KASHK WITH CAPER (CAPPARIS SPINOSA L.) EXTRACT: QUALITY DURING STORAGE
Рубрики: RESEARCH ARTICLE
Аннотация и ключевые слова
Аннотация (русский):
Introduction. Dairy products are an important part of the diet. Kashk is a traditional Iranian dairy product rich in protein. However, kashk has a high water content and is a good medium for the growth of microorganisms. The aim of this study was to investigate the effect of the ethanolic extract of caper fruit (Capparis spinosa L.) on reducing the microbial burden of kashk. Study objects and methods. The study objects were three kashk samples. The control sample was kashk without caper extract. Two experimental samples included kashk with 0.211 and kashk with 0.350 mg/mL of ethanolic caper extract. All the samples were tested for pH, sensory and antioxidant properties, colorimetric parameters, and microbial population. The experiments were performed on days 0, 7, 14, 21 and 28 of storage. Results and discussion. The results showed all the samples had pH within the standard values during the entire shelf life (3.96 to 4.53). The samples with 0.350 mg/mL of the caper extract had the lowest EC50 (12.05 μg/mL), i.e. the highest antioxidant activity. The increased concentration of the extract and storage time resulted in a decrease in L* and increase in b*, while did not impact a*. Staphylococcus aureus population increased more rapidly than Clostridium botulinum during the storage time, and the overall sensory acceptability of the kashk samples on days 0 and 7 received the highest score. Conclusion. The kashk samples containing 0.350 mg/mL of caper extract had an improved antimicrobial, antioxidant and antifungal properties and can be produced and consumed as a new functional product.

Ключевые слова:
Dairy products, plant extract, microbial population, antioxidant activity, sensory properties, shelf life
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INTRODUCTION
Nowadays, a demand for healthier food containing
essential nutrients is growing so that digestive health
is considered a key factor in producing functional food
products [1–3]. Dairy products are an important part of
the diet [4–6]. Kashk, a by-product of milk processing,
is traditionally obtained by boiling, condensing, or
drying buttermilk after buttering or lean yogurt [7].
The chemical composition of kashk includes 84.25% of
dry matter, 8.57% fat, 95.9% salt, 53.60% total protein,
11.08% ash, and 1.06% lactose. Kashk also contains
amino acids and such minerals as calcium, magnesium,
iron, sodium, and potassium [8, 9].
Generally, kashk is used as a flavoring agent [10].
Kashk results from soaking curd, adding water and salt,
grinding and sanitizing [11]. Kashk has a high microbial
contamination potential due to its high moisture and
protein content, and if contaminated, it can be very
dangerous and even lead to fatal cases. This product
is mostly exposed to Staphylococcus aureus and
Clostridium botulinum contamination [9, 12, 13].
Controlling foodborne pathogenic bacteria and
ensuring food safety is the most important issue for
those involved in food processing [14]. Due to the
detrimental effects of chemical preservatives, the need
for research into the antimicrobial effects of natural
preservatives and plant essential oils on the growth
of microorganisms in food models under laboratory
conditions has increased [13]. On the other hand, any
preservatives in kashk is prohibited to be used, while the
use of natural aromatic extracts or plants as a flavoring
agent is allowed [16].
Caper (Capparis spinosa L.) is a medicinal
plant from the Capparidaceae family and different
species of caper have different uses. Antimicrobial
effects of some species of caper on Staphylococcus
aureus, Streptococcus pyogenes, Helicobacter pylori,
Escherichia coli, and Bacillus cereus have been documented [15, 16]. The presence of stachydrine
and spermidine alkaloids, such as capparispine and
cadabicin, in the seeds, roots, flowers, and dried fruits
of caper allows using this plant as a nutritional or
pharmaceutical supplement worldwide [19–21].
The hydrophobic properties of caper extracts
increase its permeability into the cell membrane of
microorganisms, which disrupts all vital activities and
ultimately causes cell death of the microorganisms. In
addition, the extract can damage the enzymes involved
in energy regulation and synthesize constituents that
inactivate or destroy genetic materials [22, 23]. Its
antimicrobial properties are also due to the presence of
hydroxyl (OH) groups [24]. The aim of the present study
was to investigate the effect of caper fruit extract on the
quality and antimicrobial properties of kashk.
STUDY OBJECTS AND METHODS
Extraction of caper (Capparis spinosa L.).
