Introduction
Among various probiotic strains, lactic acid bacteria (LAB) have been the most extensively studied (Anal and Singh, 2007). Lactobacillus plantarum, a prominent LAB species, exhibits a wide range of bioactivities, including inhibition of pathogenic bacteria, improvement of gut microbiota, modulation of the immune system, and regulation of glucose and lipid metabolism. These features make L. plantarum a promising candidate for promoting gastrointestinal, metabolic, and cardiovascular health (Collantes et al., 2023). Nevertheless, like many other probiotic species, L. plantarum is susceptible to environmental stresses such as high temperature, low pH, salt, oxygen, and bile salts during processing and digestion, which may limit its functionality and efficacy (Ranadheera et al., 2010). One promising approach to improve probiotic viability is microencapsulation, a technique that involves embedding microorganisms within protective coatings or matrices. This not only shields probiotics from adverse conditions but also allows for targeted and controlled release at the desired site in the gastrointestinal tract. Additionally, microencapsulation can mask undesirable flavors in probiotic-containing food products (Fraj et al., 2021). Several encapsulation techniques are available, including extrusion, emulsification, spray drying, freeze drying, and cold spraying, each with its specific advantages and limitations (Luca and Oroian, 2021). Among these, freeze drying has attracted considerable attention due to its capacity to preserve the structure and bioactivity of probiotics. The selection of appropriate wall materials during freeze drying is critical for minimizing cellular damage and enhancing bacterial viability. Moreover, controlling parameters such as temperature and residual moisture is essential to maintain probiotic stability. L. plantarum thrives in slightly acidic conditions (pH 5.0–6.2), a factor that should be considered when designing preservation and storage conditions (Pramono et al., 2025). The choice of wall material plays a vital role in determining the efficiency, release profile, and functional properties of the microcapsules (Ozdemir et al., 2021). Commonly used wall materials include polysaccharides, proteins, and lipids. Among them, proteins are desirable due to their biocompatibility, low cost, availability, and desirable physicochemical characteristics (Vaziri et al., 2018). Gelatin, a widely used protein-based material, is biodegradable, biocompatible, and cost-effective, exhibiting a high molecular interaction capacity, making it suitable for encapsulation applications (Bastos et al., 2021). On the other hand, fish protein hydrolysate (FPH), derived from aquatic by-products such as skin, scales, and bones, is rich in bioactive peptides with antioxidant, antimicrobial, anti-inflammatory, immunomodulatory, and antihypertensive properties, and is widely used in the food and pharmaceutical industries (Nemati et al., 2021). These peptides, produced through enzymatic hydrolysis of fish proteins, possess low molecular weight, high solubility, and desirable bioactivity, making them effective protective agents in the microencapsulation of probiotics such as L. plantarum (González-Serrano et al., 2022). Due to its antioxidant capacity, FPH protects probiotic cells against oxidative stress during freeze-drying and storage. Furthermore, its antibacterial peptides contribute to enhanced product safety. When combined with gelatin, FPH forms a protective matrix that shields bacteria from harsh gastric conditions (Nemati et al., 2024). Additionally, the peptides and amino acids present in FPH can serve as a nutrient source for probiotics, thereby enhancing their viability (Shori, 2017). From an environmental perspective, the utilization of fish waste for FPH production represents a sustainable and cost-effective approach (Kristinsson and Rasco, 2000). Given these advantages, the present study employed a combination of commercial bovine gelatin and fish protein hydrolysate as a wall material for the microencapsulation of L. plantarum using freeze-drying technology. This research aimed to evaluate the potential of this biopolymeric blend in enhancing the viability and stability of L. plantarum under adverse environmental conditions.
Methodology
A 50g sample was mixed with 100 ml of distilled water and heated at 85°C for 20 min to inactivate endogenous enzymes. After cooling, pepsin was added (1% of total protein) and hydrolysis occurred at pH 5.8 and 58°C for 90 min. The mixture was then heated at 90°C for 10 min to inactivate the enzyme, cooled, centrifuged, and the supernatant was collected and freeze-dried (Ojagh et al., 2012). To prepare L. plantarum cultures, 1g of lyophilized powder was dissolved in 9 ml of sterile phosphate buffer, and 0.1 ml of the suspension was spread on an MRS agar plate. After anaerobic incubation at 37°C for 24 h, the cells were scraped off the plate with 2 ml of sterile phosphate buffer, and the resulting slurry was centrifuged at 13,000×g for 1 min. Gelatin (5g) and fish protein hydrolysate (5g) were dissolved in 1 liter of distilled water, maintained at 45°C for 30 min, and cooled to 25°C. A L. plantarum suspension (1×10⁸ CFU/g) was added, and the mixture was homogenized at 350 rpm for 15 min (Heinzelmann et al., 2000).
The suspension was frozen at −80°C for 24 h and then freeze-dried for 3-4 days at 30 Pa with a condenser temperature of -60°C (Machado et al., 2022). The DPPH radical scavenging activity was assessed according to the method described by Dadmehr et al. (2024). In brief, 100 µl of each sample solution was mixed with 3.9 ml of DPPH solution. Absorbance was measured at 518 nm using a spectrophotometer at 5-min intervals. Calibration was performed in the range of 5–30 min. The FRAP assay was performed as previously described by Dadmehr et al. (2024). The FRAP reagent was prepared by mixing acetate buffer (300 mM), TPTZ solution (10 nM in 40 mM HCl), and FeCl₃ (20 mM) in a 10:1:1 (v/v/v) ratio and incubated at 37°C. Then, 100 µl of the sample was added to 3 ml of the FRAP reagent, and absorbance was recorded at 593 nm. A calibration curve was constructed within 5–30 min. BHA was also analyzed under the same conditions as a reference antioxidant.

