Microbial and Chemical Characterization of Natural-Style Green Table Olives from the Gordal, Hojiblanca and Manzanilla Cultivars.

Microbial and biochemical changes in the brine during the spontaneous fermentation of Gordal, Hojiblanca and Manzanilla olive cultivars processed according to the natural style were monitored. The microbial composition was assessed through a metagenomic study. Sugars, ethanol, glycerol, organic acids and phenolic compounds were quantified by standard methods. In addition, the volatile profiles, contents of phenolic compounds in the olives and quality parameters of the final products were compared. Fermentation in Gordal brines was conducted by lactic acid bacteria (mainly Lactobacillus and Pediococcus) and yeasts (mainly Candida boidinii, Candida tropicalis and Wickerhamomyces anomalus). In Hojiblanca and Manzanilla brines, halophilic Gram-negative bacteria (e.g., Halomonas, Allidiomarina and Marinobacter) along with yeasts (mainly, Saccharomyces) were responsible for the fermentation. Higher acidity and lower pH values were reached in Gordal brines compared to Hojiblanca and Manzanilla. After 30 days of fermentation, no sugars were detected in Gordal brine, but residual amounts were found in the brines from Hojiblanca (<0.2 g/L glucose) and Manzanilla (2.9 g/L glucose and 0.2 g/L fructose). Lactic acid was the main acid product in Gordal fermentation, whereas citric acid was the predominant organic acid in the Hojiblanca and Manzanilla brines. Manzanilla brine samples showed a greater concentration of phenolic compounds than Hojiblanca and Gordal brines. After a 6-month fermentation, Gordal olives were superior compared to the Hojiblanca and Manzanilla varieties regarding product safety (lower final pH and absence of Enterobacteriaceae), content of volatile compounds (richer aroma), content of bitter phenolics (lower content of oleuropein, which resulted in less perceived bitterness) and color parameters (more yellow and lighter color, indicating a higher visual appraisal). The results of the present study will contribute to a better understanding of each fermentation process and could help to promote natural-style elaborations using the above-mentioned olive cultivars.

Microbial community and volatilome changes in brines along the spontaneous fermentation of Spanish-style and natural-style green table olives (Manzanilla cultivar).

Microbial community and volatilome of brines were monitored during the spontaneous fermentations of Spanish-style and Natural-style green table olives from Manzanilla cultivar. Fermentation of olives in the Spanish style was carried out by lactic acid bacteria (LAB) and yeasts, whereas halophilic Gram-negative bacteria and archaea, along with yeasts, drove the fermentation in the Natural style. Clear differences between the two olive fermentations regarding physicochemical and biochemical features were found. Lactobacillus, Pichia, and Saccharomyces were the dominant microbial communities in the Spanish style, whereas Allidiomarina, Halomonas, Saccharomyces, Pichia, and Nakazawaea predominated in the Natural style. Numerous qualitative and quantitative differences in individual volatiles between both fermentations were found. The final products mainly differed in total amounts of volatile acids and carbonyl compounds. In addition, in each olive style, strong positive correlations were found between the dominant microbial communities and various volatile compounds, some of them previously reported as aroma-active compounds in table olives. The findings from this study provide a better understanding of each fermentation process and may help the development of controlled fermentations using starter cultures of bacteria and/or yeasts for the production of high-quality green table olives from Manzanilla cultivar.

Production of volatile compounds by wild-type yeasts in a natural olive-derived culture medium.

The production of volatile compounds in naturally fermented green table olives from Manzanilla cultivar was investigated. A total of 62 volatile compounds were detected after 24 weeks of fermentation. To clarify the contribution of yeasts to the formation of these compounds, such microorganisms were isolated from the corresponding fermenting brines. Five major yeast strains were identified: Nakazawaea molendinolei NC168.1, Zygotorulaspora mrakii NC168.2, Pichia manshurica NC168.3, Candida adriatica NC168.4, and Candida boidinii NC168.5. When these yeasts were grown as pure cultures in an olive-derived culture medium, for 7 days at 25 °C, the number of volatiles produced ranged from 22 (P. manshurica NC168.3) to 60 (C. adriatica NC168.4). Contribution of each yeast strain to the qualitative volatile profile of fermenting brines ranged from 19% (P. manshurica NC168.3) to 48% (Z. mrakii NC168.2 and C. adriatica NC168.4). It was concluded that C. adriatica NC168.4 presented the best aromatic profile, being a solid candidate to be part of a novel starter culture to enhance the organoleptic properties of naturally fermented green table olives.

