Effect of propolis extract addition on some physicochemical, microbiological, and sensory properties of kefir drinks

Abstract Kefir drinks with sugar (5%, w/v), strawberry flavor (0.15%, v/v), and propolis extract (PE) at different ratios (0.150%, 0.225%, and 0.300%, v/v) were produced and stored at 4°C, and their physicochemical, rheological, microbiological, and sensory properties were monitored during storage. The ratio of PE and storage time had an insignificant effect on the dry matter, protein, fat contents, Commission Internationale de l'Eclairage (CIE) L* and a* color values, apparent viscosity, consistency coefficient, flow behavior index, Lactobacillus spp., Lactococcus spp., and yeast counts of kefir drinks (p > .05). The CIE b* values of kefir drinks increased with an increase in PE ratio (p < .05). All kefir samples exhibited a pseudoplastic flow behavior. Initially, the total antioxidant capacity of kefir drinks was 2.19 μmol TE/100 mL, which increased to 2.51 μmol TE/100 mL for kefir drinks with 0.225% PE. The total phenolic content and antioxidant capacity of kefir drinks with PE decreased during storage. PE addition did not influence the sensory color and taste liking scores of kefir drinks adversely until the 8th day of storage. In terms of odor liking scores, kefir drinks with 0.225% and 0.300% PE had a similar score to control drinks. Additionally, kefir drinks with 0.150% and 0.225% PE received an overall liking score similar to control drinks. Results indicated that the incorporation of PE at a ratio of 0.225% was recommended for the production of strawberry‐flavored kefir drinks with acceptable sensory characteristics and increased functional properties, and this product could be stored for up to 8 days.

grains primarily consists of Lactobacillus, Leuconostoc, Acetobacter, Streptococcus, and various yeasts (Garrote et al., 2001;Irigoyen et al., 2005).Kefir is a very valuable fermented drink for human nutrition because of its health beneficial effects.While it contains almost all nutrients in milk, it has an increased nutritional value for human body via various microorganisms in the structure of kefir grains (Karagözlü, 2003).The health benefits of kefir consumption can be attributed to the fact that it is a natural probiotic besides its high protein, mineral, and vitamin contents (Zourari & Anifantakis, 1988).Previous studies have constantly reported the health beneficial effects of kefir consumption such as its anticarcinogenic, antibacterial, antitumoral, antimicrobial, immunomodulatory, blood cholesterol and blood pressure, and digestive system regulatoryeffects (Kesenkaşetal.,2017).
In kefir production, various milk types such as cow, sheep, goat, buffalo, rice, and soy milk or a mixture of them with different contents of fat and skim milk can be used (Bensmira & Jiang, 2012;Karagözlü, 2003).Thus, the sensory and nutritional properties of kefir drinks are mostly dependent on the composition of milk, microbiological characteristics of grains and starters used, production method, storage time, and addition of different ingredients (Zourari & Anifantakis, 1988).Thermophilic and mesophilic probiotic bacteria and yeasts are mostly used together or separately as starter cultures in kefir production (Kadıoğlu, 2017;Zourari & Anifantakis, 1988).
Nowadays, the use of lyophilized cultures in industrial production saves time and facilitates production while providing hygienic and standardized production (Özcan et al., 2018).In the production of fruity kefirs, various types of flavoring agents and fruit sauces can be added to plain kefir drinks.
Propolis is a natural product produced by honey bees using substances such as resin, mucilage, and gum in plant and tree leaves.It contains more phenolic components than other bee products and has strong antioxidant activity (Mehmetoğlu et al., 2017;Viuda-Martos et al., 2008;Wang et al., 2018;Zabaiou et al., 2017;Zheng et al., 2023).Propolis, considered a natural product with medicinal properties, has been used as a supplement in the therapy of many diseases since ancient times, and PE is extremely complex in its nature because of the many chemical constituents it contains (Russo et al., 2002;Viuda-Martos et al., 2008).Recently, studies on propolis in the literature and its use as a dietary supplement have increased steadily.In general, half of raw propolis is composed of resin and plant balm while the remaining contains bee wax(30%),essentialandaromaticoils(10%),pollen(5%),andother organicsubstances(5%) (Burdock,1998;Sforcin, 2007).There are morethan850isolatedcompoundsinthecompositionofpropolis, which includes phenolic substances and their esters, caffeic acid phenyl ester (CAPE), flavonoids, terpenes, aromatic acids, aromatic aldehydes and alcohols, beta-steroids, vitamins, etc. (Koru et al., 2007;Orhan et al., 1999;Šturm & Ulrih, 2020;Viuda-Martos et al., 2008).
The high antioxidant property of propolis is derived from its polyphenols like flavonoids and phenolic acids in its composition.
Rich phenolic content of propolis creates a strong antioxidant effect and may protect human health against the damage of oxidative stress (Castaldo & Capasso, 2002;Viuda-Martos et al., 2008;Zabaiou et al., 2017).
In the literature, there are few studies on the use of propolis in the production of various milk or dairy products.Propolis was previously added to dairy products such as Ras cheese (Aly & Elewa, 2007), yogurt (Çelik, 2016;Çifci, 2015;Güney, 2016), and icecream (Mehmetoğlu,2019).In a preliminary study by Chon et al. (2020), propolis was used in kefir production among different dairy products.However, the effects of propolis on the microbiological and rheological properties, antioxidant activities, and phenolic contents of kefirs were not investigated in this preliminary study.The aim of this present study was to incorporate propolis extract into kefir drinks in order to develop a new functional product by obtaining a synergistic effect of these two products with proven health benefits.To facilitate the consumption of kefir drinks and increase its accessibility by different segments of population, propolis extract was added into strawberry-flavored kefir drinks at different ratios, and the physicochemical, rheological, antioxidant, microbiological, and sensory properties of drinks were monitored during storage at 4°C.

