Rufino Jiménez-Díaz

Purification and Genetic Characterization of Plantaricin NC8, a Novel Coculture-Inducible Two-Peptide Bacteriocin from Lactobacillus plantarum NC8

ABSTRACT A new, coculture-inducible two-peptide bacteriocin named plantaricin NC8 (PLNC8) was isolated from Lactobacillus plantarum NC8 cultures which had been induced with Lactococcus lactis MG1363 or Pediococcus pentosaceus FBB63. This bacteriocin consists of two distinct peptides, named α and β, which were separated by C2-C18 reverse-phase chromatography and whose complementary action is necessary for full plantaricin NC8 activity. N-terminal sequencing of both purified peptides showed 28 and 34 amino acids residues for PLNC8α and PLNC8β, respectively, which showed no sequence similarity to other known bacteriocins. Mass spectrometry analysis showed molecular masses of 3,587 Da (α) and 4,000 Da (β). The corresponding genes, designated plNC8A and plNC8B, were sequenced, and their nucleotide sequences revealed that both peptides are produced as bacteriocin precursors of 47 and 55 amino acids, respectively, which include N-terminal leader sequences of the double-glycine type. The mature α and β peptides contain 29 and 34 amino acids, respectively. An open reading frame, orfC, which encodes a putative immunity protein was found downstream of plNC8B and overlapping plNC8A. Upstream of the putative −35 region of plNC8B, two direct repeats of 9 bp were identified, which agrees with the consensus sequence and structure of promoters of class II bacteriocin operons whose expression is dependent on an autoinduction mechanism.

Optimization of Bacteriocin Production by Batch Fermentation of Lactobacillus plantarum LPCO10

ABSTRACT Optimization of bacteriocin production by Lactobacillus plantarum LPCO10 was explored by an integral statistical approach. In a prospective series of experiments, glucose and NaCl concentrations in the culture medium, inoculum size, aeration of the culture, and growth temperature were statistically combined using an experimental 235-2 fractional factorial two-level design and tested for their influence on maximal bacteriocin production by L. plantarum LPCO10. After the values for the less-influential variables were fixed, NaCl concentration, inoculum size, and temperature were selected to study their optimal relationship for maximal bacteriocin production. This was achieved by a new experimental 323-1 fractional factorial three-level design which was subsequently used to build response surfaces and analyzed for both linear and quadratic effects. Results obtained indicated that the best conditions for bacteriocin production were shown with temperatures ranging from 22 to 27°C, salt concentration from 2.3 to 2.5%, and L. plantarum LPCO10 inoculum size ranging from 107.3 to 107.4 CFU/ml, fixing the initial glucose concentration at 2%, with no aeration of the culture. Under these optimal conditions, about 3.2 × 104 times more bacteriocin per liter of culture medium was obtained than that used to initially purify plantaricin S from L. plantarum LPCO10 to homogeneity. These results indicated the importance of this study in obtaining maximal production of bacteriocins from L. plantarum LPCO10 so that bacteriocins can be used as preservatives in canned foods.

Induction of Plantaricin Production in Lactobacillus plantarum NC8 after Coculture with Specific Gram-Positive Bacteria Is Mediated by an Autoinduction Mechanism

Plantaricin NC8 (PLNC8), a coculture-inducible two-peptide bacteriocin from Lactobacillus plantarum NC8, has recently been purified and genetically characterized. Analysis of an 8.1-kb NC8 DNA region downstream of the PLNC8 operon revealed the presence of at least four operons involved in bacteriocin production, showing high homology to the plantaricin cluster in L. plantarum C11. However, we found a three-component regulatory operon involving a quorum-sensing mechanism. Two of these components, the induction factor (PLNC8IF) and the histidine kinase, are novel, while the response regulator is identical to PlnD from C11. Homologous expression of plNC8IF in NC8 allowed constitutive bacteriocin production. Heterologous expression of this gene in Lactococcus lactis MG1363 produced supernatants which promoted bacteriocin production in NC8. Reverse transcription-PCR studies indicated that cocultivation of NC8 with inducing cells promoted transcription of the bacteriocin and regulatory operons in NC8. An identical result was obtained after addition of an external source of PLNC8IF. We propose that the presence of specific bacteria could act as an environmental signal that is able to switch on bacteriocin production in L. plantarum NC8 via a quorum-sensing mechanism mediated by PLNC8IF.

