Microencapsulation of Lactobacillus acidophilus Using partially hydrolysed guar gum: Bead characterization and survivability under simulated gastrointestinal and thermal stress conditions

Introduction

The incorporation of probiotic microorganisms into functional foods has attracted increasing scientific, clinical, and industrial interest owing to their established roles in promoting gastrointestinal health, enhancing immune competence, and modulating host metabolic and inflammatory responses (Latif et al., 2023) [6] ; Petrariu et al., 2024 [12] ; Tukaram et al., 2025 [20] . Probiotics exert their beneficial effects through multiple mechanisms, including competitive exclusion of pathogenic microorganisms, modulation of gut microbiota composition, production of antimicrobial metabolites, enhancement of intestinal barrier integrity, and regulation of host immune signaling pathways (Huang et al., 2022) [5] ; Zhao et al., 2020 [23] ; Duhan et al., 2025 [3] . Among the diverse probiotic strains investigated to date, Lactobacillus acidophilus remains one of the most extensively studied and commercially relevant lactic acid bacteria, widely employed in fermented dairy products and nutraceutical formulations due to its documented safety, functional efficacy, International Journal of Agriculture and Food Science https://www.agriculturaljournals.com and physiological compatibility with the human gastrointestinal tract (Liu et al., 2024) [8] ; Zhu et al., 2025 [24] . Lactobacillus acidophilus is particularly valued for its ability to adhere to intestinal epithelial cells, suppress enteric pathogens, improve lactose digestion through βgalactosidase activity, and contribute to the maintenance of intestinal homeostasis (Huang et al., 2022 [5] ; Zhao et al., 2020) [23] ; Liu et al., 2024 [8] . Despite these well-recognized health benefits, the successful delivery of viable L. acidophilus cells through food matrices to the site of action remains a major technological challenge (Rodrigues et al., 2020) [15] ; Sbehat et al., 2022 [17] . The organism exhibits pronounced sensitivity to environmental stresses encountered during food processing, storage, and gastrointestinal transit, resulting in substantial viability losses prior to consumption or intestinal colonization (Bustos et al., 2025 [2] ; Vivek et al., 2023) [21] . Consequently, ensuring probiotic stability throughout the product lifecycle is a critical prerequisite for the development of efficacious probiotic and synbiotic foods (Wang et al., 2022) [22] ; Sin et al., 2025 [18] . The functional efficacy of probiotic foods is strongly dependent on the delivery of an adequate number of viable cells, generally accepted to be in the range of ≥10 7 -10 8 CFU per gram or millilitre, at the time of consumption and, more importantly, upon arrival in the intestine (Naissinger da Silva et al., 2020 [11] ; Solanki et al., 2015) [19] . During manufacturing and storage, probiotic cells are exposed to a multitude of stressors, including oxygen exposure, moisture fluctuations, osmotic stress, acidic environments, and thermal treatments associated with food processing (Mansouripour et al., 2013 [9] ; Bustos et al., 2025) [2] . Following ingestion, these stresses are further compounded by exposure to the highly acidic gastric environment (pH 1.5-3.0), digestive enzymes such as pepsin, bile salts, and intestinal peristalsis (Naissinger da Silva et al., 2020) [11] ; Moghanjougi et al., 2021 [10] .

Collectively, these factors can result in severe reductions in probiotic viability, thereby diminishing or nullifying the intended health benefits of probiotic-enriched products (Sbehat et al., 2022 [17] ; Vivek et al., 2023) [21] . Microencapsulation has emerged as an effective and versatile technological approach to enhance probiotic stability and to facilitate targeted delivery to the intestine (Rodrigues et al., 2020) [15] ; Wang et al., 2022 [22] . This technique involves entrapping probiotic cells within protective biopolymeric matrices, forming discrete microbeads or microcapsules that function as physical and biochemical barriers against environmental stresses (Moghanjougi et al., 2021) [10] ; Sbehat et al., 2022 [17] .

Encapsulation not only improves resistance to acidic and bile environments but also allows controlled release of viable cells in the intestinal tract (Vivek et al., 2023) [21] ; Sin et al., 2025 [18] . A wide range of encapsulating materials, including polysaccharides, proteins, lipids, and composite systems, have been investigated for probiotic protection (Rodrigues et al., 2020 [15] ; Wang et al., 2022) [22] . Among the critical parameters influencing encapsulation performance, bead size, matrix composition, encapsulation efficiency, and structural integrity play decisive roles in determining the protective capacity, mechanical stability, and release kinetics of encapsulated probiotics (Moghanjougi et al., 2021) [10] ; Vivek et al., 2023 [21] .

