Comparative Physicochemical, Microbiological, Antioxidant, and Sensory properties of pre‐ and post‐fermented yoghurt enriched with olive leaf and its extract

Abstract This study investigated the comparative effect of yoghurt samples enriched with different concentrations of olive leaf powder (OLP) (0.1, 0.5, 1, and 1.5 mg/ml) and its extract (OLE) (0.5, 1, 3, and 5 mg/ml) on the microbiological, sensory, physicochemical, and antioxidant properties of pre‐ and post‐fermented samples during 21 days of storage. Sensory evaluation showed that concentrations lower than 1.5 and 5 mg/ml of OLP and OLE, respectively, were acceptable. Adding OLP or OLE did not have an influence on yoghurt starter culture bacteria (p > .05). All enriched samples significantly showed higher acidity and lower pH compared with control samples (p < .05). The most important polyphenols were oleuropein (from 0.132 to 0.224 and 0.373 to 0.413 mg/g for 0.5 and 3 mg/ml of OLE, from 0.194 to 0.321 mg/g and 0.413 to 1.280 mg/g for 0.5 and 1 mg/ml of OLP, respectively) and catechin (from 0.369 to 0.382 and 0.461 to 0.477 mg/g for 0.5 and 3 mg/ml of OLE, from 0.386 to 0.405 mg/g and 0.310 to 0.710 mg/g for 0.5 and 1 mg/ml of OLP, respectively) in enriched yoghurts. Adding OLP or OLE increased shelf life, antioxidant activity percentage (AA%), and total phenol content (TPC) of enriched samples (p < .05). During 21 days of storage of all samples, TPC, AA%, and pH decreased and TTA increased.

accumulated during pruning of olive trees (Molina-Alcaide & Yanez-Ruiz, 2008). Moreover, OLs can be obtained in high quantities in olive oil industries (10%). OL can be considered as a cheap and easily available natural source of different valuable compounds, including phenolic compounds, secoiridoids, flavonoids (Jemaiet et al., 2008), and high quantities of oleuropein (60-90 mg/g dry weight) (Ansari Dogaheh et al., 2011). Numerous studies (in vivo and in vitro) have demonstrated that oleuropein has a wide variety of biological activities, including antimicrobial, anti-ischemic, antihypertensive, antiinflammatory, and anticarcinogenic properties (Zoidou et al., 2014).
In a related study, for the investigation of the effect of polyphenol-enriched yoghurt on human health, 16 nonsmoking volunteers consumed either 400 g of olive polyphenol-enriched yoghurt (containing 50 mg of encapsulated olive polyphenols) or 400 g of plain yoghurt every day for 2 weeks. The results indicated that consumption of the polyphenol-enriched yoghurt may help reduce body weight, blood pressure, low-density lipoprotein (LDL) cholesterol levels, and lipid peroxidation, and may promote growth of beneficial lactic acid bacteria (LAB) (Georgakouli et al., 2016). In this study, the aim was to enrich yoghurt, as it is a highly consumed product, with olive leaf and its extract (a rich source of polyphenols) in order to increase the functionality and shelf life of it. For this purpose, different concentrations of OLP and OLE were added to the yoghurt samples, pre-and post-fermentation. Then, the sensory evaluation, survival of lactic acid bacteria, total titratable acidity (TTA) and pH changes, antioxidant activity percentage (AA%), total phenol content (TPC), and phenolic compounds of the yoghurt samples were determined.

| Materials
Fresh cows' milk (3.6% fat) was obtained from a local milk farm in Karaj (Alborz, Iran). A starter culture (Yo-MixTM 401 LYO) containing Lactobacillus delbrueckii subsp. bulgaricus (strain ATCC 1184) and Streptococcus thermophilus was purchased from Danisco (Niebüll, Germany). All chemical compounds used in the experiments, such as de Man, Rogosa and Sharpe (MRS) agar culture medium, MRS broth, and peptone, were purchased from Merck (Darmstadt, Germany).

