Assessment of the Bacterial Content of Commercially Available Probiotic Products Containing Lactic Acid Bacteria and Their Probiotic Potentials in Jos
Abstract
Probiotic products containing lactic acid bacteria (LAB) are widely promoted for their health benefits, including support for vaginal and gastrointestinal health. However, concerns have emerged regarding the accuracy of product labeling and the viability of the claimed bacterial strains. This study is aimed at evaluating the microbial quality and probiotic potential of three commercial probiotic products marketed for vaginal health. LAB counts were determined, and isolates were identified based on standard microbiological methods. Probiotic properties were evaluated by testing tolerance to acidic pH, bile salts, phenol, and various temperatures, as well as antibacterial activity against selected pathogens. Results showed a significant discrepancy between labeled claim and actual viable counts. Product A retained only 0.96% of its labeled claim; Product B retained 0.12%, while Product C had no viable LAB. Strains isolated from Products A and B included Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium lactis, and Bifidobacterium bifidum. These isolates demonstrated varying degrees of acid, bile, and phenol tolerance, and several showed strong antagonistic activity against Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae. All isolates were resistant to cloxacillin, ceftriaxone, and ceftazidime but remained sensitive to ciprofloxacin and levofloxacin. The use of CLSI standards, while offering methodological consistency, has limitations for nonclinical strains and should be interpreted cautiously. In conclusion, only two of the three evaluated probiotic products contained viable and functionally relevant LAB strains, raising concerns about quality assurance in commercial probiotics. The results highlight the need for stricter regulatory oversight and routine postmarket validation of probiotic formulations. A limitation of this study is the exclusive use of culture-based methods, which may not detect nonculturable but viable organisms. Future studies should incorporate molecular techniques for a more comprehensive microbial assessment and to verify the presence of transferable antibiotic resistance genes.
1. Introduction
Probiotics, particularly lactic acid bacteria (LAB), have gained growing recognition for their role in supporting women’s reproductive and vaginal health, in addition to their more widely known gastrointestinal benefits [1, 2]. Defined as live microorganisms that confer health benefits to the host when administered in adequate amounts [3, 4], probiotics, especially strains from the genera Lactobacillus and Bifidobacterium, play a significant role in maintaining the balance of the vaginal microbiota [5, 6].
In a healthy vagina, Lactobacillus species dominate the microbial community, producing lactic acid and other antimicrobial substances that help maintain an acidic pH (3.8–4.5), inhibit the growth of pathogenic bacteria, and prevent infections such as bacterial vaginosis (BV), candidiasis, and urinary tract infections [7–9]. Probiotic products targeting vaginal health are designed to restore or support this protective microbiome, particularly in cases of microbial imbalance caused by antibiotics, hormonal changes, or infections [10, 11].
Given their essential role in vaginal health, probiotic strains intended for urogenital use must meet specific criteria, including the ability to adhere to vaginal epithelial cells, produce antimicrobial compounds, survive in acidic environments, and compete with pathogenic organisms [12, 13]. Despite the increasing availability of probiotic products marketed for vaginal health—ranging from oral capsules to vaginal suppositories, concerns remain about the accuracy of labeling, strain viability, and true probiotic potential [14, 15].
Many products claim to contain beneficial LAB strains, yet discrepancies between labeled and actual colony-forming units (CFUs), strain identity, and functional properties have been reported [16]. Additionally, regulatory oversight for probiotic products targeting vaginal health remains limited, contributing to the circulation of formulations with questionable efficacy and safety profiles [17, 18]. The growing reliance on probiotics for promoting health and preventing diseases calls for a robust scientific approach to guarantee the safety and effectiveness of these products. Validating the claims made by probiotic manufacturers requires comprehensive research on the viability of probiotic strains during storage, their stability in different formulations, and their safety across various population groups. These studies are crucial for differentiating products that provide real health benefits from those that may be ineffective or potentially harmful [19, 20].
