journal of functional foods 10 (2014) 336–345

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The biological activity of fermented dairy

products obtained by kombucha and
conventional starter cultures during storage

D. Hrnjez *, Ž. Vaštag, S. Milanović, V. Vukić, M. Iličić, Lj. Popović,

K. Kanurić
Faculty of Technology, University of Novi Sad, Novi Sad, Serbia

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

Article history: The effects of kombucha inoculum as a new starter culture for milk fermentation were in-
Received 3 April 2014 vestigated, during 14 days of storage. The antioxidant capacity, angiotensin converting enzyme
Received in revised form 10 June (ACE) inhibitory activity, degree of proteolysis (DP), content of vitamin C as well as sensory
2014 properties of kombucha fermented milk, were analysed and compared with fermented milk
Accepted 16 June 2014 products obtained by commercial probiotic (ABT-10) and yoghurt (YF-L812) starter cul-
Available online 12 July 2014 tures. The kombucha fermented milk product (K) showed similar trend of changes in pH,
DP and sensory properties as products obtained by probiotic (P) and yoghurt (Y) starters.
Keywords: Significant ACE inhibitory was determined in all fermented products, which increased during
Fermented dairy products storage. Kombucha product had the highest ACE activity (63.43%) at the end of storage com-
Kombucha pared with probiotic and yoghurt products. In all products, higher 2,2’-azino-bis-(3-
ACE inhibitory ethylbenzthiazoline-6-sulphonic acid) (ABTS) than 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical
Antioxidative capacity scavenging activity was determined, while both activities slightly decreased during storage.
Vitamin C Based on estimated biological activity the kombucha product can be considered as an in-
novative fermented dairy product suitable for human nutrition.
© 2014 Elsevier Ltd. All rights reserved.

1. Introduction
The consumption of yoghurt has a long tradition in many coun-
tries, with its general acceptance as a healthy and nutritious
food. Nowadays, production of yoghurt with elevated ben-
efits on human health has become one of the major focuses
in dairy industry, as current trends on the market are towards
development of functional foodstuffs, especially fermented milk
products. Over the past years, yoghurt has been proved as an
ideal food to be fortified with diverse functional ingredients,
such as vitamins, probiotics, prebiotics (Sadeq et al., 2013;
Tamime, Hassan, Farnworth, & Toba, 2007; Tripathi & Giri, 2014).
On the other hand, the use of non-conventional, functional
starter cultures holds great promises to manufacture various
yoghurts with desirable technological, nutritional and benefi-
cial health advantages (Tripathi & Giri, 2014). Recent research
studies have focus on naturally occurring bioactive compo-
nents in fermented milk products, such as bioactive peptides
(Korhonen, 2009; Urista, Fernandez, Rodriguez, Cuenca, & Jurado,
2011). Nowadays, bioactive peptides have gained increasing at-
tention for human health promotion and deferment of age-
related diseases (Kittiphattanabawona, Benjakul, Visessanguan,
& Shahidi, 2013).