| Preparationofkefirdrinks
Cow milk used in kefir production was initially heat treated at 90°Cfor5 minandthencooledtothefermentationtemperature of30°C.Later,kefirstarterculture(1 gper5 Lmilk)wasaddedto the milk, and samples were incubated at 23.5°C for about 20 h, as instructed by the manufacturer of the culture.After their pH reached4.18± 0.1,maturationprocesswascarriedoutunderrefrigeration conditions at +4 ± 1°C for 24 h.Then, kefir samples with sugar (5.00%, w/v), strawberry flavor (0.15%, v/v), and different ratios of PE (0%,0.150%,0.225%,and 0.300%, v/v) were produced, coded as A, B, C, and D, respectively.The concentrations of strawberry flavor and PE in kefir drinks were determined according to the instructions of their manufacturers.The daily consumption of water-soluble PE was recommended as 0.30 mL for children and 0.60 mL for adults by its manufacturer.Therefore,0.150and0.300%PEratioswereselectedforthedailyconsumption of 200 mL kefir drinks, which could correspond to the recommended daily consumption rates of children and adults, respectively.To suppress the bitterness of PE in kefir drinks and to facilitate their consumption, table sugar and strawberry flavor were added to samples.The ratio of sugar in kefir drinks was determined by preliminary experiments and according to the amount of sugar in commercial fruit-flavored kefir drinks currently sold in markets.Preliminary experiments revealed that kefir drinks had an unpleasant bitter taste beyond 8 days of storage at +4 ± 1°C; therefore, kefir samples were stored for a maximum of 8 days.Some physicochemical, antioxidant, rheological, microbiological, and sensory properties of kefir drinks were determined on the 1st, 4th, and 8th days of storage.

