Food Chemistry 412 (2023) 135556

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Arabic coffee infusion based kombucha: Characterization and biological

activity during fermentation, and in vivo toxicity
Jeniffer Ferreira de Miranda a, Giulia Martins Pereira Belo a, Laís Silva de Lima a,
Kelly Alencar Silva a, Thais Matsue Uekane a, Alice Gonçalves Martins Gonzalez a,
Vanessa Naciuk Castelo Branco a, Nayla Souza Pitangui b, Fabrício Freitas Fernandes c, *,
Adriene Ribeiro Lima a, *
a
Department of Bromatology, Pharmacy School, Fluminense Federal University, Niterói, RJ, Brazil
b
Department of Cell and Molecular Biology and Pathogenic Bioagents, Ribeirão Preto Medical School, University of São Paulo, SP, Brazil
c
Federal Institute of Education, Science and Technology of Mato Grosso (IFMT), Juína Campus, Linha J, s/n, Setor de Chácaras, CEP: 78320-000, Juína, MT, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: In this study, arabic coffee infusion was used to produce a fermented beverage known as kombucha. The
Fermented beverage physicochemical, antioxidant and antimicrobial activities, as well as in vivo toxicity were evaluate throughout 21
Coffea arabica L. days of fermentation. Reduction in pH and sugar levels were observed throughout the fermentation period. There
Antioxidant activity
was no significant difference in the content of total phenolic compounds between the unfermented and fer­
Antimicrobial effect
mented beverage, nor between the fermentation times, as well as in the antioxidant activity. The 5-caffeoylquinic
Galleria mellonella
acid was identified at all fermentation times evaluated, and no significant difference was observed regarding its
concentration. It showed antibacterial and antifungal activity against all strains tested. No toxic effect of the
beverages was observed in the in vivo model (Galleria mellonella) studied. These results demonstrated that coffee
infusion is a possible alternative for kombucha production since the physicochemical changes prove the meta­
bolic activity of Symbiotic Culture of Bacteria and Yeast.

1. Introduction contaminating microorganisms, which makes this beverage safe from a

The search for a healthier diet and, consequently, for foods that
contribute to the promotion of health has increased significantly. This
change in the dietary profile of the population has encouraged research
on foods and beverages related to reducing health risks, allowing the
development and creation of new food products (Santos-Buelga et al.,
2019).
A product that confers beneficial properties to health, and that has
been gaining great prominence in the world, and is currently becoming
popular in Brazil, is kombucha. Kombucha is a beverage traditionally
obtained by fermenting C. sinensis tea, added sugar, by a symbiotic
culture of bacteria and yeast (Symbiotic Culture of Bacteria and Yeast-
SCOBY), also known as “tea fungus” (Dutta & Paul, 2019).
The microbiota present in kombucha establish a strong symbiotic
relationship capable of inhibiting the growth of potentially
microbiological point of view (Jayabalan et al., 2014). In addition,
several therapeutic and health-promoting properties have been inves­
tigated in kombucha over time, such as antioxidant agent, treatment and
prevention of diabetes, reduction of cancer spread, improvement of liver
function, immune system and gastrointestinal functions, detoxifying
action, anti-inflammatory properties, among others (De Miranda et al.,
2022). Its supposed health benefits are attributed to substances with
bioactive properties, especially phenolic compounds, the main antioxi­
dant elements present in the beverage (Cardoso et al., 2020), which are
in the substrate used for fermentation and/or produced as a result of
microbial fermentation (Bhattacharya et al., 2016).
As reported in the literature, a variety of new substrates have been
used as alternatives to C. sinensis tea to obtain new kombucha beverages
with different sensory, chemical and biological characteristics.
Fermentation of kombucha culture on alternative substrates has already

* Corresponding authors at: Department of Bromatology, Pharmacy School, Fluminense Federal University, Rua Dr. Mario Vianna, 523, Santa Rosa, CEP 24241-
000, Niterói, RJ, Brazil (A. R. Lima). Federal Institute of Education, Science and Technology of Mato Grosso (IFMT), Juína Campus, Linha J, s/n, Setor de Chácaras,
CEP: 78320-000, Juína, MT, Brazil (F. F. Fernandes).
E-mail address: adrienelima@id.uff.br (A. Ribeiro Lima).

