Evaluation of Physicochemical and Antioxidant Properties of Yogurt

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Bioactive Constituents, Metabolites, and Functions

Evaluation of physicochemical and antioxidant properties of yogurt enriched by olive leaf phenolics within nanoliposomes Hamidreza Tavakoli, Omidreza Hosseini, Seid Mahdi Jafari, and Iman Katouzian J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02759 • Publication Date (Web): 15 Aug 2018 Downloaded from http://pubs.acs.org on August 16, 2018

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Journal of Agricultural and Food Chemistry

Evaluation of physicochemical and antioxidant properties of yogurt enriched by olive leaf phenolics within nanoliposomes Running title: Yogurt enriched with nanoliposomal olive phenolics

Hamidreza Tavakoli1, Omidreza Hosseini2, Seid Mahdi Jafari2*, Iman Katouzian2 1

Health Research Center, Life Style Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran

2

Department of Food Materials and Process Design Engineering, Gorgan University of Agricultural

Science and Natural Resources, Gorgan, Iran.

*Corresponding author: Tel./fax: +98 17 324 26 432. E-mail address: [email protected] (S.M. Jafari). Abstract Olive leaf extract is a rich source of phenolic compounds and oleuropein which is well-known regarding its antioxidant and antimicrobial attributes. However, the mentioned phenolic compounds will lose their beneficial properties during storage and induce undesirable aftertaste in food products. In this study, olive leaf extract-bearing nanoliposomes were produced via the ethanol injection method and using phosphatidyl choline plus cholesterol as the reagents for the wall material. Later, the prepared nanocarriers were examined in regard to their zeta potential, stability, encapsulation efficiency and particle size. Moreover, the prepared nanoliposome-loaded yogurt samples were examined considering syneresis, antioxidant activity, pH, acidity, color and the sensorial properties. The mean particle size of the fabricated nanoliposomes was in the range of 25158 nm. Also, the entire formulations had a negative charge. The encapsulation efficiency was between 70.7 to 88.2%. Besides, the application of nanoliposomes in yogurt improved the antioxidant activity and unlike the yogurt with non-encapsulated olive extract, no significant changes in color and sensorial attributes were observed and even the syneresis rate was minimized. To conclude, olive leaf phenolics can be entrapped within nanoliposomes with a considerable encapsulation efficiency for application in food products like yogurt to increase their nutritional value and public acceptance. 1 ACS Paragon Plus Environment


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Keywords: Olive leaf; nanoliposome; phenolic compounds; oleuropein; yogurt. 1. Introduction Food plant by-products and biowaste have long been considered as useless materials and a threat to the environment. Olive-mill waste and leaf are notable examples of waste which are proposed as functional components for food products 1-3. Olive leaves contain significant levels of polyphenols, particularly oleuropein. Oleuropein is well-known regarding its antioxidant, anti-inflammatory, anti-bacterial and anti-viral properties

4-5

. Currently, natural antioxidants have received great

attention both from the public and the scholars. Also, according to the vast cultivation of olives in different countries, it seems necessary to produce value-added products using the olive-mill waste and more importantly the olive leaves which have a high biological value that olive leaves contain considerable amounts of phenolic compounds

6-7

. It has been revealed

8-10

, such as oleuropein,

verbascoside, ligstroside, tyrosol and hydroxy tyrosol. Among these phenolic components, oleuropein is one of the major and key phenolic elements which has astounding antioxidant and antimicrobial attributes 11. The application of olive leaf extract and other natural polyphenols in cuisines are limited due to their bitter taste and instability in the food matrix. Also, the bioavailability of the polyphenols is rather low in the unprotected form. Encapsulation technology is a promising strategy to remove these obstacles and to preserve the bioactive food components as well as masking the undesirable taste

12-13

. Nanoliposomes are one the entrapment bodies which are widely being implemented in

the food and pharmaceutical industries. They are aggregates composed of polar lipids that tend to form bilayer structures. According to the thermodynamic rules, amphiphilic phospholipid molecules are oriented in an ordered pattern within a sphere so that the water-hating tails are oriented face to face in these spheres and the water-loving heads are adjacent to the water molecules

12, 14

.