| Preparation of OLs
Fresh young OLs were sampled from the Roughani variety (a native olive tree of Iran) in September 2017 from one of Fasa orchards at a latitude of 28.938300 (28°56′18″N) and a longitude of 53.648200 (53°38′54″E) located in Fars province of Iran. The OLs were washed several times with tap water and dried under vacuum at 25°C.
The dried OLs were ground into a fine powder by a small electric grinder (Sanyo 260 W). Then, the olive leaf powder (OLP) was passed through a stainless steel sieve (the particle size of the OLP was approximately smaller than 50 microns) to obtain homogeneous and uniformly sized fine particles. The OLP was stored in a dark bottle at 25°C.

| Preparation of OLE
At first, 10 g of OLP and 50 ml of deionized water were mixed in a flask. The flask was put on a magnetic stirrer and stirred for 24 h in a dark place at 25°C. After stirring, the sample was filtered using a filter paper into a 50-ml volumetric flask. The filtrate was passed through a sterile 0.22µm filter into a pre-sterilized container. The filtrate was kept in a dark bottle at −20°C until further use.

| Preparation of yoghurt samples
The homogenized milk was pasteurized at 90°C for 5 min, cooled to 44 ± 1°C, and poured in the sterilized bottles (500 ml). For prefermentation, defined concentrations of OLP (0.1, 0.5, 1, and 1.5 mg/ ml) and OLE (0.5, 1, 3, and 5 mg/ml) were added to a series of sterilized bottles containing 500 ml of milk and thoroughly mixed. The bottles were labeled according to the concentration of added OLP or OLE. Then, 1 ml of starter culture solution (200 g of the starter was mixed with 1 liter of milk at room temperature) was added to each bottle. Then, they were incubated at 44 ± 1°C until the pH reached at 4.5 ± 0.1 (approximately 4-5 h).
For post-fermentation, different concentrations of OLP or OLE were added to the series of sterilized bottles containing prepared yoghurt until the same concentrations of 0.1, 0.5, 1, and 1.5 mg/ml of OLP and 0.5, 1, 3, and 5 mg/ml of OLE were obtained in the final products. All samples were kept at 4°C, and the analysis was carried out after 1, 7, 14, and 21 days of cold storage. The control samples were prepared without OLP or OLE.

| Sensory analysis
Ten trained sensory panelists (comprised of five males and five females; 25-35 years old) evaluated randomly coded yoghurt samples.
Texture, color, typical yoghurt flavor, and overall acceptability were evaluated on a 5-point scale (1 = poor to 5 = excellent), while astringency, sourness, metallic taste, and bitterness were scored on a 5-point scale (1 = low intensity to 5 = high intensity). The treatments with low sensory evaluation score (defined as not acceptable) were excluded from the study (ISO 22935-1, 2, and 3).

| pH and TTA
The pH of the yoghurt samples was measured with a pH meter (Jenwy 3510). All samples were homogenized in water at 1:9 ratios before pH determination (Amirdivani & Baba, 2011). The TTA of the new products was determined according to the ISO 11869: 2012 method. TTA was calculated as follows: where V is the volume of NaOH required to neutralize the acid. All the analyses were performed in triplicate. The yoghurt samples were monitored for pH and TTA during cold storage for 1, 7, 14, and 21 days at 4°C.

| Extraction of phenolic compounds
The phenolic compounds were extracted from the enriched yoghurt samples according to the method described by Zoidou et al. (2014).
Briefly, 1 g of yoghurt sample was diluted with 1 ml of distilled water, vortexed for 1 min, and sonicated in a water bath sonicator for 15 min. Then, the samples were centrifuged at 112 g for 5 min using a refrigerated centrifuge. The clear supernatant was separated and stored at −20°C for further use.

HPLC analysis
Determination and detection of polyphenol compounds in the yoghurt samples were carried out by HPLC, a system consisting of a quaternary pump coupled to a UV detector. A conventional reversed-phase C18 column (250 mm × 4.6 mm, 5 μm) was used as the stationary phase. The gradient elution program was implemented using a system of two solvents as follows: solvent A consisted of water containing 0.2% H 3 PO 4 (v/v), and solvent B was the mixture of methanol and acetonitrile (50:50 v/v). The flow rate was constant at 1 ml/min and chromatographic analysis was performed at 25°C. Phenolic compounds were detected at 280 nm. The HPLC gradient program was as follows: 0 min, 96% A; 40 min, 50% A; 45 min, 40% A; 60 min, 0% A, 70 min, 0% A; 72 min; 96% A. Identification of the eluting peaks of phenolic compounds was performed by comparing their retention time (tR) values and the corresponding UV spectra.