* Corresponding author. Tel: +381 21485 3719; fax: +381 21450 413.
E-mail address: dajana@tf.uns.ac.rs (D. Hrnjez).
http://dx.doi.org/10.1016/j.jff.2014.06.016
1756-4646/© 2014 Elsevier Ltd. All rights reserved.
 journal of functional foods 10 (2014) 336–345
Milk proteins are well-known precursors of peptides
exerting antioxidant, antihypertensive, antimicrobial,
immunomodulatory, anticancer, mineral-binding and opioid ac-
tivity (Chibuike & Aishwarya, 2014; Korhonen, 2009;
Samaranayaka & Li-Chan, 2011). These peptides can be re-
leased during fermentation of milk by proteolytic starter cul-
tures or by addition of proteolytic enzymes. A number of these
peptides may be available in the final products, primarily de-
pending on the proteolytic capacity of microorganisms taking
part in milk fermentation. So far, in fermented milk prod-
ucts, such as cheese and yoghurt, the potential antihyperten-
sive (namely, angiotensin I converting enzyme, ACE inhibitory)
peptides have been mostly studied (Gonzalez-Gonzalez, Gibson,
& Jauregi, 2013; Papadimitriou et al., 2007).
For centuries, kombucha has been known as a consor-
tium of yeasts (Pichia, Zygosaccharomyces, Saccharomyces,
Schizosaccharomyces, Saccharomycodes, Brettanomyces,
Torulaspora and Candida) and acetic acid bacteria (Acetobacter
and Gluconobacter) (Chen & Liu, 2000; Trovatti, Serafim, Freire,
Silvestre, & Neto, 2011; Zhang, Zhang, & Xin, 2011). Other-
wise, novel research has indicated the significant presence of
lactic acid bacteria (LAB) (Marsh, O Sullivan, Hill, Ross, & Cotter,
2014; Wu, Gai, & Ji, 2004; Zhang et al., 2011). Marsh et al., (2014)
suggested that Lactobacilli are more prevalent in kombucha
than was previously understood, particularly at the later stages
of fermentation. The same authors found Lactobacillus
kefiranofaciens subsp. kefirgranum as the more abundant
genera of LAB in kombucha culture. Petrušić, Radulović,
Paunović, and Obradović (2011) also showed presence
of Lactobacillus plantarum, Streptococcus thermophilus,
Streptococcus bovis, Streptococcus lutetiensis, Brevibacterium
sp. as a constituents in kombucha inoculums on black
tea.
Kombucha has been traditionally used for fermentation of
sweetened black or green tea (Dufresne & Farnworth, 2000; Teoh,
Heard, & Cox, 2004). Kombucha fermented product contains
ethanol, carbon dioxide, a high concentration of acid (glu-
conic, acetic and lactic) and a number of other health-promoting
metabolites. Therefore, it is considered to be beneficial
beverage in cases of: digestive ailments, diabetes,
hypercholesterolaemia, high blood pressure, combating stress
and cancer as well as body vitalisation, among others (Aloulou
et al., 2012; Guttapadu, Yang, & Wieger, 2000; Houda, Amina,
& Emna, 2012; Jayabalan et al., 2011; Semantee, Ratan, &
Parames, 2013; Thummala, Ramachandran, Jagadeesan, &
Uppala, 2013). Recent studies presented the technological and
nutritional potential of kombucha as an innovative starter
culture in dairy industry (Iličić et al., 2012; Malbaša, Lončar,
Milanović, & Kolarov, 2009a; Vukic et al., 2014). As the result
of milk fermentation by kombucha inoculums, products similar
to yoghurt or kefir are produced (Iličić et al., 2013; Milanović
et al., 2008, 2012).
The aim of this study was to investigate the effects of
kombucha inoculum as a new starter culture for milk fermen-
tation, during 14 days of storage. The antioxidant capacity, an-
giotensin converting enzyme (ACE) inhibitory activity, degree
of proteolysis (DP), content of vitamin C as well as sensory prop-
erties of kombucha fermented milk, were analysed and com-
pared with fermented dairy products obtained by commercial
probiotic (ABT-10) and yoghurt (YF-L812) starter cultures.
337
2. Materials and methods
2.1. Materials
All the chemicals used for the experiments were of analyti-
cal grade. DPPH (2.2-diphenyl-1-picrylhydrazy), angiotensin-I
converting enzyme (ACE) from rabbit lung, N-hippuryl-His-
Leu hydrate were obtained from Sigma (St. Louis, MO, USA),
m-phosphoric acid (Riedel-de Haën, Seelze, Germany), aceto-
nitrile, acetic acid and vitamin C (J.T. Baker, Deventer, Holland)
HPLC grade.
2.2. Methods
2.2.1. Sample production
Fermented milk products were manufactured in laboratory con-
ditions, from pasteurised (72 °C (161 °F) for 15 seconds) and
homogenised milk with 2.8% fat (Dairy Subotica, Subotica,
Serbia).
The following starter cultures were used for milk
fermentation:
1. Kombucha inoculum – Kombucha was cultivated on black
