key: cord-0731395-pln42pwj
authors: La Torre, Chiara; Fazio, Alessia; Caputo, Paolino; Plastina, Pierluigi; Caroleo, Maria Cristina; Cannataro, Roberto; Cione, Erika
title: Effects of Long-Term Storage on Radical Scavenging Properties and Phenolic Content of Kombucha from Black Tea
date: 2021-09-08
journal: Molecules
DOI: 10.3390/molecules26185474
sha: cbaf16a2f0d08e3f8acc880d95e02b7ae172e536
doc_id: 731395
cord_uid: pln42pwj

Kombucha is a fermented beverage. Its consumption has significantly increased during the last decades due to its perceived beneficial effects. For this reason, it has become a highly commercialized drink that is produced industrially. However, kombucha is still also a homemade beverage, and the parameters which, besides its organoleptic characteristics, define the duration of its potential beneficial properties over time, are poorly known. Therefore, this study aimed to determine the effect of 9-month storage at 4 °C with 30-day sampling on the pH, total phenolic, and flavonoid contents, free radical scavenging properties of kombucha fermented from black tea. Our results highlighted that, after four months, the phenolic content decreased significantly from the initial value of 234.1 ± 1.4 µg GAE mL(−1) to 202.9 ± 2.1 µg GAE mL(−1), as well its antioxidant capacity tested by two in vitro models, DPPH, and ABTS assays. Concomitantly, the pH value increased from 2.82 to 3.16. The novel findings of this pilot study revealed that kombucha from sugared black tea can be stored at refrigerator temperature for four months. After this period the antioxidant properties of kombucha are no longer retained.

"Kombucha" is the name of a drink obtained by fermenting tea, mainly black or green, with the addition of sucrose, that acts as a substrate for fermentation, and a symbiotic culture of yeast and bacteria, known as "SCOBY" (Symbiotic Cultures of Bacteria and Yeast). The taste of this drink is slightly acidic and slightly carbonated, which makes it popular and pleasing to consumers [1] . Kombucha was first used in East Asia for its beneficial and curative effects only based on anecdotal evidences, since the Tsin dynasty began consuming it in Manchuria. It spread from China to Russia after World War I and then throughout Europe [1] .

The fermentation is due to a symbiotic culture of acetic bacteria of the genus Acetobacter and Gluconobacter and different osmophilic yeast species, including genera such as Saccharomycode, Schizosaccharomyces, Zygosaccharomyces, Brettanomyces/Dekkera, Candida, Torulospora, Koleckera, and Pichia e Mycoderma. After fermentation, the kombucha tea is filtered through a cheesecloth and is consumed as a healthy drink. It can also be bottled for commercialization [2] .

Almost forgotten for decades, kombucha became very popular again in the early 2000s, thanks to its sudden spread in Australia and in the United States. During the last decades, kombucha transitioned from a homemade fermented beverage to a soft drink produced on Figure 1 . Values of pH determinate in black tea, kombucha tea after 30 days of fermentation and all the samples during storage. Black bar refers to the starting tea. White bar refers to the kombucha sample after 30 days of fermentation (control). Values represent the three-measure mean ± standard deviation. Asterisks on the bars indicate that mean values were statistically different from the control (**** p < 0.0001).

The total phenolic content (TPC) in all kombucha samples is shown in Figure 2 . The results were expressed as µ g equivalents of gallic acid (µ g GAE) per mL of sample. Figure 1 . Values of pH determinate in black tea, kombucha tea after 30 days of fermentation and all the samples during storage. Black bar refers to the starting tea. White bar refers to the kombucha sample after 30 days of fermentation (control). Values represent the three-measure mean ± standard deviation. Asterisks on the bars indicate that mean values were statistically different from the control (**** p < 0.0001).

Five compounds were identified and quantified by HPLC analyses in all samples ( Figure 3 ). Chromatographic evolution at 280 nm is show in Figure 4A -D.

