Enhanced Storage Stability and Gastrointestinal Survival of Lactiplantibacillus plantarum by Sodium Alginate–Resistant Starch–EGCG Composite Encapsulation

ABSTRACT

The development of functional foods containing probiotics requires strategies to maintain cell viability during processing, storage, and gastrointestinal (GI) transit. In this study, Lactiplantibacillus plantarum (formerly Lactobacillus plantarum; LP) was encapsulated in sodium alginate (SA)–resistant starch (RS)–epigallocatechin gallate (EGCG) matrices using extrusion to enhance stability and delivery. Microcapsules were characterized for encapsulation efficiency (EE); process yield (PY); morphology, texture, and physicochemical properties; and structural features were examined by scanning electron microscopy (SEM), Fourier‐transform infrared spectroscopy (FTIR), and x‐ray diffraction (XRD). Storage stability was assessed for 90 days at 4°C and 25°C, and probiotic survival was evaluated under simulated GI conditions. Encapsulation produced high EE (up to approximately 97%) and PY (96% on average), and the presence of RS and EGCG enhanced the structural quality of the encapsulation matrix. SEM confirmed a compact and uniform surface in RS‐ and EGCG‐based capsules, while FTIR and XRD indicated hydrogen bonding and reduced crystallinity. During storage, the SA‐RS‐EGCG matrix maintained > 8.0 log colony‐forming unit (CFU)/g of LP at 4°C after 90 days, whereas free (unencapsulated) cells declined to < 3.5 log CFU/g. Under simulated digestion, free cells rapidly decreased, while SA‐RS‐EGCG retained approximately 85%–90% LP survival in gastric fluid and showed sustained release in intestinal fluid. These findings demonstrate that SA encapsulation provides an effective protective barrier for LP, whereas the incorporation of RS and EGCG further improves matrix structure, probiotic stability, and controlled release through complementary effects, highlighting the potential of this tri‐component delivery system for functional food applications.

Practical Applications

The composite encapsulation system developed in this study offers a practical approach to enhancing the stability of probiotic bacteria in functional foods. Using sodium alginate, resistant starch, and EGCG polyphenol together enhances both shelf life and survival during digestion, supporting the development of more effective synbiotic products for the food industry.

Abbreviations

CFU

colony‐forming unit

EE

encapsulation efficiency

PY

process yield

EGCG

epigallocatechin gallate

GI

gastrointestinal

LP

Lactiplantibacillus plantarum

PBS

phosphate‐buffered saline

RS

resistant starch

SA

sodium alginate

SEM

scanning electron microscopy

SGF

simulated gastric fluid

SIF

simulated intestinal fluid

SD

standard deviation

XRD

x‐ray diffraction

FTIR

Fourier‐transform infrared spectroscopy

1. Introduction

The global demand for functional foods has increased rapidly due to consumer awareness of the relationship between diet and health. Among the most widely studied bioactive components are probiotics, defined as live microorganisms that, when administered in adequate amounts, confer health benefits on the host (FAO/WHO ²⁰⁰²). Commonly used probiotic strains include Lactobacillus and Bifidobacterium species, which have been linked to improved gastrointestinal (GI) health, immune modulation, and metabolic regulation (Granato et al. 2010; Hill et al. ²⁰¹⁴). For probiotics to exert these effects, however, they must remain viable during processing, storage, and GI passage, and typically require a minimum of 10⁶ colony‐forming unit (CFU)/g at the time of consumption (Kechagia et al. 2013).

The survival of free probiotic cells in food matrices and during GI transit is often limited by various environmental stressors. During food processing and storage, probiotic viability may be adversely affected by oxygen exposure, moisture, and temperature conditions. In the GI tract, probiotics are further exposed to harsh physiological environments, including acidic gastric conditions and bile salts in the intestine, which can significantly reduce cell viability. Microencapsulation has emerged as an effective strategy to address these challenges by providing a protective physical barrier that enhances probiotic stability against both storage‐ and GI‐related stressors. Sodium alginate (SA), a natural polysaccharide derived from brown seaweed, is the most widely used encapsulation material due to its biocompatibility, low cost, and ability to form hydrogels in the presence of calcium ions (J. Zhang et al. ²⁰²⁴). However, alginate beads can be porous and fragile, which can compromise effectiveness under prolonged storage or acidic conditions, such as gastric pH (Huang et al. 2023; L. Wu et al. ²⁰²⁴).

To address these limitations, composite wall materials have been explored. Resistant starch (RS), a nondigestible carbohydrate, not only improves encapsulation properties by reinforcing bead structure but also functions as a prebiotic substrate that promotes probiotic growth (Etchepare et al. 2016; Muhammad et al. ²⁰²¹). RS is expected to function primarily as a physical filler within the alginate matrix, potentially contributing to increased matrix compactness and improved barrier properties through matrix filling and water‐binding effects rather than direct ionic cross‐linking. In addition, RS may provide prebiotic functionality that could further support probiotic survival. Polyphenols such as epigallocatechin gallate (EGCG), the major catechin in green tea, have also been incorporated into encapsulation systems. EGCG exhibits strong antioxidant activity and may interact with polysaccharides through hydrogen bonding and hydrophobic interactions, thereby enhancing gel strength and protecting encapsulated probiotics from oxidative and environmental stress (Pourjavid et al. 2022; Xin et al. ²⁰²⁵).

Several studies have investigated encapsulation strategies combining alginate with starches, proteins, or polyphenols. Etchepare et al. (2016) demonstrated that RS–alginate systems improved the survival of Lactobacillus acidophilus under acidic conditions, while Gheorghita et al. (2024) reported that alginate–starch capsules improved the viability of encapsulated probiotics in simulated GI fluids. Recent research on synbiotic encapsulation has highlighted the potential of multicomponent delivery systems combining probiotics with prebiotics or polyphenols (Lim et al. 2024; Wang and Mutukumira ²⁰²²). However, previous studies have mainly focused on alginate‐based encapsulation systems incorporating either RS or polyphenols individually, while relatively few have investigated the simultaneous incorporation of RS and EGCG within alginate‐based matrices (Etchepare et al. 2016; Qin et al. ²⁰²¹; Xin et al. ²⁰²⁵), particularly for the encapsulation of Lactiplantibacillus plantarum (LP). Therefore, the combined use of SA, RS, and EGCG within a single encapsulation system remains insufficiently explored. More importantly, the potential complementary effects of these components on microcapsule structural integrity, storage stability, and GI performance have not been systematically investigated.