In this study, caper fruit was collected and identified
in Khuzestan Province in southwestern Iran in 2020
(Fig. 1). The fruits were washed, dried and powdered
by using an electric mill. Then, 7 kg of dried caper
was extracted with 40 L of 70% ethanol for 24 h at
room temperature using an electric mixer. The extract
was then filtered with filter paper No.1 (repeated on
the remaining sediment). Next, all solutions were
concentrated in a vacuum rotary evaporator at 40°C, and
extraction efficiency was calculated based on [25].
Composition and antioxidant properties of caper
extract. The total amount of phenols was determined
by the Folin & Ciocalteu’s reagent, flavonoids by
aluminum chloride method, and antioxidant activity
by DPPH radical scavenging assay. Caper fruit
extracts were analyzed for quantitative and qualitative
determination of polyphenols and flavonoids using high
performance liquid chromatography reversal and diode
array detection. The apparatus was equipped with a
detector, a C18 reverse phase column (Prodigy ODS-3,
4.6×150 mm, 5 μm; Phemomenex, Torrance, CA) and a
linear converter unit. The column temperature was set
at 30 ± 1°C. Rinsing with acetonitrile aqueous solution
(97:3 ratio, both with 3% acetic acid) was performed as
the initial step. The lyophilized extract was mixed with
1 ml of mobile phase prior to analysis. Further
preparation was performed by centrifugation for
5 min at 12 rpm. Then, 20 μL of the solution was
injected directly into the high-performance liquid
chromatography system [26].
Minimum bactericidal concentration (MBC) and
minimum inhibitory concentration (MIC) of caper
extract. Staphylococcus aureus stock culture (ATCC
9144) and dried Clostridium perfringens (ATCC13124)
were taken from the Microorganism Collection Center
of Iran Scientific and Industrial Research Organization.
The stoke cultures were transferred into 10 mL Brain
Heart Infusion (BHI) broth and incubated in a shaking
incubator at 37°C for 24 h. Then, 0.1 mL of the culture
was transferred into 10 mL of BHI medium and
incubated at 37°C for another 24 h until the end of the
progressive phase.
Subsequently, diluted cultures were used to inoculate
plate agar and industrial kashk for subsequent target
inoculations. In a 96-well plate, 100 μL of the culture
medium (Müller Hinton broth) was added to all the
wells, and then 100 μL of the sample extract was added
to the first well. After mixing the culture medium and
the sample in the first well, 100 μL of it was added
to the second well and dilution continued until the
concentration of the extract in the wells was reduced
by half. Then, a uniform suspension of Staphylococcus
aureus and Clostridium perfringens half McFarland
1.5×108 CFU/mL were added to all the wells and the
96-well plate was incubated at 37°C for 24 h. At the
end, one well before the well in which turbidity was
considered minimum bactericidal concentration and the
first concentration in which turbidity was not observed
was minimum inhibitory concentration [27].
Preparation and quality evaluation of kashk
samples. Pasteurized kashk was randomly purchased
from a local manufacturer in Karaj. The experimental
samples were kashk with minimum bactericidal and
minimum inhibitory concentrations of the caper extract.
Kashk without the extract was used as control. Then,
qualitative tests of the kashk samples were performed
on days 0, 7, 14 and 28 of storage. The qualitative tests
included pH (with the help of a pH meter based on
AOAC 2000 standard), antioxidant properties (by using
DPPH radical scavenging assay), and colorimetric test
(by a HunterLab spectrophotometer based on CIELAB
system) [26, 28]. Table 1 shows the kashk samples under
study.
Microbial population in kashk during storage.