Caffeine was the main compound in all tea samples. Its initial value in black tea was 568.61 ± 0.84 µg mL −1 (Table 1) , which underwent a reduction of 37.34% after fermentation, of 40.29% after one month and of 45.37% after two months. The minimum value was reached after four months (135.36 ± 1.63 µg mL −1 ) which corresponded to a reduction of the initial value of 76.19%. After six months the caffeine content increased (674.98 ± 0.49 µg mL −1 ), reaching its maximum value (702.93 ± 0.02 µg mL −1 ) after nine months. Chlorogenic acid was the only compound that maintained its content unchanged during fermentation and over time compared to the initial value in black tea (29.60 ± 0.01 µg mL −1 ). EGCG content in tea was 20.58 ± 0.32 and remained unchanged after 30 days of fermentation and in the next three months of storage, becoming undetectable at by the fourth month. A similar trend was displayed by ferulic acid, present in a The results highlighted that the kombucha obtained after one month of fermentation, used as reference, showed the maximum TPC level which was 1.7 times higher (234.1 ± 1.4 µ g GAE mL −1 ) than the value of black tea, (137.5 ± 10.7 µ g GAE mL −1 ), respectively. In the following months (from months two to four), the TPC slowly decreased from 234.1 ± 1.4 to 223.5 ± 0.7 µ g GAE mL −1 without significant statistic difference. On the contrary, it was decreasing in a time dependent manner, from months five to nine, significantly by about 13% (202.9 ± 2.1 µ g GAE mL −1 , **** p < 0.0001) at month five, and 34% at month nine (80.8 ± 5.4 µ g GAE mL −1 ).

Five compounds were identified and quantified by HPLC analyses in all samples ( Figure 3 ). Chromatographic evolution at 280 nm is show in Figure 4A -D. Total phenolic content (TPC) (µg GAE mL −1 ) of black tea, kombucha tea after 30 days of fermentation and all the samples during storage. Black bar refers to the starting tea. White bar refers to the kombucha sample after 30 days of fermentation (control). Values represent the three-measure mean ± standard deviation. Asterisks on the bars indicate that mean values were statistically different from the white bar that represents the control (**** p < 0.0001). Caffeine was the main compound in all tea samples. Its initial value in black tea was 568.61 ± 0.84 µ g mL −1 (Table 1) , which underwent a reduction of 37.34% after fermentation, of 40.29% after one month and of 45.37% after two months. The minimum value was reached after four months (135.36 ± 1.63 µ g mL −1 ) which corresponded to a reduction of the initial value of 76.19%. After six months the caffeine content increased (674.98 ± 0.49 µ g mL −1 ), reaching its maximum value (702.93 ± 0.02 µ g mL −1 ) after nine months. Chlorogenic acid was the only compound that maintained its content unchanged during fermentation and over time compared to the initial value in black tea (29.60 ± 0.01 µ g mL −1 ). EGCG content in tea was 20.58 ± 0.32 and remained unchanged after 30 days of fermentation and in the next three months of storage, becoming undetectable at by the fourth month. A similar trend was displayed by ferulic acid, present in a smaller amount in black tea (3.15 (C) kombucha tea sample at the fifth month of storage, when the epigallocatechin gallate content was not detected whereas quercetin, absent in black tea, was identifiable; (D) kombucha tea sample at the ninth month of storage.

Total flavonoid content (TFC) underwent a decrease during fermentation ( Figure 5 ). The initial value of TFC in black tea (2.3 ± 0.2 QE mL −1 ) was reduced by 50% in kombucha tea (1.1 ± 0.3 µg QE mL −1 ) and it remained constant over time. 

Total flavonoid content (TFC) underwent a decrease during fermentation ( Figure 5 ). The initial value of TFC in black tea (2.3 ± 0.2 QE mL −1 ) was reduced by 50% in kombucha tea (1.1 ± 0.3 µ g QE mL −1 ) and it remained constant over time. 

Scavenging abilities of black tea, kombucha tea after one month of fermentation and all the samples during the storage were monitored using two common in vitro models, DPPH and ABTS assays.