tea (Camellia sinensis – oxidised, 1.5 g L–1) with saccha-
rose concentration of 70 g L–1. The tea was cooled at the
room temperature, after which inoculum from a previ-
ous fermentation was added in concentration of 10%. In-
cubation was performed at 25 ± 2 °C for 7 days (Malbaša
et al., 2009b). Kombucha inoculum in concentration of
10% (30 mL) was applied for milk fermentation. Total
number of viable cells was as follows: approximately
5 × 104 of yeast cells per cm3 of the reaction mixture and
approximately 2 × 105 of bacteria cells per cm3 of the men-
tioned mixture (Lončar, Kanurić, Malbaša, Ðurić, &
Milanović, 2013).
2. Probiotic starter culture, ABT-7 – lyophilised probiotic
culture – Probio-Tek® contains LA-5®, Lactobacillus aci-
dophilus, BB-12®, Bifidobacterium, Streptococcus thermophilus
(Chr. Hansen, Hørsholm, Denmark).
3. Yoghurt starter culture – lyophilised yoghurt culture YF-
L812 contains Streptococcus thermophilus and Lactobacil-
lus delbrueckii ssp. bulgaricus (Chr. Hansen).
Commercial starters were added according to manufactur-
er’s specification, 0.005 g 100 g−1. All samples were produced
in triplets at 42 °C. Fermentation was continued until pH = 4.5
were reached. Then samples were cooled to 4 °C, homog-
enized by mixing, packed in polypropylene glasses and stored
in refrigerator at 4 ± 1 °C. Depending on the used starter culture
different samples were produced. The samples were labelled
as K0, K7, K14, P0, P7, P14, Y0, Y7, and Y14. Letters K, Y, P
indicate used starter culture (K – kombucha, P – probiotics,
Y – yoghurt) and number 0, 7 and 14 indicate the day of
storage.
Chemical quality was tested in fermented dairy products after
production using the following methods (Carić, Milanović, &
Vucelja, 2000): dry matter (DM) (IDF/ISO 21A:1982): milk fat (MF)
according to Gerber (IDF 105:1981); total proteins (TP) (IDF
20:1962); ash (A) (IDF 90:1979);
338 journal of functional foods 10 (2014) 336–345
pH values were determined using a pH-meter (pH Spear,
Eutech Instruments Oakton, England).
Total acids content was determined as titratable acidity. After
removing CO2 from the fermentation broth, a sample of 20 mL
was taken and titrated with 0.1 mol L−1 of NaOH, while phe-
nolphthalein was used as an indicator and calculated in g L−1
lactic acid according to equation:
m lactic acid (gL−1 ) = ( VNaOH × CNaOH × Mlactic acid 1000) × 50
2.2.2. Preparation of water-soluble extracts (WSEs) of
fermented dairy products
The WSEs were prepared according to Shori and Baba (2013).
Each sample (10 g) was mixed with 2.5 mL distilled water and
the pH was adjusted to 4.0 using 1 M HCl. The yoghurt was then
incubated in water bath (45 °C) for 10 min and the precipi-
tated proteins were removed by centrifugation at 10,000 g for
10 min, 4 °C. The supernatant was collected and the pH ad-
justed to 7.0 using NaOH (0.5 M), followed by another centrifu-
gation (10,000 g, 10 min, 4 °C). The supernatant was collected
and refrigerated until further analysis.
2.2.3. Determination of the degree of proteolysis
Degree of proteolysis was measured as the percentage of con-
centration of proteins soluble in 8% TCA. Briefly, 0.85 mL of each
sample was mixed with 0.4 mL of 25% TCA and the mixture
was left at 4 °C, for 30 min. Thereafter, the mixture was cen-
trifuged at 14,100 g (Ependorf Mini Spin Plus) for 5 min. The
obtained supernatant was filtered through membrane syringe
filter with diameter pore of 0.45 µm and analysed to deter-
mine the protein content by the method described by Lowry,
Rosenbrough, Fair, and Randall (1951), using bovine serum
albumin as the standard protein. The DP value was calcu-
lated as the ratio of 8% TCA-soluble protein and total soluble
proteins, expressed as a percentage.
2.2.4. ACE inhibitory activity
The ACE inhibitory activity of the water-soluble extracts was
performed according to Yoshie-Stark, Bez, Wada, and Wasche
(2004). In each assay, a sample (at different concentration) was
incubated at 37 °C for 80 min with hippuryl-His-Leu (HHL) in
0.2 mol L−1 potassium phosphate buffer containing 300 mmol L−1
NaCl (pH 8.30) and ACE solution. The final concentrations of
the HHL and ACE were 10 mmol L−1 and 25 mU mL−1, respec-
tively. The reaction was stopped by adding 110 µL of 1 mol L−1
HCl. The hippuric acid (HA) liberated from HHL was quanti-
fied with reverse-phase high performance liquid chromatog-
raphy (RP-HPLC). 20 µL of the solution was injected directly onto
Zorbax Eclipse XDB (Agilent Technology, Santa Clara, CA, USA)-
