Journal of Functional Foods 44 (2018) 95–102

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Journal of Functional Foods

journal homepage: www.elsevier.com/locate/jff

Chemical composition and biological activity of novel types of kombucha T

beverages with yarrow
⁎
Jasmina S. Vitasa, , Aleksandra D. Cvetanovića, Pavle Z. Maškovićb, Jaroslava V. Švarc-Gajića,
Radomir V. Malbašaa
a
University of Novi Sad, Faculty of Technology Novi Sad, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia
b
University of Kragujevac, Faculty of Agronomy, Cara Dušana 34, 32000 Čačak, Serbia

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

Keywords: Kombucha beverages were produced by fermentation of new types of substrates – yarrow infusions and yarrow
Fermentation subcritical water extracts (SWE). Fermentation process parameters (pH, total acidity and yield of biomass),
Kombucha chemical composition (organic acids, total phenols and flavonoids and vitamin C content) and sensory analysis
Yarrow indicated that SWE were more suitable substrates for successful fermentation. Products obtained on infusions
Infusions
had more pronounced anticancer and antimicrobial properties whereas beverages produced on SWE had higher
Subcritical water extracts
antioxidant potential.

1. Introduction inflammation, pain, spasmodic diseases, flatulence, and dyspepsia

Kombucha is a fermented beverage which is globally consumed
because of the health benefits reported by the users. This product has
slightly acidic, carbonated and sweet taste, and is mostly prepared at
home. Fermentation process is a result of the metabolic activity of
kombucha culture (symbiosis of bacteria and yeasts) on sweetened
black or green tea, as the most common substrates. During fermentation
the cellulosic pelicle layer is also produced by acetic acid bacteria, and
this biofilm has numerous applications (Jayabalan, Malbaša, &
Sathishkumar, 2016a). Successful kombucha fermentation is conducted
in glass vessels under static conditions, on substrates that contain
source of carbon (mostly sucrose) and nitrogen (different tea compo-
nents) atom, protected from direct sunlight at room temperature. Al-
though black and green tea are the most common substrates for its
prepatration, there are reports that infusions prepared from different
medicinal plants can be used as well. Yarrow is a plant with a variety of
applications, including medical, and is also used as a livestock feed. It is
used as a food, for preparing infusions and as a spice. This herb has
sweet and bitter taste. According to the scientific evidence, there are
over a hundred active biological compounds in yarrow. Among the
most notable are achilleine, apigenin, luteolin, azulene, camphor,
coumarin, inulin, menthol, quercetin, rutin, succinic, salicylic and
caffeic acids, thujone, etc. (Dervengji, 1977). This plant is widely used
to treat various diseases including malaria, hepatitis and jaundice as
well as for the treatment of wounds, hemorrhages, headaches,
(Akram, 2013; Chandler et al., 1982; Benedek & Kopp, 2007; Lehane &
Saliba, 2008). Yarrow is commonly consumed in a form of infusion and
there is no scientific evidence about its use for kombucha preparation.
Thus, this study is a first attempt to make kombucha beverage based on
yarrow infusion as well as on its extracts obtained by subcritical water.
Subcritical water extraction is advanced extraction technique that is
gaining increasing attention nowadays in the extraction and recovery of
bioactive compounds from different natural matrices. The technique is
based on the extraction with hot water at temperatures below its critical
point while maintaining high pressures in order to keep the water in a
liquid state during the whole extraction process. These conditions alter
physico-chemical properties of water, influencing its solvating proper-
ties. The increase of the temperature of liquid water produces a series of
effects, including improved mass transfer as a result of the drop in water
surface tension that allows better penetration into sample matrix.
Moreover, the mass transfer kinetics is favoured by the disruption of
intermolecular forces (i.e., van der Waals forces, hydrogen bonds and
dipole attractions) in the sample matrix. However, the most important
effects of the increase in water temperature are the weakening of hy-
drogen bonds, resulting in a lower dielectric constant. Thus, the use of
SWE could be an alternative to the use of non-polar organic solvents in
some applications. From a green chemistry perspective, the avoidance
of organic solvents provides additional advantages in the extraction of
highly to medium polarity compounds. Due to numerous benefits that it
offers, SWE are a great candidate for production of functional

⁎
Corresponding author.
E-mail address: vitasj@uns.ac.rs (J.S. Vitas).

