and bile tolerance test: After 2 and 5 h incubation in the acidic medium (PBS buffer of pH 2.0) supplemented with 0.3% bile, the microbial strains were quantified for the number of viable cells on the respective medium after incubation at 37°C for 48 h.Acid and bile tolerance were determined as survival percentages based on the difference between the initial and final number of viable cells.
Auto-aggregation ability test: The microbial suspensions were left to stand at 37°C for 2 and 5 h, followed by the supernatant being collected and measured for optical density at 600 nm.Auto-aggregation ability was defined as the percent difference of optical density before and after 2 and 5 h autoaggregation.
Co-aggregation ability test: The microbial suspensions were mixed with two pathogenic bacteria, namely Escherichia coli and Salmonella typhi suspended in the same PBS buffer at a volumetric ratio of 1:1.The supernatant was then collected and measured for optical density at 600 nm.Co-aggregation ability was defined as the percent difference of optical density before and after 5 h coaggregation at a time interval of 1 h.

Kombucha fermentation by single-and mixed-culture
Kombucha fermentation was carried out using black tea (8 g/L) supplemented with glucose (20 g/L, 50 g/L, 100 g/L concentration) with three different microbial combinations.After being brewed for 15 min in boiling water, black tea was filtered, followed by sugar addition.The mixture was cooled to 25°C and inoculated using 10% inoculum size to reach initial microbial density of 4 log CFU/mL for single culture of acetic acid bacteria (A), binary culture of yeast -acetic acid bacteria (YA), and ternary culture of yeast -acetic acid bacteria -lactic acid bacteria (YAL).Finally, the mixtures were fermented statically under aerobic conditions at ambient temperature for 18 days to determine some physicochemical properties (pH, total soluble solids, reducing sugar content), antioxidant contents and activities, as well as antibacterial activity of fermentation broth and the characterization of bacterial cellulose films formed on the liquid surface after 7 and 14 days of fermentation.

pH, total soluble solids, reducing sugar content of fermentation broth
pH and total soluble solid (°Brix) were measured using the HI 2211-02 pH meter (Hanna Instruments, Romania) and the Master-53 M hand-held refractometer (Atago Ltd., Japan), respectively.Reducing sugar content expressed as g glucose per liter was spectrophotometrically determined based on the chromophore from the reaction of reducing sugar and DNS reagent under boiling conditions (Miller 1959).

Total phenolic content
The total phenolic content was performed according to the Folin-Ciocalteu method according to ISO 14,502-1:2005(ISO 2005) based on the reaction of phenolics with Folin-Ciocalteu reagent in an alkaline medium to form blue chromophore with maximum absorption at 765 nm.The phenolic content was calculated based on the gallic acid standard curve and expressed in mg gallic acid equivalent per liter of extracts (mg GAE/L).

Antioxidant activity -DPPH • free radical scavenging activity
Antioxidant activity was evaluated through DPPH free radical scavenging capacity based on the purple color change of DPPH solution (0.6 mM) measured at 515 nm upon reaction with phenolics and related antioxidants (Marinova and Batchvarov 2011).The DPPH antioxidant activity was calculated against the Trolox calibration curve and expressed in mg Trolox equivalent per liter of extracts (mg TE/L).

Antioxidant activity -ferric reducing antioxidant power
Ferric reducing antioxidant power (FRAP) was determined based on the chromophores formed between the working reagents (mixture of 0.3 M acetate buffer at pH 3.6, 0.01 M TPTZ prepared in 0.04 M HCl, and 0.02 M FeCl 3 .6H 2 O solution in a volumetric ratio of 10:1:1) with antioxidants (Arriola et al. 2016).Ferric reducing antioxidant activity was calculated against the Trolox calibration curve and expressed in mg Trolox equivalent per liter of extracts (mg TE/L).