In this method, each specimen was infected with
Staphylococcus aureus and Clostridium perfringens
with a microbial population of 105 CFU/mL and kept at
refrigerator temperature. To determine the population
of Staphylococcus aureus, a certain amount of sample
was diluted and sterilized on a plate. Baird-Parker agar
was added to the plate with egg yolk emulsion with
tellurite and incubated at 3 Figure 1 Caper fruit under study 7°C for 72 h. The number of Table 1 Kashk samples under study
CK0 Control kashk on day 0 (the day of production)
CK7 Control kashk on day 7 of storage
CK14 Control kashk on day 14 of storage
CK21 Control kashk on day 21 of storage
Ck28 Control kashk on day 28 of storage
EKI0 Experimental kashk with MIC on day 0 (the day
of production)
EKI7 Experimental kashk with MIC on day 7 of storage
EKI14 Experimental kashk with MIC on day 14 of storage
EKI21 Experimental kashk with MIC on day 21 of storage
EKI28 Experimental kashk with MIC on day 28 of storage
EKB0 Experimental kashk with MBC on day 0 (the day
of production)
EKB7 Experimental kashk with MBC on day 7 of storage
EKB14 Experimental kashk with MBC on day 14 of storage
EKB21 Experimental kashk with MBC on day 21 of storage
EKB28 Experimental kashk with MBC on day 28 of storage
MBC is minimum bactericidal concentration
MIC is minimum inhibitory concentration
Table 2 Phenolic, flavonoid and EC50 properties of caper extract
Treatment Extraction
efficiency, %
Total content of phenolic
compounds, mg gallic acid per g
Flavonoid content,
mg catechin per g
EC50,
mg/mL
Extract with 70% of ethanol
and 30% of water
23.20 ± 0.14 0.07 ± 2 0.01 0.20 ± 12.16 0.03 ± 1.48
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Hematian A. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. Х–Х
content and EC50 values in DPPH free radical scavenging
assay indicates the direct effect of phenols on
antioxidant activity [24]. M. Mahboubi and A. Mahboubi
extracted Capparis spinosa extract with water, ethanol,
methanol and ethyl acetate solvents and reported the
EC50 values of 500, 560, 340 and 2000 (μg/mL), while
in our study this value was 1.48 (mg/mL), indicating
the antioxidant properties of caper. Other studies also
showed that caper has a significant antioxidant activity
[17, 26, 34].
Composition and antioxidant properties of caper
extract. In recent years, phenolic compounds have
received special attention because of their biological
activities. We analyzed phenolic compounds by means
of high-performance liquid chromatography and diode
array detection (Table 3).
Four previously phenolic compounds of caper
extract, such as catechin, gallic, chlorogenic, and
o-coumaric acid, reported in [32] were also observed
in this study. Previous studies have reported rutin
as one of the main components of the extract, which
was confirmed in this study as well. Rutin has a wide
range of biological activities such as antioxidant,
antimicrobial, antidiabetic and cholesterol-lowering
effects [26, 33]. Therefore, the remarkable biological
effects of caper can be attributed to rutin, which can be a
source of bioactive compounds.
Minimum inhibitory concentration (MIC) and
minimum bactericidal concentration (MBC) of
caper extract. The microbial properties of kashk are
defined by the limit of Staphylococcus aureus and
sulfite reductive Clostridia. The results of MIC and
MBC of Capparis spinosa extract for the two target
microorganisms are shown in Table 4.
In the present study, the MBC level for
Staphylococcus aureus (0.283 mg/mL) was lower
than that for Clostridium perfringens (0.350 mg/mL).
Staphylococcus aureus lacks capsules and spores but
is resistant to drought and withstands salt up to 10%.
Clostridium perfringens is a Gram-positive, sporozoic,
and usually capsulogenic bacterium, with a higher
resistance to the extract than Staphylococcus aureus.
Various mechanisms have been proposed so far for the
antimicrobial activity of the extracts. The most accurate
theory suggests that the number and position of hydroxyl
groups are a key factor in the antimicrobial activity of
phenolic compounds, flavonoids, quercetin and their
derivatives.
Other mechanisms, such as flavonoids as a result
of the ability to form complexes with the cell wall and
inhibit the growth of microorganisms, or phenolic
compounds by enzymatic activity through reaction
with sulfhydryl groups or nonspecific interactions
with proteins prevent enzymatic and thus exhibit their
antimicrobial activity. Polyphenols are also able to form
high molecular weight soluble complexes with proteins,
thereby attaching to the bacteria and destroying the
receptors present on the bacterial cell surface [37].
Quercetin and its derivatives also inhibit bacterial
growth through the DNA gyrase inhibition [27].
Rahnavard and Razavi showed that caper extracts
demonstrated the antibacterial activity against a
variety of Gram-positive and negative bacteria,
including Staphylococcus epidermis, Staphylococcus
faecalis, Staphylococcus aureus, Micrococcus luteus,
and Bacillus cereus [21]. The aqueous extract of
caper fruit did not show any antibacterial activity,
while the ethanolic extract had the antimicrobial
effect on a variety of Streptococcus spp. and Gramnegative
bacteria [17]. The comparing of results
from different studies in this case seems complicated
because the results are influenced by factors such
as the composition and type of culture medium,
microorganism growth phase, the volume of culture
medium, pH, temperature, and incubation time. The
chemical composition, type, and the mechanism of
action of phenolic compounds also play a part in
antimicrobial activity [38]. Since one of the aims of the
present study was to increase the kashk shelf life, we
used MIC and MBC of Clostridium perfringens, which
were high, as the amount of extract used in the kashk
samples.
pH determination. The pH test of the kashk samples
is shown in Table 5. Statistical analysis of pH-dependent
parameters showed that the concentration of extract,
time, and extract × time had a significant effect on kashk
pH (P < 0.05), i.e., pH reduced with time. On the other
hand, the addition of the extract increased the pH value.