| Determinationoftotalantioxidant activity and phenolic content
Preparation of extracts as follows.In the preparation of extracts, kefir samples were mixed and homogenized, then transferred into Eppendorf®tubes.Thetubeswerethencentrifugedfor20 minat 12,225 × g by a microcentrifuge (WiseSpin, CF-10, Daihan Scientific Co. Ltd., Gang-Won-Do, Korea).Total antioxidant activity and phenolic content analyses were performed by using supernatants obtained after centrifugation.Trolox®, diammonium salt of ABTS, Folin-Ciocalteu reagent, and gallic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA) while sodium carbonate was obtained from Riedel-de Haen (Seelze, Germany).
The constant temperature was provided by a circulating water bath (Maxircu CR-12, Daihan Scientific Co. Ltd., Gang-Won-Do, Korea), and the apparent viscosity values of samples were determined at 120 rpm(111.6 s −1 ).Flow behavior index (n) and consistency coefficient (K, Pa.s n ) were calculated according to the power law model (Steffe, 1996).

| Sensoryanalyses
Hedonic scales are extensively used to measure product liking and preference.The 9-point hedonic scale may be the most informative sensory method used with a considerable success because naive consumers can understand the scale easily with minimal instruction, and results of this scale are "remarkably stable and product differences (in liking) are reproducible with different groups of subjects" (Stone & Sidel, 1992).However, our previous experience indicated that 9-point scales might be nondistinctive and/or inappropriate for all native languages and subject segments as in the use of 7-point face scales to measure children's responses to products.For this reason, sensory analyses in the present study were carried out by experienced panelists (20) on each storage day for measuring color, odor, taste,andoveralllikingswitha7-pointhedonicscale(1 = dislikeextremely and 7 = like extremely) (Bodyfelt et al., 1988).All samples were numbered with three-digit random codes and presented to each panelist in a different order.Panelists were instructed to drink water (Nazlı, Aydın, Turkey) between each sample to clean their palate.

| Statisticalanalysis
The analysis of variance (two-way ANOVA) and the Duncan multiple-comparison test were used at a significance level of α = 0.05 by means of the SAS package program (SAS System for Windows 9.0, SAS Institute Inc., Carry, NC, USA).Results were presented as mean ± standarddeviation.

| Physicochemical properties
Dry matter, fat, and protein contents of kefir drinks on the first day of storage are presented in Table 1.On average, the dry matter contents of kefir drinks with PE at different ratios were between 16.91 and 17.20% while their fat contents ranged from 2.60% to 2.67%.
Kefir drinks had a protein content in the range 2.80%-3.26%.According to Table 1, the addition of PE into kefir drinks at different ratios did not change their dry matter, fat, and protein contents significantly (p > .05).Moreover,thedrymattercontentsofplainkefir drinks without any table sugar were between 11.93 and 12.17%.
Similarly, Wszolek et al. (2001) reported the mean dry matter content of kefir samples produced by using cow's milk as 11.67%.In the study by Gül (2017), the dry matter contents of kefir produced by adding 0%-4% mint juice were reported between 10.17% and 10.85%,andthedifferencesinthedrymattercontentsofsamples containing different ratios of mint juice were found insignificant.
The dry matter contents of six different commercial kefir samples obtainedfromnationalmarketsinTurkeywereintherange9.49%-11.97%(Gursoyetal.,2020).In a study by Dinç (2008), the dry matter contents of fruit kefir drinks (n = 40)soldinthenationalmarkets of Ankara (Turkey) were found between 16.53% and 19.52%.Dry matter contents of both plain and strawberry-flavored kefir samples in our study were in good agreement with the data reported in the literature.

