Turbidimetric definition of growth limits in probiotic Lactobacillus strains from the perspective of an adaptation strategy.

The application of an adaptation strategy for probiotics, which may improve their stress tolerance, requires the identification of the growth range for each parameter tested. In this study, 4 probiotics (Lactobacillus acidophilus, Lacticaseibacillus casei, Lacticaseibacillus rhamnosus, and Lactiplantibacillus plantarum) were grown under different pH, NaCl, and sucrose concentrations at 25°C, 30°C, and 37°C. Turbidimetric growth curves were carried out and lag phase duration, maximum growth rate, and amplitude (i.e., the difference between initial and stationary phase optical density) were estimated. Moreover, cell morphology was observed, and cell length measured. The growth response, as well as the morphological changes, were quite different within the 4 species. The L. acidophilus was the most sensitive strain, whereas L. plantarum was shown to better tolerate a wide range of stressful conditions. Frequently, morphological changes occurred when the growth curve was delayed. Based on the results, ranges of environmental parameters are proposed that can be considered suboptimal for each strain, and therefore could be tested. The quantitative evaluation of the growth kinetics as well as the morphological observation of the cells can constitute useful support to the choice of the parameters to be used in an adaptation strategy, notwithstanding the need to verify the effect on viability both in model systems and in foods.

Application of pre-adaptation strategies to improve the growth of probiotic lactobacilli under food-relevant stressful conditions.

While formulating a probiotic food, it is mandatory to make sure that the viability of probiotics is adequate at the point of consumption, which can be strongly compromised by stressful conditions due to low pH and high osmolarity. In this study, three probiotic lactobacilli were subjected to different pre-adaptation conditions, and the turbidimetric growth kinetics in challenging conditions (pH 4.0-6.5, NaCl 1-7%, sucrose 0.1-0.7 M) were evaluated. Different effects were observed for Lactobacillus acidophilus, Lacticaseibacillus casei, and Lactiplantibacillus plantarum. Indeed, pre-exposition to sub-optimal conditions in terms of pH and % NaCl significantly improved the ability of L. acidophilus and L. casei to overcome the osmotic stress due to salt or sucrose, and similar effects were observed for acidic stress. L. plantarum showed to be more tolerant to the challenging conditions applied in this study. Anyway, the pre-adaptation at conditions SUB_1 (pH 4.5 and NaCl 4%) and SUB_2 (pH 5 and NaCl 2%) speeded-up its growth kinetics by reducing the length of the lag phase under sucrose stress and enhancing the maximum growth rate at the highest pH tested. Moreover, an improvement in biomass amount was observed under sucrose stress. The whole data evidenced that the application of the appropriate pre-adaptation condition could contribute to making probiotics more robust towards challenging conditions due to food matrix, processing, and storage as well as gastrointestinal transit. Further studies will be necessary to gain insight into the proteomics and metabolomics responsible for increased tolerance to stressful conditions.

An exopolysaccharide from Leuconostoc mesenteroides showing interesting bioactivities versus foodborne microbial targets.

An exopolysaccharide (EPS_B3) produced by a Leuconostoc mesenteroides strain isolated from a semi-hard Italian cheese was chemically and biologically characterized. HPLC-SEC, NMR, FT-IR and monosaccharide composition experiments were performed. Antimicrobial, antibiofilm, bifidogenic, antioxidant, and DNA-protective activity of EPS_B3 were also studied. Results revealed that EPS_B3 was a mixture of two high-molecular-weight dextran with low branching degree. Moreover, EPS_B3 displayed significant antibacterial activity against eight foodborne pathogens and inhibited biofilm formation by Listeria monocytogenes. EPS_B3 also evidenced bifidogenic activity, stimulating the growth of three probiotic bifidobacteria, and improving the tolerance of Bifidobacterium animalis subsp. lactis to oxygen stress. It also protected plasmid DNA from hydrogen peroxide damage. Only limited antioxidant capacity was observed. In conclusion, data suggest that EPS_B3 might be exploited in the context of functional foods especially for its marked antimicrobial activity as well as for the ability to improve the viability of bifidobacteria in probiotic foods. However, further studies should be carried out to assess the ability of EPS_B3 to reach intact the target site (i.e., gastrointestinal tract) to consider the possibility of use it as a new functional ingredient in foods.