Antibacterial activities -minimum inhibitory and bactericidal concentration
Minimum inhibitory concentration (MIC) values of Kombucha were evaluated by a microdilution method according to CLSI protocol M7A7-2006 (Clinical andLaboratory Standards Institute 2006).For MIC determination, sample aliquots (100 μL) were serially 2-fold diluted using 0.9% NaCl solution in a 96-well plate.Subsequently, each well containing 100 μL of Mueller-Hinton broth, 50 μL of microbial suspension (at 10 8 CFU/mL concentration), and 50 μL of Kombucha liquid (the serial dilution 500, 250, 125, 62.5, 31.3, 15.6, and 7.8 μL/mL).The last well containing 100 μL of Mueller Hinton broth, 50 μL of sterilized distilled water, and 50 μL of microbial suspension was used as control.The microplate was incubated at 37°C for 18 h, after which MIC of microbial growth was determined as the lowest concentration of Kombucha at which the microbial growth, as shown by turbidity, was not observed.Finally, minimum bactericidal concentration (MBC) was also determined by plating 20 μL of the suspension in the abovementioned well at concentrations above the MIC on Mueller-Hinton agar plates.After incubation at 37°C for 18 h, the lowest concentration without visible microbial growth was considered as the MBC.

Thickness, wet weight, dry weight, and water absorption capacity of BC films
The BC films on the surface of fermentation broth were collected and immersed in 2% NaOH (1 h, 80°C), followed by neutralization by 2% acetic acid and distilled water.The bleached films were then analyzed for wet weight and thickness using DM3025 IP54 digital micrometer (DML, UK) before being dried to constant weight at 60°C in the LO-FS100 forced convection oven (LK Lab, Korea) to determine the dry weight.The BC production was presented as g of BC on the wet and dry basis per 1 L of fermentation broth (g/L).In addition, water absorption capacity was calculated based on wet and dry weights of BC films, as illustrated in the following formula: (W o -W 1 ) × 100/W 1 ; where, W o and W 1 are the sample weights before and after drying.

Microbial enumeration
To determine the density of AAB, LAB and yeast, 0.1 mL of sample diluents was poured on YPDG, MRS, and YPDA medium.The agar plates were incubated at 30°C for 48 h, and colonies were then counted to determine the microbial density expressed in log CFU/mL.

Fermentation kinetics
Based on microbial density and glucose concentration during fermentation time, specific growth rate (μ, 1/h) and substrate utilization rate (R S , g/L/h) were computed according to the following formula: μ = (log 10 X t -log 10 X o ) × 2.303/(t 1 -t o ) and R S = (S t -S o )/t 2 ; where, X t and X o are microbial densities at the end (t 1 ) and beginning (t o ) of log phase while S t and S o are sugar concentration at the time sugar showing stable depletion (t 2 ).

Statistical analysis
All statistical techniques, including normality test, homoscedasticity of variances, one-way ANOVA, and post-hoc Tukey test, were performed at 5% significance level by using R version 4.1.2(R Core Team 2013).

Morphology and biochemical tests
Three strains of beneficial microbes, designated K1, K2, and K3, were isolated and purified from wild Kombucha; their colonial morphology, bacterial morphology, and biochemical test results are summarized in Table 1.The colony morphology of isolated strains is white or light yellow; all are round, glossy, convex, and have circular edges.However, K3 was found to have an oval shape and a little bud protruding from the mother cell when viewed under the microscope.Biochemical analysis also revealed that K3 could ferment glucose into carbon dioxide.Meanwhile, K2 microbial strain showed the ability to utilize lactose and glucose, while K1 used only glucose.In particular, K1 can form exopolysaccharide films similar to acetic acid bacteria.Similar results were also reported in the study by Wang et al. (2014) on microbial strains isolated from Kombucha, which showed the existence of two strains of yeast, two strains of AAB, and one strain of LAB.It can be concluded that K1, K2, and K3 were preliminarily identified as acetic acid bacteria, lactic acid bacteria and yeast.

Identification of microbial isolates by 16S rRNA
The results of bacterial identification by molecular analysis are presented as a phylogenetic tree in Figure 1, showing that the gene fragment amplified from bacteria K1 is similar to the gene encoding 16S rRNA of bacterial strain Komagataeibacter saccharivorans 1.1 (Accession number MN103849.1),with 99% similarity when compared with the NCBI database.The results of comparing 16S gene sequences of K2 strains showed 94% similarity with Levilactobacillus brevis (Accession number CP031208.1)while K3 strains showed 95% similarity compared with the genetic sequence of the yeast strain Saccharomyces cerevisiae (Accession number KF728798.1)from the NCBI database.Therefore, bacterial strains K1, K2, and K3 were determined to be homologous with Komagataeibacter saccharivorans, Levilactobacillus brevis and Saccharomyces cerevisiae, respectively.