https://doi.org/10.1016/j.jff.2018.02.019
Received 8 February 2017; Received in revised form 12 February 2018; Accepted 14 February 2018
Available online 23 March 2018
1756-4646/ © 2018 Elsevier Ltd. All rights reserved.
J.S. Vitas et al.
ingredients (Švarc-Gajić, 2012, chap. 11).
The aim of this paper was to investigate the possibility of production
of a new variety of kombucha beverages on yarrow infusions, which are
prepared as usual substrates, and subcritical water extracts, as unique
and innovative starting medium. The goal is to establish the basis for
novel functional food production and therefore widen the food market
by offering products with competitive quality characteristics and anti-
oxidant, antimicrobial and antiproliferative activities.
2. Material and methods
2.1. Plant material
Yarrow (Achillea millefolium L.) was collected in the area of Vlasina
Lake (Southeast region of Serbia) at an altitude of 1220 m, in August
2015. Yarrow flowers were stacked in a crate with perforated bottom,
in order to ensure air flow. Drying was performed naturally in the draft
and dark until moisture content of 10%. Dry plant material was packed
in paper bags and stored in the dark until use.
2.2. Infusions
Infusions were obtained by steeping 1.13 (Y1.13), i. e. 2.26 g
(Y2.26) of yarrow flowers in 500 mL of boiling tap water during 15 min.
2.3. Subcritical water extracts
Subcritical water extraction was performed in a homemade sub-
critical water extractor/reactor previously described by Cvetanović
et al. (2017). The extraction procedure was as follows: the operating
pressure of 45 bar, process temperatures of 115 and 140 °C, agitation
rate of 3 Hz, extraction time15 min. Extracts were prepared with dif-
ferent sample-to-solvent ratio: YI (1.13 g yarrow flowers/500 mL,
115 °C), YII (1.13 g yarrow flowers/500 mL, 140 °C) and YIII (2.26 g
yarrow flowers/500 mL, 115 °C).
2.4. Kombucha inoculum and fermentation process
The native kombucha culture contains five yeast strains
(Saccharomycodes ludwigii, Saccharomyces cerevisiae, Saccharomyces bis-
porus, Torulopsis sp. and Zygosaccharomyces sp.) and key bacterium
belongs to the strains of the genus Acetobacter (Malbaša, Lončar, Vitas,
& Čanadanović-Brunet, 2011).
Kombucha inoculum, used in this investigation, was fermentation
liquid of kombucha obtained after 7 days long fermentation on yarrow
infusion and subcritical water extract, sweetened with 35 g/500 mL
sucrose, at 25 °C. Kombucha starter was added in the amount of 10%
(v/v). Kombucha was selected as widely used microbial culture that can
produce a beverage with potential functional characteristics. Yarrow
was selected to introduce a new substrate for kombucha fermentation
and infusions and subcritical water extracts to demonstrate the influ-
ence of different production parameters on the quality of obtained
beverages.
Following kombucha beverages were produced: K-Y1.13, K-Y2.26,
K-YI, K-YII and K-YIII.
2.5. Chemicals and reagents
Folin-Ciocalteu reagent, chlorogenic acid and rutin were purchased
from Sigma-Aldrich (St. Louis, Missouri, USA). Aluminium chloride
hexahydrate, sodium nitrite, sodium hydroxide and sodium carbonate
were purchased from Merck (Darmstadt, Germany). Potassium ferri-
cyanide and ferric chloride, were obtained from Zorka (Šabac, Serbia).
Vitamin C standard and acetic acid were purchased from J.T. Baker,
The Netherlands. Ammonium-acetate was purchased from Centrohem
(Serbia), orto-phosphoric acid from Kemika (Zagreb, Croatia), lactic
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Journal of Functional Foods 44 (2018) 95–102
and oxalic acid from Laborat. Laphoma Hemikali (Skopje) and succinic,
citric and malic acid from SupelcoAnalytical (Bellefonte).
Chemicals and reagents were of analytical and HPLC grade.
2.6. pH
pH values were measured using a pH-meter (WTW series Inolab pH
720).
2.7. Total acidity
Total acidity was determined by volumetric analysis with a standard
solution of sodium hydroxide and phenolphthalein as indicator
(Malbaša, Lončar, Djurić, & Došenović, 2008). Results were expressed
as grams of acetic acid per liter of sample.
2.8. Yield of biomass
The yield of the obtained biomass was determined by mass mea-
surement. The cellulose floating pellicle layer was removed from the
fermented liquid surface, rinsed with distilled water and dried with
filter paper (Malbaša, et al., 2008). Results were expressed in grams.
2.9. Organic acids analysis
HPLC analysis of organic acids (oxalic, formic, acetic, lactic, suc-
cinic, malic and citric) was performed using reversed phase chroma-
tography, with Agilent 1100 Series HPLC, USA, according to Kordiš-
Krapež, Abram, Kač, & Ferjančić (2001), with minor modifications.
System consisted of degasser, binary pump, ZORBAX® SB-C18 column