The present study seeks to address these concerns by validating the probiotic content and potential of three commercial products marketed for vaginal health. Specifically, the study aims to (1) verify the accuracy of the labeled CFUs by comparing them with actual viable counts and (2) assess the probiotic characteristics of the LAB isolates, including their tolerance to low pH and presence of phenol, antibiotic susceptibility, and antimicrobial activity against common vaginal pathogens. By evaluating both the labeling accuracy and functional potential of LAB strains, this study contributes to quality assurance and evidence-based use of probiotics in women’s health.
2. Materials and Methods
2.1. Sample Collection
Three (3) different commercial probiotic products containing LAB species were purchased from stores in Jos metropolis to isolate LAB species. Probiotic samples were collected on the basis of their availability in stores. The products tested are shown in Table 1. Products were stored at room temperature in a cool and dry place after purchase as recommended by manufacturers. All products were analyzed prior to their expiry dates and were designated as Products A, B, and C to maintain confidentiality regarding the manufacturers’ identities.
| Product | Manufacturer’s claim | |
|---|---|---|
| Labeled organisms | Concentration | |
| A |
|
3 billion active organisms/11 mg |
| B |
|
5 billion active organisms/20 mg |
| C | Lactobacillus spores | No claim |
2.2. Sample Preparation
One capsule from each probiotic product was suspended in 10 mL of phosphate-buffered saline (PBS) and vortexed thoroughly to ensure an even distribution of cells. Serial dilutions were then prepared up to fivefold in PBS.
2.3. LAB Count
One milliliter volume of each dilution was inoculated onto deMan, Rogosa, Sharpe (MRS) agar using pour plate method. The MRS plates were incubated anaerobically at 37°C for 24 h. Colony counts were recorded and expressed as CFU per gram. The percentage of detected bacterial growth compared to the label claim was calculated by using the formula: actual count (CFU/g)/label claim (CFU/g) × 100.
2.4. Isolation and Characterization LAB Species
After enumeration of bacterial counts, pure isolates were obtained after repeated subculturing in MRS broth and on MRS agar. The pure isolates were identified as LAB based on their gram reaction, catalase test, and sugar fermentation profiles using API 50 CH system [21].
2.5. Probiotic Properties and Safety Assessment of the Isolates
The isolated LAB species were subjected to the following tests to evaluate their probiotic potential.
2.5.1. Temperature Tolerance
Temperature tolerance of all LAB isolates was tested by inoculating overnight culture on MRS agar and incubating at 30°C, 37°C, and 40°C for 24 h. Colony counts were recorded and expressed as CFU per milliliter [14].
2.5.2. Acid Tolerance
Overnight culture of the test organism was inoculated into sterile PBS, adjusted to pH value 3.0 and 2.0 using 1N hydrochloric acid (HCl). Another PBS without pH adjustment was used as control. The cultures were incubated at 37°C for 3 h. After incubation, a 0.5 mL aliquot of the bacterial suspension was inoculated onto MRS agar plates and incubated at 37°C for 24 h to measure the total viable count of the bacterial cells [14, 22].
2.5.3. Bile Salt Tolerance
Bile tolerance was tested according to the method described by [23] with modification. An overnight culture of the test organism was inoculated into MRS broth containing 0.3% bile salt. The test organism was also inoculated in MRS broth without bile, which acted as the control. Both cultures (with and without bile) were incubated at 37°C for 3 h. After incubation, a 0.5 mL aliquot of the bacterial suspension was inoculated onto MRS agar plates and incubated at 37°C for 24 h. The tolerance to bile salt was estimated by comparing viable cell counts in MRS agar with and without bile salt.
2.5.4. Antibiotic Sensitivity
Antimicrobial susceptibility testing was conducted using the Kirby–Bauer disk diffusion method on Mueller–Hinton agar [21, 24]. The antibiotics tested included the following: cloxacillin (CXC) (5 μg), ofloxacin (OFL) (5 μg), Augmentin (30 μg), ceftazidime (CAZ) (30 μg), cefuroxime (30 μg), ceftriaxone (CRX) (30 μg), erythromycin (ERY) (5 μg), gentamicin (5 μg), clindamycin (DA) (2 μg), ciprofloxacin (CIP) (5 μg), levofloxacin (LEV) (5 μg), and vancomycin (VAN) (10 μg). Bacterial suspensions, adjusted to a 0.5 McFarland standard for turbidity, were spread onto Mueller–Hinton agar plates, and antibiotic disks were placed on the surface. Plates were incubated at 37°C for 24 h, after which the diameters of the inhibition zones around the antibiotic disks were measured. Based on the size of the inhibition zones, organisms were classified as sensitive (S), intermediately resistant (I), or resistant (R) according to the Clinical and Laboratory Standards Institute (CLSI) 2019 guidelines.