In recent years, increasing attention has been directed toward the incorporation of prebiotic compounds into encapsulation matrices to create synbiotic delivery systems that combine probiotic protection with selective stimulation of beneficial gut microbiota (Salaria et al., 2013) [16] ; Ramírez-Damián et al., 2025 [13] . Partially hydrolyzed guar gum (PHGG), a water-soluble dietary fiber derived from guar gum, has gained prominence due to its low viscosity, high fermentability, and excellent gastrointestinal tolerance (Al-Asmari et al., 2025) [1] ; Reider et al., 2020 [14] . PHGG selectively promotes the growth and metabolic activity of beneficial microorganisms, Particularly Lactobacillus and Bifidobacterium species, leading to enhanced production of short-chain fatty acids and improved intestinal function (Liu et al., 2022) [7] ; Reider et al., 2020 [14] . Beyond its prebiotic effects, PHGG exhibits favourable physicochemical characteristics, including thermal stability, resistance to acidic conditions, and compatibility with a wide range of food matrices, making it a promising functional component for probiotic microencapsulation systems (Al-Asmari et al., 2025 [1] ; Sin et al., 2025) [18] . The integration of PHGG into probiotic encapsulation matrices may therefore offer a dual functional advantage: enhanced physical protection of probiotic cells during processing and gastrointestinal transit, and synbiotic stimulation of probiotic growth and activity upon release in the intestine (Salaria et al., 2013) [16] ; Ramírez-Damián et al., 2025 [13] . Although previous studies have reported improved probiotic survivability through the incorporation of various prebiotics into encapsulation systems, systematic investigations specifically evaluating the influence of PHGG on microbead characteristics, particularly bead size distribution, and its protective effects under simulated gastric, gastrointestinal, and thermal stress conditions remain limited (Moghanjougi et al., 2021) [10] ; Sbehat et al., 2022 [17] . Furthermore, probiotic responses to encapsulation are highly strain-specific, underscoring the need for targeted evaluation of technologically and clinically important strains such as Lactobacillus acidophilus NCDC-15 (Bustos et al., 2025) [2] ; Liu et al., 2024 [8] . Accordingly, the present study was undertaken to develop a PHGG-assisted microencapsulation system For Lactobacillus acidophilus NCDC-15 and to omprehensively assess its protective efficacy under conditions relevant to food processing and gastrointestinal transit. The specific objectives of the study were to (i) prepare and microencapsulate L. acidophilus NCDC-15 using PHGGbased matrices, (ii) estimate and characterize microbead size, (iii) evaluate survivability under simulated gastric and gastrointestinal conditions, (iv) and assess thermal tolerance of encapsulated cells establish the significance of observed effects. The outcomes of this investigation are expected to contribute valuable insights into the design of synbiotic encapsulation systems with enhanced probiotic stability, supporting their application in functional foods and nutritionally sensitive formulations, including infant and medical nutrition products (Solanki et al., 2015) [19] ; Wang et al., 2022 [22] .

Materials and methods

Selection of Probiotic Organism

A probiotic strain of Lactobacillus acidophilus NCDC-15 was selected for the present study due to its established probiotic functionality, gastrointestinal relevance, and frequent application in dairy-based functional foods. The culture was procured from the National Collection of Dairy Cultures (NCDC), ICAR-National Dairy Research Institute (NDRI), Karnal, India, in freeze-dried form. Upon receipt, the culture was stored under refrigerated conditions (4±1 °C) until further use.

Activation and Preparation of Probiotic Culture

The L. acidophilus NCDC-15 culture was activated following a two-step procedure to ensure optimal metabolic activity and cell viability prior to encapsulation (Moghanjougi et al., 2021) [10] ; Vivek et al., 2023 [21] . Initially, the culture was inoculated into chalk litmus milk and incubated at 37±1 °C for 18-24 h to facilitate regeneration and confirmation of acid-producing activity (Salaria et al., 2013) [16] ; Solanki et al., 2015 [19] . Following visible coagulation and acid development, the activated culture was aseptically transferred (1%, v/v) into de Man, Rogosa and Sharpe (MRS) broth and incubated at 37±1 °C for 18-24 h under anaerobic conditions (Liu et al., 2024) [8] ; Ramírez-Damián et al., 2025 [13] . This secondary activation step ensured attainment of the logarithmic growth phase, which is considered optimal for microencapsulation (Rodrigues et al., 2020) [15] ; Vivek et al., 2023 [21] . The activated culture was subsequently harvested by centrifugation at 5,000 × g for 10 min at 4 °C (Moghanjougi et al., 2021) [10] . The resulting cell pellet was washed twice with sterile phosphate-buffered saline (PBS, pH 7.0) to remove residual growth medium and resuspended in sterile PBS to obtain a concentrated probiotic suspension for encapsulation (Naissinger da Silva et al., 2020 [11] ; Sbehat et al., 2022) [17] .

