Aloe vera jelly dessert supplemented with Lactobacillus curvatus encapsulated in Plantago major mucilage and sodium alginate: Characterization of physicochemical, sensory properties and survivability against low pH, salt, heat, and cold storage

Abstract The goal of this research was to assess the free Lactobacillus curvatus (FLC) and microencapsulated L. curvatus (MLC) survivability using sodium alginate and Plantago major mucilage (PMM), as a second layer to produce probiotic aloe vera jelly dessert (AVJD). To determine bead characteristics, the aspect ratio of the bead, survival in 72°C, and cold storage were assessed as well as for AVJD, survivability of probiotics in simulated gastrointestinal condition (SGIC), and storage time. The results showed that all the beads are spherical (aspect ratio = 1.12), and under heat stress conditions, MLC showed a higher survival rate (50.15%) compared to FLC (not detected after 5 min). The number of survived probiotics in the MLC sample (8.65 log CFU/mL) was higher than FLC (7.52 log CFU/g) on the 28th day. In AVJD, the MLC survived at a minimum scientific adequate number of probiotics (6.88 log CFU/mL) on the 28th day. In SGIC, the final survival rates of FLC and MLC samples were 14.24% and 71.04%, respectively. These results suggest that using alginate and PMM is a promising method to protect L. curvatus (LC) from harsh environmental conditions and in AVJD.

of probiotics (Shrivastava & Bhatu, 2023).Some types of lactic acid bacteria are considered probiotics and can be found in various food products, such as Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus casei, and Lactobacillus reuteri (Yu et al., 2023).The LC is essential for health and food applications.
The LC is part of the microbiota of many fermented products and is characterized by bacteriogenic activity (Funck et al., 2019).
The survival of probiotics is influenced by numerous factors during the processing and storage of foods.These factors relate to unique product properties such as processing-related treatments, salt presence, sugar content, water activity, colors, titratable acidity, artificial flavors, bacteriocins, pH, hydrogen peroxide, and oxygen.
They may also be related to the following factors: conditions such as refrigeration, storage, heat treatments, incubation temperatures, production scale, packaging materials, fermentation conditions, microbiological properties such as inoculation rates and proportions, and microbial strains (Terpou et al., 2019).
Survival after passage through the gastrointestinal tract, viability during food processing, storage, and host health benefits are essential for selecting probiotics (Pourjafar et al., 2020).The harsh condition of the stomach is not tolerated by most bacteria.Thus, the selection of the exact probiotic strain is a crucial element in formulating a healthy food product (El-Sohaimy & Hussain, 2023).
Microencapsulation technology applies to preserve the viability of probiotics even under adverse conditions and to protect probiotics by creating a physical barrier from the environment.The microencapsulated spheres containing the core material are in the micro-to millimeter size range.Various microencapsulation methods such as emulsion, spray drying, and extrusion exist (Kowsalya et al., 2023).One of the other techniques used in microencapsulation is extrusion.Microencapsulation by extrusion is advantageous.
It is nontoxic, efficient to handle, and economical (Amin et al., 2021).
We hypothesized that microencapsulation could improve LC viability during storage and under harsh conditions.
Microencapsulation is an immobilization technique that encapsulates cells placed in a matrix of encapsulating material.Functionality, stability, type of release, encapsulated concentration, cost carrier biodegradable materials biocompatible, foodgrade, and capable of barrier formation are the most important criteria to select the encapsulation materials (Nami et al., 2017).
Encapsulating materials commonly used in microencapsulation techniques include sodium alginate, chitosan, gellan, xanthan gum, whey protein, starch, carrageenan, Arabic gum, cellulose, and pectin (Parsana et al., 2023).Among them, sodium alginate, the most commonly used, is insoluble at acidic pH; thus, it protects the living cells of probiotics from gastric acid as they pass through the stomach, swells in alkaline pH conditions, and releases probiotic cells in the intestine (Kowalska et al., 2022).
Using a second layer as an additional coating of alginate beads addresses these issues.The second layer may improve beads in harsh conditions (Karimi et al., 2021).For instance, the porosity of alginate beads during gastric transit is considerably reduced by applying chitosan-coated alginate beads to finally provide a more stable physicochemical appearance (Dokoohaki et al., 2019).One of the materials that can be used in microencapsulation is seed mucilage.
The mucilage could be used as a second layer to improve sodium alginate function.Plantago Major is a large genus of the Plantago family, comprising more than 250 species, widely distributed geographically in temperate and high-altitude tropical regions.It is a perennial herb with a fibrous root system, a rosette of elliptical leaves, and multiple long inflorescences (Lyu et al., 2023).The seed contains polysaccharides that are comprised arabinose, xylose, rhamnose, galactose, glucose, glucuronic acid, and galacturonic acid and can be used as active natural polymers for developing edible coatings (Noshad et al., 2021).We hypothesized that Plantago major mucilage (PMM) could be used on the bead's surface as a second layer.
The role of desserts in the menu becomes very crucial.Besides being refreshing, aloe vera jelly also has health benefits.Aloe vera contains fats, proteins, and carbohydrates that produce energy.The presence of vitamins A and C in aloe vera makes it an appropriate dessert for eye health and maintaining immunity.In contrast, other dessert products are generally sweet but often contain slight functional advantages (Wachyuni et al., 2020).So far, no studies have been conducted on the production of probiotic AVJD.Therefore, this study assessed the MLC survivability under heat, acid, and bile stress.Moreover, the effects of microencapsulated and free LC addition to AVJD as a fortified product were determined.Physicochemical and sensorial properties of AVJD and the survival ability of LC were assessed during the storage period and GI condition.

