Structural and survival analysis of Lactobacillus rhamnosus added to edible films

1 Introduction

Probiotics are non-pathogenic, gut-friendly live microorganisms that have been studied for their positive health impacts and their role in improving food quality and safety (Semwal et al., 2022; Todhanakasem et al., 2022; Coimbra et al., 2023). These microorganisms and their fermentation products are considered bio-preservatives as they inhibit or halt the growth of pathogenic bacteria (by producing organic acids, ethanol, fatty acids, hydrogen peroxide, and diacetyl; and/or by limiting their growth through space competition), act as antifungal compounds (by producing propionate, phenyl acetate, cyclic dipeptides), and produce bacteriocins (nisin, pediocin, lacticin, and enterocin) (Coimbra et al., 2023; Silva et al., 2023).

Probiotic consumption has been associated with protection of the gastrointestinal tract, immune system stimulation, symptom reduction in various diseases (irritable bowel syndrome, intestinal inflammation, lactose intolerance), reduction of blood cholesterol levels, prevention of cardiovascular diseases, improvement in the prevention of atopic allergies, prevention of various types of cancer, reduction in the incidence of diarrhea, reduction of vaginal infections, improvement in feed efficiency (weight gain), enhanced calcium absorption, and the formation or production of vitamins (Soukoulis et al., 2014; 2016; Coimbra et al., 2023; Ali et al., 2024; Nezamdoost-Sani et al., 2024).

A food product is considered to have a functional effect when it contains at least 10 million live probiotics per gram (Lee et al., 2024). However, these microorganisms must remain viable as they pass through the mouth, gastric juices, digestive enzymes, stomach, and small intestine. To confer beneficial effects, they must survive (10⁸–10¹⁰ CFU/day) in a viable state (Sogut et al., 2022; Nezamdoost-Sani et al., 2024; Sadeghi et al., 2024). Dairy products are the most commonly used vehicles for adding probiotics to foods due to the medium’s characteristics, such as the buffering effect of milk, fat concentration (protecting probiotics from direct exposure to adverse conditions), the production environment (low temperatures and suitable pH), and nutritional composition (viability and growth of microorganisms) (Penha Rodrigues Pereira et al., 2024). Yogurt, fermented milk, and cheese are the most commonly used dairy products for adding probiotics. However, there are consumption issues for people with lactose intolerance and those allergic to milk proteins. Nutritionally, there are concerns about the high concentration of cholesterol and saturated fatty acids. Additionally, cultural considerations (strict vegetarians or vegans) and religious beliefs must be taken into account (Alvarez et al., 2021; Cosme et al., 2022; Arratia-Quijada et al., 2024). Consequently, a variety of non-dairy foods have been developed as vehicles for probiotics (cereals, fruits, plant-based products, fermented cereals, meat, bakery products, and chocolates) (Alvarez et al., 2021; Arratia-Quijada et al., 2024; Penha Rodrigues Pereira et al., 2024).

A novel alternative for non-dairy products is to immobilize probiotics in bio-packaging or eco-friendly packaging. The production of edible films and coatings is an emerging technology driven by consumer awareness of synthetic or plastic packaging and the potential solution to environmental pollution (Silva et al., 2023; Tan et al., 2024). These films are used for storage and packaging, helping to ensure microbial safety, preservation against external factors (water vapor and gas and volatile compound transfer), deterioration, and protection against physical damage (impacts, pressures, or vibrations) (Radev, 2021; Coimbra et al., 2023; Silva et al., 2023). An innovation in edible films that makes the product more efficient (improving quality, appeal, and marketability) is the addition of bioactive compounds (Radev, 2021; Todhanakasem et al., 2022; Silva et al., 2023) (antioxidants, vitamins, or antimicrobials, etc.), food components (flavorings, colorants, additives, etc.), and/or being carriers of microorganisms like lactic acid bacteria (LAB) (Coimbra et al., 2023; Silva et al., 2023). Specifically, the incorporation of probiotics into biopolymeric matrices is an alternative method to control microorganisms and improve food safety, with the added potential of promoting consumer health (Todhanakasem et al., 2022). However, the production of food matrices with probiotics is challenging as a significant amount of LAB is inactivated during processing (temperature, mechanical processes, pH, osmotic stress, water activity), storage (light, exposure to acute toxic factors such as oxygen, hydrogen peroxide, and water vapor), or during interaction with the matrix (Soukoulis et al., 2014; 2016; Nezamdoost-Sani et al., 2024). Therefore, understanding the viability of probiotics during the shelf life of a food product is a challenge, as microorganisms can modify the sensory characteristics of the product and, at certain concentrations, can confer health benefits to the host (Penha Rodrigues Pereira et al., 2024). Carboxymethylcellulose (CMC) is a cellulose derivative widely used in food and pharmaceutical applications due to the high viscosity of its aqueous solutions and its excellent film-forming properties. CMC is an anionic, linear polysaccharide that is stable in water at room temperature and over a broad pH range (approximately pH 5–9). Its chemical structure contains numerous hydroxy and carboxymethyl groups, which promote interactions with water, proteins, plasticizers, and salts. These characteristics contribute to improved flexibility, mechanical performance, and structural stability of polymeric matrices, making CMC an attractive component for the development of edible films. However, the incorporation of CMC may also modify the microenvironment surrounding entrapped probiotic cells, potentially affecting their viability during storage (Smith et al., 2023; Khan et al., 2025; Lakshmi et al., 2025).

