Genomic diversity and immunomodulatory activity of Lactobacillus plantarum isolated from dairy products.

In this study, we aimed to investigate some functional characteristics and the immunomodulatory properties of three strains of Lactobacillus plantarum of dairy origin which, in a previous screening, showed to be candidate probiotics. Genome sequencing and comparative genomics, which confirmed the presence of genes involved in folate and riboflavin production and in the immune response of dendritic cells (DCs), prompted us to investigate the ability of the three strains to accumulate the two vitamins and their immunomodulation properties. The ability of the three strains to release antioxidant components in milk was also investigated. Small amounts of folate and riboflavin were produced by the three strains, while they showed a good antioxidant capacity in milk with FRAP method. The immune response experiments well correlated with the presence of candidate genes influencing in DCs cytokine response to L. plantarum. Specifically, the amounts of secreted cytokins by DCs after stimulation with cells of Lp790, Lp813 and Lp998 resulted pro-inflammatory whereas stimulation with culture supernatants (postbiotics) inhibited the release of interleukin (IL)-12p70 and increased the release of the anti-inflammatory IL-10 cytokine. This study adds further evidence on the importance of L. plantarum in human health. Understanding how probiotics (or postbiotics) work in preclinical models can allow a rational choice of the different strains for clinical and/or commercial use.

Biotyping of cultivable lactic acid bacteria isolated from donkey milk.

UNLABELLED: The diversity of lactic acid bacteria (LAB) species in donkey's milk was analysed by culture-dependent microbial techniques. Dominant strains were isolated on agar media generally used for enumerating LAB. To enrich the number of acidifying LAB present, the milk samples were incubated at 37°C for 24 h (cultured milk samples, CM). One of the CM samples was heat-treated at 63°C for 10 min before incubation at 37°C (heat-treated and cultured milk sample, TCM) to select thermophilic LAB. The microflora in these CM and TCM samples was then compared to that of the raw milk samples (RM). Among the 129 LAB isolates, 10 different species (four Enterococcus, five Streptococcus and one Pediococcus) were identified by molecular methods. Although the 10 LAB species were present in the RM samples, only three and two isolates were found in CM and TCM samples, respectively. Despite the selection protocol being set up to favour the isolation of all LAB isolates present in donkey milk, relatively few species and biotypes were isolated. No LAB isolates belonging to the most technologically important dairy starter species were detected. The possible factors related to the limited LAB diversity in donkey's milk have been discussed below. SIGNIFICANCE AND IMPACT OF THE STUDY: There is increased interest in using donkey's milk as a source of human nutrition. The large amounts of antimicrobial components and defence factors present in donkey's milk provide protection from microbial infections and distinguish donkey's milk from the milks of other mammals. However, the microbiota in donkey's milk has so far been poorly characterized, specifically with regard to the lactic acid bacteria (LAB). This study has identified cultivable, acidifying and thermoduric LAB that could be used to develop starter cultures. This is the first study to investigate the culturable LAB microbiota present in donkey's milk.

A model to assess lactic acid bacteria aminopeptidase activities in Parmigiano Reggiano cheese during ripening.

The aim of this work was to investigate in which phases of ripening of Parmigiano Reggiano cheese lactic acid bacteria aminopeptidases present in cheese extract could be involved in release of free amino acids and to better understand the behavior of these enzymes in physical-chemical conditions that are far from their optimum. In particular, we evaluated 6 different substrates to reproduce broad-specificity aminopeptidase N, broad-specificity aminopeptidase C, glutamyl aminopeptidase A, peptidase with high specificity for leucine and alanine, proline iminopeptidase, and X-prolyl dipeptidyl aminopeptidase activities releasing different N-terminal amino acids. The effects of pH, NaCl concentration, and temperature on the enzyme activities of amino acid beta-naphthylamide (betaNA)-substrates were determined by modulating the variables in 19 different runs of an experimental design, which allowed the building of mathematical models able to assess the effect on aminopeptidases activities over a range of values, obtained with bibliographic data, covering different environmental conditions in different zones of the cheese wheel at different aging times. The aminopeptidases tested in this work were present in cell-free Parmigiano Reggiano cheese extract after a 17-mo ripening and were active when tested in model system. The modeling approach shows that to highlight the individual and interactive effects of chemical-physical variables on enzyme activities, it is helpful to determine the true potential of an amino-peptidase in cheese. Our results evidenced that the 6 different lactic acid bacteria peptidases participate in cheese proteolysis and are induced or inhibited by the cheese production parameters that, in turn, depend on the cheese dimension. Generally, temperature and pH exerted the more relevant effects on the enzymatic activities, and in many cases, a relevant interactive effect of these variables was observed. Increasing salt concentration slowed down broad-specificity amino-peptidase C, glutamyl aminopeptidase A, proline iminopeptidase, and peptidase with high specificity for leucine and alanine. Interestingly, this variable did not affect broad-specificity aminopeptidase N and positively affected X-prolyl dipeptidyl aminopeptidase. The models elaborated varying pH, temperatures, and salt concentration and were a useful, low cost, and fast tool to understand the role of the main peptidases in the different phases of cheese ripening in relation to the major environmental factors influencing enzyme activity.