Probiotic properties -acid and bile tolerant activity
Bile-acid tolerance of beneficial bacteria during 4 h was investigated at pH 2 with the addition of 0.3% bile salt to determine the survival of microorganisms in simulated gastric conditions which is shown in Figure 2a.The ability to tolerate gastric acid and bile salt is crucial for probiotic bacteria to grow and thrive in the digestive tract (Tilwani et al. 2022).The results demonstrated that LAB are the most resistant to acid-bile salt compared to the other two microorganisms tested.Furthermore, the viable counts of L. brevis were consistent during 4 h of acid-bile salt exposure, ranging from 5.7 to 6.0 log CFU/mL, as opposed to the total destruction of S. cerevisiae even in the first hour and 2.0 log CFU/mL of K. saccharivorans after 4 h.Shehata et al. (2016) concluded that in MRS medium supplemented with 0.3% bile salts, the survival rate of LAB ranged from 69.8 to 88.3% after 3 h incubation in simulated gastric juice medium at pH 2.0 while other studies on probiotic strains have shown that LAB strains' ability to lower blood cholesterol was achieved by increasing bile acid synthesis based on the deconjugation of bile salts by the bile salt hydrolase enzyme (Gu et al. 2014;Hernández-Gómez et al. 2021).More specifically, LAB generates hydrolase through conjugation with free bile acids in response to bile acid attack; this enzyme also stimulates the synthesis of exopolysaccharides by LAB, which serve as a barrier to protect them from bile salts (Bustos et al. 2018;Harnentis et al. 2020).This is a significant probiotic characteristic of LAB, since its resistance to bile salts facilitates the maintenance of viable cells in the small intestine, enabling them to progress to the large intestine and demonstrate other beneficial effects (Nyiew, Kwong, and Yow 2022).

Auto-aggregation and co-aggregation
The capacity for auto-aggregation is related to intestinal adhesion and boosting microorganisms' persistence in the digestive system, all of which improve the strain's survivability and growth in a mutually beneficial interaction with had an increasingly high degree of auto-aggregation over time, from 41.6% after 2 h to 50.7% after 5 h, which were significantly higher than the other strains.Meanwhile, K. saccharivorans and S. cerevisiae exhibited constant auto-aggregation during 5 h, being 12.4% and 22.7-23.7%,respectively.Similar findings were found in a study of Orłowski and Bielecka (2006), who found that the auto-aggregation ability of probiotic lactobacilli was up to 77%.The auto-aggregative abilities of probiotics are strain-specific properties which are influenced by many factors such as cell surface charge and components, bacterial size and surrounding conditions (Grujović et al. 2022).Additionally, probiotics in the intestinal tract have the ability to co-aggregate with intestinal pathogenic microorganisms, thereby increasing the ability to inhibit the growth of pathogenic bacteria, contributing to the balance of intestinal microflora (Ciandrini, Campana, and Baffone 2017).In this study, two strains of enteropathogenic bacteria, namely Escherichia coli ATCC 8739 and Salmonella typhi ATCC 6539, were used to test the co-aggregation ability with the three isolated strains.It can be seen in Figure 2c that L. brevis was proven to be effective in co-aggregating with pathogens over time, as illustrated by the roughly double increase in co-aggregation activity from 2 h to 5 h exposure, which is consistent with the study of Collado, Meriluoto, and Salminen (2008).In contrast, S. cerevisiae and K. saccharivorans shared the same pattern in terms of low co-aggregation ability with the pathogenic bacteria (10.3-11.4%),particularly the negligible ability observed for the yeast with Salmonella pathogen.The co-aggregation ability of Lactobacillus sp. is likely the result of its good adhesion to the epithelium, which will kill or competitively inhibit pathogens (Jena et al. 2013) and form a barrier against the growth of pathogenic bacteria (Collado, Meriluoto, and Salminen 2008).

