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Microbiology and Biotechnology Letters

Research Article(보문)

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Food Microbiology (FM)  |  Probiotics in Nutrition and Health

Microbiol. Biotechnol. Lett. 2024; 52(3): 264-274

https://doi.org/10.48022/mbl.2405.05003

Received: May 10, 2024; Revised: July 16, 2024; Accepted: July 25, 2024

Survivability of Encapsulated Bifidobacterium breve BS2-PB3 during Storage and in Simulated Gastric Juice and Bile Salt

Patricia Celine, Marcelia Sugata*, and Tjie Jan Tan

Department of Biology, Faculty of Science and Technology, Universitas Pelita Harapan, Jl. MH. Thamrin Boulevard, Lippo Village, Tangerang 11580, Banten, Indonesia

Correspondence to :
Marcelia Sugata,        marcelia.sugata@uph.edu

Abstract

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Bifidobacterium breve is commonly administered as probiotics in fermented foods or supplements. Accordingly, a sufficient number of live cells must be consumed to experience the benefits of probiotics. Microencapsulation is a technique used to increase the viability and stability of probiotics when exposed to industrial processes, low/high temperature, storage periods, and gastrointestinal conditions. This study aimed to evaluate the effect of microencapsulation with alginate-chitosan and pectin-chitosan matrices on the viability and stability of B. breve BS2-PB3. Microencapsulation was performed using alginate-chitosan (2%, 0.75%) and pectin-chitosan (3%, 0.75%), followed by freeze drying. The encapsulation efficiency of both matrices was 94%, and the survival rate after freeze drying was 96%. After 8 weeks of storage, B. breve BS2-PB3 showed a stable viability, with more than 96% and 83% survival rates at 4℃ and room temperature, respectively. Both encapsulation matrices maintained bacterial viability up to 55% in simulated gastric juice (pH 2) and up to 66% in 0.3% bile salt solution. The administration of microcapsules to mice lowered the fecal pH and increased the defecation frequency. This study demonstrated that microencapsulation using alginate-chitosan and pectin-chitosan could maintain probiotic viability and stability.

Keywords: Alginate, chitosan, pectin, probiotic, shelf life

Graphical Abstract


Introduction

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Probiotics are live microorganisms, which, when given in adequate amounts of at least 1060−107 colony forming units (CFU) each day, will provide health benefits to the host [1]. Two genera of commonly used probiotics found in the human intestine are Lactobacillus and Bifidobacterium [2, 3]. Bifidobacterium spp. are gram positive bacteria that live under anaerobic conditions. Bifidobacterium breve is the predominant species in the intestines of breast-fed infants. This species has antibacterial activity against human pathogens and can increase host immunity [4, 5].

Probiotics can be consumed orally through functional foods, such as yogurt, kefir, and kimchi [6, 7]. However, probiotics could lose viability when exposed to digestive enzymes or low pH conditions in the digestive tract. In addition, as functional foods, probiotics need to go through harsh processing and storage procedures, which cause a significant decrease in their viability and stability [8, 9]. Microencapsulation is a technique used to maintain probiotic viability. It involves creating an external matrix that acts as a strong protective layer made of bio-polymers [10, 11]. Through microencapsulation, probiotic cells are trapped into the encapsulation agent to prevent damage or loss of cell viability. Microencapsulation can also control the release of bacteria in the intestines [12].

Various biopolymers, such as alginate, pectin, and chi-tosan, are commonly used for encapsulation. These carbohydrates are the preferred biopolymers due to their characteristics of nontoxicity, ability to form robust capsules, and ease of availability [10, 13]. Chitosan is generally used as an additional coating on capsules made from other biopolymers like alginate and pectin. Recent studies showed that encapsulation within alginate-chitosan and pectin-chitosan microgels can improve the stability and viability of probiotics [13−15].