Accordingly, an enclosed circular membrane is formed with a bilayer of lipid molecules, this sphere is capable of preserving the water-soluble molecules in their central cavity and to retain the hydrophobic molecules inside their bilayer membranes 15-16. 2 ACS Paragon Plus Environment

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Yogurt is one of the popular dairy products consumed all over the world and is a proper candidate for fortification and production of functional food products

17

. This dairy product has a high

nutritional value, particularly in terms of the protein and calcium content and possess therapeutic effects because of its fermented nature owing to the presence of Lactobacillus bulgaricus and Streptococcus thermophilus18. Higher dry matter of yogurt in comparison to milk raise its mineral content, especially calcium. Yogurt has beneficial characteristics like the prevention of diseases and improving the immune system which is related to the components including proteins, some vitamins, minerals, metabolic products (lactic acid, peptides, amino acids, etc.) lactose and lactic acid bacteria. Antimicrobial agents, such as acidolin, acidophilin and bactericins released by lactobacillus genus

19-20

. In addition to the high nutritional value and sensorial properties, yogurt

matrix is a suitable medium to be fortified with food bioactive ingredients like phenolics. According to the studies in this line, peptides and amino acids which are generated through the fermentation process have antioxidant properties and therefore lead to the oxidative stability of the yogurt 21. In this study nanoliposomes were fabricated using the ethanol injection method and olive leaf extract with considerable amount of oleuropein was encapsulated by these nanocarriers. Some characteristics like particles size, zeta potential, stability and encapsulation efficiency were analyzed to determine the successful production of these loaded nanovehicles. Subsequently, these nanocarriers were added to yogurt as a dairy food product to apply these bioactive materials in a nutritious food matrix and then the physicochemical attributes together with the sensorial properties were examined to determine the possibility of the production of a functional food product and mask the bitter taste of olive leaf extract in these promising cost-effective nanocarriers. 2. Materials and methods Gallic acid and Folin-Ciocalteu reagent were purchased from Sigma Aldrich (Germany). Oleuorpein was purchased from Extrasynthes (Extrasynthese, Genay, France). Furthermore, lecithin (Phosphatidyl Choline) was obtained from Heidelberg company (Germany) and phosphate buffer saline (0.1M, pH 7.0) was provided from Metrohm firm (Switzerland). All other reagents including 3 ACS Paragon Plus Environment


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chloroform, DPPH (diphenylpicrylhydrazyl), phenolphthalein, cholesterol, HCl, NaOH, methanol and ethanol were supplied by Merck Company (Germany). 2.1 Preparation of the phenolic extract For this purpose, olive leaves were collected from a known orchard in Kazeroon (Fars, Iran). Moreover, the olive mill waste was collected from Sabzineh Tak olive processing plant in Shiraz. A modified method was used to extract the phenolic components and oleuropein

22

. Briefly, 10 g of

olive mill waste and leaves were homogenized and mixed with 100 mL ethanol (80%) using a blender (Parskhazar, BG-330p, Iran) with the maximum speed of 15000 rpm for 30 s. Subsequently, it was centrifuged (SIGMA3-18k, Germany) for 20 min at 39000 g. After centrifugation, the supernatant was collected and the sediment was resuspended in 100 mL of 80 % ethanol and the ultimate volume was set to 250 mL using methanol. Finally, the dispersion was filtered by a filter paper with the pore size of 20 µm and the solvent was evaporated using a rotary evaporator (Heidolph, Schwabach, Germany) operating at 40 °C. Folin-Ciocalteu method was applied to measure the total phenolic content and the standard curve was plotted using gallic acid

23

.