C18 column (4.6 Id × 150 mm, 5 µm, 80 Å) to separate HA from
HHL. The column was eluted with 50% methanol and 0.1%
trifluoroacetic acid (in water), with flow rate of 1 mL min−1, at
22 °C. The absorbance of the eluate was measured at 228 nm.
From the chromatograms, the peak area (corresponding to HA)
was integrated, and the amount of HA in the samples was quan-
tified. The ACE inhibition activity was calculated as follows
(Eq. 2):
( A − A0 ) − (B − B0 )
ACE inhibitory activity (% ) =
( A − A0 )
where A is the amount of HA in reaction without an inhibi-
tor, B is the amount of HA in reaction with potent inhibitor,
while A0 and B0 are the respected blanks (where HCl was added
in the test tube before the enzyme solution).
2.2.5. ABTS+. radical cation decolourisation assay (TEAC)
The radical scavenging activity was determined by the ABTS+.
decolourisation assay as described by Re et al. (1999). The
(1) bleaching rate of ABTS+. solution was monitored in the pres-
ence of the prepared water-soluble extracts at 734 nm, using
T80/T80+ UV-Vis spectrophotometer (PG instruments LTD).
Briefly, 0.2 mL of sample (containing 0.5, 0.75, 1.5 and 1 mg mL−1
proteins) was added to 2 mL of diluted ABTS +. solution
(A734nm = 0.7 ± 0.02) and the absorbance reading was taken up
to 10 min. Appropriate solvent blanks were run in each assay.
In the same way, a standard Trolox curve was prepared with
known Trolox concentrations. The percentage decrease in ab-
sorbance at 734 nm at 10 min was calculated and plotted as
function of potential antioxidant or Trolox concentrations. The
Trolox equivalent antioxidant coefficient (TEAC) was calcu-
lated by dividing the absorbance percentage inhibition versus
antioxidant concentration slope by the Trolox plot slope and
was expressed as mmol TEAC per mg of proteins in hydroly-
sates (mmolTEACmg−1).
2.2.6. DPPH• (2.2-Diphenyl-1-picrylhydrazyl radical)
scavenging activity assay
The free radical scavenging activity (RSA) of protein extracts
was evaluated using the DPPH• scavenging activity assay as de-
scribed by Morales and Jimenez-Perez (2001). In brief, an aliquot
of sample (200 µL) was added to 1 mL of a daily-prepared so-
lution of DPPH• (74 mg L−1) in ethanol. The mixtures were shaken
for 5 min at room temperature, centrifuged at 12 × 4 g
(Eppendorf Mini Spin plus) for 5 min and the absorption was
measured at 520 nm (T80/T80+ UV-Vis Spectrophotometer PG
instruments LTD). The concentration of DPPH• in the reaction
mixture was calculated from the calibration curve. The DPPH•
scavenging activity, expressed as antiradical activity (RSA) was
calculated as follows (Eq. 3):
⎛ [DPPHb ] − [DPPHt ]⎞
% RSA = ⎜ ⎟⎠ × 100 (3)
⎝ [DPPHb ]
where [DPPHb] is the concentration of DPPH• in blank (in the
presence of buffer instead of protein extract) and [DPPHb] is
the concentration of DPPH• in the sample (in the presence
protein extract), after 10 min of reaction.
2.2.7. Determination of vitamin C
One gram of fermented milk sample was transferred into a
10 mL volumetric flask and filled up with 3% solution of
m-phosphoric acid, in 8% solution of acetic acid. The ob-
tained solution was mixed for 5 min and vitamin C was ex-
tracted. Then the solution was filtered through filter paper (blue
label) and through membrane syringe filter (with diameter pore
of 0.45 µm) and the filtrate was used for HPLC analysis of
vitamin C. Agilent 1100 system (USA) with C-8 column and
diode array detector (DAD) was used for determination. A mobile
phase was ammonia-acetate solution (0.1 M; pH 5.1) with flow
(2)
rate of 0.4 mL min−1 and column temperature was 37 °C. Vitamin
 journal of functional foods 10 (2014) 336–345
Table 1 – Chemical composition of milk and fermented milk products obtained by yoghurt, probiotic and kombucha
starter cultures.
Chemical Milk Probiotic starter (P)
characteristics
pH 6.8 ± 0.00 4.57 ± 0.01
TTA (lactic acid mg/mL) – 7.38 ± 0.04
Dry matter (%) 11.56 ± 0.08 12.0 ± 0.02
Milk fat (%) 2.8 ± 0.001 2.8 ± 0.002
Total proteins (%) 3.08 ± 0.02 3.07 ± 0.04
Ash (%) 0.71 ± 0.001 0.75 ± 0.0016
C was used as a standard. All analyses were performed in
triplicate.
2.2.8. Sensory analysis
Sensory analysis of the samples was carried outlying expert
committee (10–15 trained panellists selected from University
staff members). The five point system and the descriptive
method with assessing the scoring range from 1 to 5, was used.
Sensory evaluations determined by the coefficient of impor-
tance for appearance – 1, colour – 2, flavour – 3, consistency –