Characterization of Kombucha tea fermented by single-and mixed-culture pH, total soluble solids, and reducing sugar
The pH value is an useful indicator for controlling fermentation that may indicate the end of process (Malbaša, Lončar, and Djurić 2008).Change in pH during Kombucha fermentation by single acetic acid bacteria (A), binary culture of yeast-acetic acid bacteria (YA), and ternary culture of yeast-acetic acid bacteria-lactic acid bacteria (YAL) at initial glucose concentrations of 20 g/L, 50 g/L and 100 g/L is presented in Table 2.At the beginning of fermentation, the pH of black tea infusions fluctuated in the range of 4.8-5.0,which is attributed to the different acidity of the starter cultures with different microbial combinations.As can be seen, the pH decreased sharply during the first 4 days of fermentation to about 2.6-3.3 except for samples with an initial sugar concentration of 20 g/L which had a higher pH value (3.5), followed by a gradual decrease with time before the steady pH observed for the last 10 days of fermentation.During the fermentation process, LAB and AAB produce acids that lower the pH of the fermentation broth and inhibit the growth of other microorganisms after 18 days of fermentation (Kaewkod, Bovonsombut, and Tragoolpua 2019).As fermentation takes place, the change in acidity can be divided into two phases: a rapid reduction phase (0-6 days) and a slow reduction phase (6-18 days).Similar pH value changes were also noticed in the study of Kombucha from laver (Porphyra dentata) (Aung and Eun 2022) and soymilk (Xia et al. 2019), in which pH decreased abruptly to about 3.0 during the first 3 days of fermentation, remained stable for the next 5 days and gradually decreased until the end of fermentation.The pH stability of Kombucha can also be explained by the buffering capacity of the Kombucha itself, which is derived from the production of carbonic gas and hydrocarbonate ions, hence limiting the pH fluctuation of the medium (Essawet et al. 2015;Wang et al. 2022).Additionally, proteins, organic acids and minerals as well as sugars also increased the buffering capacity during fermentation (Devnani et al. 2022).Khosravi et al. (2019) explained that the chemical reaction between weak organic acids and mineral components in the fermentation broth increased its buffering capacity.Regarding total soluble solids and glucose content (Table 2), it is shown that these values decreased with fermentation time and the residual solids of mixed-culture (YA20 and YAL20) were lower than that of single culture (A20) at the end of fermentation.The result is in accordance with the study of Abuduaibifu and Tamer (2019) for goji berries Kombucha.Glucose is an important substrate for cell growth and metabolite production in most microorganisms (Khosravi et al. 2019).The total dissolved solids content decreased during fermentation due to the consumption of sugars in the medium as a substrate (Wang et al. 2022) in addition to the deposition of proteins, minerals and pigments (Zubaidah, Ifadah, and Afgani 2019).

Phenolic content and antioxidant activities
Total phenolic content and antioxidant activities measured by DPPH and FRAP assays during Kombucha fermentation by single acetic acid bacteria (A), binary culture of yeast-acetic acid bacteria (YA), and ternary culture of yeast-acetic acid bacteria-lactic acid bacteria (YAL) at initial glucose concentrations of 20 g/L, 50 g/L and 100 g/L are presented in Table 3.With regard to phenolics, the gradual increase was observed in all samples from 621.4-633.1 mg GAE/L at day 0 to 817.8-937.7 mg GAE/L at day 18.These results are consistent with the study of Ahmed, Hikal, and Abou-Taleb (2020) and Khosravi et al. (2019) on the bioactivity and antioxidant properties of Kombucha tea, which also showed that prolonging the fermentation process for 8-12 days significantly increased the phenolic content compared to Kombucha with a short fermentation time.The continuous increase in the TPC during fermentation might be attributable to the release of phenolic components under acidic conditions or catalytic action of enzymes produced by bacteria and yeasts (Khosravi et al. 2019).This can stem from the activity of the hydrolyzing enzymes including cellulases, pectinases, hemicellulose, and amylases secreted by microorganisms that catalyze the conversion of insoluble phenolic compounds to the soluble form along with the synthesis of new phenolics (Aung and Eun 2022;Xia et al. 2019).According to Bortolomedi, Paglarini, and Brod (2022) and Ivanišová et al. (2019), biologically active substances such as caffeine, rutin, chlorogenic acid, gallic acid, ascorbic acid, gluconic acid, glucuronic acid, theaflavins, catechins, coumaric acid, ferulic acid, quercetin, protocatechuic acid, ellagic acid, syringic acid, vitexin, D-saccharic acid-1,4-lactone were detected and reported in various studies on Kombucha, especially the first six compounds being present at high concentrations.
More interestingly, the antioxidant activity by DPPH assay showed different trend compared to the variation in phenolic content, which peaked after 2 days of fermentation (1191.3-1343.3mg TE/L) from the original tea (1053.1-1082.5 mg TE/L), followed by the gradual decline for the remaining days.Moreover, the change in FRAP shared the same pattern with DPPH assay with highest values (742.9-837.9mg TE/L) being noted during 4-8 days of fermentation.Similar results were reported by Ahmed, Hikal, and Abou-Taleb (2020) who concluded that the DPPH antioxidant activity of Kombucha tea gradually decreased during 12-14 days of fermentation.Regarding FRAP data, the study of Hsieh, Chiu, and Chou (2021) on fermenting Kombucha from various teas showed that FRAP reached its highest values at day 12 in all three teas (green, black and Pu'erh) and decreased sharply around day 12-16 while the study by Saimaiti et al. (2022) on the fermentation of Kombucha tea from vine tea and sweet tea reported that FRAP values initially increased and then decreased during fermentation, reaching a maximum on day 6.
The increase in fermentation time changes the composition of metabolites produced and enhances the concentration of antioxidants; however, prolonged time leads to accumulation of organic acids, which reduce the sensory value of the product as well as the content of biologically active substances (Bishop et al. 2022;Chu and Chen 2006).Overall, the increased antioxidant activity over time is thought to be dependent on the phenolic content, which is capable of scavenging free radicals or inhibiting metal ions (Ahmed, Hikal, and Abou-Taleb 2020;Hsieh, Chiu, and Chou 2021;Srihari and Satyanarayana 2012).

