# Encapsulation of Different Types of Probiotic Bacteria within Conventional/Multilayer Emulsion and Its Effect on the Properties of Probiotic Yogurt

Authors: Hamed Mahmoodi Pour; Mohammad Hossein Marhamatizadeh; Hossein Fattahi
Journal: Journal of Food Quality
 (2022)

---

## Abstract

Microencapsulation of probiotic cells within emulsion is an efficient method to enhance the viability of probiotic bacteria. In the present study, free and encapsulated probiotic cells (Lactobacillus rhamnosus and Lactobacillus plantarum) in simple and multilayer emulsions were used to produce a set of probiotic yogurts. In all samples, an increasing trend in syneresis and acidity values and a decreasing trend in pH and viability of probiotic cells were observed during the storage time. However, the changes in these parameters were more significant for free-loaded probiotic samples. Moreover, the free cells showed poor survival in the yogurt samples by decreasing the viable cell count of probiotics from 7.71–7.59 logs CFU/mL to 6.93–6.82 log CFU/mL during storage, while encapsulation in the multilayer emulsion showed an insignificant reduction from 7.65–7.59 logs CFU/mL to 7.55–7.45 log CFU/mL at the end of storage. The obtained results showed that the type of probiotic bacteria had no significant effects on the physicochemical and structural properties of samples. However, encapsulating probiotics in multilayer emulsion led to a more homogenous structure in yogurt. The sensorial properties were also not affected by the probiotic type and the encapsulation method. Consequently, the multilayer emulsion can provide an ideal delivery carrier for encapsulating probiotic bacteria in dairy products.


---

## Body

## 1. Introduction

Functional food refers to food with positive effects on the human body by lowering the risk of diseases. Recently, the demands for foods containing functional elements (e.g., probiotics) with positive impacts on preventing, controlling, or healing different health problems have increased. The new lifestyle and using ready-to-eat industrial foods and fast foods have increased the risk of disease. In this regard, probiotic-loaded foods can be considered functional foods that affect consumer health by lowering blood cholesterol and fat, developing body immunity and mineral absorption, preventing and controlling cancer, and treating gastric ulcer [1–3]. Therefore, people are looking for healthy and functional foods such as probiotics and/or synbiotic foods [4, 5].The presence of live probiotic microorganisms (MOs) at high enough concentrations (more than 107 CFU/mL) in food products causes beneficial health effects on consumers [6, 7]. These types of food products, which are known as probiotic foods, affect the consumer’s health by lowering blood cholesterol and fat, developing body immunity and mineral absorption, preventing and controlling cancer, and treating gastric ulcer. The predominant bacteria in probiotic foods is Lactobacillus [8, 9]. The viability and concentration of the probiotic MOs in the food products before consumption and at the expiry date is an important parameter. The type of starter culture, the strain of probiotic MO, the condition of storage, and the concentration of lactic acid and oxygen are the main parameters that can influence the viability of MOs in probiotic yogurts [10]. Therefore, keeping the concentration and viability of probiotic MOs up to a certain level has become an important challenge in developing probiotic yogurts [11, 12]. One of the main strategies for enhancing the MOs viability is using prebiotic carbohydrates in the food formulation. These types of foods which contain probiotic MOs and prebiotic agents are known as synbiotics [8]. Encapsulation of probiotics is also investigated recently to extend their viability and functionality during storage. To this end, Afzaal et al. [13] studied the viability of encapsulated probiotic bacteria in yogurt. Muzzafar and Sharma [14] evaluated the microencapsulation of probiotics for incorporation in cream biscuits. Chen et al. [15] evaluated the effect of xanthan-chitosan-xanthan double layer encapsulation on survival of Bifidobacterium BB01 in yogurt.The main objective of the present work was evaluating the impacts of probiotic type (Lactobacillus rhamnosus and Lactobacillus plantarum) and encapsulation method using simple and multilayer emulsions on the cell viability and physicochemical, rheological, structural, and sensorial properties of yogurts.


## 2. Materials and Methods

### 2.1. Materials

Tween 80 and MRS agar were bought from Merck Company (Darmstadt, Germany). Whey protein isolate (WPI, 98% whey protein) was purchased from BiPRO; Davisco Foods Intl., Eden Prairie, MN. Persian gum was purchased from Dena Emulsion Company (Shiraz, Iran). Olive oil was purchased from a local market in Shiraz.


### 2.2. Preparation of Bacteria

ViableL. rhamnosus and L. plantarum cultures were prepared as described in our previous work [10]. Briefly, 0.5 mL MRS broth was poured into each tube containing the freeze-dried cultures. The obtained suspension was added to 20 mL MRS broth and then maintained at 37°C for 18h. After that, the vial was centrifuged at 4000 rpm for 180 s, and the obtained sediment was collected. All these actions were performed in a sterile environment.


### 2.3. Emulsion Preparation

#### 2.3.1. Convectional Emulsion

To prepare conventional emulsion, Tween 80 (2.5%w/w) was added to distilled water (DW, 87.5%w/w). The olive oil (9%w/w) containing a pellet of bacteria (L. rhamnosus or L. plantarum, 1% w/w) was added to the aqueous phase and mixed well at 15000 rpm for 240 s by a high-speed homogenizer (Ultra-Turrax T18, IKA, Germany).


#### 2.3.2. Multilayer Emulsion

WPI (2 g) was added to DW (57.5 g) and hydrated for 12 h. Olive oil (9 g w/w) containing a pellet of bacteria (L. rhamnosus or L. plantarum, 1% w/w) was added to the prepared aqueous phase and mixed at 15000 rpm for 120 s by a high-speed homogenizer (Ultra-Turrax T18, IKA, Germany). Then, 30 g of Persian gum dispersion (3.3%, w/w) was added to the prepared emulsion and homogenized at the same condition to obtain multilayer emulsion.