(4.6x150 mm, 5-µm) and UV-DAD detector. Analysis was performed in
isocratic mode with 6 mmol/L phosphoric acid (pH 2.1) as mobile
phase. Liquid chromatography parameters were set at: column tem-
perature 28 °C, detection wavelenght 220 nm and flow rate 1.0 mL/min.
Organic acids standards were used for external standard method cali-
bration. Results were expressed in g/L.
2.10. Total phenols analysis
The Folin-Ciocalteu method (Singleton & Rossi, 1965; Kähkönen,
1999) was used to determine the total phenolics content. The reaction
mixture was prepared by mixing 0.1 mL of the sample, 7.9 mL of dis-
tilled water, 0.5 mL of Folin-Ciocalteu reagent and 1.5 mL of sodium
carbonate (20%, w/w). After incubation at room temperature for 1 h,
absorbance was measured at 750 nm. The blank was prepared by re-
placing the sample with distilled water. Chlorogenic acid was used as a
calibration standard and the results were expressed as chlorogenic acid
equivalents per mL of sample (mg CAE/mL).
2.11. Total flavonoids analysis
Flavonoids in obtained extracts and beverages were determined
using colorimetric assay based on the procedure described by Markham
(Markham, 1989). SCW extracts and infusions of yarrow as well as
kombucha beverages (1 mL) were mixed with 5% NaNO2 solution
(0.3 mL). After 5 min aluminium choride hexahydrate (10%, 0.3 mL)
was added and allowed to stand for 6 min. Sodium-hidroxide (1 mol/
dm3, 1 mL) was added to the mixture. Immediately, distilled water was
added to bring the final volume to 10 mL. The blank was prepared by
replacing the sample with distilled water. Immediately after mixing,
absorbance was measured at 510 nm. Rutin was used as a calibration
standard and results were expressed as rutin equivalents (RE) per mL of
sample (mg RE/mL).
J.S. Vitas et al.
2.12. Vitamin C analysis
Vitamin C content was determined using the HPLC technique ac-
cording to Vitas, Malbaša, Grahovac, & Lončar (2013). Samples were
filtered through 0.45 µm membrane filter and injected (20 µL) directly
in the HPLC system. Agilent 1100 Series HPLC with UV-DAD detector,
USA was used. Isocratic elution with 0.1 M ammonium-acetate
(pH = 5.1) as mobile phase at flow rate of 0.4 mL/min and C-18 column
(150 × 4.6 mm; pore diameter 5 µm) was applied. Detection wave-
length was set at 254 nm. Calibration was performed with external
standard method. Vitamin C content was expressed in mg/L.
2.13. DPPH scavenging method
For the DPPH radical-scavenging assay the procedure followed the
method of Espin, Soler-Rivas, & Wichers (2000) in which the samples
showed antioxidant activity by the reduction of purple colored DPPH to
the yellow colored diphenylpicrylhydrazine derivatives. The samples
were mixed with methanol (95%) and 90 µM DPPH to give final con-
centrations in the range of 0.01–2.00 mg/mL. The mixture was in-
cubated at room temperature for 60 min, and the absorbance was
measured at wavelength of 515 nm. Methanol was used to set zero of
absorbance. The results were expressed as inhibitory concentration at
50% (IC50), which is the concentration of the test solution for achieving
50% of the radical scavenging capacity.
2.14. Reducing power method
The antioxidant activity was also determined by the reducing power
ability following the procedure described by Oyaizu (1986). Various
concentrations of the samples (0.5–10 mg/mL) were mixed with phos-
phate buffer (1 mL, 0.2 M, pH 6.6) and 1 mL of 1% potassium ferri-
cyanide. The mixture was incubated at 50 °C. After 20 min of incuba-
tion, the reaction mixture was acidified with 1 mL of trichloroacetic
acid (10%). The mixture was further centrifuged at 3000 min−1 for
10 min. The obtained supernatant (2 mL) was mixed with double dis-
tilled water (2 mL) and 0.1% FeCl3 solution (0.4 mL). Absorbance was
measured at 700 nm. The blank was prepared by using water instead of
sample. The capacity of samples to convert Fe3+ into Fe2+ was ex-
pressed as EC50 values, which represents solution concentration for
achieving 50% of the free radical scavenging capacity.
2.15. Minimum inhibitory concentrations (MIC) determination of
kombucha beverages
MIC of the kombucha beverages and cirsimarin against the test
bacteria were determined by microdilution method in 96-multi-well
microtiter plates (Satyajit, Sarker, & Kumarasamy, 2007). All tests were
performed in Muller–Hinton broth (MHB) with the exception of the
yeast, in which case Sabouraud dextrose broth was used. A volume of
100 µL stock solutions of oil (in methanol, 200 µL/mL) and cirsimarin
(in 10% DMSO, 2 mg/mL) were pipetted into the first row of the plate.
Fifty microliters of Mueller-Hinton or Sabouraud dextrose broth (sup-
plemented with Tween 80 to a final concentration of 0.5% (v/v)) was
