Probiotic assessment of herbal mixtures sold in Awka Metropolis

 Corresponding author: Obi, C. P Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. Probiotic assessment of herbal mixtures sold in Awka Metropolis Obi CP, Umeh SO and Anaukwu CG Department of Applied Microbiology and Brewing, Faculty of Biosciences, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria. GSC Biological and Pharmaceutical Sciences, 2025, 32(03), 114–124 Publication history: Received on 26 July 2025; revised on 06 September 2025; accepted on 08 September 2025 Article DOI: https://doi.org/10.30574/gscbps.2025.32.3.0347 Abstract Herbal mixtures are among the important traditional healthcare medicines used in Awka metropolis, Nigeria, with some forms undergoing fermentation, showing the presence of microorganisms. This study aimed to assess the probiotic potential of lactic acid bacteria (LAB) isolated from 10 (ten) herbal mixture samples sold in Awka. LAB were isolated, characterized, and identified, with molecular confirmation by 16S rRNA sequencing. Their probiotic attributes were also evaluated through in vitro assays, including tolerance to acidic pH, bile, and NaCl, assessment of cell surface hydrophobicity, autoand co-aggregation abilities, cholesterol assimilation, and antimicrobial activity against common clinical pathogens( Escherichia coli, Salmonella spp and Staphylococcus aureus).Results showed the presence of Lactobacillus guizhouensis (MRSAI3), Lactobacillus helveticus (MRSAI7) and Lactobacillus terrae (MRSAI8 ).These strains demonstrated superior probiotic characteristics in the laboratory tests. They showed a high survival rate under challenging digestive conditions, such as low pH and high bile concentrations. The strains also had strong adhesion capabilities, including the ability to stick to surfaces and to other bacteria. They displayed antimicrobial activity, effectively inhibiting the growth of pathogens like E. coli (inhibition zones up to 18.06 mm). The strains were also able to assimilate cholesterol (up to 22.18%). The study confirmed that herbal mixtures are a promising source of beneficial indigenous bacteria with therapeutic potential for humans. Keywords: Herbal Mixtures; Probiotics; Lactic Acid Bacteria; In Vitro; Lactobacillus species 1. Introduction Herbal mixtures are complex mixtures of plant parts used for centuries across diverse cultures for their medicinal properties. In Nigeria, they are deeply rooted in traditional healthcare, treating a wide array of health conditions (Oladimeji, 2022). These complex mixtures, are consumed in various forms and are known to exert their therapeutic effects through a myriad of bioactive compounds (Rojas et al., 2022). Beyond their direct phytochemical composition, the microbial ecology of these mixtures, particularly through fermentation, plays a significant role in enhancing their therapeutic value. Fermentation, a metabolic process carried out by microorganisms, can increase the bioavailability of active compounds, synthesize new bioactive molecules, and improve the overall nutritional profile of herbal preparations (Ahtesham, 2016; Cruz-Casas et al., 2021). Lactic acid bacteria (LAB) are key contributors in fermentations, contributing to flavor, preservation, and health-promoting properties. Lactic acid bacteria are a diverse group of Gram-positive, non-spore-forming, facultative anaerobic bacteria, widely recognized for their role in food fermentation and as probiotics. Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host (NCCIH, 2021; Gunnars, 2018). Their beneficial effects stem from various mechanisms, including enhancing gut barrier function, modulating the immune system, producing antimicrobial compounds, competing with pathogens for nutrients and adhesion sites, and contributing to host metabolism (Harikrishnan et al., 2011). Given the traditional use of fermented foods and beverages in Nigeria, it is GSC Biological and Pharmaceutical Sciences, 2025, 32(03), 114–124 115 plausible that some herbal mixtures, especially those prepared through processes involving microbial activity, could harbor indigenous LAB strains with