#### 2.3.3. Encapsulation Efficiency

To determine the encapsulation efficiency (EE) of the probiotic bacteria in the prepared emulsions, 10 g of each sample was centrifuged at 260g (20 min at 4°C), followed by separating the water phase. The bacterial count in the sample and the separated water phase was then determined, and EE was calculated using the following equation:(1)EE%=Bacterial count of emulsion−Bacterial count of water phaseBacterial count of emulsion.


### 2.4. Yogurt Preparation

Stirred yogurt samples were produced based on the method explained by Fazilah et al. [16] with some modifications. First, milk was heated at 85°C for half an hour and then cooled down to 42°C. The starter culture (YoFlexO express 1.0, Chr. Hansen Inc., Milwaukee, WI) including Streptococcus thermophilus and Lactobacillus delbrueckii subsp bulgaricus was added to milk and inoculated at 42°C. Six different samples were prepared by adding six different probiotic bacteria (free or encapsulated) to the milk: 1. LR (free L. rhamnosus), 2. LR-ME (L. rhamnosus encapsulated within multilayer emulsion), 3. LR-E (L. rhamnosus encapsulated within conventional emulsion), 4. LP (free L. plantarum), 5. LP-ME (L. plantarum encapsulated within multilayer emulsion), and 6. LP-E (L. plantarum encapsulated within emulsion). The prepared mixtures were poured into the cups and incubated at 42 ± 1°C until reaching pH 4.2 ± 0.1. The yogurt samples were cooled and kept at 4 ± 1°C for 21 days. The percentage of dry matter (12.5%) and fat (2.5%) were relatively constant in all samples.


#### 2.4.1. Fat Determination

The fat content of the yogurts was determined based on the AACC standard method [17].


#### 2.4.2. pH and Acidity

Total titratable acidity and pH of yogurt samples were determined based on AOAC standard method [18]. Titratable acidity was reported as the percentage of lactic acid every 7 days over 21 days of the refrigeration storage period.


#### 2.4.3. Determination of Probiotic Bacteria Viability

To specify the number of viable probiotic bacteria in the yogurts, the number ofL. rhamnosus and L. plantarum colonies were counted on MRS agar after inoculating MRS agar with each of the samples and incubating at 37 ± 1°C for 3 days under an anaerobic condition [19]. The number of lactic acid bacteria was reported during 21 days of the refrigeration storage period. In addition, the starter culture count was determined based on the previous standard method [20].


#### 2.4.4. Viscosity Measurement

The viscosity of the yogurt samples was measured by a Brookfield cone and plate viscometer (DV2 Pro II, Brookfield Engineering Laboratories, USA) at 21°C. The applied shear rate was 80.64 s−1, and the spindle type was CP51 [21].


#### 2.4.5. Syneresis Measurement

Syneresis measurements were carried out according to the method of Gilbert et al. [22] with some modifications. Each sample was weighed, equilibrated at 10°C for 120 min, and centrifuged at 10°C (238 × g, 10 min). The expelled serum was then collected and weighed. The ratio between the expelled serum and the yogurt mass was reported as syneresis.


#### 2.4.6. Color Measurement

Brightness (L∗), redness-greenness (a∗), and yellowness-blueness (b∗) of the surface of samples were determined using a digital camera (Canon PowerShot A540, 6 megapixels resolution) as reported by Ścibisz et al. [23]. Each sample was placed in a box (50 × 50 × 60 cm3) equipped with a natural daylight source (6500 K), and the camera was placed on the top of the box at the distance of 25 cm from the sample. L∗, a∗, and b∗ were calculated by Adobe Photoshop® CS6.


#### 2.4.7. Surface Electron Micrograph

The surface of each lyophilized yogurt was studied by a scanning electron microscope (TESCAN Vega3, Czech Republic). Micrographs of samples were taken at an accelerating voltage of 20.0 kV after coating samples with a gold layer by a sputter coater (Desk Sputter Coater DSR1, Nanostructural Coating Co., Iran).


#### 2.4.8. Sensory Properties

Sensory evaluation was conducted by 10 well-trained panelists from Pegah Fars Company (Shiraz, Iran). This test was performed according to the 5-point standard hedonic method (1 = dislike extremely, 2 = dislike moderately, 3 = neither like nor dislike, 4 = like moderately, and 5 = like extremely). For this purpose, the samples were kept at 4°C for 1 week prior to sensory tests. The prepared yogurts with random codes were given to the panelists in a white plastic cup to evaluate the characteristics including aroma, taste, color, texture, strength, and general acceptance [24]. Sensory evaluation was conducted in testing booths with different light sources: day light illumination for color assessment and red light for other organoleptic properties. Panelists rinsed their mouths with tap water before tasting and also between each sample.


### 2.5. Statistical Analysis

All tests were done in the form of completely random blocks and repeated 3 times. Comparison of means was done with Duncan’s multiple range test at the significance level of 5%. Data were reported as mean ± standard deviation. Additionally, the quantitative data were statistically analyzed using SAS Statistical Software, version 9.1 (SAS Institute Inc., 2000; Cary, NC, USA).


## 2.1. Materials

Tween 80 and MRS agar were bought from Merck Company (Darmstadt, Germany). Whey protein isolate (WPI, 98% whey protein) was purchased from BiPRO; Davisco Foods Intl., Eden Prairie, MN. Persian gum was purchased from Dena Emulsion Company (Shiraz, Iran). Olive oil was purchased from a local market in Shiraz.


## 2.2. Preparation of Bacteria

ViableL. rhamnosus and L. plantarum cultures were prepared as described in our previous work [10]. Briefly, 0.5 mL MRS broth was poured into each tube containing the freeze-dried cultures. The obtained suspension was added to 20 mL MRS broth and then maintained at 37°C for 18h. After that, the vial was centrifuged at 4000 rpm for 180 s, and the obtained sediment was collected. All these actions were performed in a sterile environment.