Bacteriocin Production in Vancomycin-Resistant and Vancomycin-Susceptible Enterococcus Isolates of Different Origins

ABSTRACT Bacteriocin production was determined for 218Enterococcus isolates (Enterococcus faecalis[93] and E. faecium[125]) obtained from different origins (human clinical samples [87], human fecal samples [78], sewage [28], and chicken samples [25]) and showing different vancomycin susceptibility patterns (vancomycin resistant, all of them vanA positive [56], and vancomycin susceptible [162]). All enterococcal isolates were randomly selected except for the vancomycin-resistant ones. A total of 33 isolates of eight different bacterial genera were used as indicators for bacteriocin production. Forty-seven percent of the analyzed enterococcal isolates were bacteriocin producers (80.6% of E. faecalis and 21.6% ofE. faecium isolates). The percentage of bacteriocin producers was higher among human clinical isolates (63.2%, 81.8% of vancomycin-resistant isolates and 60.5% of vancomycin-susceptible ones) than among isolates from the other origins (28 to 39.3%). Only one out of the 15 vancomycin-resistant isolates from human fecal samples was a bacteriocin producer, while 44.4% of fecal vancomycin-susceptible isolates were. The bacteriocin produced by the vanA-containing E. faecium strain RC714, named bacteriocin RC714, was further characterized. This bacteriocin activity was cotransferred together with thevanA genetic determinant to E. faecalis strain JH2-2. Bacteriocin RC714 was purified to homogeneity and its primary structure was determined by amino acid sequencing, showing an identity of 88% and a similarity of 92% with the previously described bacteriocin 31 from E. faecalis YI717. The presence of five different amino acids in bacteriocin RC714 suggest that this could be a new bacteriocin. The results obtained suggest that the epidemiology of vancomycin resistance may be influenced by different factors, including bacteriocin production.

Production of plantaricin NC8 by Lactobacillus plantarum NC8 is induced in the presence of different types of gram-positive bacteria

Lactobacillus plantarum NC8 was shown to produce plantaricin NC8 (PLNC8), a recently purified and genetically characterized inducible class IIb bacteriocin, only when it was co-cultured with other gram-positive bacteria. Among 82 strains belonging to the genera Bacillus, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Listeria, Pediococcus, Staphylococcus, and Streptococcus, 41 were shown to induce PLNC8 production in L. plantarum NC8. There was apparently no relationship between the sensitivity of the strains and their ability to induce the bacteriocin, indicating that the inducer and sensitive phenotypes may not be linked. In some instances, induction was promoted by both living and heat-killed cells of the inducing bacteria. However, no PLNC8-inducing activity was found in the respective cell-free, pure culture supernatants. Inducer strains also promoted the production of a PLNC8-autoinducing activity by L. plantarum NC8, which was found only in the cell-free co-culture supernatants showing inhibitory activity. This PLNC8-autoinducing activity was diffusible, heat resistant, and of a proteinaceous nature, and was different from the bacteriocin itself. Taken together, the results suggest that the presence of specific gram-positive bacteria acts as an environmental stimulus activating both PLNC8 production by L. plantarum NC8 and a PLNC8-autoinducing activity, which in turn triggers or maintains bacteriocin production in the absence of inducing cells.

Bactericidal action of oleuropein extracted from green olives against Lactobacillus plantarum

The phenolic compound oleuropein extracted from green olives was shown to be bactericidal against nine strains of Lactobacillus plantarum isolated from green olive fermentation brines. Heat‐treated oleuropein also demonstrated a strong bactericidal effect but not alkali‐treated oleuropein, which allowed survival of most of the strains tested. The bactericidal effect was accompanied by changes in the typical bacillary structure and Gram‐positive stain of L. plantarum.

Formation of lactic acid bacteria-yeasts communities on the olive surface during Spanish-style Manzanilla fermentations.

This work examines the formation of poly-microbial communities adhered to the surface of Manzanilla olive fruits processed according to the Spanish style. The experimental design consisted of four pilot fermenters inoculated with four Lactobacillus pentosus strains, plus another fermenter which was not inoculated and fermented spontaneously. Lactic acid bacteria and yeasts were analysed in depth on olive epidermis throughout fermentation by plate count, molecular techniques and scanning electron microscopy. Data show that in all cases high population levels (above 8 log(10) CFU per olive) were reached for both groups of microorganisms at the second week of fermentation and that these counts never fell below 6 log(10) CFU per olive during the 3 months that fermenters were monitored. In situ observation of olive epidermis slices revealed a strong aggregation and adhesion between bacteria and yeasts by the formation of a matrix which embedded the microorganisms. Geotrichum candidum, Pichia galeiformis and Candida sorbosa were the main yeast species isolated from these biofilms at the end of fermentation (confirmed by RFLP analysis of the 5.8S-ITS region), while molecular characterization of lactobacilli isolates by means of RAPD-PCR with primer OPL(5) showed in many cases a high similarity in their banding profiles with the inoculated strains. Results obtained in this survey show the importance of studying the olive epidermis throughout fermentation, because ultimately, olives are ingested by consumers.