Microencapsulation is often followed by freeze drying, a low-pressure method of drying that involves freezing and sublimation processes to reduce the water content and improve the stability of probiotics during storage [5, 12]. This method involves three steps: 1) freezing the substance; 2) primary drying, which involves lowering the pressure to sublimate frozen water into gas; and 3) secondary drying to remove the remaining water. The final water content after additional drying is only between 2% and 10% [12, 16−18]. The moisture content of freeze-dried probiotic microcapsules is crucial for maintaining bacterial stability during storage. Microorganisms typically exhibit enhanced survival at lower water activity levels. Therefore, freeze drying is commonly employed to stabilize microorganisms, such as probiotics, by halting the cellular metabolic activity, thereby preserving their original composition and physical properties [19].

Previous studies conducted by Juvi et al. [20] and Sugata et al. [21] investigated the efficacy of alginate-chitosan encapsulation in enhancing the survivability of encapsulated Lactobacillus sp. against various environmental conditions. Similar to Lactobacillus, Bifidobacterium are also susceptible to reduced viability in the digestive tract when subjected to harsh environments, such as an extremely low pH. This study aims to evaluate the effects of microencapsulation using pectin-chitosan and alginate-chitosan matrices on the viability of B. breve BS2-PB3 isolated from breast milk. The impact of supplemented encapsulated BS2-PB3 on the digestive tract is also assessed using BALB/c mice as animal models.

Materials and Methods

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Fig. 1 presents the flowchart summarizing the experimental procedure in this study.

Figure 1.Methodology flowchart.

Media and culture preparation

This study used 1 L of Trypticase Phytone Yeast (TPY) agar consisting of 17.6 g Tryptone Soya Broth (Himedia, India), 5 g bacteriological peptone (Liofilchem, Italy), 5 g D(+)glucose monohydrate (Merck, USA), 2.5 g yeast extract (Condalab, Spain), 1 ml Tween-80 (Merck), 0.5 g L-cysteine (Now Foods, USA), 2 g dipotassium phos-phate (Merck), and 15 g bacteriological agar (Oxoid, UK). B. breve BS2-PB3 was originally isolated from local breastmilk [22]. The culture stock was maintained by storing the liquid culture at -40℃ in TPY media supplemented with 25% glycerol (Merck). To grow the bacteria, the culture stock was streaked on TPY agar and incubated at 37℃ for 72 h anaerobically. The anaerobic condition was provided by using an anaerobic atmosphere generation sachet (Thermo Fisher Scientific, USA). Subsequently, the growth curve of B. breve BS2-PB3 was made by measuring the optical density of the bacteria liquid culture at 600 nm (OD600) and counting the number of viable cells (CFU/ml) at 0, 16, 24, 40, 48, 64, and 72 h. At the exponential phase, the cells were harvested by centrifugation at 4000 × g for 10 min.

Microencapsulation and freeze drying

B. breve BS2-PB3 was encapsulated within two different biopolymers, namely, alginate and pectin, using an extrusion method [5, 16]. Briefly, 2% (w/v) sodium alginate solution and 3% (w/v) low-methoxyl pectin solution were prepared separately, autoclaved, and then cooled to room temperature. The B. breve BS2-PB3 cell suspension was mixed with each biopolymer solution (2% alginate or 3% pectin) with a ratio of 1:10 (v/v). Each mixture was extruded with a syringe needle (1 ml, 26G) (OneMed, Indonesia) into 4% (w/v) CaCl2 (Merck) under gentle stirring. The wet microcapsules formed were allowed to harden for 30 min. After hardening, the alginate and pectin microcapsules containing B. breve BS2-PB3 were removed from the solution using filter paper. The chi-tosan solution was prepared by dissolving chitosan in 0.1 M acetic acid. The microcapsules were then coated by dispersing alginate and pectin microcapsules in 0.75% (v/v) chitosan solution under gentle stirring for 1 h and subsequently removed by filtration. Next, the microcapsules were stored in -40℃ overnight, and the frozen microcapsules were freeze-dried using Christ Alpha 1-2 LDplus (Martin Christ, Germany) under the following condition: 48 h and 0.67 mbar at -40℃.