4 and taste – 10. The single complex indicator which reflects
the overall sensory quality expressed as % of the maximum
quality was obtained by adding the single score. Dividing this
value by the sum of coefficients of interest (20) a weighted
average score is obtained, which expresses the overall quality
of the product.
2.2.9. Colour measurement
The colour of fermented dairy products is determined by pho-
toelectric tristimulus colourimeter CHROMAMETER CR-400,
Konica Minolta CRA33 extension. It works by measuring re-
flected colour and colour differences of different surfaces values
of the fermented milk products (whiteness (L-), red/greenness
(a-), and yellow/blueness (b-)). The software SpectroMagic
NXPROQCver 2.0 was used for the results processing.
2.2.10. Statistics
All chemical analyses and assays were performed in tripli-
cate for all produced samples (n) and values were expressed
as average value. Univariate treatments of the data were per-
formed by analysis of variance (ANOVA), using “Statistica 9”
software program. Duncan’s multiple range tests were per-
formed to evaluate significant differences among the studied
parameters. Differences among analysed parameters of pro-
duced samples with different starter cultures were consid-
ered statistically significant when P ≤ 0.05.
3. Results
3.1. Physicochemical characteristics of fermented
milk products
The chemical composition of milk and obtained fermented dairy
products are shown in the Table 1. Regardless the starter cul-
tures, the fermented milk products were not significantly dif-
ferent in dry matter, fat, proteins and ash content.
339
Yoghurt starter (Y) Kombucha starter (K)
4.52 ± 0.03 4.54 ± 0.02
7.24 ± 0.04 7.11 ± 0.02
11.91 ± 0.07 11.60 ± 0.09
2.8 ± 0.001 3.0 ± 0.001
2.98 ± 0.06 2.77 ± 0.05
0.73 ± 0.003 0.69
At the end of the milk fermentation, the pH values in the
products varied from 4.52 to 4.57 (Table 1). In the first week
of storage the pH was not changed significantly, while all
samples had a sharp decrease in pH during the second week
(Fig. 1a). The decrease in pH was followed by simultaneous in-
crease in titratable acidity (expressed as lactic acid content)
(data not showed). In general, higher acidification rate was ob-
served in the P and K samples compared with Y sample. At
the end of the storage, the pH values in the P, K and Y prod-
ucts reached 4.00, 4.10 and 4.35, respectively, which indicate
the products’ stability.
In part, the post-acidification of the fermented milk prod-
ucts is undesirable process; hence it could be consequence of
metabolism of microorganisms. Some microorganisms (e.g.
some lactic acid bacteria) can remain active even at cold storage
(Considine, Healy, Kelly, & McSweeney, 2000). In accordance with
the literature, these results also revealed that type of starter
culture affects the different post-acidification rate of final prod-
ucts, although further analysis is required to evaluate the re-
sponsible microorganisms. Nevertheless, the results indicated
that kombucha inoculum could ensure manufacturing of fer-
mented milk product with similar physicochemical charac-
teristics and acidity with respect to conventional starter
cultures. The significant influence (P ≤ 0.05) of combined effect
of starter culture and day of storage on pH value was
observed.
3.2. Degree of proteolysis
The extent of proteolysis was evaluated based on the index
of 8% TCA soluble proteins and expressed as degree proteoly-
sis (DP). The combined effect of storage time and starter cul-
tures on DP is presented in Fig. 1b.
Yoghurt as a type of fermented dairy product is not ex-
pected to have high extent of proteolysis, which is in accor-
dance with the obtained results. Compared with the raw milk,
by the end of fermentation the DP increased from 1.7 ± 0.02
in raw milk to 2.25 ± 0.034% in Y, 2.35 ± 0.041% in P and
2.56 ± 0.151% in the K product. During the cold storage the value
of DP increased in all samples, with significant variations among
them (Fig. 1b). The probiotic product had a continuous in-
crease of DP during the whole storage period, reaching the final
value of 3.56 ± 0.162% after 14 days of storage.
The kombucha and yoghurt products showed very similar
trend in the change of DP, it increased during the first week
and without significant change during the second week of
storage. The final values of DP were 2.77 ± 0.039 and
2.61 ± 0.071% in the K and Y samples, respectively.
340 journal of functional foods 10 (2014) 336–345