Antibacterial activity of Kombucha
The antibacterial activity of Kombucha after 7 and 14 days of fermentation using different microbial combinations against some pathogenic  microorganisms is shown in Table 4.It is evident that all Kombucha tea samples fermented with different sugar concentrations and microbial combinations exhibited antibacterial action, as illustrated by MIC/MBC values being increased over fermentation time from day 7 to day 14.Specifically, the Kombucha tea samples using a single culture of K. saccharivorans could inhibit pathogenic bacteria almost equally at all three sugar concentrations (20, 50, 100 g/L glucose) with MIC/MBC values being in the range of 250-500 μL/mL.However, after 14 days of fermentation, the antibacterial activity of the single cultures increased.It is speculated that when the fermentation time is prolonged, the microorganisms continue to be active and produce organic acids at higher levels that reduce the pH of the environment, as well as biologically active compounds that disrupt the cell wall structure of the pathogenic bacteria.
In contrast to the same activity of single cultures (A20, A50, A100) at day 7, the antibacterial activity of mixed-cultures increased with increasing sugar content.In addition, the mixed-culture method showed good inhibitory and bactericidal ability against fungi (Candida albicans) and Gram-positive bacteria (Staphylococcus aureus) with notable increase over fermentation time.According to Battikh et al. (2013), the antibacterial properties of black tea were enhanced throughout the Kombucha fermentation process, which can be attributed to the tea acidification resulting from microbial metabolism.In addition, proteins, hydrolyzed and newly formed tea phenolics, and other microbial metabolites such as bacteriocins and enzymes also contributed to the increased antibacterial activity (Battikh et al. 2013) based on their inhibition of biosynthesis of cell wall components, proteins and nucleic acids (Lopes, Oliveira Santos, and Prentice-Hernández 2021;Reygaert 2014).
The antibacterial activity of Kombucha is influenced by many factors including acidity, fermentation time and the content of antibacterial substances, mainly inherent phenolic compounds (such as catechins) present in tea leaves and those produced during fermentation (Nyiew, Kwong, and Yow 2022).Among the newly formed compounds, verbascoside contributes greatly to the antibacterial ability of Kombucha (Cardoso et al. 2020) in addition to the bacteriocins produced by LAB (Pei et al. 2020;Matei et al. 2018).In terms of acidic compounds, acetic acid produced by AAB is considered an agent that inhibits the growth of pathogenic bacteria even at low concentration (Halstead et al. 2015;Ryssel et al. 2009).

