INTRODUCTIONNowadays, a demand for healthier food containingessential nutrients is growing so that digestive healthis considered a key factor in producing functional foodproducts [1–3]. Dairy products are an important part ofthe diet [4–6]. Kashk, a by-product of milk processing,is traditionally obtained by boiling, condensing, ordrying buttermilk after buttering or lean yogurt [7].The chemical composition of kashk includes 84.25% ofdry matter, 8.57% fat, 95.9% salt, 53.60% total protein,11.08% ash, and 1.06% lactose. Kashk also containsamino 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 microbialcontamination potential due to its high moisture andprotein content, and if contaminated, it can be verydangerous and even lead to fatal cases. This productis mostly exposed to Staphylococcus aureus andClostridium botulinum contamination [9, 12, 13].Controlling foodborne pathogenic bacteria andensuring food safety is the most important issue forthose involved in food processing [14]. Due to thedetrimental effects of chemical preservatives, the needfor research into the antimicrobial effects of naturalpreservatives and plant essential oils on the growthof microorganisms in food models under laboratoryconditions has increased [13]. On the other hand, anypreservatives in kashk is prohibited to be used, while theuse of natural aromatic extracts or plants as a flavoringagent is allowed [16].Caper (Capparis spinosa L.) is a medicinalplant from the Capparidaceae family and differentspecies of caper have different uses. Antimicrobialeffects of some species of caper on Staphylococcusaureus, Streptococcus pyogenes, Helicobacter pylori,Escherichia coli, and Bacillus cereus have been documented [15, 16]. The presence of stachydrineand spermidine alkaloids, such as capparispine andcadabicin, in the seeds, roots, flowers, and dried fruitsof caper allows using this plant as a nutritional orpharmaceutical supplement worldwide [19–21].The hydrophobic properties of caper extractsincrease its permeability into the cell membrane ofmicroorganisms, which disrupts all vital activities andultimately causes cell death of the microorganisms. Inaddition, the extract can damage the enzymes involvedin energy regulation and synthesize constituents thatinactivate or destroy genetic materials [22, 23]. Itsantimicrobial properties are also due to the presence ofhydroxyl (OH) groups [24]. The aim of the present studywas to investigate the effect of caper fruit extract on thequality and antimicrobial properties of kashk.STUDY OBJECTS AND METHODSExtraction of caper (Capparis spinosa L.).In this study, caper fruit was collected and identifiedin Khuzestan Province in southwestern Iran in 2020(Fig. 1). The fruits were washed, dried and powderedby using an electric mill. Then, 7 kg of dried caperwas extracted with 40 L of 70% ethanol for 24 h atroom temperature using an electric mixer. The extractwas then filtered with filter paper No.1 (repeated onthe remaining sediment). Next, all solutions wereconcentrated in a vacuum rotary evaporator at 40°C, andextraction efficiency was calculated based on [25].Composition and antioxidant properties of caperextract. The total amount of phenols was determinedby the Folin & Ciocalteu’s reagent, flavonoids byaluminum chloride method, and antioxidant activityby DPPH radical scavenging assay. Caper fruitextracts were analyzed for quantitative and qualitativedetermination of polyphenols and flavonoids using highperformance liquid chromatography reversal and diodearray detection. The apparatus was equipped with adetector, a C18 reverse phase column (Prodigy ODS-3,4.6×150 mm, 5 μm; Phemomenex, Torrance, CA) and alinear converter unit. The column temperature was setat 30 ± 1°C. Rinsing with acetonitrile aqueous solution(97:3 ratio, both with 3% acetic acid) was performed asthe initial step. The lyophilized extract was mixed with1 ml of mobile phase prior to analysis. Furtherpreparation was performed by centrifugation for5 min at 12 rpm. Then, 20 μL of the solution wasinjected directly into the high-performance liquidchromatography system [26].Minimum bactericidal concentration (MBC) andminimum inhibitory concentration (MIC) of caperextract. Staphylococcus aureus stock culture (ATCC9144) and dried Clostridium perfringens (ATCC13124)were taken from the Microorganism Collection Centerof Iran Scientific and Industrial Research Organization.The stoke cultures were transferred into 10 mL BrainHeart Infusion (BHI) broth and incubated in a shakingincubator at 37°C for 24 h. Then, 0.1 mL of the culturewas transferred into 10 mL of BHI medium andincubated at 37°C for another 24 h until the end of theprogressive phase.Subsequently, diluted cultures were used to inoculateplate agar and industrial kashk for subsequent targetinoculations. In a 96-well plate, 100 μL of the culturemedium (Müller Hinton broth) was added to all