Enhancing Protoplast Formation of the Probiotic Lactobacillus acidophilus

Abstract

Lactobacillus acidophilus is a widely researched probiotic bacterium with broad applications in health and biotechnology; however, its protoplast formation has not been extensively investigated. This study aimed to optimize conditions for L. acidophilus protoplast formation. Freeze-dried cells were suspended in 20 mM HEPES buffer (pH 7) supplemented with sucrose (1.0 M, 1.5 M, and 2.0 M) to induce hyperosmotic conditions, yielding a final cell density of 10⁸ cells/mL. The suspensions were treated with 125 µg/mL lysozyme and incubated at 37 °C for 30 min, 1 h, or 2 h. Prior to enzymatic treatment, the buffer, lysozyme, and cell suspensions were equilibrated at either 22 °C (room temperature) or 37 °C. Phase contrast microscopy was used to evaluate protoplast formation across all treatment combinations, and a three-way ANOVA was conducted to assess the effects of buffer molarity, incubation time, and temperature. Protoplasts are valuable tools for genetic manipulation, cell fusion, and cell wall studies, yet optimized protocols for their generation in L. acidophilus are lacking. The highest protoplast yield with minimal lysis was observed under 2.0 M sucrose conditions after 2 h of incubation, particularly when all components were equilibrated at 37 °C. Prolonged lysozyme exposure increased lysis, especially at lower buffer molarities. Elevated buffer molarity conferred a protective effect by maintaining cell integrity during enzymatic digestion. These findings highlight the importance of osmotic strength and thermal equilibration in optimizing protoplast formation and provide a reproducible framework for controlled enzymatic treatments in L. acidophilus.

1. Introduction

Probiotics are living microorganisms that, when consumed in adequate amounts, have been shown to offer a range of health benefits to both humans and animals [1]. Among these probiotics, lactic acid bacteria (LAB), such as Lactobacillus acidophilus, has been shown to be heavily researched for their beneficial effects on the gut microflora and their important role in human health and nutrition. Additionally, probiotics have been shown to serve a major role in the food industry through fermented food production, flavor enhancements, and food preservation [2]. Probiotic strains, when added to food products, have been shown to alter texture, taste, aroma, and increase yields and shelf life of products when administered in a sufficient and safe amount [3]. Additionally, L. acidophilus has been reported to contribute to gut health by modifying the intestinal lumen and producing antimicrobial molecules that inhibit harmful pathogenic bacteria from adhering to the gut lining. In a previous study, L. acidophilus was shown to lower the mean duration of C. difficile-associated diarrhea in 73 infants aged 3–24 months [4], suggesting that it may even reduce the duration of C. difficile-associated diarrhea in vulnerable populations [4,5].

Current public interest in probiotics and the Lactobacilli genus has grown rapidly in recent years. This shift most likely is attributed to the rising awareness of personal health and health education, along with a surge in demand for functional foods and supplements. As a result, this has created a huge market for brands and manufacturers to design novel probiotic products and supplements. In 2023, global retail sales of probiotics were reported to be valued at over USD 87 billion globally, with the market projected to grow at a compound annual growth rate (CAGR) of 14.1% from 2024 through 2030, reaching a projected value of USD 220.14 billion by 2030 [6]. Despite the increase in public interest, maintaining probiotic viability during production and digestion has been shown to be a significant challenge. Probiotics are exposed to a myriad of potential physical and chemical stress factors from processing to metabolic activity, such as low pH, digestive enzymes, temperature fluctuations, and osmotic shifts. Additionally, Probiotics must survive the acidic gastric environment and digestive enzymes (pH, acid, and bile resistance) to reach the intestines to promote health benefits. Although Lactobacillus species are considered intrinsically resistant to acid, there are differences between species and strains and their sensitivity at pH values below 3.0 [5,7,8].

In response to these challenges during processing and post-consumption of probiotics, various methods have been implemented to improve probiotic survivability and function. Selective breeding, strain screening, and, more recently, genetic and metabolic engineering have been employed to enhance strain performance. Techniques such as conjugation, transformation, transduction, and protoplast fusion are being used to introduce or enhance desirable traits in probiotic strains.

