In vitro multi-parameter evaluation and Z-score-based selection of autochthonous probiotic candidates from Nile tilapia (Oreochromis niloticus)

Autochthonous probiotics isolated from the host gastrointestinal tract are considered superior candidates due to their host-adapted characteristics. However, a standardized quantitative approach for integrating multiple in vitro probiotic parameters into a single ranking system for Nile tilapia remains limited. This study aimed to evaluate and rank autochthonous bacterial isolates from the intestine of Nile tilapia ( Oreochromis niloticus ) using multiple in vitro criteria and Z-score normalization. Nine parameters were evaluated and categorized into safety aspects (hemolytic activity and antibiotic resistance), functional properties (antagonistic activity, cell adhesion, cell-surface hydrophobicity, and auto-aggregation), and growth performance (pH tolerance, bile salt tolerance, and specific growth rate at multiple temperatures). The findings were integrated and standardized using Z-score normalization to rank the most promising candidates for subsequent in vivo trials. Thirty-four autochthonous bacteria were isolated from Nile tilapia intestine, of which 15 isolates demonstrated antagonistic activity against at least one tested pathogen ( Aeromonas hydrophila and/or Streptococcus iniae ). Among these 15 isolates, six showed susceptibility to all 12 tested antibiotics (no resistant profile), and ten exhibited non-hemolytic activity on both sheep and tilapia blood agar. Significant differences ( P < 0.05) among isolates were observed in cell-surface hydrophobicity, auto-aggregation, and specific growth rate. All isolates tolerated 6% bile salts, with six isolates tolerating up to 12%, and all Bacillus strains survived exposure to pH 2 after 24 h. Z-score normalization integrating all nine parameters identified Bacillus sp. CHP02 (Z = 1.48), Bacillus sp. RP01 (Z = 1.44), and Bacillus sp. RP00 (Z = 1.30) as the most promising probiotic candidates. These strains warrant further investigation in in vivo trials to confirm their probiotic efficacy in Nile tilapia aquaculture.