Standard solutions
The stock solution of 1 mg/ml was prepared from the phenolic compound standard. Then, by diluting appropriate volumes of the stock solution with methanol, working standards were obtained at lower concentrations (0.02, 0.04, 0.1, 0.2, and 0.4 mg/ml). Stock solution and working standards were stored in a refrigerator at 4°C.

| Determination of TPC
The TPC was determined by the colorimetric assay according to the method described by Mohammed and Manan (2008). A mixture containing yoghurt aliquot extract (200 μl), deionized water (800 μl of), and Folin-Ciocalteu reagent (100 μl) was prepared and incubated for 3 min at room temperature. Then, 300 μl of sodium carbonate (20%) was added to the mixture and incubated for 2 h at room temperature under dark conditions. The absorbance of the mixture was determined using UV-Vis spectrophotometer (Perkin Elmer Lmbda 25) at 765 nm. A blank sample was prepared with distilled water instead of the aliquot extract.
In addition, the gallic acid standard curve was prepared (from 0 to 100 mg/L) and TPC was expressed in mg of gallic acid equivalent/g dry matter. The analysis of samples was made in triplicate, and data are presented as mean ± standard deviation (SD).

| Determination of antioxidant activity (AA %)
The antioxidant activity percentage (AA %) of the yoghurt extract was determined according to the procedure reported by Robert et al. (1999). For this purpose, 2.5 mg of 2, 2-diphenyl-1-picrylhydrazyl (DPPH) reagent was dissolved in 100-ml methanol. This stock solution was kept in a dark place at ambient temperature. A quantity of 0.1 ml of yoghurt extract was mixed with 3.9 ml of methanolic DPPH solution. The control sample was prepared with the same volume of methanol and deionized water instead of the sample. Absorbance was measured at 515 nm by a UV-Vis spectrophotometer (Perkin Elmer Lmbda 25). The measurements were carried out in triplicate.

Data analysis
Statistical analysis was performed with SPSS Version 11.0 statistic software package and Duncan's test for mean comparison was adopted to highlight significant differences among the yoghurt samples. Data were significant if the p-value was found to be <0.05.
Data are expressed as means ± standard deviation (SD).

| Sensory analysis
Sensory properties of the final products play the most important role in their daily consumptions. Therefore, organoleptic assessment was performed by 10 trained panelists at the Institute of Standards and Industrial Research of Iran (ISIRI) according to the ISO 22935-1 method. Results of the organoleptic assessment of the enriched yoghurt samples are presented in Table 1.
It was observed that concentrations of 5 mg/ml of OLE and 1.5 mg/ml of OLP were not accepted, pre-or post-fermentation, due to unacceptable sensory specifications, including color, bitter flavor, and undesirable texture (p < .05). The results showed that increase of OLE concentration to 5 mg/ml was made to sense a potential flavor of olive leaf.

| Microbiological analysis
Viable bacteria counts were performed on days 1, 7, 14, and 21 in triplicate. Table 2 shows the enumeration of L. delbrueckii subsp. The enumeration of S. thermophilus in the enriched yoghurt products, as well as in control samples, during storage for 21 days is presented in Table 3. It was observed that adding OLP or OLE did not significantly affect the total count of the S. thermophilus in the enriched yoghurt samples in comparison with control yoghurt samples at the end of each studied period (p > .05). Also, it was observed that the colonies of the S. thermophilus had existed in all enriched samples over 21 days of storage and no significant decrease was observed after the 21-day storage (p > .05). The findings of the current study revealed that S. thermophilus bacteria were more stable in comparison with L. delbrueckii subsp. bulgaricus after 21 days of storage in all enriched yoghurt samples.
In addition, the viable counts of S. thermophilus increased slightly or remained constant with increase of OLP or OLE concentration, pre-or post-fermentation.