https://doi.org/10.1016/j.foodchem.2023.135556
Received 1 October 2022; Received in revised form 17 January 2023; Accepted 21 January 2023
Available online 24 January 2023
0308-8146/© 2023 Elsevier Ltd. All rights reserved.
J. Ferreira de Miranda et al.
been related, such as herbal infusions, flowers, fruit juices, cow’s milk,
soybean, marshmallow, a non-conventional food plant (PANC) (Emil­
janowicz & Malinowska-Pańczyk, 2019; Silva et al., 2021) among
others, which showed satisfactory results regarding its kinetics and
biological properties.
In this context, coffee is a good option for diversification of substrates
for kombucha production, because besides being one of the most
consumed beverages in the world, it represents a source of bioactive
compounds. Different constituents of coffee have been suggested as
potentially chemoprotective in different chemical and biological sys­
tems, due to bioactive compounds present in the beverage, such as
caffeine, chlorogenic acids, and products resulting from the Maillard
reaction originating in the roasting of the bean, such as melanoidins
(Ludwig et al., 2014).
Knowing that microbial fermentation results in the enhancement of
biological effects in beverages containing polyphenols, and that there is
very little scientific information about the use of coffee as a possible
substrate for obtaining kombucha, this study aimed to produce kom­
bucha beverages using coffee infusion and evaluate the physicochemical
profile, antioxidant and antimicrobial activity, as well as possible toxic
effects in vivo.
2. Materials and methods
2.1. Preparation of fermented coffee beverage
The 100% Arabic coffee (Coffea arabica L.), from the Southern region
of Minas Gerais (Brazil), medium roast and fine grind, crystal sugar and
mineral water were purchased in the local market in Niterói, RJ (Brazil).
Coffee concentration used for infusion preparation was defined
based on previous tests with 10, 5 and 2% of coffee (w/v), this was
submitted to 14 days of fermentation with kombucha culture. Only the
infusion fermented with 2% of coffee showed a pH within the estab­
lished range for kombucha (pH ≥ 2.5 to pH ≤ 4.2) (De Miranda et al.,
2022).
Three liters of coffee infusion in the ratio of 2% (w/v) coffee and 5%
(w/v) sucrose (Jayabalan et al., 2014) were prepared in triplicate. The
infusion was stirred for 5 min with a glass rod, and then filtered through
Whatman #1 filter paper. This beverage was used as a base for preparing
the fermented beverages and aliquots were frozen at − 20 ◦ C to be used
as a control and thus establish a comparison with the fermented
beverages.
The coffee infusion intended for the preparation of the fermented
beverage was cooled to approximately 25 ◦ C and distributed in 600 mL
glass containers, one for each day of fermentation to be monitored.
Symbiotic kombucha culture (5%, w/v) and 10% (v/v) of the previous
fermented were added (Jayabalan et al., 2014; De Miranda et al., 2022).
The glass containers were covered with paper towels to protect the
drinks from insects and still allow oxygen to enter. The beverages were
incubated at 25 ± 3 ◦ C (Cardoso et al., 2020) and evaluated at
fermentation times of 0, 6, 12, 15, and 21 days.
The resulting beverages were filtered on Whatman #1 paper filter to
remove cellulose residue. For the analyses of bioactive compounds,
antioxidant and antimicrobial activity, the samples were centrifuged at
7000 rpm for 15 min, and the supernatant was sterilized by filtration
through a 0.22 µm filter (Millipore®). Subsequently all samples were
frozen (- 20 ◦ C) in closed containers for analysis. The samples were
coded as CI for the unfermented coffee infusion and as CIK for the coffee
infusion kombucha with their respective fermentation times (CIK0, CIK6,
CIK12, CIK15 and CIK21).
2.2. Physical-chemical characterization
2.2.1. pH and total acidity
The pH was determined by direct reading in a potentiometer (Mult-
007, Ion®, Brazil) periodically calibrated with pH 4.0 and 7.0 buffer
2
Food Chemistry 412 (2023) 135556
solutions at 25 ◦ C, according to AOAC (1992).
The titratable acidity of the fermented beverages, expressed as a
percentage of acetic acid, was determined by diluting 5 mL of homog­
enized sample in 50 mL of distilled water with the addition of 2 drops of
phenolphthalein solution. Afterwards titration was performed with a
0.1 N NaOH solution. The potentiometer (Mult-007, Ion®, Brazil) was
used during the titration for better accuracy of the phenolphthalein
turning point at pH 8.2 to 8.4 (IAL, 2008).
2.2.2. Total soluble solids and determination of reducing sugars
Soluble solids were monitored by reading in a digital refractometer
(Nova Instruments Equipment Laboratories Ltda, Brazil), with a scale of
0 to 45◦ Brix, calibrated with distilled water.
The determination of reducing sugars was performed by the 3,5-dini­
trosalicylic acid (DNS) method according to Miller (1959), with modi­
fications. Initially acid hydrolysis of the samples was performed since
they were prepared with sucrose, a non-reducing sugar. One milliliter of
each sample was dispensed into test tubes, then 1 mL of 2 N HCl was
added and subjected to a water bath at 65 ◦ C for 10 min. After the time,
3 mL of 1 N NaOH was added to each tube in order to stop the hydrolysis.
After homogenization, 1 mL of the hydrolysate was transferred to a 25
mL volumetric flask, where it was diluted by making up the volume with
distilled water. After preparing the samples, the DNS test was per­
formed. One milliliter of the diluted samples was transferred to Folin Wu
tubes, where 1 mL of the DNS reagent was added. Subsequently, the
tubes were homogenized and submitted to a water bath at 100 ◦ C for 5
min. After cooling the tubes under running water, the absorbance was
read in a spectrophotometer (Vis Spectro 560, Germany) at 540 nm. The
concentration of reducing sugars present in the samples was calculated
based on the straight line equation generated from the glucose (0.9 g/L)
calibration curve.
2.2.3. Colorimetry
The colorimetric analyses of the samples were performed by reflec­
tance using a spectrophotometer (Colorview 9000 Byk-Gardner,
Columbia, MD) with QC Manager Software. A CIE Illuminant D65 was
used in all the colour measurements. The colour measured was
expressed by the CIE L*, a*, b* (CIELAB) colour space values (Silva et al.,
2021).
2.3. Bioactive compound analysis
2.3.1. Total phenolics determination
The concentration of total phenolic compounds was determined by
means of the Folin-Ciocalteu (FC) colorimetric assay using gallic acid as
standard, as described by Silva et al. (2021). The total content of
phenolic compounds was expressed as µg gallic acid equivalent per mL
of sample (µg GAE /mL) and calculated based on the straight-line
equation generated from the calibration curve at concentrations 10,
20, 30, 40, 60, and 80 µg GAE/mL.
2.3.2. Determination of 5-caffeoylquinic acid and caffeic acid
The identification and quantification of 5-caffeoylquinic acid (5-
CQA) and caffeic acid in the beverages were performed by the high-
performance liquid chromatography (HPLC) method. Initially, the
samples were freeze-dried and subjected to a liquid–liquid extraction
process. A volume of 24 mL of methanol and 36 mL of ultrapure water
were added to each sample and shaken at room temperature in a bench-
top shaker incubator (Lucadema®, Brazil) for 30 min at 200 rpm for
resuspension of the samples. Afterwards, the solution was transferred to
a 100 mL volumetric flask by filtration through a Whatman #1 paper
filter. One milliliter of each solution, carrez 1 and carrez 2, were added
to the contents of the flask for precipitation of proteins and other high
molecular weight molecules, the volume was made up to mark with
ultrapure water. The solution was stirred for 5 s and allowed to stand for
10 min for colloidal precipitation. After the procedures, the samples
J. Ferreira de Miranda et al.
were filtered on Whatman #1 paper filter, and the resulting solution was
submitted to the chromatographic method (Farah et al., 2005).
The analysis was performed in an HPLC equipment (Shimadzu®,
Japan) composed of a LC-20AT quaternary pump, SPD-M20A diode
array detector (DAD), CBM-20A control system, DGU-20A5 degasser
and SIL-20AC automatic injector. Chromatographic separation of the
compounds was achieved using a C18 reversed-phase column (Shim-
pack VP-ODS, 5 µm, 250 mm × 4.6 mm, Shimadzu®) and a mobile phase
containing 80% of 10 mM citric acid solution, pH adjusted to 2.5 with 2
N hydrochloric acid, and 20% of methanol (Eluent A), and methanol
(Eluent B), with flow rate of 1 mL/min, and run time of 39 min (Farah
et al., 2005). The 5-CQA and caffeic acid were monitored at 330 nm. The
chromatographic data were integrated in the LC Solution software
(Shimadzu®).
Commercial standards of 5-CQA (Sigma®, 95.0% purity) and caffeic
acid (Sigma®, 98.0% purity) were used for the identification and
quantification of chromatographic peaks by comparison with retention
time and by external standardization, respectively. The concentration of
5-CQA and caffeic acid was calculated based on the straight-line equa­
tion generated from the calibration curve at the concentrations 5, 10, 25,
50, 75, and 100 mg/L.
2.4. Antioxidant activity
