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Biomanufacturing

Biomanufacturing - Poster Session E

Poster B1 - Vitalizing Food-Safe Antimicrobial Alternatives for Cultured Meat using Escherichia coli

Saturday, October 26, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Lab-grown, cultured meat begins to address sustainability concerns of traditional industrial harvesting of meat and raising animals thus becoming an increasingly promising solution to environmental concerns of meat production. Antimicrobial agents have been traditionally used in the food industry to prevent or control the growth of harmful microorganisms in food. Traditionally, Pen-Strep is used in mammalian cell culture media to avoid bacterial contamination—an important concern of safe experimentation and preservation of tissue-engineered meat. Nisin, an antibacterial peptide, may be used as an alternative agent FDA Food-Safe preservative in human foods. Nisin is currently used as a natural preservative commercially (ex. canned dairy puddings containing semolina and tapioca). With the motivation to mitigate the impending climate issue catalyzed by industrial meat harvesting, this study examines Nisin’s viability to reduce the contamination of lab-produced meat through the experimentation of Nisin with QM7 cells.

Materials and

Methods:  Nisin Cell Culture Procedure:
QM7 cells (quail myoblast cell line) cultured in QM7 Growth Media (DMEM/F12, 1% Pen/Strep, 10% Fetal Bovine Serum). The cells were measured at 10,000 viable cells and were inoculated into 24 total wells in a 96-well plate. The plated, cultured cells were treated with Nisin Media (4mg Nisin/ 4 Liters Pen/Strep-Free Growth Media (200 μl per well)). Each row was treated with varying concentrations (0 μg/ml, 1 μg/ml, 10 μg/ml, 25 μg/ml, 50 μg/ml, 100 μg/ml, 1000 ug/ml).
Imaging Procedures:
Nisin-treated cells were held in an incubator for 24 hours at 37°C. Post-incubation, the metabolic activity of the cells was analyzed using the standard protocol of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay at 540 mm wavelength as an indication of cell viability, proliferation, and cytotoxicity. An additional Nisin cytotoxicity analysis was conducted using Live/Dead Assay to validate the presence of live and dead cells. Suspensions of live QM7 cells were stained and analyzed through fluorescence microscopy according to the LIVE/DEAD Viability protocol. Imaging validated live cells (green), while cell nuclei are stained with Hoechst (blue) and dead cells (red).

Results, Conclusions, and Discussions: 

Results:
Cultured QM7 cells show no indication of necrosis when exposed to Nisin at varying concentrations indicating its capacity for antimicrobial activity when exposed to, within 24 hours of treatment. The presence of QM7 stained with green (live cells) and blue (cell nuclei) indicates the presence of viable QM7 cells after being treated with Nisin with a consistent percentage viability of >99%. The metabolic activity of the cells, as demonstrated by the results of the MTT assay, did not indicate a notable impact on activity when exposed to varying concentrations of Nisin from control to the highest concentration of 1000 ug/ml. The absorbance reading of the MTT assay showed results along and above the threshold of 540mm, indicating a standard increase in cell proliferation as well as supporting viability.
Discussion and

Conclusions:
The results of this study indicate that Nisin can be used as a safe alternative to Pen-Strep in mammalian cell culture methods. In the exploration of Nisin’s candidacy for the preservation of cellularly cultured meat, Nisin poses no significant differences in cellular metabolic activity, with no dependence on concentration, when cells are treated. Using Nisin in cultured meat production offers a safer and more consumer-friendly alternative to traditional antibiotics. With specificity in targeting harmful bacteria, Nisin ensures the safety and quality of cultured meat. Furthermore, Nisin's established use in food preservation aligns with consumer preferences for natural additives, enhancing the overall appeal and market acceptance of cultured meat products. By improving shelf life and reducing the risk of antibiotic resistance, Nisin contributes to a more sustainable and health-conscious approach to meat production. In future studies, we will assess the effectiveness of Nisin on gram-positive bacteria individually and when exposed to QM7 cells.

Acknowledgements (Optional):