José Luis Ruiz-Barba

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.

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.

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.

A novel Lactobacillus pentosus-paired starter culture for Spanish-style green olive fermentation.

A new starter culture consisting of two Lactobacillus pentosus strains was developed and successfully used for Spanish-style green olive fermentations in an industrial study. The inoculum, consisting of L. pentosus LP RJL2 and LP RJL3 strains, was inoculated in 10,000 kg glass fiber containers at 10⁶ CFU/ml and 10⁵ CFU/ml, final concentration respectively, in five different olive processing plants in the south of Spain. As a control, uninoculated fermentors were also used. In all inoculated fermentors, the paired starter rapidly colonized the brines to dominate the natural microbiota and persisted throughout fermentation. A decline in pH to reach about 5.0 was achieved in the first 15-20 days, reaching about 4.0 at the end of the process. The lactic acid concentration in brines increased rapidly in the first 20 days of fermentation (0.3-0.4 g/100 ml) to give values higher than 0.8 g/100 ml at the end of the process. In contrast, increasing lactic acid concentration was slower in uninoculated than in the inoculated brines, and the final concentrations were lower. Although reaching similar values at the end of the process, the decline in pH in uninoculated fermentors was slower than in the inoculated ones. These results show the efficacy of the new starter culture to control the lactic acid fermentation of Spanish-style green olives.

Enterocin C, a class IIb bacteriocin produced by E. faecalis C901, a strain isolated from human colostrum.

Enterocin C (EntC), a class IIb bacteriocin was purified from culture supernatants of Enterococcus faecalis C901, a strain isolated from human colostrum. Enterocin C consists of two distinct peptides, named EntC1 and EntC2, whose complementary action is required for full antimicrobial activity. The structural genes entC1 and entC2 encoding enterocins EntC1 and EntC2, respectively, and that encoding the putative immunity protein (EntCI) are located in the 9-kb plasmid pEntC, harboured by E. faecalis C901. The N-terminal sequence of both antimicrobial peptides revealed that EntC1 (4284 Da) is identical to Ent1071A, one of the two peptides that form enterocin 1071 (Ent1071), a bacteriocin produced by E. faecalis BFE 1071. In contrast, EntC2 (3867 Da) presents the non-polar alanine residue at position 17 (Ala(17)) instead of the polar threonine residue (Thr(17)) in Ent1071B, the second peptide constituting Ent1071. In spite of peptide similarities, EntC differs from Ent1071 in major aspects, including the complementary activity among its constitutive peptides and its wider inhibitory spectrum of activity. Different amphiphilic alpha-helical conformations between EntC2 and Ent1071B could explain both, acquired complementary activity and increased antimicrobial spectrum.

The locus responsible for production of plantaricin S, a class IIb bacteriocin produced by Lactobacillus plantarum LPCO10, is widely distributed among wild-type Lact. plantarum strains isolated from olive fermentations

The genes plsA and plsB encoding for production of plantaricin S (Pls), a two-peptide bacteriocin produced by Lactobacillus plantarum LPCO10, are commonly distributed among wild-type Lact. plantarum strains isolated from olive fermentations. Among 68 independent isolates from different olive processing plants in South Spain, 15 of them were shown to produce bacteriocins that were active against other lactic acid bacteria, as well as spoilage and pathogenic bacteria. On the basis of PCR amplification and hybridization with specific probes, the Pls operon was detected in all the bacteriocin producer strains but not in the non-producer ones. Purification and subsequent amino acid sequencing of the bacteriocin produced by some of the 15 isolates yielded both the alpha and beta peptides from Pls. These results suggest that bacteriocin production contributes an ecological advantage for the wild-type Lact. plantanum strains in the colonization of the spontaneous, traditional olive fermentation process.

Coculture with specific bacteria enhances survival of Lactobacillus plantarum NC8, an autoinducer-regulated bacteriocin producer, in olive fermentations

Bacteriocin production in Lactobacillus plantarum NC8 is activated by coculture with specific bacteriocin production-inducing bacterial strains. The system is further regulated by a three-component regulatory system involving a specific autoinducer peptide (PLNC8IF). We have used L. plantarum NC8 as a starter culture in Spanish-style green olive fermentations and examined the influence of coculturing in its survival. We found that L. plantarum NC8 greatly enhanced its growth and survival in the olive fermentations when co-inoculated with two specific bacteriocin-production inducing strains, i.e. Enterococcus faecium 6T1a-20 and Pediococcus pentosaceus FBB63, when compared to singly-inoculated fermentations. In addition, a constitutive bacteriocin-producer NC8-derivative strain was used as a control in the olive fermentations and showed also better viability than the parental NC8 strain. Our results suggest the involvement of bacteriocin production in the viability enhancement found in both cases. We postulate that the presence of specific bacteria is recognized by L. plantarum NC8 as an environmental stimulus to switch a specific adaptive response on, most probably involving bacteriocin production. The design of novel bacteriocin-producing starter cultures for food fermentations should consider their constitutive versus regulated character.