Abstract
Helicobacter pylori (H. pylori) infection is a widespread gastric infectious disease, posing significant challenges due to the increasing prevalence of antibiotic resistance. This study aimed to evaluate the synergistic antibacterial activity of Takifugu obscurus skin peptides (TSPs) and the cell-free supernatant of Lactobacillus plantarum WUH3 (LCFS) in developing a potential green and efficient dietary supplement therapy. Using enzymatic hydrolysis and ultrafiltration techniques, the most bioactive peptide fraction, TSPb (1-3 kDa), was identified. The effects of TSPb and LCFS-both individually and in combination-on H. pylori biofilm function, membrane morphology, and internal structure were systematically analyzed using urease activity, N-phenyl naphthylamine (NPN) uptake, nucleic acid leakage, scanning electron microscopy (SEM), and infrared (IR) spectroscopy. The results showed that both LCFS and TSPb significantly inhibited H. pylori urease activity, with inhibition rates of 53.60% and 54.21% at 24 h, respectively, and the highest inhibition rate of 74.64% was observed with their combined treatment. SEM, NPN fluorescence, and nucleic acid leakage analyses revealed distinct mechanisms of action for each treatment. LCFS treatment caused membrane surface loosening and morphological deformation, while TSPb induced the formation of localized membrane pores. IR spectroscopy further confirmed that the combined treatment led to a more severe disruption of the lipid and protein structure within the bacterium. Overall, compared to individual treatments, the combination of TSPb and LCFS exhibited enhanced intracellular penetration and a more significant effect on bacterial viability. This study successfully identified TSPb as a highly bioactive peptide and elucidated its potential synergistic antibacterial mechanism with LCFS. These findings provide scientific evidence for the development of functional antimicrobial foods and gastric health dietary supplements, offering a promising strategy for the prevention and management of H. pylori infections.