2.5.5. Antagonistic Activity Against Some Pathogens
Antagonistic activity of the LAB strains was performed against three vaginal isolated pathogens (Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae) using the agar well diffusion test as described by [25]. The LAB isolate was inoculated in MRS broth and incubated at 37°C for 24 h. A 200 μL of the test pathogen was seeded onto the surface of Mueller–Hinton agar plates. Wells were bored into the agar. A 100 μL of CFS (cell-free supernatant) obtained by centrifugation of the test culture (LAB strains) at 3000 rpm for 15 min was added into the wells and then incubated at 37°C for 24 h. The antagonistic activity of the LAB strain was determined by measuring the diameter of the zone of inhibition around the wells.
2.5.6. Phenol Tolerance
As described by [23], overnight culture of the test organism was inoculated into MRS broth supplemented with 0.5% v/v phenol. The test organism was also inoculated into MRS broth without phenol, which acted as the control. Both cultures (with and without phenol) were incubated at 37°C for 3 h. After incubation, 0.5 mL aliquot each was inoculated onto MRS agar plates and incubated at 37°C for 24 h. The cell viability was measured by counting the number of colonies and expressed in CFUs per milliliter.
2.5.7. Hemolytic Activity
Hemolytic activity was evaluated using the protocol described by [26]. A blood agar plate containing 5% (w/v) sterile blood was infected with a colony of the test organism, and the plate was then incubated at 37°C for 48 h. Following incubation, β-hemolysis (a clear zone), α-hemolysis (a greenish zone), or γ-hemolysis (no zone) was detected on the plate.
2.6. Statistical Analysis
A descriptive analysis was performed using chi-square at a 95% confidence interval to determine the prevalence of BV and its associated risk factors. The analysis was conducted using SPSS software Version 26.
3. Results
The result for LAB count of the probiotic products is presented in Table 2. For Product A, the label claim was 3 billion cells (3.0 × 109) per capsule; the expected cell count per gram was 2.71 × 108, while the LAB count was 2.60 × 105CFU/mL achieving only 0.96% of the label claim. Product B achieved only 0.12% of the label claim, while Product C had no CFUs.
| S/N | Label claim | |||
|---|---|---|---|---|
| Product | CFU/capsule | Expected CFU/g | LAB count (CFU/g) | Percentage of label claim |
| A | 3.0 × 109 | 2.70 × 108 | 2.60 × 105 ± 3.2 × 104 | 0.96% |
| B | 5.0 × 109 | 2.50 × 108 | 2.93 × 105 ± 2.7 × 104 | 0.12% |
| C | No label claim | Not stated | No growth | |
Table 3 presents the bacterial species isolated from the three products, A and B. Product C had no growth. Four bacterial species were isolated from Product A including Lactobacillus acidophilus (A1), Lactobacillus plantarum (A2), Lactobacillus rhamnosus (A3), and Bifidobacterium lactis (A4). Three species were isolated from Product B, namely, L. acidophilus (B1), L. plantarum (B2), and Bifidobacterium bifidum (B3). No species was isolated from Product C.
| Product | LAB species isolated | Isolate ID | Label claim |
|---|---|---|---|
| A |
|
|
14 species |
| B |
|
|
8 species |
| C | — | Lactobacillus spores | |
3.1. Probiotic Properties and Safety Assessment of the Isolates
The bacterial count of the isolates at three different temperatures 30°C, 37°C, and 40°C is shown in Table 4. The highest counts were observed at 37°C, while lower counts were recorded at 30°C and 40°C, with the lowest growth at 40°C.