Ultimately, the equation obtained from the standard curve was as follows: Y=5.1075 X + 184.9

R2=0.988

(1)

2.2 Oleuropein measurement via high performance liquid chromatography (HPLC) An Azura HPLC system (Knauer, Berlin, Germany) equipped with a UV–Vis photodiode-array detector (DAD 2. one langmuir, Knauer) and LC pump (P 6.1L) was applied to determine the oleuropein content in olive waste and leaves. The polyphenols separation was performed with a 5µm ODS3 reversed-phase Prodigy column (250 × 4.6 mm; Phenomenex, USA) using solvent A (water/acetic acid, 97/3, V/V) and solvent B (methanol) under the gradient condition at 25 °C and 60 min. The flow rate and injection volume were 1 mL/min and 20 µL, respectively. The standard solution of oleuropein was added to the device at different concentrations (200-1000 ppm) and the wavelength of the photodetector was set at 280 nm

24

. This experiment was performed in 2

replications. 4 ACS Paragon Plus Environment

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2.3 Fabrication of nanoliposomes loaded with olive phenolics A modified method was applied (ethanol injection procedure)

25

. At first, phosphatidyl choline

(lecithin) and cholesterol were mixed with the ratio of 4:1, and labeled as (PC). Then, 10 g of PC mixture was blended with different concentrations of the phenolic extract from 1 g (F1) to 5 g (F5) (as shown in Table 1). The mixture was then dissolved in 35-39 g of ethanol and immediately agitated with the same volume (35-39 g) of phosphate buffer saline (PBS, pH=6, 0.05M) and stored for 30 min. The aqueous phase then represents a milky color as a result of the formation of nanoliposomes. Afterwards, ethanol was evaporated using a rotary evaporator at the temperature of 40 °C and the remaining volume was set at 50 g so that the phenolic content of final nanoliposomes was adjusted to 2, 4, 6, 8, and 10 % (W/W). Finally, the liposome system was homogenized by a rotor-stator device (Heidolph, Schwabach, Germany) at the rate of 10,000 rpm for 10 min. Table 1 2.4 Determining the stability of nanoliposomes 5 mL of each sample was placed in a 10 mL test tube and centrifuged at 3500 rpm for 15 min in order to disrupt the dispersion and to acquire a 2-phasic mixture

26

. Then, nanoliposome stability

(NS) was calculated by the following equation: %NS=(Fev/Iev)×100

(2)

Fev: final volume of the nanoliposome Iev: initial volume of the nanoliposome 2.5 Determination of encapsulation efficiency At first, the free (unencapsulated) phenolic compounds were separated from the entrapped parts via centrifugation at 3000 rpm for 15 min. Afterwards, chloroform was added into the encapsulated phase so that the liposomal membrane could be solved and the phenolic compounds are liberated. Ultimately, the concentration of phenolic compounds in each sample was determined based on gallic acid standard curve as mentioned in Section 2.1. The encapsulation efficiency (EE) was measured using the following equation: 5 ACS Paragon Plus Environment


EE%= ((Ctotal-Cfree)/ Ctotal) × 100

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(3)

where Ctotal is the concentration of the total phenol content in the liposomal suspension and Cfree is the concentration of the free phenolic content. 2.6 Analysis of particle size and zeta potential The Zetasizer apparatus (Malvern, UK) was applied to determine the size and zeta potential of the samples. First, the samples were diluted with distilled water (1:100) and then they were taken in vertical cylindrical cuvettes (10 mm diameter). The measurement of zeta potential and size was done at 25°C 19. The average size of the nanostructures was recorded in terms of number, intensity, and volume distribution). 2.7 Formulation of yogurt enriched with polyphenol-bearing nanoliposomes Pasteurized yogurt with 3% fat, 3.4% protein and 5.3% carbohydrate were prepared from Ramak dairy plant (Shriaz, Iran). First, milk was cooled to 42 °C after the pasteurization process. Later the commercial starter culture (YSC) at the concentration of 5% was added into the cooled milk and incubated for 3 h at the temperature of 41 °C. After this process, the product was kept in refrigerator for 24 h. Then, 15 g of the nanoliposome formulation containing 10% phenolics (F5) and a control sample (1.5 g phenolic extract without encapsulation) was added separately to 100 g of yogurt samples and stored at 4°C in the refrigerator. The characteristics of the produced yogurts were examined during the 21-day period of storage. Also, the yogurt without nanoliposomes and phenolic extract was considered as the control sample. 2.7.1 Evaluation of the antioxidant activity by DPPH method 2.5 ml distilled water was added to 10 g of yogurt (pH was set to 4 by adding HCl 1 M) and stirred. The yogurt was incubated at 45 °C for 10 min and then centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant was separated and the pH was set to 7 by adding NaOH. Centrifugation was done again at 10,000 rpm for 10 min at 4 °C and about 250 µL of the sample was added into 3 mL of the ethanolic solution of DPPH (60 µM) and stirred vigorously. Later, it was stored for 5 minutes at the ambient temperature and the absorption was recorded at 517 nm (Aextract). In the control 6 ACS Paragon Plus Environment