Microencapsulation of Probiotic Organism Preparation of Encapsulation Matrix

The encapsulation matrix was prepared using food-grade biopolymers as mentioned in Table 1. Sodium alginate (3%, w/v) served as the primary gelling agent, corn starch (2%, w/v) was incorporated to enhance structural rigidity and matrix density, and partially hydrolyzed guar gum (PHGG) was added at two different concentrations (3% and 5%, w/v) to evaluate its effect on bead characteristics and probiotic survivability (Salaria et al., 2013; Al-Asmari et al., 2025) [1,16] ; Sin et al., 2025 [1,18] . All matrix components were dissolved in sterile distilled water with continuous stirring at ambient temperature until a homogeneous solution was obtained. The activated probiotic suspension was then aseptically mixed into the matrix solution to achieve uniform cell distribution.

Formation of Microcapsules

The activated Lactobacillus acidophilus NCDC-15 culture was harvested by centrifugation and the resulting cell pellet was resuspended in sterile phosphate-buffered saline to obtain a standardized cell concentration of approximately ≥10 8 -10 9 /g, as determined by plate count analysis (Moghanjougi et al., 2021 [10] ; Naissinger da Silva et al., 2020) [11] . This concentration range was selected to ensure sufficient probiotic loading within the microcapsules while maintaining matrix integrity and uniform bead formation (Rodrigues et al., 2020) [15] ; Vivek et al., 2023 [21] .

A calculated quantity of the standardized probiotic suspension was aseptically incorporated into a predetermined volume of the encapsulation matrix composed of sodium alginate, corn starch, and partially hydrolyzed guar gum (PHGG) at different concentration levels (Salaria et al., 2013) [16] ; Ramírez-Damián et al., 2025 [13] . The probiotic-polymer mixture was homogenized under constant gentle stirring for 15-20 min at ambient temperature to achieve uniform cell distribution and to minimize air entrapment, which could otherwise compromise bead integrity and encapsulation efficiency (Wang et al., 2022) [22] ; Sbehat et al., 2022 [17] .

The resulting probiotic-loaded polymer suspension was subsequently atomized through a pneumatic spray nozzle under constant operating pressure into a chilled (4±1 °C) 0.1 M calcium chloride solution. The extrusion-spray process facilitated immediate ionic crosslinking of sodium alginate with calcium ions, leading to the formation of spherical microcapsules (Moghanjougi et al., 2021) [10] ; Wang et al., 2022 [22] . Rapid gelation in the chilled calcium chloride bath minimized cell leakage and thermal or mechanical stress on the probiotic cells during bead formation (Rodrigues et al., 2020) [15] ; Vivek et al., 2023 [21] . The formed microcapsules were allowed to harden in the calcium chloride solution for 20 min to ensure complete gelation and structural stabilization of the encapsulation matrix (Sbehat et al., 2022) [17] . Following hardening, oversized or irregularly shaped beads were removed manually to obtain a uniform bead size distribution (Moghanjougi et al., 2021) [10] . The remaining microcapsules were collected by filtration using Whatman No. 1 filter paper, rinsed with sterile distilled water to remove residual calcium ions, and transferred into sterile containers (Rodrigues et al., 2020) [15] . The freshly prepared microcapsules were stored under refrigerated conditions (4±1 °C) until further analysis (Solanki et al., 2015) [19] .

Assessment of Survivability of Microencapsulated Probiotic Organism Survivability under Simulated Gastric pH Conditions

Simulated gastric environments were prepared using sterile buffer solutions adjusted to pH 1.0, 1.5, and 2.0 using 1 N HCl (Naissinger da Silva et al., 2020) [11] ; Moghanjougi et al., 2021 [10] . Microencapsulated probiotic beads were suspended in the respective solutions and incubated at 37±1 °C for a predetermined exposure period (Sbehat et al., 2022) [17] . At the end of incubation, samples were withdrawn, neutralized, and the beads were disintegrated using sodium citrate solution to release entrapped cells (Rodrigues et al., 2020) [15] . Viable counts were determined by the standard plate count method using MRS agar, followed by incubation at 37±1 °C for 48 h (Vivek et al., 2023) [21] .