The present study aimed to address this gap by developing microcapsules containing LP using SA‐RS matrices with EGCG co‐encapsulation. The objective was to evaluate whether the incorporation of RS and EGCG into SA‐based microcapsules could improve the encapsulation performance of probiotics, particularly in terms of storage stability and GI tolerance of LP. In this system, SA served as the primary encapsulating material by forming a calcium‐crosslinked hydrogel network, while RS was expected to reinforce the capsule structure and provide potential prebiotic functionality through matrix filling and water‐binding effects rather than direct ionic cross‐linking. EGCG was incorporated as a bioactive compound with potential antioxidant and protective functions. Although the encapsulation process itself provides a physical barrier for probiotic protection, the combination of SA, RS, and EGCG may provide complementary functionalities that contribute to improved microcapsule stability and probiotic survival. Therefore, this tri‐component delivery system was hypothesized to exhibit enhanced protective performance compared with single‐ or bi‐component formulations.

2. Materials and Methods

2.1. Materials

SA was obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). RS (Hi‐Maize 260) was purchased from Ingredion Singapore Pte Ltd. EGCG (purity 97.6%) was supplied by Zhejiang Delekang Food Co., Ltd. (Zhejiang, China). Anhydrous calcium chloride was obtained from Tianjin Tianli Chemical Reagent Co., Ltd. (Tianjin, China). Phosphate‐buffered saline (PBS; pH 7.4) was purchased from Wuhan Sewell Biotechnology Co., Ltd. (Wuhan, China), and 0.85% (w/v) physiological saline (NaCl) was obtained from Hunan Kelun Pharmaceutical Co., Ltd. (Hunan, China). De Man, Rogosa, and Sharpe (MRS) agar and broth were purchased from Beijing Aoboxing Biotechnology Co., Ltd. (Beijing, China). Pepsin (from porcine stomach mucosa, activity 1:3000; optimal pH 2–4), pancreatin (from porcine pancreas, ≥ 4000 U/g trypsin activity, ≥ 7000 U/g amylase activity, and ≥ 4000 U/g lipase activity), and porcine bile salts (bile acid content ≥ 60%) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Lactiplantibacillus plantarum strain LP‐36 was obtained from Hangzhou Bairui Biotechnology Co., Ltd. (Hangzhou, China).

2.2. Activation and Preparation of Bacterial Suspension

A total of 0.1 g of freeze‐dried LP powder was dissolved in 10 mL of sterile physiological saline (0.85% NaCl). Following dissolution, 0.5 mL of the suspension was inoculated into 50 mL of MRS broth and incubated at 37°C for 24–48 h. The culture was then streaked onto MRS agar plates and incubated at 37°C for 24–48 h to obtain isolated colonies. A single well‐isolated colony was selected to ensure strain purity before encapsulation and subsequently inoculated into MRS broth and cultivated at 37°C for 16 h to obtain an active culture. The culture was centrifuged at 4000 rpm (1790 × g, Cence LP500‐A, rotor radius 10 cm) for 10 min at 4°C, and the pellet was washed twice with sterile physiological saline before being resuspended in 20 mL of sterile saline. The activation procedure was performed according to previously reported methods for LP (Chun et al. 2014; Ni et al. ²⁰²³) with minor modifications, including an additional agar plating purification step and optimization of cultivation parameters for subsequent encapsulation experiments. The resulting bacterial suspension contained approximately 9–10 log CFU/mL, as confirmed by plate counting on MRS agar (37°C, 48 h). This activated suspension was used for subsequent microencapsulation.

2.3. Preparation of LP Microcapsules

Microcapsules containing LP were prepared using the extrusion technique, adapted from Etchepare et al. (2016) and Pérez‐Ruiz et al. (2024), with minor modifications. The formulation composition, calcium chloride concentration, and gelation conditions were adjusted, and RS and EGCG were incorporated into the alginate matrix during encapsulation. The wall material solution was prepared by dissolving SA and RS at a mass ratio of 1:1 to obtain final concentrations of 2% (w/v) for each component. This ratio was selected based on previous studies and practical considerations related to encapsulation performance and processability. A concentrated bacterial suspension of LP (9–10 log CFU/mL) was then mixed with the wall material solution at a 1:2 (v/v) ratio and gently stirred for 15 min to ensure homogeneity. The resulting mixture was extruded dropwise through a syringe needle into 100 mL of 2.5% (w/v) CaCl₂ solution and allowed to solidify for 30 min. The formed microcapsules were subsequently filtered and washed twice with sterile 0.85% saline to remove residual CaCl₂ and surface‐associated bacteria, yielding wet microcapsules. Blank microcapsules (SA and SA‐RS without LP) were prepared using the same procedure but without adding the bacterial suspension. The slight difference in total volume between formulations was considered unlikely to substantially influence bead formation, as the bacterial suspension represented only a small fraction of the total system. Therefore, the blank microcapsules were considered appropriate control samples for physicochemical and textural analyses.

2.4. Preparation of Co‐Encapsulated Microcapsules

The preparation of co‐encapsulated microcapsules followed the same extrusion procedure described in Section  2.3 , except that a bacterial suspension (9–10 log CFU/mL), EGCG solution (0.1 mg/mL), and SA‐Hi‐Maize RS solution (2% w/v each, mixed 1:1) were combined at a 1:1:2 (v/v) ratio before extrusion. A corresponding blank SA‐RS‐EGCG sample (without LP) was also prepared using the same ratios to serve as a nonbacterial control. The absence of bacterial suspension resulted in a minor difference in total volume, which was not expected to substantially affect the overall formulation characteristics or bead formation behavior.

2.5. Encapsulation Properties

2.5.1. Encapsulation Efficiency

A total of 1.0 g of LP wet microcapsules was weighed, and 10 mL of PBS was added. The mixture was homogenized using a high‐speed homogenizer (10,000 rpm, 30 s) and subsequently incubated in a shaker at 37°C for 30 min. A 1 mL aliquot of the homogenized sample was then diluted with sterile saline, spread onto an MRS agar plate, and incubated under aerobic conditions at 37°C for 48 h for colony counting (Cai et al. 2014; Y. Wu and Zhang ²⁰¹⁸; Zou et al. ²⁰¹¹).