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sample (Acontrol), distilled water was used instead of yogurt water

27

. In the end, the radical

scavenging capacity was calculated from this formula: Radical scavenging capacity= ((Acontrol-Aextract)/Acontrol)×100

(4)

2.7.2 Measurement of pH and acidity Firstly, 10 mL yogurt sample was added to a beaker containing 10 mL distilled water and after stirring, the electrode of pH meter (Metrohm-827, Switzerland) which was calibrated by buffers of pH= 4 and 7 was placed into the sample and the pH was recorded

28

. The acidity assay was done

according to the National Standard of Iran No. 2852. Accordingly, 9 g of the yogurt sample was weighed in a beaker and water was added equally. Later, the dispersion was titrated by NaOH 0.1 N plus phenolphthalein 1% and this process was continued until a light pink color was appeared and remained stable for 5 s. Acidity (in terms of lactic acid) was calculated by dividing the volume of the consumed NaOH by 10. 2.7.3 Determination of syneresis 20 g of the yogurt sample was poured into the centrifugal tube and placed in a centrifuge at 500 rpm for 5 min. Later, the transparent supernatant was transferred into a small beaker and the volume was determined using a pipette. The measurement of syneresis was done according to the equation below 29. % Syneresis: VE / Y

(5)

herein, VE is the weight of the transparent supernatant and Y is the weight of the yogurt sample. 2.7.4 Color analysis of yogurt samples L* index alterations were examined at stable conditions using image processing. Image analysis was carried out via Image J software 30. The photos were taken by a DMC-FS42 Panasonic camera and were saved in JPG format. Afterwards, the photos were converted from RGB to Lab mode via the color space converter plugin in ImageJ and the L* index was measured. 2.7.5 Sensory analysis of yogurt samples

7 ACS Paragon Plus Environment


After educating the panelists, 20 people (both men and women) with the average age of 25-35 years were selected as panelists. At first, samples were placed in 3 different plates (A, B and C and the sensorial valorization was done by a hedonic test based on the 5-point scoring form (1. Very Bad, 2. Bad, 3. Normal, 4. Good, 5. Excellent) and ultimately, the score of each sample was analyzed by SAS. Thereby, attributes like color, taste, texture and overall acceptability were evaluated for the samples

31

. In addition, all the necessary circumstances and same conditions of temperature, light

and environment were prepared for the panelists and they were directed to drink water prior to testing each sample 32. 2.8 Statistical analysis The final results were analyzed by Analysis of Variance (ANOVA) and means were compared by Duncan test at 5% level of probability. The data was analyzed using the SAS 9.1.3 Portable software and the graphs were plotted via Excel 2016. 3. Results and discussion 3.1 Total phenolics and oleuropein content of samples In this study, the phenolic content of the olive leaf extract (composed of mainly oleuropein together with other compounds like luteolin-4’-O-glucoside, luteolin-7’-O-glucoside, luteolin, tyrosol and hydroxy tyrosol) was about 35.71 ± 1.15 mg/g sample. The chromatogram of the olive leaf extract (diluted twice) is depicted in Fig. 1. It should be noted that the negative peak in the chromatogram denotes the negative absorption of some compounds at the selected wavelength 33. Fig. 1 Considering the surfaces area below the curves (1364.050 and 1370.296) along with the obtained values for oleuropein (230.87 and 232.09), the oleuropein level present in the twice-diluted extract was 231.48 µg/mL and as a result, the ultimate level of oleuropein was 23.148 mg/mL of extract. 3.2 Encapsulation efficiency of olive leaf phenolics within nanoliposomes Fig. 2A demonstrates the impact of different concentrations of olive leaf phenolics on the encapsulation efficiency within nanoliposomes. There was not a significant difference (P>0.05) 8 ACS Paragon Plus Environment