Microbial biogeography of Spanish-style green olive fermentations in the province of Seville, Spain.

The aim of this study is to examine the biogeography of the microbial communities associated to the Spanish-style green olive fermentations in the province of Seville (Andalucía, south-western Spain). Also, to understand how microorganisms colonize and persist in non-sterile food fermentations across a specific table olive producing area, i.e. a specific "agroecosystem". The microbial diversity, bacteria and yeast, in 30 ten-ton fermenters of three different fermentations yards (patios) along the olive fermentation was studied. A total of 951 microbial isolates were obtained which were clustered according to their RAPD profile. A total of 376 distinct genotypes were identified, belonging to 57 different microbial species, 41 bacterial and 16 yeast species. Up to 16 bacterial species had not been described before in table olives. Only the species Lactobacillus pentosus showed a ubiquitous presence in all 30 fermenters. Pediococcus parvulus, Lactobacillus collinoides/paracollinoides, Lactobacillus coryniformis, Lactobacillus plantarum, Pichia manshurica and Candida thaimueangensis were found in every patio. Cosmopolitan strains, up to 15, were shared by the three patios and belonged to the species L. pentosus (12 strains), P. parvulus (1), L. collinoides/paracollinoides (1) and P. manshurica (1). To expand our biodiversity analyses to the "regional" level, we have compared our results with those obtained from two previously studied patios of similar characteristics and in the same geographical area. PERMANOVA analysis of the microbial community composition revealed significant differences among different patios in their structure at every fermentation stage. In contrast, SIMPER analyses showed that, as fermentation progressed, the overall dissimilarities among patios were reduced. Discriminant species were identified for each fermentation stage. Among these, L. pentosus and P. parvulus were "eu-constant" species, while L. collinides/paracollinoides and Marinilactibacillus psychrotolerans group were "constant" species that could be considered microbial key taxa based on the occurrence stability index. The characteristic and, presumably, well adapted microbiota associated to the Spanish-style olive fermentations at the specific geographic area described here is a valuable natural resource which should be preserved conveniently. To our knowledge, this is the first study on the microbial biogeography of table olive fermentations, both at the species and strain levels.

Expression of genes involved in metabolism of phenolic compounds by Lactobacillus pentosus and its relevance for table-olive fermentations.

Genes with the potential to code for enzymes involved in phenolic compound metabolism were detected in the genome of Lactobacillus pentosus IG1, isolated from a green olive fermentation. Based on homology, these genes could code for a 6-P-ß Glucosidase, two different Tannases, a Gallate decarboxylase and a p-Coumaric decarboxylase. Expression of up to seven of these genes was studied in L. pentosus IG1 (olive fermentation) and CECT4023T (corn silage), including responses upon exposure to relevant phenolic compounds and different olive extracts. Genes potentially coding Tannase, Gallate decarboxylase and p-Coumaric acid decarboxylase significatively increased their expression upon exposure to such compounds and extracts, although it was strain dependent. In general, both the genetic organization and the characteristics of gene expression resembled very much those described for Lactobacillus plantarum. In accordance to the observed induced gene expression, metabolism of specific phenolic compounds was achieved by L. pentosus. Thus, methyl gallate, gallic acid and the hydroxycinamic acids p-coumaric, caffeic and ferulic were metabolized. In addition, the amount of phenolics such as tyrosol, oleuropein, rutin and verbascoside included in a minimal culture medium was noticeably reduced, again dependent on the strain considered.

Purification and genetic characterization of gassericin E, a novel co-culture inducible bacteriocin from Lactobacillus gasseri EV1461 isolated from the vagina of a healthy woman.