The concentrations of the alginate (2% w/v), pectin (3%w/v), and chitosan (0.75% w/v) used were selected based on previous research demonstrating their efficacy in producing protective microcapsules [13, 20, 24]. The concentration of alginate (2%) was lower than that of pectin (3%) due to practical constraints. That is, solutions with alginate concentrations above 2% become too viscous to flow through the syringe, hence impeding the microcapsule formation. The chitosan concentration (0.75%) was chosen to provide optimal coating without interfering with the core structure of the microcapsules. These concentrations were determined through preliminary experiments that assessed the balance between the protective capabilities and the practical feasibility in microcapsule production.

Encapsulation efficiency and viability assessment

To determine the number of viable bacteria in the microcapsules, 1 g of wet microcapsules and 0.1 g dried microcapsules were re-suspended in 9 ml and 9.9 ml, respectively, of a 0.1 M sodium citrate solution (pH 6). The solution was homogenized using a vortex and incubated for 1 h. After serial dilutions of dissolved microcapsules, the number of viable cells in the solution was determined using a plate count assay on TPY agar after anaerobic incubation at 37℃ for 72 h. The encapsulation yield (EY) was calculated as follows:

Encapsulation yield (%):NN0×100

where, N is the number of viable cells in the microcapsules, and N0 is the number of cells used for encapsulation.

The survival rate after freeze drying was calculated as follows:

Survival rate (%):NN0×100

where, N is the number of viable cells in the freeze-dried microcapsules, and N0 is the number of viable cells in the wet microcapsules.

Long-term storage (shelf life)

After drying, the microcapsules and the probiotic liquid cultures were stored at room temperature (±25℃) and 4℃ for 8 weeks. The viable probiotic cells in the microcapsules and liquid culture were enumerated using a plate count assay on TPY agar at weeks 2, 4, and 8. The survival rate was calculated as follows:

Survival rate (%):NN0×100

where, N is the number of viable cells at week-N of storage, and N0 is the number of viable cells on the first day of storage.

Viability in the simulated gastric juice and bile salt solution

The simulated gastric juice (SGJ) was prepared by resuspending 3 g/l pepsin (Himedia) in 0.5% (w/v) sodium chloride (Merck). The pH was adjusted to 2.0. Subsequently, 0.3% (w/v) bile salt (Oxoid) solution was prepared by resuspending 3 g/l bile salt in sterile water. Bile salts with a 0.3% concentration were used in this experiment because the concentration reflects conditions in the human digestive system [23]. One milliliter of the cell suspension and 0.1 g of the microcapsules were added to 9 ml and 9.9 ml SGJ/bile salt solutions, respectively. The mixture was homogenized and incubated at 37℃ for 3 h with agitation at 110 rpm. To determine the number of viable cells (CFU/ml) after exposure to the SGJ/bile salt, the samples were obtained at constant intervals (t = 0, 1, 2, 3 h) and spread-plated on TPY agar, followed by anaerobic incubation at 37℃ for 72 h.

In vivo study: probiotic supplementation in mice

Six-week-old male BALB/c mice were obtained from Universitas Gadjah Mada, Yogyakarta. Prior to the experiment, the mice were acclimatized for 1 week, fed once a day with water available ad libitum, and placed at room temperature (±25℃) with a 12 h light-dark cycle. All protocols for this study were approved by the Health Research Ethics Committee, Faculty of Health Sciences, Universitas Pelita Harapan, Indonesia (0029/PE.KEPK-FIKes-UPH/V/2023).

The mice were randomly categorized into two groups: control and treatment. The control group was fed pellets without probiotics, while the treatment group was fed pellets supplemented with probiotics. Each group contained three BALB/c mice. The pellets were made by mixing 0.5 g commercial mice feed (Indonesia) and 0.1 g dried alginate-chitosan microcapsules without and with probiotics (1010 CFU/ml). One formulated pellet was given once daily in the morning for 21 days. After the pellet was consumed, 5.4 g of commercial feed was given to each mouse. Water was available ad libitum throughout the whole experiment. During 21 days of supplementation, the mice feces were collected, counted, and weighed every day. The pH of the feces was also checked using pH paper after 1 g of feces was diluted in 10 ml distilled water.