Regarding probiotics, many bacterial strains can be used, but the most commonly employed belong to the genera Lactobacillus and Bifidobacterium (Alonso García et al., 2019; Sáez-Orviz et al., 2021; Temiz and Özdemir, 2021). L. rhamnosus is a facultative lactic acid bacterium, rod-shaped, non-spore-forming, anaerobic, and gram-positive. The reported benefits of L. rhamnosus include improved gut health, prevention of gastrointestinal infections, and reduction of certain allergic symptoms (Temiz and Özdemir, 2021). Iglesias et al. (2018) and Temiz and Özdemir (2021) conducted coatings of strawberries based on L. rhamnosus and gelatin enriched with inulin, determining that these coatings can increase the shelf life under refrigeration (lower counts of molds, yeast, and mesophilic bacteria) and functionality (reduce weight loss, decomposition, and preserve total phenolic content and antioxidant activity of stored fruit) of strawberries. Therefore, the primary aim was to develop starch-based edible films containing different proportions of carboxymethylcellulose (CMC) as carriers of L. rhamnosus and to evaluate their mechanical properties, microstructural characteristics, and probiotic survival during storage. Specifically, the study sought to determine how increasing the CMC content influences film flexibility, matrix structure, and the viability of the incorporated probiotic.

2 Materials and methods

2.1 Materials and strain

All reagents and culture media employed in this study were of analytical grade and obtained from commercial suppliers. Lactobacillus rhamnosus NRRL B-442 (maintained as frozen stocks at −80 °C in MRS broth supplemented with glycerol) was obtained from the microbial collection of the Faculty of Chemical Sciences, BUAP. The strain is deposited in the Agricultural Research Service (ARS) Culture Collection (NRRL) and has been previously used as a probiotic model microorganism in studies evaluating survival in food systems and edible matrices. Its documented tolerance to food-processing and storage conditions makes it suitable for evaluating probiotic incorporation into edible films. Prior to its use, the strain was activated through two successive transfers in Man–Rogosa–Sharpe (MRS BIOXON, Mexico City, Mexico) broth and incubated at 37 °C for 24 h to ensure optimal viability.

2.2 Preparation of edible films

Three edible film formulations were prepared: starch and starch with CMC (CTR Scientific, Mexico City, Mexico), each with and without L. rhamnosus. Film-forming solutions (FFS) were prepared by dissolving starch (1% w/w) or starch (1% w/w) and CMC (0.5% w/w), together with sorbitol (1% w/w). The concentrations of starch (1%, w/v) and CMC (0.5%, w/v) refer to the stock polymer solutions used during film preparation. The designations starch/CMC (60:40) and starch/CMC (50:50) correspond to the final proportions of each polymer in the film-forming dispersion. A 0.125N sodium hydroxide (CTR Scientific, Mexico City, Mexico) solution was used only during polymer dispersion and solubilization and then dispersion was cooled and adjusted to a cell-compatible pH (4.5) before L. rhamnosus addition (Bertuzzi et al., 2007). Control films without L. rhamnosus were evaluated.

Prior to film preparation, L. rhamnosus was cultured in MRS broth and incubated at 37 °C overnight. Based on previous growth studies performed under identical culture conditions, this incubation period corresponds to the late exponential growth phase and was therefore selected for inoculum preparation. A 10 mL aliquot of the overnight culture was transferred to fresh MRS broth and grown at 37 °C to the end of the exponential phase of growth (12 h). Subsequently, 1 mL was inoculated to the different FFS to obtain a target inoculum of 10⁸ UFC/mL. FFS were homogenized under aseptic conditions at 20,000 rpm for 1 min (IKA T18, basic, Chicago, Ill., U.S.A.) and poured into 60 mm inner diameter sterile Petri dishes. Films were prepared with 7 mL of FFS per Petri dish (1 film). After drying under 5 psi vacuum at 30 °C for 12 h. Films were kept in sealed Petri dishes at two temperatures, 5 °C and 22 °C at 50% relative humidity during 24 days of storage.