The antioxidant activity was evaluated by the 2,2-diphenyl-1-picryl­
hydrazyl (DPPH) radical scavenging activity (%DPPH) and by the
maximum effective concentration (EC50), according to Silva et al.
(2021).
2.5. Antimicrobial activity
2.5.1. Microbial strains and cultivation
Bacterial strains of food importance, Listeria monocytogenes
CLIST2033 (CDCF6254), enteropathogenic Escherichia coli (EPEC)
INCQS 00185 (CDC 0127a), obtained from the Collection of Reference
Microorganisms in Health Surveillance (CMRVS) of the National Insti­
tute for Quality Control in Health (INCQS) of FIOCRUZ, Staphylococcus
aureus (ATCC 13565) and enterohemorrhagic E. coli (EHEC) O157:H7
(RJ 691/1) were used to evaluate antibacterial activity of unfermented
and fermented coffee infusion. The strains frozen at − 20 ◦ C were reac­
tivated on Soy Tryptone Agar (TSA) plates (Kasvi®, Brazil), and incu­
bated at 35 ◦ C for 24 h.
The fungi tested are etiological agents of high-incidence mycoses
(Singulani et al., 2019), and were kindly provided by the Immuno­
chemistry and Glycobiology Laboratory of the Medicine School of São
Paulo University (Ribeirão Preto, Brazil). Candida albicans (ATCC
90028), C. neoformans (H99) and Cryptococcus gattii (R265) were
maintained by cultivation on Sabouraud agar medium (Kasvi®, Brazil)
for 7 days at 30 ◦ C.
2.5.2. Antibacterial and antifungal activity
Antibacterial activity was evaluated using a broth microdilution
method, where the minimum inhibitory concentration (MIC) and the
minimum bactericidal concentration (MBC), the lowest concentration of
a substance capable of killing a particular strain of bacteria, were
determined. The analyses were performed in triplicate in 96-well
microplates (CLSI, 2012).
Isolated colonies were transferred from the TSA plates to tubes
containing tryptone soy broth (TSB) (Kasvi®, Brazil), and incubated
under stirring at 35 ◦ C for 24 ± 2 h. The inoculum was standardized
until it reached a turbidity equivalent to 0.5 on the McFarland scale (108
CFU/mL). A dilution to 105 CFU/mL was then prepared, so that the final
concentration of bacteria in each well was approximately 104 CFU/mL.
Serial dilutions were prepared by adding 100 µL of a sample in 100
µL of TSB broth. The concentrations of the samples tested ranged from
500 to 1 µL/mL. Subsequently, 100 µL of the standardized bacterial
3
Food Chemistry 412 (2023) 135556
culture was inoculated into all wells except the negative control wells
(TSB broth). The final volume of each well was 200 μL, and after
micropipetting, the microplates were capped and incubated at 35 ◦ C for
24 ± 2 h.
Once the incubation period was over, all wells were visually
observed to check for microbial growth (CLSI, 2012; Cardoso et al.,
2020). Turbidity or the presence of precipitate were considered to be
indicative of growth. MBC was determined from wells that did not show
turbidity, indicative of bacterial inhibition. An aliquot of the contents of
the wells, which showed no growth, was plated with TSA and incubated
at 35 ◦ C for 24 ± 2 h in order to determine the presence or absence of
microbial colonies. MBC was considered as the lowest concentration at
which there was no growth of colonies in TSA. All tests were performed
with the controls Chloramphenicol (CLO) at concentrations ranging
from 100 to 0.2 µg/mL, TSB broth, and TSB broth with added bacterial
inoculum.
Antifungal activity was also evaluated by the microdilution method
in 96-well microplates, as stated by the document M27-A3 (CLSI, 2017),
with determination of MIC and minimum fungicidal concentration
(MFC), according to the procedure described above.
The antifungal activity was verified in Sabouraud medium. A sus­
pension of 108 cells/mL, of each fungal strain was diluted to 5 × 106
cells/mL distributed into the wells. The microplates were incubated at
37 ◦ C for 24 h. The MIC was determined by reading the optical density
(OD) at 600 nm. The determination of MFC was performed qualitatively,
by checking the presence or absence of fungal growth after treatment
with the samples in solid Sabouraud medium incubated at 37 ◦ C for 3
days. Amphotericin (AmB), Sabouraud medium and untreated fungal
growth were used as controls. The AmB concentrations used as controls
in the experiment were 8 to 0.015625 (µg/mL).
2.6. In vivo toxicity
To evaluate the in vivo toxicity effect, the Galleria mellonella model
was employed (Megaw et al., 2015). The larvae with an average weight
of 150 mg were divided into 5 groups with a total of 10 larvae, namely: a
control group treated with 10 mM phosphate buffered saline, pH 7.2
(PBS); a group treated with coffee infusion (CI); a group treated with
coffee infusion and SCOBY inoculum without fermentation (CIK0); and
two groups treated with coffee-infused kombucha for 15 and 21 days
(CIK15 and CIK21). Prior to application of the samples, asepsis of the
larvae was performed with swab and 70% ethanol, then 10 µL of each
sample was inoculated into the hind paw using an insulin syringe.
Subsequently, the larvae were incubated at 37 ◦ C and observed daily for
10 days to verify changes in pigmentation, locomotion, and larval death
for the elaboration of the survival curve. The assay was performed in
replicates of independent experiments.
2.7. Statistical analysis
Independent triplicates of non-fermented and fermented beverages
were prepared, and all analyzes were performed in triplicate. Results
were expressed as means ± standard deviations (SD). The means of the
sample groups for analyzed parameters were compared using a one-way
analysis of variance (ANOVA) and of Tukey’s multiple comparison. The
correlation between pH and titratable acidity, total phenolic content and
the antioxidant activity for the DPPH radical (%) were performed by
Pearson’s Correlation. The analyses were performed using GraphPad
Prim® version 9 software, and the significance level was set at 5%.
3. Results and discussion
3.1. Physical-chemical characterization
The graphical representation below (Fig. 1) shows the variations of
pH parameters, titratable acidity in acetic acid, soluble solids (◦ Brix) and
J. Ferreira de Miranda et al.
Fig. 1. pH variation, total soluble solids (◦ Brix), titratable acidity (TA) in acetic acid (A) and reducing sugars of coffee infusion kombucha (CIK) over the 21-day
fermentation period (B).
reducing sugars of CIK over the fermentation time. According to the data
obtained, a reduction in pH values and a gradual increase acidity are
observed. At the end of 21 days of fermentation, the pH ranged from
4.52 (CIK0) to 3.25 (CIK21), reducing rapidly in the first 6 days followed
by a slight decrease until the end of fermentation, and showed an acetic
acid content equivalent to 0.72% (7.2 g/L), with a significant difference
between CI and CIK at all times evaluated. But from day 6 of fermen­
tation there was no difference in pH between CIK (there was no differ­
ence between CIK6, CIK12, CIK15 and CIK21). The increase in organic acid
production is related to the decrease in pH of the fermented beverage
(Fig. 1A), indicated by a strong negative correlation between these two
factors. These results can be explained by the production of organic
acids by the microbial consortium, which metabolizes sucrose producing
various acids, mainly acetic and glycuronic acid, contributing to the
increased acidity of the medium (Jayabalan et al., 2014), thus being a
characteristic of SCOBY-fermented beverages (Jayabalan, Marimuthu &
Swaminathan, 2007).
These results are in agreement with the results of other studies pre­
sent in the literature (Cardoso et al., 2020; Silva et al., 2021; Zou et al.,
2021), as well as with the study of Watawana, Jayawardena, and Wai­
sundara (2015), who also used coffee as a substrate for kombucha
4
Food Chemistry 412 (2023) 135556
production. According to this study, the coffee sample had an initial pH
value of 5.0, which decreased to 4.1 after 7 days of fermentation.
According to the regulations establishing quality parameters for
kombucha (De Miranda et al., 2022), the beverage can be considered
safe for consumption, from a microbiological point of view, when the pH
value corresponds to the range pH ≥ 2.5 to pH ≤ 4.2. In this sense, when
making a comparison with our results, CIK has a pH parameter that
indicates it is a safe beverage from day 6 of fermentation on. The pH is
the most important parameter in the fermentation of this beverage,
because the organic acids formed are attributed as the main responsible
for the functional activities, in particular the antimicrobial activity
(Villarreal et al., 2018).
Soluble solids present in sugary drinks are represented mainly by
sugars and organic acids and measured in ◦ Brix. Given the results, a
reduction in soluble solids was observed (Fig. 1A), ranging from 5.17
(CIK0) to 3.96 ◦ Brix (CIK21) after 21 days of fermentation. Similarly, a
gradual reduction in reducing sugars was also seen throughout the
fermentative process (Fig. 1B). The CIK0 had 51.4 g/L of reducing
sugars, and after 21 days of fermentation it had 39.2 g/L (CIK21), a 24%
reduction; this difference was significant. Since sugar is the main carbon
source used by SCOBY, this reduction may be associated with the
J. Ferreira de Miranda et al. Food Chemistry 412 (2023) 135556