Survivability under Simulated Intestinal Bile Salt Conditions

To simulate intestinal stress, bile salt solutions were prepared at concentrations of 1.0%, 1.5%, and 2.0% (w/v) (Naissinger da Silva et al., 2020 [11] ; Wang et al., 2022) [22] . Microencapsulated probiotic beads were exposed to these solutions at 37±1 °C (Moghanjougi et al., 2021) [10] . Following exposure, viable cell counts were determined using the plate count method after appropriate dilution and bead disintegration (Sbehat et al., 2022) [17] . The survivability of encapsulated cells was compared with nonencapsulated controls to quantify the protective effect of the encapsulation system (Vivek et al., 2023) [21] .

Survivability during Thermal Treatment

Thermal stability of microencapsulated L. acidophilus NCDC-15 was assessed to simulate processing conditions commonly encountered in dairy and functional food manufacturing (Mansouripour et al., 2013 [9] ; Rodrigues et al., 2020) [15] . Microcapsules were subjected to heat treatments at 75 °C for 30 s and 80 °C for 30 s using a thermostatically controlled water bath (Solanki et al., 2015) [19] ; Vivek et al., 2023 [21] . Immediately after heating, samples were cooled rapidly in an ice bath to arrest thermal damage (Mansouripour et al., 2013) [9] . Viable counts were determined as described previously and expressed as log CFU/g (Sbehat et al., 2022) [17] .

Statistical Analysis

All experiments were conducted in triplicate, and results were expressed as mean±standard deviation. The data obtained from survivability studies under simulated gastric pH, bile salt concentrations, and thermal treatments were subjected to appropriate statistical analysis to determine the significance of observed differences. Analysis of variance (ANOVA) was performed using suitable SPSS statistical software, and mean comparisons were carried out at a significance level of p<0.05.

Results and discussions Effect of PHGG on Survivability of Microencapsulated Probiotic Organism

The survivability of Lactobacillus acidophilus NCDC-15 under simulated gastrointestinal stress conditions was evaluated to elucidate the protective effect of partially hydrolyzed guar gum (PHGG) when incorporated into the microencapsulation matrix. Sodium alginate-starch microcapsules containing different levels of PHGG (3% and 5%) were compared with non-encapsulated control cells across a range of simulated gastric pH values (1.0, 1.5, and 2.0). The results demonstrate a clear pH-dependent decline in probiotic viability, with PHGG-containing microcapsules consistently exhibiting superior survivability relative to control cells.