| Isolate ID | Temperature tolerance | ||
|---|---|---|---|
| 30°C | 37°C | 40°C | |
| A1 (L. acidophilus) | 3.2 × 102 | 5.4 × 104 | 1.5 × 102 |
| A2 (L. plantarum) | 6.4 × 101 | 8.9 × 103 | 1.7 × 101 |
| A3 (L. rhamnosus) | 1.3 × 102 | 2.2 × 104 | 3.4 × 101 |
| A4 (B. lactis) | 2.4 × 102 | 7.7 × 103 | 1.0 × 102 |
| B1 (L. acidophilus) | 2.4 × 102 | 2.9 × 104 | 3.1 × 101 |
| B2 (L. plantarum) | 1.4 × 102 | 9.4 × 103 | 2.3 × 101 |
| B3 (B. bifidum) | 2.5 × 101 | 4.4 × 103 | 1.4 × 101 |
The tolerance of the isolates to various stress conditions (acid tolerance, bile salt tolerance, and phenol tolerance) is presented in Table 5. At pH 2, A3 and B3 exhibited the highest survival rates, 64.5% and 55.0%, respectively, showing strong resistance to highly acidic conditions. A1 also demonstrated moderate tolerance with a 35.7% survival rate, while A4 and B1 had very low survival rates, indicating poor resistance to stomach acid. At pH 3, most isolates showed improved survival. A3 (83.9%) and B3 (85.0%) continued to exhibit high acid tolerance, while A1 and B1 significantly improved to 78.6% and 77.8%, respectively. A4, which was more S at pH 2, showed a better survival at pH 3, though the tolerance was still lower compared to other isolates. The isolates showed varying degrees of bile tolerance, with survival rates ranging from 42.5% to 80.0%. A1 and B1 showed the highest survival rates, with 71.4% and 80.0%, respectively. A4 has the lowest survival rate at 42.5%. For phenol tolerance, B1 showed the highest tolerance, with a survival rate of 71.1%, followed closely by A1 at 69.1%. B2 and B3 exhibited moderate phenol tolerance, with survival rates of 56.4% and 57.5%, respectively. A3 had the lowest phenol tolerance, with a survival rate of 31.9%.
| Isolate ID | Control | Acid tolerance at pH 2 | Survival rate (%) | Acid tolerance at pH 3 | Survival rate (%) |
|---|---|---|---|---|---|
| A1 (L. acidophilus) | 4.2 × 103 | 1.5 × 103 | 35.7 | 3.3 × 103 | 78.6 |
| A2 (L. plantarum) | 3.5 × 103 | 6.5 × 102 | 18.6 | 9.5 × 102 | 27.1 |
| A3 (L. rhamnosus) | 3.1 × 103 | 2.0 × 103 | 64.5 | 2.6 × 103 | 83.9 |
| A4 (B. lactis) | 4.0 × 103 | 3.6 × 102 | 9.0 | 2.0 × 103 | 50.0 |
| B1 (L. acidophilus) | 4.5 × 103 | 1.7 × 102 | 3.8 | 3.5 × 103 | 77.8 |
| B2 (L. plantarum) | 5.5 × 103 | 1.0 × 103 | 18.2 | 3.1 × 103 | 56.4 |
| B3 (B. bifidum) | 4.0 × 103 | 2.2 × 103 | 55.0 | 3.4 × 103 | 85.0 |
The isolates showed varying degrees of bile tolerance, with survival rates ranging from 42.5% to 80.0%. A1 and B1 showed the highest survival rates, with 71.4% and 80.0%, respectively. A4 has the lowest survival rate at 42.5%. For phenol tolerance, B1 showed the highest tolerance, with a survival rate of 71.1%, followed closely by A1 at 69.1%. B2 and B3 exhibited moderate phenol tolerance, with survival rates of 56.4% and 57.5%, respectively. A3 had the lowest phenol tolerance, with a survival rate of 31.9% (Table 6).