thewells, and then 100 μL of the sample extract was addedto the first well. After mixing the culture medium andthe sample in the first well, 100 μL of it was addedto the second well and dilution continued until theconcentration of the extract in the wells was reducedby half. Then, a uniform suspension of Staphylococcusaureus and Clostridium perfringens half McFarland1.5×108 CFU/mL were added to all the wells and the96-well plate was incubated at 37°C for 24 h. At theend, one well before the well in which turbidity wasconsidered minimum bactericidal concentration and thefirst concentration in which turbidity was not observedwas minimum inhibitory concentration [27].Preparation and quality evaluation of kashksamples. Pasteurized kashk was randomly purchasedfrom a local manufacturer in Karaj. The experimentalsamples were kashk with minimum bactericidal andminimum inhibitory concentrations of the caper extract.Kashk without the extract was used as control. Then,qualitative tests of the kashk samples were performedon days 0, 7, 14 and 28 of storage. The qualitative testsincluded pH (with the help of a pH meter based onAOAC 2000 standard), antioxidant properties (by usingDPPH radical scavenging assay), and colorimetric test(by a HunterLab spectrophotometer based on CIELABsystem) [26, 28]. Table 1 shows the kashk samples understudy.Microbial population in kashk during storage.In this method, each specimen was infected withStaphylococcus aureus and Clostridium perfringenswith a microbial population of 105 CFU/mL and kept atrefrigerator temperature. To determine the populationof Staphylococcus aureus, a certain amount of samplewas diluted and sterilized on a plate. Baird-Parker agarwas added to the plate with egg yolk emulsion withtellurite and incubated at 3 Figure 1 Caper fruit under study 7°C for 72 h. The number of Table 1 Kashk samples under studyCK0 Control kashk on day 0 (the day of production)CK7 Control kashk on day 7 of storageCK14 Control kashk on day 14 of storageCK21 Control kashk on day 21 of storageCk28 Control kashk on day 28 of storageEKI0 Experimental kashk with MIC on day 0 (the dayof production)EKI7 Experimental kashk with MIC on day 7 of storageEKI14 Experimental kashk with MIC on day 14 of storageEKI21 Experimental kashk with MIC on day 21 of storageEKI28 Experimental kashk with MIC on day 28 of storageEKB0 Experimental kashk with MBC on day 0 (the dayof production)EKB7 Experimental kashk with MBC on day 7 of storageEKB14 Experimental kashk with MBC on day 14 of storageEKB21 Experimental kashk with MBC on day 21 of storageEKB28 Experimental kashk with MBC on day 28 of storageMBC is minimum bactericidal concentrationMIC is minimum inhibitory concentrationTable 2 Phenolic, flavonoid and EC50 properties of caper extractTreatment Extractionefficiency, %Total content of phenoliccompounds, mg gallic acid per gFlavonoid content,mg catechin per gEC50,mg/mLExtract with 70% of ethanoland 30% of water23.20 ± 0.14 0.07 ± 2 0.01 0.20 ± 12.16 0.03 ± 1.48405Hematian A. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. Х–Хcontent and EC50 values in DPPH free radical scavengingassay indicates the direct effect of phenols onantioxidant activity [24]. M. Mahboubi and A. Mahboubiextracted Capparis spinosa extract with water, ethanol,methanol and ethyl acetate solvents and reported theEC50 values of 500, 560, 340 and 2000 (μg/mL), whilein our study this value was 1.48 (mg/mL), indicatingthe antioxidant properties of caper. Other studies alsoshowed that caper has a significant antioxidant activity[17, 26, 34].Composition and antioxidant properties of caperextract. In recent years, phenolic compounds havereceived special attention because of their biologicalactivities. We analyzed phenolic compounds by meansof high-performance liquid chromatography and diodearray detection (Table 3).Four previously phenolic compounds of caperextract, such as catechin, gallic, chlorogenic, ando-coumaric acid, reported in [32] were also observedin this study. Previous studies have reported rutinas one of the main components of the extract, whichwas confirmed in this study as well. Rutin has a widerange of biological activities such as antioxidant,antimicrobial, antidiabetic and cholesterol-loweringeffects [26, 33]. Therefore, the remarkable biologicaleffects of caper can be attributed to rutin, which can be asource of bioactive compounds.Minimum inhibitory concentration (MIC) andminimum bactericidal concentration (MBC) ofcaper extract. The microbial properties of kashk aredefined by the limit of Staphylococcus aureus andsulfite reductive Clostridia. The results of MIC andMBC of Capparis spinosa extract for the two targetmicroorganisms are shown in Table 4.In the present study, the MBC level forStaphylococcus aureus (0.283 mg/mL) was lowerthan that for Clostridium perfringens (0.350 mg/mL).Staphylococcus aureus lacks capsules