Among these techniques, protoplast formation has emerged as a powerful tool for enabling precise cellular and genetic modifications. Protoplast formation, the removal of the bacterial cell wall, facilitates genetic manipulation by increasing cell permeability, enabling processes such as gene transfer, cell fusion, and delivery of metabolites or biomolecules. These applications have been extensively utilized in industrial microbiology and biotechnology for strain improvement, vaccine development, and fundamental cell biology studies [9,10]. Despite earlier studies establishing methods for protoplast for mation in various Lactobacillus species, relatively few recent investigations have focused specifically on probiotic strains such as Lactobacillus acidophilus, which can present unique challenges. While protocols exist for related species, including L. delbrueckii and L. casei, they exhibit strain-dependent variability and modest regeneration efficiencies, highlighting the need for species-specific optimization [11,12]. Optimizing protoplast formation protocols in probiotic bacteria is therefore essential to harness their full potential for genetic engineering and industrial applications. Specifically, establishing reproducible methods for protoplast generation in L. acidophilus is essential to expand its use in genetic engineering, functional genomics, and biotechnological innovation.

Many probiotic bacterial cells are highly sensitive to environmental fluctuations, including changes in osmotic pressure, enzyme concentration, and temperature, all of which influence their survival and viability during protoplast formation. In a previous study performed in the 1970s, Bachmann [13] demonstrated that hyperosmotic buffers containing high concentrations of sucrose could prevent premature lysis. However, the ideal conditions for generating stable protoplasts in L. acidophilus have not yet been fully established.

Developing an optimized protocol may be vital for procedures requiring longer enzymatic exposure. Enhancing protoplast stability under these conditions could directly support advances in genetic engineering, targeted drug delivery, and adaptive strain development for both medical and industrial microbiology [14].

The objective of the study was to determine the optimal combination of buffer molarity, temperature, and incubation time, using an optimized amount of lysozyme concentration (125 µg/mL) required for L. acidophilus to produce the highest rate of protoplast. Identifying the best environmental conditions that promote high yield of protoplasts and reduce cell lysis, will contribute to a more robust and reproducible protocol for probiotic applications.

2. Materials and Methods

2.1. Bacterial Strain and Growth Conditions

Lactobacillus acidophilus La-14^® was purchased from CHR Hansen (Milwaukee, WI, USA). A stock cell suspension of Lactobacillus acidophilus (10⁸ CFU/mL) was prepared by placing 0.03 g of frozen L. acidophilus cells into a sterile 15 mL Falcon tube and reconstituting with 10 mL of 20 mM HEPES buffer (pH 7.0). HEPES buffer at pH 7.0 was used to maintain conditions optimal for lysozyme activity and cell stability. This pH falls within the optimal enzymatic range (6.2–7.5) for lysozyme and lactic acid bacteria. The tube was gently shaken 20 times to ensure homogenous suspension. Cell viability was visually confirmed under phase-contrast microscopy prior to lysozyme treatment to ensure batch consistency. L. acidophilus La-14^® was selected for its commercial relevance, robust growth characteristics, and established safety profile as a probiotic strain in food and supplement applications [7].

2.2. Preparation of Protoplast Formation Buffer

HEPES [20 mM, pH 7.0] (H3375; Sigma-Aldrich, MilliporeSigma®, St. Louis, MO, USA) was prepared and sterilized by vacuum filtration through a 0.2 μm Nalgene™ Rapid-Flow™ PES membrane filter (Thermo Fisher Scientific, Waltham, MA, USA) and supplemented with either 1.0 M, 1.5 M, or 2.0 M sucrose to create hyperosmotic conditions. Lysozyme from chicken egg white (RPI crystallized; Thermo Fisher Scientific, Waltham, MA, USA) was used for the enzymatic digestion of the L. acidophilus cell wall. For lysozyme buffer concentration preparation, 5 µg of lysozyme was dissolved in 5 mL deionized water (diH₂O) and filter sterilized using a Choice™ PES (polyethersulfone) syringe filter (0.2 μm, 50 mm; Thermo Scientific™, Thermo Fisher Scientific, Waltham, MA, USA; Cat. No. 5640020) to yield a 1 µg/mL lysozyme-HEPES working solution. A total of 125 µg/mL of lysozyme solution was then mixed with 1.0 M, 1.5 M, or 2.0 M HEPES buffer under sterile conditions to produce the stock protoplast formation buffers.

2.3. Protoplast Formation and Treatment Conditions

Cells were washed twice with protoplast formation buffer (HEPES + 1 M sucrose, pH 7.0) and resuspended in 5 mL of freshly prepared buffer containing lysozyme (final concentration 125 µg/mL). Equal volumes (500 µL each) of the washed cell suspension and HEPES buffer (1.0 M, 1.5 M, or 2.0 M sucrose) were combined under sterile conditions.