Statistical analysis

All values are expressed herein as the mean ± standard deviation. The statistical differences were analyzed by one-way analysis of variance at a significance level of p ≤ 0.05 (95% confidence interval), followed by a Tukey’s honest significant difference (HSD) test. All analyses were performed using Excel 16.75 and online web statistical calculators (https://astatsa.com/).

Results and Discussion

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Growth curve

The BS2-PB3 growth curve was used to determine the phase of bacterial growth. In this study, the cells were harvested after they reached the exponential phase because the encapsulation procedure required a high number of cells. Based on the growth curve in Fig. 2, the exponential phase of BS2-PB3 grown in TPY broth was between 16 h and 48 h after inoculation. Therefore, BS2-PB3 was grown for 24 h, and the cells were harvested for encapsulation.

Figure 2.Growth curve of the B. breve BS2-PB3 grown in a TPY broth.

Microencapsulation and freeze drying

B. breve BS2-PB3 was encapsulated within either alginate (2%) or pectin (3%) microgels using an extrusion method. The wet microcapsules containing probiotic were then coated with chitosan (0.75%). The alginate-chitosan microcapsules were transparent and spherical, with a 1.1 mm diameter, while the pectin-chitosan microcapsules were white and spherical, with a 1.2 mm diameter (Fig. 3). The pectin-chitosan microcapsules were slightly bigger than the alginate-chitosan ones because the percentage of pectin used was higher, consequently increasing the viscosity of the pectin solution. The ionic bonds between the Ca2+ ion in calcium chloride and guluronic acid (GulA) on alginate and galacturonic acid (GalA) on pectin chain allowed the microcapsules to form [8].

Figure 3.Wet microcapsules containing B. breve BS2-PB3: (A) alginate-chitosan and (B) pectin-chitosan.

The wet microcapsules were frozen overnight at -40℃and subsequently freeze-dried for 48 h. The freeze-dried microcapsules are shown in Fig. 4. The dried microcapsules from both biopolymers were similar in size, with diameters ranging from 0.9 to 1.0 mm. The alginate-chitosan dried microcapsules appeared white, while the pectin-chitosan ones were yellow. Drying methods, such as freeze drying (also known as lyophilization), are commonly used to increase the stability and viability of probiotics. By removing water, which is essential for microbial growth and metabolism, drying can prolong the probiotics’ shelf life. Freeze drying involves freezing the sample and reducing the surrounding pressure, allowing the frozen water (ice) to directly sublimate from solid to vapor. This process typically reduces the water content in probiotics to a very low level, that is, often between 2% and 10%, thus inhibiting the cell metabolic activity [18, 24].

Figure 4.Dried microcapsules containing B. breve BS2-PB3: (A) alginate-chitosan and (B) pectin-chitosan.

Viability assessment

The effect of the encapsulation process on the BS2-PB3 viability was investigated (Table 1). Both the alginate-chitosan and pectin-chitosan matrices showed an EY of more than 94%, and no significant difference (p > 0.05) was observed in the EY between the two matrices. The high EY indicated that numerous living cells were successfully trapped into the encapsulation matrices, and that the needle extrusion method was an effective method for encapsulation. Li et al. [25] and Moghanjougi et al. [26] reported that encapsulation using pectin and alginate matrices has an encapsulation success percentage of approximately 89−99%. A high EY was achieved because the extrusion method provided gentle processing conditions that did not involve high temperatures, extreme pH conditions, or organic solvents that can stress the cells [27, 28].

Table 1 . Viability of B. breve BS2-PB3 before and after microencapsulation and freeze drying.

