Bifidobacterium animalis subsp. lactis HN019 live probiotics and postbiotics: production strategies and bioactivity evaluation for potential therapeutic properties.

Introduction: B. animalis subsp. lactis HN019 is a commercially available well-characterized probiotic with documented effects on human health, such as the ability to enhance the immune function and to balance the intestinal microbiome. Therefore, optimizing the manufacturing process to improve sustainability, increasing biomass yields and viability, and avoiding animal -derived nutrients in the medium to meet vegan consumer's needs, is currently of interest. Besides the established use of live probiotic cells, alternative supplements indicated as postbiotics, like non-viable cells and/or probiotics derived bioactive molecules might be considered as potential next generation biotherapeutics. In fact, advantages of postbiotics include fewer technological limitations, such as easier production processes and scale-up, and even higher specificity. Methods: In this work, medium design together with different fermentation strategies such as batch, fed-batch and in situ product removal on lab-scale bioreactors were combined. Medium pretreatment by ultrafiltration and protease digestion was performed to reduce polysaccharidic contaminants and facilitate the purification of secreted exopolysaccharides (EPS). The latter were isolated from the fermentation broth and characterized through NMR, GC-MS and SEC-TDA analyses. The expression of TLR-4, NF-kb and IL-6 in LPS challenged differentiated CaCo-2 cells treated with EPS, live and heat-killed B. lactis cells/broth, was evaluated in vitro by western blotting and ELISA. Zonulin was also assessed by immunofluorescence assays. Results and Discussion: The titer of viable B. lactis HN019 was increased up to 2.9 ± 0.1 x 1010 on an animal-free semidefined medium by applying an ISPR fermentation strategy. Medium pre-treatment and a simple downstream procedure enriched the representativity of the EPS recovered (87%), the composition of which revealed the presence of mannuronic acid among other sugars typically present in polysaccharides produced by bifidobacteria. The isolated EPS, live cells and whole heat inactivated broth were compared for the first up to date for their immunomodulatory and anti-inflammatory properties and for their ability to promote intestinal barrier integrity. Interestingly, EPS and live cells samples demonstrated immune-stimulating properties by downregulating the expression of TLR-4 and NF-kb, and the ability to promote restoring the integrity of the intestinal barrier by up-regulating the expression of zonulin, one of the tight junctions forming proteins. Postbiotics in the form of heat killed broth only reduced NF-kb expression, whereas they did not seem effective in the other tested conditions.

Optimization of growth of Levilactobacillus brevis SP 48 and in vitro evaluation of the effect of viable cells and high molecular weight potential postbiotics on Helicobacter pylori.

Several Levilactobacillus brevis strains have the potential to be used as probiotics since they provide health benefits due to the interaction of live cells, and of their secreted products, with the host (tissues). Therefore, the development of simple fermentation processes that improve cell viability to reduce industrial production costs, and at the same time the characterization and biological evaluation of cell-free postbiotics that can further promote application, are of great interest. In the present study, small scale batch fermentations on semi defined media, deprived of animal derived raw materials, were used to optimize growth of L. brevis SP48, reaching 1.2 ± 0.4 × 1010 CFU/ml of viable cells after 16 h of growth. Displacement, competition, and inhibition assays compared the effect, on Helicobacter pylori, of L. brevis cells to that of its partially purified potentially postbiotic fraction rich in exopolysaccharides and proteins. The expression of pro and anti-inflammatory biochemical markers indicated that both samples activated antimicrobial defenses and innate immunity in a gastric model. Moreover, these compounds also acted as modulators of the inflammatory response in a gut in vitro model. These data demonstrate that the high molecular weight compounds secreted by L. brevis SP48 can contrast H. pylori and reduce inflammation related to intestinal bowel disease, potentially overcoming issues related to the preservation of probiotic viability.

Limosilactobacillus fermentum from buffalo milk is suitable for potential biotechnological process development and inhibits Helicobacter pylori in a gastric epithelial cell model.

Probiotics are living microorganisms that give beneficial health effects while consumed, and each strain possesses diverse and unique properties and also different technological characteristics that affect its ability to be produced at large scale. Limosilactobacillus fermentum is a widely studied member of probiotics, however, few data are available on the development of fermentation and downstream processes for the production of viable biomasses for potential industrial applications. In the present study a novel L. fermentum strain was isolated from buffalo milk and used as test example for biotechnological process development. The strain was able to produce up to 109 CFU/mL on a (glucose based) semi-defined medium deprived of animal-derived raw materials up to the pilot scale (150 L), demonstrating improved results compared to commonly used, although industrially not suitable, media rich of casein and beef extract. The study of strain behavior in batch experiments indicated that the highest concentration of viable cells was reached after only 8 h of growth, greatly shortening the process. Moreover, initial concentrations of glucose in the medium above 30 g/L, if not supported by higher nitrogen concentrations, reduced the yield of biomass and increased production of heterolactic fermentation by-products. Biomass concentration via microfiltration on hollow fibers, and subsequent spray-drying allowed to recover about 5.7 × 1010CFU/gpowder of viable cells, indicating strain resistance to harsh processing conditions. Overall, these data demonstrate the possibility to obtain and maintain adequate levels of viable L. fermentum cells by using a simple approach that is potentially suitable for industrial development. Moreover, since often exopolysaccharides produced by lactobacilli contribute to the strain's functionality, a partial characterization of the EPS produced by the newly identified L. fermentum strain was carried out. Finally, the effect of L. fermentum versus H. pylori in a gastric epithelial cell model was evaluated demonstrating its ability to stimulate the response of the immune system and displace the infective agent.