added to the other wells. A volume of 50 µL from the first test wells was
pipetted into the second well of each microtiter line and then 50 µl of
scalar dilution was transferred from the second to the twelfth well. Ten
microliters of resazurin indicator solution (prepared by dissolution of a
270-mg tablet in 40 mL of sterile distilled water) and 30 µL of nutrient
broth were added to each well. Finally, 10 µL of bacterial suspension
(106CFU/mL) and yeast spore suspension (3 × 104 CFU/mL) was added
to each well. For each strain, the growth conditions and the sterility of
the medium were checked. Standard antibiotic amracin was used to
control the sensitivity of the tested bacteria, whereas nystatin was used
as a control against the tested yeast. Plates were wrapped loosely with
cling film to prevent dehydration. The plates were placed in an
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Journal of Functional Foods 44 (2018) 95–102
incubator at 37 °C for 24 h for the bacteria and at 28 °C for 48 h for the
yeast. Subsequently, colour change was assessed visually. Any colour
change from purple to pink or colorless was recorded as positive. The
lowest concentration at which colour change occurred was taken as the
MIC value.
2.16. Cytotoxic activity determination by MTT assay of kombucha
beverages
The influence of kombucha beverages on the growth of malignantly
transformed cell lines was evaluated by MTT (3-[4,5-dimethylthiazol-2-
yl]-2,5 diphenyl tetrazolium bromide) assay. The following cell lines
have been used: RD (cell line derived from human rhabdomyosarcoma),
Hep2c (cell line derived from human cervix carcinoma-HeLa derivative)
and L2OB (cell line derived from murine fibroblast). Cells were seeded
(2 × 105 cells/mL; 100 µL/well) in 96-well cell culture plates in nu-
trient medium [minimum essential medium (MEM) Eagle supplemented
with 5% of Hep2c, RD and L2OB] and grown at 37 °C in humidified
atmosphere for 24 h. Then, corresponding extract (stock solution: 5 mg
of extract dissolved in 1 mL of absolute ethanol) and control (absolute
ethanol) diluted with nutrient medium to desired concentrations were
added (100 µL/well) and cells were incubated at 37 °C in humidified
atmosphere for 48 h. Pure nutrient medium (100 µL) represented po-
sitive control for each cell line. After incubation period, supernatants
were discarded MTT [dissolved in D-MEM (Dulbecco’s modification of
Eagle’s medium) in concentration of 500 µg/mL] was added in each
well (100 µL/well). Immediately after, all wells were incubated at 37 °C
in humidified atmosphere for 4 h. Reactions were halted by adding 100
µL of Sodium Dodecyl Sulfate-SDS (10% in 10 mM HCl). After overnight
incubation at 37 °C, absorbance was measured at 580 nm using a
spectrophotometer (Ascent 6–384 [Suomi], MTX Lab Systems Inc.,
Vienna, VA 22182, USA). The number of viable cells per well (NVC)
was calculated from a standard curve plotted as cell numbers against
A580. Corresponding cells (grown in flasks), after cell count by hae-
mocytometer, were used as standards. Standard suspensions were
plated in serial dilution, centrifuged at 800 rpm for 10 min and then
treated with MTT/D-MEM and 10% SDS/10 mM HCl solutions in the
same way as the experimental wells (ut supra). The number of viable
cells in each well was proportional to the intensity of the absorbed light,
which was then read in an ELISA plate reader at 580 nm. Absorbance
(A) at 580 nm was measured 24 h later. Cell survival (%) was calculated
by dividing the absorbance of a sample with cells grown in the presence
of various concentrations of the investigated extracts with control op-
tical density (the A of control cells grown only in nutrient medium), and
multiplying by 100. The blank absorbance was always subtracted from
the absorbance of the corresponding sample with target cells. IC50
concentration was defined as the concentration of an agent inhibiting
cell survival by 50%, compared with a vehicle-treated control. The
results of the measurements are expressed as the percentage of positive
control growth taking the cis-diamminedichloroplatinum (cis-DDP)
determined in positive control wells as the 100% growth (Mosmann,
1983; Dighe, Shiradkara, Rohomb, & Dighe, 2011; Baviskar,
Khadabadia, Deore, & Shiradkar, 2012).
2.17. Sensory analysis
Sensory analysis was performed according to Malbaša, Vitas,
Lončar, Grahovac, & Milanović (2014), with some modifications. De-
scriptive test and 5-point category scale (1-the lowest and 5-the highest)
were used. Appearance, colour, taste and odour were evaluated.
All analyses in this investigation were performed after production of
the beverages and they were repeated three times. All results are given
as mean ± standard deviation.
J.S. Vitas et al. Journal of Functional Foods 44 (2018) 95–102

