1. Sample Collection and Isolation of LAB
A total of 20 sediment and seawater samples were collected along the Alexandrian Mediterranean coast, Egypt, under aseptic condition using 50 ml sterile Falcon tubes and kept at 4°C. LAB were isolated from the collected samples on de Man, Rogosa, and Sharpe (MRS) agar media (Difco, Detroit, MI, USA) by using the spread plate technique and incubated at 30°C for 48 hrs under anaerobic conditions generated by AnaeroPack (Mitsubishi™ AnaeroPack-Anaero, Oxoid) (El Khateeb et al., 2022). Gram-positive isolates with catalase-negative activity were preserved on MRS agar slants at 4°C for routine use or maintained in 30% glycerol at − 80°C for long period storage.
2. Antimicrobial assay of selected LAB isolates
LAB isolates were inoculated in MRS broth, incubated at 30°C for 48 hrs, and cells were collected by centrifugation (6,000 ×g for 15 min, 4°C). Cell-free supernatants were adjusted to pH 7.0 by using 1N NaOH, filter-sterilized through 0.45 µm pore size filters (Sartorius, Göttingen, Germany), and considered “crude extract” (Rajaram et al., 2010). The antimicrobial activity of all crude extracts of the LAB isolates was tested against seven bacterial strains, including Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Klebsiella pneumonia ATCC 13883, Pseudomonas aeruginosa ATCC 27853, Salmonella typhimurium ATCC 14025 and Bacillus coagulans JCM 2257 and Enterococcus faecalis JCM 5803, using the agar well diffusion method (Hamdan et al., 2016). Mueller-Hinton agar media were inoculated with 1% overnight cultures of the target bacteria. After solidification, wells of 8 mm in diameter were punched out using sterile cork borers. Each well was loaded with 100 µL of the crude extract, and the plates were placed in a refrigerator for 2 hrs to allow for the diffusion of the designated extract. Subsequently, all plates were incubated aerobically at 37°C for 24 hrs and the diameter of the inhibition zones around each well was recorded in mm.
3. Identification of LAB
The putative LAB isolate, which exhibited strong antimicrobial activity, was identified using the API 50 CHL test kit (BioMérieux, Lyon, France), as described by the manufacturer, and scored after 24 and 48 hrs incubation at 30°C. Interpretation of the fermentation profiles was facilitated using the computer-aided database API-WEB™ V.5.0 software (Ameen et al., 2020). Additionally, the selected LAB isolate was identified by 16S rRNA gene sequencing following the method of (El Khateeb et al., 2022). The obtained sequences were compared to those in the NCBI database using BLAST algorithm online tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
4. Extraction and purification of bacteriocin
The selected LAB isolate was inoculated in MRS broth and incubated at 30ºC for 24 hrs. Cells were harvested by centrifugation (6,000 ×g for 15 min, 4°C), and the supernatant was filter-sterilized and adjusted to pH 7.0. The cell-free culture supernatant was saturated with 70% ammonium sulfate and the mixture was stored with continuous stirring for 24 hrs at 4ºC to precipitate out proteins and was later centrifuged at 8,000 ×g at 4°C for 15 min. The collected precipitate was resuspended in 0.1 M potassium phosphate buffer (pH 7.2) and dialyzed against the same buffer for 12 hrs at 4ºC using tubular cellulose acetate membrane (1000 Da cut-off, Sigma-Aldrich, Germany) (Rajaram et al. 2010). The crude bacteriocin extract was freeze-dried and kept for further purification.