Bacterial cellulose biosynthesis
Cellulose is a structural component of the cell walls of plants, algae and fungi, and it is also synthesized by many acetic acid bacteria (Gomes, Iouko Ida, and Aparecida Spinosa 2022).Currently, cellulose produced from bacteria is considered an eco-friendly raw material because of its biodegradability and harmlessness to human health (Jayasekara and Ratnayake 2019).Bacterial cellulose has been found to be structurally sound, hydrophilic, and mechanically robust (Lina et al. 2020) with potential application in moist burn pads, artificial blood vessels, food preservation films, and electronic microchips (Bandyopadhyay et al. 2019;Chadha et al. 2022).During fermentation of Kombucha, yeast growth helps AAB to form biofilm on the surface of the medium, which exposes AAB to the oxygen required for growth, thus changing the composition of the original tea medium (Bishop et al. 2022;May et al. 2019).The changes in wet weight, dry weight and thickness of cellulose film formed on day 7 and day 14 of fermentation by single acetic acid bacteria (A), binary culture of yeast-acetic acid bacteria (YA), and ternary culture of yeast-acetic acid bacteria-lactic acid bacteria (YAL) at initial glucose concentrations of 20 g/L, 50 g/L and 100 g/L are shown in Table 5.In general, it can be observed that glucose concentration of 50 g/L was the appropriate substrate concentration for biocellulose production, as illustrated by the highest wet and dry weight of cellulose films regardless of the inoculum combination.In addition, the inoculation of mixed-culture at 100 g/L glucose led to the significant decrease in cellulose production yield.Interestingly, despite the low productivity, bacterial cellulose resulting from glucose 100 g/L exhibited superior water absorption capacity, about 3.2-9.7 times higher than that of 50 g/L glucose.
In terms of appearance (Table S1), it was demonstrated that a medium with a glucose concentration of 50 g/L produced the toughest, most uniformly colored cellulose film with the most stable physicochemical features.On day 7 and 14, the A50 cellulose film exhibited the best visual and tactile qualities, including a white film and a smooth surface.The films produced on day 7 by both YA50 and YAL50 were sharp but rough around the edges whereas both surface smoothness and structural integrity were improved after 14 days.In addition, the thickness of cellulose films from 50 g/L glucose regardless of inoculation methods (A50, YA50, YAL50) was shown to be superior to other samples although the ternary culture (YAL50) exhibited thinner films compared to A50 and YA50 (Table 5).
According to the findings, AAB's increase in cellulose production would not be significantly affected by the addition of sugar at the optimal concentration.In the case of low sugar concentration, it only provides just enough carbon source for acetic acid bacteria to survive, but not to increase the bacterial population.When the sugar concentration is too high, microorganisms produce large amounts of organic acids, which inhibit the growth of ethanol-producing yeasts which provide ethanol for the metabolism of AAB for the conversion of ethanol into acetic acid and extracellular cellulose.According to Tapias et al. (2022), the relationship between AAB and yeast improves cellulose synthesis based on cooperative and competitive interactions that benefit both groups of microorganisms including cellulose production yield.Furthermore, an increase in substrate concentration to 100 g/L resulted in a decrease in cellulose synthesis efficiency.This can be attributed to the fact that microorganisms shift their preference to grow biomass in the elevated nutrient levels, as evidenced by the higher microbial density observed in the final 7 days of fermentation.

Sugar assimilation
Microbial growth is a series of biochemical reactions through the synthesis of cellular matter and the production of extracellular products (Moradali and Bernd 2020).Sugar assimilation rate of microbial isolates during fermentation by single acetic acid bacteria (A), binary culture of yeast-acetic acid bacteria (YA), and ternary culture of yeast-acetic acid bacteria-lactic acid bacteria (YAL) at initial glucose concentrations of 20 g/L, 50 g/L and 100 g/L is shown in Table 6.It can be seen that the increase in substrate concentration led to the increase in sugar assimilation; specifically, the values for single culture at 20 g/L glucose (0.08699) rose to 0.11629 and 0.29148 for 50 g/L and 100 g/L, respectively.The most interesting finding is that the inoculation of binary and ternary combination significantly resulted in the increased sugar assimilation (0.53866-0.66588),especially at high substrate concentration of 100 g/L.

