LACTATE CONTENT, TOTAL POLYPHENOLS AND ANTIOXIDANT ACTIVITY OF SELECTED COMMERCIAL YOGURTS FROM THE SERBIAN MARKET

: One of the most popular dairy products worldwide is yogurt, which flavor is highly influenced by lactic acid content. Yogurts with fruit preparations contain polyphenols, secondary plant metabolites with proven antioxidant properties. As there is no data regarding neither lactate nor polyphenol content in commercial yogurts in Serbia, this study aimed to determine lactate and polyphenol levels, as well as antioxidant activity in selected yogurts. A total of 15 plain and 5 fruit yogurts with strawberry preparations were analyzed. Lactate contents were from 0.83 to 1.33%. Three plain yogurts in plastic containers differed in lactate content. There was no difference in lactate content among the same plain yogurts packed in plastic and Tetra Pack containers. Fruit yogurts with strawberry preparations differed in total polyphenol content in the range from 6.84 to 29.11mg GAE/100 g and antioxidant properties were determined by reducing power test (from 0.22 to 0.79) and DPPH assay (from 28.13 to 87.23%), while there was no difference regarding lactate. Our results provided new information about the levels of lactate, total polyphenols and antioxidant activity of selected commercial yogurts available on the Serbian market.


INTRODUCTION
Yogurt has been used traditionally in the Balkan region (Teneva Angelova, Balabanova, Boyanova & Beshkova, 2018) and it is popular worldwide, due to its health benefits and sensory properties (Chen et al., 2017;Ścibisz, Ziarno & Mitek, 2019;Deshwal, Tiwari, Kumar, Raman & Kadyan, 2021;Tolu & Altun, 2021). It is produced from milk, which undergoes initial treatment, standardization, homogenization, heat treatment, fermentation, and cooling. Fermentation is the most important step of the process for the formation of the texture and flavor of yogurt. It is based on the activity of starter cultures Streptococcus thermophilus subsp. Thermophiles (STT) and Lactobacillus delbrueckii subsp. Bulgaricus (LDB), gram positive bacteria. STT are aerotolerant and thermotolerant, which enables them to grow faster at the beginning. They hydrolyze lactose from milk into glucose and galactose, of which the first is converted into pyruvate. It is then converted into lactic acid, by the activity of lactic acid dehydrogenase. According to TS 1330 Yogurt Standard, lactic acid in yogurt should be in the range of 0.6-1.6% (Tolu & Altun, 2021) however, there is big variability in the reported amount of lactic acid in analysed yogurts (Fernandez-Garcia and McGregor, 1994;Ekinci & Gurel, 2008;Güler & Park, 2011;Cruz et al., 2012;Vénica, Perottim & Bergamini, 2014). A decrease of oxygen, as well as the release of amino acids by the activity of SST, enables faster growth of LDB, resulting in higher production of lactic acid. At pH of 4.3-4.7, fermentation is stopped by cooling to 5 °C (Sfakianakis & Tzia, 2014). However, due to the acid tolerance of LDB, slow rate post-fermentation acidification can be continued during shelf life, and that can also be influenced by packaging material (Deshwal et al., 2021). Lactic acid is a nonvolatile compound which highly contributes to the acidic taste of yogurt (Güler & Park, 2011;Chen et al., 2017).
The aims of this study were manifolds: 1) to determine lactic acid content in selected commercial plain and fruit preparations-added yogurt available on the Serbian market; 2) to evaluate whether the package influenced lactate content in plain yogurt; 3) to analyze total polyphenol levels in fruit yogurts with strawberry preparations and their contribution to antioxidant activities.

Material sampling
A total of 20 samples (15 plain and 5 fruit yogurts with strawberry preparations) were purchased in a supermarket (in Belgrade, Serbia) in September 2021. The sample planning and their collection and preparation were according to instructions given in the EuroFIR guide (Oseredzuk, Salvini, Roe & Moller, 2009). Characteristics of the yogurts that were available on labels are provided in Table 1.
Three commercially available yogurts, in both plastic (polypropylene 200 ml cup with aluminium foil closure) and Tetra Pak (250 ml) containers, were chosen to analyze whether the types of packaging material could have influenced lactate content.
Samples were stored at +4 ⁰C for 7 days (total of six days after opening).

