Abstract
The current study aimed to validate the safety of electrohydrodynamically encapsulated Lactiplantibacillus plantarum CRD7 and Lacticaseibacillus rhamnosus CRD11 in accordance with guidelines of FAO/WHO and ICMR/DBT. In vitro assays such as mucin degradation, hemolysis of blood cells, antimicrobial susceptibility pattern, possession of virulence factors, biogenic amine, and ammonia production were assessed. In results, the cross-streak and co-culture techniques revealed that CRD7 and CRD11 were compatible in vitro. Upon visual inspection through scanning electron and fluorescence microscopy, the integrity of bacterial cell membrane was confirmed even after the encapsulation process. CRD7 and CRD11 were non-hemolytic and showed negative responses to gelatinase, urease, and DNase activities. Non-mucinolytic activity of CRD7 and CRD11 was verified by measuring cell growth rate (p < 0.05) in different modified media followed by SDS-PAGE. High-performance liquid chromatography analysis revealed that both the strains did not produce biogenic amines (putrescine, cadaverine, histamine, and tyramine). Neither of the Lactobacillus strains produced ammonia after growing in BHI broth for 5 days at 37 °C. L-lactate production was highest (p < 0.05) in CRD11 (8.83 g/L), followed by CRD7 (8.16 g/L), whereas the lowest (p < 0.05) D-lactate was registered for encapsulated CRD11 (0.33 g/L) and CRD7 (0.49 g/L). The antibiogram profile determined through minimum inhibitory concentration showed that CRD7 and CRD11 were sensitive to key antibiotics suggested by EFSA except for gentamycin and kanamycin. PCR data on virulence genes demonstrated that both strains were safe for probiotic use. Moreover, CRD7 and CRD11 strains caused insignificant (p > 0.05) changes in the cell viability of Caco-2 cells as estimated by MTT (98.94-99.50%) and NR uptake (95.42-97.03%) assays and showed sensitivity to human serum. According to the results of these evaluated attributes, it is concluded that L. plantarum CRD7 and L. rhamnosus CRD11 are safe, non-toxic to human epithelial cells, and thus may be potentially suitable for various food/feed applications.