Based on standard, pH higher than 4.5 is considered Table 5 pH and DPPH radical scavenging ability of kashk samples during 28-day storage
Time, days pH EC50, μg/mL
CK EKI EKB CK EKI EKB
0 0.03 ± 4.53ab 0.02 ± 4.54a 0.04 ± 4.56a 0.5 ± 32.74c 0.7 ± 15.11j 0.5 ± 12.05k
7 0.02 ± 4.36c 0.01 ± 4.51b 0.03 ± 4.53ab 0.2 ± 33.28c 0.5 ± 17.10f 0.2 ± 14.07h
14 0.01 ± 3.99f 0.03 ± 4.41c 0.05 ± 4.42c 0.7 ± 33.47c 0.9 ± 17.40f 0.4 ± 14.24h
21 0.01 ± 3.29j 0.04 ± 4.05e 0.02 ± 4.11d 1.1 ± 35.03b 1.3 ± 18.05e 0.5 ± 15.10j
28 0.04 ± 2.96h 0.01 ± 3.94f 0.01 ± 3.96f 0.8 ± 40.12a 0.7 ± 20.15d 0.8 ± 17.05f
CK: control kashk, EKI: experimental kashk with MIC (0.211 mg/mL of caper extract), and EKB: experimental kashk with MBC (0.350 mg/mL of
caper extract)
Letters a‒h indicate significant differences
Table 6 Colorimetric characteristics of kashk samples
Time,
days
L* a* b*
CK EKI EKB CK EKI EKB CK EKI EKB
0 0.4 ± 67.20a 0.5 ± 67.17a 1.2 ± 67.15a 0.06 ± 3.05a 0.06 ± 3.05a 0.05 ± 3.05a 1.2 ± 22.66e 1.0 ± 22.55e 1.4 ± 22.50e
7 0.9 ± 67.08a 1.4 ± 67.07a 1.0 ± 67.07a 0.10 ± 3.00a 0.02 ± 3.09a 0.01 ± 3.06a 0.6 ± 24.39d 0.4 ± 24.41ce 0.5 ± 23.35de
14 0.6 ± 65.17c 0.8 ± 66.06b 0.4 ± 66.26b 0.04 ± 3.07a 0.04 ± 3.11a 0.03 ± 3.09a 1.3 ± 24.99dc 0.5 ± 25.20c 0.9 ± 24.05d
21 0.5 ± 65.10c 0.9 ± 65.05c 1.3 ± 65.07c 0.03 ± 3.03a 0.02 ± 3.12a 0.05 ± 3.10a 0.7 ± 25.15c 0.9 ± 25.75bc 1.1 ± 25.65bc
28 1.1 ± 60.15e 2.4 ± 61.10d 7.1 ± 61.44d 0.08 ± 3.10a 0.03 ± 3.00a 0.04 ± 3.04a 1.0 ± 27.20a 0.4 ± 26.04bc 0.6 ± 26.20b
CK: control kashk, EKI: experimental kashk with MIC (0.211 mg/mL of caper extract), and EKB: experimental kashk with MBC (0.350 mg/mL of
caper extract) Letters a‒e indicate significant differences 
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Faraji et al. stated that addition of 0.5–2% ethanolic
extract of Allium stipitatum over a 21-day storage resulted
in an increase in L* (from 53.3 to 70.2), no change in a*
(around 3.49), and increased b* (from 17.05 to 49.56) [40].
Hosseini and Ansari reported that the addition of
modified Tapioca starch to kashk over a 60-day storage
increased L*, but did not impact a* and b* [29].
The effect of microbial population on shelf life
of kashk samples. Bacterial population changes of
Staphylococcus aureus and Clostridium perfringens
in the control and experimental kashk samples (with
the caper extract) during 28 days of storage at 4°C
are shown in Fig. 2. The initial concentration of
Staphylococcus aureus in the control sample was
4.91 CFU/mL, which remained unchanged until the
end of storage. By the end of storage (on day 28), the
initial amount of Staphylococcus aureus had reduced to
0.6 CFU/mL in the experimental sample with MIC
(0.211 mg/mL of the caper extract), and to 0.3 CFU/mL
in the kashk with MBC (0.350 mg/mL).