BACKGROUND: Lactobacillus gasseri is one of the dominant Lactobacillus species in the vaginal ecosystem. Some strains of this species have a high potential for being used as probiotics in order to maintain vaginal homeostasis, since they may confer colonization resistance against pathogens in the vagina by direct inhibition through production of antimicrobial compounds, as bacteriocins. In this work we have studied bacteriocin production of gassericin E (GasE), a novel bacteriocin produced by L. gasseri EV1461, a strain isolated from the vagina of a healthy woman, and whose production was shown to be promoted by the presence of certain specific bacteria in co-culture. Biochemical and genetic characterization of this novel bacteriocin are addressed. RESULTS: We found that the inhibitory spectrum of L. gasseri EV1461 was broad, being directed to species both related and non-related to the producing strain. Interestingly, L. gasseri EV1461 inhibited the grown of pathogens usually associated with bacterial vaginosis (BV). The antimicrobial activity was due to the production of a novel bacteriocin, gassericin E (GasE). Production of this bacteriocin in broth medium only was achieved at high cell densities. At low cell densities, bacteriocin production ceased and only was restored after the addition of a supernatant from a previous bacteriocin-producing EV1461 culture (autoinduction), or through co-cultivation with several other Gram-positive strains (inducing bacteria). DNA sequence of the GasE locus revealed the presence of two putative operons which could be involved in biosynthesis and immunity of this bacteriocin (gaeAXI), and in regulation, transport and processing (gaePKRTC). The gaePKR encodes a putative three-component regulatory system, involving an autoinducer peptide (GaeP), a histidine protein kinase (GaeK) and a response regulator (GaeR), while the gaeTC encodes for an ABC transporter (GaeT) and their accessory protein (GaeC), involved in transport and processing of the bacteriocin. The gaeAXI, encodes for the bacteriocin gassericin E (GasE), a putative peptide bacteriocin (GaeX), and their immunity protein (GaeI). CONCLUSIONS: The origin of the strain (vagina of healthy woman) and its ability to produce bacteriocins with inhibitory activity against vaginal pathogens may be an advantage for using L. gasseri EV1461 as a probiotic strain to fight and/or prevent bacterial infections as bacterial vaginosis (BV), since it could be better adapted to live and compete into the vaginal environment.

Diversity and enumeration of halophilic and alkaliphilic bacteria in Spanish-style green table-olive fermentations.

The presence and enumeration of halophilic and alkaliphilic bacteria in Spanish-style table-olive fermentations was studied. Twenty 10-tonne fermenters at two large manufacturing companies in Spain, previously studied through both culture dependent and independent (PCR-DGGE) methodologies, were selected. Virtually all this microbiota was isolated during the initial fermentation stage. A total of 203 isolates were obtained and identified based on 16S rRNA gene sequences. They belonged to 13 bacterial species, included in 11 genera. It was noticeable the abundance of halophilic and alkaliphilic lactic acid bacteria (HALAB). These HALAB belonged to the three genera of this group: Alkalibacterium, Marinilactibacillus and Halolactibacillus. Ten bacterial species were isolated for the first time from table olive fermentations, including the genera Amphibacillus, Natronobacillus, Catenococcus and Streptohalobacillus. The isolates were genotyped through RAPD and clustered in a dendrogram where 65 distinct strains were identified. Biodiversity indexes found statistically significant differences between both patios regarding genotype richness, diversity and dominance. However, Jaccard similarity index suggested that the halophilic/alkaliphilic microbiota in both patios was more similar than the overall microbiota at the initial fermentation stage. Thus, up to 7 genotypes of 6 different species were shared, suggesting adaptation of some strains to this fermentation stage. Morisita-Horn similarity index indicated a high level of codominance of the same species in both patios. Halophilic and alkaliphilic bacteria, especially HALAB, appeared to be part of the characteristic microbiota at the initial stage of this table-olive fermentation, and they could contribute to the conditioning of the fermenting brines in readiness for growth of common lactic acid bacteria.

PCR-DGGE assessment of the bacterial diversity in Spanish-style green table-olive fermentations.

The bacterial ecology associated to Spanish-style green olive fermentations has been studied, attending to its dynamics along the time and its distribution, by a culture-independent approach based on PCR-DGGE. Forty-three 10-tonne fermenters were selected from the fermentation yards (patios) of two large table-olive manufacturing companies in southern Spain. The fermenting brines of 20 of these fermenters were previously analyzed through culture-dependent methods, allowing comparisons of both methodologies. A statistical analysis of DGGE banding profiles obtained using bacteria universal primers demonstrated significant evidences of discrimination of bacterial communities by location (patio) and fermentation stage. Specific microbial "fingerprints" could be established for these variables. At least 17 bacterial species were detected, most of them previously isolated from the same fermenters. Most of these species belonged to the lactic acid bacteria (LAB) group. Dominance of species within the Lactobacillus plantarum group was confirmed. Marinilactibacillus sp. and Propionibacterium olivae, which were not isolated in the previous culture-dependent study, were detected. Alkalibacterium sp. and Halolactibacillus sp. were detected for the first time in table olive fermentations. Using Lactobacillus-group specific primers, significant clustering within the DGGE banding profiles was observed, allowing discrimination regarding the actual fermentation stage. These results corroborated the previous culture-dependent study, and added the detection of Alkalibacterium sp. and Pediococcus acidilactici. The species Alkalibacterium sp., Marinilactibacillus sp. and Halolactibacillus sp. are characterized by their ability to carry out lactic acid fermentation under alkaline conditions and thus ascribed within the halophilic and alkaliphilic lactic acid bacteria (HALAB). Their ubiquitous presence suggests that they could play an important role in Spanish-style olive fermentations, especially at the initial fermentation stage. Thus, they could contribute to brine conditioning before L. plantarum group-driven lactic acid fermentation takes place.