significant probiotic potential. Despite the known benefits, the microbial quality of traditional herbal mixtures is often unregulated, raising concerns about the co-occurrence of pathogenic microorganisms due to unhygienic practices during preparation and handling (Olaniran et al., 2022). However, while many studies focus on the contamination aspects of herbal medicines, some researchers explore their probiotic potential. Qosimah et al. (2023) reviewed the synergistic potential of probiotics and herbal supplements as antibacterial, antioxidant, and immunomodulatory agents. Fadare (2021) demonstrated the in vitro antibacterial synergy between probiotic Lactobacillus strains and garlic extract against Salmonella species. Studies on LAB isolation from traditional fermented foods in Nigeria have identified strains with promising probiotic traits, including acid and bile tolerance, adhesion to epithelial cells, and antimicrobial activity against common pathogens (Chopade et al., 2019; Prabhurajeshwar and Chandrakanth, 2019). These findings showed the potential of traditional foods and, herbal preparations, as reservoirs for probiotic strains. Therefore, this study focuses on the probiotic assessment of bacterial isolates from herbal mixtures sold in Awka metropolis. 2. Materials and Methods 2.1. Sample Collection Ten (10) samples of various herbal mixture preparations were randomly purchased from different retail shops across Awka Metropolis, Anambra State, Nigeria. Samples were grouped as, A1 to A10, whereby A1, A2,A3,A4,A5,A6,A7,A8,A9 and A10 represent Herbal diarrhoea, Herbal cough and catarrh, Herbal stomach ulcer, Herbal pile pain management, Herbal anthritis , Herbal Man power, Herbal hypertension ,Herbal washing and setting, Herbal Herbal infection cure and Herbal malaria and typhoid respectively. which were herbal mixtures that were randomly purchased from different sellers from places (Amawbia, Ifite and Unizik temporary sites) in Awka, Metropolis. 2.2. Sterilization of Glassware and Media Preparation All glassware were thoroughly washed, sterilized in a hot air oven at 120oC for 2 hours, and stored (Sangari et al., 2022). Culture media (De Mann, Rogosa, and Sharpe (MRS) agar and broth for LAB isolation and enumeration) were prepared according to manufacturers' instructions, sterilized by autoclaving at 121oC for 15 minutes, and aseptically dispensed. 2.3. Examination of the Herbal Mixtures: pH Determination The pH values of all collected herbal mixture samples were determined using a digital pH meter, following the protocol by Vijayakumar and Adedeji (2017). 2.4. Microbial Examination: Determination of Lactobacillus Counts One milliliter of each herbal sample was serially diluted, and a 10−3 dilution was plated onto MRS agar using pour plate method. Plates were incubated anaerobically at 37oC for 48 hours for Total Lactic Acid Bacteria Counts (TLC) (Osuntokun et al., 2022). 2.5. Isolation and Identification of Lactic Acid Bacteria (LAB) Developed colonies from MRS agar plates were subcultured by streaking on fresh MRS agar to obtain pure cultures. Pure cultures were obtained and stored on agar slants at 4oC. LAB isolates were characterized based on colonial morphology, Gram staining, and biochemical tests including catalase test and sugar fermentation (glucose, lactose, sorbitol, inositol, xylitol, and mannitol) (Osuntokun et al., 2022). 2.6. Molecular Identification of LAB Isolates Genomic DNA was extracted from selected LAB isolates. The 16S rRNA gene was amplified using PCR, sequencing was performed using the BigDye Terminator kit on a 3510 ABI sequencer (Inqaba Biotechnological, Pretoria, South Africa). Obtained sequences were edited using Trace edit and compared with the NCBI database using BLASTN for species identification (Kumar et al., 2016; Raheem and Adil, 2024). GSC Biological and Pharmaceutical Sciences, 2025, 32(03), 114–124 116 2.7. In vitro Determination of Probiotic Properties Probiotic properties were assessed following the methods of Huqin et al. (2018). 