Survivability of Lactobacillus acidophilus NCDC-15 in Simulated Gastric pH

Exposure to highly acidic gastric conditions represents one of the most critical barriers to probiotic survival following oral administration. As expected, a pronounced reduction in viable counts of L. acidophilus was observed with decreasing pH and increasing incubation time (Table 1). However, the magnitude of viability loss varied significantly depending on the presence and concentration of PHGG in the encapsulation matrix. At pH 1.0, the most severe condition evaluated, nonencapsulated control cells exhibited a rapid decline in viability, with counts decreasing from an initial value of 8.71 log CFU/g to 5.26, 5.11, and 4.49 log CFU/g after 1, 2, and 3 h of incubation, respectively. This sharp reduction reflects the extreme sensitivity of L. acidophilus to strong acid stress, which disrupts membrane integrity, denatures intracellular proteins, and impairs enzymatic activity. In contrast, microcapsules containing 3% and 5% PHGG showed significantly improved resistance to acidic stress, retaining 5.49 and 6.82 log CFU/g, respectively, even after 3 h of exposure. The higher PHGG concentration consistently conferred greater protection, indicating a dosedependent effect. A similar but less severe trend was observed at pH 1.5, where control cells showed a gradual reduction in viability from 8.71 log CFU/g to 5.62 log CFU/g after 3 h of incubation. In comparison, encapsulated cells containing 3% and 5% PHGG maintained substantially higher viable counts (6.86 and 7.10 log CFU/g, respectively) over the same exposure period. The improved survivability at pH 1.5 compared with pH 1.0 emphasizes the strong influence of gastric acidity on probiotic stability, while reinforcing the protective role of PHGG-based encapsulation. At pH 2.0, which more closely resembles postprandial gastric conditions, the decline in viability was comparatively moderate. Control cells showed a reduction from 8.71 to 6.10 log CFU/g after 3 h, whereas encapsulated cells containing 5% PHGG retained 7.30 log CFU/g. Notably, microcapsules with 5% PHGG demonstrated minimal loss in viability during the initial 1-2 h of exposure, suggesting that the encapsulation matrix effectively buffered acid diffusion and delayed proton penetration into the microcapsules. The enhanced protective effect of PHGG is further substantiated by percent survivability data (Table 2, Figure 1). At pH 1.0, only 60.39% of non-encapsulated cells survived after 1 h, decreasing to 51.55% after 3 h. In contrast, survivability of encapsulated cells containing 5% PHGG remained as high as 78.94% after 3 h, representing a substantial improvement over both the control and the 3% PHGG formulation. Similar trends were observed at pH 1.5 and 2.0, where PHGG-containing microcapsules consistently exhibited higher survivability across all incubation periods. The improved acid tolerance observed in PHGG-containing microcapsules can be attributed to multiple synergistic mechanisms such as the incorporation of PHGG likely increased matrix density and viscosity, thereby reducing acid diffusion into the microcapsules (Salaria et al., 2013) [16] ; Wang et al., 2022 [22] ; Sin et al., 2025 [18] . Moreover, PHGG may exert a buffering effect within the microcapsule microenvironment, moderating the internal pH of entrapped cells (Reider et al., 2020) [14] ; Liu et al., 2022 [7] . In addition, PHGG may act as a prebiotic fiber that interacts with the bacterial cell surface, contributing to stabilization of membrane structures, preservation of protein functionality, and enhancement of cellular stress tolerance under acidic conditions (Bustos et al., 2025 [2] ; Al-Asmari et al., 2025) [1] . The results clearly demonstrate that PHGG-assisted microencapsulation significantly enhances the survival of L. acidophilus NCDC-15 under simulated gastric conditions, with the 5% PHGG formulation showing the highest protective efficacy (Moghanjougi et al., 2021 [10] ; Vivek et al., 2023) [21] . Importantly, viable counts remained above the minimum therapeutic threshold (≥10 6 -10 7 CFU/g) even after prolonged exposure to acidic environments, indicating the potential of the encapsulation system for effective probiotic delivery and functional performance in acidic food matrices and gastrointestinal transit (Naissinger da Silva et al., 2020) [11] ; Solanki et al., 2015 [19] ; Sbehat et al., 2022 [17] . Values represent mean percent survivability calculated relative to initial viable counts (0 h). Different superscripts ( a-c ) within the same pH and incubation time indicate significant differences (p<0.05). Values represent mean percent survivability relative to initial counts.

Survival of Lactobacillus acidophilus NCDC-15 under Simulated Gastrointestinal Conditions

Survival in the intestinal environment is a critical determinant of probiotic efficacy, as bile salts exert strong detergent-like effects on bacterial cell membranes, leading to membrane destabilization, leakage of intracellular constituents, and subsequent loss of cell viability. The survivability of L. acidophilus, expressed as log CFU/ml, is provided in Table 3. Across all bile salt concentrations, a pronounced time-and concentration-dependent decline in viable counts was observed. However, the magnitude of viability loss differed markedly among treatments, with PHGG-containing microcapsules consistently exhibiting superior survivability compared with the control.

At 1.0% bile salt, the viable count of control cells decreased significantly (p<0.05) from an initial value of 8.71 log CFU/ml to 5.44 log CFU/ml after 3 h of incubation. In contrast, microcapsules containing 3% and 5% PHGG retained significantly higher counts of 6.10 and 6.46 log CFU/ml, respectively, at the same exposure time. Statistical analysis revealed significant differences among treatments after 1, 2, and 3 h of incubation, as indicated by elevated Fvalues and distinct superscripts, confirming the protective effect of PHGG against bile-induced stress. A similar trend was observed at 1.5% bile salt, where bile toxicity was more pronounced. After 3 h of incubation, control cells showed a drastic reduction in viability to 4.62 log CFU/ml, whereas microencapsulated cells containing 3% and 5% PHGG maintained significantly higher counts of

