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Background Several species in Lactobacillaceae family were recognized as potential probiotic bacteria. In this group of lactic acid bacteria, species are taxonomically closed and usually share similar 16S rRNA gene, thus, instead of so their identification and discrimination are too difficult. Method In the present study, virtual restriction fragment length polymorphism (RFLP) is instead of was used as a tool to discriminate between the closely related species Lactiplantibacillus plantarum (L plantarum), Lactiplantibacillus paraplantarum (L paraplantarum), and Lactiplantibacillus pentosus (L pentosus); Latilactobacillus sakei (L sakei), Latilactobacillus curvatus(L curvatus), and Latilactobacillus graminis (L graminis); Lacticaseibacillus casei (L casei), Lacticaseibacillus paracasei (L paracasei), Lacticaseibacillus zeae, and Lacticaseibacillus rhamnosus; Lactobacillus gasseri (L gasseri) and Lactobacillus johnsonii (L johnsonii). In silico comparative analysis of 16S rRNA sequences digested by 280 restriction enzymes was performed in order to search the key enzymes which gives different profiles. Results Results revealed that L casei, L paracasei, L zeae, and Lb rhamnosus could be separated from each other on the basis of AlwI, BpuEI, BsgI, BsrDI, BstYI, EarI, MluCI, and NsPI RFLP. Results showed also that different RFLP patterns were obtained from L sakei, L graminis and L curvatus by using both AflI and NspI endonucleases (in separated restriction) and L plantarum, L paraplantarum, and L pentosus were distinguished each one from the other by MucI, NspI, and TspDTI PCR-RFLP. Lb gasseri and L johnsonii were also separated on the basis of Mse I, Taq I, and Dra I RFLP. Conclusion In this study, we proved that too closely related species could be separated in virtual analysis on basis of their 16S rRNA RFLP patterns using key restriction enzymes method.

Lactic acid bacteria play an important role in milk coagulation and cheese ripening. To select strains showing interesting industrial features, two indigenous lactobacilli (Lactobacillus brevis and Lactobacillus plantarum) were studied for aminopeptidase activity. Cell and cells free extract were tested for leucyl aminopeptidase activity on the chromogenic leucyl-p-nitroanilide substrate. Intracellular and membrane enzymes were solubilized with glycine /lyzozyme treatment then purified by ammonium sulphate precipitation followed by Sephadex G100 and diethylaminoethyl (DEAE) ions exchange chromatography's separation. The molecular weight of denatured proteins was estimated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). Effects of several parameters, pH, temperature, some ions and inhibitors on purified enzyme activity were studied. Cellular aminopeptidase activity was higher for CHTD 27 strain than BH14 strain. No aminopeptidase activity was noted in the cell free extract. The results of chromatography sephadex G100 combined to those of electrophoresis allowed suggesting a dimer structure for the native enzyme. The Lb CHTD27 purified enzyme showed maximal activity at pH 6.6 and at 40°C. This enzyme was partially inhibited by ethylenediamine acetic acid (EDTA) and Cu 2+ ions but increased by Na 2+ and Co 2+ ions. The aminopeptidase extracted from Lb BH14 was inhibited by EDTA and phenylmethanesulfonylfluoride or phenylmethylsulfonyl fluoride (PMSF), its maximal activity was observed at pH 7.5 and 40°C. In addition to other characteristics as proteolysis and autolysis, in this paper we showed that both studied strains were also able to degrade peptide with specific peptidases which are important characters in cheese manufacturing.

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