Kombucha exhibited good antioxidant activity against DPPH radical during the storage at all tested concentrations when compared to black tea ( Figure 6 ). The results of the DPPH radical assay were also expressed as the Trolox equivalent antioxidant capacity (TEAC) using Trolox as reference standard. The TEAC values are reported in Table 2 . During fermentation, %IDPPH of kombucha tea at the highest and lowest concentrations (55.1 ± 1.3 at 200 µ L, and 10.8 ± 0.8 at 10 µ L, respectively) increased by about 70% compared to that of black tea, which was 16.26 ± 0.7 (2.6 ± 0.1 µ g TE mL −1 ) at 200 µ L, and 3.1 ± 0.1 (0.8 ± 0.1 µ g TE mL −1 ) at 10 µ L. At the highest concentration, a 13% decrease was observed during the first three months of storage with respect to the control value, while it reduced by 30% after four months. At the fifth month it dropped drastically to a value of 

Scavenging abilities of black tea, kombucha tea after one month of fermentation and all the samples during the storage were monitored using two common in vitro models, DPPH and ABTS assays.

Kombucha exhibited good antioxidant activity against DPPH radical during the storage at all tested concentrations when compared to black tea ( Figure 6 ). The results of the DPPH radical assay were also expressed as the Trolox equivalent antioxidant capacity (TEAC) using Trolox as reference standard. The TEAC values are reported in Table 2 . During fermentation, %I DPPH of kombucha tea at the highest and lowest concentrations (55.1 ± 1.3 at 200 µL, and 10.8 ± 0.8 at 10 µL, respectively) increased by about 70% compared to that of black tea, which was 16.26 ± 0.7 (2.6 ± 0.1 µg TE mL −1 ) at 200 µL, and 3.1 ± 0.1 (0.8 ± 0.1 µg TE mL −1 ) at 10 µL. At the highest concentration, a 13% decrease was observed during the first three months of storage with respect to the control value, while it reduced by 30% after four months. At the fifth month it dropped drastically to a value of 23.2 ± 6.1% (3.0 ± 0.7 µg TE mL −1 ), corresponding to a reduction of 58% of the control value (**** p < 0.0001). The scavenging ability of kombucha declined to 18.9 ± 0.4% (2.9 ± 0.1 µg TE mL −1 ) after nine months. 23.2 ± 6.1% (3.0 ± 0.7 µ g TE mL −1 ), corresponding to a reduction of 58% of the control value (**** p < 0.0001). The scavenging ability of kombucha declined to 18.9 ± 0.4% (2.9 ± 0.1 µ g TE mL −1 ) after nine months. 

The inhibition percentage of ABTS is reported in Figure 7 and the corresponding TEAC values against ABTS are reported in Table 3 . Kombucha tea samples showed lower inhibitory abilities on ABTS• + radical cation at the highest concentrations (100 and 200 µ L) than those against DPPH radical. After one month of fermentation %IABTS of tea Kombucha at the highest concentrations (200 and 100 µ L) increased by about 54% (47.4 ± 1.3, 9.1 ± 0.3 µ g TE mL −1 ) and 40.6% (26.6 ± 0.5, 6.1 ± 0.1 µ g TE mL −1 ) respectively, as compared to the black tea at the same concentrations, that is 21.8 ± 0.7 corresponding to 4.8 ± 0.1 µ g TE Figure 6 . %I DPPH of Trolox, black tea, kombucha tea after 30 days of fermentation and all the samples during storage. White bar refers to %I DPPH of kombucha tea after 30 days of fermentation (control), the others refer to kombucha samples over nine months of storage. Asterisks on the bars indicate that mean values were statistically different from the control (* p < 0.05, *** p < 0.001, **** p < 0.0001). 

