Pangenome Analysis and Genome-Guided Probiotic Evaluation of Cyclic Dipeptides Producing Levilactobacillus Brevis DY55bre Strain From a Lactic Acid Fermented Shalgam to Assess Its Metabolic, Probiotic Potentials, and Cytotoxic Effects on Colorectal Cancer Cells

Abstract

This study investigates the genetic, metabolic, and probiotic characteristics of Levilactobacillus brevis DY55bre, a strain isolated from the traditional Turkish fermented beverage, shalgam. Whole-genome sequencing revealed a circular genome of 2.485 Mb with a GC content of 45.72%, predicted 2791 genes, and multiple CRISPR-Cas systems. Pangenome analysis demonstrated an open structure, with 18.9% core genes and 103 strain-specific genes, highlighting its genetic diversity. The DY55bre exhibits heterofermentative carbohydrate metabolism due to the presence of the araBAD operon and the lack of 1-phosphofructokinase (pfK) and fructose-1,6-bisphosphate aldolase enzymes. Probiotic evaluation revealed firm survival under simulated gastrointestinal conditions, including resistance to acidic pH (as low as 3.0) and bile salts (up to 1%), along with significant adhesion to intestinal epithelial cell lines (HT29;59.3%, Caco-2;87%, and DLD-1;60.8%). The strain exhibited high auto-aggregation (84.55%) and cell surface hydrophobicity (56.69%), essential for gut colonization. Safety assessments confirmed its non-hemolytic nature and absence of horizontally acquired antibiotic resistance genes. Notably, GC-MS analysis identified bioactive cyclic dipeptides, Cyclo(D-Phe-L-Pro) and Cyclo(L-Leu-L-Pro), which demonstrated cytotoxic effects against colorectal cancer cell lines, with IC50 values of 7.71 mg/mL for HT29 and 3.19 mg/mL for DLD-1. The cell-free supernatant exhibited antimicrobial activity against pathogens, likely due to the synergistic effects of cyclic dipeptides, organic acids, and other metabolites. Antioxidant assays revealed significant ABTS+ (76.63%) and DPPH (34.25%) radical scavenging activities, while cholesterol assimilation tests showed a 27.29% reduction. These findings position the DY55bre as a promising candidate for functional foods, nutraceuticals, and therapeutic applications, warranting further in vivo validation.