Cytotoxic effects of Lactiplantibacillus plantarum i-ul4 postbiotics cultivated in different media against mcf-7 human breast cancer cells

Postbiotics are receiving vast attention for their potential benefits in cancer therapy. The cytotoxicity of postbiotics produced by Lactiplantibacillus plantarum I-UL4 have been demonstrated on breast cancer cells and were affected by the components of the selective de Man Rogosa Sharpe (MRS) growth medium. The aim of this study was to compare the cytotoxic activities of postbiotics produced by L. plantarum I-UL4 on breast cancer cells using different media formulated by Plackett Burman statistical design (PBD). This study prepared 16 types of postbiotics in two forms: freeze-dried (FP) and liquid postbiotics (LP). The bacteriocin inhibitory activities of LP were determined by agarwell diffusion assay using Pediococcus acidilactici 4-46 as an indicator for quality assessment prior to cytotoxicity determination. The cytotoxicity of FP was subsequently determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay using MCF-7 breast cancer cells and MCF10A non-malignant breast epithelial cells as a control, respectively. The effects of medium components on the bacteriocin inhibitory and cytotoxic activities of postbiotics were determined by PBD. The cytotoxicity mechanism on MCF-7 breast cancer cells was elucidated with Annexin V Fluorescein-5-isothiocyanate assay for the selected postbiotics. The protein concentrations of selected liquid and freeze-dried media and their respective postbiotics were compared by Bradford and Modified Lowry assays. Different bacteriocin inhibitory activities were detected for different postbiotics produced by L. plantarum I-UL4 cultivated in different media. The highest bacteriocin inhibitory activity was achieved by the reported LPRMRS postbiotic. Yeast extract from all the formulated media contributed to the highest percentage of bacteriocin inhibitory activity of postbiotics (80.42%). Similarly, the results of cytotoxic activities indicated clearly that the medium composition and the MTT incubation time greatly influenced the cytotoxic profile of FP on MCF-7 cells. Yeast extract only demonstrated a positive effect on the cytotoxic activities of FP at 72 hr of incubation. Most minerals contributed to the cytotoxic activities of FP throughout the incubation period. FM9 and FM12 media and their respective FP were selected for the subsequent study of postbiotic cytotoxicity mechanisms on MCF-7 cells due to their potent cytotoxicity. FP12 induced higher apoptotic MCF-7 cells as compared to FP9 from 24 hr to 72 hr. In comparison with the reported control media, minute toxicity was observed for all FP, except for FPRMRS. All FP induced lower toxicity towards MCF10A cells from 24 hr to 72 hr of incubation as compared to the cytotoxic activities against MCF-7 cells, The reported freeze-dried FM (FFM) media and its respective FP (FPFM) induced higher percentage of late apoptotic MCF-7 cells as compared to MCF10A cells at 48 hr and 72 hr. Different protein concentrations were detected for the selected liquid and freeze-dried media and their amino acids. In conclusion, the medium composition greatly affected the cytotoxic activity of postbiotics produced by L. plantarum I-UL4.

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