To assess the effect of temperature, the cell suspension, HEPES buffer, and lysozyme were each pre-incubated separately for 10 min at either 22 °C (room temperature) or 37 °C before mixing. The pre-tempered solutions were then gently mixed for 20 s and incubated at 37 °C for 30 min, 1 h, or 2 h. All treatments were conducted in sterile, autoclaved glassware. Deionized water (diH₂O) used for buffer preparation was filter-sterilized when containing sucrose or HEPES.

2.4. Experimental Design

The experimental design followed a 3 × 2 × 3 factorial (Figure 1), to evaluate the effects of buffer molarity (1.0 M, 1.5 M, or 2.0 M sucrose in 20 mM HEPES, pH 7.0), solution temperature (22 °C or 37 °C), and incubation time (30 min, 1 h, or 2 h) on L. acidophilus protoplast formation. A total of 18 treatment combinations were evaluated (n = 3 microscope fields per treatment) using phase-contrast microscopy.

Figure 1. Experimental design for optimizing L. acidophilus protoplast yield. The experiment was designed to obtain and optimize the yield of protoplasts by systematically varying several parameters. Cells were incubated with lysozyme in HEPES buffer containing sucrose at either 37 °C or 22 °C for 10 min. Buffer molarity (HEPES), sucrose concentration, and incubation time at 37 °C were further tested to assess their effects on protoplast yield.

All treatment groups received lysozyme at a final concentration of 125 µg/mL for enzymatic digestion. Each component: cell suspension, HEPES buffer at three different molarities (1.0 M, 1.5 M, or 2.0 M), and lysozyme, was pre-incubated independently for 10 min at the assigned temperature (37 °C or 22 °C) before mixing. Following pre-incubation the solutions were gently mixed by pipetting to form a uniform suspension and incubated at 37 °C for the designated timepoints (30 min, 1 h, 2 h) Negative controls consisted of L. acidophilus cells incubated under identical condition without lysozyme treatment.

The experimental design used to optimize the conditions of the formation of Lactobacillus acidophilus protoplasts is shown in Figure 1. The buffer morality, lysozyme (125 µg) exposure/incubation time, and solution temperature were independent variables, and the survival rate of each strain was a dependent variable.

2.5. Microscopy and Quantification of Protoplasts

After incubation, 10 µL of each treated suspension was placed on microscope slides and examined under phase-contrast microscopy (Leica DM6 B, Hamamatsu sCMOS, 40× magnification). Three independent fields were evaluated per treatment.

Protoplast yield percentages were calculated by counting intact protoplast versus lysed or aggregated cells across three microscope fields per treatment. Percentages represent the proportion of protoplast relative to the total observed cells (intact + lysed + aggregated):

$$\mathrm{Protoplast}\mathrm{Yield}\left(\%\right)={\displaystyle \frac{\mathrm{Intact}\mathrm{Protoplasts}}{\mathrm{Total}\mathrm{Cells}\left(\mathrm{Intact}+\mathrm{Lysed}+\mathrm{Aggregated}\right)}}\times 100$$

2.6. Statistical Analysis

A three-way ANOVA (Analysis of Variance) was performed to determine interactions among each independent variables, buffer molarity (1.0 M, 1.5 M, 2.0 M), solution temperature (22 °C, 37 °C), and lysozyme incubation time (30 min, 1 h, 2 h) on the dependent variable, protoplasts formation (% yield). The p-value < 0.05 was statistically different.

3. Results and Discussion

Main effects for buffer molarity, lysozyme incubation time, and solution temperature on protoplast formation are shown in Table 1.

Table 1. ANOVA summary of main effects on protoplast formation (n = 3 replicates per condition).

Variables	Df	Sum Sq	Mean Sq	F-Value	p-Value
Molarity	2	192	96.2	108.2	5.98 × 10−16
Time (Lysozyme)	2	4716	2358.2	2652.9	<2.0 × 10−16
Temperature	1	1	1.5	1.7	0.202

Both molarity and time had statistically significant main effects (p < 0.001) on protoplast formation, whereas, temperature showed no statistically significant main effect on protoplast formation (p = 0.202) (Table 1).

Interactions between the main effects of buffer molarity, lysozyme incubation time, and solution temperature on protoplast formation are shown in Table 2.

Table 2. ANOVA summary of interaction effects on protoplast formation (n = 3 replicates per condition).