MatrixCell count (109 CFU/ml)Encapsulation yield (%)Survival rate (%)
Encapsulated cellsEncapsulated cells after drying
Alginate-chitosan1.97 ± 0.031.91 ± 0.0194.80 ± 0.50a96.60 ± 1.13a
Pectin-chitosan1.97 ± 0.111.89 ± 0.0494.10 ± 1.30a96.40 ± 0.92a


The survival rate of the dried microcapsules in the alginate-chitosan and pectin-chitosan matrices after drying was more than 96% and not significantly different (p > 0.05). Several studies showed that the percentage of bacterial survivability after freeze drying generally ranged between 86% and 98% [17, 26, 29]. The percentage of survivability in this study was quite high, proving the ability of the alginate-chitosan and pectin-chitosan matrices to protect bacterial cells during the freezing and drying process. The carbohydrates in the microcapsule shielded probiotics from direct exposure to harsh conditions, such as the freeze-drying process, thereby reducing the cell damage and improving the survival rates. Additionally, the microcapsules created a physical barrier around the probiotic cells that slowed the penetration of ice crystals into the cells during freezing and subsequent freeze drying. By reducing the rate of the ice crystal formation inside the cells, the microcapsules may help mitigate the damage caused by the intracellular ice formation and increase the cell survivability [30]. Both biopolymers demonstrated excellent protective properties, resulting in a high cell survivability after microencapsulation and freeze drying. However, pectin can be costly when used in large quantities. Conversely, while alginate is more economical, its use is challenging because the formation of the alginate solution must be carefully controlled to ensure even mixing without forming lumps.

Polysaccharides are the primary focus of encapsulation research, with alginate and starch being the most commonly utilized materials across various microencapsulation techniques. Among protein-based materials, various forms of whey protein are predominantly used in spray-freezing methods. Other protein options, such as gelatin, egg albumin, and vegetable proteins, are also viable due to their accessibility and affordability. However, research on using lipids as encapsulation materials for probiotics remains relatively limited.

Shelf life

The survival rate of the encapsulated BS2-PB3 within the alginate-chitosan and pectin-chitosan matrices during storage at room temperature (±25℃) and 4℃ was monitored for 8 weeks. The encapsulated cells within both matrices exhibited a higher viability than the suspension cells throughout the storage period (Figs. 5 and 6). However, the lower temperature of 4℃ better preserved the cell viability compared to room temperature. This was attributed to the slowing down of the cell metabolism at lower temperatures, which inhibited growth and maintained the morphological, physiological, and genetic stabilities of the bacteria [31−33].

Figure 5.BS2-PB3 shelf life at room temperature (±25℃). Each value is written as the mean ± standard deviation of the three replicate tests. The data were statistically analyzed using Tukey’s honest significant difference (HSD). Values with different letters represent a statistically significant difference (p ≤ 0.05).

Figure 6.BS2-PB3 shelf life at 4℃. Each value is written as the mean ± standard deviation of the three replicate tests. The data were statistically analyzed using Tukey’s honest significant difference (HSD). Values with different letters represent a statistically significant difference (p ≤ 0.05).

The survivability of BS2-PB3 within the alginate-chitosan and pectin-chitosan matrices showed no significant differences (p > 0.05) in either wet or dried microcapsules when stored at room temperature (Fig. 5), with similar results observed at 4℃ (Fig. 6). When compared to the cell suspension without encapsulation, BS2-PB3 experienced a significant decrease (p < 0.05) in viability after 14 days of storage, exhibiting a greater decrease at room temperature (±25℃) compared to 4℃. These results aligned with those in other studies on encapsulated probiotics. For instance, B. breve encapsulated with pectin showed a significantly high number of viable cells when stored at low temperature (4℃ and -20℃) after 13 weeks [25]. Similarly, B. animalis BB-12 encapsulated with alginate and pectin showed a high survivability after 30 days of storage at 4℃ [26].