The encapsulation efficiency (EE) was calculated according to the method described by Y. Wu and Zhang (2018) as the percentage of viable cells recovered from the microcapsules after disruption relative to the initial cell suspension. All measurements were performed in triplicate. EE was calculated using the following equation:

$$\begin{array}{ccc}\hfill \mathrm{EE}\left(\%\right)& =& \left(\mathrm{Viable}\mathrm{cell}\mathrm{recovered}\mathrm{from}\mathrm{disrupted}\mathrm{microcapsules}/\right.\hfill \\ & & \left.\mathrm{Viable}\mathrm{cell}\mathrm{count}\mathrm{of}\mathrm{initial}\mathrm{bacterial}\mathrm{suspension}\right)\hfill \\ & & \times 100\hfill \end{array}$$

2.5.2. Process Yield

Process yield (PY) was determined according to the method described by Sun et al. (2023). The total dry solids used for microcapsule preparation were recorded prior to extrusion (W, g). After bead formation, the samples were prefrozen at −80°C and subsequently freeze‐dried for 24 h under vacuum conditions until constant weight was achieved. The recovered dry mass of microcapsules was measured (T, g). PY was expressed as the percentage of recovered microcapsule mass relative to the initial solids using the following equation:

$$\mathrm{PY}\left(\%\right)=\left(T/W\right)\times 100$$

Blank beads (SA, SA‐RS, SA‐RS‐EGCG) were freeze‐dried and weighed using the same procedure to ensure consistency among formulations. All measurements were performed in triplicate.

2.6. Bead Appearance and Particle Size

The visual appearance of the microcapsules was documented using a smartphone camera under standardized imaging conditions, including fixed distance, angle, and zoom settings for all samples. More than 50 randomly selected beads from each formulation were analyzed using Image J software after scale calibration. A reference object of known dimensions was included in each image to establish the pixel‐to‐length ratio, which was applied consistently during particle size analysis. Individual bead diameters were measured, and the mean particle size was calculated according to Chan et al. (2017), with the modification that macroscopic digital imaging was used instead of microscopic imaging. The analysis primarily focused on particle size determination, while bead appearance and overall shape were qualitatively evaluated based on visual observation.

2.7. Physicochemical Analysis

2.7.1. Water Activity, Moisture Content, and Hygroscopicity

Water activity (a _w) of freeze‐dried microcapsules was measured using a water activity meter (AquaLab Series 4TE, Decagon Devices, USA) at 25°C. About 1.0 g of the sample was equilibrated in the chamber until a stable reading was obtained.

Moisture content (%) was determined gravimetrically (AOAC 934.01). Approximately 2.0 g of sample was dried in a hot‐air oven at 105°C until it reached constant weight (two consecutive weights differing by < 0.001 g), and the percentage of moisture was calculated from the weight loss.

Hygroscopicity was determined following the procedure described by Fritzen‐Freire et al. (2012), with slight modification. Approximately 1 g of sample was placed in a desiccator containing a saturated NaCl solution (75% relative humidity, 25°C) until the sample weight stabilized (mass variation < 0.001 g between two successive weighings, typically within 5–7 days). Hygroscopicity was expressed as grams of water absorbed per 100 g of dry solids. All measurements were carried out in triplicate using independent batches, and results are expressed as mean ± standard deviation (SD). Significant differences were assessed by one‐way analysis of variance (ANOVA) (p < 0.05).

2.8. Texture Analysis

Texture profile analysis (TPA) of hydrogel microbeads was performed using a TA.XT Plus texture analyzer (Stable Micro Systems Ltd., UK), according to the method reported by Kong et al. (2025) with minor modifications. A P/0.5 cylindrical probe was selected for testing, and measurements were carried out in TPA mode. The trigger force was set at 5 g. The pretest, test, and posttest speeds were all maintained at 1.00 mm/s. Samples were subjected to a compression ratio of 50%, with a 5.00 s interval between the two compression cycles. All measurements were carried out in triplicate on independent batches of beads, and results are expressed as mean ± SD. All analyses were conducted at room temperature.

2.9. Scanning Electron Microscopy Analysis

The morphology of freeze‐dried microcapsules was observed according to the methods of Etchepare et al. (2016) using Phenom Pro scanning electron microscope. The freeze‐dried microcapsules were mounted on aluminum stubs using double‐sided conductive carbon tape and gently cleaned with N₂ to remove loose particles. The samples were sputter‐coated with gold prior to imaging. Surface morphology was examined using a Phenom Pro scanning electron microscope operated at an accelerating voltage of 10 kV.

2.10. Fourier‐Transform Infrared Spectroscopy

Fourier‐transform infrared spectra were recorded using a Fourier‐transform infrared spectroscopy (FTIR) spectrometer (Shimadzu, Kyoto, Japan) to analyze SA, EGCG, and the microencapsulated formulations (SA‐LP, SA‐LP‐EGCG, SA‐RS, SA‐RS‐LP, and SA‐RS‐LP‐EGCG). Freeze‐dried samples were finely ground using a ceramic mortar and pestle and mixed with spectroscopic‐grade potassium bromide (KBr) at a ratio of 1:100 (w/w). The mixtures were compressed into transparent discs under vacuum, and spectra were collected in the range of 500–4000 cm^{− 1} at a resolution of 4 cm^{− 1} with 32 scans per sample. Sample preparation and analysis followed the protocol by Hu et al. (2021).

2.11. X‐Ray Diffraction Analysis

The crystalline structure of the encapsulation materials and microcapsule composites was analyzed using an x‐ray diffractometer (SmartLab 9 kW, Rigaku, Japan) equipped with a Cu Kα radiation source (λ = 1.5406 Å). Diffraction patterns were collected in the 2θ range of 5°–60° with a step size of 0.02° and a scan rate of 2°/min under operating conditions of 45 kV and 200 mA. Freeze‐dried samples were finely powdered, placed on a flat sample holder, and gently pressed to ensure a uniform surface. Data were processed using Rigaku software to identify characteristic peaks and evaluate the degree of crystallinity, following the approach described by J. Zhang et al. (2024) with minor modifications.