| Color parameters
The color parameters of kefir drinks with PE at different ratios during storage are given in  3).Since the flow behaviorindicesbetween0 < n < 1,kefirdrinkswithPEatdifferent ratios exhibited a non-Newtonian pseudoplastic flow behavior.Similarly, a variety of kefir drinks were reported to show a pseudoplastic flow behavior in the literature (Doğan, 2011;Gursoy et al., 2020;Kök-Taşetal.,2013;Tratnik et al., 2006).
The mean apparent viscosity values of plain kefir samples produced from cow milk were reported as 101, 89, and 75 mPa.sby Tratnik et al. (2006) on the 1st, 5th, and 10th days of storage, respectively.The average viscosities of plain, fruit, and diet kefir drinks marketed in Ankara (Turkey) were determined as 104.4,120, and 111 mPa.s,respectively (Uslu,2010).Kök-Taşetal.(2013) reported the apparent viscosities of kefir drinks with a fat content of 0.1% between2.02and2.47Pa.swhenkefirdrinkswerestoredforupto 21 days.In a study by Doğan (2011), different ratios of honey (10, 20, and 30, w/w) were added to kefir, and an increase in honey concentration decreased the consistency coefficients of kefir drinks.
The apparent viscosities of kefir drinks at a shear rate of 50 s −1 were found between 15.39 and 16.35 mPa.s while all samples exhibited a pseudoplastic behavior.Irigoyen et al. (2005)

| Total phenolic content and antioxidant activity
The phenolic content and antioxidant activity values of kefir drinks with PE at different ratios during storage at 4°C are shown in Table 4.
Thetotalantioxidantcapacityofkefirsamples(100 mL)wasbetween 1.97 and 2.51 μmol TE while their total phenolic content (100 mL)  Adding different ratios of raw propolis powder into ayran (0%-0.75%,w/v)andbuttermilk(1%-3%,w/v),Çelik(2016) reported that increasing the ratio of propolis powder in both products resulted in an increase in their DPPH radical scavenging activities and phenolic contents, which decreased during storage.Mehmetoğlu (2019)   and stated that the antioxidant activities and phenolic contents of pudding samples significantly increased with an increase in the ratio of propolis while there was a decrease in these values during storage.Santos et al. (2019) produced probiotic yogurt samples by using PE (0.05%, w/v), pasteurized strawberry pulp (10%, w/v), and sugar (12.6%) and determined their total phenolic contents between 5.49 and5.73 mgGAE/g,whichdecreasedduring28 daysofstorage.Studying the potential use of propolis as an alternative to food additives like sodium benzoate and potassium sorbate in the preservation of orange juice, Yang et al. (2017) reported that the use of propolis could effectively protect the antioxidant capacity of orange juice.The results of this present study were in good agreement with literature data.