The inhibition percentage of ABTS is reported in Figure 7 and the corresponding TEAC values against ABTS are reported in Table 3 . Kombucha tea samples showed lower inhibitory abilities on ABTS• + radical cation at the highest concentrations (100 and 200 µL) than those against DPPH radical. After one month of fermentation %I ABTS of tea Kombucha at the highest concentrations (200 and 100 µL) increased by about 54% (47.4 ± 1.3, 9.1 ± 0.3 µg TE mL −1 ) and 40.6% (26.6 ± 0.5, 6.1 ± 0.1 µg TE mL −1 ) respectively, as compared to the black tea at the same concentrations, that is 21.8 ± 0.7 corresponding to 4.8 ± 0.1 µg TE mL −1 at 200 µL and 15.8 ± 1.5 or 3.4 ± 0.2 µg TE mL −1 at 100 µL. After one month of storage, the abilities of the samples at 200 (42.3 ± 0.5, 9.1 ± 0.3 µg TE mL −1 ) and 100 µL (25.0 ± 0.8, 5.5 ± 0.1 µg TE mL −1 ) were lowered by 11% and 6%, respectively, compared to the control. The antioxidant capacity of the control at the highest concentration underwent further but progressive reduction up to 27% in the first three months of storage, but it was reduced by 73% (%I ABTS 12.7 ± 0.9, or 3.4 ± 0.2 µg TE mL −1 ) at the fifth month of storage and 85% (%I ABTS 7.0 ± 0.4, or 1.6 ± 0.1 µg TE mL −1 ) at the ninth month.

lecules 2021, 26, 5474 9 mL −1 at 200 µ L and 15.8 ± 1.5 or 3.4 ± 0.2 µ g TE mL −1 at 100 µ L. After one month of stor the abilities of the samples at 200 (42.3 ± 0.5, 9.1 ± 0.3 µ g TE mL −1 ) and 100 µ L (25.0 ± 5.5 ± 0.1 µ g TE mL −1 ) were lowered by 11% and 6%, respectively, compared to the cont The antioxidant capacity of the control at the highest concentration underwent further progressive reduction up to 27% in the first three months of storage, but it was redu by 73% (%IABTS 12.7 ± 0.9, or 3.4 ± 0.2 µ g TE mL −1 ) at the fifth month of storage and 8 (%IABTS 7.0 ± 0.4, or 1.6 ± 0.1 µ g TE mL −1 ) at the ninth month. 

The consumption of kombucha has increased over the last decades due to its perceived beneficial effects. For this reason, it has become a highly commercialized drink, industrially produced but is still also a homemade beverage. To evaluate the effects of long-term storage of kombucha on radical scavenging properties and its phenolic content, we kept the tea samples at refrigerator temperature in the dark. Accordingly, to the literature, the pH values of black tea in the kombucha decreased after 30 days of fermentation due to the metabolic activity of tea fungus yeasts and acetic acid bacteria that produce mainly acetic acid [15] .

In fact, the pH value of sweetened black tea was 5.59, and it dropped to 2.82 in the kombucha beverage obtained after 30 days. Then, the changes in the first five months of storage were less than 0.2 units, while at the sixth month, the pH value of sample significantly increased up to 3.24, and it remains constant at these values at less than 0.05 units for the last three months. This pH rise is most likely due to the subsequent use of acids by bacteria as a carbon source in the absence of sugar in the tea [16, 17] . The final pH of kombucha samples (3.16), after nine months of storage, is still in the safe pH range of 2.5 to 4.2 for human consumption [18, 19] .

Total phenolic and flavonoid contents were also monitored after fermentation and during storage. The results highlighted that kombucha after one month of fermentation showed the highest total phenolic content level, which was 1.7 times higher than the value of black tea, as a consequence of the action of microbial enzymes from bacteria and yeasts in an acidic environment, which hydrolyzes complex tea polyphenols into smaller molecular weight phenolic compounds causing an increase in polyphenol concentration [19] . Total phenolic content is reduced after four months of storage. A similar trend was seen for the total flavonoid content but earlier than the total phenolic content.

In contrast to Ning et al. [20] , HPLC analysis pointed out that chlorogenic acid maintained its content unchanged either during fermentation and over time compared to the initial value in black tea. On the other hand, all the other monitored phenolics dropped at the fifth month. This agrees with the literature in which the strategy to prolong phenolic content is studied [20] . Conversely, total flavonoid content was constantly lower in black tea, most likely due to the microbial activity of the SCOBY during fermentation.