2.12. Storage Stability of Encapsulated LP

Freeze‐dried microcapsules were stored at 4°C and 25°C for 90 days, and samples were analyzed at 0, 30, 60, and 90 days. Samples were placed in sealed airtight containers under ambient atmospheric conditions without vacuum treatment, with limited headspace to reduce oxygen exposure. At each sampling point, 1 g of microcapsules was dissolved in 9 mL of sodium citrate solution (0.1 M, pH 6.5) and gently agitated for 10–15 min until complete dissolution. The suspension was serially diluted in PBS, plated on MRS agar, and incubated at 37°C for 48 h. Viable cell counts were expressed as log CFU/g on a dry weight basis. The procedure was adapted from Etchepare et al. (2016) and Mirzaei et al. (2012).

2.13. Viability of Free and Encapsulated LP Under Simulated GI Conditions

The GI tolerance of LP was evaluated using a static in vitro digestion model with minor modifications (Afzaal et al. 2020; Zanjani et al. ²⁰¹⁴). Simulated gastric fluid (SGF) was prepared by dissolving 0.32 g pepsin (approximately 960 U/mL activity, porcine source) and 0.20 g NaCl in 100 mL of distilled water, followed by adjustment to pH 2.0 using 0.1 M HCl (Afzaal et al. 2020; Zanjani et al. ²⁰¹⁴). Simulated intestinal fluid (SIF) was prepared by dissolving 0.10 g pancreatin (trypsin ≥ 4000 U/g, amylase ≥ 7000 U/g, lipase ≥ 4000 U/g) and 0.08 g porcine bile salts (≥ 60% bile acid content) in PBS (pH 7.4). Both SGF and SIF were preheated at 37°C for 20 min and filtered through a 0.22 µm membrane. During the gastric digestion phase, independent digestion groups were prepared for each sampling time point (0, 30, 60, 90, and 120 min). At each designated time point, the pH of the digestion mixture was adjusted to 7.0 to terminate gastric digestion, after which the entire digestion mixture was directly disrupted to fully release the encapsulated bacteria. Subsequently, 1 mL of the resulting suspension was serially diluted, and viable cell counts were determined using the plate count method. After 120 min of gastric digestion, the pH of the system was adjusted to 7.0 using NaOH solution prior to intestinal digestion. Subsequently, 10 mL of SIF was added, and the mixture was vortexed thoroughly before further incubation at 37°C with shaking at 100 rpm. Independent digestion groups were also prepared for each intestinal digestion time point (1, 2, 3, and 4 h). At each designated time point, 1 mL of the digestion medium was directly withdrawn, serially diluted, and plated to determine the viable counts of free bacteria released into the intestinal fluid. During the intestinal phase, the release of viable cells was determined by enumerating the bacteria present in the surrounding SIF, representing cells released from the microcapsules at each sampling time.

Survival rate (%) during the gastric phase was calculated as:

$$\mathrm{Survival}\mathrm{rate}\left(\%\right)=\left(N_t/N_0\right)\times 100$$

where N₀ is the initial viable cell count before exposure to SGF, and N_t is the viable cell count at time t during gastric digestion.

Release rate (%) during the intestinal phase was calculated as:

$$\mathrm{Release}\mathrm{rate}\left(\%\right)=\left(N_r/N_0\right)\times 100$$

where N_r is the viable cell count recovered from the SIF at time t, and N₀ is the initial viable cell count before digestion.

2.14. Statistical Analysis

All experiments were performed in triplicate, and results are expressed as mean ± SD. Statistical analysis was conducted using one‐way ANOVA to evaluate differences among treatments at each sampling time point. This approach was selected to assess the effect of formulation at each time point, while time‐dependent changes were interpreted descriptively. Differences were considered statistically significant at p < 0.05. When significant differences were observed, multiple comparisons among treatments were performed using Tukey's post hoc test, and groups were distinguished based on their mean values, with different lowercase letters indicating statistically significant differences among treatments at the same sampling time point (p < 0.05). Statistical analyses were performed using Origin software (OriginLab Corporation, Northampton, MA, USA).

3. Results and Discussion

3.1. Encapsulation Efficiency and Process Yield of LP

The EE and PY of LP in different microcapsule formulations were presented in Table 1. The EE of the microcapsules was high overall, with the SA‐RS‐EGCG formulation reaching 97.0%, while the SA‐RS formulation exhibited a comparatively lower EE of 85.5%. These results are consistent with previous studies, which demonstrated that alginate–starch‐based carriers can achieve EE values above 90% for lactic acid bacteria (J. Zhang et al. ²⁰²⁴). The addition of EGCG did not negatively affect the EE, which aligns with findings by Xin et al. (2025), where tea polyphenols incorporated within alginate‐pectin matrices also maintained high EE values for LP. However, it is important to note that the impact of EGCG on probiotics is concentration‐dependent. At concentrations comparable to those used in the present study, EGCG can be compatible or even protective within encapsulation matrices (Qin et al. 2021), whereas higher concentrations have been reported to exert antimicrobial effects through mechanisms such as membrane disruption (Ikigai et al. 1993; Steinmann et al. ²⁰¹³). In the present study, the selected EGCG concentration was not associated with an apparent reduction in the viability of LP, as reflected by the high EE and stable viable cell counts observed after encapsulation. These findings suggest that the applied EGCG level was compatible with probiotic survival under the conditions investigated.

TABLE 1.

Encapsulation efficiency (EE) and process yield (PY) of LP microcapsules (mean ± SD, n = 3).

Sample	Specific count (log CFU/g)	EE (%)	PY (%)
SA‐RS	8.91 ± 0.02b	85.5 ± 0.5b	96.34 ± 0.5b
SA‐RS‐EGCG	9.15 ± 0.01a	97.0 ± 0.3a	96.73 ± 0.4a

Note: Different superscript letters within the same column indicate significant differences (p < 0.05).

Abbreviations: EE, encapsulation efficiency; EGCG, epigallocatechin gallate; PY, process yield; RS, resistant starch; SA, sodium alginate.

The high PY values indicate minimal loss of wall materials during bead formation, confirming efficient microcapsule production across all formulations. Notably, the incorporation of RS and EGCG did not adversely affect the recovery of microcapsules, suggesting that the multicomponent formulation remained compatible with the extrusion encapsulation process. Further characterization results presented in subsequent sections suggest that the SA‐RS‐EGCG formulation may also contribute to improved microcapsule stability and probiotic protection.