Genetic diversity and dynamics of bacterial and yeast strains associated to Spanish-style green table-olive fermentations in large manufacturing companies.

We have genotyped a total of 1045 microbial isolates obtained along the fermentation time of Spanish-style green table olives from the fermentation yards (patios) of two large manufacturing companies in the Province of Sevilla, south of Spain. Genotyping was carried out using RAPD-PCR fingerprinting. In general, isolates clustered well into the relevant phylogenetic dendrograms, forming separate groups in accordance to their species adscription. We could identify which bacterial and yeast genotypes (strains) persisted throughout the fermentation at each patio. Also, which of them were more adapted to any of the three stages, i.e. initial, middle and final, described for this food fermentation. A number of genotypes were found to be shared by both patios. Fifty seven of these belonged to five different bacterial species, i.e. Lactobacillus pentosus, Lactobacillus paracollinoides/collinoides, Lactobacillus rapi, Pediococcus ethanolidurans and Staphylococcus sp., although most of them (51) belonged to L. pentosus. Four yeast genotypes were also shared, belonging to the species Candida thaimueangensis, Saccharomyces cerevisiae and Hanseniaspora sp. Two genotypes of L. pentosus were found to be grouped with those of two strains used in commercially available starter cultures, one of them bacteriocinogenic, which were used up to three years before this study in these patios, demonstrating the persistence of selected strains in this environment. Biodiversity was assessed though different indexes, including richness, diversity and dominance. A statistically significant decrease in biodiversity between the initial and final stages of the fermentation was found in both patios. However, values of biodiversity indexes in the fermenters were very similar, and no significant differences were found in the total biodiversity between both patios. This study allowed us to identify a range of well adapted strains (genotypes), especially those belonging to the lactic acid bacteria, which could be useful to improve safety and quality of table olive fermentations.

Propionibacterium olivae sp. nov. and Propionibacterium damnosum sp. nov., isolated from spoiled packaged Spanish-style green olives.

Five strains of Gram-stain-positive bacteria were isolated from anomalous fermentations occurring in post-packaging of sealed airtight food-grade plastic pouches of Spanish-style green olives. These isolates could be grouped into two sets, which showed a similarity in their respective 16S rRNA gene sequences of 98.40 and 98.44 % with Propionibacterium acidipropionici NCFB 563 and 98.33 and 98.11 % with Propionibacterium microaerophilum M5(T), respectively, and a similarity of 99.41 % between them. The 16S rRNA gene phylogeny revealed that the isolates grouped into two statistically well-supported clusters separate from P. acidipropionici NCFB 563 and P. microaerophilum M5(T). Enzymic activity profiles as well as fermentation patterns differentiated these two novel bacteria from other members of the genus Propionibacterium. Finally, phenotypic, genotypic and phylogenetic data, supported the proposal of two novel species of the genus Propionibacterium, for which the names Propionibacterium olivae sp. nov. (type strain, IGBL1(T) = CECT 8061(T) = DSM 25436(T)) and Propionibacterium damnosum sp. nov. (type strain, IGBL13(T) = CECT 8062(T) = DSM 25450(T)) are proposed.

Microbial diversity and dynamics of Spanish-style green table-olive fermentations in large manufacturing companies through culture-dependent techniques.

