| Summary: | Preservation of probiotics by lyophilization is considered a method of choice for developing stable products. However, both direct consumption and reconstitution of dehydrated probiotic preparations before application “compromise” the survival and functional characteristics of the microorganisms under the stress of the upper gastro-intestinal tract. We evaluated the impact of different food additives on the viability, mucin adhesion, and zeta potential of a freeze-dried putative probiotic, <i>Lactiplantibacillus</i> (<i>Lp</i>.) <i>plantarum</i> HAC03. HAC03-compatible ingredients for the formulation of ten rehydration mixtures could be selected. Elevated efficacy was achieved by the B-active formulation, a mixture of non-protein nitrogen compounds, sugars, and salts. The survival of <i>Lp. plantarum</i> HAC03 increased by 36.36% compared rehydration with distilled water (4.92%) after passing simulated gastro-intestinal stress conditions. Cell viability determined by plate counting was confirmed by flow cytometry. B-active formulation also influenced <i>Lp. plantarum</i> HAC03 functionality by increasing its adherence to a Caco-2 cell-line and by changing the bacterial surface charge, measured as zeta potential.Hydrophobicity, mucin adhesion and immunomodulatory properties of <i>Lp. plantarum</i> HAC03 were not affected by the B-active formulation. The rehydration medium also effectively protected <i>Lp. plantarum</i> ATCC14917, <i>Lp. plantarum</i> 299v, <i>Latilactobacillus sakei</i> (<i>Lt.</i>) HAC11, <i>Lacticaseibacillus (Lc.) paracasei</i> 532, <i>Enterococcus faecium</i> 200, and <i>Lc. rhamnosus</i> BFE5263.
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