## 2.3. Emulsion Preparation

### 2.3.1. Convectional Emulsion

To prepare conventional emulsion, Tween 80 (2.5%w/w) was added to distilled water (DW, 87.5%w/w). The olive oil (9%w/w) containing a pellet of bacteria (L. rhamnosus or L. plantarum, 1% w/w) was added to the aqueous phase and mixed well at 15000 rpm for 240 s by a high-speed homogenizer (Ultra-Turrax T18, IKA, Germany).


### 2.3.2. Multilayer Emulsion

WPI (2 g) was added to DW (57.5 g) and hydrated for 12 h. Olive oil (9 g w/w) containing a pellet of bacteria (L. rhamnosus or L. plantarum, 1% w/w) was added to the prepared aqueous phase and mixed at 15000 rpm for 120 s by a high-speed homogenizer (Ultra-Turrax T18, IKA, Germany). Then, 30 g of Persian gum dispersion (3.3%, w/w) was added to the prepared emulsion and homogenized at the same condition to obtain multilayer emulsion.


### 2.3.3. Encapsulation Efficiency

To determine the encapsulation efficiency (EE) of the probiotic bacteria in the prepared emulsions, 10 g of each sample was centrifuged at 260g (20 min at 4°C), followed by separating the water phase. The bacterial count in the sample and the separated water phase was then determined, and EE was calculated using the following equation:(1)EE%=Bacterial count of emulsion−Bacterial count of water phaseBacterial count of emulsion.


## 2.3.1. Convectional Emulsion

To prepare conventional emulsion, Tween 80 (2.5%w/w) was added to distilled water (DW, 87.5%w/w). The olive oil (9%w/w) containing a pellet of bacteria (L. rhamnosus or L. plantarum, 1% w/w) was added to the aqueous phase and mixed well at 15000 rpm for 240 s by a high-speed homogenizer (Ultra-Turrax T18, IKA, Germany).


## 2.3.2. Multilayer Emulsion

WPI (2 g) was added to DW (57.5 g) and hydrated for 12 h. Olive oil (9 g w/w) containing a pellet of bacteria (L. rhamnosus or L. plantarum, 1% w/w) was added to the prepared aqueous phase and mixed at 15000 rpm for 120 s by a high-speed homogenizer (Ultra-Turrax T18, IKA, Germany). Then, 30 g of Persian gum dispersion (3.3%, w/w) was added to the prepared emulsion and homogenized at the same condition to obtain multilayer emulsion.


## 2.3.3. Encapsulation Efficiency

To determine the encapsulation efficiency (EE) of the probiotic bacteria in the prepared emulsions, 10 g of each sample was centrifuged at 260g (20 min at 4°C), followed by separating the water phase. The bacterial count in the sample and the separated water phase was then determined, and EE was calculated using the following equation:(1)EE%=Bacterial count of emulsion−Bacterial count of water phaseBacterial count of emulsion.


## 2.4. Yogurt Preparation

Stirred yogurt samples were produced based on the method explained by Fazilah et al. [16] with some modifications. First, milk was heated at 85°C for half an hour and then cooled down to 42°C. The starter culture (YoFlexO express 1.0, Chr. Hansen Inc., Milwaukee, WI) including Streptococcus thermophilus and Lactobacillus delbrueckii subsp bulgaricus was added to milk and inoculated at 42°C. Six different samples were prepared by adding six different probiotic bacteria (free or encapsulated) to the milk: 1. LR (free L. rhamnosus), 2. LR-ME (L. rhamnosus encapsulated within multilayer emulsion), 3. LR-E (L. rhamnosus encapsulated within conventional emulsion), 4. LP (free L. plantarum), 5. LP-ME (L. plantarum encapsulated within multilayer emulsion), and 6. LP-E (L. plantarum encapsulated within emulsion). The prepared mixtures were poured into the cups and incubated at 42 ± 1°C until reaching pH 4.2 ± 0.1. The yogurt samples were cooled and kept at 4 ± 1°C for 21 days. The percentage of dry matter (12.5%) and fat (2.5%) were relatively constant in all samples.


### 2.4.1. Fat Determination

The fat content of the yogurts was determined based on the AACC standard method [17].


### 2.4.2. pH and Acidity

Total titratable acidity and pH of yogurt samples were determined based on AOAC standard method [18]. Titratable acidity was reported as the percentage of lactic acid every 7 days over 21 days of the refrigeration storage period.


### 2.4.3. Determination of Probiotic Bacteria Viability

To specify the number of viable probiotic bacteria in the yogurts, the number ofL. rhamnosus and L. plantarum colonies were counted on MRS agar after inoculating MRS agar with each of the samples and incubating at 37 ± 1°C for 3 days under an anaerobic condition [19]. The number of lactic acid bacteria was reported during 21 days of the refrigeration storage period. In addition, the starter culture count was determined based on the previous standard method [20].


### 2.4.4. Viscosity Measurement

The viscosity of the yogurt samples was measured by a Brookfield cone and plate viscometer (DV2 Pro II, Brookfield Engineering Laboratories, USA) at 21°C. The applied shear rate was 80.64 s−1, and the spindle type was CP51 [21].


### 2.4.5. Syneresis Measurement

Syneresis measurements were carried out according to the method of Gilbert et al. [22] with some modifications. Each sample was weighed, equilibrated at 10°C for 120 min, and centrifuged at 10°C (238 × g, 10 min). The expelled serum was then collected and weighed. The ratio between the expelled serum and the yogurt mass was reported as syneresis.


### 2.4.6. Color Measurement

Brightness (L∗), redness-greenness (a∗), and yellowness-blueness (b∗) of the surface of samples were determined using a digital camera (Canon PowerShot A540, 6 megapixels resolution) as reported by Ścibisz et al. [23]. Each sample was placed in a box (50 × 50 × 60 cm3) equipped with a natural daylight source (6500 K), and the camera was placed on the top of the box at the distance of 25 cm from the sample. L∗, a∗, and b∗ were calculated by Adobe Photoshop® CS6.