consumption of this compound during the fermentation process. During content between CI and CIK after 21 days, nor between the fermentation
fermentation, yeasts hydrolyze sucrose into glucose and fructose, times, as well as in the DPPH radical scavenging activity (Table 2). There
metabolizing them into ethanol. Acetic bacteria, in turn, use ethanol to was no significant correlation between total phenolic compound content
produce acetic acid, and glucose to produce glycuronic acid and bacte­ and DPPH scavenging ability (%) of all beverages was non-significant.
rial cellulose (Gomes et al., 2018). The half maximum effective concentration (EC50) was also calculated
Table 1 presents the results of the color determination analysis. The and determined in the range of 121.97 ± 25.37 to 142.19 ± 16.73 mL/L,
color was expressed in parameters of the scale developed by the Com­ however, no significant difference was found between the beverages
ission Internationale de l’Éclairage (CIE) L*a*b*. The L* coordinate rep­ evaluated (Table 2).
resents how light or dark the sample is (luminosity), with values Coffee is especially characterized by the presence of important
between 0 (totally black) and 100 (totally white). The a* coordinate can contents of caffeine, diterpenes, trigonelline, chlorogenic acids (CGA),
assume values between − 80 and + 100, whose extremes correspond to and melanoidins. Chlorogenic acids represent the main bioactive con­
green and red, respectively. The b* coordinate can vary from − 50 to + stituents responsible for the antioxidant properties of coffee, with 5-CQA
70, with intensity from blue to yellow. The use of polar coordinates as the main class of CGA (Ludwig et al., 2014). The 5-CQA was identified
allows for a more adequate interpretation of color variations: C* or in all analyzed samples according to the retention time of the standard
chromaticity, which provides a measure of color intensity or saturation, (13 min), however, caffeic acid was not identified in any of the analyzed
and hue angle (h◦ ), which corresponds to tone (Ferreira & Spricigo, samples, perhaps because its concentrations were below the detection
2017). limit of the method. Table 2 presents the results of the 5-CQA concen­
In CIK, a significant increase in the a* coordinate was observed trations of the beverages, also with no significant differences observed.
regarding with CI, ranging from − 0.1 (CI) to 1.22 (CIK21), this was These results differ from the data in the literature. In general, ac­
statistically significant. The b* coordinate showed oscillation cording to studies already conducted evaluating the antioxidant poten­
throughout the fermentation period, ranging from 8.06 to 7.06, however tial of beverages fermented with SCOBY, both with C. sinensis and other
there was no significant difference between CI and CIK21. The highest alternative substrates, the fermentative process may be able to enhance
degree of saturation (C*) was observed on the 6th day of fermentation, this activity, mainly due to the increased content of phenolic com­
however after 21 days the saturation decreased to 7.17 being lower in pounds. Several studies point out that the complexed forms of phenolic
the CIK21 than in the CI, this difference was not significant. Regarding compounds can be broken down into more simplified forms or smaller
Luminosity (L*), there was no significant difference between CI and CIK molecules due to the acidic environment and by the action of enzymes
during fermentation. The hue (h◦ ) also decreased throughout fermen­ released by bacteria and yeasts in the symbiotic kombucha culture,
tation, reaching the lowest value (80.08) on day 21, showing a signifi­ increasing their total content (Bhattacharya et al., 2016; Gamboa-
cant difference between CI and CIK21 (Table 1). According to Watawana, Gómez et al., 2016; Jayabalan et al., 2008; Silva et al., 2021).
Jayawardena, and Waisundara (2015), the variation in the colors of According to a study by Jayabalan, Marimuthu, and Swaminathan
kombucha drinks may be related to the biotransformation processes of (2007), the catechin isomers (epicatechin, epicatechin-3-gallate, epi­
phenolic compounds, in which the process of polymerization (joining of gallocatechin, and epigallocatechin-3-gallate), the main phenolic com­
molecules) or depolymerization (degradation) of these compounds may pound present in tea, showed variable concentrations during
occur. This process can increase or decrease the content of phenolic fermentation of kombuchas prepared from green tea, black tea, and
compounds and end up modifying the color. waste tea brewing. Degradation of the isomers and a marked increase of
these compounds were observed on the 12th day of fermentation. A fact
3.2. Bioactive compounds and antioxidant activity also verified by Vitas et al. (2018), who, when developing a new variety
of kombucha from the flowers of Achillea millefolium (milfoil), found that
Phenolic compounds are side products of plant metabolism (Liu, all the fermented beverages produced had higher levels of phenolic
2013), and have high antioxidant capacity, as they are able to neutralize compounds compared to the unfermented substrates. Silva et al. (2021)
free radicals and reactive oxygen species (Jayabalan et al., 2008). In produced kombucha from the infusion of wax mallow flowers (Malva­
kombucha, antioxidant activity is associated with the presence of these viscus arboreus) flowers and observed a 145% increase in total phenolic
compounds in the substrate used for fermentation (Malbaša et al., 2011). content after 14 days of fermentation. According to these authors, these
The results regarding the concentration of total phenolics, as well as the results can be explained by the biotransformation/degradation process
antioxidant activity against DPPH● radical neutralization, and 5-CQA of these compounds by kombucha activity (low pH and enzyme pro­
concentration of CI and CIK are presented in Table 2. duction by bacteria and yeasts present in the beverage). In these studies,
There was not any significant difference regarding the total phenolic the increase in phenolic compound content was correlated with
increased antioxidant activity.
Watawana, Jayawardena and Waisundara (2015) used three types of
Table 1
Color analysis of coffee infusion (CI) and coffee infusion kombucha (CIK) on
coffee beverage (fine-ground, coarse-ground and instant coffee) with a
days 0 (CIK0), 6 (CIK6), 12 (CIK12), 15 (CIK15) and 21 (CIK21) of fermentation concentration of 1% coffee and 10% sucrose, as substrate for fermen­
according to the color parameters of CIELAB scale. tation with SCOBY in order to investigate whether the antioxidant po­
tential of coffee could be enhanced. After the 7-day fermentation period,
Parameters CI CIK0 CIK6 CIK12 CIK15 CIK21
a significant increase in phenolic compounds, CGA, caffeic acid, and
Lightness 79.15 79.12 79.04 78.42 78.87 ± 79.15
caffeine was observed, especially in the kombucha sample produced
(L*) ± 0.01a ± 0.06a ± 0.08a ± 1.12a 1.68a ± 0.05a
Coordinate − 0.1 ± 1.32 ± 0.62 ± 0.65 ± 0.77 ± 1.22 ±
with fine-ground coffee drink (FGC). The same was verified in the
a* 0.01b 0.21a 0.72a 0.35a 0.01a 0.35a antioxidant activity analyses. Antioxidant capacity was enhanced after
Coordinate 8.06 ± 6.97 ± 8.44 ± 6.45 ± 7.39 ± 7.06 ± fermentation, with FGC standing out in all antioxidant assays tested
b* 0.02ab 0.19c 0.80a 0.71c 0.48bc 0.39bc (ORAC, EC50, and superoxide radical scavenging), which was correlated
Chroma 8.05 ± 7.10 ± 8.47 ± 6.49 ± 7.43 ± 7.17 ±
with the increased content of bioactive compounds. However, the study
(C*) 0.00ab 0.15bc 0.84a 0.74c 0.48bc 0.32bc
Hue (h◦ ) 90.70 79.28 86.32 84.37 84.00 ± 80.08 by Watawana, Jayawardena and Waisundara (2015) does not specify the
± 0.50a ± 1.95b ± ± 2.52 0.43bcd ± species of coffee used, nor the degree of roasting of the bean. This in­
4.02ac formation is relevant, since the concentration of compounds can vary
cd
3.25bd