| Mucilage extraction
Deionized water was used to prepare water: seed ratio of 60:1 at pH 6.8 to extract PMM from whole seeds.The 0.1 M HCl or NaOH solution was used for adjusting pH in the water bath (75 ± 2.0°C).
The water was preheated to a specific temperature before the seeds were added.The seed and water slurry were stirred with a motorized stirring paddle throughout the process (1.5 h).The seeds were passed through an extractor to isolate the mucilage from the expanded seeds, where a rotating plate scraped the mucilage from the surface of the seeds.The collected mucilage was then filtered, dried in an oven (overnight at 45°C), ground, and sieved through an 18 mesh sieve.The final dry powder is packaged and stored in a cool and dry place.

| Bacterial cultivation
The LC (PTCC 1955) was obtained from the Persian Type Culture Collection.Among the Lactobacilli, LC is resistant to erythromycin.
The LC was cultivated in selective MRS broth supplemented with 4 μg/mL erythromycin (Sigma) under microaerophilic and capnophilic conditions using Anaerocult® C Merk Gas Pack at 37°C for 48 h.

| LR microencapsulation procedures
The LC broth culture was centrifuged at 2800 g for 10 min and then washed with 0.1% sterile peptone water (peptic digest of animal tissue and sodium chloride).The final volume of 5 mL was considered for the centrifuged washed cell.The estimated total bacteria count in the MRS broth was about 2.25 × 10 9 CFU/mL.To perform the microencapsulation, an extrusion technique was applied.The 5 mL of centrifuged bacterial culture (9.3 × 10 11 CFU/mL) was added to 15 mL of 1.5% sodium alginate solution.The bacterial suspensions were injected through a 0.11 mm needle into a sterile 0.1 mol/L CaCl 2 .It was subsequently placed in a refrigerator for 12 h before washing the microspheres with 0.1% peptone water and separated into (0.2, 0.4, 0.6, and 0.8% w/w) mucilage solution and gently shaken on the apparatus at 100 rpm for 40 min with orbital shaker (two-stage method).
The beads were then washed several times with 0.1% sterile peptone water.The morphology and aspect ratio of 20 microspheres were assessed by a digital microscope (Olympus BX51, Japan) and analyzed with Micromeasure software version 1.07.To calculate the aspect ratios, Equation (1) was finally applied: The encapsulation yield (EY) was calculated and shown in Equation ( 2): where N is the number of viable cells (CFU/g of microsphere) after forming the microencapsulated cells and N0 is the number of viable cells (CFU/g of mixed alginate) taken initially to produce the beads (Pourakbar et al., 2023).

| Scanning electron microscopy
The microspheres were subsequently vacuumed and examined with a scanning electron microscope (JSM-5800LV, JEOL, Japan).
Cross-sections of lyophilized beads and AVJD were initially coated with gold.The outer surface and internal structure of the freezedried beads were observed.