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between F1, F2, and F3 formulations in terms of encapsulation efficiency which was in the range of 85.1 to 88.2 %. The nanoliposomes containing olive leaf phenolics (containing mainly oleuropein plus other compounds like luteolin-4’-O-glucoside, luteolin-7’-O-glucoside, luteolin, tyrosol and hydroxy tyrosol) at F4 and F5 formulations had a lower encapsulation efficiency which was 77.4 % from 70.7 %, respectively. In the same way, other scholars

34

reported a reduction in encapsulation

efficiency with the enhancement of green tea polyphenols concentration in liposomal matrix from 60% to 40%. Nakayama et al 35 proved that epigallocatechin gallate at low concentrations ceases the permeance of calcein from liposomes, whereas at high concentrations, the membrane of the liposomes breaks down. In another study, Takahashi et al

36

encapsulated curcumin in liposomal

systems with an encapsulation efficiency of 68% using the microfluidizer device. Moreover, Gibis et al 37 reported that with the encapsulation of grape seed extract into liposomes, almost 80% of the extract binds to the surface of the liposome membrane instead of incorporating into the liposomal membrane. Furthermore, in a study nanoliposomes were prepared via microfluidization technique and the scholars reported that by the increase in core material content and maintaining the same level of wall materials, the encapsulation efficiency is lowered

38

. It was illustrated that several

factors influence encapsulation efficiency including the type of wall material, payload, ratio of core to wall material, method of encapsulation, size of particles and the total solid content 39. Fig. 2 3.3 Stability of nanoliposomes loaded with olive leaf phenolics The results of nanoliposomal stability (Fig. 2B) indicate there are some differences in formulated nanoliposome samples in terms of stability against the executed centrifugal force. In this test, the reported values demonstrate the serum separation level; the low level of separated aqueous phase indicates higher stability. According to the study of Taylor et al

40

, the instability of liposomes may be attributed to the

collision of particles due to Brownian motion and their fusion and in terms of thermodynamics; this is because of the tendency of system to lower the energy in the bilayer structure of the liposomes. 9 ACS Paragon Plus Environment


The major reason for the instability of the system is the fusion of the vesicles plus their collision leading to the fusion of the liposome membranes. Nisin was encapsulated inside the liposomes in the fermented milk system and it was reported that the physical stability of the liposomes is dependent on medium factors, liposome size, number of the layers, phospholipid structure and the production method of the liposomes

41

. Also, it was proved that the implementation of

phospholipids in the preparation of liposomes is significantly effective in their final stability 36. We found that the formulation with lowest (2%, F1) and highest (10%, F5) content of olive leaf phenolics had the maximum and minimum stability based on centrifugal stability test, respectively. In response to the constant level of phosphatidyl choline, with the increase of extract, the loading level and encapsulation efficiency were lowered and at the centrifugation step, they expose low stability. The extract of Gossypium hirsutum was encased by nanoliposomes and it was reported that by increasing the extract in the formulation, the stability of the system was lowered 42. Besides, Ostwald ripening is a significant factor responsible for the instability of the emulsion systems and nanostructures. This phenomenon is more common in systems with nano-sized particles (Gutiérrez et al., 2008). One factor which influences this process is the changes in the size of particles, moreover, the loaded extract inside the nanoliposomes affect their final size and therefore these factors can influence the rate of Ostwald ripening and consequently, instability within the nanoliposome network. The applied phenolic compounds in the extract (including mostly oleuropein, tyrosol and hydroxy tyrosol) contain hydroxyl groups which are dissociated under the experiment conditions into negatively charged O- groups and these groups are able to form hydrogen and ionic bonds with positively charged choline head moieties of phospholipids in the nanoliposome structure favoring the process of Ostwald ripening

