Microencapsulation of Lactobacillus acidophilus LA‐5 and Bifidobacterium animalis BB‐12 in pectin and sodium alginate: A comparative study on viability, stability, and structure

Abstract The present study aimed at examining whether the microencapsulation of Lactobacillus acidophilus LA‐5 and Bifidobacterium animalis BB‐12 inside hydrogels could prolong their survival in freeze‐drying conditions, stored at 4℃ and in the gastrointestinal medium. Microencapsulation was performed by emulsion with a syringe, while sodium alginate and high methoxyl pectin were used as a carrier material. A relatively high efficiency of encapsulation was obtained (>92%). Z‐Average and pdI in samples were not significant (p < .05). In different treatments, changes in the number of bacteria after freeze‐drying, 30 days of storage, and gastrointestinal conditions, compared to each other, were significant (p < .05). However, the survival rate after a reduction during storage was higher than 106 cfu/g, indicating the suitability of the microencapsulation process. The surface of microcapsules observed by a scanning electron microscope (SEM) confirmed the success of encapsulation. Finally, a lower decrease in the count of microencapsulated was observed in comparison to the free cells.


| INTRODUC TI ON
The most common probiotics introduced into functional foods are Lactobacillus and Bifidobacteria species, known as a nonpathogenic resident of the intestine, playing an important role in preventing the colonization of pathogens and the adjustment of host safety response de Lara Pedroso et al., 2012;Sohrabpour et al., 2021). Bifidobacterium lactis BB-12 and Lactobacillus acidophilus LA-5 are two commercial probiotic strains widely used as adjuvant cultures and generally known as "safe" (GRAS) . L. acidophilus shows antimicrobial effect due to the formation of organic acids and bacteriocin. It is also resistant to bile acid and has an antibiotic effect on intestinal pathogens such as Escherichia coli, L. acidophilus can attach to the intestine and survive for 2 days in the gastrointestinal juice. Bifidobacteria are used because they produce low acid and consume more lactic acid during storage. They have probiotic properties such as anti-cancer activity, folic acid synthesis, improvement of the nutritional value of food, and induction of immunoglobulin production. B. animalis is mostly selected for fermented dairy products because of its beneficial effects on human health and oxygen and acid tolerance compared with other species. B. animalis BB12 is capable of simultaneously producing conjugated linoleic acid, exopolysaccharides, and bacteriocins as postbiotics (Amiri, Aghamirzaei, et al., 2021;. Probiotics must be resistant to food operating, storage, and intestinal conditions to reach their intended location and show health effects with a minimum amount 10 6 -10 7 cfu/g Mularczyk et al., 2021;Rezazadeh-Bari et al., 2019;Vallejo-Castillo et al., 2020).
Microencapsulation is an acceptable method for probiotics protection, which provides high survival and high performance due to controlled release. Extrusion, spray-drying, and emulsion are the most common methods for probiotic microencapsulation; extrusion and spray-drying are less used, owing to probiotic susceptibility to applied temperatures and large particle size Liu et al., 2017;Martin et al., 2013;Nasri et al., 2020;Ohlmaier-Delgadillo et al., 2021;Saini et al., 2020;dos Santos et al., 2019).
Microencapsulation by emulsification/internal ionic gelation is a suitable method for the production of water in oil emulsion particles, described for the stabilization of unstable materials (Holkem et al., 2017). One advantage of this method is that smaller particles (less than 100 μm) do not alter the sensory properties of the product. This method requires no special equipment and sophisticated techniques, and due to its simple formulation and low cost, it has high cell viability and porous particles (Amine et al., 2014;Gebara et al., 2013;Holkem et al., 2016).
Alginate is the major compound used for the microencapsulation of probiotics, mainly because of its safety, good gelling properties (temperature and pH), and biocompatibility. Alginate is degraded in low pH, allowing the release of probiotics in digestive conditions (Amine et al., 2014;Han et al., 2018;Martin et al., 2013;Pupa et al., 2021;Qi et al., 2020;Sánchez-Portilla et al., 2020).
Pectin is a nontoxic and cheap polymer that forms a gel structure in the presence of divalent metal ions such as calcium. In the encapsulation process, the use of high methoxyl pectin is more efficient; high molecular weight and high gelling power provide small microparticles (Awasthi, 2011;Fathi et al., 2014;Panghal et al., 2019).
Particle size is an important factor, since large grains may produce sandy texture in the product, while small grains do not provide sufficient protection for bacteria. Therefore, probiotics should be trapped in a limited range of particle sizes to minimize the problems associated with cell survival and food texture (Machado et al., 2020).
Resistance to gastrointestinal conditions depends on the strain and species. The selection of carrier matrix can improve survival and significantly increase the number of live bacteria reaching the colon (Yonekura et al., 2014).
Therefore, the objective of this study was to produce probiotic microcapsules of L. acidophilus LA-5 and B. animalis BB-12 with emulsion technique in the sodium alginate and pectin with freezedrying and the evaluation of cell survival after the process and stability under gastrointestinal simulation conditions and their viability during 30 days of storage at a refrigerated temperature.

