Modeling the antimicrobial effects of olive mill waste extract, rich in hydroxytyrosol, on the growth of lactic acid bacteria using response surface methodology.

The objective of this study is to assess the inhibitory effects of an aqueous extract from olive oil mill waste (alperujo) on the growth of a lactic acid bacteria (LAB) cocktail consisting of various strains of Lactiplantibacillus pentosus and Lactiplantibacillus plantarum species. For this purpose, response surface methodology was employed using two independent variables (pH levels 3.5-5.55; hydroxytyrosol concentration ranging from 0.93-2990 ppm). The response variable was the average inhibition per treatment on the LAB cocktail (expressed as a percentage). The developed model identified significant terms, including the linear effect of hydroxytyrosol and pH, their interaction, and the quadratic effect of pH. Maximum inhibition of the LAB cocktail was observed at progressively higher concentrations of hydroxytyrosol and lower pH values. Therefore, complete inhibition of LAB in the synthetic culture medium could only be achieved for concentrations of 2984 ppm hydroxytyrosol at a pH of 3.95. These findings suggest that extracts derived from "alperujo" could be utilized as a natural preservative in acidified foods with a bitter flavor and antioxidant requirements.

Accomplished High-Resolution Metabolomic and Molecular Studies Identify New Carotenoid Biosynthetic Reactions in Cyanobacteria.

Cyanobacteria and microalgae are characterized by a rich and varied profile of chlorophyll (8-20 mg/g) and carotenoid (>2.2 mg/g) pigments, being noteworthy material for natural pigment production in the food industry. We propose a systematic workflow that uses high-performance liquid chromatography (HPLC) coupled with high-resolution tandem mass spectrometry in a broadband collision-induced dissociation mode (bbCID) acquisition mode to simultaneously obtain MS and MSn spectra. Metabolomic studies showed for the first time the presence of carotenoids with 5,6-epoxy-groups (5,6-epoxy- and 5,8-furanoid ß-cryptoxanthin), carotenoids from the α-branch (5,8-furanoid-2'-3'-didehydro α-cryptoxanthin), and 2'-dehydrodeoxomyxol in cyanobacteria. To support the new findings, an in silico search retrieved the putative sequences of carotenogenic enzymes involved in the corresponding biosynthetic pathways (ZEP, NSY, CrtL-b and CrtR) in the analyzed cyanobacteria species. Consequently, high-throughput metabolomics studies assisted by molecular analysis offer a powerful tool for providing insights into the characterization of bioactive compounds and their metabolism in cyanobacteria and microalgae.

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.

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

Three isolates originating from Spanish-style green-olive fermentations in a manufacturing company in the province of Seville, Spain, were taxonomically characterized by a polyphasic approach. This included a phylogenetic analysis based on 16S rRNA gene sequences and multi-locus sequence analysis (MLSA) based on pyrH, recA, rpoA, gyrB and mreB genes. The isolates shared 98.0 % 16S rRNA gene sequence similarity with Vibrio xiamenensis G21T. Phylogenetic analysis based on 16S rRNA gene sequences using the neighbour-joining and maximum-likelihood methods showed that the isolates fell within the genus Vibrio and formed an independent branch close to V. xiamenensis G21T. The maximum-parsimony method grouped the isolates to V. xiamenensis G21T but forming two clearly separated branches. Phylogenetic trees based on individual pyrH, recA, rpoA, gyrB and mreB gene sequences revealed that strain IGJ1.11T formed a clade alone or with V. xiamenensis G21T. Sequence similarities of the pyrH, recA, rpoA, gyrB and mreB genes between strain IGJ1.11T and V. xiamenensis G21T were 86.7, 85.7, 97.3, 87.6 and 84.8 %, respectively. MLSA of concatenated sequences showed that strain IGJ1.11T and V. xiamenensis G21T are two clearly separated species that form a clade, which we named Clade Xiamenensis, that presented 89.7 % concatenated gene sequence similarity, i.e. less than 92 %. The major cellular fatty acids (>5 %) of strain IGJ1.11T were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C16 : 0 and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c). Enzymic activity profiles, sugar fermentation patterns and DNA G+C content (52.9 mol%) differentiated the novel strains from the closest related members of the genus Vibrio. The name Vibrio olivae sp. nov. is proposed for the novel species. The type strain is IGJ1.11T ( = CECT 8064T = DSM 25438T).

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)).

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.

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.

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.

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.

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.