Selection of lyoprotectants for the stabilization of the freeze-dried probiotic Limosilactobacillus reuteri CRL 1098 by an accelerated inactivation assay.

Abstract

The high potential of the probiotic L. reuteri CRL 1098, as hypocholesterolemic and vitamin B₁₂ producer strain, would require addressing freeze-drying studies of the microorganism as well as its stability during storage. L. reuteri CRL 1098 was freeze-dried with glutamate, maltodextrin and trehalose as lyoprotectants and an accelerated inactivation assays under isothermal conditions (50, 60 and 70°C) was used to evaluate the stability of lyophilizates. Results obtained were analyzed with informatic GInaFiT Ap-inn and traditional predictive tools (D/z and Arrhenius models) for describing both, the inactivation process and the bacterial behaviour. The predicted shelf life of the lyophilized probiotic to obtain 1 × 10⁶ UFC/g was experimentally tested at 30°C. Regardless of the lyoprotectant used, the survival of L. reuteri CRL1098 fitted acceptably to the weilbulian model of Marfat in all heat treatments; the data described curves with up dawn concavities (p < 1) and allowed to identify a more sensitive bacterial population through the first decimal reduction time (δ-value) specially at 50°C in presence of maltodextrin. From the second reduction time, survivors of L. reuteri CRL 1098 fitted to first kinetic order reactions (lineal distribution of data), so D-values (decimal reduction time), k-values (inactivation rate constant), and z-values (intrinsic resistance) as well as the activation energy Ea, were calculated through D/z model and Arrhenius model. From these, a resistant subpopulation of L. reuteri CRL 1098 could be described, mainly when trehalose was used as lyoprotectant, which was characterized by high z-values. These results correlated with higher D-values but lower k-values and Ea in trehalose, which did not show the best stabilizing property along the storage tested at 30°C. Maltodextrine proved to be the best stabilizer lyoprotectant, especially when the shelf life is predicted at low temperatures of conservation.