3.2. Bead Appearance and Particle Size

All microcapsule formulations exhibited generally spherical shapes, indicating successful gelation and extrusion (Figure 1a–f). In the case of sodium SA alone, beads were observed to be less uniform and slightly translucent and exhibited the smallest mean diameter (Figure 1a), which may be associated with the structural characteristics of alginate hydrogels, as previously described in the literature (L. Zhang et al. ²⁰²²). In contrast, incorporation of RS‐produced beads with greater uniformity, and visually appeared to exhibit more homogeneous and regular structures (Figure 1d–f), consistent with its reported ability to reinforce alginate matrices, which may contribute to the observed differences in bead appearance. The SA‐RS‐LP‐EGCG group appeared to display more defined and regular morphology (Figure 1f), although this is presented as a qualitative observation based on visual inspection rather than a quantitative assessment. Similar reinforcement by polyphenols has been attributed in other studies to intermolecular hydrogen bonding and hydrophobic interactions between EGCG and polysaccharides (Qin et al. 2021), which may contribute to the observed differences in bead appearance. Quantitative particle size analysis supported these visual observations, confirming significant differences in mean bead diameter among the different formulations (Figure 1g). Collectively, these findings suggest that the incorporation of RS and EGCG influenced the appearance and particle size characteristics of alginate‐based microcapsules, which may be relevant to their performance as probiotic delivery systems.

FIGURE 1.

Morphology and particle size distribution of microcapsules prepared with different formulations. Images (a–f) show representative microcapsules from (a) SA, (b) SA‐LP, (c) SA‐LP‐EGCG, (d) SA‐RS, (e) SA‐RS‐LP, and (f) SA‐RS‐LP‐EGCG. The bar chart (g) presents the mean particle diameter (µm) of more than 50 microcapsules per group (mean ± SD, n = 3). Different lowercase letters above the bars indicate statistically significant differences among formulations (p < 0.05).

3.3. Water Activity, Moisture Content, and Hygroscopicity

The physicochemical properties of freeze‐dried microcapsules are presented in Table 2. Low a _w, moisture content, and hygroscopicity are critical parameters influencing the stability of probiotic formulations during storage, as they directly affect microbial viability, oxidation processes, and the potential for moisture‐induced degradation. a _w ranged from 0.28 in SA to 0.18 in SA‐RS‐LP‐EGCG, with all values below 0.30, which is generally considered favorable for the storage stability of probiotic formulations. Alginate‐only beads (SA) showed the highest a _w (0.28), moisture content (5.92%), and hygroscopicity (18.4%). This trend is consistent with earlier reports indicating that alginate gels can exhibit relatively open and less densely cross‐linked structures depending on formulation conditions, such as Ca^{2 +} concentration and gelation parameters (Chávarri et al. 2010). Incorporation of LP was associated with slightly reduced values, which may reflect denser gel packing around cells as observed in similar encapsulation systems (Ni et al. 2023). Formulations containing EGCG (SA‐LP‐EGCG) showed significantly lower a _w (0.24), moisture content (4.91%), and hygroscopicity (15.2%) compared with SA and SA‐LP (p < 0.05). The decrease in moisture content may be associated with interactions between EGCG and the polymer matrix, particularly through hydrogen bonding, which can influence matrix packing and water mobility during freeze‐drying. In addition, such interactions may contribute to reduced hygroscopicity by limiting water vapor uptake in the dried matrix (Qin et al. 2021). As freeze‐drying and hygroscopicity represent distinct processes, namely water removal during dehydration and water uptake during storage, these parameters reflect different aspects of microcapsule behavior and should therefore be interpreted separately. In general, higher a _w and moisture levels can accelerate biochemical reactions and reduce probiotic viability, while high hygroscopicity can lead to moisture uptake during storage, promoting structural collapse and loss of encapsulation integrity. Therefore, reducing these parameters is essential for maintaining long‐term stability of probiotic microcapsules, particularly under typical storage conditions in sealed containers exposed to ambient humidity.

TABLE 2.

Water activity (a _w), moisture content, and hygroscopicity of freeze‐dried microcapsules (mean ± SD, n = 3).

Sample	Water activity	Moisture content (%)	Hygroscopicity (%)
SA	0.28 ± 0.01a	5.92 ± 0.21a	18.40 ± 0.30a
SA‐LP	0.27 ± 0.01b	5.34 ± 0.18b	17.00 ± 0.50b
SA‐LP‐EGCG	0.24 ± 0.01c	4.91 ± 0.22c	15.20 ± 0.40c
SA‐RS	0.22 ± 0.01d	4.48 ± 0.15d	14.30 ± 0.50d
SA‐RS‐LP	0.20 ± 0.01e	4.32 ± 0.12e	13.80 ± 0.30e
SA‐RS‐LP‐EGCG	0.18 ± 0.01f	3.95 ± 0.10f	12.10 ± 0.20f

Note: Different superscript letters within a column indicate significant differences at p < 0.05.

The addition of RS further improved stability, with SA‐RS showing a _w of 0.22, moisture content of 4.48%, and hygroscopicity of 14.3%. RS has been reported to modify the structural properties of alginate matrices, primarily through filler effects and matrix packing rather than direct ionic cross‐linking, which may influence water retention and moisture‐related properties (Mirzaei et al. 2012; Etchepare et al. ²⁰¹⁶). Among all formulations, SA‐RS‐LP‐EGCG exhibited the most stable profile, with the lowest a _w (0.18), moisture (3.95%), and hygroscopicity (12.1%). Similar improvements in physicochemical stability have been reported in phenolic‐rich delivery systems (Rajagukguk et al. 2022). Collectively, these results indicate that the combination of RS and EGCG may contribute to improved physicochemical stability of probiotic microcapsules. This improved physicochemical stability is expected to contribute to better preservation of viable cells during storage by limiting moisture‐related degradation and enhancing resistance to environmental stress.