| Heat resistance of MLC and FLC
The heat resistance of FLC and MLC (8.75 to 9.28 log CFU/mL) was studied at 72°C (temperature degree for milk pasteurization) in MRS broth medium for 1, 2, 3, 4, and 5 min.After heat treatment, the samples were cooled with tap water.For releasing bacteria, sterile sodium citrate buffer 0.1 M (pH = 6.2) was used to finally collect the sample.The viability of the bacteria was determined by serial dilution and cultivation methods.

| Survival in salt and acid stress
The glycine-HCl buffer (pH 1.5) containing 15% NaCl was chosen as salt and acid stress condition.The MLC and FLC were added to this medium.The pour-plate technique in MRS selective medium was used to enumerate the effective cells in the samples at 0, 30, 60, and 90 min.The plates were finally incubated at 37°C for 48 h.

| MLC and FLC survival during storage in MRS
The MLC and FLC were kept in MRS broth and stored at 4°C for 28 days, respectively.The viability of LC cells was enumerated in the 1, 7, 14, 21, and 28 days of storage time.

| Preparation of probiotic AVJD
Control AVJD was prepared from 100 g of AVJD powder (Deraje Co, Yazd, Iran).The preparation was then boiled in 500 mL of water for 10 min and stirred with constant stirring.The endpoint of heating was determined by reaching a Brix value of 65 0 using a refractometer.The mixture was then transferred to 75°C, poured into sterilized glass bottles, and allowed to stand.The FLC and MLC jellies were prepared using the same method.Equal final viable numbers (~9 log CFU/g) of bacteria in MLC and FLC forms were placed in AVJD and then poured into sterile glass containers.The physicochemical, sensory, and microbial analyses of all produced jellies were assessed during storage at 4°C. (1)

| Titratable acidity and pH
One gram of AVJD was taken and dissolved in 20 mL of distilled water in a conical flask, two to three drops of 2% phenolphthalein indicator were added, and then titrated with standardized 0.1 N NaOH till pink color appeared.The titratable acidity can be calculated as follows: where Ta = titratable acidity; B = ml of 0.1 N NaOH; W = sample weight (Sikder and Ahmed 2019).
A digital pH meter was finally used to measure the pH of the control and probiotic AVJD (GPRT-1400-AN; Greisinger Electronic GmbH, Germany).

| Survival of MLC and FLC in AVJD during the storage time
MRS agar was supplemented with 4 μg/mL erythromycin (Sigma).
It was used to evaluate the FLC-AVJD and MLC-AVJD survivability during storage time.The AVJDs were stored at 4°C for 28 days, and LC count was carried out at 1, 7, 14, 21, and 28 days of storage.For the sampling of supplemented AVJDs, 1 and 0.1 mL of MLC-AVJD and FLC-AVJD were appropriately mixed with 9 and 0.9 mL of sterile sodium citrate 0.1M (pH = 6.2), respectively, and cultured in erythromycin MRS agar.

| Survival of FLC and MLC exposed to simulated gastrointestinal condition
The method of Dokoohaki et al. (2019) was employed to analyze both simulated gastric fluid (SGF) and intestinal fluid (SIF).One and 0.1 g from AVJD containing FLC and MLC in 1 and 21 days of production were chosen for this assessment.The FLC and MLC were added to flasks containing SGF and incubated at 37°C in a shaker incubator (Shaker incubator, Labtron, Tehran, Iran), for 2 h (gastric phase).They were then added to SIF at pH 4.3-5.2.The LC counts were performed on triplicate samples after 30 min, 2, 4, and 6 h (each in three different flasks of the same experiment) using corresponding volumes (0.01 to 1 mL change).Aliquots of 0.01 and 0.1 mL of each preparation were immersed into erythromycin MRS agar before being incubated under microaerophilic conditions at 37°C for 72 h (Dokoohaki et al., 2019).
The survival rate was calculated using Equation (4): N, the number of live probiotics (CFU/mL) after digestion and treatment of the simulated gastric and intestinal fluids and N0, the number of live probiotics (CFU/mL) before digestion and treatment in the simulated gastric and intestinal fluids.

| Sensory evaluation of AVJD
Forty-five trained panelists, who reported being consumers of AVJD products, were selected among students, faculty, and staff at Fars Agriculture Research and Education center, Iran, to evaluate each AVJD sample.The panel comprised men (38.5%) and women (61.5%) with an average age of 35-40 years.A 5-point hedonic scale was applied to the AVJD sensory assessment.Panelists rated flavor, color, texture, and general acceptability.Compared to AVJD on the market, 5 means "very much like", and 1 means "not at all" were applied for scoring.The panelists eventually assessed samples in the first and every 7 days during storage.