Fig. 1 – The changes during the storage period in: (a) pH value, (b) degree of proteolysis (DP). (c) IC50 values of the
angiotensin converting enzyme inhibitory activity; (d) content of vitamin C.

The results showed that interaction of factors starter
culture*day of storage had significant (P < 0.005) impact on DP
(Fig. 1b). As the extent of proteolysis mainly depends on the
proteolytic enzymes of starter cultures (Shah & Ravula, 2001),
it is evident that probiotic starter more likely produce protease/
peptidases compared with K and Y samples. However, lower
DP values in K and Y samples may be considered as the result
of symbiosis occurring among the bacteria populations. For in-
stance, S. thermophilus may be utilising the peptides and amino
acids liberated by L. bulgaricus and thus instead of being ac-
cumulated, they are further consumed (Slocum, Jasinski,
Anantheswaran, & Kilara, 1988).
Generally, it was observed that the probiotic starter induced
the highest rate of changes in both, pH and proteolysis, the
yoghurt starter had the lowest, while the product with
kombucha inoculum had values of these parameters within
this range. This could be additional conformation that the
kombucha inoculum is suitable for fermented milk produc-
tion, compared with conventional starter cultures.
3.3. ACE inhibitory activity
ACE (EC 3.4.15.1) plays a central role in the regulation of blood
pressure through the production of the potent vasoconstric-
tor, angiotensin-II, and the degradation of the vasodilator, bra-
dykinin. Captopril and other synthetic ACE inhibitors, used in
the clinical treatment of hypertension, could have some un-
desirable effects to humans (Acharya, Sturrock, Riordan, &
Ehlers, 2003). Therefore, natural ACE inhibitory food compo-
nents have gained great importance. Food protein derived pep-
tides, such as casokinins and lactocinins, have shown great
promise as functional food ingredients with potential to control
hypertension.
The ACE inhibitory activity of WSEs was examined by means
of the protein concentration range from 0.2 to 1 mg mL−1 in the
test tube. In all samples ACE inhibitory activity was deter-
mined at day 0, and further increased during the whole storage
period. After production, the kombucha starter showed lower
ACE inhibitory activity (46.27 ± 0.694%; P < 0.05) than the prod-
 journal of functional foods 10 (2014) 336–345
Table 2 – Radical scavenging activity (mean ± SD) of water-soluble extracts (WSE) measured by DPPH (2,2-diphenyl-1-
picrylhydrazyl) and ABTS [2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] methods during the storage.
Day of storage AADPPH %
Yoghurt Probiotic
0 day 9.4586 ± 0.301a
13.2299 ± 0.043b
7 day 9.8844 ± 0.381a 19.4951 ± 0.645b
14 day 6.1131 ± 0.301a 4.95742 ± 0.043b
a,b,c
Means in the same row with different alphabets are significantly different (P < 0.05).
ucts with yoghurt and probiotic starters (48.48 ± 0.724 and
51.70 ± 1.034%, respectively).
After 7 days of storage all samples had similar values of ACE
inhibitory activity, showing an increase after the second week.
The sample with kombucha starter had the highest ACE in-
hibition (IC50 = 0.25 mg mL−1) on the 14th day of storage, while
there were no significant differences between Y14 and P14
sample, reaching IC50 approximately 0.33 mg mL−1 (Fig. 1c). The
calculated IC50 values are in accordance with the results pre-
sented by other research (Donkor, Henriksson, Vasiljevic, &
Shah, 2007; Hernandez-Ledesma, Miralles, Amigo, Ramos, &
Recio, 2005; Papadimitriou et al., 2007). Donkor et al. (2007) also
reported an increase of the inhibitory activity after the second
week of storage for both probiotic and control yoghurt, even
though in the probiotic sample the activity decreased after the
first week.
In this study, different starter cultures led to differences in
ACE inhibitory activity in fermented products (Fig. 1c). This con-
firms that proteinase and peptidase activity of starter cul-
tures may affect the milk protein breakdown to various extents
and thus can yield a wide range of peptides with functional
properties, such as ACE inhibitory activity. Nevertheless, there
was not a clear correlation between the trend of changes in
DP and ACE inhibitory activity in any product, but both these
parameters increased during storage. The ACE inhibitory pep-
tides are known to be di-, tri- and oligopeptides. Conse-
quently, it is likely possible that during storage the short ACE
inhibitory peptides were derived from initially produced longer
peptides, while their concentration did not reflect on the TCA
soluble index used for DP quantification. Also it should be taken
into consideration that not only peptides exert ACE inhibi-
tory activity. The concentration of ionic Ca also could affect
the ACE inhibition, depending on its concentration (Gonzalez-
Gonzalez et al., 2011). Some polyphenol compounds could also
have this activity and this should be further investigated. Even
observed weak in vitro antihypertensive activities, milk derived
bioactive peptides are considered as ‘pro-drugs’ that serve as
precursors of ACE inhibitors during physiological proteolysis
(Chibuike & Aishwarya, 2014). This study, however, indicate that
all used starter cultures may have potential in development
of antihypertensive fermented milk product.
3.4. Antioxidant activity
The RSA of the water-soluble extracts of fermented milk prod-
ucts was evaluated by the ABTS+. and DPPH radical assays. Both
assays indicate the ability of substances to act as electron
donors or H atom donors in free radical reactions. The attempt
341
ABTS %
Kombucha Yoghurt Probiotic Kombucha
17.8832 ± 0.172c
50.6542 ± 0.323a
47.4733 ± 0.21b
41.0446 ± 0.123c
12.3175 ± 0.373c 54.8991 ± 0.541a 50.1879 ± 0.78b 36.2129 ± 0.112c
7.4817 ± 0.344c 51.7405 ± 0.810a 43.0556 ± 0.25b 39.1016 ± 0.189c
of free radical on biological molecules such as proteins, lipids
and DNA may be considered as the onset of several chronic
diseases (Chandrasekara & Shahidi, 2011). The ability of
kombucha fermented milk product to scavenge different radi-
cals: hydroxyl radical, superoxide radical anion and reducing
power were previously attributed to the phenolics, flavo-
noids, vitamin C and vitamins B presented in the kombucha
inoculums (Jayabalan, Subathradevi, Marimuthu, Satishkumar,
& Swaminathan, 2008).
All analysed samples showed significant scavenging ability
against ABTS+ radical cation. The highest activity was deter-
mined in the yoghurt (TEAC value 6.74 mmol mg−1) product, fol-
lowed by probiotic (6.34 mmol mg −1 ) and kombucha
(5.40 mmol mg−1) products. After the first week of storage a slight
increase of the activity was observed in the Y and P products.
Conversely after the second week of storage the activity de-
creased in all samples and significantly lower values were re-
corded compared with day 0. The highest TEAC value
(7.49 mmol mg−1) was observed in the sample Y7.
The DPPH RSA in K sample (Table 2) was significantly higher
(P > 0.05) after production (17.88 ± 0.17%) than in Y and P samples
(9.46 ± 0.3 and 13.23 ± 0.04% respectively). Kombucha starter used
for milk fermentation improved DPPH RSA during refriger-
ated period due to its own antioxidative capacity (Jayabalan
et al., 2008; Vitas, Malbasa, Lončar, & Grahovac, 2013). The trend
of DPPH activity differs among the samples, but as the ABTS
RSA, it decreased during the second week of storage. The
kombucha samples showed the highest DPPH RSA (P < 0.05) after
14 days of storage (7.48 ± 0.34%) compared with Y14 and P14
samples (6.11 ± 0.3 and 4.96 ± 0.04%, respectively).
Using ABTS radical cation, the free RSA of all samples were
between 36% and 55%, while with DPPH radicals the activi-
ties were below 18%. The difference between the DPPH and
ABTS RSA could be partially due, to previously reported dis-
advantages of DPPH if used for hydrophilic antioxidants, such
as protein hydrolysates (Chang, Wu, & Chiang, 2007; Tang et al.,
2010). This mostly refers to the fact that the DPPH can be dis-
solved only in organic media (especially in alcoholic media),
which can lead to precipitation of proteins whose activity is
analysed. On the contrary, ABTS can be solubilised in aqueous
and organic media; thus, radical scavenging activities of both
hydrophilic and lipophilic compounds can be measured (Tang
et al., 2010). Our results, which are in agreement with these
findings, suggest that the ABTS method was more appropri-
ate than the DPPH assay for the measurement of antioxidant
activity of fermented milk WSEs.
Overall, our results revealed that various starter cultures had
different influences on RSA of fermented products, mea-
342 journal of functional foods 10 (2014) 336–345