### 2.4.7. Surface Electron Micrograph

The surface of each lyophilized yogurt was studied by a scanning electron microscope (TESCAN Vega3, Czech Republic). Micrographs of samples were taken at an accelerating voltage of 20.0 kV after coating samples with a gold layer by a sputter coater (Desk Sputter Coater DSR1, Nanostructural Coating Co., Iran).


### 2.4.8. Sensory Properties

Sensory evaluation was conducted by 10 well-trained panelists from Pegah Fars Company (Shiraz, Iran). This test was performed according to the 5-point standard hedonic method (1 = dislike extremely, 2 = dislike moderately, 3 = neither like nor dislike, 4 = like moderately, and 5 = like extremely). For this purpose, the samples were kept at 4°C for 1 week prior to sensory tests. The prepared yogurts with random codes were given to the panelists in a white plastic cup to evaluate the characteristics including aroma, taste, color, texture, strength, and general acceptance [24]. Sensory evaluation was conducted in testing booths with different light sources: day light illumination for color assessment and red light for other organoleptic properties. Panelists rinsed their mouths with tap water before tasting and also between each sample.


## 2.4.1. Fat Determination

The fat content of the yogurts was determined based on the AACC standard method [17].


## 2.4.2. pH and Acidity

Total titratable acidity and pH of yogurt samples were determined based on AOAC standard method [18]. Titratable acidity was reported as the percentage of lactic acid every 7 days over 21 days of the refrigeration storage period.


## 2.4.3. Determination of Probiotic Bacteria Viability

To specify the number of viable probiotic bacteria in the yogurts, the number ofL. rhamnosus and L. plantarum colonies were counted on MRS agar after inoculating MRS agar with each of the samples and incubating at 37 ± 1°C for 3 days under an anaerobic condition [19]. The number of lactic acid bacteria was reported during 21 days of the refrigeration storage period. In addition, the starter culture count was determined based on the previous standard method [20].


## 2.4.4. Viscosity Measurement

The viscosity of the yogurt samples was measured by a Brookfield cone and plate viscometer (DV2 Pro II, Brookfield Engineering Laboratories, USA) at 21°C. The applied shear rate was 80.64 s−1, and the spindle type was CP51 [21].


## 2.4.5. Syneresis Measurement

Syneresis measurements were carried out according to the method of Gilbert et al. [22] with some modifications. Each sample was weighed, equilibrated at 10°C for 120 min, and centrifuged at 10°C (238 × g, 10 min). The expelled serum was then collected and weighed. The ratio between the expelled serum and the yogurt mass was reported as syneresis.


## 2.4.6. Color Measurement

Brightness (L∗), redness-greenness (a∗), and yellowness-blueness (b∗) of the surface of samples were determined using a digital camera (Canon PowerShot A540, 6 megapixels resolution) as reported by Ścibisz et al. [23]. Each sample was placed in a box (50 × 50 × 60 cm3) equipped with a natural daylight source (6500 K), and the camera was placed on the top of the box at the distance of 25 cm from the sample. L∗, a∗, and b∗ were calculated by Adobe Photoshop® CS6.


## 2.4.7. Surface Electron Micrograph

The surface of each lyophilized yogurt was studied by a scanning electron microscope (TESCAN Vega3, Czech Republic). Micrographs of samples were taken at an accelerating voltage of 20.0 kV after coating samples with a gold layer by a sputter coater (Desk Sputter Coater DSR1, Nanostructural Coating Co., Iran).


## 2.4.8. Sensory Properties

Sensory evaluation was conducted by 10 well-trained panelists from Pegah Fars Company (Shiraz, Iran). This test was performed according to the 5-point standard hedonic method (1 = dislike extremely, 2 = dislike moderately, 3 = neither like nor dislike, 4 = like moderately, and 5 = like extremely). For this purpose, the samples were kept at 4°C for 1 week prior to sensory tests. The prepared yogurts with random codes were given to the panelists in a white plastic cup to evaluate the characteristics including aroma, taste, color, texture, strength, and general acceptance [24]. Sensory evaluation was conducted in testing booths with different light sources: day light illumination for color assessment and red light for other organoleptic properties. Panelists rinsed their mouths with tap water before tasting and also between each sample.


## 2.5. Statistical Analysis

All tests were done in the form of completely random blocks and repeated 3 times. Comparison of means was done with Duncan’s multiple range test at the significance level of 5%. Data were reported as mean ± standard deviation. Additionally, the quantitative data were statistically analyzed using SAS Statistical Software, version 9.1 (SAS Institute Inc., 2000; Cary, NC, USA).


## 3. Results and Discussion

### 3.1. Encapsulation Efficiency of Emulsions

An effective encapsulation system for probiotics should improve the viability of probiotics and protect them from harsh conditions during processing and gastrointestinal digestion. The encapsulation efficiency of LR-ME, LR-E, LP-ME, and LP-E samples were 98.43 ± 0.42, 99.02 ± 0.31, 97.89 ± 0.89, and 98.05 ± 0.66%, respectively. The results showed that there were no significant differences between the viability of probiotic bacteria in different samples.


### 3.2. Fat Content

The amount of fat in yogurt can affect the pH value, acidity, viscosity, structure, and sensorial properties [20]. The results showed that the probiotic and encapsulation type had no significant effects on the fat content of yogurt samples (Figure 1). The fat contents of samples were in the range of 2.51 to 2.54%. Similar results were also reported by Ningtyas et al. [25] and Ranadheera et al. [26].Figure 1
Fat content of probiotic yogurt. LR (L. rhamnosus), LR-ME (encapsulated L. rhamnosus in multilayer emulsion), LR-E (encapsulated L. rhamnosus in emulsion), LP (L. plantarum), LP-ME (encapsulated L. plantarum in multilayer emulsion), and LP-E (encapsulated L. plantarum in emulsion).