| Texture of AVJD
Texture analyzer (Brookfield CT3 Texture Analyzer, 4500, USA) was used for TPA analysis of AVJD in the first and the last days of storage time.Using the TA11/1000 cylindrical probe, 40% of the AVJD cubes were compressed to twice their original height.The sample had a height of 30 mm and a diameter of 20 mm.A penetration depth of 20 mm was measured at the following speeds: 1 mm/s for the pretest, 1 mm/s for the test, and 10 mm/s for the posttest.Parameters such as hardness (g), cohesiveness, adhesiveness (mJ), chewiness (mJ), springiness (mm), and gumminess (g) were measured from the device.All these measurements were performed in at least three replicates at 25 ± 3°C for each sample.

| Statistical analysis
Data were statistically analyzed using SPPS software (SPSS Ver 21.0).All statistical analyses were performed using one-way ANOVA and Duncan's multiple range test (p < .05).

| Size, morphology, color, and encapsulation efficiency of beads
Macroscopic and microscopic images (Figure 1a-h) demonstrate the spherical particles (aspect ratio of 1.12) produced by the extrusion method.The spherical nature of the encapsulated beads prevented cell overgrowth within the encapsulated beads (Karimi et al., 2021).was also pointed out that the protective characteristic of encapsulation can be removed by reducing in diameter, whereas enhancing the capsule diameter effectively reduces the digestibility of pancreatic enzymes (Afzaal et al., 2019).
The number of viable cells was 10.28 Log CFU/g in beads after forming the microencapsulated cells and 11.52 Log CFU/g in mixed alginate initially taken to produce the beads.So, the encapsulation yield was 89.23%.The formation of the alginate and oligosaccharides arabinogalactans combination may be one of the reasons for the high encapsulation yield.The presence of polysaccharides in the seed coat makes it gummy under warm and humid conditions (Noshad et al., 2021).This is thought to reduce the porosity of the bead's surface and prevent bacteria from escaping into the environment.Furthermore, encapsulation efficiency is directly associated with the variety of hydrogel matrices (Afzaal et al., 2020).
For instance, it was reported that supplementing okra mucilage as a second layer in beads could increase the microencapsulation yield (90.14%) (Pourakbar et al., 2023).

| Scanning electron microscopy
The shape of the microsphere was analyzed by scanning electron mi-   The soft and homogenous shapes of the capsules resemble the findings of our study.An improved resistance to harsh conditions was remarkable by using this type of bead (Pourjafar et al., 2020).Moreover, numerous changes in the structural properties including surface characteristics, size, humidity, and configuration of microcapsules reported in previous studies.The rigidity of microcapsules is attributed to adding PMM which intensifies the hydrogel network to finally fill the remaining gaps (Hu et al., 2021).The uniform and smooth microstructure of the wall composition due to optimal proportion and adding 0.8% PMM was interesting.In addition, the distribution of Lactobacillus occurred in all samples.The penetration of bacterial cells into the matrix of multilayered microcapsules was confirmed by SEM images.Concomitant to increase the PMM concentration in the second layer of the bead, the smoother outer layer was observed.

| Heat tolerance
We investigated the effect of temperature on FLC and MLC viability and tolerability (Figure 3a).As a result, MLC showed a higher survival rate (50.15%) than FLC (not detected within 5 min).Therefore, the temperature tolerance for MLC (72°C for 5 min) was higher than the Lactobacillus acidophilus (10 7 -10 8 cfu/mL) was achieved, which is the advised therapeutic level in functional foods (Zeashan et al., 2020).
The temperature was a key factor influencing probiotic viability.The association of exposure time, temperature, and viability of encapsulated probiotics was shown in previous studies (Dokoohaki et al., 2019;Karimi et al., 2021).For example, the reduction of Lactobacillus plantarum in microencapsulated form (0.8 log CFU reduction) after heat treatment at 50°C for 5 min was fewer than the free form (5.7 log CFU reduction) (Ni et al., 2023).
F I G U R E 2 Scan electron microscopy of microencapsulated Lactobacillus curvatus (MLC) 0.2% (a), 0.4% (b), 0.6% (c), 0.8% (d) of plantago major mucilage, respectively, used in microencapsulation.et al. (2004) found that LC resists low salt concentrations (2%, wt/ vol).However, high concentrations (6% wt/vol) of sodium chloride significantly inhibited LC growth.Primarily, NaCl exhibited an inhibiting role due to acting as a water activity (aw) lowering agent (Verluyten et al., 2004).In addition, high degrees of acid resistance are presented by the LC.It usually possesses acid resistance mechanisms like F1F0-ATPase, glutamate decarboxylases, or arginine deiminase (ADI) systems, which protect it against acid (Janßen et al., 2020).Therefore, the decrease in the number of bacteria may be related to the sensitivity of these bacteria to salt in the environment (Luan et al., 2022).