Values are expressed as mean ± standard deviation from triplicate experiments. among coffee species and with the degree of roasting of the bean,
Different letters in the same line indicate significantly different values, ANOVA, contributing to products with different characteristics, which can make
Tukey post-test (p < 0.05). it difficult to compare studies on the same substrate (Butt & Sultan,

5
J. Ferreira de Miranda et al. Food Chemistry 412 (2023) 135556

Table 2
Total phenolic content, DPPH radical scavenging activity, maximum effective concentration (EC50) and 5-caffeoylquinic acid (5-CQA) of coffee infusion (CI) and
coffee infusion kombucha (CIK) over 21 days of fermentation.
CI CIK0 CIK6 CIK12 CIK15 CIK21

Total phenolics (µg GAE*/mL) 510.82 ± 1.34a 551.33 ± 3.93a 521.71 ± 30.93a 541.67 ± 11.44a 571.12 ± 32.83a 542.91 ± 63.66a
%DPPH 70.61 ± 3.97a 73.46 ± 4.99a 69.02 ± 6.28a 68.86 ± 4.90a 72.59 ± 3.58a 66.82 ± 7.85a
EC50 (mL/L) 142.19 ± 16.73a 121.97 ± 25.37a 136.34 ± 27.51a 132.39 ± 18.61a 123.71 ± 13.96a 141.03 ± 22.93a
5-CQA (µL/mL) 162.31 ± 13.71ª 175.35 ± 14.62ª 143.91 ± 31.42ª 167.57 ± 1.89ª 169.96 ± 2.82ª 175.42 ± 53.52ª

Values are expressed as mean ± standard deviation from triplicate experiments. *Gallic acid equivalents.
Different letters in the same line indicate significantly different values, ANOVA, Tukey post-test (p < 0.05).

2011). day. Also, bacteriostatic activity against L. monocytogenes and entero­

Despite the divergences found, in comparison with literature data, it
is important to emphasize that several factors can influence the
fermentation process and contribute to obtaining fermented beverages
with different physicochemical and biological characteristics. The va­
riety and amount of raw material, preparation method, as well as carbon
source, inoculum, fermentation time and temperature, pH, oxygen, are
all factors that can contribute to the variability in compositions among
beverages (Villarreal et al.,2018).
Furthermore, coffee beverage already has a high antioxidant ca­
pacity as it is rich in bioactive compounds. On the report of a study by
Bobková et al. (2020), the antioxidant capacity of an Arabic coffee
beverage (at a concentration of approximately 6%), according to the
DPPH sequestering activity, can vary from 46.86 to 78.55%, depending
on the location of coffee production and degree of bean roasting.
Although fermentation did not enhance this activity, all the beverages
evaluated showed a high percentage of DPPH sequestration, around
70%, and can be considered antioxidant.
3.3. Antimicrobial activity
Antibacterial and antifungal activity of the beverages against
microbiological pathogens are presented in Table 3.
At time 0, no activity was observed against all bacteria tested. From
the 6th day of fermentation, CIK exhibited activity against all bacterial
strains studied. CIK showed bactericidal activity against S. aureus from
the 6th day of fermentation, and against the other strains on the 12th
pathogenic E. coli from the 6th day of fermentation. CI exhibited
bacteriostatic activity only against S. aureus.
Regarding antifungal activity, CIK showed fungicidal activity against
C. albicans, C. gattiie, C. neoformans on the 21st day of fermentation, and
inhibitory activity against C. neoformans on the 12th and 15th day. CI
did not show activity against any of the tested fungi.
Antimicrobial activity in kombucha beverages produced from both
C. sinensis and alternative substrates have already been reported by
several studies. The drink is known for its antimicrobial properties
against various pathogenic microorganisms. This activity can be
attributed to the presence of acetic acid, and a variety of other organic
acids, which can influence antimicrobial activity by cytoplasmic acidi­
fication that inhibits cell growth (Četojević-Simin et al, 2012) by
phenolic compounds already present in raw material and formed during
fermentation (Jayabalan, Marimuthu & Swaminathan, 2007; Jayabalan
et al., 2008), as well as by bacteriocins, high molecular weight proteins
and enzymes, which are produced during fermentation (Battikh et al.,
2011; Bhattacharya et al., 2016).
Battikh, Bakhrouf and Ammar (2012) compared the antimicrobial
activity of kombucha produced from black tea to kombuchas produced
from five plants with medicinal properties (Thymus vulgaris L., Lippia
citriodora, Rosmarinus officinalis, Foeniculum vulgare and Mentha piperita).
Antimicrobial activity, by means of the agar diffusion method (wells),
was investigated against Staphylococcus epidermidis, S. aureus, Micro­
coccus luteus, E. coli, Pseudomonas aeruginosa, Salmonella Typhimurium,
L. monocytogenes, C. albicans, Candida krusei, Candida tropicalis, Candida

Table 3
Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC) and Minimum Fungicide Concentration (MFC) of coffee infusion (CI) and
coffee infusion kombucha (CIK) on days 0 (CIK0), 6 (CIK6), 12 (CIK12), 15 (CIK15) and 21 (CIK21) of fermentation, against bacterial and fungal strains.
Bacterial Samples MIC (µL/ MBC (µL/ Fungal Samples MIC (µL/ MFC (µL/
mL) mL) mL) mL)

Staphylococcus aureus (ATCC13565) CI 500 ND Candida albicans (ATCC CI ND ND

CIK0 ND ND 90028) CIK0 ND ND
CIK6 500 500 CIK6 ND ND
CIK12 250 500 CIK12 ND ND
CIK15 250 500 CIK15 ND ND
CIK21 250 500 CIK21 500 500
Listeria monocytogenes (CLIST2033) CI ND ND Cryptococcus gattii (R265) CI ND ND
CIK0 ND ND CIK0 ND ND
CIK6 500 ND CIK6 ND ND
CIK12 250 500 CIK12 ND ND
CIK15 250 ND CIK15 ND ND
CIK21 250 ND CIK21 500 500
Escherichia coli enteropatogênica (INCQS CI ND ND Candida neoformans (H99) CI ND ND
00185) CIK0 ND ND CIK0 ND ND
CIK6 500 ND CIK6 ND ND
CIK12 250 500 CIK12 500 ND
CIK15 250 ND CIK15 500 ND
CIK21 250 ND CIK21 500 500
Escherichia coli O157:H7 (RJ 691/1) CI ND ND
CIK0 ND ND
CIK6 500 ND
CIK12 250 500
CIK15 250 ND
CIK21 250 ND

ND - Not detected.

