This study was conducted to evaluate the probiotic properties of Pediococcus pentosaceus OZF isolated from human breast milk. The results obtained so far suggest that the strain is resistant to low pH, bile salt, pepsin and pancreatin, so it could survive while passing through the upper part of the gastrointestinal tract and reveal its potential probiotic action on host organism. The strain was non-pathogenic (γ-hemolytic), produced anti-Listerial bacteriocin, exhibited a strong autoaggregating phenotype (85.71%) and demonstrated 6.26 and 12.99% coaggregation with Salmonella enterica serotype Typhimurium SL 1344 and Escherichia coli LMG 3083 (ETEC), respectively. The degree of adhesion of Ped. pentosaceus OZF to the human Caco-2 cell line was investigated and when compared to the adhesion of pathogenic strains tested, it was shown to inhibit the growth of human enterotoxigenic E. coli LMG 3083 (ETEC) and of Salm. Typhimurium SL 1344. Ped. pentosaceus OZF seems to adhere to human intestinal cells via mechanisms that involve different combinations of carbohydrate and lipid factors on the bacteria and eukaryotic cell surface. The percentage of adhesion to n-hexadecane was 34% showing that the surface was rather hydrophilic. Higher affinity displayed by Ped. pentosaceus OZF for chloroform demonstrates the basic property of a cell, which may be due to the presence of carboxylic groups on the cell surface.

A collection of pediocin AcH amino acid substitution mutants was generated by PCR random mutagenesis of DNA encoding the bacteriocin. Mutants were isolated by cloning mutagenized DNA into anEscherichia coli malE plasmid that directs the secretion of maltose binding protein-pediocin AcH chimeric proteins and by screening transformant colonies for bactericidal activity againstLactobacillus plantarum NCDO955 (K. W. Miller, R. Schamber, Y. Chen, and B. Ray, 1998. Appl. Environ. Microbiol. 64:14–20, 1998). In all, 17 substitution mutants were isolated at 14 of the 44 amino acids of pediocin AcH. Seven mutants (N5K, C9R, C14S, C14Y, G37E, G37R, and C44W) were completely inactive against the pediocin AcH-sensitive strains L. plantarum NCDO955,Listeria innocua Lin11, Enterococcus faecalisM1, Pediococcus acidilactici LB42, and Leuconostoc mesenteroides Ly. A C24S substitution mutant constructed by other means also was inactive against these bacteria. Nine other mutants (K1N, W18R, I26T, M31T, A34D, N41K, H42L, K43N, and K43E) retained from <1% to ∼60% of wild-type activity when assayed against L. innocua Lin11. One mutant, K11E, displayed ∼2.8-fold-higher activity against this indicator. About one half of the mutations mapped to amino acids that are conserved in the pediocin-like family of bacteriocins. All four cysteines were found to be required for activity, although only C9 and C14 are conserved among pediocin-like bacteriocins. Several basic amino acids as well as nonpolar amino acids located within the hydrophobic C-terminal region also were found to be important. The mutations are discussed in the context of structural models that have been proposed for the bacteriocin.

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All Gram‐negative bacteria tested were normally resistant to the bactericidal action of pediocin P of Pediococcus pentosaceous Pep1, which had been isolated from vacuum‐packaged sausages. However, when they were subjected to sublethal stress (exposure to physical and chemical stresses such as freezing, heating and acid treatment) and were treated with pediocin P, gram‐negative bacterial cells that were normally resistant to pediocin P developed sensitivity to it and pediocin P reduced the viability of bacterial cells surviving sublethal stresses, although the extent of reduction varied with strain and type of treatment. When the combined effect of ethylenediaminetetraacetic acid (EDTA), sublethal heat and pediocin P was studied by adding EDTA and pediocin P to cell suspensions right before heating them, there was a considerable reduction in the number of viable cells, even at the lowest concentration of pediocin P tested. The results of our preliminary studies showed that sublethal injury can make the gram‐negative bacterial cells sensitive to pediocin P.

Pediococcus pentosaceus Pep1 is a vacuum-packaged Turkish sausage isolate which produces a potentially novel bacteriocin of the pediocin (anti-Listeria) family of peptides designated as pediocin P. Curing experiments and plasmid profile analysis indicated that both bacteriocin immunity and production determinants were linked and encoded by 9.0 MDa plasmid, pHD1.0. Attempts to transform purified plasmid pHD1.0 into recipient Escherichia coli JM109 cells by electroporation were successful but none of the E. coli JM109 cells were able to express and/or release pediocin P. However, P. pentosaceus PC, a plasmid-cured variant of P. pentosaceus Pep1 was successfully transformed with pHD1.0 by electroporation and Bac-Bacs P. pentosaceus PC cells restarted to express and/or release pediocin P again as indicated by the presence of zone of growth inhibition of L. plantarum NCDO 955 around colonies.

Nondenaturing polyacrylamide gel electrophoresis revealed the presence of diversity among bacteriocins produced by strains of Bacillus sphaericus. Bacteriocin bands of six strains (pathogenic and non pathogenic) were found to be located just below the stacking gel. However, in two other strains (1 pathogenic and 1 collection strain) more than one protein band with bacteriocin activity were seen in the middle of resolving gel. In bacteriocin-treated cultures, electron-microscopy studies revealed the growth of lysedswollen ghost cells, and loss of viability among sensitive strains.

The main goal of this study was to develop an improved oral delivery system for Pediococcus pentosaceus OZF, a promising probiotic bacterium, and to assess its viability under simulated gastrointestinal (GI) tract model by comparing the efficiency of microbiological and molecular approaches. Encapsulation was carried out using extrusion method and as a result, encapsulation system including 0.75 % lactulose, 1.8 % sodium alginate, 0.1 M CaCl2, and 5 min gelling time was shown to have a significantly protective effect against pH 2.0 acid stress over 3 h. However, completely loss of viability was exhibited by free OZF cells under similar conditions. To provide an additional barrier for capsules, coating process was investigated using different biopolymers, and the survival rates of free and encapsulated OZF cells upon expose to simulated GI conditions were detected by conventional culture techniques and propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR) method. No significant differences between the biopolymers were detected, except the chitosan which leads totally 85 % protection and extra 25 % improvement in the survival of OZF cells compared to uncoated capsules. In conclusion, our findings indicated that chitosan-coated capsules provided an important protective effect on the viability of OZF cells against the GI system conditions encountered during the transit of food. In addition, this study was found successful in monitoring the viable OZF cells in capsules using PMA-qPCR method.