At lower temperature, the BS2-PB3 viability after 2 weeks of storage significantly decreased in the wet microcapsules, but not in the dried ones. The cell survivability in the wet microcapsules exceeded 90%, while the survival rate in the dried microcapsules exceeded 99%, even after 4 weeks of storage. However, the cell viability in the dried microcapsules significantly decreased to 96.7 ± 0.57% for alginate-chitosan and 96.3 ± 0.76% for pectin-chitosan after 8 weeks of storage. These findings demonstrate that the drying process can enhance the cell survivability throughout the storage period. The main objective of the encapsulation and drying processes was to reduce the capsule’s water content, leading to the inactivation of the cell metabolism and the preservation of the cell viability [5, 12].

The high stability and viability of probiotics during storage are critical considerations in the food industry. Our findings on the enhanced stability of encapsulated probiotics have significant implications for both industrial applications and consumer health. From an industrial perspective, the improved storage stability of encapsulated probiotics can lead to the extended shelf life of probiotic products, potentially reducing waste and increasing cost-effectiveness. This could enable the incorporation of probiotics into a wider range of food products, including those with longer storage periods or more challenging storage conditions. For consumers, a higher probiotic viability at the time of consumption may translate to improved health benefits. The ability to maintain the probiotic viability at room temperature, albeit for shorter periods compared to refrigerated storage, can enhance the accessibility of probiotic products in regions with limited cold chain facilities. However, this study has limitations that warrant consideration. Our experiments were conducted under controlled laboratory conditions, which may not fully replicate the complex environments encountered in food matrices or during product distribution. Additionally, we also focused on specific strains and encapsulation materials, and the results may vary with different probiotic species or encapsulation techniques.

Cell survivability in SGJ

The viability of the suspension and the encapsulated cells in SGJ was evaluated. After 3 h of SGJ exposure, the suspension cells exhibited a significant reduction in viability (p < 0.05), with 10% survival rate (Table 2). This result suggests that BS2-PB3 is susceptible to the stomach’s acidic environment, necessitating an encapsulating system to protect the cells during gastrointestinal transit. BS2-PB3 encapsulated with alginate-chitosan and pectin-chitosan demonstrated a survival rate of approximately 55%, which is fivefold higher than the suspension cells. This enhanced survival suggests that the microencapsulated BS2-PB3 may better withstand gastrointestinal conditions, potentially allowing probiotics to colonize the intestine and exert positive effects on human health.

Table 2 . Survival of B. breve BS2-PB3 in the simulated gastric juice.

MatrixViable cell number (109 CFU/ml)Survival rate (%)
0 h1 h2 h3 h
Alginate-chitosan1.95 ± 0.011.51 ± 0.011.21 ± 0.011.08 ± 0.0155.29 ± 0.38a
Pectin-chitosan1.92 ± 0.011.54 ± 0.051.20 ± 0.011.06 ± 0.0155.28 ± 0.13a
None (Suspension cells)1.93 ± 0.011.07 ± 0.050.68 ± 0.050.20 ± 0.0110.17 ± 0.33b


Previous studies reported that microencapsulation using alginate and pectin, followed by chitosan coating, can maintain probiotic viability better than suspension cells under gastrointestinal conditions [13, 34]. In the alginate-chitosan matrix, the Ca2+ ions form specific bindings during capsule formation without causing chain shifts. Conversely, in pectin chains, the Ca2+ ions bind to the GalA residues at random positions, resulting in chain shifts during the capsule formation. Despite these structural differences, both matrices demonstrated efficacy in shielding probiotics from adverse environmental conditions. The alginate-chitosan microcapsules reportedly provided protection against SGJ because alginate can shrink at a low pH, preventing bacterial release from the microcapsules [17, 35]. Similarly, pectin is known to be resistant to low pH and can provide a physical barrier, especially when coated with chitosan [26].