| Materials
The lyophilized culture of B. animalis subsp lactis BB-12 and L. aci-

Microencapsulation in sodium alginate
Microencapsulation of bacteria in sodium alginate was carried out according to the emulsion method developed by Holkem et al. (2016) with some modifications. First, sodium alginate solution (2% w/v) was prepared in deionized water and after sterilization with an autoclave (C73981, Webwco, Germany) stored in a refrigerator for 24 hr, so that alginate particles were well absorbed.
The next day, to coincide with the environment temperature, alginate solution was transferred to the outside of the refrigerator. Then, in a sterile condition, 5 ml of microbial suspension was mixed with 20 ml of sodium alginate, then added by sterile syringe as a dropper into a solution containing 99 g of rapeseed oil and 1 g of Tween 80 (previously sterilized), blended using a magnetic stirrer (RS3001, MLW, Germany) at 750 rpm and placed in the same round for 20 min until the mixture was completely emulsified in the oil phase. After that, 40 ml of sterilized calcium chloride solution (0.1 M) was added to the emulsion solution by syringe as a dropper and then emulsion was mixed on a magnetic stirrer for 5 min at 100 rpm. Due to the contact of alginate with calcium solution, the capsule wall was formed and beads were sedimented at bottom of the container. After completion of mixing time, 40 ml of sterile peptone water was added to separate the phases, and the solution was stabled for 30 min. After the complete sedimentation, the oily layer was poured out and microcapsules were separated by centrifugation at 324g, and temperature of 4℃ for 10 min. The beads were rinsed twice with sterile physiology serum (0.9%) to remove residual particles. In the end, microcapsules were kept in sterile-sealed containers with peptone water at refrigerator temperature until later use.

Microencapsulation in pectin
Microencapsulation in pectin was performed using the emulsion method provided by Gebara et al. (2013) with some modifications.
About 2 g of pectin powder was added to 100 ml of distilled water twice at 70℃ and was stirred continuously with a magnetic stirrer until it was completely dissolved. The solution was sterilized by filtration set (Millipore, Merck, Germany) with a filter paper size of 0.88 μm. Other steps were similar to the alginate method, with a difference that 0.8 M solution of calcium chloride was used.

| Probiotic cell count
Microcapsule cell counting was fulfilled by the method provided by Holkem et al. (2016) with some modifications. One ml of microcapsules was added to 9 ml of sterile sodium citrate solution (2% w/v, pH 7), and it was homogenized by a stomacher (Circulator400, Seward, UK) at 260 rpm for 4 min. During this process, beads were destroyed and bacterial cells were released. Serial dilution step with sterile peptone water solution (0.1%) was performed using pour plate method in MRS Agar medium. Finally, the number of bacteria was counted after 37 hr of incubation at 37℃. For free cell count, the pour plate technique was performed according to the method provided by de Lara Pedroso et al. (2012) with some modifications. It should be noted that Bifidobacterium was inoculated in the MRS Agar medium and incubated in anaerobic jars using the anaerobic gas pack system. All plates were done in two repetitions.