3.4. Texture Analysis

Texture properties of probiotic‐loaded microbeads are presented in Table 3. Significant differences (p < 0.05) were observed among formulations, demonstrating the influence of matrix composition on mechanical behavior. Beads containing RS (SA‐RS, SA‐RS‐LP, SA‐RS‐LP‐EGCG) exhibited higher hardness compared to alginate‐only systems, with SA‐RS‐LP‐EGCG reaching the maximum value (approximately 126.65 g). This confirms the reinforcing role of starch in improving gel rigidity through denser polymer entanglement (Mirzaei et al. 2012). The additional presence of EGCG may also contribute to increased hardness through non‐covalent interactions, such as hydrogen bonding, between polyphenolic groups and the polymer matrix. In the context of probiotic delivery, increased hardness is advantageous as it enhances the structural integrity of microcapsules, allowing them to better withstand mechanical stress during processing and storage, as well as harsh conditions encountered during GI transit. Adhesiveness values were more negative in RS‐based samples, particularly SA‐RS‐LP (−1.78 g·s), indicating higher adhesiveness. This suggests that the incorporation of RS influences the surface interaction properties of the beads. EGCG‐containing formulations (SA‐LP‐EGCG, SA‐RS‐LP‐EGCG) exhibited intermediate adhesiveness values, suggesting that polyphenol–polymer interactions may influence surface properties (Makarewicz et al. 2021). The variation in adhesiveness among formulations reflects differences in surface interaction properties, which may influence particle aggregation and handling behavior. Springiness and cohesiveness values remained relatively stable across groups, suggesting that these properties were less influenced by formulation changes. This behavior may be influenced by interactions within the polymer network, including possible polyphenol–polysaccharide interactions in EGCG‐containing formulations, which can restrict chain mobility and tighten the gel network (Cortés‐Ferré et al. ²⁰²⁵).

TABLE 3.

Texture profile analysis of probiotic‐loaded alginate‐based beads prepared with different combinations of sodium alginate (SA), resistant starch (RS), LP, and EGCG. Values are expressed as mean ± standard deviation (n = 3).

Sample	Hardness (g)	Adhesiveness (g·s)	Springiness	Cohesiveness	Gumminess (g)	Chewiness (g)	Resilience
SA	116.12 ± 2.89b	−1.05 ± 0.01b	0.75 ± 0.06a	0.59 ± 0.03a	69.14 ± 5.01a b	51.61 ± 5.64a b	0.23 ± 0.01a
SA‐LP	108.27 ± 3.87c	−0.57 ± 0.39a	0.77 ± 0.01a	0.59 ± 0.01a	64.33 ± 1.46b	50.30 ± 3.39a b	0.23 ± 0.01a
SA‐LP‐EGCG	111.61 ± 2.73b c	−1.26 ± 0.78b	0.66 ± 0.03b	0.56 ± 0.02a b	62.77 ± 3.21b	41.40 ± 3.23c	0.20 ± 0.01b
SA‐RS	120.84 ± 1.31b	−1.51 ± 1.15b	0.71 ± 0.04a b	0.55 ± 0.03b	68.04 ± 1.72a b	48.24 ± 1.75b	0.21 ± 0.01b
SA‐RS‐LP	123.55 ± 5.96a b	−1.78 ± 0.23c	0.73 ± 0.03a b	0.58 ± 0.02a b	71.29 ± 3.49a	52.40 ± 4.56a	0.21 ± 0.01b
SA‐RS‐LP‐EGCG	126.65 ± 2.23a	−1.39 ± 0.60b c	0.70 ± 0.03a b	0.57 ± 0.03a b	71.90 ± 4.26a	53.70 ± 2.22a	0.20 ± 0.02b

Note: Different superscript letters within a column indicate significant differences (p < 0.05).

Gumminess and chewiness were highest in SA‐RS‐LP‐EGCG (71.90 and 53.70 g, respectively), reflecting improved structural compactness and mechanical stability. These results align with previous reports where dual biopolymer systems improved bead integrity under stress conditions (Dong et al. 2013). Collectively, the texture results indicate that incorporation of RS and EGCG influenced the mechanical properties of alginate‐based microcapsules, particularly hardness and adhesiveness. These changes may be relevant to the physical stability and handling characteristics of the microcapsules and could potentially influence their performance as probiotic delivery systems. Taken together, the results from bead appearance, particle size, physicochemical properties, and texture analysis suggest that incorporation of RS and EGCG influenced the structural and functional characteristics of alginate‐based microcapsules, which may be relevant to their potential application as probiotic delivery systems.

3.5. Scanning Electron Microscopy Analysis

Scanning electron microscopy (SEM) analysis revealed distinct morphological differences between the two formulations (Figure 2). Microcapsules prepared with SA‐LP‐EGCG exhibited relatively porous and irregular surfaces, which is consistent with the characteristic weak and permeable alginate network reported previously (L. Zhang et al. ²⁰²²). In contrast, the incorporation of RS (SA‐RS‐LP‐EGCG) resulted in beads with smoother and more compact surfaces, as well as visually denser internal structures, suggesting improved matrix integrity. Similar structural improvements in dual‐biopolymer systems have been shown to enhance encapsulation performance and probiotic stability (Sultana et al. 2000). These findings suggest that RS and EGCG contributed to the formation of a more robust alginate‐based matrix, which may help improve the protection of LP during processing and storage.

FIGURE 2.

SEM images showing surface and internal morphology of SA‐LP‐EGCG and SA‐RS‐LP‐EGCG microcapsules, with the top row representing SA‐LP‐EGCG and the bottom row representing SA‐RS‐LP‐EGCG, and panels showing overall morphology, cut cross‐sections, and high‐magnification surface views.

3.6. Fourier‐Transform Infrared Spectroscopy

The FTIR spectra of SA, EGCG, and the microencapsulated formulations (SA‐LP, SA‐LP‐EGCG, SA‐RS, SA‐RS‐LP, SA‐RS‐LP‐EGCG) revealed characteristic absorption bands corresponding to functional groups of the encapsulating components (Figure 3). SA displayed a broad O–H stretching band at 3304 cm^{− 1} and a carboxylate asymmetric stretching at 1600–1650 cm^{− 1}, typical of its anionic polysaccharide backbone (J. Zhang et al. ²⁰²⁴). The carboxylate groups (COO⁻) are primarily involved in Ca^{2 +} mediated ionic cross‐linking of alginate chains (egg‐box model), and changes in their position or intensity may reflect variations in cross‐linking interactions within the gel network. EGCG exhibited O─H stretching at 3357 cm^{− 1} along with aromatic C═C skeletal vibrations at 1691 and 1612 cm^{− 1}, confirming its polyphenolic structure (Zhao et al. 2022). RS contributions were reflected in RS‐containing formulations, which showed characteristic O─H stretching near 3312–3320 cm^{− 1} and polysaccharide C–O/C–O–C bands around 1020–1080 cm^{− 1}, consistent with starch‐polysaccharide interactions (Vaitkeviciene et al. 2022).

FIGURE 3.

