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The aim was to study the effect of sublimation temperature (from –20 °C to –10 °C) and sublimation time (from 18 to 40 h) on the viability of Lactobacillus fermentum K73, the moisture content and the texture features by response surface methodology. The stability of the freeze-dried powders was studied; these powders were stored at 4 °C, 25 °C, and 37 °C for 36 days. Four different matrices were used: culture medium (8% total solids), the mixture of this solution with maltodextrin: whey (0.6: 0.4), with maltodextrin or whey (40% total solids). Results showed that the sublimation conditions to increase cell viability (>8.755 log CFU/g) and to decrease moisture content (<4.087% [wet basis]) in the mixtures were –10 °C and 18 h. The image analysis successfully correlated sublimation temperature and time with the structural characteristics of the powders, cell viability, and moisture content. The shelf life study showed that the specific rate of cell viability loss was lower when the freeze-dried powders were stored at 4 °C, being the denatured whey solution the best cryoprotectant matrix during the freeze-drying process and shelf life. [ABSTRACT FROM AUTHOR]