| Encapsulation efficiency
The efficiency of encapsulation, showing the number of living microorganisms during the microencapsulation process, was calculated using Equation 1 (Maleki et al., 2020): Where EE% is the percentage of the efficacy of capsulation; N denotes the number of cells released from capsules (cfu/g) and N 0 represents the number of live cells used for encapsulation (cfu/g).

| Evaluation of the stability of microcapsules to Freeze-Drying
To evaluate of freeze-drying effect, on the same day, a portion of microcapsules was frozen at −18℃ for 24 hr. The frozen microcapsules were dried in a vacuum dryer (FD-5005-BT, Dena industry, Iran).

| Stability of microencapsulated bacteria during storage
The microencapsulated bacteria were stored in sterile peptone water in a 1:1 ratio at 4℃ for 30 days, and the survival rate was assessed using the method outlined in the previous sections (Martin et al., 2013).

| Survival of probiotics after exposure in gastrointestinal conditions
The test was carried out using the method developed by Maleki et al. (2020) with a few changes. One gram of freshly prepared beads was added to 10 ml simulated gastric juice (GJ) (HCl 0.08 M containing 0.2% NaCl, pH 1.55), without pepsin and incubated at 37℃ for 0, 60, and 120 min. After incubation, 1 ml of the above solution was removed and placed in 9 ml of simulated intestinal juice (IJ) without bile salts (KH 2 PO 4 , pH 7.43) and incubated at 37℃, for 50, 100, and 150 min. After incubation, 1 ml of the solutions was pure plated using the method described in the probiotic cell count section.

| Particle size analysis
Dynamic light scattering (DLS) is a physical method used to determine the distribution and other particles in solutions and suspensions based on their Brownian motion. First, 2 ml of samples was poured into a cuvette and diluted with distilled water twice for distillation. Then, the cuvette was placed in a dynamic diffraction analyzer (Nano ZS ZEN 3,600, Malvern, UK), and parameters were measured using visible light with a wavelength of 633 nm at 25℃. (1) % EE = N N 0 × 100

| Experimental design and data analysis
All experiments were carried out in a completely randomized design with three replications. Analysis of variance was done at α = 0.05, and the least significant difference test was used to confirm the difference between the means at p < .05 using Microsoft Excel 2016 software.

| Encapsulation efficiency
The results of variance analysis showed that the efficiency of encapsulation was not significant in different samples (p > .05) (Figure 1(a)).
The encapsulation efficiency obtained in this study was similar to (Gebara et al., 2013;Holkem et al., 2016;Krasaekoopt et al., 2004) results. They reported, respectively, the average efficiency of 89, 84, and 99% for pectin microcapsules and sodium alginate containing L. acidophilus and B. animalis by the internal gelatinization method.
It was observed that the size of pectin microcapsules was higher than that of sodium alginate, probably related to wall material and the high viscosity of 2% (W) of pectin solution relative to the same therefore, it seemed to be a practical and appropriate method.

| Evaluation of the stability of microcapsules to Freeze-Drying
In different treatments, changes in the number of bacteria after freeze-drying and compared with each other were significant (p < .05) (Figure 2). B. animalis BB-12 was more susceptible to freezedrying than L. acidophilus LA-5, and pectin microcapsules exhibited higher resistance to freeze-drying conditions; however, reduction of

| Stability of microencapsulated bacteria during storage
The probiotic of about 10 6 cfu/g (Holkem et al., 2017). However, in this study, the survival rate during storage time was higher than this value.

