Giordano, Irene (2024) Balancing health and taste:sonication and microencapsulation strategies for the metabolic attenuation of probiotics in the development of stable plant-based probiotic beverages. [Tesi di dottorato]

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As consumer preferences shift toward dairy alternatives as vehicles for probiotics, there is growing interest in modulating probiotic performance to attenuate the "probiotic off-flavors" associated with rapid acidification. Attenuation is a strategy developed to counteract this phenomenon by controlling and/or modulating probiotic metabolism while preserving probiotic viability. Attenuation can be induced by sublethal stress, such as low-intensity sonication, or by physical separation through microencapsulation. Lacticaseibacillus casei ATCC 393 and Limosilactobacillus reuteri DSM 17938 were used as model probiotics to develop an efficient attenuation strategy. Conventional, single cell and transcriptional analyses were performed to evaluate the cultivability, viability, metabolic activity and genetic survival strategy of the tested probiotic. Specific analyses were also carried out to evaluate the effect of the sonication treatment on probiotic functional and adhesion properties. Finally, the feasibility of the attenuation strategy was tested in three real food systems. The obtained results demonstrate the central role of the intensity of the sonication treatment in altering probiotic physiology. Sonicated Lc. casei ATCC 393 showed a delay in acidification and growth rate, an improvement in surface and adhesion properties, and an impairment in morphology and membrane structure. The size of the viable (detected by single cell analysis) and cultivable subpopulation suggested the induction of the Viable But Not-Cultivable status in response to the more intense treatment. Transcriptional changes suggested a metabolic shift towards alternative use of pyruvate, preservation of ATP and DNA structure. Furthermore, a strong genetic stress response was observed for the shorter treatment, while a transcriptional adaptation was observed for the longer treatment. The comparison between different microcapsules revealed that the attenuation efficacy of the system depends on microcapsule permeability and barrier properties. Only the combined use of alginate and chitosan effectively delayed the Lc. casei ATCC 393 acidification rate. In addition, an improvement of the attenuation strategy was obtained by encapsulating the probiotic subject to the more intense sonication treatment in chitosan-coated alginate microcapsules. Li. reuteri DSM 17938 showed a similar response to sonication treatments for both acidification rate and cultivability. In addition, sonication decreased the strain survival when exposed to acidic environment while increased the strain survival when exposed to bile salts. Different results were obtained for the two attenuated strains added to the different plant-based beverages, underlining the need to properly evaluate the combination probiotic-food matrix. Although the obtained results are specific and dependent on the experimental plans, the multiparametric nature of both technologies opens the possibility to improve the strategy applied.

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