2.8. Tolerance to Acidic pH LAB isolates were grown in MRS broth which was adjusted to pH 5.0, 3.0, and 2.0 with HCl, incubated anaerobically at 37oC for 48 hours. Survival rates were determined by viable counts on MRS agar. 2.8.1. Bile Tolerance This was assessed by using MRS broth supplemented with varying concentrations of bovine bile (0.1 ml, 0.5 ml, 1.0 ml, 2.0 ml). Isolates were incubated anaerobically at 37oC for 48 hours. Survival rates were determined by viable counts. 2.8.2. NaCl Tolerance This was done by inoculating isolates into MRS broth supplemented with 3%, 6.5%, and 10% (w/v) NaCl and observing growth after 48 hours incubation at 37oC. 2.9. Growth at Different Temperatures The specific growth rates and duplication times were determined by monitoring optical density changes of LAB isolates in MRS broth at 30oC and 35oC over 24 hours. 2.10. Cell Surface Hydrophobicity This was determined by measuring the adherence of bacterial cells to hexadecane/xylene (Botthoulath et al., 2018). Absorbance of the aqueous phase was measured at 620 nm. 2.10.1. Auto aggregation This was done by measuring the decrease in optical density (OD) of bacterial suspensions at 600 nm over time (3 and 24 hours) due to cell sedimentation (Chopade et al., 2019). 2.10.2. Co-aggregation The ability of LAB isolates to co-aggregate with clinical pathogens (Escherichia coli, Staphylococcus aureus, and Salmonella sp.) was measured by mixing bacterial suspensions and observing the reduction in OD at 570 nm after 4 hours of incubation (Prabhurajeshwar and Chandrakanth, 2019). 2.10.3. Assimilation of Cholesterol Lactic acid bacteria (LAB) strains were grown in modified MRS broth containing 0.3% oxgall and 100 µg/mL watersoluble cholesterol. Cholesterol content in the supernatant after 12, 24, and 48 hours was determined spectrophotometrically at 550 nm after extraction and reaction with o-phthalaldehyde (Jenny et al., 2023). 2.11. Assay for Antimicrobial Activity of Isolated LAB on Clinical Isolates Cell-free supernatants of LAB isolates were obtained by centrifugation and filtration. Their antimicrobial activity was determined by the agar well diffusion assay against clinical isolates of Escherichia coli, Staphylococcus aureus, and Salmonella sp. (Santos et al., 2016). Inhibition zone diameters were measured after incubation. 3. Results The result of the Probiotic Assessment of Herbal Mixtures Sold in Awka Metropolis as shown below showed the herbal mixtures purchased from Awka Metropolis,Table 1 presents the pH profile of ten different herbal samples (A1-A10), which is an important factor for assessing the chemical composition and its suitability for microbial growth. This is followed by Table 2, which showed the Total Lactobacillus count (TLC) in each herbal sample, providing a quantitative measure of the microbial load of these beneficial bacteria.Then table 3 revealed the morphological and biochemical characterization of the isolates whereby standard microbiological tests, such as Gram reaction, motility, and sugar fermentation, was used to confirm the isolates. The molecular analysis identified the key isolates as Lactobacillus guizhouensis (MRSAI3), Lactobacillus helveticus (MRSAI7), and Lactobacillus terrae (MRSAI8). Table 4 and Table 6 quantitatively assess the isolates' in vitro survival under simulated gastrointestinal stress, specifically examining their GSC Biological and Pharmaceutical Sciences, 2025, 32(03), 114–124 117 tolerance to low pH and high bile concentrations. Table 5(Sodium chloride tolerance of the isolates) and figure 1 and 2(growth at different temperatures) further support the isolates' robustness by examining their tolerance to varying salt concentrations and temperatures. Table 8 measures the isolates' cholesterol assimilation potential, a key therapeutic trait, while Table 9 quantifies their antimicrobial activity by measuring the diameter of inhibition zones against test pathogens. Finally, Figures 3, 4, and 5 showed the isolates' cell surface hydrophobicity, autoaggregation, and coaggregation capabilities, which are essential for adherence and colonization of the intestinal mucosa. Table 1 pH of the herbal mixtures (Mean ± SD) Sample Mean ± SD A1 3.45 ± 0.025 A2 5.40 ± 0.132 A3 2.93 ± 0.076 A4 3.05 ± 0.050 A5 3.93 ± 0.076 A6 4.05 ± 0.050 A7 5.72 ± 0.076 A8 3.65 ± 0.050 A9 4.82 ± 0.104 A10 3.92 ± 0.076 Key • A1 =Herbal diarrhoea A2 = Herbal cough and catarrh • A3 = Herbal stomach ulcer A4 = Herbal pile pain management • A5 = Herbal anthritis A6 = Herbal Man power • A7 = Herbal hypertension A8 = Herbal washing and setting • A9 = Herbal infection A10 = Herbal malaria and typhoid Microbial Examination: Determination of Lactobacillus Counts(logcfu/ml) Table 2 The Lactobacillus loads of herbal samples Sample TLC A1 0.94±0.01 A2 1.25±0.02 A3 2.17±0.01 A4 0.88±0.03 A5 0.99±0.01 A6 1.37±0.04 A7 1.14±0.02 A8 1.19±0.01 A9 1.22±0.01 A10 1.61±0.07 Key • A1 =Herbal diarrhoea A2 = Herbal cough and catarrh GSC Biological and Pharmaceutical Sciences, 2025, 32(03), 114–124 118 • A3 = Herbal stomach ulcer A4 = Herbal pile pain management • A5 = Herbal anthritis A6 = Herbal Man power • A7 = Herbal hypertension A8 = Herbal washing and setting • A9 = Herbal infection A10 = Herbal malaria and typhoid Table 3 Morphological and Biochemical Characteristics of the Lactobacillus species Parameter MRSAI1 MRSAI2 MRSAI3 MRSAI5 MRSAI7 MRSAI8 Appearance Cream White White Light cream White Cream Edge Entire Entire Entire Entire Entire Entire Elevation Convex Convex Convex Convex Convex Convex Gram Reaction + + + + + + Shape Rods Rods Rods Rods Rods Rods Motility - - - - - - Catalase - - - - - - Indole - - - - - - Methyl red + + + + + + Citrate - - - - - - Glucose + + + + + + Lactose + + + + + + Sorbitol +/- + +/- +/- + +/- Arabinose + +/- +/- + + +/- Inositol + +/- + + + +/- Xylitol +/- +/- + + + + Key; - =Negative;+ = Positive;The molecular analysis identified the notable Lactobacillus isolates as Lactobacillus guizhouensis (MRSAI3), Lactobacillus helveticus (MRSAI7) and Lactobacillus terrae (MRSAI8) Table 4 Effect of Low pH on the Growth of the Isolates Isolate pH = 2.0 (S% at 3h) pH = 2.0 (S% at 5h) pH = 3.0 (S% at 5h) pH = 5.0 (S% at 3h) pH = 5.0 (S% at 5h) MRSAI1 95.88±1.18 93.03±4.35 92.98±2.09 87.59±11.95 89.48±1.94 MRSAI2 41.95±0.99 41.95±0.99 90.51±2.07 87.59±1.93 89.47±1.93 MRSAI3 86.13±1.97 88.66±2.01 92.87±2.12 90.06±2.02 91.77±2.03 MRSAI5 27.96±0.70 43.51±1.03 92.63±2.11 73.43±1.78 79.12±1.86 MRSAI7 93.58±2.04 94.00±2.17 95.81±2.22 92.72±2.16 92.72±2.16 MRSAI8 90.29±2.05 92.46±2.11 96.52±2.24 88.10±2.07 89.00±2.07 Key: S% = Percentage of Survival GSC Biological and Pharmaceutical Sciences, 2025, 32(03), 114–124 119 Table 5 Sodium Chloride Tolerance of the Isolates Isolates Percentage of Nacl 1 2 3 4 5 6 7 MRSAI1 + + + + + + - MRSAI2 + + + + + + +/- MRSAI3 + + + + + + + MRSAI5 + + + + + + - MRSAI7 + + + + + + + MRSAI8 + + + + + + + + = Presence of growth - =No growth 3.1. Growth at Different Temperatures Figure 1 Growth at 30oC Figure 2 Growth at 35oC GSC Biological and Pharmaceutical Sciences, 2025, 32(03), 114–124 120 Table 6 Effect of Bile on the Growth of the Isolates Isolates S% (0.1mL) S% (0.5mL) S% (1.0mL) S% (2.0mL) MRSAI1 93.33±2.17 92.36±2.12 91.38±2.13 90.10±2.07 MRSAI2 65.26±1.61 24.47±0.61 24.47±0.61 24.47±0.61 MRSAI3 90.78±2.20 85.67±2.02 53.38±1.29 27.96±0.69 MRSAI5 88.86±2.11 71.18±1.74 66.97±1.63 44.48±1.09 MRSAI7 94.66±2.21 90.34±2.18 88.10±2.07 85.67±2.02 MRSAI8 91.38±2.13 90.1±2.07 88.10±2.07 85.67±2.02 S% = Percentage of Survival 3.2. The Invitro Survival in Gastric Juice The result of the Invitro Survival of the lactic acid bacteria isolates in Gastric Juice are shown in the table below Table 7 The Invitro Survival in Gastric Juice Isolate CFU/mL after 3hours CF/mL after 5hours MRSAI1 78.33±1.88 40.13±0.96 MRSAI2 31.33±0.76 16.63±0.40 MRSAI3 78.33±1.88 30.37±0.71 MRSAI5 20.57±0.51 13.70±3.78 MRSAI7 16.63±0.40 10.77±0.25 MRSAI8 81.23±1.96 36.23±0.86 3.3. The Cholesterol Assimilation Potential of the Isolates The result of the cholesterol assimilation potential of the lactic acid bacteria isolates are shown in the table below Table 8 Cholesterol Assimilation Potential of the Isolates Isolate 12 hours (A%) 24 hours (A%) 48 hours (A%) MRSAI1 8.37±0.19 12.46±0.31 16.31±0.41 MRSAI2 2.48±0.06 8.13±0.19 9.95±0.25 MRSAI3 8.86±0.22 12.55±0.31 19.00±0.47 MRSAI5 5.04±0.13 5.93±0.15 11.46±0.28 