sured by either ABTS or DPPH method. As there was a lack of

correlation between DP and radical activities, bioactive pep-
tides cannot be assumed as only components responsible for
antioxidant activity. At least, they had not been in the con-
centration in which they influenced the overall antioxidant ca-
pacity of extracts. This suggests that some other components
such as phenol compounds, and vitamins should be further
investigated as potential contributors to the antioxidative ca-
pacity, as those components are suggested as important an-
tioxidants (Samaranayaka & Li-Chan, 2011; Zhong, Ma, &
Shahidi, 2012).

3.5. Vitamin C

Natural antioxidants such as vitamin C and tea extracts have

Fig. 2 – Sensory analyses of fermented dairy products
already been investigated and commercialised as alterna-
during storage period.
tives to synthetic antioxidants in food (Shahidi, 2000). In con-
tribution to the antioxidative potential, the content of vitamin
C was analysed in fermented dairy products. The highest
content of vitamin C after production was recorded in sample
K0 (0.5457 ± 0.017 mg 100 g−1) followed by P0 (0.4404 ± 0.014 mg
3.7. Colour assay
100 g−1) and Y0 (0.4078 ± 0.013 mg 100 g−1). Fermentation by
In addition to sensory evaluation, the colour measurements
komucha showed an increase of vitamin C due to the pres-
were performed on the samples by means of the CIE and CIE
ence of vitamin C in kombucha inoculum in concentration of
Lab colour system. Reflectance spectroscopy quantifies the
0.5096 ± 0.02 mg 100 g−1. Refrigerated storage had profound effect
colour in terms of tristimulus values, which are calculated in
on vitamin C content and it decreased in all samples, by 18.2,
a CIE L, a, and b colour spaces. The L* serves as the psycho-
8.7 and 26.8% in probiotic, yoghurt and kombucha samples, re-
metric correlation of perceived lightness ranging from white
spectively. These results are consistent with the research re-
(100) to black (0), which can be described using the terms strong,
ported by Oberman (1985) who found that fermentation by lactic
moderate and pale, along a grey scale.
acid bacteria resulted in a marked decline in content of vitamin
In contrary to the sensory evaluation (which is more sub-
C. In spite of the decline of vitamin C content after 7 and 14 days
jective methods), significant differences in colour (CIE and CIE
of storage, it still remained significantly higher (P < 0.05) in the
Lab system) between samples obtained by probiotic, yoghurt
sample obtained by kombucha starter than the samples pro-
and kombucha starters are evident (Table 3). Brightness (Y) of
duced using probiotic and yoghurt starter cultures. (Fig. 1d).
samples was from 63.51% in K0 up to 67.03% in Y0 and it was
It is important to emphasise that, sample K14 (sample on the
not significantly (P < 0.05) changed during the storage period.
14th day of storage) did not have significantly (P < 0.05) differ-
The whiteness values (L) of all fermented dairy samples were
ent content of vitamin C compared with sample produced by
not changed during 14 days of storage but it was significantly
yoghurt starter after production (Y0). This high content of
(P < 0.05) higher in samples obtained by the yoghurt and
vitamin C in kombucha product during the storage may con-
probiotic starters than the kombucha starter.
tribute to the antioxidant activity of the new type of fer-
Red/greenness (a-) values of all samples increased after
mented dairy product. Furthermore, significant correlation
7 days of storage and it declined though prolonged storage
between the highest values of antioxidant activities and the
period until the 14th day. The yellowness/blueness (b) value of
quantity of vitamin C in all samples was noticed. The samples
samples had similar trend such as a values. Storage time had
obtained by kombucha had the highest correlation between
a significant effect on the b values of the products. The b values
the RSADPPH and vitamin C content (R2 = 0.997).
of samples significantly declined during storage (P < 0.05). It is
evident that kombucha samples had the highest values of b
3.6. Sensory analysis coefficient and the smallest values of the a coefficient due to
colours of added inoculums.
The sensory evaluations of fermented dairy products are shown
in Fig. 2. No significant differences were observed in appear-
ance, flavour, taste, and overall preference scores between the
three groups of fermented dairy products. Even though, the con- 4. Conclusion
sistency and taste score was greater in Y0 and P0 samples than
in the K0 sample. The kombucha fermented dairy product had Fermented dairy product obtained by kombucha inoculum is
a characteristic, distinctive mild sour, refreshing taste and con- appropriate new product which could increase assortment of
spicuous aroma. However, prolonged storage period up to functional food. During the storage, the fermented milk product
14 days showed that kombucha inoculums affected the con- obtained by kombucha (K) showed similar trend in changes of
sistency and flavour which were reduced in Y and P samples, pH, degree of proteolysis (DP) and sensory properties as prod-
but increased in K sample. ucts with conventional probiotic (P) and yoghurt (Y) starters.
 journal of functional foods 10 (2014) 336–345 343