Preparation of yogurts extracts
Methanolic and 80% ethanolic extracts of fruit-added yogurts were prepared. Briefly, 2 g of fruit-added yogurts were measured and an appropriate volume of organic solvents was added (8 ml), vigorously mixed for 1 min and left for 12 hours at 4 °C. The next day, extracts were vortexed three times for 1 min and then centrifuged at 5000 rpm for 10 min. Extracts were separated from precipitates and their volumes were measured and further used for the determination of polyphenols and antioxidant activities during the same day.

Determination of lactic acid
Lactic acid content was determined according to Borshchevskaya, Gordeeva Kalinina and Sineokii (2016). Briefly, tenfold dilution of yogurts samples in distilled water was made. After centrifugation at 3000 rpm for 10 min, 50 μl of supernatant was added to 950 µL of 0.2% iron (III)-chloride solution and absorbance was measured at 390 nm after 5 min of incubation using a spectrophotometer UV-1800 (Shimadzu Corporation, Kyoto, Japan).
The standards were prepared by diluting lactate (Sigma-Aldrich, Germany) in range from 0.25-10.0 g/L and linear curve equation obtained y = 0.12x + 0.014, with r 2 = 0.99.

Total polyphenol content
Total polyphenol content was determined using a slightly modified Folin-Ciolcateu (FC) method (Blainski, Lopes & de Mello, 2013). In brief, 125 µL of the sample was mixed with 125 µL of FC reagent and incubated for 6 minutes, at room temperature.
Then 1000 µL of 7% Na2CO3 was added and the mixture was incubated for 90 minutes, in the dark, at room temperature. Subsequently, 500 µL of distilled water was added and the absorbance was measured at 760 nm, with gallic acid used as standard.

DPPH test
Measurement of 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging ac-tivities (1,1-diphenyl-2-picrylhydrazyl) was performed according to the slightly modified method of Blois (1958). Briefly, 640 µL of 0.15 mM DPPH solution was added to the 160 µL mL of the sample, the mixture was in-cubated for 30 min at room temperature and the absorbance was measured at 517 nm. DPPH scavenging activity (%) was determined according to the following formula: where ABS blank is absorption of blank sample and ABS sample is absorption reading of sample.

Reducing power test
The assessment of reducing power was performed according to the method by Oyaizu (1986), reported by Kim, Cho, Yeon, Choi and Lee (2019), with slight modifications. In brief, we mixed 250 μL of yogurt samples, 50 μL of 0.2 M phosphate buffer (pH 6.6) and 50 μL of 1% K 3 Fe(CN) 6 and incubated it for 20 min at 50 °C. Then we added 50 μL of 10% trichloroacetic acid and centrifuged the mixture at 865×g for 10 min. The amount of 300 µL of supernatant was mixed with 400 µL of distilled water and 100 µL 0.1% FeCl 3 and incubated for 10 min at room temperature. Subsequently, the absorbance was measured at 700 nm.

Statistical analysis
Six replicates were made for each of the samples and the results were presented as mean values and standard deviation. Differences between means were analyzed using ANOVA and Tukey's post-hoc test. Pearson test was used for the assessment of the correlation between total polyphenols and antioxidant activity. Statistically significant results were those with the p value less than 0.05. Data analysis was performed using the software GraphPad Prism (version 9, San Diego, California).

