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Propionibacterium freudenreichii is used as a cheese-ripening starter and as a probiotic. Its reported physiological effects at the gut level, including modulation of bifidobacteria, colon epithelial cell proliferation and apoptosis, and intestinal inflammation, rely on active metabolism in situ. Survival and activity are thus key factors determining its efficacy, creating stress adaptation and tolerance bottlenecks for probiotic applications. Growth media and growth conditions determine tolerance acquisition. We investigated the possibility of using sweet whey, a dairy by-product, to sustain P. freudenreichii growth. It was used at different concentrations (dry matter) as a culture medium. Using hyperconcentrated sweet whey led to enhanced multistress tolerance acquisition, overexpression of key stress proteins, and accumulation of intracellular storage molecules and compatible solutes, as well as enhanced survival upon spray drying. A simplified process from growth to spray drying of propionibacteria was developed using sweet whey as a 2-in-1 medium to both culture P. freudenreichii and protect it from heat and osmotic injury without harvesting and washing steps. As spray drying is far cheaper and more energy efficient than freeze-drying, this work opens new perspectives for the sustainable development of new starter and probiotic preparations with enhanced robustness. [ABSTRACT FROM AUTHOR]