2.3 Structural characterization

2.3.1 Mechanical properties

All films were tested after 12 days of storage at 22 °C. Film thickness (μm) was determined on every film formulation averaging measurement at 5 points of each film using a micrometer (Scala, 111-B, Mexico). EFs were fixed on a circular acrylic support of 3 cm in diameter. With the texture analyzer (Texture Technologies Corp. TA/XT2i, Scarsdale, USA), a graph of force versus time was prepared. Mechanical properties were calculated from the resulting force-time curves. The breaking force (N) was determined at the point of highest value of the curve, the elongation or elasticity (mm) and the tensile strength, distance of the film before breaking (N/mm²). For this, a cylindrical needle of 0.14 cm in diameter was used at a speed of 1 mm/s and a distance traveled of 10 mm (Zivanovic et al., 2005).

2.3.2 Color measurements

Optical properties of the films were assessed using a colorimeter (Colorgard System/05) operating in transmittance mode. The color parameters were expressed according to the Hunter scale (L, a, b*). Five measurements were made for each repetition and type of film made (Ojagh et al., 2010).

2.3.3 Structural analysis

Structural analysis was carried out using atomic force microscopy (AFM), employing a Nanosurf Naio-AFM microscope (Nanosurf, Liestal, Switzerland) provided with silicon carbide tips, and the measurements were done in contact mode. The acquisition and analysis data were performed with the Naio Control Software Version 3.1. All measurements were done in air, under static force operating mode with a set point of 55 nN. Structural analysis was performed with five different samples and four regions of each sample. The shown images are representative of each sample. AFM images were qualitatively evaluated to identify differences in surface morphology, pore formation, and apparent roughness among formulations. The data are representative of 1,000 mm² in total and are reported in mm². (Aguilar-Sánchez et al., 2019).

2.4 Evaluation of probiotic survival

Edible films were stored at 5 °C and 22 °C for 0, 2, 4, 8, 12 and 24 days. At each sampling time, films were aseptically removed from the plates and immersed in tubes containing 9 mL of 0.1% sterile peptone water. These were vortexed vigorously to suspend the adhering cells. Serial dilutions were prepared and viable counts (CFU/g) were determined by the pour-plate method on MRS agar followed by incubation at 37 °C for 48 h. Every test was performed in triplicate.

2.5 Modeling of survival kinetics

Growth or inhibition curves were generated from experimental data by plotting log (N/No), where N was the average number of CFU/g at time (t) and No was the average number of CFU/g at the initial time point. Microbial growth or inactivation curves were fitted using the Weibull model (Equation 1) through nonlinear regression (KaleidaGraph 3.51, Synergy Software, Reading, PA, USA)

log N N o = − b t n (1)

The equations were rewritten following the original Weibull formulation commonly used for microbial survival analysis, and the parameters were defined as follows: N is the viable cell count at time t, N₀ is the initial viable cell count, b is the scale (inactivation) parameter, n is the shape parameter, and t is storage time. The Weibull model corresponds to an upward concave survival curve when n > 1, a downward concave curve when n < 1, and a linear curve when n = 1. The model yields two parameters: b, which indicates the rate of bacterial inactivation, and n, which indicates how the microorganisms survive or die.

The parameters n and b were used to calculate the frequency distribution of resistance. The mode of distribution represents the treatment time at which most of the population dies or is inactivated. The mean value represents the average inactivation time with its variation. Once the values of b and n were determined, the resistance frequency curves were plotted using the following equation (Equation 2):where t _*c* is a measure of the resistance or sensitivity of the organism (Peleg and Cole, 1998).

f t c = b n t c n − 1 ⁡ exp ⁡ − b t c n (2)

2.6 Statistical analysis

For mechanical properties, all determinations were performed in triplicate, and each experiment was performed twice. The results were analyzed through analysis of variance using Minitab v.16 software (Lead Technologies Inc., USA). Statistical differences between results were determined using Tukey’s comparison test (α = 0.05). For L. rhamnosus survival a two-way analysis of variance was performed to compare edible films type and storage temperature.