Microbial growth
Microbial growth is the most common cause of changes in food ingredients; therefore, the growth patterns of microorganisms as well as the internal and external factors affecting their growth have been studied extensively (Gomes et al. 2018;Lazo-Vélez et al. 2018;Wang et al. 2021).Microbial densities of three isolated strains during fermentation by single acetic acid bacteria (A), (a)  (Figure 3, Table S2), K. saccharivorans thrived during 0-4 days of fermentation and then entered the stationary phase, followed by the death phase at day 6 (A20) and day 5 (YA20 and YAL20).Interestingly, after 7 days of fermentation, the density of K. saccharivorans tended to be stable during the 7-9 days of fermentation.It can be explained by the fact that K. saccharivorans adhere to the cellulosic films formed on the surface of the fermentation broth which acts as protective barriers for their growth in the presence of oxygen within the headspace.In addition, the death rate of K. saccharivorans accelerated in mixed-culture regardless of initial sugar concentration, as depicted by their remarkably lower density than that of single culture over the remaining fermentation time (up to 18 days).The same trend was also observed for yeast with almost no viability at the end of fermentation (Figure 4, Table S3).It is noteworthy that the changes in densities of LAB upon increasing sugar concentration showed negligible variation (Figure 5, Table S4).As for specific growth rate which is the most important parameter to be controlled during fermentation as it represents microbial proliferation (Metsoviti et al. 2019), Table 6 shows that the presence of yeast in the fermentation broth triggered the growth of AAB, particularly at higher sugar concentration of 100 g/L with specific growth rate of 0.10268-0.10316.Furthermore, the presence of AAB also increased the growth rate of yeast (0.1041-0.10579) while the growth of LAB showed no effect.

Interaction between AAB, LAB, and yeast
AAB, LAB, and yeasts use glucose for metabolism and growth (Maslanka and Zadrag-Tecza 2019;Wang et al. 2021), and some studies have also demonstrated a dual relationship between AAB and yeast (Dutta and Paul 2019).Ethanol produced from the fermentation of glucose and fructose by yeast is converted by AAB into organic acids, especially acetic acid, which in turn stimulates yeast to produce more ethanol, thereby simplifying metabolic pathway of acetic acid production and promoting the growth of AAB (Bishop et al. 2022).The mutually supportive interaction of symbiotic fermentation is one of the reasons leading to the interaction that increases the density of yeast and bacteria (Landis et al. 2022).Besides, LAB ferments glucose to lactic acid, acetic acid, mannitol, and pyruvate, creating a microbiologically stable fermentation medium and providing lactate as the carbon source for the indispensable growth of AAB (De Vuyst and Leroy 2020).Lactic acid bacteria have a supporting role in providing a part of lactate for AAB to use as a carbon source, along with producing organic acids to maintain a low pH of the fermentation solution, avoiding the invasion of pathogenic bacteria (De Vuyst and Leroy 2020).LAB growth can even be facilitated by yeast's carbon dioxide production and subsequently by releasing vitamins and other nutrients from autolytic yeast cells when fermented (Agyirifo et al. 2019).Therefore, the substrate assimilation rate was always higher in binary and ternary cultures than in single culture.Depletion of glucose energy sources and continuous increase of ethanol, lactic acid, and acetic acid concentrations cause a decrease in the number of yeast, lactic acid bacteria and acetic acid bacteria (De Vuyst and Weckx 2016).
The term "symbiotic" is commonly used to refer to the microbial interactions that take place in Kombucha tea; this goes with the essential need that the relationships are mutually beneficial, as the word "reciprocal" would imply (Landis et al. 2022;May et al. 2019).To be more specific, when two or more species of microorganisms coexist, mutual or antagonistic interactions can occur because each microorganism requires different nutrients and optimal growing conditions, resulting in different growth rates.The yeast and bacteria in Kombucha tea are involved in metabolic activities that utilize the substrates in different and complementary ways.Yeast hydrolyzes sucrose into glucose and fructose which are in turn converted to ethanol through glycolysis for AAB to produce acetic acid (Amarasinghe, Weerakkody, and Waisundara 2018).It is postulated in our data that yeast and AAB appeared to be in a close cooperative interaction whereas LAB showed no interaction, either beneficial or harmful, with the other microorganisms.This finding is in accordance with the study of Devanthi et al. (2021) on the interaction between yeast strains (Dekkera bruxellensis) and K. intermedius bacteria derived from Kombucha and molasses in single-and mixed-culture.As stated by Tran et al. (2020), the relationship between AAB and yeast is non-strict parasitism in that although yeast provides oxidizable substrates, AAB can still grow in the absence of yeast.Additionally, AAB influences the organic acid profile while yeast is responsible for microbial dynamics, chemical composition and sensory characteristics of Kombucha (de Noronha et al. 2022).