and spores butis resistant to drought and withstands salt up to 10%.Clostridium perfringens is a Gram-positive, sporozoic,and usually capsulogenic bacterium, with a higherresistance to the extract than Staphylococcus aureus.Various mechanisms have been proposed so far for theantimicrobial activity of the extracts. The most accuratetheory suggests that the number and position of hydroxylgroups are a key factor in the antimicrobial activity ofphenolic compounds, flavonoids, quercetin and theirderivatives.Other mechanisms, such as flavonoids as a resultof the ability to form complexes with the cell wall andinhibit the growth of microorganisms, or phenoliccompounds by enzymatic activity through reactionwith sulfhydryl groups or nonspecific interactionswith proteins prevent enzymatic and thus exhibit theirantimicrobial activity. Polyphenols are also able to formhigh molecular weight soluble complexes with proteins,thereby attaching to the bacteria and destroying thereceptors present on the bacterial cell surface [37].Quercetin and its derivatives also inhibit bacterialgrowth through the DNA gyrase inhibition [27].Rahnavard and Razavi showed that caper extractsdemonstrated the antibacterial activity against avariety of Gram-positive and negative bacteria,including Staphylococcus epidermis, Staphylococcusfaecalis, Staphylococcus aureus, Micrococcus luteus,and Bacillus cereus [21]. The aqueous extract ofcaper fruit did not show any antibacterial activity,while the ethanolic extract had the antimicrobialeffect on a variety of Streptococcus spp. and Gramnegativebacteria [17]. The comparing of resultsfrom different studies in this case seems complicatedbecause the results are influenced by factors suchas the composition and type of culture medium,microorganism growth phase, the volume of culturemedium, pH, temperature, and incubation time. Thechemical composition, type, and the mechanism ofaction of phenolic compounds also play a part inantimicrobial activity [38]. Since one of the aims of thepresent study was to increase the kashk shelf life, weused MIC and MBC of Clostridium perfringens, whichwere high, as the amount of extract used in the kashksamples.pH determination. The pH test of the kashk samplesis shown in Table 5. Statistical analysis of pH-dependentparameters showed that the concentration of extract,time, and extract × time had a significant effect on kashkpH (P < 0.05), i.e., pH reduced with time. On the otherhand, 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 storageTime, days pH EC50, μg/mLCK EKI EKB CK EKI EKB0 0.03 ± 4.53ab 0.02 ± 4.54a 0.04 ± 4.56a 0.5 ± 32.74c 0.7 ± 15.11j 0.5 ± 12.05k7 0.02 ± 4.36c 0.01 ± 4.51b 0.03 ± 4.53ab 0.2 ± 33.28c 0.5 ± 17.10f 0.2 ± 14.07h14 0.01 ± 3.99f 0.03 ± 4.41c 0.05 ± 4.42c 0.7 ± 33.47c 0.9 ± 17.40f 0.4 ± 14.24h21 0.01 ± 3.29j 0.04 ± 4.05e 0.02 ± 4.11d 1.1 ± 35.03b 1.3 ± 18.05e 0.5 ± 15.10j28 0.04 ± 2.96h 0.01 ± 3.94f 0.01 ± 3.96f 0.8 ± 40.12a 0.7 ± 20.15d 0.8 ± 17.05fCK: control kashk, EKI: experimental kashk with MIC (0.211 mg/mL of caper extract), and EKB: experimental kashk with MBC (0.350 mg/mL ofcaper extract)Letters a‒h indicate significant differencesTable 6 Colorimetric characteristics of kashk samplesTime,daysL* a* b*CK EKI EKB CK EKI EKB CK EKI EKB0 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 ± 22.50e7 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 ± 23.35de14 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 ± 24.05d21 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 ± 25.65bc28 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 ± 26.20bCK: control kashk, EKI: experimental kashk with MIC (0.211 mg/mL of caper extract), and EKB: experimental kashk with MBC (0.350 mg/mL ofcaper extract) Letters a‒e indicate significant differences 407Hematian A. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. Х–ХFaraji et al. stated that addition of 0.5–2% ethanolicextract of Allium stipitatum over a 21-day storage resultedin 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 ofmodified Tapioca starch to kashk over a 60-day storageincreased L*, but did not impact a* and b* [29].The effect of microbial population on shelf lifeof kashk samples. Bacterial population changes ofStaphylococcus aureus and Clostridium perfringensin the control and experimental kashk samples (withthe caper extract) during 28 days of storage at 4°Care shown in Fig. 2. The initial concentration ofStaphylococcus aureus in the control sample was4.91 CFU/mL, which remained unchanged until theend of storage. By the end of storage (on day 28), theinitial amount of Staphylococcus aureus had reduced to0.6 CFU/mL in the experimental sample with MIC(0.211 mg/mL of the caper extract), and to 0.3 CFU/mLin the kashk with MBC (0.350 mg/mL).The initial bacterial count of Clostridium perfringensin the control was 4.50 CFU/mL, which had notincreased significantly by