| MLC and FLC survival during storage in MRS
Details of MLC and FLC survival are shown in Figure 3c.Following 28 days of storage, a substantial reduction in the viable cell count ( ̴ 2.66 log CFU reduction) was seen in FLC.The MLC count was nearly constant during storage time.It was previously reported that the number of viable probiotics in microencapsulated samples was higher than in the free form during storage (Ni et al., 2023).The encapsulated Lactobacillus plantarum with alginate-gelatine gel viable number decreased by 2.1 log CFU/mL during storage.In contrast, the free probiotic had more reduction in viable number after 6 days of storage (1.51 log CFU/mL) (Ni et al., 2023).This result was in contrast with our result.One of the reasons may be related to the time of sampling and storage medium.The storage time in their research was shorter than mine.Moreover, the storage medium in their research was normal saline, while in our research, it was MRS broth.
Therefore, enrichment media could extend probiotic viability during storage time (Ni et al., 2023).

| Titratable acidity and pH
The content of acidity and pH are shown in Figure 4a,b.Total acidity and pH are the main characteristics used to evaluate the AVJD that help in the desirable balance of sugary acids necessary for a pleasant taste (Curi et al., 2023).While the pH had no significant difference among the samples at the end of storage (p > .05), the acidity of MLC and FLC was significantly more significant than the C sample (p ≤ .05).
In the C sample, a significant decrease in pH during storage time was observed (p ≤ .05).The remaining microorganisms in the AVJD, which are not damaged during the production process, are one of the reasons for these changes.However, pH and acidity are not consistently or poorly correlated in certain foods (Paulson & Stevens 1974).
The higher acidification at the end of storage is probably associated with a unique fermentation of LC.However, this did not adversely affect the product (Funck et al., 2019).High acidity in the ice cream containing free and encapsulated bacteria was seen.It can be related to the production of organic acid by probiotic bacteria (Maleki et al., 2023).Based on the obtained results, the acidity of FLC had a more significant value (p ≤ .05)than MLC because of the decreasing activity of MLC.The size of the alginate layer is one of the aspects that act on the metabolic activity of microencapsulated bacteria in products.The more layers of encapsulation led to less acid released.In this study, we used two layers as a wall for encapsulation.Furthermore, the layers formed around the bacteria may slow food intake, reducing the proportion of organic acids (Ghasemnezhad et al., 2017).A similar result was found by the other researchers (Saniani et al., 2023).

| Survival of MLC and FLC in AVJD during the storage time
Cell damage and loss of viability can occur throughout food processing and storage.A successful encapsulation process should therefore ensure the viability of the probiotics through the various stages of processing and maintain their activity during storage (Neuenfeldt et al., 2022).In comparison with FLC, the MLC survival was more significant than FLC during the storage stability test at 4°C.An increase in the storage stability of MLC was observed (Figure 5a).During the storage time, the survival rate of all supplemented AVJD began to decline.
There was a difference between the FLC and the MLC count in the 28 days of storage time.FLC had a marked decrease in viable counts  Lactobacillus plantarum.The data showed that the count of free cells reduced from 8.67 log CFU/mL to 1.27 log CFU/mL with a viability of 14.68% after 60 days of storage at 4°C.However, the survival rate of encapsulated bacteria was 60% (Li et al., 2023).It differs from the current research results.One of the reasons for this difference is due to the types of bacteria and wall materials.Encapsulation methods can improve the stability of probiotics during storage.This process is suggested as an effective procedure to protect probiotics during storage.
Various factors such as the extent of metabolization, pH reduction, and treatment type are influencing the viability of probiotic bacteria to recommend the use of prebiotics in a formulation (Shahraki et al., 2023).