6
J. Ferreira de Miranda et al.
parapsilosis, Candida glabrata, Candida dubliniensis and Candida sake. The
beverages were fermented for 21 days, and all bacteria tested were
sensitive to fermented beverages resulting from C. sinensis, L. citriodora
and Me. piperite. F. vulgare kombucha inhibited the activity of
S. epidermidis, Mi. luteus, P. aeruginosa, S. Typhimurium and
L. monocytogenes, and the infusion only showed no activity against
S. Typhimurium. T. vulgaris kombucha did not exhibit any antibacterial
activity. Regarding antifungal activity, fermented beverages from
R. officinalis and T. vulgaris were not active against all the fungi tested.
C. albicans, C. dubliniensis and C. tropicalis were sensitive to kombuchas
from black tea, F. vulgare, Me. piperita and L. citriodora. C. parapsilosis
was sensitive to fermented beverages from F. vulgare and Me. piperita,
while C. sake was inhibited by kombucha from F. vulgare and
L. citriodora. The fermented F. vulgare beverage acted moderately on
C. krusei. Furthermore, this study observed that kombuchas from
L. citriodora, R. officinalis, F. vulgare and Me. piperita showed higher
antibacterial and antifungal activity compared to traditional black tea
kombucha (Battikh et al., 2012).
The infusion of lemon balm (Melissa officinalis L.) fermented with
SCOBY, showed positive results regarding antimicrobial activity.
Četojević-Simin et al. (2012) examined the antimicrobial activity of
lemon balm kombucha (Melissa officinalis L.) against potentially patho­
genic bacteria, yeasts and fungi using the agar diffusion method (wells).
The strains of P. aeruginosa, Proteus mirabilis, E. coli, Salmonella enter­
itidis, Erwinia carotovora, S. aureus, Bacillus cereus, Sarcina Lutea,
Saccharomyces cerevisiae, Candida pseudotropicalis, Rhodotorula sp.,
Aspergillus niger, Penicillium aurantiogriseum and Aspergillus flavus were
tested. According to the results, lemon balm kombucha was able to
inhibit the activity of P. aeruginosa, P. mirabilis, E. coli, S. enteritidis,
E. carotovora, S. aureus and B. cereus.
As seen in Table 3, CI exhibited bacteriostatic activity against
S. aureus, indicating that coffee bioactive compounds may be associated
with this activity. There is evidence that coffee extracts or beverages can
exert activity against S. aureus (Daglia et al., 2007), enterobacteria
(C. freundii, E. aerogenes, E. cloacae, E. coli, K. oxytoca, P. hauseri,
P. mirabilis, Serratia marcescens, Salmonella enterica) (Almeida et al,
2006), B. cereus, P. aeruginosa, S. Typhimurium (Rufián-Henares & De La
Cueva, 2009), L. innocua and L. monocytogenes (Ludwig et al., 2014;
Mueller et al., 2011).
According to Almeida et al. (2006), caffeic acid, trigonelline,
caffeine, chlorogenic acids, and protocatechuic acid are the potential
antimicrobial agents, natural from coffee (without the bean roasting
process), against several strains of enterobacteria, especially against
Salmonella enterica. Bioactive compounds can exert antimicrobial ac­
tivity by altering the structure and function of the cytoplasmic mem­
brane, disrupting the active transport and flow of protons and electrons,
and inhibiting enzymes by oxidizing compounds (Almeida et al., 2006).
However, the main contributors to the antimicrobial activity of
coffee have been found to be related to compounds formed by the
roasting process, since little or no antimicrobial activity has been
observed in analyses performed on green coffee (Mueller et al., 2011).
Mueller et al. (2011) evaluated the antimicrobial activity of coffee
against E. coli and L. innocua and observed that at higher concentrations
the coffee beverage was able to exhibit inhibitory and microbicidal ac­
tivity against L. innocua and E. coli, respectively; however, this activity
was not observed in beverages prepared with green coffee. Within this
perspective, Maillard reaction products, especially melanoidins, are
supposed to be the main compounds involved in the antimicrobial
mechanisms of coffee (Daglia et al., 2007).
Melanoidins are formed from carbohydrates, proteins/amino acids
and phenolic compounds during coffee roasting (Maillard reaction)
(Mueller et al., 2011; Rufián-Henares & De La Cueva, 2009). The anti­
microbial activity of coffee melanoidins can be attributed to their ability
to permeabilize external and internal membranes of microorganisms,
and to interfere with microbial cell synthesis processes (Mueller et al.,
2011). In addition, they are involved in metal chelation mechanisms, an
7
Food Chemistry 412 (2023) 135556
important factor in mediating antibacterial activity of this compound
(Mueller et al., 2011; Rufián-Henares & De La Cueva, 2009). According
to Rufián-Henares and De La Cueva (2009), at low concentrations,
melanoidins can exert bacteriostatic activity mediated by iron chelation,
limiting the availability of iron needed by bacteria; and at high con­
centrations, melanoidins can have bactericidal activity by removing
Mg2+ ions from the outer membrane, promoting cell membrane
disruption. However, no mechanism of the antimicrobial action of coffee
melanoidins is well understood.
There are still many controversies and doubts about the main com­
pounds and mechanisms involved in antimicrobial activity of coffee,
what requires further study, and it also makes it difficult to establish a
correlation of the antimicrobial activity with other coffee-based prod­
ucts. With regard to kombucha produced from coffee infusion, no report
has been found in the literature regarding its antimicrobial property,
and this work may be the first to investigate such activity.
According to the results obtained, the fermentation of coffee infusion
with SCOBY potentiated antimicrobial effects of the coffee beverage,
which after fermentation became effective against all strains tested.
However, the results are inconclusive regarding the factors that
contributed to this antimicrobial action. The main compounds or
mechanisms involved in the antimicrobial activity of CIK are still not
known, and further studies are needed. A more detailed study on
biotransformation processes of the chemical compounds during the
fermentation process is needed. Identify and quantify the remaining
chemical compounds, both of those that already come from the coffee
infusion and those that were probably produced during the fermentation
process. Chemical characterization of the metabolites present in the
fermented beverage would be essential to better understand possible
processes and mechanisms involved in the beverage’s beneficial
properties.
3.4. In vivo toxicity
Toxicity was evaluated after treatment of G. mellonella larvae with
the tested samples. The larvae were observed for 10 days to verify the
presence or absence of melanization, a very common process during the
immune response of insects, occurring as a result of stress or infection
(Megaw et al., 2015). In addition, the death/survival of the larvae, as
well as the presence or absence of melanization, were also analyzed. In
this experiment, all larvae treated with all samples survived and did not
show melanization, therefore, we can say that the studied samples do
not show toxicity (Fig. 2).