| Isolate ID | Control | Bile tolerance (0.3%) | Survival rate (%) | Phenol tolerance at 0.5% | Survival rate (%) |
|---|---|---|---|---|---|
| A1 (L. acidophilus) | 4.2 × 103 | 3.0 × 103 | 71.4 | 2.9 × 103 | 69.1 |
| A2 (L. plantarum) | 3.5 × 103 | 2.3 × 103 | 65.7 | 1.9 × 103 | 54.2 |
| A3 (L. rhamnosus) | 3.1 × 103 | 1.8 × 103 | 58.1 | 9.9 × 102 | 31.9 |
| A4 (B. lactis) | 4.0 × 103 | 1.7 × 103 | 42.5 | 1.9 × 103 | 47.5 |
| B1 (L. acidophilus) | 4.5 × 103 | 3.6 × 103 | 80.0 | 3.2 × 103 | 71.1 |
| B2 (L. plantarum) | 5.5 × 103 | 3.5 × 103 | 63.6 | 3.1 × 103 | 56.4 |
| B3 (B. bifidum) | 4.0 × 103 | 2.3 × 103 | 57.5 | 2.3 × 103 | 57.5 |
Antibiotic sensitivity of the isolates is presented in Table 7. All the LAB strains showed complete resistance to CXC, CAZ, and CRX. Variable sensitivity to OFL, VAN, ERY, and DA was observed. Most strains were S to both LEV and CIP, though intermediate resistance was seen in some strains, particularly B2 and B3.
| Isolate ID/antibiotics | A1 | A2 | A3 | A4 | B1 | B2 | B3 |
|---|---|---|---|---|---|---|---|
| CXC (5 μg) | R | R | R | R | R | R | R |
| OFL (5 μg) | S | I | R | S | I | I | S |
| AUG (30 μg) | S | S | I | S | R | S | S |
| CAZ (30 μg) | R | R | R | R | R | R | R |
| CRX (30 μg) | R | R | R | R | R | R | R |
| GEN (5 μg) | I | S | S | R | S | S | I |
| CTR (30 μg) | R | R | I | R | R | R | 1 |
| ERY (5 μg) | R | R | I | R | I | I | R |
| DA (2 μg) | S | I | I | R | R | R | I |
| VAN (10 μg) | I | S | S | S | I | R | S |
| LEV (5 μg) | S | S | R | S | S | I | I |
| CIP (5 μg) | R | S | S | I | S | I | S |
The antibacterial activity of the LAB strains varied across the tested pathogens (E. coli, S. aureus, and K. pneumoniae). Strain A3 exhibited the strongest inhibition, particularly against K. pneumoniae (17.7 mm) and S. aureus (17.0 mm). Strains A4, B1, B2, and B3 showed moderate to strong antibacterial effects, with strain B3 showing consistent inhibition across all pathogens, especially S. aureus (16.0 mm). Strains A2 and B2 displayed moderate inhibition, particularly against K. pneumoniae (Table 8).
| Isolate ID | Diameter of zone of inhibition (mm) | ||
|---|---|---|---|
| A1 (L. acidophilus) | E. coli | S. aureus | K. pneumonia |
| A2 (L. plantarum) | 13.1 | 14.0 | 14.6 |
| A3 (L. rhamnosus) | 15.2 | 17.0 | 17.7 |
| A4 (B. lactis) | 15.4 | 12.2 | 13.6 |
| B1 (L. acidophilus) | 14.2 | 15.0 | 14.7 |
| B2 (L. plantarum) | 13.8 | 13.9 | 15.1 |
| B3 (B. bifidum) | 14.1 | 16.0 | 15.4 |
Strain A1 had the highest optical density (0.70), indicating the greatest bacterial growth, followed by B3 (0.66), A3 (0.65), and B1 (0.56). Strains A2 (0.36) and B2 (0.39) showed lower optical density values, suggesting comparatively reduced bacterial growth. All strains show no hemolytic activity (Table 9).