Purification of the lyophilized protein was carried out by RP-HPLC (Agilent 1200 series Technologies Inc., USA) using C18 reverse-phase column, following the method of (Zhang et al., 2018). Eluted fractions were collected and tested for antibacterial activity by the spot-on-lawn assay described by Oliveira et al. (2017). In brief, 10 µl of each fraction was spotted onto a double-layered Muller Hinton agar plates inoculated with 1% of Klebsiella pneumonia ATCC 13883 and incubated at incubation condition 30ºC for 24 hrs, where the appearance of inhibition zones around each spot was examined and expressed in arbitrary unit per milliliter (AU/ml) using the following formula (Hamdan et al., 2016):
$$\:\text{A}\text{r}\text{b}\text{i}\text{t}\text{r}\text{a}\text{r}\text{y}\:\text{u}\text{n}\text{i}\text{t}\:(\text{A}\text{U}/\text{m}\text{l})\:=\:\left(\text{z}\text{o}\text{n}\text{e}\:\text{o}\text{f}\:\text{i}\text{n}\text{h}\text{i}\text{b}\text{i}\text{t}\text{i}\text{o}\text{n}\:\right(\text{m}\text{m})/\text{v}\text{o}\text{l}\text{u}\text{m}\text{e}\:\text{o}\text{f}\:\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}\:\text{l}\text{o}\text{a}\text{d}\text{e}\text{d})\:\text{x}\:1000$$
5. Measurement of molecular weight and concentration of bacteriocin
In each purification step, bacteriocin concentration was calculated by the method of Bradford (Bradford, 1976) using bovine serum albumin (BSA) as a protein standard. Additionally, the molecular weight of the protein compounds was determined by 15% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) as described by Ge et al. (2016). The gel was stained with Coomassie brilliant blue R-250 (Sigma-Aldrich, Germany) and the molecular weight of the protein band was compared with standard markers (10–170 kDa).
6. Amino acid sequence analysis
The selected protein band was excised from the SDS-PAGE gel and eluted in phosphate buffered saline (PBS) following the technique which described by (Castellanos-Serra et al., 1997). Amino acid sequencing was performed by Edman degradation analysis using an Applied Biosystems (ABI) model 477A protein sequencer with an online ABI model 120A phenyl thiohydantoin amino acid analyzer (Cornwell III et al., 1988). The obtained amino acid sequences were compared with those in the GenBank database using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) for identification of the bacteriocin.
7. In vitro Cytotoxicity Activity
The cytotoxicity of the pure bacteriocins was assessed in vitro on three human cancer cell lines; A549 (human lung carcinoma), HepG2 (human liver hepatocellular carcinoma), and MCF-7 (human breast carcinoma) using the microculture tetrazolium (MTT) assay (Razak et al., 2019). Cells (1.0 × 105) were treated with varying concentrations of the pure compound (ranging from 10 µg/mL to 10 mg/mL) in a 96-well microplate and incubated at 37°C for different durations (12, 24, and 48 hours). Subsequently, 20 µL of MTT solution (5 mg/mL) was added to each well and incubated for 4 hours at 37°C, then the addition of 200 µL of Dimethyl sulfoxide (DMSO) to dissolve the purple MTT-formazan crystals. The absorbance of each well was measured at 560 nm using a microtiter plate reader (ELISA reader). Cell viability percentages in each well were calculated using the following formula:
$$\:\text{C}\text{e}\text{l}\text{l}\:\text{V}\text{i}\text{a}\text{b}\text{i}\text{l}\text{i}\text{t}\text{y}\:\text{\%}\:\text{=}\left(\frac{{\text{A}}_{\text{c}}\text{-}{\text{A}}_{\text{t}}}{{\text{A}}_{\text{c}}}\right)\text{×100}$$
Where; "Ac" is the absorbance of the untreated cells, and "At" is the absorbance of the treated cells.
8. Antioxidant Activity against DPPH
By following Gulcin & Alwasel (2023), DDPH solution of 6 × 10− 5 M was prepared in methanol. A volume of 1 ml of pure bacteriocin was added to 1 ml of methanolic DPPH radical solution. The sample was incubated at room temperature in the dark for 30 min after mixing vigorously, and the absorbance was measured at 517 nm. Activity was calculated using the following formula:
$$\:\text{D}\text{P}\text{P}\text{H}\left(\text{\%}\right)=\left(\frac{{\text{A}}_{\text{b}\text{l}\text{a}\text{n}\text{k}}-{\text{A}}_{\text{t}\text{e}\text{s}\text{t}}}{{\text{A}}_{\text{b}\text{l}\text{a}\text{n}\text{k}}}\right)\times\:100$$
Where; "Ablank" is the absorbance of the untreated cells, and "Atest" is the absorbance of the treated cells.