Cell survivability in the bile salt solution

Bile salts behave like detergents and can damage the DNA when they enter bacterial cells, causing a lower probiotics survival in the digestive tract [36, 37]. After being exposed to bile salts for 3 h, the alginate-chitosan and pectin-chitosan encapsulated cells showed a survival rate of approximately 67%. Only 44.25% of the cells survived in the suspension. The percentages of cell survivability in the two microcapsule matrices and in the suspension were significantly different (p ≤ 0.05) (Table 3). These results aligned with the findings of Chávarri et al. [34], Koo et al. [38], and Bepeyeva et al. [13], suggesting that microencapsulation can protect cells against damage from bile salts.

Table 3 . Survival of B. breve BS2-PB3 in 0.3% bile salt solution.

MatrixViable cell number (109 CFU/ml)Survival rate (%)
0 h1 h2 h3 h
Alginate-chitosan1.92 ± 0.011.64 ± 0.101.50 ± 0.101.29 ± 0.0167.20 ± 0.46a
Pectin-chitosan1.91 ± 0.011.62 ± 0.111.49 ± 0.111.28 ± 0.0166.84 ± 0.17a
None (Suspension cells)1.91 ± 0.011.62 ± 0.011.33 ± 0.010.85 ± 0.0144.25 ± 0.52b


BS2-PB3 maintained a higher viability after exposure to bile salt compared to after exposure to gastric juice. This bile salt resistance might be caused by the ability of BS2-PB3 to produce the bile salt hydrolase (BSH) that catalyzes the amide bond hydrolysis in conjugated bile acids, causing the release of free amino acids. Previous studies found that several Bifidobacterium species, including B. breve, have BSH-encoding genes [36, 39−41]. Another mechanism for surviving bile salt exposure was the efflux system, which, with the help of multidrug transporters, pumps the accumulated bile acids in the bacterial cytoplasm to the external environment [37, 42]. Bile efflux transporter genes have been found in Bifidobacterium species (e.g., Bbr_0838 gene in B. breve [43] and BL0920 gene in B. longum [44]).

Microencapsulation enhances the bile resistance of probiotics through several mechanisms. The encapsulation material (e.g., alginate or pectin) forms a protective barrier around the probiotic cells, shielding them from the detrimental effects of bile salts, including membrane disruption and oxidative stress, which are known to reduce probiotic viability in the gastrointestinal tract. The encapsulation process may also stabilize the cell membrane and intracellular components, thereby enhancing the probiotic’s ability to withstand bile exposure. The controlled release of probiotics from the microcapsules in the gastrointestinal tract ensures a prolonged bile salt exposure, promoting adaptation and survival of the probiotic cells. The chitosan coating further enhances the viability by facilitating the electrostatic interactions between its amine groups and the carboxylic groups of the bile salts, thereby reducing the bile salt permeability into microcapsules [45]. Moreover, the encapsulation materials may create a favorable microenvironment within the capsule, potentially buffering the pH changes or sequestering the bile salts and providing additional protection to the encapsulated probiotics. These mechanisms collectively contribute to the improved bile resistance observed in the microencapsulated probiotics, highlighting the potential of microencapsulation as a strategy to enhance probiotic efficacy and viability in the gastrointestinal tract.

In vivo study

In this preliminary in vivo experiment, the alginate-chitosan microcapsules were selected for investigation. While our previous results showed no significant difference in survivability between the alginate-chitosan and pectin-chitosan treatments, the choice of alginate-chitosan for further study was based on several practical considerations. Alginate is more commonly used than pectin in the food industry because of its favorable gelation properties, widespread availability, versatility, stability, regulatory approval, and mechanical strength. Additionally, this approach allows for a focused investigation that can serve as a foundation for future comparative studies with other encapsulation systems, including pectin-chitosan.

The microcapsules, with or without BS2-PB3, were mixed with mice feed and formed into pellets (Fig. 7) before being fed to the mice for voluntary consumption. Probiotics are generally given to mice through oral administration using a syringe. However, this method requires technical expertise; otherwise, it can set off a stress response that can result in esophageal or stomach injury, pneumonia, and even death. Previous studies reported that oral administration with voluntary consumption shows lower stress levels compared to the syringe method [46, 47].

