Effect of Exopolysaccharide produced by Levilactobacillus brevis OR921362 on HEK 293 and HeLa cell lines

 

Kondamudi Suman, Aruna Bandi

St Francis College for Women, Umanagar, Begumpet, Hyderabad – 500016, India.

 

ABSTRACT:

The current research involved isolating exopolysaccharide (EPS) producer, Levilactobacillus brevis OR921362 from fermented cucumber using modified MRS media. LB-EPS (exopolysaccharide produced by the isolate) has exhibited antimicrobial activity and dose-response against indicator organisms like P aeroginosa MTCC10307, E coli MTCC452, K pneumoniae MTCC4031, S aureus MTCC3160.  The minimum inhibitory concentration analysis of LB-EPS was investigated using Azithromycin as a standard drug. At the highest concentration of 1000μg/ml LB-EPS has exhibited 65%, 86%, 82%, and 81% inhibition rate, whereas the standard drug has displayed 46%, 79%, 64%, and 78% inhibition against indicator organisms P aeroginosa MTCC10307, E coli MTCC452, K pneumoniae MTCC4031, S aureus MTCC3160 respectively. LB-EPS was further studied for cytotoxicity assessment against the HEK 293 and HeLa cell lines. The percentage of decrease in viability with an increase in concentration of LB-EPS was observed, determining 319.87±2.64 µg/ml as the IC50 value. On HeLa cell line LB-EPS at the highest concentration, the percentage of viability and inhibition was observed to be 33.15% and 66.85% respectively with an IC50 value of 63.75±0.927µg/ml. For a comparative study, Cisplatin was used as a standard drug. Statistically, there was a significant difference in the antitumor activity at different LB-EPS and standard drug concentrations with p<0.05.

 

 

INTRODUCTION: 

A broad range of diversified polymeric forms of carbohydrates are produced by different bacteria particularly Lactic acid bacteria and Bifidobacteria in the process of fermentation¹. Significant interest has been gained in the recent times on exopolysaccharides (EPS) due to their potential applications in pharmacological and biomedical areas². Recent researchers have investigated that EPS have potential health benefits such as anticancer, immunostimulatory activity, antibiofilm activity, and antiviral activity^3-6.

 

Contemporary inventions are related to low-risk medications to treat cancer and especially using antitumor properties of LAB as an important area of study⁷. As stated by Riaz et al⁸ in 2017, LAB EPS may have a scope of non-toxic, active, anti-tumor activities. Chemotherapy is usually the method employed in the process of treating one of the major health issues. Minor to unexpected life-threatening effects can be a consequence of the activities⁹. The vigorous research on biomaterials produced from different sources like EPS from LAB is drawing interest that has anti-tumour activities¹⁰. Substituting of synthetic antitumour agents by LAB EPS can be a future hope as they have less cytotoxicity and side effects^11-13. As stated by Bomfim et al¹⁴, and Neha et al¹⁵, EPS extracted from LAB have beneficial applications as antimicrobial substances, and antioxidant activities and provide high scope in food industries too.

 

MATERIALS AND METHODS:

Isolation and Identification of LAB:

The EPS-producing Levilactobacillus brevis OR921362 was isolated using deMan Rogosa Sharpe (MRS) media¹⁶ from a fermented cucumber sample. EPS was extracted from the bacterial culture using the ethanol precipitation method¹⁷ and subjected to dialysis. The EPS recovered after dialysis was lyophilized and used for further studies. 

 

Minimum inhibitory Concentration (MIC):

The antibacterial activity of Levilactobacillus brevis OR921362 was studied using a 24hr culture and its respective purified EPS against indicator organisms (P aeroginosa MTCC10307, E coli MTCC452, K pneumoniae MTCC4031, S aureus MTCC3160, B subtilis MTCC1133) by agar spot method^18,19 with few modifications. Further MIC was determined by the microtiter broth dilution method²⁰. The 96-well plate was labelled horizontally as AC (antibiotic control), 1000μg/ml, 500μg/ml, 250μg/ml, and 125 μg/ml for LBEPS leaving one well empty the preceding wells were labelled similarly for the standard drug. The last well was labelled as GC (growth control). All the wells were suspended with 100μl of Muller Hinton Broth (MHB) followed by serial dilution of LBEPS and Azithromycin in the respective wells. The concentration of LBEPS taken was 5mg/ml. The standard drug selected for the current study was Azithromycin, 5mg/ml. To the prepared serial dilutions 100 μl of 24 hr culture (0.5Mac Farland standard) of the test organism (s) were added and incubated at 37°C for 24 hr. AC and GC were maintained meticulously. The respective indicator organism cultures were diluted to 0.5 Mac Farland standard. All the experiments were carried out in triplicates. Statistically, analysis of variance (ANOVA) was performed with the aid of GraphPad Prism 10.0.3 to identify the significance of the research work carried out, where p<0.05 was considered significant and p>0.05 as insignificant.