Interaction Factors	Df	Sum Sq	Mean Sq	F-Value	p-Value
Molarity × Temperature	2	1279	639.5	719.4	<2 × 10−16
Molarity × Time	4	732	182.9	205.8	<2 × 10−16
Temperature × Time	2	183	91.5	102.9	1.28 × 10−15
3-way interaction (M × T × T)	4	1517	379.2	426.7	<2 × 10−16

Significant interactions were observed between Molarity × Time (p < 0.05) indicating that molarity’s effect depends on how long cells are exposed to lysozyme, Temperature × Time (p < 0.05) showed that temperature’s effect varies with time, and a strong three-way interaction (Molarity × Time × Temperature, p < 0.05), indicated that the effect of incubation time on protoplast formation depends on both osmotic buffer strength and incubation temperature (Table 2).

Although temperature alone did not produce a statistically significant main effect on protoplast formation (p = 0.202), its significant interaction with both buffer molarity and lysozyme incubation time suggests an amplifying effect. This is likely due to both lysozyme and L. acidophilus being optimal at physiological temperatures (~37 °C). Therefore, while temperature may not act independently, it enhances the effects of enzymatic activity and osmotic balance when aligned with the appropriate molarity and exposure duration. This highlights the importance of considering synergistic effects among variables rather than assessing temperature in isolation.

The lower protoplast yields observed when solutions were pre-incubated at 22 °C likely result from reduced lysozyme activity and decreased membrane fluidity in L. acidophilus at sub-physiological temperatures. These findings suggest that pre-tempering all components, including the buffer, lysozyme, and bacterial suspension to 37 °C prior to combination and incubation, may play a critical role in enhancing protoplast formation. Stabilizing the system at physiological temperature before enzymatic treatment appears to optimize conditions for efficient cell wall digestion and protoplast integrity.

To examine the interactive effects of sucrose buffer molarity (1.0 M, 1.5 M, and 2.0 M), lysozyme incubation time (30 min, 1 h, 2 h), and temperature (37 °C and 22 °C) on protoplast formation in L. acidophilus, a three-way ANOVA was conducted. Significant interactions were observed among all three factors (Molarity × Time × Temperature, p < 0.001), indicating that the effect of one factor depended on the levels of the others.

To further explore these interactions, separate one-way ANOVAs were performed within each molarity group across time. These analyses revealed that protoplast formation (%) varied significantly in a molarity-dependent manner, as shown in Figure 2 and Figure 3.

Figure 2. Percentage of protoplast formation at 37 °C under varying molarity concentrations (1.0 M, 1.5 M, and 2.0 M) and incubation times following lysozyme treatment (125 µg/mL). No significant differences were observed across molarity concentrations or timepoints. Data are expressed as mean ± standard error. All treatments share the same letter because no statistically significant differences were observed among molarity or timepoints at 37 °C (p > 0.05).

Figure 3. Percentage of protoplast formation at 22 °C under varying molarity concentrations (1.0 M, 1.5 M, and 2.0 M) and incubation times following lysozyme treatment (125 µg/mL). A significant decrease was observed at 1.5 M (p < 0.05), while changes at 1.0 M and 2.0 M, including between 30 min and 1 h at 1.0 M, were not statistically significant (p > 0.05). Data are expressed as mean ± standard error. Different letters indicate significant differences at p < 0.05.

At 37 °C, the percentage of protoplast was not significantly different across the molarity concentrations (1.0 M, 1.5 M, and 2.0 M) and timepoints (30 min, 1 h, 2 h). A decrease in the overall protoplast production is shown at 1.0 M and 1.5 M with increasing incubation time in lysozyme (125 µg/mL). However, at 2.0 M protoplast percent yield initially decrease from 91% at 30 min to 86% at 1 h, followed by an increase of 87% at 2 h.

Figure 3 displays the percentage of protoplast formation at 22 °C under the same molarity concentrations and incubation timepoints under lysozyme treatment (125 µg/mL). At both 1.0 M and 1.5 M, a decrease in percentage of protoplast formation was observed over time, with yields decreasing from approximately 91% (at 30 min) to 63% (at 2 h) for 1.0 M and from 92% (at 30 min) to 41% (at 2 h) for 1.5 M, respectively. The decrease at 1.5 M was more substantial and statistically significant (p < 0.05). In contrast, protoplast formation at 22 °C remained relatively stable over time at 2.0 M, with yields of 90%, 83%, and 78% at 30 min, 1 h, and 2 h, respectively, showing no significant decrease with prolonged lysozyme exposure (125 µg/mL).