43

. Moreover, increase in size of

the nanoliposomes may occur as a result of the initial oxidation products which degrade to aldehydes and other moieties 44. Accordingly, it was indicated that the entrance of extract into the liposomal network not only affect the size of the liposomes but also influences the particle size distribution. By the increase in the latter parameter, the system undergoes instability 45. 10 ACS Paragon Plus Environment

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3.4 Particle size and zeta potential of nanoliposomal olive leaf phenolics The results related to the size of nanoliposomes at different concentrations of the olive leaf extract are depicted in Fig. 3. In the measured samples, the size of particles was in the range of 25-158 nm; with the augmentation of olive leaf extract from 2% to 10%, the size of the nanoliposomes increased. Similarly, a nanoliposome matrix was developed to protect the bioactive components of clove and eugenol extract and it was demonstrated that after loading them inside the nanoliposomes, their size increased considerably 46. Indeed, as the concentration of eugenol extract increased from 7.5 to 15 mg/mL, the size of nanoliposomes enhanced considerably. The authors reported the employed phenols were oriented in the vicinity between the polar groups (choline molecule), the glycerol structure plus the first atoms of the acyl chains. They also reported that eugenol is able to from a powerful interaction with liposomes regarding the hydrophobic interaction between its allyl group (CH2-CH=CH2) plus the acyl moieties of dipalmitoyl phosphatidylcholine. In our study, lower concentration of phenolic extract leads to the formation of smaller nanoparticles as seen in Fig. 3. Also, as the main constituents of the polyphenol extract include oleuropein, tyrosol and hydroxy tyrosol which are basically packed in the aqueous core of the nanoliposomes owing to the hydrophilic nature and OH groups of these compounds. Fig. 3 The size reduction of the particles to the nano-scale significantly alters the physicochemical properties as well as improving their biological activities. The increase in the surface to volume ratio leads to the increase in bioavailability and allows the transmission of these structures across the cell membranes 13. In relation to the size of polyphenol-bearing nanoliposomes, nanoliposomes were prepared containing green tea poly phenols with the mass ratio of (1:8) using the method of ethanol injection and microfluidization 25. Furthermore, it was reported that size is not a key factor in determining the characteristics of nanoparticles since no significant quality difference was observed among the particles with different sizes

47

. IUPAC reported that with the usage of UF

filters and/or preparation of stable solutions, the upper limit of the size of nanoparticles is 11 ACS Paragon Plus Environment


considered as 500 nm. Considering the study of Solans et al 48, particles in the size range of 500 nm demonstrate the attributes of nanoparticles and therefore these scholars reported 500 nm as the maximum value of the size of nanoparticles. Thereby, in this study, all the fabricated nanoliposomes are within the nanoscale range. Zeta potential of the nanoliposome samples (F1, F3, and F5) at different concentrations of olive leaf phenolics (containing mainly oleuropein plus other compounds like luteolin-4’-O-glucoside, luteolin-7’-O-glucoside, luteolin, tyrosol and hydroxy tyrosol) were -0.3, -6.9, and -23.1 mV, respectively. Considering the given data, all formulations had a negative charge. The higher content of polyphenolic compounds increased the negative charge of the vesicles which may be due to the negative charge of the olive leaf polyphenols (-12 mV) after the relative ionization of the compounds under the pH of the system. However, at the low levels of phenolic extract as most of the compounds are packed in the aqueous core, lower zeta potential is observed due to encapsulation. In addition, it was reported that phenolic compounds not only can be incorporated within the liposomes but also can be adhered or absorbed onto the surface of the liposomal membrane

37

. The zeta potential is also dependent on the temperature and ionic strength of the

environment 49. Zeta potential is an index that determines the interaction rate between the colloidal particles as well as the stability of the vesicle-based suspensions. Indeed, zeta potential is a function of the surface charge of the lipid vesicles, absorbed layers on the surface and the nature of the environment in which the liposomes are dispersed. The more surface charge of liposomes leads to the repulsion of these carriers which hinders the aggregation of the vesicles and increases the stability of the liposomes. Generally, if the zeta potential of the whole colloidal system is higher than ±30 mV, the particles are stable regarding the electrostatic repulsion 50. Also, according to a research by Zhang et al 51, the presence of repulsion force on the surface of the particles decrease their aggregation and to reach this goal the value of zeta potential must be lower or higher than -25 and +25 mV