Table 3 – Effect of different starter culture on the color of fermented dairy products during storage assayed by (a) CIE
system, (b) CIE LAB system.
(a)
Sample CIE system
Y (%) λ (nm)
1 day 7 day 14 day 1 day 7 day 14 day
Milk 66.08 ± 0.04 – – 566.57 ± 0.03 – –
P 66.91 ± 0.018 67.46 ± 0.05 66.98 ± 0.04 569.19 ± 0.03 568.53 ± 0.04 569.79 ± 0.01
Y 67.03 ± 0.32 67.22 ± 0.06 66.76 ± 0.19 569.39 ± 0.04 568.65 ± 0.05 569.65 ± 0.01
K 63.51 ± 0.03 64.76 ± 0.08 64.49 ± 0.09 571.72 ± 0.01 571.05 ± 0.01 571.54 ± 0.04

(b)
Sample CIE LAB
L A B
1 day 7 day 14 day 1 day 7 day 14 day 1 day 7 day 14 day
Milk 85.04 ± 0.02 – – –3.3 ± 0.02 – – 5.86 ± 0.03 – –
P 85.46 ± 0.09 85.73 ± 0.03 85.50 ± 0.02 −2.94 ± 0.02 3.09 ± 0.01 2.67 ± 0.01 6.90 ± 0.02 6.67 ± 0.02 6.79 ± 0.01
Y 85.52 ± 0.17 85.62 ± 0.03 85.38 ± 0.02 −2.92 ± 0.02 3.00 ± 0.02 2.75 ± 0.04 7.03 ± 0.02 6.58 ± 0.02 6.85 ± 0.02
K 83.71 ± 0.17 84.36 ± 0.04 84.22 ± 0.01 −1.97 ± 0.01 2.21 ± 0.02 2.06 ± 0.02 7.09 ± 0.02 6.93 ± 0.02 7.12 ± 0.01

Significant ACE inhibitory activity was determined in all fer-
mented products, which increased during the storage. Still, the
samples obtained by kombucha inoculum had the highest ACE
inhibitory (63. 43%) after 14 days of storage. This study, however,
indicated that all used starter cultures may have potential in
development of antihypertensive fermented milk product. The
high content of vitamin C in kombucha fermented dairy prod-
ucts during the storage contributed to the DPPH antioxidative
capacity of the new type of fermented dairy product. In all
samples, higher 2,2’-azino-bis-(3-ethylbenzthiazoline-6-
sulphonic acid) (ABTS) than 1,1-diphenyl-2-picrylhydrazyl
(DPPH) radical scavenging activity was established, while both
activities slightly decreased after the second week of storage.
The differences in colour, flavour and consistency were ob-
served among the three groups of fermented dairy products
after 14 days of storage. Even more, after 14 days of storage
kombucha fermented dairy product had the highest overall
sensory quality. Based on these results, the kombucha inocu-
lum could ensure the manufacture of fermented dairy product
with similar physicochemical, distinctive sensory and pro-
nounced bioactive characteristics with respect to conven-
tional yoghurt starters.
Acknowledgements
Authors want to thank Ministry of Education, Science and
Technological Development of Republic of Serbia for the fi-
nancial support of research presented in this article, Project
No. 46009.
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