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Growing interests in replacing conventional preservatives and antibiotics in food and pharmaceutical industries have driven the exploration of bacterial metabolites, especially those from strains with generally recognized as safe (GRAS) status, such as lactic acid bacteria (LAB). In this study, a supernatant cocktail derived from multiple LAB strains was prepared and its bioactivities-antimicrobial, antibiofilm, antioxidant, cytotoxicity, and stability-were thoroughly investigated. The cocktail's main components were identified using thermal and protease treatments, gas chromatography coupled to mass spectrometry (GC-MS), and flame ionization detection (GC-FID). The results demonstrated that the supernatant cocktail had a broad inhibition spectrum and was effective against food-related bacterial indicators with the highest activity observed on Bacillus cereus ATCC9634 (inhibition zone sizes 12.33 mm) and the lowest on Enterococcus faecium DSM 13590 (3.31 mm). It showed dose- and time-dependent delaying effects on the growth of tested fungi. Regarding the antibiofilm activity, it was more effective in preventing biofilm formation (40% biofilm mass reduction) than in degrading preformed biofilm (20% reduction). Additionally, the cocktail showed antioxidant capacity of 10.1 ± 0.3 g Trolox equivalent (TE)/kg and dose-dependent cytotoxicity on HEK-293 and HT-29 cell lines. The main bioactive compounds in this cocktail are organic acids (particularly acetic acid), volatiles, and bacteriocin-like compounds. The antimicrobial capacity of this supernatant cocktail was highly reproducible across different fermentation batches, and it remained highly stable at 4 °C. Overall, these findings provided novel insights into the functional potentials of LAB metabolites, broadening their application as customizable biopreservatives for food and pharmaceutical industry., Competing Interests: Declarations. Conflict of Interest: The authors declare no competing interests., (© 2024. The Author(s).)

This study is focused on the analysis of regional variation of the chemical compositions of three Clinopodium menthifolium subsp. ascendens (Jord.) Govaerts Tunisian accession, as well as their inhibition toward fungi and insect pests. The diversity of the chemical constituents and biological activities in front of the aforementioned variations was found to be remarkable. Essential oils were obtained by hydrodistillation of the aerial parts and analyzed by gas chromatography-mass spectrometry (GC-MS). A total of 41, 42, and 30 compounds were identified respectively from Clinopodium menthifolium essential oils harvested from three Tunisian regions, namely Ain-Draham (ADEO), Babouch (BEO), and Tabarka (TEO). All analyzed oils were rich in oxygenated monoterpenes with different major constituents. Piperitenone (34.5%), cis-piperitone oxide (26.1%), and pulegone (47.9%) were the dominant compounds in the three volatile oils, respectively. The antifungal activity was investigated in vitro using six targeted fungal strains (Aspergillus flavus, Aspergillus terreus, Candida albicans, Microsporum canis, Microsporum gypseum, and Trichophyton mentagrophytes). The toxicity and repellency of essential oils were evaluated against the stored product pest Tribolium confusum. The tested samples were differently effective toward the target fungi and the pest depending on the variability of their chemical compositions. BEO exhibited the highest fungitoxic properties toward A. terreus mold, M. canis dermatophyte, and C. albicans yeast (the MIC values ranged from 40 to 400 μg mL -1 ). In addition, the data showed that TEO repelled T. confusum moderately (PR = 42.5% at 2 h after exposure). Concerning the contact treatment, both ADEO and BEO were proved to possess slightly toxic effects toward T. confusum pest (% of mortality 27.5-32.5% at 5% concentration). The results showed that the geographic origin greatly influenced the chemical composition and the associated bioactivities of Clinopodium menthifolium subsp. ascendens.