International Journal of Agriculture and Food Science https://www.agriculturaljournals.com 5.86 and 6.10 log CFU/ml, respectively. At the highest bile salt concentration (2.0%), representing severe intestinal stress conditions, probiotic survivability declined substantially in all treatments; nevertheless, PHGGcontaining microcapsules continued to confer significant protection. After 3 h of exposure, viable counts in the control declined to 4.36 log CFU/ml, whereas 3% and 5% PHGG microcapsules retained 4.67 and 5.74 log CFU/ml, respectively. The consistently higher survivability observed at 5% PHGG across all incubation periods indicates a clear concentration-dependent protective effect of PHGG under extreme bile stress. No significant differences were observed among treatments at 0 h for any bile salt concentration, confirming uniform initial probiotic loading. However, with increasing incubation time, significant differences (p<0.05) emerged between control and PHGG-containing microcapsules, particularly at higher bile salt concentrations. In some cases, differences between control and 3% PHGG were not significant during early incubation periods at lower bile concentrations, suggesting that higher PHGG levels are required to impart optimal protection under more severe bile concentrations. The enhanced bile tolerance observed in PHGG-containing microcapsules can be attributed to multiple synergistic mechanisms. Incorporation of PHGG likely increased the density and compactness of the encapsulation matrix, thereby limiting bile salt diffusion into the microcapsule core (Salaria et al., 2013) [16] ; Wang et al., 2022 [22] ; Sin et al., 2025 [18] . Additionally, PHGG may interact with bile salts, reducing their effective detergent activity within the microenvironment surrounding the encapsulated cells (Reider et al., 2020 [14] ; Liu et al., 2022) [7] . As a fermentable prebiotic fiber, PHGG may also contribute to stabilization of bacterial cell membranes and enhancement of stress resistance through modulation of cellular physiology and energy metabolism, collectively improving probiotic survivability under bile stress conditions (Bustos et al., 2025) [2] ; Al-Asmari et al., 2025 [1] ; Vivek et al., 2023 [21] . Heating is a common unit operation in the manufacture of several dairy products, including milk powders, and represents a major challenge to the survival of probiotic microorganisms (Mansouripour et al., 2013) [9] ; Rodrigues et al., 2020 [15] . High temperatures can induce irreversible damage to bacterial cell membranes, denature intracellular proteins, disrupt enzymatic systems, and impair metabolic activity, leading to substantial losses in probiotic viability (Bustos et al., 2025 [2] ; Vivek et al., 2023) [21] . To mitigate these adverse effects, microencapsulation has been proposed as an effective strategy to enhance the thermal stability of probiotic cultures during processing by providing a physical barrier against heat transfer and dehydration stress (Mansouripour et al., 2013) [9] ; Wang et al., 2022 [22] . Thermal treatment resulted in a marked reduction in viable counts across all treatments; however, the magnitude of reduction varied significantly depending on the presence and concentration of PHGG in the encapsulation matrix, indicating a concentration-dependent protective effect of PHGG-assisted microencapsulation under heat stress conditions (Salaria et al., 2013) [16] ; Solanki et al., 2015 [19] ; Vivek et al., 2023 [21] . In control samples, the initial viable count of 8.71 log CFU/ml declined sharply to 4.22 and 4.14 log CFU/ml following exposure to 75 °C and 80 °C, respectively, indicating high sensitivity of free L. acidophilus cells to heat shock. In contrast, microencapsulated cells exhibited significantly higher thermal tolerance. Capsules containing 3% PHGG retained 5.26 and 5.07 log CFU/ml after treatment at 75 °C and 80 °C, respectively, whereas capsules containing 5% PHGG showed the highest protection, with viable counts of 6.20 and 5.62 log CFU/ml under the same conditions (Table 4). At 75 °C, survivability of control cells was limited to 48.45%, while capsules containing 3% and 5% PHGG exhibited survivability of 61.60% and 71.77%, respectively. A similar trend was observed at 80 °C, where PHGG-based microcapsules consistently maintained higher survivability compared with the control. These results clearly demonstrate a concentration-dependent protective effect of PHGG against thermal stress, with the 5% PHGG formulation providing the most effective protection. Statistical analysis of the thermal survivability data (Table 4) revealed no significant differences among treatments at the initial stage, confirming uniform initial probiotic loading. However, following thermal exposure, highly significant differences (p<0.05) were observed between control and PHGG-containing microcapsules at both 75 °C and 80 °C. The improved thermal stability of L. acidophilus observed in PHGG-containing microcapsules can be attributed to several complementary mechanisms such as the encapsulation matrix acting as a physical barrier, reducing direct heat transfer to the entrapped cells and thereby moderating the rate of temperature increase within the microcapsule core (Mansouripour et al., 2013) [9] ; Rodrigues et al., 2020 [15] ; Wang et al., 2022 [22] . In addition, the incorporation of PHGG likely enhances matrix cohesiveness and moisture retention, thereby reducing dehydration-induced cellular damage during heat exposure (Salaria et al., 2013) [16] ; Solanki et al., 2015 [19] ; Vivek et al., 2023 [21] . Moreover, PHGG may stabilize bacterial cell membranes and intracellular proteins by creating a protective microenvironment that limits thermal denaturation and oxidative stress, collectively contributing to improved probiotic survival under thermal processing conditions (Bustos et al., 2025) [2] ; Al-Asmari et al., 2025 [1] .