Galleria mellonella larvae are commonly used to study the virulence
of microbial pathogens, to measure the efficacy of antimicrobial agents,
to study in vivo toxicity of preservatives, and to test new antimicrobial
drugs (Trevijano-Contador & Zaragoza, 2019). Maguire, Duggan, and
Kavanagh (2016) used G. mellonella larvae model to measure the toxicity
of a variety of food preservatives, such as potassium nitrate, potassium
sorbate, sodium benzoate, sodium nitrate, sodium chloride, sodium ni­
trite, and sodium acetate. The results showed a strong positive correla­
tion with results obtained using cell culture and mammalian models
(Maguire, Duggan, & Kavanagh, 2016). Allegra et al., (2018) investi­
gated the suitability of G. mellonella larvae to measure chemical toxicity
compared to cell culture systems. Cell culture systems overestimated the
toxicity of chemicals, especially in low-toxicity chemicals. In contrast,
the toxicity test on G. mellonella larvae was found to be reliable for
chemicals of low toxicity. These studies demonstrate the feasibility of
using this in vivo model for toxicity evaluation, proving to be an
attractive alternative.
So far only the study by Silva et al. (2021) used this technique to
analyze the toxicity of kombucha drinks. Similar to our results, Silva
et al. (2021) also observed no signs of toxicity in larvae treated with
green tea kombucha and marshmallow kombucha after 7 days of
treatment.
J. Ferreira de Miranda et al.
Fig. 2. Galleria mellonella larvae treated with coffee-infused kombucha (CIK) at fermentation times 0 (CIK0), 15 (CIK15) and 21 (CIK21) days, and with coffee infusion
(CI) and phosphate-buffered saline controls (PSB) (A). Graph of toxicity absence of the evaluated samples (B).
4. Conclusion
The results presented and discussed allow us to conclude that the
elaboration of a kombucha based on coffee infusion, under the prepa­
ration conditions presented, is feasible, since the changes in the physi­
cochemical characteristics, such as reduction in the contents of total
soluble solids and reducing sugars and increase acidity expressed as
acetic acid proved the metabolic activity of SCOBY. Besides, according
to the pH value, CIK can be considered safe from a microbiological point
of view since pathogenic microorganisms are hardly able to grow in
acidic foods.
Regarding the concentration of phenolic compounds, antioxidant
activity and concentration of 5-caffeoylquinic acid, no significant dif­
ferences were observed between the non-fermented and fermented
beverage, nor between the fermentation intervals. On the other hand,
fermentation of coffee infusion potentiated the antimicrobial action of
the beverage, observed against most of the tested strains. The beverages
evaluated showed no toxic effect on the G. mellonella larvae model
studied.
The current study presents important data that can cooperate with
future studies on the biochemical transformations that occur in the
fermentation of coffee with SCOBY, considering mainly the chemical
characterization of the metabolites formed in this process.
CRediT authorship contribution statement
Jeniffer Ferreira de Miranda: Investigation, Formal analysis,
Writing – original draft, Visualization, Writing – review & editing.
Giulia Martins Pereira Belo: Investigation. Laís Silva de Lima:
Investigation. Kelly Alencar Silva: Investigation, Resources, Writing –
review & editing. Thais Matsue Uekane: Investigation, Resources,
Writing – review & editing. Alice Goncalves Martins Gonzalez: Re­
sources, Investigation, Writing – review & editing. Vanessa Naciuk
Castelo Branco: Formal analysis, Resources, Investigation, Writing –
review & editing. Nayla Souza Pitangui: Resources, Investigation.
Fabrício Freitas Fernandes: Investigation, Writing – review & editing,
Resources. Adriene Ribeiro Lima: Conceptualization, Project admin­
istration, Supervision, Investigation, Resources, Formal analysis,
Writing – original draft, Writing – review & editing, Visualization.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
No data was used for the research described in the article.
8
Food Chemistry 412 (2023) 135556
Acknowledgements
The authors received financial support from Fundação de Amparo à
Pesquisa do Estado do Rio de Janeiro (FAPERJ, Brazil), through Project
E-26/200 930/2017. We are also grateful to the Fundação de Amparo à
Pesquisa do Estado de São Paulo (FAPESP, Brazil), the Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil), and to
the Conselho Nacional de Desenvolvimento Científico e Tecnológico
(CNPq, Brazil) for their continued support.
References
Allegra, E., Titball, R. W., Carter, J., & Champion, O. L. (2018). Galleria mellonella larvae
allow the discrimination of toxic and non-toxic chemicals. Chemosphere, 198,
469–472. https://doi.org/10.1016/j.chemosphere.2018.01.175
Almeida, A. A. P., Farah, A., Silva, D. A. M., Nunan, E. A., & Glória, M. B. A. (2006).
Antibacterial activity of coffee extracts and selected coffee chemical compounds
against enterobacteria. Journal of Agricultural and Food Chemistry, 54(23),
8738–8743. https://doi.org/10.1021/jf0617317
AOAC - Association of Official Analytical Chemistry. (1992). Official Methods of Analysis
of the Association of Analytical Chemistry.12 ed. Washington, DC, 1992. 1115p.
Battikh, H., Bakhrouf, A., & Ammar, E. (2012). Antimicrobial effect of Kombucha
analogues. LWT - Food Science and Technology, 47(1), 71–77. https://doi.org/
10.1016/j.lwt.2011.12.033
Battikh, H., Chaieb, K., Bakhrouf, A., & Ammar, E. (2011). Antibacterial and antifungal
activities of black and green kombucha teas. Journal of Food Biochemistry, 37(2),
231–236. https://doi.org/10.1111/j.1745-4514.2011.00629.x
Bhattacharya, D., Bhattacharya, S., Patra, M. M., Chakravorty, S., Sarkar, S.,
Chakraborty, W., et al. (2016). Antibacterial activity of polyphenolic fraction of
kombucha against enteric bacterial pathogens. Current Microbiology, 73(6), 885–896.
https://doi.org/10.1007/s00284-016-1136-3
Bobková, A., Hudáček, M., Jakabová, S., Belej, Ľ., Capcarová, M., Čurlej, J., et al. (2020).
The effect of roasting on the total polyphenols and antioxidant activity of coffee.
Journal of Environmental Science and Health - Part B Pesticides, Food Contaminants, and
Agricultural Wastes, 55(5), 495–500. https://doi.org/10.1080/
03601234.2020.1724660
Butt, M. S., & Sultan, M. T. (2011). Coffee and its consumption: Benefits and risks. Critical
Reviews in Food Science and Nutrition, 51(4), 363–373. https://doi.org/10.1080/
10408390903586412
Cardoso, R. R., Neto, R. O., dos Santos D’Almeida, C. T., do Nascimento, T. P.,
Pressete, C. G., Azevedo, L., Martino, H. S. D. L., Cameron, C., Ferreira, M. S. L., & de
Barros, F. A. R. (2020). Kombuchas from green and black teas have different
phenolic profile, which impacts their antioxidant capacities, antibacterial and
antiproliferative activities. Food Research International, 128. https://doi.org/
10.1016/j.foodres.2019.108782
Četojević-Simin, D. D., Velićanski, A. S., Cvetković, D. D., Markov, S. L., Mrdanović, J.Ž.,
Bogdanović, V. V., et al. (2012). Bioactivity of Lemon Balm Kombucha. Food and
Bioprocess Technology, 5(5), 1756–1765. https://doi.org/10.1007/s11947-010-0458-