| Survival in simulation gastrointestinal condition
Figure 5b shows the survival of FLC and MLC under GI conditions over time.The cell viability decreased, and the survival rate reached 14.24% and 71.04% for FLC and MLC samples, respectively.PMM showed a protective effect as a proper physical barrier.The alginate bead surface had a porous structure.The biopolymer network formed in PMM-alginate may be responsible for the slow diffusion of GI fluid through the wall material and decreased outflow of the entrapped probiotics from the microcapsules to the exterior environment of the gastrointestinal tract (Parsana et al., 2023).The viability of probiotics under harsh conditions of upper gastrointestinal tract is affected by the pore size of alginate-based microcapsules.The recommended pore size of the microcapsule is smaller than the size of hydrogen ions (<1 nm) and enzymes (<5 nm) (Khorshidi et al., 2021).
The results of this research are supported by the study of Shafizadeh et al. (2020).In their study, flaxseed mucilage was used for co-encapsulating probiotic L. casei in the alginate (ALG) matrix.
The viability of microencapsulated bacteria was higher than that of free cells under GI conditions (Shafizadeh et al., 2020).

| Sensory evaluation
The sensory properties of AVJD were evaluated and are shown in

| Texture of AVJD
The assessment of texture parameters showed that with increasing storage time, hardness increased significantly (p ≤ .05) in all samples (Table 2).Among the samples, MLC had the highest hardness significantly (p ≤ .05) at the 28th of the storage period.However, significant changes in structural parameters were expected due to modification of the AVJD protein crosslinking pattern.It seems that the incorporated beads make the gel more rigid, probably because the amount of water absorbent in the network decreases and the solid structure (beads) increases.An increase in hardness (1.95-6.80)N was also seen in gelatin desserts with microencapsulated Lactobacillus fermentum when stored at 4°C for 25 days (Amani et al., 2017;Dokoohaki et al., 2019).The result of this research fits with the report of Didar (2023) who attributed the higher hardness of enriched white chocolate with co-encapsulated Lactobacillus acidophilus (La-5) (Didar, 2023).
Adhesiveness (also known as stickiness) is determined by the action necessary to overcome the attractive forces between the surface of the food and the body part that comes into contact (e.g., tongue, teeth, palate).Adhesiveness increased significantly during the storage period in all samples (p ≤ .05).Among the samples, the C sample had the highest significant adhesiveness in the 28th of storage time (p ≤ .05).As an essential parameter, hardness and adhesiveness are positively correlated with microcapsule mechanical strength.Adhesiveness value is related to surface properties and the combination effects of adhesiveness and cohesiveness forces.
Moreover, they are associated with the molecular structure of the product.Studies have shown that stronger jellies have higher adhesive strength value (Yusof et al., 2019).In this research, adding FLC and MLC to AVJD raised the adhesiveness significantly (p ≤ .05).Such increased adhesiveness of condensed milk-based desserts was also reported (Aragon-Alegro et al., 2007).
Cohesiveness is described as a large number of broken protein connections during stressful circumstances and springiness (the ability to recover its initial condition after applying a deformation) (Sekhavatizadeh et al., 2022).Cohesiveness decreased significantly (p ≤ .05)with increasing storage time.Among the samples, the most    textural properties of food, like springiness (Kouamé et al., 2023).
For instance, the large protein particles in MLC-AVJD may increase springiness (Figure 7).The springiness increased during the storage time.Consistent with current results, a previous study showed springiness increased after a more extended incubation period (Mudgil et al., 2017).
Gumminess is closely connected with the statutes of hardness and , sodium citrate, peptone water, and MRS Agar were purchased from Merk company (Merck, Darmstadt, Germany).Sodium alginate was provided by Sigma Company (Sigma, Steinheim, Germany).The Plantago major seed was provided by Ahura Med, Marvdasht, Fars, Iran.It was collected from growing plants in Pasargad city, Fars province (South of Iran).Further plant identification was performed by the herbarium of the Fars Research Center for Agriculture and Natural Resources (FRCANR), Shiraz, Iran.A voucher specimen is deposited in the herbarium of the FRCANR, Shiraz, Iran.
Similar results were reported byCastro-Rosas et al. (2021) who produced alginate microspheres obtained by extrusion.Various factors affect the sphericity of microspheres.These include molecular weight of alginate, intrinsic viscosity, agitation speed, degree of polymerization, concentration of alginate, and air pressure applied to the system.There is a direct association between increasing agitation, higher pressure, and a greater chance of breakage of beads(Castro-Rosas et al., 2021).
in the microscopic images.Pourjafar et al. (2020) reported a similar morphology for L. acidophilus and L. rhamnosus covered with a singlelayer coating (chitosan) and double (chitosan and Eu S100).A smooth appearance was noticed on the surface of the double-coated capsules.
free form.In similar research,Karimi et al. (2021) found that microencapsulated Lactobacillus reuteri had a survival rate (73.25%) after 5 min exposure to heat.The difference between our results andKarimi et al. (2021) studies may be related to the type of wall material and the number of bead layers(Karimi et al., 2021).The use of encapsulating bacteria increases thermal resistance because of a protective layer.Belyani et al. (2023) reported the effect of temperature changes on the stability of microencapsulated Lactobacillus acidophilus La-5 along different temperatures of 55-60°C.The encapsulating bacteria had a more resistance than the free one(Belyani et al., 2023).Particularly, an excellent survival ability to heat stress (72°C) was observed in the case of encapsulated Lactobacillus acidophilus with sodium alginate-soy protein isolate.The minimum level of