### 3.3. pH

Yogurt production has relied on the activity of two homofermentative bacteria as well as the production of lactic acid and other aromatic compounds. The production of acid is the main reason for coagulation during fermentation. One of the main reasons for the unilateral selection of starters for yogurt preparation is their ability for acid generation during 4 to 5 h of incubation [20]. The survival of probiotic bacteria in foods is influenced by the pH of the food. The pH of yogurt during storage is shown in Figure 2. Based on the results, the pH values of all samples significantly (p<0.05) reduced during storage. Similarly, Tseng and Zhao [27] reported a pH reduction during the storage of yogurts. The results attained by Temiz et al. [28] and Turgut and Cakmakci [29] also found that the pH of fruit yogurts decreases during storage. According to Figure 2, the samples containing free bacteria showed lower pH values than those with encapsulated bacteria. In addition, among probiotic bacteria, L. rhamnosus had stronger effects on pH reduction. The effect of free probiotic bacteria on reducing postacidification was more pronounced compared to the encapsulated ones [14, 29]. The pH reduction might be attributed to the production of s-galactosidase enzyme by the starters. Moreover, the application of remaining carbohydrates by survived microorganisms over the storage time can lead to the production of lactic acid, formic acid, and CO2 [30].Figure 2
pH content of probiotic yogurt. LR (L. rhamnosus), LR-ME (encapsulated L. rhamnosus in multilayer emulsion), LR-E (encapsulated L. rhamnosus in emulsion), LP (L. plantarum), LP-ME (encapsulated L. plantarum in multilayer emulsion), and LP-E (encapsulated L. plantarum in emulsion). Capital letters indicate significant differences between samples at the same storage time and different time; lowercase letters indicate significant differences between the same samples at different storage times.


### 3.4. Acidity

The titratable acidity values of functional yogurt samples are shown in Figure3. The acidity of all functional yogurts has been significantly increased with a stable pace over the storage period. The results were consistent with those reported by Bakirci and Kavaz [31], Singh and Muthukumarappan [32], Temiz et al. [28], and Tseng and Zhao [27]. Temiz et al. [28], Singh and Muthukumarappan [32], and Ertem and Cakmakci [33] also found that the acidity of yogurt increased during storage. The pH reduction and increase of titratable acidity during the storage time can be attributed to the activity of starter microorganisms. Encapsulation of probiotic bacteria had considerable impacts on the acidity of samples. Free bacteria increased acidity more than encapsulated bacteria. Among probiotic bacteria, L. rhamnosus had a more significant effect on acidity. Similar results were reported by Chen et al. [15], who evaluated the effects of encapsulation of Bifidobacterium BB01 on the acidity of yogurt. In fact, the consumption of sugar and producing organic acids by microorganisms resulted in pH reduction and acidity increment [34].Figure 3
Acidity (% lactic acid) of probiotic yogurt. LR (L. rhamnosus), LR-ME (encapsulated L. rhamnosus in multilayer emulsion), LR-E (encapsulated L. rhamnosus in emulsion), LP (L. plantarum), LP-ME (encapsulated L. plantarum in multilayer emulsion), and LP-E (encapsulated L. plantarum in emulsion). Capital letters indicate significant differences between samples at the same storage time and different time; lowercase letters indicate significant differences between the same samples at different storage times.


### 3.5. Viability of Probiotic and Starter Bacteria

Although there are many standards for the number of probiotic bacteria in yogurt, the acceptable number is usually reported as 107 CFU/g [35]. The number of probiotic bacteria was enumerated every 7 days from the first day of production until 3 weeks. Figure 4 shows the number of L. rhamnosus and L. plantarum during the storage. As can be seen, the survival of bacteria has been significantly decreased during the storage time. A similar observation was also reported by Eskandari et al. [36]. Encapsulation had a significant effect on the improving viability of probiotic bacteria, and samples encapsulated with multilayer emulsion had the highest viability. Encapsulated probiotic bacteria within emulsions can endure adverse conditions. These results are in agreement with those of Iqbal et al. [37] that showed survival of probiotic bacteria in yogurt. Also, Muzzafar and Sharma [14] reported the efficiency of different coating types on the survival of encapsulated bacteria compared to free bacteria in food products. Similarly, Qi et al. [38] declared higher survival of probiotics after encapsulation in yogurt. The number of encapsulated bacteria reduced by just 1.76 log CFU/g while the number of bacteria in the free form reduced significantly to 4.82 log CFU/g. Pradeep Prasanna and Charalampopoulos [39] also observed a considerable reduction of the number of free probiotics (3.67 log CFU/g) in comparison with the encapsulated form. According to Figure 4, the amount of survived population at the end of the storage period in encapsulated samples was more than 107 CFU/g. The results were consistent with those reported by Salwa et al. [40]. The results of the starter count were reported in Figure 5. The bacteria count significantly reduced during storage time. This reduction was due to the consumption of the existing sugar by bacteria and the production of lactic acid. Also, after 14 days of storage, the samples containing free probiotic bacteria showed lower cell viability. The fact could be related to the lower level of pH.Figure 4
Viability ofL. rhamnosus and L. plantarum of probiotic yogurt. LR (L. rhamnosus), LR-ME (encapsulated L. rhamnosus in multilayer emulsion), LR-E (encapsulated L. rhamnosus in emulsion), LP (L. plantarum), LP-ME (encapsulated L. plantarum in multilayer emulsion), and LP-E (encapsulated L. plantarum in emulsion). Capital letters indicate significant differences between samples at the same storage time and different time; lowercase letters indicate significant differences between the same samples at different storage times.Figure 5
Starter count of probiotic yogurt. LR (L. rhamnosus), LR-ME (encapsulated L. rhamnosus in multilayer emulsion), LR-E (encapsulated L. rhamnosus in emulsion), LP (L. plantarum), LP-ME (encapsulated L. plantarum in multilayer emulsion), and LP-E (encapsulated L. plantarum in emulsion). Capital letters indicate significant differences between samples at the same storage time and different time; lowercase letters indicate significant differences between the same samples at different storage times.


