Enzyme-assisted extraction of flavanones from citrus pomace: Obtention of natural compounds with anti-virulence and anti-adhesive effect against Salmonella enterica subsp. enterica serovar Typhimurium

Food poisoning has been recognized as a global challenge, causing illness in millions of people. Salmonella enterica subsp. enterica serovar Typhimurium is a major foodborne pathogen causing disease in humans by expressing virulence genes to invade the host epithelial cells. Thus, the search for natural compounds with anti-virulence and anti-adhesive effect could help to mitigate Salmonella infection. This study aimed to determine the effect of flavanones on S. Typhimurium virulence and adhesion to Caco-2 cells. Flavanones were obtained from citrus pomace (CP) by enzyme-assisted (CP-E) and conventional hydroalcoholic (CP–H) extraction methods, which produced extracts rich in aglycones and glycosylated flavanones, respectively. CP-E and aglycone standards (naringenin and hesperetin) decreased bacterial growth (25–90%), motility (50–90%), and adhesion to Caco-2 cells (25–50%). CP-H and glycosylated flavanones (naringin and hesperidin) affected only bacterial adhesion (decreased 20–75%). Agglutination assays demonstrated the binding of extracts and pure flavanones to S. Typhimurium adhesion structures. Fimbrial gene expression (fimA and fimZ) was downregulated in the presence of CP-H, CP-E, and aglycones. Both CP extracts decreased the expression of genes associated with cell invasion (hilA, ssrB and sopD). These results suggest that CP extracts and flavanones inhibit S. Typhimurium adhesion by interacting with fimbriae and flagella structures and downregulating fimbrial and virulence genes. CP-E and aglycone flavanones, rarely found in nature, exhibited superior inhibitory effects on bacterial viability, motility, adhesion and virulence gene expression than glycosylated flavanones. The outcomes of this research advance the understanding of how aglycone and glycosylated flavanones impact S. Typhimurium virulence activity and adhesion, contributing to their application as functional food ingredients for the prevention of adhesion, invasion, and the development of this pathogen in the gut.