The results of DPPH inhibition properties of kombucha tea directly depend on the tea constituents and the components produced during fermentation time (30 days). The decrease of antioxidant capacity during storage was most likely related to microbial transformation of the compounds responsible for the maximum scavenging ability into less potential scavenging structures. On the other hands, the inhibition percentage of ABTS assay showed lower inhibitory abilities in respect to ABTS• + radical action.

The antioxidant activities of our tested samples likely depend on the composition and the chemical nature of phenolic compounds [21, 22] . Then, during the storage, changes in the composition of antioxidant compounds of kombucha tea might result from the formation of certain compounds as in the case of quercetin in our results, thus leading to a lower antioxidant activity [22] .

It is important to mention here, that during the COVID-19 pandemic of 2019-2020 the consumption of fermented food, especially beverages, increased in several countries [17, 23, 24] . In particular, the consumption of industrial kefir and kombucha increased [15, 25] and the latter was reported, in the magazine Forbes, as the drink of 2020 [26] . Although, as source of bioactive components that could be beneficial for human health, there is no evidence about systematic human trials being done using kombucha tea [27] and some toxicity related to kombucha consumption has been reported so far when kept in a ceramic pot for six months or in lead-glazed earthenware at refrigerator temperature [28, 29] .

The kombucha starter (Figure 8 ) was obtained from Kefiralia (Burumart Commerce S.L, Arrasate, Spain) and was maintained in sugared black tea. Dimethylsulphoxide (DMSO), absolute ethanol and methanol, formic acid, and acetonitrile HPLC-grade were purchased from Carlo Erba (Milan, Italy), Folin-Ciocâlteu reagents, sodium carbonate, DPPH, ABTS, potassium persulfate (K 2 S 2 O 8 ), aluminium chloride (AlCl 3 ), potassium acetate, chloroform, and ethyl acetate were purchased from Sigma Aldrich (Milan, Italy). 

The kombucha starter (Figure 8 ) was obtained from Kefiralia (Burumart Commerce S.L, Arrasate, Spain) and was maintained in sugared black tea. Dimethylsulphoxide (DMSO), absolute ethanol and methanol, formic acid, and acetonitrile HPLC-grade were purchased from Carlo Erba (Milan, Italy), Folin-Ciocâlteu reagents, sodium carbonate, DPPH, ABTS, potassium persulfate (K2S2O8), aluminium chloride (AlCl3), potassium acetate, chloroform, and ethyl acetate were purchased from Sigma Aldrich (Milan, Italy). 

Black tea (3 g) was immersed into 1 L of boiling water and infused for about 15 min. Then it was filtered through a sterile sieve. This was repeated for three times and 1 L of each preparation was kept into sterilized glass jars. Commercial sucrose (7%) was then added to the hot drink and, after cooling to room temperature, the infusion was inoculated with a commercial kombucha SCOBY (150 g) size (15 × 2 × 10 cm). The jars were covered with a clean cloth. The fermentation was carried out in the dark at 25 ± 2 °C for 30 days and, at the end of this time, the kombucha tea samples were filtered through a cheesecloth and transferred to three amber jars.

The jars containing kombucha tea were placed in a refrigerator (T = 4 °C) for nine months. Sampling was performed every 30 days by taking an aliquot (100 mL) which was analyzed. The tea fermented for one month was used as the control for the kombucha tea samples stored at 4 °C for longer times-up to nine months. pH values, content of total polyphenol compounds, qualitative and quantitative profile of the main tea polyphenols, content of total flavonoids, and free radical scavenging activities of each sample were determined.

The pH values of all the kombucha tea samples were measured using an electronic pH meter (Hanna Instruments, George Washington Hwy, Smithfield, RI, USA) calibrated at pH 4.0 and 7.0.

The total polyphenolic content (TPC) compounds in the tea samples were quantified by the Folin-Ciocâlteu colorimetric method as previously described [30], with appropriate 

Black tea (3 g) was immersed into 1 L of boiling water and infused for about 15 min. Then it was filtered through a sterile sieve. This was repeated for three times and 1 L of each preparation was kept into sterilized glass jars. Commercial sucrose (7%) was then added to the hot drink and, after cooling to room temperature, the infusion was inoculated with a commercial kombucha SCOBY (150 g) size (15 × 2 × 10 cm). The jars were covered with a clean cloth. The fermentation was carried out in the dark at 25 ± 2 • C for 30 days and, at the end of this time, the kombucha tea samples were filtered through a cheesecloth and transferred to three amber jars.