### 3.6. Viscosity

Viscosity is one of the parameters that can be changed by the production of the acid components by different bacteria [41]. The effects of probiotic bacteria type (L. rhamnosus and L.plantarum) and encapsulation methods (simple and multilayer) on the viscosity of samples are presented in Figure 6. The results indicated that free bacteria decreased the viscosity of samples due to the production of acid components which affects the structure of samples. Our results were in accordance with those reported by Afzaal et al. [13] and Tarrega et al. [42]. The viscosity of LR, LR-ME, LR-E, LP, LP-ME, and LP-E samples were 65.86, 116.48, 88.97, 77.53, 124.56, and 93.25 mPs, respectively. After incorporating multilayer emulsion in the yogurt sample, the presence of protective layers around bacteria led to a lower acidic components release. Moreover, Figure 6 shows the shear thinning behavior of all samples by increasing the shear rate.Figure 6
Viscosity of probiotic yogurt. LR (L. rhamnosus), LR-ME (encapsulated L. rhamnosus in multilayer emulsion), LR-E (encapsulated L. rhamnosus in emulsion), LP (L. plantarum), LP-ME (encapsulated L. plantarum in multilayer emulsion), and LP-E (encapsulated L. plantarum in emulsion).


### 3.7. Syneresis

The effects of probiotic bacteria type (L. rhamnosus and L. plantarum) and encapsulation methods (simple and multilayer) on the syneresis of samples are presented in Figure 7. It was indicated that free bacteria increased the syneresis of samples due to the production of high acid components. Moreover, the syneresis increment can be also related to the fact that the probiotics use solid materials and convert them into various metabolites. Similarly, Afzaal et al. [13] also reported higher syneresis value in yogurt after treatment with free probiotics compared to encapsulated ones.Figure 7
Syneresis of probiotic yogurt. LR (L. rhamnosus), LR-ME (encapsulated L. rhamnosus in multilayer emulsion), LR-E (encapsulated L. rhamnosus in emulsion), LP (L. plantarum), LP-ME (encapsulated L. plantarum in multilayer emulsion), and LP-E (encapsulated L. plantarum in emulsion). Capital letters indicate significant differences between samples at the same storage time and different time; lowercase letters indicate significant differences between the same samples at different storage times.


### 3.8. Color

Color is one of the main properties of yogurt that can affect the consumer acceptability [43]. Figure 8 indicated that probiotic bacteria type (L. rhamnosus and L. plantarum) and encapsulation methods (simple and multilayer) had no significant effects on the L∗, a∗, and b∗ of samples.Figure 8
Color parameters of probiotic yogurt. LR (L. rhamnosus), LR-ME (encapsulated L. rhamnosus in multilayer emulsion), LR-E (encapsulated L. rhamnosus in emulsion), LP (L. plantarum), LP-ME (encapsulated L. plantarum in multilayer emulsion), and LP-E (encapsulated L. plantarum in emulsion).


### 3.9. SEM

The structure of the sample was evaluated by scanning electron microscopy. Figure9 showed that probiotic bacteria types (L. rhamnosus and L. plantarum) had no significant effects on the structure of samples. However, samples incorporating multilayer emulsion were more homogenous due to the presence of WPI and Persian gum. Similar morphology was also reported by Fazilah et al. [44] and El Kadri et al. [45] after addition of double emulsion to yogurt samples.Figure 9
SEM micrograph parameters of probiotic yogurt. LR (L. rhamnosus), LR-ME (encapsulated L. rhamnosus in multilayer emulsion), LR-E (encapsulated L. rhamnosus in emulsion), LP (L. plantarum), LP-ME (encapsulated L. plantarum in multilayer emulsion), and LP-E (encapsulated L. plantarum in emulsion).


### 3.10. Sensory Properties

Color, odor, texture, taste, strength, and overall acceptance of different yogurt samples were investigated by panelists. The effects of probiotic bacteria type (L. rhamnosus and L. plantarum) and encapsulation methods (simple and multilayer) on the sensorial aspects of yogurts are presented in Figure 10. It was revealed that probiotic bacteria type and encapsulation methods had no significant effects on the sensory properties of samples. Kailasapathy [46] also reported the constant sensorial properties of yogurt after encapsulation of probiotic bacteria.Figure 10
Sensorial properties parameters of probiotic yogurt. LR (L. rhamnosus), LR-ME (encapsulated L. rhamnosus in multilayer emulsion), LR-E (encapsulated L. rhamnosus in emulsion), LP (L. plantarum), LP-ME (encapsulated L. plantarum in multilayer emulsion), and LP-E (encapsulated L. plantarum in emulsion).


## 3.1. Encapsulation Efficiency of Emulsions

An effective encapsulation system for probiotics should improve the viability of probiotics and protect them from harsh conditions during processing and gastrointestinal digestion. The encapsulation efficiency of LR-ME, LR-E, LP-ME, and LP-E samples were 98.43 ± 0.42, 99.02 ± 0.31, 97.89 ± 0.89, and 98.05 ± 0.66%, respectively. The results showed that there were no significant differences between the viability of probiotic bacteria in different samples.


