| Summary: | Here, the olive leaf extract (OLE) rich in polyphenols was employed as a prebiotic agent, together with <i>Lactobacillus reuteri</i> and <i>Bacillus clausii</i>, to develop synbiotics. The prebiotic effect of olive leaf extract on the probiotic strains was tested at concentrations of 0, 50, 100, 400, and 1000 μg mL<sup>−1</sup>, and also 20 and 40 mg mL<sup>−1</sup>. Olive leaf extract at 40 mg mL<sup>−1</sup> showed the best prebiotic activity on <i>L. reuteri</i> and <i>B. clausii</i>. A basal diet and two experimental synbiotic-containing diets were prepared. The synbiotic diets were manufactured by adding to the basal diet 5 × 10<sup>6</sup> CFU g<sup>−1</sup> <i>L. reuteri</i> + 5 × 10<sup>6</sup> CFU g<sup>−1</sup> <i>B. clausii</i> + 0.25 mg g<sup>−1</sup> OLE and 1 × 10<sup>7</sup> CFU g<sup>−1</sup> <i>L. reuteri</i> + 1 × 10<sup>7</sup> CFU g<sup>−1</sup> <i>B. clausii</i> + 0.25 mg g<sup>−1</sup> OLE. The diets were administered to the freshwater crayfish <i>Astacus astacus</i> (1.35 ± 0.04 g) in an 84-day feeding trial. The diet containing 5 × 10<sup>6</sup> CFU g<sup>−1</sup> <i>L. reuteri</i> + 5 × 10<sup>6</sup> CFU g<sup>−1</sup> <i>B. clausii</i> + 0.25 mg g<sup>−1</sup> OLE significantly improved (<i>p</i> < 0.05) final weight, specific growth rate, body condition, and survival rate. A significant growth of <i>Enterobacteriaceae</i>, which include strains with proven beneficial activities for intestinal health and general animal welfare, significantly increased in crayfish fed with synbiotics. The obtained results could be suitable for functional feed development in crayfish farming.
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