Table 1 3. Results and discussion

pH, total acidity and yield of biomass of yarrow kombucha beverages and initial sub-
strates.
3.1. The effect of fermentation process on pH, total acidity and yield of
Sample pH Total acidity, g acetic acid/L Yield of biomass, g biomass

Y1.13 7.62 ± 0.00 0.03 ± 0.00 / Value of pH, total acidity and yield of biomass are standard para-
Y2.26 7.35 ± 0.00 0.06 ± 0.00 /
meters that indicate the success of the production process (Jayabalan,
YI 5.40 ± 0.00 0.06 ± 0.00 /
YII 4.81 ± 0.00 0.06 ± 0.00 / Malbaša, Lončar, Vitas, & Sathishkumar, 2014).pH value of traditional
YIII 5.04 ± 0.00 0.09 ± 0.00 / kombucha products, after 7 days long fermentation period, is 2.95 for
K-Y1.13 3.51 ± 0.00 1.26 ± 0.00 1.05 ± 0.00 kombucha on black tea and 3.21 for green tea kombucha (Malbaša
K-YT2.25 3.32 ± 0.00 4.97 ± 0.02 0.93 ± 0.00 et al., 2011). These values are in accordance to the investigation of
K-YI 2.60 ± 0.00 10.76 ± 0.02 2.86 ± 0.00
Jayabalan, Marimuthu, & Swaminathan (2007), in which black tea and
K-YII 2.46 ± 0.00 11.88 ± 0.02 1.39 ± 0.00
K-YIII 2.39 ± 0.00 17.76 ± 0.00 3.39 ± 0.00 green tea were used. In this investigation, all produced beverages had
lower pH values than used substrates which indicated the metabolic
activity of kombucha on the yarrow SWE and infusions (Table 1). SWE
itself had lower pH than infusions and fermentation was more pro-
nounced on SWE. This suggested that SWE is more suitable for kom-
bucha fermentation. The change in pH units was more pronounced for

Fig. 1. Organic acids content of yarrow kombucha beverages.