| Isolate ID | Optical density at 600 nm | Biofilm formation capacity | Hemolytic activity |
|---|---|---|---|
| A1 (L. acidophilus) | 0.70 | Strong | No hemolysis |
| A2 (L. plantarum) | 0.36 | Moderate | No hemolysis |
| A3 (L. rhamnosus) | 0.65 | Strong | No hemolysis |
| A4 (B. lactis) | 0.50 | Strong | No hemolysis |
| B1 (L. acidophilus) | 0.56 | Strong | No hemolysis |
| B2 (L. plantarum) | 0.39 | Moderate | No hemolysis |
| B3 (B. bifidum) | 0.66 | Strong | No hemolysis |
4. Discussion
4.1. Discrepancy Between Labeled and Actual Viable Counts
The results revealed significant inconsistencies between the labeled claims and actual viable LAB counts of the probiotic products. Product A achieved only 0.96%, Product B achieved 0.12%, and Product C had no viable count. These findings are consistent with previous studies that have reported a widespread lack of compliance with probiotic label claims, often attributed to factors such as inadequate storage, exposure to heat, and prolonged shelf life, all of which can compromise bacterial viability [18, 27]. Additionally, the formulation process, such as encapsulation or freeze-drying, might not have adequately protected the bacterial cells during production [28]. Of particular concern is the claim that Product C contains Lactobacillus spores which is misleading, as Lactobacillus species are non–spore-forming bacteria. This fundamental error, coupled with the complete absence of viable CFUs in the product, raises serious concerns about the authenticity and quality of the formulation. The lack of viable organisms suggests that the product may never have contained live bacteria or that they were rendered nonviable due to poor manufacturing, storage, or labeling practices. Such discrepancies not only undermine consumer trust but also pose potential health risks, particularly for individuals relying on probiotics for therapeutic or preventive support.
Also, the method used for quantifying viable bacterial cells might have influenced the low recovery rates in Products A and B. Although the LAB count method used in this study is a culture-based approach, it is widely regarded as the most reliable method for quantifying viable and functional probiotic organisms. While plate counts are the most widely used method for enumerating viable bacteria, they often underestimate the total number of cells due to the inability of some bacteria to form colonies under standard growth conditions [29, 30]. However, they remain the global standard because culturable organisms are considered functionally relevant for probiotic efficacy.
4.2. Identification of Bacterial Strains
Strains identified from Products A and B included L. acidophilus, L. plantarum, L. rhamnosus, B. lactis, and B. bifidum. These are species commonly found in both vaginal and gut ecosystems. Their presence supports the intended function of the products; however, this was only evident for Products A and B. The complete absence of bacterial growth in Product C raises serious concerns about the authenticity of its label claims or the possibility of bacterial death due to suboptimal manufacturing or storage conditions [31, 32]. The discrepancy between the listed and isolated organisms may also reflect variations in product quality or degradation of viable strains over time. Although this study relied on culture-based methods for bacterial identification, which are widely accepted for evaluating viable probiotic content, such methods have limitations, as not all strains may grow optimally under uniform incubation conditions. Therefore, incorporating molecular techniques such as polymerase chain reaction (PCR) and metagenomic sequencing in future studies could allow for a more comprehensive and accurate assessment of both culturable and nonculturable bacterial species present in probiotic products [33, 34].
4.3. Probiotic Potential of the Isolates
All isolates showed optimal growth at 37°C, consistent with the human body temperature and indicative of their potential to survive and function in vivo. LAB species thrived best at body temperature, an essential trait for colonization and competitive exclusion of pathogens. However, reduced growth at 30°C and 40°C suggests that temperature variations during storage or transport may significantly affect product efficacy, highlighting the need for cold chain maintenance in the probiotic supply system [35].
The acid tolerance of probiotic strains is a critical determinant of their ability to survive the harsh gastric environment and reach the target site of action in the host. This means that acid tolerance is an essential quality for probiotics [35, 36]. At pH 2, only Strains A3 and B3 showed high survival rates (64.5% and 55.0%, respectively), indicating strong acid resistance and suggesting their potential for effective gastric transit. Strain A1 displayed moderate acid tolerance (35.7%), while A4 and B1 demonstrated poor survival, indicating vulnerability under highly acidic conditions. When the pH was raised to 3.0, survival rates improved across all strains. A3 and B3 maintained their high acid tolerance (83.9% and 85.0%, respectively), and Strains A1 and B1 also showed significant enhancement in survival (78.6% and 77.8%, respectively), indicating that a slight reduction in acidity substantially improves viability. These findings are consistent with previous studies reporting strain-specific differences in acid tolerance among LAB [35].