| Microbiological properties
Lactobacillus spp., Lactococcus spp., and yeast-mold counts of kefir drinks with PE at different ratios during storage at 4°C are shown in
studied the rheological properties of kefir with kefir grains at different ratios duringstorage(28 days)at5 ± 1°C,andreportedthattheirviscosity values decreased significantly during storage.In a study by Gursoy et al. (2020), the apparent viscosity values of commercial kefir samples(4,10,and25°C)werebetween0.48and1.76mPa(111.6 s −1 ) at three different temperatures while their flow behavior indices varied from 0.18 to 0.44, indicating a pseudoplastic flow behavior.The results of the present study were in good agreement with the data reported in the literature.
rangedfrom11.55to13.98 mgGAE.Thetotalantioxidantcapacity and phenolic content of kefir samples increased with an increase in the ratio of PE, and they decreased slightly during storage.While the lowest total antioxidant capacity was found for the kefir samples A andBonthe8thdayofstorage,itwas2.51 μmolTE/100 mLforthe kefir sample D on the 1st day of storage.In general, changes in the total phenolic contents of kefir samples were in parallel with their total antioxidant activity values.TA B L E 3 Changesintherheologicalpropertiesofkefirdrinkswithpropolisextractduringstorage.
in ice cream production and reported its adverse effect on the sensory properties of ice cream samples while the most liked one was the control sample.Adding microencapsulated PE into cake samples at different ratios (2.5%, 5.0%, 7.5%, and 10.0%, w/w),Acun (2021) found that cakes with more than 5.0% PE were not preferred by panelists.Results in the present study were generally in good agreement with the literature.Since the ratio of PE added to strawberry-flavored kefir drinks was relatively low in comparison to previous studies, their sensory liking scores were similar to those of the control drinks.4| CON CLUS IONTheadditionofPEintokefirdrinksandstoragetimeofupto8 days at 4°C did not have a significant effect on the dry matter, protein, and fat contents of drinks.The pH values of drinks decreased while their titratable acidity values increased with an increase in storage time.The addition of PE into strawberry-flavored kefir drinks and storage time did not have a significant effect on their L* and a* color values, but b* color values increased as the ratio of PE increased.Generally, the addition of PE and storage time did not change the rheological properties of kefir samples, which showed a pseudoplastic flow behavior.It was found that the total antioxidant capacity and phenolic content of kefir drinks increased with an increase in the ratio of PE while these values decreased during storage.The addition of PE did not influence the sensory color and taste liking scores of kefir drinks, and the overall liking scores of kefirdrinkswithPEataratioofupto0.225%weresimilartothose of the control drinks.The addition of PE had an insignificant effect on the total Lactobacillus spp., Lactococcus spp., and yeast counts of kefir drinks.Results indicated that it was possible to produce a strawberry-flavored kefir drink with 0.225% PE, which could be
Gursoy et al. (2020)teincontentsofkefirdrinkswithpropolisextract.and protein contents of kefir drinks are highly dependent on the type of milk used in kefir production.Gursoy et al. (2020)reported the fat contents of six different commercial kefir drinks be- a A:Control,B:Kefirdrinkwith0.150%propolisextract,C:Kefirdrinkwith0.225%ofpropolisextract,andD:Kefirdrinkwith0.300%ofpropolis extract.bDifferentsuperscriptswithin the same column indicate that means are significantly different (p < .05).FatThe pH and titratable acidity values of kefir drinks with PE at different ratios during storage are given in Table2.The pH values of kefir drinks were between 3.96 and 4.10 while their titratable acidity valuesrangedfrom0.74%and0.83%duringstorage.Theaddition of PE did not influence the pH and acidity (lactic acid) values of kefir samples significantly (p > .05)butthepHvaluesofkefirdrinksdecreased(p < .05)whiletheiracidityvaluesincreasedduringstorage(p < .05).Similar to our study, using different kefir grains, Garrote

Table 2 .
The addition of PE did not have a Rheological properties of kefir drinks with PE at different ratios during storage are given in Table3.On average, the appar- significant effect on the L* color values of strawberry-flavored kefir drinks (p > .05).During storage, the L* color values of kefir samples variedbetween84.45and85.50,andtheinteractionofPEratiowithstoragetime on the L* color values of kefir drinks was also found insignificant (p > .05).TheindividualeffectofPEadditiononthea* color values of kefir drinks was insignificant (p > .05).On the other hand, the addition of PE into kefir drinks increased the b* (yellowness) color values of samples significantly (p < .05),andthisvaluevariedbe-tween5.37and7.12duringstorage.Itisconsideredthatthissituation is caused by the yellowish color of PE.Additionally, it was found that the individual effect of storage time on the L*, a*, and b* color values of kefir samples was insignificant (p > .05).Addingpropolistoyogurt samplesatdifferentratios(0.25%-0.75%),Çifci(2015)reportedthat Hunter L and color values of yogurt samples decreased and b color values increased as storage time and the propolis ratio increased.TA B L E 2 ChangesinthepH,titratableacidity,andcolorparametersofkefirdrinkswithpropolisextractduringstorage. a A:Control,B:Kefirdrinkwith0.150%propolisextract,C:Kefirdrinkwith0.225%ofpropolisextract,andD:Kefirdrinkwith0.300%ofpropolis extract.bDifferent superscripts within the same column indicate that means are significantly different (p < .05).3.3 | Rheological properties

Sample code a Storage time (day) Total phenolic content b (mg GAE/100 mL) Total antioxidant activity (μmol TE/100 mL)
b Different superscripts within the same column indicate that means are significantly different (p < .05).

Table 5 .
The ratio of PE did not have a statistically significant effect on the total Lactobacillus spp., Lactococcus spp., and yeast counts (log