Analytical and biological characterization of saponin-based phytobiotics for poultry health

The rise of antibiotic resistance and the EU's 2006 ban on antimicrobial growth promoters have intensified the search for sustainable alternatives in poultry production. Plant saponins have attracted considerable interest as phytobiotic candidates due to their antimicrobial and anticoccidial properties, yet their industrial adoption has been hampered by the absence of validated analytical methods for complex formulations and a limited understanding of how their structure drives biological activity. This doctoral thesis tackled both challenges through three integrated experimental studies. First, a vanillin–sulfuric acid UV-Vis spectrophotometric method was developed and validated per ICH Q2(R2) guidelines to quantify saponins in lipid-based microencapsulated Yucca schidigera extracts. The method outperformed conventional gravimetric approaches in selectivity (92.6% vs. 66.4% recovery) while meeting regulatory standards for precision and accuracy. Second, LC-HRMS profiling of Y. schidigera identified 15 saponins, and multivariate modeling (PLS-DA, ridge regression) pinpointed schidigerasaponins D3 and D4—both bearing a smilagenin aglycone with three glucose units—as the primary bioactive markers against Clostridium perfringens and Eimeria tenella. This suggested that glycosylated saponins may outperform their deglycosylated counterparts against Gram-positive bacteria and protozoan parasites. Third, comparing acid hydrolysis effects in Y. schidigera and fenugreek revealed strikingly opposite outcomes: hydrolysis boosted antimicrobial activity in the heavily glycosylated fenugreek saponins, yet reduced both antimicrobial and anticoccidial efficacy in the moderately glycosylated Y. schidigera saponins. Since both hydrolyzed extracts converged to similar sapogenin profiles, aglycone identity alone could not explain the difference—pointing instead to glycosylation pattern as the key determinant. These findings support an "optimal glycosylation window" hypothesis: roughly three hexose units strikes the best balance between aqueous solubility and membrane permeability. This framework enables structure-guided selection of botanical sources, advancing saponin-based phytobiotics from empirical screening toward rational, evidence-based development.