Bile tolerance, a key probiotic trait, was highest in Strains A1 and B1, indicating their potential to survive in the small intestine where bile salts are present [37]. Strain A4, which exhibited poor bile tolerance, may be less effective as a probiotic in vivo. These findings are consistent with other studies suggesting that strains with strong bile tolerance are more likely to persist in the intestinal environment and provide health benefits [38].
Phenol tolerance varied among the strains, with B1 showing the highest resistance. Phenol, a toxic compound produced by gut bacteria during protein fermentation, was best tolerated by Strain B1, followed by A1. Strains capable of tolerating phenol are better suited for survival in the human gut and vagina and may reduce the harmful effects of these toxic metabolites [39]. Although Strain A3 demonstrated strong acid tolerance, its low phenol tolerance may limit its effectiveness in environments with higher phenol concentrations.
Antibiotic resistance among LAB strains is a growing concern, as probiotics with resistance genes can potentially transfer these genes to pathogenic bacteria in the gut [40]. In this study, all strains were R to CXC, CAZ, and CRX, which are in line with recent research highlighting the prevalence of antibiotic resistance in commercial probiotics [41, 42]. The strains showed sensitivity to fluoroquinolones such as LEV and CIP, which are commonly used to treat bacterial infections. While resistance to certain antibiotics may be intrinsic to LAB strains and not necessarily transferable, the potential for horizontal gene transfer of acquired resistance genes remains a safety concern, particularly when probiotics are used in large populations. Screening of probiotic strains is highly recommended to ensure they do not harbor transmissible resistance determinants [43].
The antibacterial activity of the LAB strains against pathogens such as E. coli, S. aureus, and K. pneumoniae is an important aspect of their probiotic potential. Strain A3 exhibited the strongest antibacterial activity, particularly against K. pneumoniae and S. aureus, consistent with studies showing that LAB strains produce bacteriocins and other antimicrobial compounds that inhibit pathogen growth [44]. Strain B3 also demonstrated consistent inhibition across all pathogens, indicating its potential as a broad-spectrum probiotic. However, the variability in antibacterial activity among the strains suggests that not all LAB isolates are equally effective against pathogens, which has been corroborated by other studies [45].
The absence of hemolytic activity in the tested strains is a positive feature, ensuring they do not damage red blood cells, aligning with safety standards for probiotics [46]. This trait is shared with most established probiotic strains, and any hemolytic activity observed in other studies would require careful strain selection to avoid harmful effects [47].
All strains demonstrated biofilm formation, which can enhance their effectiveness by providing protection against pathogens and antibiotics. Biofilm formation is common among LAB strains and contributes to their resilience in hostile environments [48]. Recent studies show that stronger biofilm-forming strains have improved resistance to gastrointestinal conditions [47], and biofilm formation is critical for LAB survival and colonization in the gut and vaginal mucosa [49].
4.4. Limitations of Study
Several limitations should be acknowledged in this study. First, the reliance on culture-based methods may have led to an underestimation of the bacterial species present in the probiotic products, as some strains may have been nonculturable under the conditions used. Second, the study did not assess the long-term viability of the probiotics, which is crucial for determining their effectiveness over time. Future studies should incorporate molecular techniques such as qPCR or metagenomic sequencing to provide a more comprehensive analysis of the bacterial composition in probiotic products [50]. Furthermore, in vivo studies would be necessary to validate the probiotic potential of these strains, as in vitro assays may not fully capture the complexities of the gut and vagina environment.
5. Conclusion
This study revealed significant discrepancies between probiotic product label claims and actual bacterial counts, emphasizing the need for stricter quality control within the probiotic industry. The probiotic potential of the isolated strains varied, with some demonstrating strong resistance to acid and bile, as well as antibacterial activity and antibiotic resistance. The challenges in assessing probiotic efficacy were further highlighted by the limitations of culture-based methods, indicating a need for additional molecular research. These findings contribute to the ongoing discussion regarding the quality and effectiveness of probiotics in commercial products and suggest avenues for future research and product development.
Conflicts of Interest
The authors declare no conflicts of interest.
Funding
No funding was received for this manuscript.
Open Research
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.
