Transcriptomic analysis reveals differential gene expression patterns of Lacticaseibacillus casei ATCC 393 in response to ultrasound stress.

In recent years, there has been a growing interest in modulating the performance of probiotic, mainly Lactic Acid Bacteria (LAB), in the field of probiotic food. Attenuation, induced by sub-lethal stresses, delays the probiotic metabolism, and induces a metabolic shift as survival strategy. In this paper, RNA sequencing was used to uncover the transcriptional regulation in Lacticaseibacillus casei ATCC 393 after ultrasound-induced attenuation. Six (T) and 8 (ST) min of sonication induced a significant differential expression of 742 and 409 genes, respectively. We identified 198 up-regulated and 321 down-regulated genes in T, and similarly 321 up-regulated and 249 down-regulated in ST. These results revealed a strong defensive response at 6 min, followed by adaptation at 8 min. Ultrasound attenuation modified the expression of genes related to a series of crucial biomolecular processes including membrane transport, carbohydrate and purine metabolism, phage-related genes, and translation. Specifically, genes encoding PTS transporters and genes involved in the glycolytic pathway and pyruvate metabolism were up-regulated, indicating an increased need for energy supply, as also suggested by an increase in the transcription of purine biosynthetic genes. Instead, protein translation, a high-energy process, was inhibited with the down-regulation of ribosomal protein biosynthetic genes. Moreover, phage-related genes were down-regulated suggesting a tight transcriptional control on DNA structure. The observed phenomena highlight the cell need of ATP to cope with the multiple ultrasound stresses and the activation of processes to stabilize and preserve the DNA structure. Our work demonstrates that ultrasound has remarkable effects on the tested strain and elucidates the involvement of different pathways in its defensive stress-response and in the modification of its phenotype.

Microencapsulation and sonication: A multiple physical approach to attenuate the probiotic Lacticaseibacillus casei ATCC 393.

Modulation of probiotic performances represents a tool to avoid the probiotic off-flavor in probiotic food. Microencapsulation and sonication were evaluated in slowing down the Lacticaseibacillus casei ATCC 393 induced acidification. Firstly, the influence of alginate concentration and chitosan coating on acidification rate were tested. Microcapsule morphology and the entrapment efficacy were also evaluated. Then, two time of exposure to ultrasound, 6 and 8 min, were applied for L. casei attenuation. Finally, sonicated cells were encapsulated. ΔpH after 6 and 24 h of incubation at 37 °C revealed that chitosan-alginate microcapsules and the 8-min sonicated probiotic presented a significant delayed acidification. When all the systems were compared, the encapsulation of 8-min sonicated L. casei in chitosan-alginate microcapsules significantly improved the results obtained with the single technologies. These results suggest that by modulating the operating parameters and combining these two technologies an increasingly efficient attenuation system can be developed.

Ultrasound Attenuation Improves Some Surface Properties of the Probiotic Strain Lacticaseibacillus casei ATCC 393.

Ultrasound attenuation has been recently proposed as a tool to modulate probiotic metabolism. The study aimed to characterize the response of the probiotic Lacticaseibacillus casei ATCC 393 to sonication. Two ultrasound treatments were tested (57 W, duty cycle 50%, 6 or 8 min). Attenuation was assessed as a pH decrease in MRS broth after 6 and 24 h of incubation at 37 °C. Cultivability was evaluated by plate count immediately after sonication and by growth index on overnight cultures. Surface changes were determined by auto-aggregation, hydrophobicity, biofilm production tests, and by membrane damages. The 6 min treatment induced a temporary attenuation, while a prolongated exposure to sonic waves caused major attenuation effects (ΔpH 0.97 after 24 h). Both sonication treatments affected probiotic cultivability with a significant (p < 0.05) reduction of plate counts and an alteration of the growth index. Although auto-aggregation was negatively affected upon sonication, the hydrophobicity and biofilm production were improved with no significant differences (p > 0.05) between the sonicated samples. Moreover, sonicated L. casei ATCC 393 resulted in increased membrane permeability. These results suggest that ultrasound technology can be successfully used to modulate the L. casei ATCC 393 fermentative metabolism and to improve its surface properties.

Application of ultrasound and microencapsulation on Limosilactobacillus reuteri DSM 17938 as a metabolic attenuation strategy for tomato juice probiotication.

Counteracting probiotic-induced physicochemical and sensory changes is a challenge in the development of probiotic beverages. The aim of the study is to apply ultrasound and microencapsulation for the attenuation of Limosilactobacillus reuteri DSM 17938 to avoid change in a probiotic tomato juice. Preliminarily, six ultrasound treatments were applied. Probiotic survival in acid environment (pH 2.5) and bile salts (1.5 g/l) after ultrasound treatment was also studied. The probiotic was inoculated in tomato juice in four forms: free cells (PRO-TJ), sonicated-free cells (US-TJ), untreated-microencapsulated (PRO-MC-TJ) and sonicated-microencapsulated cells (US-MC-TJ). Probiotic viability and pH were monitored during 28 days of storage at 4 and 20 °C. Sensory analysis was performed for PRO-TJ and US-MC-TJ sample (4 °C). Ultrasound (57 W for 6 min) did not affect cell survival and transitorily modulated probiotic acidifying capacity; it reduced probiotic survival in acidic environment but increased probiotic survival in bile salts solution. Ultrasound was effective in maintain pH value of tomato juice but only at 4 °C. Instead, microencapsulation with sodium-alginate leads to a more stable probiotic juice, particularly at 20 °C. Finally, probiotication slightly modified some sensory attributes of the juice. This study shows the potential of ultrasound and microencapsulation as attenuation strategies and highlights the need for process optimization to increase ultrasound efficacy.

Effect of Microencapsulation on Survival at Simulated Gastrointestinal Conditions and Heat Treatment of a Non Probiotic Strain, Lactiplantibacillus plantarum 48M, and the Probiotic Strain Limosilactobacillus reuteri DSM 17938.

Cells of the probiotic strain Limosilactobacillus reuteri DSM 17938 and of the non-probiotic strain Lactiplantibacillus plantarum 48M were microencapsulated in alginate matrix by emulsion technique. Survival of microorganisms in the microcapsules was tested against gastrointestinal (GI) simulated conditions and heat stress. Results demonstrated that the microencapsulation process improved vitality of Lactiplantibacillus plantarum 48M cells after GI conditions exposure, allowing survival similarly to the probiotic Limosilactobacillus reuteri DSM 17938. Moreover, microencapsulation was able to protect neither Limosilactobacillus reuteri DSM 17938 nor Lactiplantibacillus plantarum 48M cells when exposed to heat treatments. Microencapsulated Limosilactobacillus reuteri DSM 17938 cells were still able to produce reuterin, an antimicrobial agent, as well as free cells.