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.