Culture Conditions Determine the Balance between Two Different Exopolysaccharides Produced by Lactobacillus pentosus LPS26

ABSTRACT Lactobacillus pentosus LPS26, isolated from a natural fermentation of green olives, produces a capsular polymer constituted of two exopolysaccharides (EPS): EPS A, a high-molecular-weight (high-Mw) polysaccharide (1.9 × 106 Da) composed of glucose and rhamnose (3:1), and EPS B, a low-Mw polysaccharide (3.3 × 104 Da) composed of glucose and mannose (3:1). Fermentation experiments in a chemically semidefined medium with different carbon sources (glucose, fructose, mannitol, and lactose) showed that all of them except fructose supported EPS A production rather than EPS B production. The influence of temperature and pH was further analyzed. As the temperature dropped, increased synthesis of both EPS was detected. The control of pH especially enhanced EPS B production. With regard to this, the maximum total EPS production (514 mg liter−1) was achieved at a suboptimal growth temperature (20°C) and pH 6.0. Continuous cultures showed that EPS A, synthesized mainly at low dilution rates, is clearly dependent on the growth rate, whereas EPS B synthesis was hardly affected. EPS production was also detected in supplemented skimmed milk, but no increase on the viscosity of the fermented milk was recorded. This could be linked to the high proportion of the low-Mw polysaccharide produced in these conditions in contrast to that observed in culture media. Overall, the present study shows that culture conditions have a clear impact on the type and concentration of EPS produced by strain LPS26, and consequently, these conditions should be carefully selected for optimization and application studies. Finally, it should be noted that this is, to our knowledge, the first report on EPS production by L. pentosus.

Biofilm formation on abiotic and biotic surfaces during Spanish style green table olive fermentation

In this work, the establishment of polymicrobial communities on the surfaces which come into contact with the brine during Spanish style Gordal cv. green olive fermentation when subjected to spontaneous or controlled processes (inoculated with Lactobacillus pentosus LPCO10 or 128/2) was studied. Scanning electron microscopy showed that L. pentosus and yeast populations were able to form mixed biofilms throughout the fermentation process on both abiotic (glass slide) and biotic (olive skin) surfaces. The biofilm architectures in both supports were completely different: on the glass slides only aggregates of L. pentosus and yeasts without any polymeric matrix surrounding them were found while on the skin of the fruits, true mature biofilms were observed. During fermentation, the lactic acid bacteria (LAB) population on the olives remained similar while that of yeasts increased progressively to reach similar levels at the end of the process (8-9 log CFU/cm(2)). Molecular analysis showed that different populations of L. pentosus and yeasts were the only microbial members of the biofilm formed during fermentation, regardless of inoculation. Hence, the green olive surface provides an appropriate environmental condition for the suitable development and formation of complex biofilms during controlled or natural table olive processing.

Bacteriocin production and competitiveness of Lactobacillus plantarum LPCO10 in olive juice broth, a culture medium obtained from olives

A culture medium, named olive juice broth, which resembles the natural environment of Lactobacillus plantarum in the traditional Spanish-style green olive fermentation was obtained from green olives. In this medium, the bacteriocin-producing L. plantarum LPCO10 strain was able to produce bacteriocin throughout the incubation time (15 days). Bacteriocin purification from olive juice broth was achieved by a protocol including ammonium sulphate precipitation of cell-free, L. plantarum LPCO10 culture supernatants, and cation-exchange, hydrophobic-interaction and reversed-phase chromatographies. In a series of mixed cultures in olive juice broth, L. plantarum LPCO10 was able to dominate the bacteriocin-sensitive L. plantarum 128/2 strain, whereas the non-bacteriocin-producing, LPCO10 strain derivative, L. plantarum 55-1 strain did not show such capability. These results indicated that olive juice broth may be a valuable experimental substitute for olive fermentation brine in gaining more knowledge about the role of the bacteriocin-producing L. plantarum strains in the control of the Spanish-style green olive fermentation.