## 3.2. Fat Content

The amount of fat in yogurt can affect the pH value, acidity, viscosity, structure, and sensorial properties [20]. The results showed that the probiotic and encapsulation type had no significant effects on the fat content of yogurt samples (Figure 1). The fat contents of samples were in the range of 2.51 to 2.54%. Similar results were also reported by Ningtyas et al. [25] and Ranadheera et al. [26].Figure 1
Fat content of probiotic yogurt. LR (L. rhamnosus), LR-ME (encapsulated L. rhamnosus in multilayer emulsion), LR-E (encapsulated L. rhamnosus in emulsion), LP (L. plantarum), LP-ME (encapsulated L. plantarum in multilayer emulsion), and LP-E (encapsulated L. plantarum in emulsion).


## 3.3. pH

Yogurt production has relied on the activity of two homofermentative bacteria as well as the production of lactic acid and other aromatic compounds. The production of acid is the main reason for coagulation during fermentation. One of the main reasons for the unilateral selection of starters for yogurt preparation is their ability for acid generation during 4 to 5 h of incubation [20]. The survival of probiotic bacteria in foods is influenced by the pH of the food. The pH of yogurt during storage is shown in Figure 2. Based on the results, the pH values of all samples significantly (p<0.05) reduced during storage. Similarly, Tseng and Zhao [27] reported a pH reduction during the storage of yogurts. The results attained by Temiz et al. [28] and Turgut and Cakmakci [29] also found that the pH of fruit yogurts decreases during storage. According to Figure 2, the samples containing free bacteria showed lower pH values than those with encapsulated bacteria. In addition, among probiotic bacteria, L. rhamnosus had stronger effects on pH reduction. The effect of free probiotic bacteria on reducing postacidification was more pronounced compared to the encapsulated ones [14, 29]. The pH reduction might be attributed to the production of s-galactosidase enzyme by the starters. Moreover, the application of remaining carbohydrates by survived microorganisms over the storage time can lead to the production of lactic acid, formic acid, and CO2 [30].Figure 2
pH content of probiotic yogurt. LR (L. rhamnosus), LR-ME (encapsulated L. rhamnosus in multilayer emulsion), LR-E (encapsulated L. rhamnosus in emulsion), LP (L. plantarum), LP-ME (encapsulated L. plantarum in multilayer emulsion), and LP-E (encapsulated L. plantarum in emulsion). Capital letters indicate significant differences between samples at the same storage time and different time; lowercase letters indicate significant differences between the same samples at different storage times.


## 3.4. Acidity

The titratable acidity values of functional yogurt samples are shown in Figure3. The acidity of all functional yogurts has been significantly increased with a stable pace over the storage period. The results were consistent with those reported by Bakirci and Kavaz [31], Singh and Muthukumarappan [32], Temiz et al. [28], and Tseng and Zhao [27]. Temiz et al. [28], Singh and Muthukumarappan [32], and Ertem and Cakmakci [33] also found that the acidity of yogurt increased during storage. The pH reduction and increase of titratable acidity during the storage time can be attributed to the activity of starter microorganisms. Encapsulation of probiotic bacteria had considerable impacts on the acidity of samples. Free bacteria increased acidity more than encapsulated bacteria. Among probiotic bacteria, L. rhamnosus had a more significant effect on acidity. Similar results were reported by Chen et al. [15], who evaluated the effects of encapsulation of Bifidobacterium BB01 on the acidity of yogurt. In fact, the consumption of sugar and producing organic acids by microorganisms resulted in pH reduction and acidity increment [34].Figure 3
Acidity (% lactic acid) of probiotic yogurt. LR (L. rhamnosus), LR-ME (encapsulated L. rhamnosus in multilayer emulsion), LR-E (encapsulated L. rhamnosus in emulsion), LP (L. plantarum), LP-ME (encapsulated L. plantarum in multilayer emulsion), and LP-E (encapsulated L. plantarum in emulsion). Capital letters indicate significant differences between samples at the same storage time and different time; lowercase letters indicate significant differences between the same samples at different storage times.


## 3.5. Viability of Probiotic and Starter Bacteria

Although there are many standards for the number of probiotic bacteria in yogurt, the acceptable number is usually reported as 107 CFU/g [35]. The number of probiotic bacteria was enumerated every 7 days from the first day of production until 3 weeks. Figure 4 shows the number of L. rhamnosus and L. plantarum during the storage. As can be seen, the survival of bacteria has been significantly decreased during the storage time. A similar observation was also reported by Eskandari et al. [36]. Encapsulation had a significant effect on the improving viability of probiotic bacteria, and samples encapsulated with multilayer emulsion had the highest viability. Encapsulated probiotic bacteria within emulsions can endure adverse conditions. These results are in agreement with those of Iqbal et al. [37] that showed survival of probiotic bacteria in yogurt. Also, Muzzafar and Sharma [14] reported the efficiency of different coating types on the survival of encapsulated bacteria compared to free bacteria in food products. Similarly, Qi et al. [38] declared higher survival of probiotics after encapsulation in yogurt. The number of encapsulated bacteria reduced by just 1.76 log CFU/g while the number of bacteria in the free form reduced significantly to 4.82 log CFU/g. Pradeep Prasanna and Charalampopoulos [39] also observed a considerable reduction of the number of free probiotics (3.67 log CFU/g) in comparison with the encapsulated form. According to Figure 4, the amount of survived population at the end of the storage period in encapsulated samples was more than 107 CFU/g. The results were consistent with those reported by Salwa et al. [40]. The results of the starter count were reported in Figure 5. The bacteria count significantly reduced during storage time. This reduction was due to the consumption of the existing sugar by bacteria and the production of lactic acid. Also, after 14 days of storage, the samples containing free probiotic bacteria showed lower cell viability. The fact could be related to the lower level of pH.Figure 4
Viability ofL. rhamnosus and L. plantarum of probiotic yogurt. LR (L. rhamnosus), LR-ME (encapsulated L. rhamnosus in multilayer emulsion), LR-E (encapsulated L. rhamnosus in emulsion), LP (L. plantarum), LP-ME (encapsulated L. plantarum in multilayer emulsion), and LP-E (encapsulated L. plantarum in emulsion). Capital letters indicate significant differences between samples at the same storage time and different time; lowercase letters indicate significant differences between the same samples at different storage times.Figure 5
Starter count of probiotic yogurt. LR (L. rhamnosus), LR-ME (encapsulated L. rhamnosus in multilayer emulsion), LR-E (encapsulated L. rhamnosus in emulsion), LP (L. plantarum), LP-ME (encapsulated L. plantarum in multilayer emulsion), and LP-E (encapsulated L. plantarum in emulsion). Capital letters indicate significant differences between samples at the same storage time and different time; lowercase letters indicate significant differences between the same samples at different storage times.