The initial bacterial count of Clostridium perfringens
in the control was 4.50 CFU/mL, which had not
increased significantly by day 28 of storage. In the
kashk with MIC (0.211 mg/mL of the caper extract), the
initial amount of Clostridium perfringens reduced to
4 CFU/mL, and in the kashkl with MBC (0.350 mg/mL),
to 3.50 CFU/mL. The high susceptibility of
Staphylococcus aureus to the caper extract can be
related to phenolic compounds present in the extract [11].
Although the extract under study also reduced
the initial amount of Clostridium perfringens in
kashk, the reduction was less than a logarithmic cycle
indicating its resistance to the extract. The decrease in
microbial load in the whole system could be due to the
intrinsic acidic pH of kashk, but after day 21 a different
trend was observed for Clostridium perfringens in
the control sample. The overall results showed that
with the increasing of storage time of the extractcontaining
kashk samples, Staphylococcus aureus
bacterial population decreased more rapidly than that
of Clostridium botulinum, but none reached zero.
Golestan et al. investigated the antimicrobial properties
of the ethanolic extract of Allium stipitatum against
Clostridium botulinum and Staphylococcus aureus in
kashk. They found that Staphylococcus aureus count
decreased more rapidly with increasing storage time, it
had reached zero by the end of day 21 of storage [13].
Sensory properties of kashk. Figure 3 illustrates
the effect of the caper extract on the sensory properties
of the kashk samples based on the evaluation of
panelists. The results showed that the highest scores of
texture, smell, taste, oral feeling, adhesion, and general
acceptability had the kashk samples on days 0, 7 and 14
of storage. With time, namely after 48 days of storage,
the samples had the lowest overall acceptability.
The results also showed that the texture of the
samples containing the extract received the highest
score on day 14, while the control sample on day 14 had
a lower score. However, the odor of the control sample
had a higher score comparedc to that of the experimental
kashk. There was no significant difference between the
samples regarding taste and oral sensation. It is worth
to note that the adhesion factor score remained maximal
for all the samples during the storage time. The general
acceptability of the kashk samples received the highest
scores on days 0 and 7, and the lowest scores, on day 48
in all the three samples.
Golestan et al. demonstrated that peppermint
essential oil at concentrations of 1500 and 2500 ppm
and Mentha pulegium essential oil at a concentration of
2500 ppm had significant effects on taste of kashk [13].
Kashk samples containing 1500 ppm of Mentha
pulegium essential oil and control samples were
introduced as suitable samples, but kashk samples with
Figure 2 Dependence of Staphylococcus aureus concentration
in kashk samples on storage time. CK: control kashk, EKI:
experimental kashk with MIC (0.211 mg/mL of caper extract),
and EKB: experimental kashk with MBC
(0.350 mg/mL of caper extract)
Figure 3 Dependence of Clostridium perfringens
concentration in kashk samples on storage time. CK: control
kashk, EKI: experimental kashk with MIC (0.211 mg/mL
of caper extract), and EKB: experimental kashk with MBC
(0.350 mg/mL of caper extract)
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Hematian A. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. Х–Х
Mentha pulegium essential oil at the concentrations of
1500 and 2500 ppm were acceptable for panelists.
Agulló et al. studied the effect of modified Tapioca
starch on sensory properties (i.e., texture, smell, taste,
oral sensation, adhesion and general acceptance) of
kashk [29]. Their results showed that the type of starch
affected the sensory properties. The sample with 1.5%
hydroxypropylated tapioca starch had the best result and
the control sample received the lowest score.
CONCLUSION
The results of the study showed that the caper
(Capparis spinose L.) extract had no adverse effects on
pH of kashk during storage time, and the kashk sample
with the extract at the concentration of 0.350 mg/mL
had the lowest EC50 (12.05 mg/mL), or the highest
antioxidant activity on day 0 of storage. The increased
extract concentration and storage time resulted in a
decrease in L* and an increase in b*, while they did not
influence a*.
The number of bacteria had gradually decreased
in the kashk samples with both concentrations of the
extract by the end of a 28-day storage. With increasing
the storage time, Staphylococcus aureus bacterial
population declined compared to Clostridium botulinum.
The sensory evaluation results showed that the
texture of the extract-containing samples had a higher
score, which was even higher by day 14, and the control
sample had a lower score on the same day. But in terms
of smell the control samples were superior to the extractcontaining
samples.
In general, we can conclude that the kashk samples
containing 0.350 mg/mL of caper extract had improved
antimicrobial, antioxidant, and antifungal properties and
can be considered as a new functional product.
CONTRIBUTION
Authors are equally related to the writing of the
manuscript and are equally responsible for plagiarism.
CONFLICT OF INTEREST
The authors declare no conflict of interest.

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