| Probiotic bacterial survival in gastrointestinal conditions
The encapsulation such as pH, presence or absence of enzymes, and various wall materials can provide different results.

| Particle size analysis
The results of analysis of Z-Average and pdI in different samples were insignificant (p > .05). Due to the use of a similar type of syringe in the production of microcapsules, the type of coating material did not affect particle size. In general, the microcapsules of pectin are larger than sodium alginate, and microcapsules B. animalis BB-12 also has a larger particle size than L. acidophilus LA-5 (Figure 1b,c). These results were consistent with SEM images, confirming the largeness of pectin microcapsules containing B. animalis BB-12 compared with other particles. In the study by Sandoval-Castilla et al. (2010), the size of alginate microcapsules was smaller than pectin.
Due to the use of a low diameter syringe, the average diameter of particles was small in comparison to results obtained in other studies for the microencapsulation by emulsion method. Barbosa et al. (2015) reported the mean diameter of alginate microcapsules containing Lactobacillus corvatus by emulsion method at about 266 and 473 μm. The diameter of Bifidobacterium in sodium alginate microcapsules by emulsion method performed by Hansen et al. (2002) and Holkem et al. (2017) was reported to be about 19, 67, and 54 μm, respectively. In addition, the diameter of L. acidophilus and B. bifidum microcapsules in sodium alginate 2% by Krasaekoopt et al. (2004) was reported to be 1.6 μm, roughly similar results.
The low mean diameter of particles can be attributed to the high efficiency and effect of the presence of probiotics in microparticles, and this effect can be ascribed to a change in the zeta potential of microcapsules, as reported by Martin et al. (2013). They reported that alginate microcapsules containing probiotics had a smaller size than nonprobiotic microcapsules. The application of the emulsion method to produce microcapsules could control the size of gelatinization and similar microparticles; the diameter of microparticles was controlled by the concentration and viscosity of sodium alginate and pectin solutions and the mixture of emulsion (Hansen et al., 2002).
The size of microparticles affects the efficiency of encapsulation and food texture. The diameters smaller than 100 μm are preferred for most applications for better protection against the gastrointestinal tract (Holkem et al., 2017;Mirtič et al., 2018).

| Morphological characteristics
According to Figure 4a,b, elliptic microcapsules were similar to the results obtained by Jagannath et al. (2010). The rugged surface of microcapsules indicates the presence of probiotics inside the capsules (Martin et al., 2013). It was also observed that the immobilization of cells in sodium alginate produced semispherical microcapsules (shape BS) with rigid surface and spongy structure The cavities all over the microcapsules were due to the rapid submersion of frozen water from the microcapsule matrix during the freeze-drying process, leading to porosity in places where there were ice crystals. Particles were accumulated together because of their fineness. These images were similar to results obtained by Holkem et al. (2016). Generally, wrinkles and cracks are the results of the mechanical stress caused by nonuniform drying of various parts of the liquid droplets in the early stages of drying. High molecular weight polymers dry quickly to prevent the release of internal vapors, resulting in increased bubble formation in the matrix of wall materials, expanding the internal space of the microcapsule, and creating more concavity (Maleki et al., 2020). Comparison of pectin and alginate microcapsules showed that alginate beads were relatively spherical, while pectin beads had a geometrically shaped plate; this phenomenon was related to the difference in the cross-links created in each case.

| CON CLUS ION
Microencapsulated bacteria show many advantages over free cells, including protection, high volume of productivity, improved control process, protection of cells against damages, and reduced sensitivity to contamination. However, the stabilization of probiotic cells requires some specific processes with complex stages of food production and increased cost. The results of this study about the effectiveness of encapsulation to protect probiotics were controversial, and the high diversity of parameters under evaluation made it difficult to find the best method of encapsulation.

ACK N OWLED G M ENT
The authors are grateful for the financial support from the Urmia University.

CO N FLI C T O F I NTE R E S T
The authors have declared no conflicts of interest for this article.

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