6
CLSI - Clinical and Laboratory Standars Institute. (2012). Methods for Dilution
Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically. CLSI
document M07-A9. Clin. Lab. Standars Inst. 32, 18.
CLSI - Clinical and Laboratory Standars Institute. (2017). Reference Method for Broth
Dilution Antifungal Susceptibility Testing of Yeasts. CLSI document M27-A2. Clin.
Lab. Standars Inst. 22, 15.
Daglia, M., Papetti, A., Grisoli, P., Aceti, C., Spini, V., Dacarro, C., et al. (2007). Isolation,
identification, and quantification of roasted coffee antibacterial compounds. Journal
of Agricultural and Food Chemistry, 55(25), 10208–10213. https://doi.org/10.1021/
jf0722607
Ferreira, M. D., & Spricigo, P. C. (2017). Colorimetria-princípios e aplicações na
agricultura. Parte 4. Análises não destrutivas. In Instrumentação Pós-colheita em Frutas
e Hortaliças (pp. 208–220). Embrapa.
J. Ferreira de Miranda et al.
Dutta, H., & Paul, S. K. (2019). Kombucha drink: Production, quality, and safety aspects.
In Production and Management of Beverages (pp. 259–288). Elsevier Inc.. https://doi.
org/10.1016/b978-0-12-815260-7.00008-0
Emiljanowicz, K. E., & Malinowska-Pańczyk, E. (2019). Kombucha from alternative raw
materials–The review. Critical Reviews in Food Science and Nutrition, 60(19),
3185–3194. https://doi.org/10.1080/10408398.2019.1679714
Farah, A., De Paulis, T., Trugo, L. C., & Martin, P. R. (2005). Effect of roasting on the
formation of chlorogenic acid lactones in coffee. Journal of Agricultural and Food
Chemistry, 53(5), 1505–1513. https://doi.org/10.1021/jf048701t
Gamboa-Gómez, C. I., González-Laredo, R. F., Gallegos-Infante, J. A., Pérez, M. M. L.,
Moreno-Jiménez, M. R., Flores-Rueda, A. G., et al. (2016). Antioxidant and
angiotensin-converting enzyme inhibitory activity of Eucalyptus camaldulensis and
Litsea glaucescens infusions fermented with kombucha consortium. Food Technology
and Biotechnology, 54(3), 367–373. https://doi.org/10.17113/ftb.54.03.16.4622
Gomes, R. J., Borges, M. de F., Rosa, M. de F., Castro-Gómez, R. J. H., & Spinosa, W. A.
(2018). Acetic acid bacteria in the food industry: Systematics, characteristics and
applications. Food Technology and Biotechnology, 56(2), 139–151. https://doi.org/
10.17113/ftb.56.02.18.5593
IAL– Instituto Adolfo Lutz. (2008). Chemical and physical methods for food analysis,
fourth ed. Instituto Adolfo Lutz, Sao Paulo.
Jayabalan, R., Marimuthu, S., & Swaminathan, K. (2007). Changes in content of organic
acids and tea polyphenols during kombucha tea fermentation. Food Chemistry, 102
(1), 392–398. https://doi.org/10.1016/j.foodchem.2006.05.032
Jayabalan, R., Subathradevi, P., Marimuthu, S., Sathishkumar, M., & Swaminathan, K.
(2008). Changes in free-radical scavenging ability of kombucha tea during
fermentation. Food Chemistry, 109(1), 227–234. https://doi.org/10.1016/j.
foodchem.2007.12.037
Jayabalan, R., Malbaša, R. V., Lončar, E. S., Vitas, J. S., & Sathishkumar, M. (2014).
A review on kombucha tea-microbiology, composition, fermentation, beneficial
effects, toxicity, and tea fungus. Comprehensive Reviews in Food Science and Food
Safety, 13(4), 538–550. https://doi.org/10.1111/1541-4337.12073
Liu, R. H. (2013). Health-promoting components of fruits and vegetables in the diet.
Advances Nutrition, 4(3). https://doi.org/10.3945/an.112.003517
Ludwig, I. A., Clifford, M. N., Lean, M. E. J., Ashiharad, H., & Crozier, A. (2014). Coffee:
Biochemistry and potential impact on health. Food & Function, 5, 1695–1717.
https://doi.org/10.1039/c4fo00042k
Maguire, R., Duggan, O., & Kavanagh, K. (2016). Evaluation of Galleria mellonella larvae
as an in vivo model for assessing the relative toxicity of food preservative agents. Cell
Biology and Toxicology, 32(3), 209–216. https://doi.org/10.1007/s10565-016-9329-
x Journal of Food Processing and Preservation, 39(6), 2596–2603. https://doi.org/
Malbaša, R. V., Lončar, E. S., Vitas, J. S., & Čanadanović-Brunet, J. M. (2011). Influence
of starter cultures on the antioxidant activity of kombucha beverage. Food Chemistry,
127(4), 1727–1731. https://doi.org/10.1016/j.foodchem.2011.02.048
Food Chemistry 412 (2023) 135556
Megaw, J., Thompson, T. P., Lafferty, R. A., & Gilmore, B. F. (2015). Galleria mellonella as
a novel in vivo model for assessment of the toxicity of 1-alkyl-3-methylimidazolium
chloride ionic liquids. Chemosphere, 139, 197–201. https://doi.org/10.1016/j.
chemosphere.2015.06.026
Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing
sugar. Analytical Chemistry. Washington, US, 31(3), 426–428.
De Miranda, J. F., Ruiz, L. F., Silva, C. B., Uekane, T. M., Silva, K. A., Gonzalez, A. G. M.,
et al. (2022). Kombucha: A review of substrates, regulations, composition, and
biological properties. Journal of Food Science, 1–25. https://doi.org/10.1111/1750-
3841.16029
Mueller, U., Sauer, T., Weigel, I., Pichner, R., & Pischetsrieder, M. (2011). Identification
of H2O2 as a major antimicrobial component in coffee. Food and Function, 2(5),
265–272. https://doi.org/10.1039/c0fo00180e
Rufián-Henares, J. A., & De La Cueva, S. P. (2009). Antimicrobial activity of coffee
melanoidins - A study of their metal-chelating properties. Journal of Agricultural and
Food Chemistry, 57(2), 432–438. https://doi.org/10.1021/jf8027842
Santos-Buelga, C., et al. (2019). Plant phenolics as functional food ingredients. Advances
in Food and Nutrition Research. Academic Press. ISSN 1043-4526.
Silva, K. A., Uekane, T. M., de Miranda, J. F., Ruiz, L. F., da Motta, J. C. B., Silva, C. B.,
et al. (2021). Kombucha beverage from non-conventional edible plant infusion and
green tea: Characterization, toxicity, antioxidant activities and antimicrobial
properties. Biocatalysis and Agricultural Biotechnology, 34, Article 102032. https://
doi.org/10.1016/j.bcab.2021.102032
Singulani, J. L., Galeane, M. C., Ramos, M. D., Gomes, P. C., dos Santos, C. T., de
Souza, B. M., et al. (2019). Antifungal activity, toxicity, and membranolytic action of
a mastoparan analog peptide. Frontiers in Cellular and Infection Microbiology., 9, 419.
https://doi.org/10.3389/fcimb.2019.00419
Trevijano-Contador, N., & Zaragoza, O. (2019). Immune response of Galleria mellonella
against human fungal pathogens. Journal of Fungi, 5(1), 1–13. https://doi.org/
10.3390/jof5010003
Villarreal, S. A., Beaufort, S., Bouajila, J., Souchard, J., & Taillandier, P. (2018).
Understanding Kombucha Tea Fermentation : A Review To cite this version : HAL Id :
hal-01945573.
Vitas, J. S., Cvetanović, A. D., Mašković, P. Z., Švarc-Gajić, J. V., & Malbaša, R. V. (2018).
Chemical composition and biological activity of novel types of kombucha beverages
with yarrow. Journal of Functional Foods, 44, 95–102. https://doi.org/10.1016/j.
jff.2018.02.019
Watawana, M. I., Jayawardena, N., & Waisundara, V. Y. (2015). Enhancement of the
functional properties of coffee through fermentation by “Tea Fungus” (Kombucha).
10.1111/jfpp.12509
Zou, C., Li, R. Y., Chen, J. X., Wang, F., Gao, Y., Fu, Y. Q., et al. (2021). Zijuan tea- based
kombucha: Physicochemical, sensorial, and antioxidant profile. Food Chemistry, 363,
3–10. https://doi.org/10.1016/j.foodchem.2021.130322