Results

Discussion and conclusion

The results showed that the microencapsulated wall containing Lactobacillus, combined with gelatin and hydrolyzed protein, had a moisture content of 7.5% and a water activity of 0.32 ± 0.02, indicating the physical stability and good shelf life of the system. The low water activity, which prevents the growth of unwanted microorganisms and chemical degradation, plays a crucial role in protecting probiotics. Low moisture also prevents the fragility of the structure (Hasniah, 2022). The use of gelatin and hydrolyzed protein has created a resistant and controlled matrix in water retention, increased mechanical stability and resistance to environmental conditions (Mushtaq et al., 2022). Examination of the surface structure of the microencapsulated particles using scanning electron microscopy (SEM) indicated the success of the microencapsulation process with a combination of gelatin and hydrolyzed fish protein. The particles had a relatively uniform and spherical structure and their surface showed a dense texture without cracks. It has been confirmed that the stability of probiotics decreases under oxidative conditions. Therefore, the use of agents with antioxidant activity can help increase the survival of microencapsulated probiotics (Dadmehr et al., 2024). The compounds resulting from protein hydrolysis, depending on the type of initial protein and the hydrolysis method, can include free amino acids, small peptides, and in some cases, larger protein molecules remaining (Ramezani et al., 2018). Gelatin and bioactive peptides have been reported as natural antioxidants, which were used as a protective layer in this study (Yaghoubzadeh et al., 2019; Nurilmala et al., 2020). The antioxidant activity of gelatin is due to its peptides, which contain high amounts of arginine, tyrosine, and phenylalanine and are thought to have higher antioxidant activity (Shiao et al., 2021). These coatings may enhance the antioxidant activity of microencapsulated L. plantarum. The results obtained from the antioxidant tests showed that the radical scavenging activity and reducing power of the microencapsulated samples increased significantly with increasing concentration. In the DPPH test, the percentage of free radical scavenging increased from 10.25% at a concentration of 15.625 mg/ml to 25.33% at a concentration of 250 mg/ml. Similarly, in the FRAP test, the reducing power value increased from 0.297 µmol Fe²⁺/g at a concentration of 625.15 mg/ml to 187.5 µmol Fe²⁺/g at a concentration of 250 mg/ml. This dose-dependent increase indicates the presence of bioactive compounds with antioxidant properties in the coating formulation. Hydrolyzed fish proteins are rich in peptides with functional groups (such as -NH₂ and -COOH) that can react with free radicals. (Harnedy and FitzGerald, 2012) In addition, the gelatinous structure of the matrix may also play a role in stabilizing the antioxidant compounds (Shahidi and Ambigaipalan, 2015). The test results are in line with similar studies. For example, Wang et al. (2017) in a similar study showed that microcapsules containing peptide-coated probiotics showed higher DPPH and FRAP activity at higher concentrations. Also, the study by Mehdipour Biregani and Ahari (2021) reported that hydrolysates derived from fish protein have a high ability to absorb free radicals. The results obtained indicate that the developed microencapsulation system not only increased the stability of L. plantarum, but also provided significant antioxidant properties, which can be used as a bioactive carrier in functional products. Wang et al. reported that microencapsulation of L. plantarum with gelatin increased its stability and antioxidant properties under different conditions (Wang et al., 2019). In another study, Cui et al. (2021) showed that the use of marine-derived peptides in the coating of microcapsules led to a significant improvement in antioxidant activity in DPPH and ABTS assays (Cui et al., 2021). They attributed this effect to the presence of amine and hydrophobic active groups in the coating composition. Razavi et al. (2020) reported that coating L. plantarum with gum arabic and alginate increased antioxidant activity, but the extent of this increase was less than that of protein coatings such as gelatin and FPH. Also, Hebert et al. (2017) pointed out the synergistic effects between probiotic bacteria and marine bioactive peptides, which led to the enhancement of biological functions, including antioxidant properties (Hebert et al., 2017). The results of this study are consistent with the study of Wang et al. (2019) who showed that gelatin microencapsulation can significantly increase the antioxidant activity of L. plantarum. Also, Cui et al. (2021) reported that the combination of marine peptides with probiotic bacteria resulted in high iron (Fe³⁺) reduction potential and enhanced antioxidant function, which is consistent with the results of the FRAP assay. In addition, the study conducted by Razavi et al. (2020) who investigated the antioxidant activity of L. plantarum microencapsulated with gum arabic and alginate confirmed a dose-dependent increase in FRAP; however, the reducing power was reported to be lower compared to protein functional groups in peptides derived from protein sources, which play an important role in electron transfer in the FRAP assay (Hebert et al., 2017). In the present study, the reduction in water activity due to the use of a combination of gelatin and hydrolyzed rainbow trout proteins can help create a limiting environment for destructive reactions (such as oxidation and enzymatic degradation), which is consistent with the results of previous studies. Microencapsulation of L. plantarum with a combination of gelatin and hydrolyzed fish protein not only increases bacterial survival under oxidative conditions, but also plays an effective role in enhancing antioxidant properties through chemical interactions with radicals and metal ions. This feature allows the use of this compound in the food and pharmaceutical industries as a natural and safe alternative to synthetic antioxidants such as BHA.

Conflict of Interest

The authors declare that they have no conflict of interest
Acknowledgment
This research was supported by Iranian Fisheries Science Research Institute (IFSRI).