Characteristics of the collected samples
The samples of plain yogurts showed high variability for fat content being in a range from 0.50-3.20g/100 g and consequently in saturated fat from 0.35-2.58 g/100 g (Table 1). At the same time, in yogurts P1, P6 and P12 probiotic bacteria were added and other had only starter cultures. The total energy value of fruit-added yogurts was higher compared to the plain ones (mean value 77.8±9.3 kcal/100 g compared to 48.9±9 kcal/100 g, respectively) due to higher added sugar content as there was no direct association between sugar amounts and percentage of fruit preparation. Furthermore, the content of fruit varied from 7-18%, therefore the consumption of 200 g of fruit yogurt would lead to a dietary intake of 14-36 g of strawberry preparation.
Additionally, there were no statistically significant differences when comparing plain yogurts' samples P8, P10 and P12 in plastic (polypropylene cup with aluminium foil closure) and Tetra Pak containers. Although Saint-Eve, Lévy, Le Moigne, Ducruet and Souchon (2008) observed differences in the postacidification of yogurts stored in glass and polypropylene containers, indicating the influence of different package materials, our result suggests that both plastic (polypropylene) and Tetra Pak containers could have a similar influence on quality maintenance considering lactate content (Fig. 1).
Furthermore, there was no change in lactate levels 6 days after the opening of samples in Tetra Pak containers after storage at +4 °C in the refrigerator (Fig. 2). The period of 7 days was chosen as it is stated on the label of Tetra Pak samples that they can be used within this period. As samples in polypropylene 200 ml containers are usually opened and consumed immediately, our results indicate that yogurts in Tetra Pack containers will not increase lactate concentration if they are stored for 7 days, which could be important information for consumers. However, our observations are limited due to the very small number of samples. The samples of commercial fruit yogurts with strawberry preparations investigated in this study contained from 7% to 18% of strawberry preparations. There was no statistically significant difference in the lactate content among the fruit-added yogurts samples, with mean values in a range from 0.98 to 1.33% (Fig. 1) Additionally, the fruit yogurts with strawberry preparations investigated in our study did not contain lower levels of lactate compared to the plain yogurt counterparts (Fig. 1). Moreover, sample S1 had higher lactate concentration compared to those of plain yogurts' samples P3 (p = 0.01) and P7 (p = 0.03) and post-fermentation production of lactate occurring in fruit yogurts (Ścibisz et al., 2019) could, at least partly, contribute to observed, levels of lactate in our fruit yogurts' samples. . Lactate levels (%) of plain yogurts in Tetra Pak containers (P8-12tp) determined the first day and the following six days after opening, stored at +4 °C To our best knowledge, there is only one recent study investigating lactate content in fruit yogurts with strawberry preparations (Ścibisz et al., 2019) reporting lower lactate levels (about 0.6%) than those observed in our study, but it is worth noting the yoghurts in the mentioned study by Ścibisz and coworkers contained more fruits (about 20% of strawberry preparation). The number of investigated samples in our study is small (n = 5) and lactate content could significantly vary, so it may be important to routinely monitor its content.