Conclusion

The present study demonstrates that microencapsulation using a sodium alginate-corn starch matrix supplemented with partially hydrolysed guar gum is an effective strategy for enhancing the stability of Lactobacillus acidophilus NCDC-15 under conditions relevant to food processing and gastrointestinal transit. Incorporation ofpartially hydrolysed guar gum significantly improved probiotic survivability against severe gastric acidity, bile salt stress, and thermal treatments, with the 5% partially hydrolysed guar gum formulation consistently providing superior protection in a concentration-dependent manner. Importantly, encapsulated probiotic cells maintained viable counts above the recommended therapeutic threshold, indicating their potential to exert functional health benefits upon consumption. The combined protective and prebiotic functionality of partially hydrolysed guar gum highlights the value of this synbiotic microencapsulation system for improving probiotic delivery and efficacy. Overall, the findings support the application of partially hydrolysed guar gum-based microencapsulation in the development of stable probiotic and synbiotic foods, nutraceuticals, and specialized products such as infant and medical nutrition formulations, where probiotic viability is critical to product performance.

References

1. Al-Asmari F, Abdelshafy AM, Neetoo H, Zhang Y. Natural gums (gum Arabic, guar gum and xanthan gum) as a promising source of prebiotics: A review on their functional roles and food applications. International Journal of Biological Macromolecules. 2025;318:145101. doi:10.1016/j.ijbiomac.2025.145101.
2. Bustos AY, Taranto MP, Gerez CL, Agriopoulou S, Smaoui S, Varzakas T, et al. Recent advances in the understanding of stress resistance mechanisms in probiotics: Relevance for the design of functional food systems. Probiotics and Antimicrobial Proteins. 2025;17(1):138-58. doi:10.1007/s12602-024-10273-9.
3. Duhan P, Gupta B, Ahmed M, Bansal P. Gut microbiome engineering with probiotics: current trends and future directions. Discover Applied Sciences. 2025;7(11):1354. doi:10.1007/s42452-025-07622-w.
4. G. L, Elena M. Synbiotic microencapsulation of Lactobacillus strains from Mexican fermented beverages for enhanced probiotic functionality. Molecules. 2024;30(5):1185. doi:10.3390/molecules30051185.
5. Huang R, Wu F, Zhou Q, Wei W, Yue J, Xiao B, et al. Lactobacillus and intestinal diseases: Mechanisms of action and clinical applications. Microbiological Research. 2022;260:127019. doi:10.1016/j.micres.2022.127019.
6. Latif A, Shehzad A, Niazi S, Zahid A, Ashraf W, Iqbal MW, et al. Probiotics: Mechanism of action, health benefits and their application in food industries. Frontiers in Microbiology. 2023;14:1216674. doi:10.3389/fmicb.2023.1216674.
7. Liu T, Hu X, Chen P, Zhang R, Zhang S, Chang W, et al. Effect of partially hydrolyzed guar gum on the composition and metabolic function of the intestinal flora of healthy mice. Journal of Food Biochemistry. 2022;46(12):e14508. doi:10.1111/jfbc.14508.
8. Liu Y, Nawazish H, Farid MS, Abdul Qadoos K, Habiba UE, Muzamil M, et al. Health-promoting effects of Lactobacillus acidophilus and its technological applications in fermented food products and beverages. Fermentation. 2024;10(8):380. doi:10.3390/fermentation10080380.
9. Mansouripour S, Esfandiari Z, Nateghi L. The effect of heat process on the survival and increased viability of probiotic by microencapsulation: A review. Annals of Biological Research. 2013;4(4):83-87.