Figure 7.Reformulated mice feed pellets.

During the treatment period, the fecal samples were collected from each mouse, and the number of fecal droppings was counted and weighed. Their pH was also measured. As shown in Table 4, the fecal pH in the control groups remained stable, while the treatment group exhibited a significant decrease in pH by week 2, with no further decrease by week 3. These results were consistent with findings of Park et al. [48], which demonstrated that Lactobacillus supplementation increased the production of short-chain fatty acids (SCFAs), particularly acetic acid, resulting in a lowered fecal pH. Although we did not directly measure the SCFA concentrations, the observed pH reduction strongly suggests enhanced SCFA production. An increased SCFA production is a crucial mechanism through which the probiotics can modulate the gut environment, potentially offering various health benefits, such as improved gut barrier function and enhanced mineral absorption. These findings highlight the potential of our encapsulated BS2-PB3 to beneficially alter the gut environment through SCFA production, supporting its efficacy as a probiotic supplement. Future studies directly measuring the SCFA concentrations in fecal samples would further validate this mechanism.

Table 4 . Mice fecal pH, weight, and droppings.

TimepHWeight (g)Number of droppings
ControlTreatmentControlTreatmentControlTreatment
Week 16.1 ± 0.45a5.8 ± 0.53a0.72 ± 0.05a0.91 ± 0.16a17 ± 0.17a22 ± 0.40a
Week 26.0 ± 0.00a5.0 ± 0.00b0.78 ± 0.01a1.14 ± 0.01b18 ± 0.35a30 ± 0.92b
Week 36.1 ± 0.45a5.0 ± 0.00b0.78 ± 0.01a1.14 ± 0.01b18 ± 0.72a32 ± 0.65b

Data are written as the means ± standard deviation of the three replicate tests. The data were statistically analyzed using Tukey’s honest significant difference (HSD). The significant difference (p ≤ 0.05) within each column is indicated by a different letter.

The fecal weight and the number of droppings in both groups increased from week 1 to 3 (Table 4). Notably, a significant increase in the fecal droppings was observed in the treatment group after 2 weeks. Importantly, the probiotic administration did not cause diarrhea in mice, as their feces were neither loose, watery, or bloody (data not shown). Each mouse typically produces between 30 and 50 droppings daily [49]. In our study, the control group exhibited a lower number of droppings than this typical range, which may be attributed to several factors, including diet composition, gut microbiota composition, water intake, experimental conditions, and strain-specific variations. Despite the lower-than-typical dropping count in the control group, the number of droppings in the control group remained relatively stable through-out the study. The significant increase observed in the treatment group after the probiotic supplementation suggests that BS2-PB3 can improve the mice’s digestive system function, albeit within what appears to be a modified baseline for our specific experimental conditions. These results highlight the importance of considering study-specific factors when interpreting the fecal output data. Future investigations should aim to elucidate the exact causes of the lower baseline fecal count observed in our control group potentially through a more detailed monitoring of the dietary intake, gut transit time, and microbiome composition.

Conclusion

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In this study, B. breve BS2-PB3 was successfully encapsulated with two matrix combinations, that is, 2%alginate and 3% pectin, which were both coated with 0.75% chitosan. The EY and the survival rate after freeze drying exceeded 90% for both the alginate-chitosan and pectin-chitosan matrices. Encapsulation with both matrices and freeze drying improved the shelf life of BS2-PB3 over a 21-day storage period, particularly at a lower temperature. Both matrices maintained the BS2-PB3 viability above 55% and 66% when exposed to SGJ and bile salts, respectively. Additionally, the supplementation of the BS2-PB3-containing microcapsules improved the digestive system of the BALB/c mice.

Acknowledgments

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This work was funded by the Centre of Research and Community Development Universitas Pelita Harapan (039/LPPM-UPH/I/2023).

Conflict of Interest

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The authors have no financial conflicts of interest to declare.

References

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