## 3.6. Viscosity

Viscosity is one of the parameters that can be changed by the production of the acid components by different bacteria [41]. The effects of probiotic bacteria type (L. rhamnosus and L.plantarum) and encapsulation methods (simple and multilayer) on the viscosity of samples are presented in Figure 6. The results indicated that free bacteria decreased the viscosity of samples due to the production of acid components which affects the structure of samples. Our results were in accordance with those reported by Afzaal et al. [13] and Tarrega et al. [42]. The viscosity of LR, LR-ME, LR-E, LP, LP-ME, and LP-E samples were 65.86, 116.48, 88.97, 77.53, 124.56, and 93.25 mPs, respectively. After incorporating multilayer emulsion in the yogurt sample, the presence of protective layers around bacteria led to a lower acidic components release. Moreover, Figure 6 shows the shear thinning behavior of all samples by increasing the shear rate.Figure 6
Viscosity of probiotic yogurt. LR (L. rhamnosus), LR-ME (encapsulated L. rhamnosus in multilayer emulsion), LR-E (encapsulated L. rhamnosus in emulsion), LP (L. plantarum), LP-ME (encapsulated L. plantarum in multilayer emulsion), and LP-E (encapsulated L. plantarum in emulsion).


## 3.7. Syneresis

The effects of probiotic bacteria type (L. rhamnosus and L. plantarum) and encapsulation methods (simple and multilayer) on the syneresis of samples are presented in Figure 7. It was indicated that free bacteria increased the syneresis of samples due to the production of high acid components. Moreover, the syneresis increment can be also related to the fact that the probiotics use solid materials and convert them into various metabolites. Similarly, Afzaal et al. [13] also reported higher syneresis value in yogurt after treatment with free probiotics compared to encapsulated ones.Figure 7
Syneresis of probiotic yogurt. LR (L. rhamnosus), LR-ME (encapsulated L. rhamnosus in multilayer emulsion), LR-E (encapsulated L. rhamnosus in emulsion), LP (L. plantarum), LP-ME (encapsulated L. plantarum in multilayer emulsion), and LP-E (encapsulated L. plantarum in emulsion). Capital letters indicate significant differences between samples at the same storage time and different time; lowercase letters indicate significant differences between the same samples at different storage times.


## 3.8. Color

Color is one of the main properties of yogurt that can affect the consumer acceptability [43]. Figure 8 indicated that probiotic bacteria type (L. rhamnosus and L. plantarum) and encapsulation methods (simple and multilayer) had no significant effects on the L∗, a∗, and b∗ of samples.Figure 8
Color parameters of probiotic yogurt. LR (L. rhamnosus), LR-ME (encapsulated L. rhamnosus in multilayer emulsion), LR-E (encapsulated L. rhamnosus in emulsion), LP (L. plantarum), LP-ME (encapsulated L. plantarum in multilayer emulsion), and LP-E (encapsulated L. plantarum in emulsion).


## 3.9. SEM

The structure of the sample was evaluated by scanning electron microscopy. Figure9 showed that probiotic bacteria types (L. rhamnosus and L. plantarum) had no significant effects on the structure of samples. However, samples incorporating multilayer emulsion were more homogenous due to the presence of WPI and Persian gum. Similar morphology was also reported by Fazilah et al. [44] and El Kadri et al. [45] after addition of double emulsion to yogurt samples.Figure 9
SEM micrograph parameters of probiotic yogurt. LR (L. rhamnosus), LR-ME (encapsulated L. rhamnosus in multilayer emulsion), LR-E (encapsulated L. rhamnosus in emulsion), LP (L. plantarum), LP-ME (encapsulated L. plantarum in multilayer emulsion), and LP-E (encapsulated L. plantarum in emulsion).


## 3.10. Sensory Properties

Color, odor, texture, taste, strength, and overall acceptance of different yogurt samples were investigated by panelists. The effects of probiotic bacteria type (L. rhamnosus and L. plantarum) and encapsulation methods (simple and multilayer) on the sensorial aspects of yogurts are presented in Figure 10. It was revealed that probiotic bacteria type and encapsulation methods had no significant effects on the sensory properties of samples. Kailasapathy [46] also reported the constant sensorial properties of yogurt after encapsulation of probiotic bacteria.Figure 10
Sensorial properties parameters of probiotic yogurt. LR (L. rhamnosus), LR-ME (encapsulated L. rhamnosus in multilayer emulsion), LR-E (encapsulated L. rhamnosus in emulsion), LP (L. plantarum), LP-ME (encapsulated L. plantarum in multilayer emulsion), and LP-E (encapsulated L. plantarum in emulsion).


## 4. Conclusion

It was observed that encapsulation of probiotics within both types of delivery systems enhanced the cell viability in yogurt samples. Sensorial characteristics and physicochemical parameters of the probiotic yogurts were also influenced by the encapsulation method. Based on the results, multilayer emulsion was an effective tool to preserve the viability of bacteria at the recommended effectiveness level (above 107 CFU/g) in food products with low negative effects on the physicochemical and sensorial properties.



---

*Source: 7923899-2022-03-31.xml*