day 28 of storage. In thekashk with MIC (0.211 mg/mL of the caper extract), theinitial amount of Clostridium perfringens reduced to4 CFU/mL, and in the kashkl with MBC (0.350 mg/mL),to 3.50 CFU/mL. The high susceptibility ofStaphylococcus aureus to the caper extract can berelated to phenolic compounds present in the extract [11].Although the extract under study also reducedthe initial amount of Clostridium perfringens inkashk, the reduction was less than a logarithmic cycleindicating its resistance to the extract. The decrease inmicrobial load in the whole system could be due to theintrinsic acidic pH of kashk, but after day 21 a differenttrend was observed for Clostridium perfringens inthe control sample. The overall results showed thatwith the increasing of storage time of the extractcontainingkashk samples, Staphylococcus aureusbacterial population decreased more rapidly than thatof Clostridium botulinum, but none reached zero.Golestan et al. investigated the antimicrobial propertiesof the ethanolic extract of Allium stipitatum againstClostridium botulinum and Staphylococcus aureus inkashk. They found that Staphylococcus aureus countdecreased more rapidly with increasing storage time, ithad reached zero by the end of day 21 of storage [13].Sensory properties of kashk. Figure 3 illustratesthe effect of the caper extract on the sensory propertiesof the kashk samples based on the evaluation ofpanelists. The results showed that the highest scores oftexture, smell, taste, oral feeling, adhesion, and generalacceptability had the kashk samples on days 0, 7 and 14of storage. With time, namely after 48 days of storage,the samples had the lowest overall acceptability.The results also showed that the texture of thesamples containing the extract received the highestscore on day 14, while the control sample on day 14 hada lower score. However, the odor of the control samplehad a higher score comparedc to that of the experimentalkashk. There was no significant difference between thesamples regarding taste and oral sensation. It is worthto note that the adhesion factor score remained maximalfor all the samples during the storage time. The generalacceptability of the kashk samples received the highestscores on days 0 and 7, and the lowest scores, on day 48in all the three samples.Golestan et al. demonstrated that peppermintessential oil at concentrations of 1500 and 2500 ppmand Mentha pulegium essential oil at a concentration of2500 ppm had significant effects on taste of kashk [13].Kashk samples containing 1500 ppm of Menthapulegium essential oil and control samples wereintroduced as suitable samples, but kashk samples withFigure 2 Dependence of Staphylococcus aureus concentrationin 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 perfringensconcentration in kashk samples on storage time. CK: controlkashk, EKI: experimental kashk with MIC (0.211 mg/mLof caper extract), and EKB: experimental kashk with MBC(0.350 mg/mL of caper extract)408Hematian A. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. Х–ХMentha pulegium essential oil at the concentrations of1500 and 2500 ppm were acceptable for panelists.Agulló et al. studied the effect of modified Tapiocastarch on sensory properties (i.e., texture, smell, taste,oral sensation, adhesion and general acceptance) ofkashk [29]. Their results showed that the type of starchaffected the sensory properties. The sample with 1.5%hydroxypropylated tapioca starch had the best result andthe control sample received the lowest score.CONCLUSIONThe results of the study showed that the caper(Capparis spinose L.) extract had no adverse effects onpH of kashk during storage time, and the kashk samplewith the extract at the concentration of 0.350 mg/mLhad the lowest EC50 (12.05 mg/mL), or the highestantioxidant activity on day 0 of storage. The increasedextract concentration and storage time resulted in adecrease in L* and an increase in b*, while they did notinfluence a*.The number of bacteria had gradually decreasedin the kashk samples with both concentrations of theextract by the end of a 28-day storage. With increasingthe storage time, Staphylococcus aureus bacterialpopulation declined compared to Clostridium botulinum.The sensory evaluation results showed that thetexture of the extract-containing samples had a higherscore, which was even higher by day 14, and the controlsample had a lower score on the same day. But in termsof smell the control samples were superior to the extractcontainingsamples.In general, we can conclude that the kashk samplescontaining 0.350 mg/mL of caper extract had improvedantimicrobial, antioxidant, and antifungal properties andcan be considered as a new functional product.CONTRIBUTIONAuthors are equally related to the writing of themanuscript and are equally responsible for plagiarism.CONFLICT OF INTERESTThe authors declare no conflict of interest.