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J.S. Vitas et al.
products obtained on infusions and amounted approximately 4 pH units
in comparison to 2–3 pH units for SWE. The amount of the herb also
influenced the fermentation in the way that higher amount caused in-
tensification of the process. This pointed out that extracted compounds
may have caused stronger kombucha activity. Yarrow kombucha bev-
erages on SWE had pH values that were lower in comparison to tradi-
tional products, while products on infusions had slightly higher pH
values.
The established pattern for total acidity (Table 1) was the same as
for the pH values. Total acidity of the substrates was very low, in the
range from 0.03 to 0.09 g CH3COOH/L and total acidity of kombucha
beverages was significantly lower for products obtained on infusions.
The amount of the used herb was the reason for higher intensity of the
fermentation, in both types of beverages. Acidity of K-YI and K-YII was
slightly different, with K-YII showing higher values. K-YIII had the
highest total acidity. Traditional kombucha products, obtained after
7 days of fermentation, have total acids content of 5.23 (black tea) and
6.55 (green tea) g of acetic acid per liter of the beverage (Malbaša et al.,
2011). Beverages obtained on yarrow SWE had considerably higher
total acidity ranging from 10.76 to 17.79 g acetic acid/L and sample K-
Y1.13 had much lower total acids content compared to traditional
products. Only results for sample K-Y2.26 showed content which was in
accordance to black tea kombucha beverages.
The major characteristic of kombucha beverage visible to the naked
eye is the formation of the cellulose pellicle layer that floats on the
surface of the product. Initial substrates do not possess biomass, be-
cause it is the result of the Acetobacter xylinum activity (Jayabalan et al.,
2014). In this investigation, fermentation was performed in 600 mL
beakers, with 10 cm diameter. Cellulosic pellicle is formed depending
on the dimension of the used bioreactor, as well (Jayabalan et al.,
2014). Yield of biomass (Table 1) was not meaningfully different for
beverages obtained on infusions. Results for the biomass indicated more
intensive fermentation process on SWE with higher herb content.
Lowest value was measured for sample K-YII and highest for K-YIII.
3.2. Organic acids
Organic acids are common constituents of beverages and foodstuffs.
These compounds affect sensory properties and chemical and micro-
biological stability of food. Organic acids also play an important role in
biological processes by its involvement in metabolic pathways, as in-
termediate or final products (González & González, 2013). Oxalic,
formic, acetic, lactic, succinic, malic, and citric acid were examined in
all samples. Results showed that lactic acid was not present in any of
the samples, although it was quantified in traditional kombucha pro-
ducts, but in very low concentrations (Jayabalan et al., 2007). Analysis
showed that SWE did not contain any of the examined compounds and
infusions do not contain lactic, acetic, malic and citric acid. Results of
HPLC analysis of organic acids in kombucha beverages are presented in
Fig. 1.
Oxalates are produced as a result of carbohydrates oxidation by
bacteria. Oxalic acid was determined in infusions yielding
0.14 ± 0.00 g/L (Y1.13) and 0.16 ± 0.00 g/L (Y2.26) and content
was slightly higher in sample with higher herb content. Kombucha
beverages showed considerably higher oxalic acid content in compar-
ison to the corresponding initial substrates (Fig. 1a). This indicated that
kombucha activity caused the production of oxalic acid, which was
more abundant in beverages produced on YI, YII and YIII and was in
2.53–3.45 g/L range. Higher herb content influenced positively the
oxalic acid production in these types of beverages.
Formic acid anion is produced by carbon dioxide reduction in re-
action catalyzed by the formate dehydrogenase. Infusions contained
0.04 ± 0.00 g/L (Y1.13) and 0.06 ± 0.00 g/L (Y2.26) of formic acid.
SWE yarrow kombucha beverages had formic acid in 1.51–2.33 g/L
range (Fig. 1b).
Acetic acid is the most characteristic product of kombucha
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Journal of Functional Foods 44 (2018) 95–102
fermentation and it was determined in all kombucha beverages. It is
produced by acetic acid bacteria from ethanol (Jayabalan et al., 2007).
Herb content in the initial substrate had pronounced positive influence
on acetic acid production (Fig. 1c). Acetic acid ranged from 8.12 to
15.10 g/L in SWE kombucha beverages. Only results for sample K-Y1.13
were in accordance with acetic acid content in traditional products,
after 6 days of fermentation (Jayabalan et al., 2007). All other bev-
erages had expressively higher contents.
Succinic acid is the result of the metabolic activity of yeasts (Ye,
Yue, & Yuan, 2014) and it was determined in samples Y1.13
(0.09 ± 0.00 g/L) and Y2.26 (0.05 ± 0.00 g/L) and the content was
higher in the sample with lower herb content. The same pattern was
established for kombucha beverages obtained on infusions. Succinic
acid production was more pronounced on YI, YII and YIII samples,
amount increased with the increase in herb content and was in the
range from 0.16 to 0.30 g/L (Fig. 1d).
Malic acid can be produced by yeasts (Ye, et al., 2014). Kombucha
caused the production of malic acid and the content was higher in
sample K-Y2.26 (0.22 ± 0.00 g/L) in comparison to K-Y1.13
(0.10 ± 0.00 g/L) indicating positive influence of herb content. The
highest malic acid content showed sample K-YIII (1.59 ± 0.00 g/L)
also indicating positive influence of herb content. Sample K-YII had
slightly lower content than K-YI (Fig. 1e).
Citric acid can be synthesized by yeasts, but then it is taken into the
cell and catabolized (Ye, et al., 2014). Citric acid was determined only
in K-YII (0.04 ± 0.00 g/L) and K-YIII (0.07 ± 0.00 g/L) sample and K-
YIII showed higher citric acid content indicating positive influence of
herb content. Citric acid was not detected in traditional beverages, after
6 days of fermentation (Jayabalan et al., 2007).
Oxalic, formic, acetic and malic acid showed the same pattern in
content.
3.3. Total phenols
Phenolic compounds are plant secondary metabolites with a variety
of important functions. Besides, these compounds are important as
technofuctional and health-promoting constituents of food (Kammerer,
Kramer, & Carle, 2013). Phenolics are regarded as antioxidants because
of their ability to scavenge radical species (Jayabalan, Malbaša, &
Sathishkumar, 2016b). Scientific evidence suggest that the most
common phenols in yarrow are chlorogenic acid, vicenin-2, luteolin-
3′,7-di-O-glucoside, luteolin-7-O-glucoside, rutin, apigenin-7-O-gluco-
side, luteolin and apigenin (Benetis, Radušienė1, & Janulis, 2008).
Depending on the preparation techniques different extracts may contain
diferent phenols in different concentrations. Literature data suggest that
subcritical water extraction is superior extraction approach in com-
parison to other techniques for phenol isolation (Cvetanović, Švarc-
Gajić, Mašković, Savić, & Nikolić, 2015). In current study, all produced
beverages had higher total phenols contents in comparison to corre-
sponding initial substrates. This suggested that kombucha activity
caused degradation of complex phenolic compounds which lead to the
increase in their total content (Jayabalan et al., 2016b). The higher
amount of the added herb lead to the increase in total phenols content
(Fig. 2a). The content of total phenolics was higher in SWE than in
infusions. This trend was also established for kombucha beverages.
Differencies in total phenols contents between infusions and kombucha
produced on infusions was small, but they were more pronounced for
SWE and beverages obtained from SWE. For initial substrates the con-
tent was in the 0.12–0.25 mg ECA/mL range, and in 0.14–0.34 mg ECA/
mL range for kombucha products. Traditional products contained
around 90 (green tea kombucha) and 80 (black tea kombucha) µg gallic
acid equivalent, after 6 days of fermentation (Jayabalan, Subathradevi,
Marimuthu, Sathishkumar, & Swaminathan, 2008).
J.S. Vitas et al. Journal of Functional Foods 44 (2018) 95–102