3 Results and discussion

3.1 Mechanical properties

The mechanical and optical properties of starch and starch/CMC edible films with and without L. rhamnosus are presented in Table 1. Significant differences (p < 0.05) were observed among formulations, indicating that both film composition and probiotic incorporation influenced film characteristics. Control starch films exhibited the greatest puncture force and tensile strength, suggesting a more compact and resistant polymeric network. The incorporation of L. rhamnosus into starch films increased film thickness from 102.4 to 130.3 μm while significantly reducing puncture force (31.7%), tensile strength (44.8%), and elongation (48.8%), indicating that bacterial cells disrupted the continuity of the starch matrix.

TABLE 1. Mechanical and color properties of starch and starch/CMC edible films added with Lactobacillus rhamnosus and controls (without Lactobacillus rhamnosus).

Thickness.

Penetration force.

Tensile strength.

Elongation.

−Means followed by a different superscript letter within a column for each are significantly different (p < 0.05).

The addition of CMC reduced film thickness and increased flexibility compared with starch films. Control starch/CMC (60:40) and starch/CMC (50:50) films showed significantly greater elongation values (7.2 and 8.3 mm, respectively) than starch controls (4.3 mm), confirming the plasticizing and flexibilizing effect of CMC. However, probiotic incorporation significantly decreased puncture force, tensile strength, and elongation in both CMC-containing formulations. For example, tensile strength decreased from 954.2 to 535.4 N/mm² in the starch/CMC (60:40) films and from 711.6 to 438.1 N/mm² in the starch/CMC (50:50) films after the incorporation of L. rhamnosus. These results demonstrate that CMC improved film flexibility but reduced mechanical resistance relative to starch films. Moreover, the presence of probiotic cells altered the polymeric network, leading to thicker films with lower mechanical performance. This behavior suggests that bacterial cells act as discontinuities within the matrix, interfering with polymer–polymer interactions and reducing the cohesion of the film structure.

Similarly, Piermaria et al. (2015) reported that the presence of Lactobacillus causes discontinuities in the matrix, resulting in decreased tensile strength (requiring less force to break the films). These findings suggest that the inclusion of microorganisms may alter the plasticizing effect of glycerol. These authors also noted that in a matrix with glycerol, the presence of microorganisms leads to significantly lower elongation values. Tan et al. (2024) demonstrated that the inclusion of Lactobacillus decreases the film’s stretchability, due to bacterial interference and interaction between macromolecules during the film formation process, leading to decreased polymer network cohesion and, consequently, reducing the film’s ability to stretch. This contrasts with the findings of Semwal et al. (2022), who described that the incorporation of probiotic cells improves the rigidity of the caseinate edible film (due to the low pH in the matrix produced by the lactic acid of the trapped bacteria) and changes the elongation at break, suggesting that bacterial cells reduce the film’s elasticity but with better plasticity (flexible but not brittle), allowing the film to withstand more tension and greater extensibility before breaking.

3.2 Color

CMC-containing films exhibited higher L* values than starch films, indicating greater brightness. The lower a* and b* values observed in the CMC formulations suggest reduced red and yellow color intensity and a less opaque appearance. These results indicate that increasing the CMC content modifies the optical properties of the films and produces a lighter appearance. Bhowmik et al. (2024) indicated that high a and b values indicate dark red and yellow tones, which are suitable for developing food packaging materials. This is consistent with the findings of Coimbra et al. (2023), as starch films with added probiotics also showed a slight increase in yellowish and reddish tones.

3.3 Structural analysis

AFM observations suggest that starch films (Figure 1A) exhibited a more homogeneous surface appearance and fewer visible structural discontinuities than the starch/CMC formulations. Although no pronounced pores were observed, small surface irregularities were present, resulting in a denser and more compact film structure. Additionally, microbial cells appear more compact and orderly within the film structure. In contrast, films containing CMC (Figures 1B,C) showed L. rhamnosus cells to be more dispersed, forming pores and spaces throughout the films, resulting in rougher surfaces and the formation of non-uniform aggregates. This is consistent with the findings of Li et al. (2020), who related the dense and smooth (homogeneous) surface of starch and CMC films (cassava starch/CMC) to good interaction between starch and its components. Irregularities or particles were associated with starch gelatinization. They also proposed that the surface roughness of the starch-CMC films with added probiotics increases with the concentration of LAB due to the aggregation of lactic acid bacteria. The presence of pores may influence permeability to O₂, CO₂, and water vapor. The formation of holes was observed by Ebrahimi et al. (2018), who described that adding LAB to edible films (CMC) increases space formation compared to the control film (only CMC), as microorganisms embed in the film matrix. This is similar to the findings of Huang et al. (2024), who stated that the addition of probiotics suggests a film with a rougher and denser surface, relating these characteristics to the integration of probiotics (embedding probiotics and film fibers). Additionally, Singh et al. (2019) mentioned that during cellular diffusion in the film matrix, bacteria are expected to accumulate in larger pores and structural defects, resulting in a less homogeneous distribution of bacteria. In general, viable bacteria can be effectively trapped in the cross-linked cellulose-based film, and their release largely depends on the film composition and the medium’s pH (Singh et al., 2019).