The jars containing kombucha tea were placed in a refrigerator (T = 4 • C) for nine months. Sampling was performed every 30 days by taking an aliquot (100 mL) which was analyzed. The tea fermented for one month was used as the control for the kombucha tea samples stored at 4 • C for longer times-up to nine months. pH values, content of total polyphenol compounds, qualitative and quantitative profile of the main tea polyphenols, content of total flavonoids, and free radical scavenging activities of each sample were determined.

The pH values of all the kombucha tea samples were measured using an electronic pH meter (Hanna Instruments, George Washington Hwy, Smithfield, RI, USA) calibrated at pH 4.0 and 7.0.

The total polyphenolic content (TPC) compounds in the tea samples were quantified by the Folin-Ciocâlteu colorimetric method as previously described [30] , with appropriate modifications. The fermented tea sample (0.1 mL) was transferred in an amber glass vial and was added by 2 mL of distilled water, 0.5 mL of the Folin-Ciocâlteu reagent (diluted 1:10 with distilled water), and 0.4 mL of a 7.5% sodium carbonate solution (Na 2 CO 3 ), up to a final volume of 3 mL. The mixture was shaken under constant magnetic stirring for 30 min, at room temperature in the dark. The absorbance was measured at 760 nm using a spectrophotometer Jasco UV-550. Three analyses were carried out for each sample. Gallic acid was used as the standard in order to plot the calibration curve. For the linearity study, an eight-point calibration curve was constructed using different concentrations of gallic acid stock solutions (range 0.5-0.01 mg mL −1 ). A linear correlation was found between absorbance of the blue complex at 760 nm and concentration of gallic acid in the range 0.5-0.01 mg mL −1 (y = 3.6607x − 0.0036). The coefficient (R 2 ) obtained from the linear regression was 0.9998, indicating an excellent linear correlation between the data. The total phenolic content (TPC) was expressed as µg equivalents of gallic acid (µg GAE) per mL of kombucha.

Five compounds in kombucha tea samples were identified and quantified by reversedphase high performance liquid chromatography coupled with diode array detector (HPLC-DAD) [31] . The samples were filtered through a membrane filter (0.45 µm) into HPLC vials and analyzed as such. An aliquot (10 µL) of each sample was injected into a Shimadzu (Kyoto, Japan) HPLC system equipped with a diode array detector (SPD-M10Avp). The chromatographic separation was performed on a Mediterranea SEA C-18 column (4.6 mm i.d. × 25 cm, 5 µm). The mobile phase was a 0.1% formic acid (A) and acetonitrile (B) mixture. The gradient used was the following: 0 min, 10% B; 20 min, 22% B; 40 min, 40% B; 45 min, 10% B, 51 min, 10% B. The flow rate and column temperature were maintained as 0.6 mL min −1 and at room temperature, respectively. Detection was made at the absorption maxima of the pure standard compounds: caffeine was detected at 273 nm, EGCG at 280 nm, ferulic acid at 325 nm, chlorogenic acid at 327 nm, and quercetin at 365 nm, and identification was made by comparison of the retention times and characteristic UV-Vis spectra of pure standard compounds used as references. Individual components were analyzed quantitatively by the external standard method. The calibration curves for standards (caffeine, EGCG, ferulic acid, chlorogenic acid, and quercetin) were prepared with six appropriate concentrations. The limit of detection (LOD) and the limit of quantification (LOQ) for each standard were calculated as follows: LOD = 3(S y /S) and LOQ = 10(S y /S), where S y is the standard deviation of the response of the curve and S is the slope of the calibration curve.