In Figure 4, the control sample of L. acidophilus is displayed by phase-contrast microscopy. The control was exposed to the same pre-incubation and incubation temperatures, and HEPES minus the sucrose-induced hyperosmotic conditions and lysozyme treatment. Phase-contrast images of percentage of protoplasts produced under different treatment conditions are shown in Figure 5 and Figure 6, where L. acidophilus La-14^® was exposed to three different sucrose molarity treatments (1.0 M, 1.5 M, and 2.0 M), each pre-incubated at two solution temperatures (37 °C and 22 °C) and all treated at a fixed lysozyme concentration of 125 µg/mL at three timepoints (30 min, 1 h, 2 h).

Figure 4. Control. Phase-contrast image of untreated Lactobacillus acidophilus. All visible cells correspond to the species. The yellow arrows indicate representative short rod morphologies characteristic of the control condition.

Figure 5. Lactobacillus acidophilus protoplast formation under varying osmotic and incubation conditions at 37 °C. (A–C) Cells treated in 1 M sucrose buffer for 30 min, 1 h, and 2 h, respectively. (D–F) Cells treated in 1.5 M sucrose buffer for 30 min, 1 h, and 2 h, respectively. (G–I) Cells treated in 2 M sucrose buffer for 30 min, 1 h, and 2 h, respectively. Green arrows indicate intact protoplasts; red arrows indicate lysed or aggregated cells. Percentages shown within each panel represent the proportion of protoplasts observed under each treatment condition.

Figure 6. Microscopic visualization of Lactobacillus acidophilus protoplast formation under varying sucrose molarity and incubation times at 22 °C. (A–C) Cells treated with 1 M sucrose buffer for 30 min, 1 h, and 2 h, respectively. (D–F) Cells treated with 1.5 M sucrose buffer for 30 min, 1 h, and 2 h, respectively. (G–I) Cells treated with 2 M sucrose buffer for 30 min, 1 h, and 2 h, respectively. Green arrows indicate intact spherical protoplasts, while red arrows highlight lysed or aggregated cells. Percentages shown within each panel represent the proportion of protoplasts observed under each treatment condition.

At 37 °C, 1.0 M and 1.5 M osmotic conditions, Figure 5A,D show a high initial yield of protoplasts after 30 min, indicated by transformation from rod shaped to a spherical shape noted by the green arrows (92% and 91%, respectively). However, both show a decrease with increased incubation time, as seen in Figure 5B (83%) and Figure 5E (77%) after 1 h displaying more cell lysis and aggregation (noted by the red arrows). In Figure 5C (71%) and Figure 5F (74%), at 1.0 M and 1.5 M osmotic conditions there was a further decline in percent yield of protoplast and increased cell lysis at 2 h incubation in lysozyme (125 µg/mL). This was most likely due to extended exposure to lysozyme, which can increase cell lysis over time. This trend aligns with lysozyme’s known time-dependent enzyme activity on cell wall digestion.

Cells treated with 2.0 M maintained protoplast integrity across all timepoints, with similar yields of 91% at 30 min (Figure 5G), 86% at 1 h (Figure 5H) and 87% at 2 h (Figure 5I). These results suggest that a higher molarity provides an osmotic protection effect against extended enzymatic degradation. Bachmann [13] similarly reported that hyperosmotic buffers reduce protoplast lysis during enzymatic wall digestion, although only a general range was reported for protoplast stability. Our findings provide quantitative insights into how varying sucrose molarity directly affects protoplast stability in L. acidophilus La-14, with 2.0 M sucrose providing superior protection against cell lysis during extended lysozyme exposure at different temperatures.

At 22 °C, (Figure 6A–I) show protoplast formation following a similar pattern but with lower overall yields, especially at longer incubation times. At 1.0 M, protoplast production decreased from 91% (30 min, Figure 6A) to 63% (2 h, Figure 6C). A similar decline was seen in 1.5 M, with a decrease from 92% (30 min, Figure 6D) to 82% (1 h, Figure 6E) to 41% by 2 h (Figure 6F) These results indicate that enzyme activity at ambient temperatures (22 °C), may reduce the efficacy of protoplast formation due to temperature-dependent lysozyme activity and optimal growth temperature of L. acidophilus (37 °C) thereby altering cell membrane permeability.

Prior studies have shown that lysozyme exhibits higher activity at near-physiological temperatures (37 °C), which may explain the better preservation of protoplasts under warmer conditions [11]. With increased molarity to 2.0 M (Figure 6G–I), similar protective effects were observed where protoplast production maintained a consistent yield of 93% from 30 min (Figure 6C) to 77% at 2 h (Figure 6I), suggesting the significant role hyperosmotic conditions play on protoplast survival under long-term exposure to lysozyme.