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respectively. Although, some studies revealed that even at low zeta potential (nearly 0), liposomes may remain stable 25. 3.5 Addition of olive leaf extract-loaded nanoliposomes into yogurt 3.5.1 Syneresis of yogurt samples Table 2 shows the syneresis of yogurt samples during 21 days of storage. As can be seen, the syneresis rate significantly decreased with the addition of nanoencapsulated extract to the yogurt sample other than the first day of storage in comparison to the control sample. As the total dry matter of the yogurt increases, the porosity of yogurt is decreased which in turn enhances the stability of yogurt and minimize the syneresis rate. Herein, with the addition of nanoliposomes because of the presence of lecithin, the absorption of water is accelerated and therefore the syneresis process is retarded

19

. By the increase in the rate of free extract, yogurt samples revealed high

syneresis which probably is due to the increase in the active water content of yogurt

28

plus the

reduction in the water holding capacity 52. In fact, during the 21-day storage period, the reduction in pH and its effect on casein micelles lead to the minimization in the syneresis rate 53. Table 2 The inhibition of phase separation and lowering the rate of syneresis during the storage of yogurt is one the main goals in the dairy industry. Syneresis mainly occurs due to modification and disruption in the protein network of yogurt. Other factors influencing the syneresis of yogurt include shear execution, shrinkage in the structure and the reduction in the bonding energy of whey proteins to the casein network during storage 54-55. 3.5.2 Antioxidant activity of yogurt samples Catalase and super oxidase enzymes, casein and lactic acid bacteria exhibit antioxidant properties that are present in the yogurt network

56

. According to Fig. 4, it can be perceived that the

antioxidant activity of the yogurt samples with free and encapsulated olive leaf extract is considerably higher than simple yogurt. Fig. 4 13 ACS Paragon Plus Environment


Phenolic compounds are well-known regarding their antioxidant properties

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57

. Here, oleuropein

together with other olive leaf phenolics (a combination of luteolin-4’-O-glucoside, luteolin-7’-Oglucoside, luteolin, tyrosol and hydroxy tyrosol) exhibit antioxidant properties and raise the antioxidant activity of yogurt samples regardless of being in the encapsulated or the free form. It can be concluded that the encased extract exposes antioxidant properties just like the free form of extract, however, the radical scavenging level of the encapsulated form was a bit lower than the free form at the first day of storage. After some days, the antioxidant activity of the yogurt with free extract revealed a descending trend in the antioxidant activity owing to the destruction of the phenolic compounds during time with fermentation and the effect of yogurt bacteria 27. Also, Gad and co-workers reported the reduction in the antioxidant activities of yogurt enriched with 10% palm date extract during storage

58

. In Fig. 4, it is clear that the antioxidant activity of the yogurt

samples containing nanoliposomal phenolics is increased during time due to the controlled release of the phenolic components from the nanoliposomal network. Moreover, it can be noted that the radical scavenging capacity of the control sample is changed during storage which may be contributed to proteolysis and formation of organic acids and modifications in α-amylase and αglucosidase 27 together with the metabolic performance and antimicrobial activity. 3.5.3 pH and acidity of yogurt samples Modifications in pH and acidity of yogurt samples during 21 days of storage at 4 °C was examined and the results are summarized in Fig. 5. As it is shown, pH of the samples was lowered during storage and acidity was enhanced which is the same for the entire samples. Fig. 5 The results of this study indicated that storage time had a significant effect (P0.05).


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Moreover, another research group reported that oleuropein and olive phenolics (tyrosol and hydroxy tyrosol) do not alter pH and acidity of milk and yogurt during storage 59. 3.5.4 Color results of yogurt samples In this study, the L index was applied to examine the lightness/whiteness of the yogurt samples via image processing as higher values of L indicate bright samples. Some scanned images are provided in Fig. 6. The highest intensity of white color was seen in the control yogurt (without the free/encapsulated yogurt) and storage time had a significant effect on lowering the L index (P

Evaluation of Physicochemical and Antioxidant Properties of Yogurt

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