We have studied the microbiota associated to Spanish-style green olive fermentations, attending to its dynamics along the time. Twenty 10-tonne fermenters were selected from two large table-olive manufacturing companies in southern Spain. While culture-dependent methodology was used to isolate the microorganisms, molecular methods were used to identify the isolates. A total of 1070 isolates were obtained, resulting in 929 bacterial and 141 yeast isolates. Thirty seven different bacterial species were isolated, belonging to 18 different genera, while 12 yeast species were isolated, belonging to 7 distinct genera. This fermentation was dominated by the species Lactobacillus pentosus, while its accessory microbiota was variable and depended on the fermentation stage and the actual fermentation yard ("patio"). It was noticeable the abundance of lactic acid bacteria isolates, belonging to 16 different species. Twenty bacterial species were isolated for the first time from Spanish-style green olive fermentations, while 17 had not been described before in any table olive preparation. The genera Brachybacterium, Paenibacillus, Sporolactobacillus, Paracoccus and Yersinia had not been cited before from any table olive preparation. Saccharomyces cerevisiae and Candida thaimueangensis appeared to dominate the yeast microbiota. Candida butyri/asseri and Rhodotorula mucilaginosa had not been described before from Spanish-style preparations, while Saturnispora mendoncae was isolated for the first time from any table olive preparation. Biodiversity was analysed through different alpha and beta indexes which showed the evolution of the microbiota over time.

Enterococcus olivae sp. nov., isolated from Spanish-style green-olive fermentations.

Six strains of a hitherto unknown, Gram-stain-positive coccus were recovered from samples of Spanish-style green-olive fermentations. The 16S rRNA gene sequences from these isolates shared 98.7% and 98.5% of their nucleotide positions with those from Enterococcus saccharolyticus subsp. taiwanensis 812(T) and from E. saccharolyticus subsp. saccharolyticus ATCC 43076(T), respectively. The sequence of the rpoA gene in the isolates was 95% similar to that of E. saccharolyticus CECT 4309(T) ( = ATCC 43076(T)). The 16S rRNA and rpoA gene phylogenies revealed that the isolates grouped in a statistically well-supported cluster separate from E. saccharolyticus. Enzyme activity profiles as well as fermentation patterns differentiated the novel bacteria from other members of the Enterococcus genus. Finally, phenotypic, genotypic and phylogenetic data supported the identification of a novel species of the genus Enterococcus, for which the name Enterococcus olivae sp. nov. is proposed. The type strain is IGG16.11(T) ( = CECT 8063(T) = DSM 25431(T)).

Garvicin A, a novel class IId bacteriocin from Lactococcus garvieae that inhibits septum formation in L. garvieae strains.

Lactococcus garvieae 21881, isolated in a human clinical case, produces a novel class IId bacteriocin, garvicin A (GarA), which is specifically active against other L. garvieae strains, including fish- and bovine-pathogenic isolates. Purification from active supernatants, sequence analyses, and plasmid-curing experiments identified pGL5, one of the five plasmids found in L. garvieae [M. Aguado-Urda et al., PLoS One 7(6):e40119, 2012], as the coding plasmid for the structural gene of GarA (lgnA), its putative immunity protein (lgnI), and the ABC transporter and its accessory protein (lgnC and lgnD). Interestingly, pGL5-cured strains were still resistant to GarA. Other putative bacteriocins encoded by the remaining plasmids were not detected during purification, pointing to GarA as the main inhibitor secreted by L. garvieae 21881. Mode-of-action studies revealed a potent bactericidal activity of GarA. Moreover, transmission microscopy showed that GarA seems to act by inhibiting septum formation in L. garvieae cells. This potent and species-specific inhibition by GarA holds promise for applications in the prevention or treatment of infections caused by pathogenic strains of L. garvieae in both veterinary and clinical settings.

Induction of bacteriocin production by coculture is widespread among plantaricin-producing Lactobacillus plantarum strains with different regulatory operons.

We describe the bacteriocin-production phenotype in a group of eight singular bacteriocinogenic Lactobacillus plantarum strains with three distinct genotypes regarding the plantaricin locus. Genotyping of these strains revealed the existence of two different plantaricin-production regulatory operons, plNC8-plNC8HK-plnD or plnABCD, involving three-component systems controlled each of them by a specific autoinducer peptide (AIP), i.e. PLNC8IF or PlnA. While all of the strains produced antimicrobial activity when growing on solid medium, most of them halted this production when cultured in broth, thus reflecting the functionality of regulatory mechanisms. Antimicrobial activity in broth cultures was re-established or enhanced when the specific AIP was added to the culture or by coculturing with specific bacterial strains. The latter trait appeared to be widespread in bacteriocinogenic L. plantarum strains independently of the regulatory system used to regulate bacteriocin production or the specific bacteriocins produced. The induction spectrum through coculture, i.e. the pattern of bacterial strains able to induce bacteriocin production, was characteristic of each individual L. plantarum strain. Also, the ability of some bacteria to induce bacteriocin production in L. plantarum by coculture appeared to be strain specific. The fact that induction of bacteriocin production by coculturing appeared to be a common feature in L. plantarum can be exploited accordingly to enhance the viability of this species in food and feed fermentations, as well as to contribute to probiotic functionality when colonising the gastrointestinal tract.