10. Moghanjougi ZM, Bari MR, Khaledabad MA, Amiri S, Almasi H. Microencapsulation of Lactobacillus acidophilus LA-5 and Bifidobacterium animalis BB-12 in pectin and sodium alginate: A comparative study on viability, stability, and structure. Food Science and Nutrition. 2021;9(9):5103-11. doi:10.1002/fsn3.2470.
11. Naissinger da Silva M, Tagliapietra BL, Flores VDA, Pereira dos Santos Richards NS. In vitro test to evaluate survival in the gastrointestinal tract of commercial probiotics. Current Research in Food Science. 2020;4:320-5. doi:10.1016/j.crfs.2021.04.006.
12. Petrariu O, Barbu IC, Niculescu A, Constantin M, Grigore GA, Cristian R, et al. Role of probiotics in managing various human diseases, from oral pathology to cancer and gastrointestinal diseases. Frontiers in Microbiology. 2024;14:1296447. doi:10.3389/fmicb.2023.1296447.
13. Ramírez-Damián M, Garfias-Noguez C, BermúdezHumarán LG, Sánchez-Pardo ME. Synbiotic microencapsulation of Lactobacillus strains from Mexican fermented beverages for enhanced probiotic International Journal of Agriculture and Food Science https://www.agriculturaljournals.com functionality. Molecules. 2025;30(5):1185. doi:10.3390/molecules30051185.
14. Reider SJ, Moosmang S, Tragust J, Trgovec-Greif L, Tragust S, Perschy L, et al. Prebiotic effects of partially hydrolyzed guar gum on the composition and function of the human microbiota -results from the PAGODA trial. Nutrients. 2020;12(5):1257. doi:10.3390/nu12051257.
15. Rodrigues F, Cedran M, Bicas J, Sato H. Encapsulated probiotic cells: Relevant techniques, natural sources as encapsulating materials and food applications -A narrative review. Food Research International. 2020;137:109682. doi:10.1016/j.foodres.2020.109682.
16. Salaria A, Thompkinson DK, Kumar MH, Sabikhi L. Prebiotics in the microencapsulating matrix enhance the viability of probiotic Lactobacillus acidophilus LA1. International Journal of Fermented Foods. 2013;2(1):33-45.
17. Sbehat M, Mauriello G, Altamimi M. Microencapsulation of probiotics for food functionalization: an update on literature reviews. Microorganisms. 2022;10(10):1948. doi:10.3390/microorganisms10101948.
18. Sin PY, Tan SH, Farida Asras MF, Lee CM, Lee TC, Karim MR, et al. Exploring dual-coating strategies for probiotic microencapsulation using polysaccharide and protein systems. Discover Food. 2025;5(1):235. doi:10.1007/s44187-025-00540-1.
19. Solanki AK, Parmar MP, Kadiya KS, Thompkinson DK, Patel KS. Physicochemical and microbiological properties of probiotic microencapsulated enriched infant formula. Indian Journal of Dairy Science. 2015;68(2):144-147.
20. Tukaram KS, Sopanrao SR, Kacharu LO. Unveiling The Therapeutic Potential of Probiotics: A Review. Journal of Future Foods. 2025. doi:10.1016/j.jfutfo.2025.09.011.
21. Vivek K, Mishra S, Pradhan RC, Nagarajan M, Kumar PK, Singh SS, et al. A comprehensive review on microencapsulation of probiotics: Technology, carriers and current trends. Applied Food Research. 2023;3(1):100248. doi:10.1016/j.afres.2022.100248.
22. Wang X, Gao S, Yun S, Zhang M, Peng L, Li Y, et al. Microencapsulating alginate-based polymers for probiotics delivery systems and their application. Pharmaceuticals. 2022;15(5):644. doi:10.3390/ph15050644.
23. Zhao W, Liu Y, Kwok L, Cai T, Zhang W. The immune regulatory role of Lactobacillus acidophilus: An updated meta-analysis of randomized controlled trials. Food Bioscience. 2020;36:100656. doi:10.1016/j.fbio.2020.100656.
24. Zhu J, Sun Y, Dong Y, Zhao Y, Gai Z, Fang S. Efficacy and safety of lactobacillus acidophilus LA85 in preventing antibiotic associated diarrhea: a randomized, placebo controlled study. Food Science and Nutrition. 2025;13(6):e70490. doi:10.1002/fsn3.70490.