Figure
Figure 3b illustrates the viability of FLC and MLC in acid and salt solutions.The viability of both the FLC and MLC decreased with increased exposure time.At pH 1.5 and salt 15%, the FLC and MLC decreased viable number ( ̴ 3.11 log CFU/mL) after 3 h of incubation.Verluyten

4
Acidity(a)  and pH (b) values of free Lactobacillus curvatus (FLC) and microencapsulated Lactobacillus curvatus (MLC) and control (C) AVJD during storage time at 4°C in AVJD.Data (mean ± standard deviation) are from three replications.

Figure
Figure6a-d.MLC showed the highest textural values probably due to the release of wall material (PMM) in AVJD.The color and acceptability scores were at least in MLC, which can be associated with the development of grainy texture because of MLC wall materials.Moreover, PMM and alginate provide a highly viscous medium to enhance MLC texture.According to another report and in confirming the result of this research, sensory evaluation demonstrated that judges perceived the presence of beads in probiotic food sensed(Saniani et al., 2023).
Texture parameters in C, MLC, and FLC jellies during 28 days of storage at 4°C.

| 3387 SEKHAVATIZADEH
et al. significant value (p ≤ .05) of cohesiveness was related to FLC at the end of storage time.Springiness is the speed and extent of deformed material recovery to its initial condition after removing a specific force.In our study, springiness increased significantly (p ≤ .05) in all samples except C-AVJD during storage time.The MLC had the most significant springiness value among the samples in the 28th of storage (p ≤ .05).The particle diameter could affect the microstructural and cohesiveness.The MLC revealed the most significant (p ≤ .05)value in F I G U R E 7 The SEM image of AVJD contains microencapsulated Lactobacillus curvatus (MLC), free Lactobacillus curvatus (FLC), and Control (c), during storage time.C in 1st (a); FLC in 1st (b); MLC in 1st (c); C in 28th (d); FLC in 28th (e); MLC in 28th (f) of storage time.
(Castro-Rosas et al., 2021)e of The average of the alginate layer and PMM diameter was 3271.44 ± 47.8 to 3354.0 ± 154.44 μm and 35.33 ± 3.00to 48.89 ± 1.03 μm, respectively.This average increases with the PMM concentration of the extrusion-derived beads significantly (p < .05).In another research, the range size of beads was varied from 100 μm to 5 mm in diameter.This parameter affects the organoleptic properties of foods, and large microspheres can be disadvantageous when incorporated into food systems(Castro-Rosas et al., 2021).It Li et al. (2023)f storage, dropping from 8.93 log CFU/mL to 4.91 log CFU/mL, with a viability percentage of 54.98%.However, the reduction of MLC was 2.19 log CFU/mL, and the survival rate was 77.85% during storage.Similarly,Li et al. (2023)used protein-isolated fibrils, sodium alginate, carboxymethylcellulose, and xanthan gum to protect