High-salt brines compromise autoinducer-mediated bacteriocinogenic Lactobacillus plantarum survival in Spanish-style green olive fermentations.

The effect of NaCl on plantaricin production by five Lactobacillus plantarum strains was investigated. Plantaricin production in these strains was found to be regulated by three-component regulatory systems ruled by two different autoinducer peptides (AIPs), either PLNC8IF or Plantaricin A. Bacteriocin activity exhibited by these strains came to a halt in liquid medium containing NaCl concentrations of or above 2%. In contrast, bacteriocin activity was still observed when the producing strains were growing on solid medium containing up to 4% NaCl. Bacteriocin activity in liquid medium containing up to 2% NaCl could be restored by coculturing the producing strains with a selected plantaricin-production inducing strain of Lactococcus lactis. Growth of these bacteriocinogenic L. plantarum strains was monitored in traditional Spanish-style green olive fermentations. Survival of these strains could not be enhanced when provided with a range of plantaricin-production inducing mechanisms previously described, such as constitutive AIP production or coinoculation with a specific bacteriocin-production inducing strain of L. lactis. Our results suggest that it is advisable the use of constitutive bacteriocin producers, or at least non-AIP-dependant ones, as starters for olive fermentations due to the intrinsic physical characteristics of this food fermentation, especially the high salt concentration of the brines currently used.

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.

Genome sequence of Lactobacillus pentosus IG1, a strain isolated from Spanish-style green olive fermentations.

Lactobacillus pentosus is the most prevalent lactic acid bacterium in Spanish-style green olive fermentations. Here we present the draft genome sequence of L. pentosus IG1, a bacteriocin-producing strain with biotechnological and probiotic properties isolated from this food fermentations.

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.

Carotenoid production in Lactobacillus plantarum.

Eighteen strains of Lactobacillus plantarum from different origins were screened for carotenoid production, as many of them exhibited a deep yellow pigmentation when cultured as isolated colonies on MRS-agar plates. We found that most of them produced significative amounts of the yellow C(30) carotenoid 4,4'-diaponeurosporene in the range 1.8 to 54 mg/kg of dry cell weight. Although some of the strains produced just trace amounts of this carotenoid, PCR studies showed that all of them harbored the genes crtM and crtN which, inferred from homology, had been predicted in the three L. plantarum complete genome sequences currently available. Our results suggest the full functionality of a C(30) carotenoid biosynthesis pathway in this species, driven by the operon crtNM. DNA sequencing of the entire crtNM operon in the maximum carotenoid-producing strain found in this study, i.e. L. plantarum CECT7531, was accomplished. Genes crtM and crtN were annotated as dehydrosqualene synthase and dehydrosqualene desaturase, respectively, in this strain.

Knockout of three-component regulatory systems reveals that the apparently constitutive plantaricin-production phenotype shown by Lactobacillus plantarum on solid medium is regulated via quorum sensing.

It has been found that many bacteriocins from lactic acid bacteria (LAB) are only produced in broth cultures when specific growth conditions are achieved and a dedicated three-component regulatory system, involved in a quorum sensing (QS) mechanism, is switched on. Surprisingly, bacteriocin production in LAB occurs in an apparently constitutive manner on solid media. This study addresses the question of constitutive versus regulated bacteriocin production on solid media in two different QS-regulated plantaricin-producing strains: Lactobacillus plantarum NC8 and L. plantarum WCFS1. Construction of knockout mutants for their respective regulatory operons revealed that bacteriocin production is controlled through a QS mechanism in both strains, on solid as well as in liquid media. These results could be extensible to other bacteriocins from LAB which are only produced on agar plates and not in broth cultures. Our findings suggest that QS-regulated bacteriocin production in LAB has evolved for competing on solid supports rather than in liquid media. In practice, this could be of major importance in vegetable fermentations, where the solid substrate itself provides an enormous surface where bacteria can attach to and produce biofilms. Therefore, QS-regulated bacteriocinogenic LAB growing in biofilms are under the optimum conditions to produce bacteriocins. Selection of strains to be used as starter cultures for vegetable fermentations should take into account these facts.