Peptidase activity in various species of dairy thermophilic lactobacilli.

AIMS: The aim of the present work was to evaluate the enzymatic potential manifested by aminopeptidase activity of different thermophilic Lactobacillus biotypes and to measure the influence of cell growth phase on enzyme expression. METHODS AND RESULTS: The activities were evaluated by the hydrolysis of beta-naphthylamide substrates for both whole and mechanically disrupted cells of L. helveticus, L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis strains, collected from both the exponential and the stationary growth phase. In general, activities were higher for cells in the exponential rather than in the stationary phase and the disrupted cells showed higher activities than the whole cells. The highest activity expressed by all strains corresponded to X-prolyl-dipeptidyl aminopeptidase while a moderate activity was observed towards Arg-betaNa, Lys-betaNa and Leu-betaNa. The lowest activity was observed for Pro-betaNa. CONCLUSIONS: It may be inferred that the cell structure and the cell physiology are crucial to define the level of efficiency of expression for aminopeptidase activity. The two species may be characterized by a different enzymatic system that hydrolyses N-terminal leucine. SIGNIFICANCE AND IMPACT OF THE STUDY: The differences of peptidase activities in L. helveticus and L. delbrueckii species acquires an importance to comprehend their role in the biochemical events occurring in cheese ripening.

Differentiation of Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus delbrueckii subsp. lactis by SDS-PAGE of cell-wall proteins.

AIMS: In the present study, a method based on SDS-PAGE fingerprinting of surface layer proteins was developed to identify Lactobacillus delbrueckii subsp. bulgaricus and subsp. lactis dairy isolates. METHODS AND RESULTS: The two subspecies, identified by species-specific PCR, were characterized by different SDS-PAGE cell-wall protein profiles; subspecies bulgaricus showed one band of about 31 kDa which, in some cases, was observed at a doublet, and subspecies lactis showed one band of about 21 kDa or 18 kDa. CONCLUSION: The sensitivity of this procedure for discriminating between the two subspecies was very high. The different types of SDS-PAGE profile for cell-wall proteins of the strains studied in this work did not seem to be correlated to the different dairies of origin. SIGNIFICANCE AND IMPACT OF THE STUDY: The method appears to be an efficient taxonomic tool. It has the advantage of easy gel interpretation over fingerprinting of whole-cell protein extracts, and may be used as an alternative to established PCR-based techniques which, though rapid and safe, require expensive instruments and reagents.

Presence of peptidase activities in different varieties of cheese.

Lactic acid bacteria (LAB) counts, PepX activity towards H-Phe-Pro-beta NA, and aminopeptidase activity towards H-Arg-beta NA. HCl, H-Lys-beta NA, H-Leu-beta NA, H-Pro-beta NA, H-Glu-beta NA derivatives have been evaluated in 32 commercial samples of cheese, one processed cheese, and one yoghurt. The presence of intracellular exo-peptidase activities in cheese extracts free from bacterial cells was detected, even after 1 year of ripening. An inverse ratio between the presence of viable lactic microflora and peptidase activity in the cheese extracts was observed. The importance of LAB starter exo-peptidases in the degradation of casein oligopeptides, and the key role of autolysis in the release of peptidases in the cheese, are discussed.