Total flavonoid content (TFC) was determined by a colorimetric method as described previously [32] . Briefly, 0.30 mL of the sample solution were diluted with 1.68 mL of distilled water. Then, 0.9 mL of MeOH, 0.06 mL of a 10% AlCl 3 solution, and 0.06 mL of 1 M solution of potassium acetate were added to the solution. The mixture was allowed to stand for 30 min at room temperature, under constant magnetic stirring, in the dark, and then the absorbance was measured against the blank at 420 nm using a spectrophotometer Jasco UV-550. Three analyses were carried out for each sample. Quercetin was used as the standard in order to plot an eight-point calibration curve. The linearity range of calibration curve was 10-0.001 µg mL −1 (y = 0.084x − 0.0019). The coefficient (R 2 ) obtained from the linear regression was 0.9984, indicating a good linear correlation between the data. The results were expressed as µg of quercetin equivalents (µg QE) per mL of kombucha [31] .

Two different in vitro assays, DPPH and ABTS, were used to evaluate the changes over time in free-radical scavenging abilities of all kombucha tea samples.

The scavenging activity on DPPH radical was determined by the colorimetric method previously described [25] with slight modification. To different volumes of each sample (10, 50, 100, 200 µL), 0.1 mL of DPPH solution (1 mM) and 2.8 mL of MeOH were added. After an incubation time of 30 min, under magnetic stirring, at room temperature and in the dark, the reduction of DPPH free radical was measured by reading the absorbance at 517 nm using a spectrophotometer Jasco UV-550. The experiments were carried out against a blank (3 mL of MeOH) and a control (2.9 mL of MeOH, 0.1 mL DPPH solution). Each sample was tested in triplicate. The antioxidant activity was given as a percentage of free radical inhibition (%I DPPH ), according to the formula: %I DPPH = [(absorbance of the control − absorbance of the sample)/absorbance of the control)] × 100.

The results were expressed also as µg of Trolox equivalents per mL of tea samples (µg TE mL −1 ).

ABTS• + radical cation is well soluble in both aqueous and organic solvents, so this method can be extensively used to determine antioxidant activity for both hydrophilic and lipophilic compounds [33] . This radical cation was formed by a reaction between 7 mM ABTS solution and 2.45 mM potassium persulfate (K 2 S 2 O 8 ), and then allowing the mixture to stand for 16 h in darkness at room temperature. It remains stable for the following 48 h, and is characterized by an intense green/blue color. ABTS• + solution was diluted in methanol until the absorbance reached the value of 0.70 ± 0.02 at 734 nm. Different volumes of each tea broth sample (10, 50, 100, 200 µL) were mixed with ABTS solution (3 mL), and the absorbance was recorded at 734 nm after 10 min of incubation at room temperature in the dark, against a blank and a control. Each sample was tested in triplicate. The radical scavenging activity was given as percentage of ABTS• + radical inhibition (%I ABTS ), according to the following formula: %I ABTS = [(absorbance of the control − absorbance of the sample)/absorbance of the control)] × 100.

The results were expressed as µg of Trolox equivalents per mL of tea kombucha samples (µg TE mL −1 ).

Each tea broth from three different preparations, was analyzed in triplicate and, the results were expressed as mean ± standard deviation (SD). One-way ANOVA method and a Holm-Sidak comparison method via GraphPad Prism 8 were used. The significance was established at p values < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).

The polyphenol content of kombucha during long-term storage decreases significantly from the fifth month on and becomes one-third of the initial value after nine months. Therefore, a period of up to four months ensures the preservation of polyphenols of kombucha tea and their antioxidant activities. The results of this pilot study highlighted that the "shelf life" of kombucha stored at refrigerator temperature could be no longer than four months, as only during this period the preservation of polyphenol content and its antioxidant activities are ensured.

Author Contributions: Methodology and writing-original draft preparation, C.L.T.; coordinating the work and writing-review and editing, A.F., E.C.; software, P.C.; validation, P.P., R.C. and M.C.C.; conceptualization and supervision, E.C. All authors have read and agreed to the published version of the manuscript. 

The data presented in this study are available on request from the corresponding author.

The authors declare no conflict of interest.

Sample Availability: Samples of the compounds are available from the authors. 

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