FTIR spectra of EGCG, sodium alginate (SA), and microcapsules prepared with different formulations.

The addition of LP altered the FTIR spectrum of SA‐LP, causing the O–H stretching band to shift to 3216 cm^{− 1}, which may result from hydrogen bonding interactions between hydroxyl/carboxyl groups of alginate and surface proteins or polysaccharides of the bacterial cells. The SA‐LP‐EGCG formulation exhibited a shift toward 3400 cm^{− 1}, suggesting additional hydrogen bonding from the hydroxyl groups of EGCG. Similarly, SA‐RS and SA‐RS‐LP beads showed bands at 3312 and 3320 cm^{− 1}, indicating the integration of RS within the alginate matrix. As RS does not directly participate in Ca^{2 +} ‐mediated cross‐linking, its presence may influence the network structure through matrix packing and interactions with alginate chains, which can be reflected in the observed spectral shifts. The SA‐RS‐LP‐EGCG system exhibited the most prominent shifts, with O–H stretching at 3340 cm^{− 1} and COO⁻ stretching at 1654 cm^{− 1}, suggesting the presence of intermolecular interactions, including hydrogen bonding, within the composite matrix. The persistence of aromatic C═C vibrations near 1600 cm^{− 1} in EGCG‐containing formulations further confirmed the successful incorporation of the polyphenol (Xin et al. 2025).

Overall, the observed peak shifts, broadening, and intensity variations suggest intermolecular interactions between alginate, RS, EGCG, and LP within the composite matrix. These findings are consistent with previous FTIR studies on polysaccharide‐based encapsulation systems and support the successful incorporation of the different components within the microcapsule matrix (Dou et al. 2014).

3.7. X‐Ray Diffraction Analysis

The x‐ray diffraction (XRD) analysis provided valuable insights (Figure 4) into the crystalline structure and interactions among SA, RS, EGCG, and their encapsulated formulations with LP (Figure 4). SA exhibited broad diffraction peaks at approximately 2θ = 13° and 21.5°, characteristic of its semicrystalline and largely amorphous polysaccharide structure (J. Zhang et al. ²⁰²⁴). RS displayed distinct crystalline reflections at around 2θ values of 15°, 17°, 20°–22°, and 23°, consistent with the B‐type crystallinity pattern typical of native RS2, in agreement with previous reports (Sajilata et al. 2006). In contrast, EGCG exhibited distinct diffraction peaks, indicating its crystalline nature.

FIGURE 4.

XRD patterns of SA, RS, EGCG, LP, and microencapsulated formulations.

When encapsulated systems were examined, structural modifications were evident. The SA‐LP sample exhibited a broad halo centered at approximately 2θ = 21.5° with reduced intensity compared to pure SA, suggesting a reduction in structural order and crystallinity, which may result from combined effects of alginate gelation, Ca^{2 +}‐mediated cross‐linking, freeze‐drying, and the presence of bacterial cells. The addition of EGCG (SA‐LP‐EGCG) further attenuated this peak without introducing new crystalline reflections, suggesting that EGCG–alginate interactions may contribute to reduced structural order, although this effect may also be influenced by factors such as molecular dispersion within the matrix and amorphization during freeze‐drying. Similar reductions in polymer crystallinity by polyphenols through hydrogen bonding have been reported previously (Xie et al. 2024).

In RS‐containing formulations (SA‐RS and SA‐RS‐LP), the characteristic RS peaks at 15°, 17°, and 23° were retained but with reduced intensity, suggesting partial amorphization due to integration with the alginate matrix. This effect was even more pronounced in the SA‐RS‐LP‐EGCG system, where both bacteria and EGCG contributed to further disruption of crystalline domains (Sajilata et al. 2006; J. Zhang et al. ²⁰²⁴).

Overall, the XRD results suggest that co‐encapsulation contributed to partial amorphization and changes in the structural organization of the composite matrix containing alginate, RS, EGCG, and LP The reduced crystallinity observed in RS‐ and EGCG‐containing formulations may reflect increased molecular dispersion and intermolecular interactions within the matrix. These findings are consistent with the differences observed in morphology, physicochemical properties, and texture characteristics described in earlier sections, supporting the formation of a structurally modified multicomponent encapsulation system.

3.8. Storage Stability of Encapsulated LP

At 4°C, encapsulated cells retained significantly higher viability compared to free cells across all formulations (Figure 5b). Statistical comparisons were performed among different formulations at each sampling time point. In addition, all samples exhibited a progressive decrease in viability over storage time, with more pronounced reductions observed in free cells compared to encapsulated formulations. Viable cell counts were determined before and after freeze‐drying using plate counting and expressed on a dry‐weight basis, allowing direct comparison of cell survival during lyophilization. Free LP declined rapidly from approximately 9.0 to 3.2 log CFU/g by Day 90, representing a reduction of more than 5.5 log units, whereas the encapsulated samples such as SA‐RS‐LP and SA‐RS‐LP‐EGCG showed minimal reductions (less than 1 log unit) and maintained counts above 8.4 log CFU/g, confirming the protective role of the matrix. This enhanced stability can be attributed to the barrier properties of SA and the ability of RS to reduce oxygen and moisture diffusion by filling gaps within the gel network, thereby creating a more compact and less permeable bead structure (De Prisco and Mauriello ²⁰¹⁶; Etchepare et al. ²⁰¹⁶). The presence of EGCG further contributed antioxidative protection, with its hydroxyl‐rich polyphenolic structure capable of scavenging reactive oxygen species and stabilizing bacterial membranes, consistent with previous reports on polyphenol‐polysaccharide systems (Qin et al. 2021; L. Zhang et al. ²⁰²²). At 25°C, degradation was accelerated across all samples (Figure 5a), yet encapsulation still mitigated losses relative to free cells; the latter declined to roughly 2.8 log CFU/g by Day 90, indicating near‐complete inactivation, whereas SA‐RS‐LP‐EGCG maintained 7.45 log CFU/g, corresponding to only a 1.55 log reduction. This stabilization effect can be linked to the combined functions of RS, acting both as a prebiotic substrate and wall filler to enhance compactness and reduce porosity (Fritzen‐Freire et al. ²⁰¹²), and EGCG, which provides antioxidative and membrane‐protective functions (Makarewicz et al. 2021). Among all formulations, SA‐RS‐LP‐EGCG consistently demonstrated the highest storage stability at both temperatures. The influence of RS and EGCG became more apparent during extended storage, while differences among formulations were less evident at earlier stages. This time‐dependent behavior may be associated with gradual moisture migration, oxidative stress, and changes in matrix properties during storage, where RS‐ and EGCG‐containing systems exhibited improved retention of probiotic viability over time. These findings are generally consistent with previous studies reporting improved probiotic stability in multicomponent encapsulation systems containing polysaccharides and polyphenolic compounds (Qin et al. 2021; L. Zhang et al. ²⁰²²). The improved stability observed in these formulations is also consistent with their lower water activity, moisture content, and hygroscopicity, which may help limit moisture‐related degradation during storage. Based on these results, encapsulated microcapsules could be effectively stored at 4°C for at least 90 days while maintaining high viable cell counts (> 8 log CFU/g). At 25°C, although storage stability was reduced, the microcapsules still retained relatively high viability after 90 days, particularly in the SA‐RS‐LP‐EGCG formulation, suggesting that refrigerated storage is preferable for long‐term preservation.