Conclusions
It is noted that pH, total soluble solids, and reducing sugars were all decreased after 18 days of fermentation using various starter cultures and sugar concentrations.Meanwhile, although phenolic content showed continuous increase throughout fermentation, DPPH and FRAP based antioxidant activities all showed elevated activity during the first 2-8 days of fermentation and then reduced during the remaining days.Additionally, this study highlighted the use of a combination of AAB-LAB-yeast to produce Kombucha with enhanced antibacterial activity.It is concluded that S. cerevisiae and K. saccharivorans appeared to be in a close interactive relationship whereas L. brevis showed less interaction with the other microorganisms.The susceptibility of Gram-negative and Gram-positive bacteria to Kombucha suggests a potential application of this beverage as a novel antibacterial product along with its antioxidant activities.Additionally, the utilization of starter cultures containing solely AAB or in combination with yeast demonstrates the prospects of controlling the product quality at an industrial scale.

Figure 1 .Figure 2 .
Figure 1.Phylogenetic trees based on gene sequences of acetic acid bacteria K1 (a), lactic acid bacteria K2 (b), and yeast K3 (c) isolated from kombucha tea and reference strains in the gene bank database, using Neighbor-Joining analysis.

Table 2 .
Changes in total soluble solid (°Brix), pH, and reducing sugar content (g glucose/L) of kombucha during 18-day fermentation by single acetic acid bacteria (A), binary culture of yeast-acetic acid bacteria (YA), and ternary culture of yeast-acetic acid bacteria-lactic acid bacteria (YAL) at initial glucose concentrations of 20 g/L, 50 g/L và 100 g/L.results were presented as mean (standard deviation) of triplicates and different letters in the same columns indicate that the mean values were significantly different at 95% confidence level.A -Komagataeibacter saccharivorans, Y -Saccharomyces cerevisiae, L -Levilactobacillus brevis.

Figure 3 .
Figure 3. Changes in microbial densities (log CFU/mL) of komagataeibacter saccharivorans during 14-day fermentation by single acetic acid bacteria (A), binary culture of yeast-acetic acid bacteria (YA), and ternary culture of yeast-acetic acid bacteria-lactic acid bacteria (YAL) at initial glucose concentrations of (a) 20 g/L, (b) 50 g/L and (c) 100 g/L.

Figure 4 .
Figure 4. Changes in microbial densities (log CFU/mL) of Saccharomyces cerevisiae during 14-day fermentation by binary culture of yeast-acetic acid bacteria (YA), and ternary culture of yeast-acetic acid bacteria-lactic acid bacteria (YAL) at initial glucose concentrations of (a) 20 g/L, (b) 50 g/L and (c) 100 g/L.

Figure 5 .
Figure 5. Changes in microbial densities (log CFU/mL) of Levilactobacillus brevis during 14-day fermentation by ternary culture of yeast-acetic acid bacteria-lactic acid bacteria (YAL) at initial glucose concentrations of 20 g/L, 50 g/L và 100 g/L.

Table 1 .
Cellular morphology and biochemical tests of three microbial strains isolated from kombucha tea.
-and + denote negative and positive results, respectively.

Table 3 .
Changes in total phenolic content (TPC), antioxidant activity (DPPH, FRAP) of kombucha during 18-day fermentation by single acetic acid bacteria (A), binary culture of yeast-acetic acid bacteria (YA), and ternary culture of yeast-acetic acid bacteria-lactic acid bacteria (YAL) at initial glucose concentrations of 20 g/L, 50 g/L và 100 g/L.The results were presented as mean (standard deviation) of triplicates and different letters in the same columns indicate that the mean values were significantly different at 95% confidence level.

Table 4 .
MIC/MBC of kombucha tea after 7 and 14 days of fermentation by single acetic acid bacteria (A), binary culture of yeast-acetic acid bacteria (YA), and ternary culture of yeast-acetic acid bacteria-lactic acid bacteria (YAL) at initial glucose concentrations of 20 g/L, 50 g/L và 100 g/L.

Table 5 .
Physical properties of wet bacterial cellulose films formed within kombucha tea after 6-and 12-day fermentation by single acetic acid bacteria (A), binary culture of yeast-acetic acid bacteria (YA), and ternary culture of yeast-acetic acid bacteria-lactic acid bacteria (YAL) at initial glucose concentrations of 20 g/L, 50 g/L The results were presented as mean (standard deviation) of triplicates and different letters in the same rows indicate that the mean values were significantly different at 95% confidence level.