 

In vitro studies on cell line cultures using LB-EPS:

The MTT Cell Proliferation Assay²¹ was performed to determine the anti-tumor activity of EPS²². The proliferation rate of cells and the cell viability reduction experiment were carried out by Synteny Life Science Private Limited, India. Chemicals were procured from Sigma Chemicals Co. (St. Louis, MO), and Fetal Bovine Serum (FBS) was procured from Gibco. 25cm ² and 75 cm² flask and 96 well plated were procured from Eppendorf India. The Cancer cell lines were procured from NCCS, Pune, and the cells were maintained in MEM supplemented with 10% FBS and the antibiotics penicillin/streptomycin (0.5 mL^-1), in an atmosphere of 5% CO ₂ /95% air at 37⁰C.

Cytotoxicity studies on HEK 293 and Antitumor activity on HeLa cell lines:

The cytotoxicity studies were carried out on HEK 293 (Human Embryo Kidney) cell lines and antitumor activity was studied on HeLa (Henrietta Lacks) cell line cultures. With three independent experiments, six concentrations of compounds in triplicates, the viability of the cell was evaluated by the MTT Assay. Cells were trypsinized, and a trypan blue assay was performed to assess the number of viable cells in the cell suspension. The cells were then counted using a hemocytometer and seeded at a density of 5.0 x 10³ cells per well in 100µl of culture medium in a 96-well plate. The plate was incubated overnight at 37°C. After initial incubation, the old media was replaced with 100µl of fresh media containing different concentrations of the test compound, with each concentration placed in separate wells. After 48 hours, the media was discarded, and fresh media containing MTT solution (0.5mg/mL) was added to each well. The plates were then incubated at 37°C for 3 hours. During this incubation, metabolically active cells reduced the MTT salt to formazan crystals, which precipitated in the wells. The optical density of the solubilized formazan crystals in DMSO was measured at 570nm using a microplate reader. The percentage growth inhibition was calculated using the following formula. %Inhibition = 100(Control – Treatment)/Control. The IC₅₀ value was determined by using a linear regression equation i.e., y =mx+c.  Here, y = 50, m and c values were derived from the viability graph.

 

RESULTS:

Isolation and identification:

The lactic acid bacteria isolated from fermented cucumber was identified by 16s rRNA typing as Levilactobacillus brevis OR921362. The extracted EPS from Levilactobacillus brevis OR921362 was labelled as LB-EPS and used for further studies.  

 

Minimum inhibitory Concentration (MIC):

Levilactobacillus brevis OR921362 and its EPS (LB-EPS) were observed to show antibacterial activity towards indicator organisms like P aeroginosa MTCC10307, E coli MTCC452, K pneumoniae MTCC4031, S aureus MTCC3160²³. MIC studies have shown a dose-response activity with increasing concentrations of LB-EPS (Table 1). It was studied against P aeroginosa MTCC10307 (Figure 1), E coli MTCC452 (Figure 2), K pneumoniae MTCC4031 (Figure 3), S aureus MTCC3160 (Figure 4) and observed that with an increase in the concentration of LB-EPS, there was an increase in the inhibitory effect against the indicator organisms.             

 

Table 1. Minimum Inhibitory Concentration of LBEPS. Azithromycin as a standard drug was used in the current study.

	P aeroginosa MTCC10307			E.coli MTCC452		K pneumoniae MTCC4031		S aureus MTCC3160
Concentration	EPS		Azithromycin	EPS	Azithromycin	EPS	Azithromycin	EPS	Azithromycin
	Inhibition in %
125μg/ml	58	38		80	44	59	54	70	71
250μg/ml	60	40		83	48	74	57	72	72
500μg/ml	62	42		86	50	81	63	73	75
1000μg/ml	65	46		86	79	82	64	81	78

 

 

Figure 1. Effect of LB-EPS on P aeroginosa MTCC10307 

 

 

Figure 2. Effect of LB-EPS on E coli MTCC452

 

 

Figure 3. Effect of LB-EPS on K pneumoniae MTCC4031

 

 

Figure 4. Effect of LB-EPS on S aureus MTCC3160

 

In vitro studies to determine the anti-cervical cancer activity of LB-EPS:

 

Cytotoxicity stdies and Antitumor activity:

For safe declaration of LB-EPS usage as a drug, cytotoxicity studies were initially performed. Cytotoxicity studies were carried out on HEK 293 cell line culture. At the highest concentration of LB-EPS (100μg/ml) percentage of viability was observed to be 83.92%, whereas at the lowest concentration (5µg/ml) it was 98.58% (Figure 5) (Table 2). From the observations it was inferred that, the inhibition rate on HEK 293 cells is acceptable, determining 319.87±2.64µg/ml as IC50 value (Figure 6). Human papillomavirus, being considered as the causative agent, Cervical cancer is the fourth most common cancer worldwide. In the current study, LB-EPS from Levilactobacillus brevis OR921362 was considered for investigation of its activity against cervical cancer cell lines. The HeLa cell lines were selected and a dose-response was observed with a gradual decrease in viability proportionate to an increase in concentration of LB-EPS (Figure 7). At the highest concentration, 100μg/ml the percentage of viability and inhibition was observed to be 33.15% and 66.85% respectively (Figure 7). At the lowest concentration of 5μg/ml, the percentage of viability and inhibition was observed to be 94.26% and 5.74% respectively (Figure 8) (Table 2). The IC50 value for this condition was noted to be 63.75±0.927µg/ml. For a comparative study, Cisplatin was used as a standard drug. Statistically after performing Tukey’s Honest Significant Difference post hoc test, it was enumerated as p<0.05 and there was a significant difference among the antitumor activity at different concentrations of LB-EPS. The study was limited to cell line cultures due to economic constraints.

 

 

Figure 5. Cytotoxicity studies on Human Embryonic Kidney cell line culture (HEK293) by LB-EPS                       

Table 2. Effect of LB-EPS on HEK293 and HeLa cell lines with statistical significance, ± indicating standard deviation of mean values (all experiments were run in triplicates)

	HEK293		HeLa
Concentration (µg)	% Viability	Absorbance at 570nm	% Viability	Absorbance at 570nm
5	98.58	0.97±0.004	94.26	0.49±0.003
10	95.83	0.94±0.006	86.79	0.45±0.003
25	92.88	0.91±0.004	68.97	0.36±0.008
50	87.38	0.86±0.008	48.09	0.25±0.008
100	83.92	0.82±0.005	33.15	0.17±0.005

 

 

Figure 6. Graphical representation of cytotoxicity studies on HEK293 cell line culture

 

 

Figure 7. Observations of LBEPS activity on HeLa cell line culture (Henrietta Lacks) showing a decrease in viability with an increase in concentration from 5μg-100μg.

 

Figure 8. Graphical representation of LBEPS activity on HeLa cell line culture showing dose response

 

DISCUSSION:

The indicator organisms used for antimicrobial activity such as P aeroginosa MTCC10307, E coli MTCC452, K pneumoniae MTCC4031, S aureus MTCC3160 were further analysed for Minimum inhibitory concentration, and a dose-response was observed with an increase in the concentration of LB-EPS. The Exopolysaccharide produced by Lactobacillus kefiranofaciens DN1 isolated from kefir has also been reported to show dose response activity towards S. Enteritidis and L. monocytogenes²⁴. Azithromycin was used as a standard drug for comparative study as it is considered as a broad-spectrum antibiotic²⁵. and the study includes both Gram positive and Gram-negative microorganisms similar to Samanje²⁶, and Shanthi studies²⁷. Cytotoxicity studies against HEK 293 cell line culture have shown 83.92% viability at the highest concentration of LBEPS considered (100μg/ml) indicating its acceptance as a drug. Earlier research on EPS against cancer cell lines mostly included Colon cancer (Caco-2) or Colorectal cancer (HT-29) cell line²⁸ culture producing significant effects. The current research was a study using HeLa (cervical cancer) cell line as comparatively less research was done in this area and the EPS used was extracted from Lactobacillus gasseri. EPS of Lactobacillus gasseri in the year 2017 was studied for anti-cervical cancer activity and observed to produce inhibition against HeLa cell line cultures. Earlier studies have shown 73-77% cell death against HeLa cell lines at 400 µg/ml concentration of EPS produced by Lactobacillus gasseri isolated from the human vagina²⁹. Previous investigations on HeLa cells with a density of 1x10⁶ cells/well and EPS concentration of 100μg/ml have shown 36% inhibition. The current study with a cell density of 5.0 X 10 3 cells / well and EPS concentration of 100μg/ml has shown inhibition of 66.85%. Other than EPS, invitro study on HeLa cell lines was earlier performed using Peronema canescens Jack leaves extract³⁰, ethanolic extract of Allium cepa L³¹, ethanol and ethyl acetate extract of Kenikir³², Adhatoda vasica³³, sodium copper chlorophyllin from endangered medicinal plant Rhinacanthus nasutus³⁴, and pentane-2,4-dione extracted from the leaves of Cordia sebestena³⁵.

 

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Received on 07.11.2024      Revised on 17.03.2025 Accepted on 24.05.2025      Published on 01.12.2025 Available online from December 06, 2025 Research J. Pharmacy and Technology. 2025;18(12):5793-5797. DOI: 10.52711/0974-360X.2025.00835 © RJPT All right reserved
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