FIGURE 5.

Storage stability of free LP and encapsulated formulations (SA‐LP, SA‐LP‐EGCG, SA‐RS‐LP, SA‐RS‐LP‐EGCG) over 90 days at two temperatures. (a) Viability during storage at 25°C; (b) viability during storage at 4°C. Values are expressed as mean log CFU/g (n = 3) ± SD. Different lowercase letters indicate significant differences (p < 0.05) among treatments at the same storage time.

3.9. In Vitro GI Tolerance

The simulated GI assay demonstrated that encapsulation significantly enhanced the survival of LP compared to free cells (Figure 6a; p < 0.05). During the gastric phase, viable counts represent the total cells recovered after disruption of the microcapsules, whereas during the intestinal phase, counts correspond to cells released into the surrounding medium, as described in Section 2.13. Under these conditions, free cells showed a steep decline, dropping to below 50% viability after 120 min, reflecting their known sensitivity to low pH and pepsin. In contrast, encapsulated formulations retained substantially higher survival. SA‐LP preserved approximately 70% viability, while SA‐RS‐LP provided greater protection (around 85%), consistent with previous studies showing that RS reduces bead porosity and enhances barrier effects against acid penetration (Etchepare et al. 2016; Muhammad et al. ²⁰²¹).

FIGURE 6.

Survival and release behavior of free and encapsulated LP under simulated gastrointestinal conditions. (a) Survival (%) during gastric digestion (pH 2.0, 120 min); (b) release (%) during intestinal digestion (pH 6.8, 4 h) are shown for free cells, SA‐LP, SA‐LP‐EGCG, SA‐RS‐LP, and SA‐RS‐LP‐EGCG formulations. Values represent mean ± SD (n = 3). Different lowercase letters indicate significant differences (p < 0.05) among treatments at the same sampling time.

The incorporation of EGCG further improved protection, with SA‐LP‐EGCG and SA‐RS‐LP‐EGCG showing the highest survival rates during gastric digestion. Notably, SA‐RS‐LP‐EGCG retained nearly 90% viability after 120 min, suggesting a combined contribution of RS and EGCG to improved probiotic survival during gastric digestion. This is supported by recent reports indicating that starch‐alginate matrices form denser gels through hydrogen bonding and hydrophobic interactions, while polyphenols may contribute to protection through both antioxidant activity and interactions with the matrix that enhance barrier properties and limit diffusion under gastric conditions (Gheorghita et al. 2024; Pourjavid et al. ²⁰²²).

In the intestinal phase, free cells continued to decline, decreasing to approximately 50% of the initial recoverable fraction after 4 h, consistent with bile salt sensitivity (Figure 6b). Encapsulated systems displayed a gradual increase in release rate of viable cells, with SA‐LP and SA‐LP‐EGCG reaching around 60%–70% release, while RS‐based systems (SA‐RS‐LP and SA‐RS‐LP‐EGCG) achieved about 80% release (Saniani et al. 2023; Zanjani et al. ²⁰¹⁴; Wang and Mutukumira ²⁰²²). The sustained release in RS‐containing formulations likely reflects structural changes in the starch–alginate matrix under intestinal conditions, where swelling and partial matrix relaxation may facilitate diffusion pathways and promote progressive release of viable cells. Similar controlled‐release behavior has been reported in alginate‐starch capsules and synbiotic matrices (Zanjani et al. 2014; Wang and Mutukumira ²⁰²²).

Collectively, these findings indicate that incorporation of RS and EGCG was associated with improved survival of encapsulated LP under simulated GI conditions. During the intestinal phase, RS‐containing formulations exhibited higher release rates of viable cells compared with alginate‐only formulations, particularly after prolonged incubation. Among all formulations, SA‐RS‐LP‐EGCG showed the highest survival during gastric digestion and the highest release rate during intestinal digestion. These results support the potential application of multicomponent alginate‐based microcapsules for probiotic delivery in functional food systems.

4. Conclusion

This study demonstrated that encapsulation of LP in SA–RS–EGCG matrices improved probiotic stability under storage and simulated GI conditions. Structural characterization suggested that incorporation of RS and EGCG modified the physicochemical and mechanical properties of the microcapsules, which was associated with improved probiotic stability and altered release behavior during intestinal digestion. Among the tested formulations, SA‐RS‐LP‐EGCG exhibited the best overall performance, maintaining high viable cell counts during 90 days of storage and showing the highest release rate of viable cells under simulated intestinal conditions. These findings suggest that incorporation of RS and EGCG may help improve the functional performance of alginate‐based microcapsules for probiotic delivery applications. In addition, RS and EGCG have been reported in previous studies to possess potential prebiotic and antioxidant properties, respectively, which may further support the application of these materials in probiotic encapsulation systems. Overall, the present study supports the potential of multicomponent alginate‐based microcapsules for improving probiotic stability in functional food applications. Future studies should further evaluate the performance of these systems in real food matrices and in vivo conditions.

Author Contributions

Dawei Chang: supervision, methodology, conceptualization, writing – review and editing, formal analysis, funding acquisition. Sendi Sara: methodology, conceptualization, data curation, writing – original draft, formal analysis, visualization. Youling L. Xiong: writing – review and editing, conceptualization. Yujiao Sun: resources, writing – review and editing, funding acquisition. Li Feng: funding acquisition.

Conflicts of Interest

The authors declare no conflicts of interest.