3.4 Survival analysis

Figure 2 illustrates the survival of L. rhamnosus in different types of films. The initial population of L. rhamnosus in all film formulations was approximately 8.1 log (CFU/g). For starch films stored at 5 °C, probiotic populations remained stable throughout storage. Whereas starch/CMC (60:40) and starch/CMC (50:50) films decreased to 7.4 and 6.1 log (CFU/g), respectively. At 22 °C, final populations were 7.5, 7.1, and 6.1 log CFU/g, respectively. This is consistent with the findings of Ebrahimi et al. (2018), who determined that by the end of 42 days of storage (at 25 °C), there was a loss of 10³ CFU/g in CMC and glycerol films.

The Weibull model provided a suitable fit to all survival curves, with a mean R² value of 0.954. The two-way ANOVA indicates significant differences (p < 0.05) for the two factors analyzed (film type and temperature) as well as their interaction for the b and n values (Table 2). Comparison of the Weibull parameters indicated that starch films stored at 5 °C exhibited the highest probiotic stability, whereas the starch/CMC formulations, particularly at 22 °C, showed significantly faster inactivation rates. These results confirm that both film composition and storage temperature influenced L. rhamnosus survival. As the storage temperature increases, there is an increase in the death rate of L. rhamnosus. Figure 3 confirms that death frequencies are higher at 22 °C, almost 3 to 4 times their value compared to storage at 5 °C, affecting the initial hours of storage. For films containing CMC (condition) represented in Figure 3C, the frequency remains constant over time. The resistance frequency distributions derived from the Weibull model provide information about the heterogeneity of the probiotic population. Peaks located at shorter resistance times indicate that a larger proportion of cells are more susceptible to environmental stress and are inactivated more rapidly. Conversely, distributions shifted toward longer resistance times indicate a more resistant population. In the present study, films stored at 22 °C exhibited frequency distributions concentrated at shorter resistance times, whereas films stored at 5 °C showed broader distributions shifted toward longer resistance times, indicating greater stability of L. rhamnosus under refrigerated storage conditions.

TABLE 2. Weibull model parameters (mean ± standard deviation) of Lactobacillus rhamnosus added to starch and starch/CMC edible films at two different storage temperatures.

^a,bMeans followed by a different superscript letter within a column for each are significantly different (p < 0.05).

4 Conclusion

The incorporation of L. rhamnosus significantly influenced the properties of starch-based edible films. Probiotic addition increased film thickness and reduced puncture force, tensile strength, and elongation compared with the corresponding control formulations, indicating that bacterial cells altered the integrity of the polymeric network. Increasing the proportion of carboxymethylcellulose (CMC) improved film flexibility but was also associated with greater reductions in probiotic viability during storage. AFM observations suggested that starch films exhibited a more homogeneous microstructure, whereas starch/CMC films showed a rougher and more heterogeneous surface with greater pore formation. These structural differences may have contributed to the distinct survival patterns observed among formulations.

Among the evaluated systems, starch films stored at 5 °C provided the highest stability for L. rhamnosus, maintaining viable populations throughout the 24-day storage period. In contrast, starch/CMC formulations showed greater losses in viability, particularly at 22 °C. These findings support the hypothesis that increasing the CMC fraction modifies the film microstructure and mechanical behavior but may reduce the protective capacity of the matrix toward entrapped probiotic cells. Overall, starch-based edible films demonstrated the best balance between mechanical performance and probiotic stability, highlighting their potential as carriers of beneficial microorganisms in functional food-packaging applications. Future studies should incorporate quantitative microstructural analyses and evaluate barrier properties to better understand the relationship between film composition, structure, and probiotic survival.

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Footnotes

Edited by: Shuva Bhowmik, University of Otago, New Zealand

Reviewed by: Maisah Meyhr D’Carmo Sodré, Universidade Estadual de Santa Cruz, Brazil

Natalia Burlaga, Poznan University of Technology, Poland