Enhanced performance of the 2.0 M buffer at both temperatures suggests a buffering and protective role of high sucrose concentrations, particularly over longer exposure times. This aligns with earlier hypotheses on protoplast stabilization via osmotic strength and expands on them by applying the concept specifically to L. acidophilus, a species whose protoplast behavior has not been extensively characterized [13].

Protoplasts were easily distinguishable via phase-contrast microscopy (Figure 5 and Figure 6). Intact protoplasts appeared as enlarged, spherical cells, while lysed cells presented as fragmented cellular debris. This distinction is supported by prior studies on bacterial envelope visualization using phase-contrast imaging [14].

37 °C was more favorable due to the natural growth temperature of L. acidophilus, and the molarity buffer at 2.0 M sucrose provided significant protection against cell lysis, maintaining protoplast viability over time. These results suggest that protoplast efficacy and viability depend on both lysozyme enzymatic activity (temperature-dependent) and osmotic protection (linked to sucrose molarity). High molarity buffers exert osmotic pressure that stabilizes the cytoplasmic membrane after cell wall removal, reducing the risk of osmotic shock and maintaining membrane integrity [13]. In the absence of a rigid peptidoglycan layer, cells become highly susceptible to lysis due to internal turgor pressure. The inclusion of sucrose at higher concentrations (e.g., 2.0 M) in the HEPES buffer provides a hypertonic environment, reducing the risk of lysis and maintaining membrane integrity. Our results reinforce this concept, with 2.0 M consistently providing superior protection during extended lysozyme exposure across both tested temperatures.

Although temperature was not a significant main effect in our ANOVA, appears to modulate protoplast formation through its influence on lysozyme activity and bacterial physiology. Lysozyme functions optimally near 37 °C, which accelerates cell wall digestion and may improve protoplast yield at earlier timepoints. Additionally, L. acidophilus is thermophilic, with an optimal growth temperature around 37 °C. Experiments at this temperature not only enhances enzymatic action but also supports better membrane fluidity, metabolic health, and cellular stress response, all of which may contribute to improved protoplast integrity and viability.

Optimization of protoplast formation in probiotic strains like L. acidophilus opens opportunities for novel applications in probiotic engineering, including the development of designer probiotics engineered to carry therapeutic, metabolic, or adhesive functions. Safe strain modification using protoplast fusion or homologous recombination is increasingly feasible and may be more acceptable than plasmid insertions or chemical mutagenesis. Furthermore, while protoplast fusion is an artificial approach, it conceptually parallels natural mechanisms of horizontal gene transfer such as transformation and conjugation, which facilitate genetic exchange and evolution in bacteria. By enabling direct cytoplasmic mixing and recombination between parental strains, protoplast fusion offers a controlled means to harness similar genetic outcomes in vitro. Thus, this work not only enhances our understanding of protoplast biology but may also contribute to the development of next-generation probiotics for industrial, clinical, or dietary use.

L. acidophilus La-14^® was selected due to its relevance in therapeutic and industrial applications, widespread commercial use, and rigorous safety and functional characterization. It’s compatibility with human microbiota, for health benefits, makes it a strong candidate for probiotic enhancement studies that can potentially be tailored for targeted delivery, improved viability to stressors, or enhanced metabolic function.

Future studies may improve many biotechnological applications, such as protoplast fusion, gene transfer, and mucosal delivery systems, where sustained protoplast viability is critical [10,13]. Longer survivability under enzymatic exposure may enhance transformation efficiency, reduce production losses, and create more resilient lactic acid bacteria for food biotechnology, pharmaceutical delivery, or strain enhancement [11].

4. Conclusions

In conclusion, the study evaluated the effects of lysozyme exposure time, buffer molarity, and solution temperature on protoplast formation in L. acidophilus bacteria. Results showed that prolonged lysozyme exposure reduced protoplast formation yield due to cell lysis. However, a 2.0 M sucrose-buffered HEPES solution molarity played a significant role in providing a protective effect, maintaining cell integrity, and supporting higher protoplast survival even after extended enzymatic treatment. These findings suggest the critical role of osmotic conditions and temperature conditioning in minimizing cell damage during protoplast generation. Optimizing these conditions is essential for developing reproducible and efficient protoplast protocols in L. acidophilus. Such protocols could facilitate future applications in genetic engineering, strain improvement, and functional genomics, enabling broader use of this probiotic in biotechnological and industrial microbiology contexts.