Total polyphenols and antioxidant activity in fruit yogurts with strawberry preparations
The concentrations of polyphenols were analyzed in both methanolic and ethanolic extract to enable a more efficient comparison of our results with available data from other studies since some authors have been using methanolic extract, and other ethanolic extracts. The total polyphenol contents did not differ comparing methanolic and 80% ethanolic extracts and their levels were in a range from 6.84 to 29.11 mg GAE/100g (Table 2). Significant differences among the samples of fruit yogurts with strawberry preparations regarding their total polyphenol content were found (   , 2021). All aforementioned factors could have been affected the total content of polyphenols in yogurts from this study. At this moment, to our best knowledge, there is no data regarding the total polyphenol content of fruit yogurts with strawberry preparations neither in the most comprehensive polyphenol data base Phenol explorer nor in the Serbian food composition database. So, the obtained results could be useful to extend existing databases, which might be important for future public health investigations (Ocke et al., 2021). However, in several studies, the total polyphenol concentrations and antioxidant activity of strawberries and their extracts have been analyzed (Aaby et al., 2012;Mahmood et al., 2012;Nowicka et al., 2019;Salazar-Orbea et al., 2021). The reported levels for total polyphenols in these studies have shown high variability ranging from 8.45 to 208.58 mg/100g of total polyphenols. As the average daily intake of polyphenols is about 1000 mg/daily (Olas, 2018;Fraga et al., 2019), dietary intake of commercial fruit yogurts with strawberry preparations could provide substantial amounts of these antioxidants substances, especially, for the consumer who prefer commercial products over fresh fruits. However, the total polyphenol content showed no correlation with fruit preparations percentage. Polyphenols are considered to be the main contributors to antioxidant activity in fruit yogurts with strawberry preparation (Citta et al., 2017), but since they are present only in plants, in the current study, their concentrations were not determined in the samples of plain yogurt.
Numerous diseases, such are cardiovascular, neurodegenerative and cancer can result from oxidative stress (Basu et al., 2016;Olas, 2018). Antioxidative activity can be measured using various tests that are cost-effective and easily reproducible. They are based on one of the reactions: hydrogen atom transfer or electron transfer (Huang, Ou & Prior, 2005). The DPPH test is based on the transfer of hydrogen, which is donated by antioxidants to DPPH free radicals. On the other hand, the reducing power assay is based on an electron transfer reaction from potassium ferricyanide (Fe +3 ) to a substance with antioxidant potential (Bhalodia, Nariya, Acharya & Shukla, 2013). Literature data shows the common use of these tests alone or in combination for the assessment of the antioxidant activity of various plants (Nowicka et al., 2019;Dinkçi, Aktaş, Akdeniz & Sirbu, 2021;Pavlović et al., 2021;Batinić et al., 2022). Results for antioxidative activity determined by the DPPH test and reducing power for methanolic and ethanolic extracts are presented in Table 2. In our study, the sample with the highest polyphenol content (S4) showed the highest antioxidant activity measured by the DPPH test and reducing power test (Table 2). However, this sample did not contain the highest percentage of fruit preparation (Table 1). On the other hand, the fruit yogurt with the lowest amount of fruit preparation contained fewer polyphenols than other samples and had the lowest DPPH value (Tables 1 and 2). The control sample (plain yogurts) had lower values for DPPH (2.84±0.11%) and reducing power (0.07±0.02). There were statistically significant correlations between total polyphenols and DPPH in both methanolic (r 2 = 0.9128; p = 0.03) and 80% ethanolic (r 2 = 0.9537; p = 0.01) extracts. On the other hand, they were not demonstrated for reducing power assay regarding both methanolic (r 2 = -0.1161; p = 0.8) and ethanolic extracts (r 2 = -0.1234; p = 0.8). However, the main limitation of our study is the small number of analyzed samples. In contrast, Nowicka et al. (2019) reported a low correlation between total polyphenols and DPPH (r 2 =0.038), but higher for the ABTS test (r 2 =0.444) in 90 different strawberry cultivars.
Numerous literature data point out that the effects of strawberry polyphenols' dietary intake were dose-dependent (Alvarez-Suarez et al., 2011;Basu et al., 2016;Huang et al., 2022). Huang et al. (2022) reported that strawberry polyphenols (flavonols, flavanols and phenolic acid), in a dose from16 to 64 μg GAE/mL), decreased NO production and the expression of proinflammatory proteins iNOS and c-FOS in RAW 264.7 macrophage treated with lipopolysaccharides. Therefore, fruit yogurts containing strawberry polyphenols could be potentially used as part of an anti-inflammatory diet, depending on the level of certain polyphenols, but further human intervention studies are necessary. Additionally, strawberry polyphenols and anthocyanins dose-dependently weakened gastric lesions in rats (Alvarez-Suarez et al., 2011). A human intervention study reported that a high daily dose of strawberries (50 g) elevated whole blood glutathione in obese subjects compared to a daily intake of 25 g of strawberries (Basu et al., 2016). Thus, the content of fruit preparation and polyphenols in commercial yogurts on the Serbian market could be of critical importance for their health effects.
There are not much literature data about commercial fruit yogurts considering their total polyphenols content and antioxidant activity. Pereira, Barros and Ferreira (2013) investigated 14 samples of yogurts available in Portugal, but there were not any only strawberry-added. One of the analyzed samples in their study contained 6% of strawberry preparation, which is lower compared to our samples but it was labeled as "strawberry-kiwi" yogurt (Pereira et al., 2013). However, the DPPH and reduction power values from ethanolic extract were presented in different units than ours disabling direct comparison of the results. Also, Moldovan, Iasko and David (2016) investigated total polyphenols and the antioxidant activity in methanolic extracts of 12 commercial yogurts available in Romania, among which three were "strawberry yogurts", which contained about 400-500 µg GAE/ mL (approximately 40 mg GAE/100 g), which are far higher values compared to our results. So, our data may represent the first and most valuable information for consumers about the polyphenolic content and antioxidant activity of fruit yogurts with strawberry preparations available on the Serbian market.

CONCLUSIONS
Plain yogurts available in Serbian markets, either in plastic or Tetra Pak containers, did not differ in lactate content. Additionally, there was no difference in lactate content comparing strawberry-added and plain yogurts. Finally, statistically significant differences regarding the content of total polyphenols and antioxidant activities of fruit yogurts with strawberry preparations were observed. It can be suggested that labeling fruit-added yogurts for the content of polyphenols may be a useful strategy to better describe and provide far better information to consumers regarding antioxidant activity and consequently potential health benefits.