Exploring Ultrasound-Induced Metabolic Attenuation in Lacticaseibacillus casei ATCC 393: A Combined Approach Using Traditional Methods and Flow Cytometry

Irene Giordano¹Mohammed Salman¹Stefania Arioli²Diego Mora²Gianluigi Mauriello^1*

• ¹Department of Agriculture, School of Agriculture and Veterinary Medicine, University of Naples Federico II, Portici, Italy
• ²Department of Food, Environmental and Nutritional Sciences,University of Milan, Milano, Italy

Attenuation technologies applied to probiotics aim to modulate specific metabolic pathwaysparticularly acidification-while maintaining cell viability. Although ultrasound represents an emerging tool in this context, its precise mechanism of actions on probiotic cells are not well understood. The study aimed to establish a suitable method to uncover the effects of ultrasound attenuation on probiotics. Lacticaseibacillus casei ATCC 393 was exposed to sonication for 6 and 8 minutes in water suspension. Morphological changes, cultivability, acidification capacity and growth recovery were assessed using culture-dependent methods. Flow cytometry (FCM) coupled with fluorescent staining was used to assess membrane integrity (as a marker of viability) and esterase activity (as a marker of metabolic activity). Finally, plate count and FCM data were compared to estimate the overall ultrasound effect. A reduction in cell size was observed and confirmed by decreased forward and side scatter signals. Acidification was dependent on the intensity applied and only the 8-min treatment induced a prolonged modulation over 24 h. Esterase activity was similarly affected by both sonicating times, whereas membrane integrity reduction was dependent on the treatment intensity. The probiotic demonstrated the ability to restore growth, with recovery time proportionally increasing with the duration of ultrasound treatment. Direct comparisons of the viable, culturable and metabolic active subpopulations indicate that they are similarly affected by 6 min of sonication. On the contrary, 8 min of sonication increased sample heterogenies generating three different subpopulations. The lack of overlap between viable and culturable clusters suggested that the cells sonicated for 8-min entered the viable but nonculturable state. These results provide insight into the intensity-dependent effects of ultrasound on probiotic functionality and demonstrate the value of integrative analytical approaches (FCM combined with traditional methods) for characterizing bacterial responses to attenuation strategies.