Fig. 3. Vitamin C content of yarrow kombucha beverages and initial substrates.

content, 1.14 mg/L and 1.11 mg/L, respectively. Beverages obtained on

SWE had lower vitamin C content in comparison to products obtained
on infusions, suggesting that infusions are more suitable substrate for
bacteria activity. Samples K-YI and K-YIII had similar vitamin C content
(0.69 mg/L and 0.64 mg/L) while K-YII had lower content (0.48 mg/L).
The content of the added herb had no influence on vitamin C produc-
tion. Traditional beverages (Malbaša et al., 2011) have higher vitamin
C content (15.19 mg/L black tea kombucha and 7.88 mg/L green tea
kombucha) than new types of kombucha products examined in this
paper. This can be attributed to the fact that black and green tea are
more suitable for kombucha fermentation in the context of vitamin C
synthesis. Also, two different methods were employed for vitamin C
Fig. 2. Total phenols and flavonoids content of yarrow kombucha beverages and initial
analysis. Enzyme test for vitamin C determination in traditional kom-
substrates.
bucha products was used, while HPLC was employed in this in-
vestigation and represents far more sensitive technique.
3.4. Flavonoids

Huge interest in terms of this group of compounds is a consequence 3.6. Biological activity
of its strong biological activity. Apart from apigenin and its glicosides,
yarrow flower also contains a large amount of rutin, resveratrol, nar- Antioxidant, antimicrobial and antiproliferative activities of pro-
ingin, naringenin and myricetin. Also, morin, kaempferol and quercetin duced kombucha beverages, using in vitro tests, were determined.
may occur in this plant (Keser, Celik, Turkoglu, Yilmaz, & Turkoglu, Antioxidant activity in this investigation was determined by DPPH
2013). Total flavonoids content, in this study, was lower in kombucha radical scavenging and reducing power method.
beverages than in corresponding initial substrates and this pattern was
more pronounced in samples obtained on SWE (Fig. 2b). This suggests
that kombucha caused degradation of flavonoids, whose amount in 3.6.1. DPPH scavenging ability
infusions and SWE was higher in samples which contained more herb. DPPH radical scavenging test is the most common method for an-
Flavonoids content was slightly higher in samples YI (0.10 ± 0.00 mg tioxidant activity evaluation. DPPH radical is stable and synthetic.
ER/mL) and YII (0.11 ± 0.00 mg ER/mL) when compared to Y1.13 Results for AADPPH are given in Table 2. Kombucha beverages produced
(0.09 ± 0.00 mg ER/mL). The amount of the added herb had negative on SWE showed considerably higher AADPPH in comparison to kom-
influence on flavonoids content in kombucha beverages produced on bucha products on infusions. IC50 value was in 0.02–1.12 mg/mL range
SWE and positively for products obtained on infusions (Fig. 2b). Fla- for produced beverages. The higher amount of the added herb caused
vonoids content in kombucha beverages was in the range from 0.04 to the increase in AADPPH of products on infusions, while for products on
0.17 mg ER/mL. SWE this value was in very close range. Traditional kombucha bev-
erages also inhibited DPPH radicals by 37.00% (kombucha with black
3.5. Vitamin C tea) and 63.00% (kombucha with green tea), after 7 days of fermenta-
tion (Malbaša et al., 2011). Jayabalan et al. (2008) also measured the
Vitamins are naturally present in low concentrations in food. In AADPPH of traditional kombucha beverages and determined that after
human organism vitamins exhibit health-promoting effects and are re- 6 days of fermentation black tea kombucha inhibited around 70.00%
sponsible for normal metabolic pathways and different physiological and green tea kombucha around 80.00% of DPPH radicals.
functions (Russell, 2013). Vitamin C was analyzed in this investigation
(Fig. 3.) since it is the most common vitamin that can be found in Table 2
kombucha beverages (Malbaša, et al., 2011; Jayabalan et al., 2016b). Antioxidant activity of yarrow kombucha beverages.
This vitamin has strong antioxidant activity due to its redox po-
Sample IC50 (mg/mL) EC50 (mg/mL)
tential (Russell, 2013). In kombucha fermentation it is assumed that
vitamin C is derived from glucose and synthetized by bacteria (Dutton, K-Y1.13 1.12 ± 0.01 > 10 ± 0.00
1980). Vitamin C content in all substrates showed small differences and K-YT2.25 0.50 ± 0.00 9.50 ± 0.30
K-YI 0.04 ± 0.01 0.74 ± 0.00
it was in the range of 0.38–0.42 mg/L. The content was higher in the
K-YII 0.02 ± 0.00 0.29 ± 0.01
obtained kombucha beverages which suggested that kombucha caused K-YIII 0.03 ± 0.00 0.53 ± 0.01
its synthesis. Samples K-Y1.13 and K-Y2.26 had almost the same

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Table 3
Antimicrobial activity of yarrow kombucha beverages.

Microbial strains MIC values (µg/mL)

K-Y1.13 K-Y2.26 K-YI K-YII K-YIII A N

Staphylococcus aureus ATCC 25,923 78.13 78.20 312.50 78.13 78.25 0.97 /
Klebsiella pneumoniae ATCC 13,883 19.53 19.53 312.50 156.25 78.13 0.49 /
Escherichia coli ATCC 25,922 39.10 78.25 78.13 134.60 312.50 0.97 /
Proteus vulgaris ATCC 13,315 78.13 78.13 312.50 156.25 156.40 0.49 /
Proteus mirabilis ATCC 14,153 156.25 156.25 39.10 156.40 78.13 0.49 /
Bacillus subtilis ATCC 6633 39.10 19.53 78.13 156.25 9.77 0.24 /
Candida albicans ATCC 10,231 78.13 156.40 312.50 39.10 39.10 / 1.95
Aspergillus niger ATCC 16,404 39.10 156.25 78.13 39.10 312.50 / 0.97