| pH and TTA values
pH and TTA are known key factors that impact the shelf life and acceptability of dairy products. Table 4 shows the pH values of yoghurt samples enriched with OLP or OLE, which were determined every week over a period of 21 days. The results showed that pH values of all products significantly dropped during the storage period (p < .05). Also, the addition of OLP at different concentrations, pre-or post-fermentation, had a slight effect on the pH of enriched samples when compared to that of plain yoghurt samples (p > .05).
Increase of OLP or OLE concentration resulted in a slight decrease in the pH of yoghurt samples, pre-or post-fermentation. The pH values of all products dropped (p < .05) due to the activity of the lactic acid bacteria during the storage. Table 5

| Identification of phenolic compounds
The limits of detection (LOD) and quantitation (LOQ) were obtained for vanillin, vanillic acid, caffeic acid, oleuropein, apigenin, luteolin, tyrosol, and catechin. It was observed that the LOD and LOQ ranged approximately from 10 to 30 and 30 to 90 µg/g, respectively. Table 6    TPC were higher when OLP or OLE was added post-fermentation in comparison with those of pre-fermentation. A positive relationship between the concentration of OLP or OLE and TPC was observed.

| Evaluation of TPC in the enriched yoghurt
Besides the starter culture activities, the time, and temperature of fermentation might have affected this condition.

| Evaluation of antioxidant activity (AA%)
DPPH assay was performed to determine the antioxidant activity percentage (AA%) of the yoghurt samples. The results showed that the antioxidant activity values of enriched samples were higher than those of control samples (

| DISCUSS ION
In this study, OLP and OLE were added before and after fermen-     Echium plantagineum, did not influence the viability of lactic acid bacteria, which were higher than 10 7 cfu/g at 21 days of storage (Dal . Yoghurt containing olive leaf hot water extract (0, 0.1%, 0.2%, and 0.4% (w/v)) stored for 15 days at 4°C showed no significant effect on the population of lactic acid bacteria when compared to that of control yoghurt (Cho et al., 2020). Yoghurt sup-        during fermentation, the viable cell counts of LAB did increased, but increase of red ginseng extract concentration resulted in a decrease of LAB cell counts (Jung et al., 2016). In similar studies, the addition of hazelnut skin (Bertolino et al., 2015) and freeze-dried stevia to the yoghurt had suggested a significant effect on pH and TTA of the final product relative to control samples. However, adding oleuropein, which is a natural antioxidant (Zoidou et al., 2014), and tea (Jaziri et al., 2009)  Also, yoghurt fortified with phenolic compounds extracted from strawberry press residues was studied, and results showed that TPC obtained was 4640.0 ± 23.93 mg GAE/100 g dry extract (Ivanov & Dimitrova, 2019). The TPC increased when the yoghurt sample was enriched with different additives, including, freeze-dried stevia (Carvalho et al., 2018) and peppermint, dill, basil (Amirdivani & Baba, 2011), when compared to the blank yoghurt. The TPC values of yoghurt fortified with red cactus pear peel powder and its mucilage powder were 348.0 ± 4.8 mg GAE/100 g and 410.6 ± 3.9 mg GAE/100 g, respectively (Hernández-Carranza et al., 2019). Total phenol content (TPC) of yoghurt fortified at 0%, 1%, 3%, and 5%

Concentrations of added OLP
of green olive powder was obtained 4.30, 4.52, 5.85, and 6.96 (mg GAE/kg), which on day 15 of storage, TPC caluculated 3.67, 4, 4.82 and 5.60 (mg GAE/kg), respectively (Cho et al., 2017).    extract was reduced during storage and the increase of olive leaf hot water extract concentration led to an increased antioxidant activity (Cho et al., 2020). The DPPH radical scavenging activity of yoghurt fortified with green olive leaf powder decreased after a storage of 14 days, and also an increase in antioxidant activity percentage was observed with increasing green olive leaf powder concentrations (Cho et al., 2017).

| CON CLUS IONS
Olive leaf powder and OLE are the rich sources of phenolic compounds; OLP is a healthy raw material that contains nutraceutical compounds. Yoghurt supplemented with OLP and OLE can be clas- or OLE can be considered as the commercial nutraceutical products.

ACK N OWLED G EM ENT
We acknowledge the support of the Iranian National Standards Organization, which provided materials and instrumental facilities.

CO N FLI C T O F I NTE R E S T
There is no conflict of interest regarding the publication of this manuscript.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.