MRSAI7 1.54±0.04 6.7±0.17 12.54±0.31 MRSAI8 10.01±0.25 13.94±0.35 21.69±0.56 Key: A % = Percentage Assimilation GSC Biological and Pharmaceutical Sciences, 2025, 32(03), 114–124 121 Figure 3 Cell Surface Hydrophobicity of the test isolates A% = percentage of Absorbance Figure 4 Auto aggregation Potential of the Isolates Figure 5 Coaggregation Potential of the Isolates GSC Biological and Pharmaceutical Sciences, 2025, 32(03), 114–124 122 Table 9 Diameter Zones of Inhibition of Lactic acid bacteria Against the Test Pathogens Lactic acid bacteria Mean Diameter Zone (Ⴟ± SD) mm MSAI1 EMBAI6 SSAI3 MRSAI1 11.22±0.11 17.62±0.12 14.56±0.07 MRSAI2 8.12 ± 0.07 17.08±0.33 11.74±0.12 MRSAI3 12.46±0.21 18.06±0.11 15.26±0.12 MRSAI5 9.86±0.11 15.87±0.17 13.22±0.08 MRSAI7 10.44±0.07 17.44±0.11 13.18±0.11 MRSAI8 11.82±0.17 17.88±0.22 14.37±0.81 4. Discussion This study assessed the probiotic potential of lactic acid bacteria (LAB) isolated from herbal mixtures sold in Awka metropolis, it showed the presence of beneficial microorganisms within these herbal products. The consistent presence of LAB across various samples, as indicated by total Lactobacillus counts, suggests that certain herbal preparations, possibly due to their fermentation processes or raw material composition, naturally harbor these bacteria. This aligns with knowledge that traditional fermented foods and beverages are rich sources of diverse microbial strains (CruzCasas et al., 2021). The in vitro assessment of LAB isolates also showed that several strains possess key probiotic characteristics. Their remarkable tolerance to acidic pH (as low as pH 2.0) and bile salts is critical, as these conditions are major barriers to microbial survival in the gastrointestinal tract (Huqin et al., 2018). Isolates such as MRSAI1, MRSAI3, MRSAI7, and MRSAI8 exhibited particularly robust survival rates, suggesting their ability to reach and colonize the intestines effectively. This acid and bile tolerance is a criterion for probiotic selection. Furthermore, the ability of several isolates to tolerate high NaCl concentrations indicates their resilience to various environmental stresses. The assessment of adhesion-related properties, including cell surface hydrophobicity, autoaggregation, and coaggregation, showed significant findings.Whereby high autoaggregation capacities among all LAB isolates signify their ability to self-associate and potentially form biofilms in the gut, which is important for sustained colonization and interaction with the host (Chopade et al., 2019; Ivshina et al., 2022). More importantly, the coaggregation abilities of isolates like MRSAI3 and MRSAI8 with common pathogens such as E. coli, S. aureus, and Salmonella sp. Showed a direct mechanism by which these LAB could exert antagonistic effects, preventing pathogen adhesion and colonization. The significant antimicrobial activity demonstrated by the cell-free supernatants of the LAB isolates, especially MRSAI3, further showed their probiotic potential. The production of organic acids, bacteriocins, and other antimicrobial compounds is a primary mechanism by which LAB inhibit the growth of pathogenic bacteria (Santos et al., 2016). The high inhibition zones (mm) against clinical isolates of S. aureus, E. coli, and Salmonella sp. suggest that these LAB strains could be effective in managing or preventing bacterial infections in the gut. Additionally, the observed cholesterol assimilation capabilities of isolates like MRSAI8 and MRSAI3 point towards an added health benefit, as certain probiotic strains can contribute to maintaining healthy cholesterol levels (Jenny et al., 2023). The molecular analysis identified the notable Lactobacillus isolates as Lactobacillus guizhouensis (MRSAI3), Lactobacillus helveticus (MRSAI7) and Lactobacillus terrae (MRSAI8). 5. Conclusion In conclusion, this study showed the ability of the indigenous LAB from herbal mixtures in Awka metropolis as promising probiotic candidates. This research contributes to the scientific understanding of traditional products and opens avenues for making use of local microbial biodiversity of herbal mixtures for health benefits.