3.6.2. Reducing power
Results of reducing power analysis are presented in Table 2. This
test indicates the electron transfer capability and therefore the anti-
oxidative potential (Jayabalan et al., 2008). Kombucha beverages on
SWE showed higher reducing power than kombucha products on in-
fusions. The higher herb content caused decrease in reducing power
values, but K-YII (0.29 mg/mL) had the highest value. EC50 value
ranged from 0.29 to over 10 mg/mL. After 6 days of fermentation,
traditional kombucha products demonstrate reducing power around
0.20 (absorbance at 700 nm) (green tea kombucha) and approximately
0.05 (absorbance at 700 nm) for black tea kombucha (Jayabalan et al.,
2008).
3.6.3. Antimicrobial activity
Results of antimicrobial activity for kombucha beverages are given
in Table 3. Previous investigations suggested that traditional kombucha
products (Battikh, Chaieb, Bakhrouf, & Ammar, 2013), as well as
kombucha beverages obtained by fermentation of medicinal herb
(thyme, lemon verbena, rosemary, fennel and peppermint) extracts had
antibacterial and antifungal potential (Battikh, Bakhrouf, & Ammar,
2012) evaluated using agar diffusion method. Antimicrobial activity
resulted from produced organic acids, plant-derived phenolic com-
pounds, as well as enzymes, proteins and bacteriocins (Battikh, et al.,
2013; Battikh, et al., 2012). To our knowledge, these results represent
the first examination of the minimum inhibitory concentrations for
yarrow kombucha beverages. All produced beverages exhibited anti-
microbial activity. Investigation included testing of the samples to-
wards 6 bacterial (Staphylococcus aureus, Klebsiella pneumoniae, Escher-
ichia coli, Proteus vulgaris, Proteus mirabilis and Bacillus subtilis) and 2
yeast strains (Candida albicans and Aspergillus niger). The highest anti-
microbial potential had the sample K-Y1.13 and the lowest the sample
K-YI. Products obtained on infusions had more pronounced anti-
microbial effect. K-YI showed remarkably higher activity towards Pro-
teus mirabilis and K-YIII towards Bacillus subtilis in comparison to other
samples. Beverages K-YII and K-YIII had the highest activity towards
Candida albicans and samples K-Y1.13 and K-YII to Aspergillus niger. MIC
values were in the range from 9.765 to 312.5 μg/mL which is con-
sidered to be very good antimicrobial activity in comparison to the
standard antibiotic Amracin (for bacteria) and Nystatin (for yeasts)
(Cvetanović et al., 2015). Kombucha beverages showed good anti-
microbial ability and the obtained results indicated that these products
have potential to be used as antimicrobial agents.
3.6.4. Antiproliferative activity
Srihari, Arunkumar, Arunakaran, & Satyanarayana (2013) de-
termined that traditional black tea kombucha beverage inhibited the
growth and reduced the posibillity of metastasis in prostate cancer.
Results of antiproliferative activity for yarrow kombucha products are
given in Fig. 4. Examination of antitumor activity included testing of
kombucha bevrages towards three different cell lines: cell line derived
from human rhabdomyosarcoma (RD), cell line derived from human
cervix carcinoma Hep2c (HeLa) and cell line derived from murine fi-
broblast (L2OB). All samples exhibited antiproliferative activity. Pro-
ducts obtained on infusions were most effective to Hep2c cells and
beverages produced on SWE had the highest activity to RD cells. An-
tiproliferative activity was higher for products on infusions.
3.7. Sensory analysis
Results of descriptive test showed that produced yarrow kombucha
beverages had sensory characteristics that were in accordance to sen-
sory quality of traditional kombucha products (Malbaša et al., 2008).
All samples had formed cellulosic pellicle that was floating on the
surface of the beverage because of the produced carbon dioxide. Colour,
odour, taste and appearance were greatly influenced by the initial
substrate. Beverages produced on SWE had acidic and pleasant taste
and odour characteristic for the used extracts. Taste and odour of K-
Y1.13 and K-Y2.26 were acidic, but not so pleasant and resemblant to
the used extract. Colour of K-YI was yellow, while colour of K-YII and K-
YIII was brown and dark-brown, respectively. K-Y1.13 and K-Y2.26
were brown, as well. In all beverages yeasts were on the bottom of the
fermentation vessel and they were also attached to the lower side of the
cellulosic layer. Samples K-YI, K-YII and K-YIII had clear appearance
and K-Y1.13 and K-Y2.26 were blurry.
Results of five point category scale test are given in Fig. 5. Colour
and taste of beverages on SWE were given the highest mark (5) and
colour and taste of products on infsuions were marked with 3. Re-
garding odour, kombucha beverages on SWE showed higher quality
(average mark 4.67) in comparison to products on infusions (mark 3).
Average mark of appearance was 4.33 for beverages on SWE and 2 for
products on infusions. The results indicated that best sensory char-
acteristics had samples obtained on SWE yarrow extract (average sen-
sory mark 5) and the lowest sensory quality showed samples produced
on yarrow infusions (average sensory mark 3).
4. Conclusions
Kombucha beverages were successfully produced on yarrow infu-
sions and subcritical water extracts.
The most suitable substrate, according to fermentation process
parameters (pH, total acidity, yield of biomass) was subcritical water
extract obtained at 115 °C, from 2.26 g yarrow flowers in 500 mL water.
Organic acids (oxalic, formic, acetic, succinic and malic) content
was higher in beverages produced from subcritical water extracts.
Vitamin C values were higher in beverages produced on infusions.
Total phenols and flavonoids contents depended directly on herb
amount.
Yarrow kombucha beverages produced by fermentation on sub-
critical water extracts showed higher antioxidant activity, but lower
antimicrobial and antiproliferative activity in comparison to products
obtained on infusions.
Kombucha beverages produced from subcritical water extracts of

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45
40
35

IC50 (μg/mL)
Hep2c cells
30
25 RD cells
20
L2OB cells
15
10
5
0

K-Y1.13

K-Y2.26

K-YI

K-YII

K-YIII

Cis-DDP
sample
Fig. 4. MTT assay graph of yarrow kombucha beverages.

radical. Journal of Agricultural and Food Chemistry, 48, 648–656.

Fig. 5. Sensory analysis of yarrow kombucha beverages.
yarrow had the highest sensory score.
Acknowledgement
This investigation is financially supported by Ministry of Education,
Science and Technological Development, Republic of Serbia (Grant III
46009).
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