Suchergebnisse

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. ; This study was supported by the Spanish Government through the MEC Projects AGL2003-00642 and AGL2006-00763. ; Peer reviewed

In this study, we describe a very simple and rapid method for extraction of both plasmid and chromosomal DNA from colonies or pellets of bacteria and yeast that is suitable for subsequent PCR. Our method involves only chloroform as the cell disrupting and extracting agent, also contributing to DNA preservation from inherent and contaminating nucleases. ; This study was supported by the Spanish government through MCYT projects AGL2000-1611-C03-01 and AGL2003-00642. ; Peer reviewed

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 heatkilled 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. ; This work was supported by the Spanish Government through MCYT project AGL2000-1611-CO3-01. A.M. was the recipient of a grant from MCYT, Spain. ; Peer reviewed

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. ; This work was supported by the Spanish Government through MCYT project AGL2000-1611-CO3-01. A.M. was the recipient of a grant from MCYT, Madrid, Spain. ; Peer reviewed

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 and , 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 PLNC8 and PLNC8 , respectively, which showed no sequence similarity to other known bacteriocins. Mass spectrometry analysis showed molecular masses of 3,587 Da ( ) and 4,000 Da ( ). 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 and 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. ; This work was supported by the Spanish Government through MCYT project AGL2000-1611-CO3-01. A.M. was the recipient of a grant from MCYT, Spain. ; Peer reviewed

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 23 5-2 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 32 3-1 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. ; This work was supported in part by the Spanish government through MCYT projects AGL2000-1611-CO3-01 and AGL2000-1539-CO2-01 and by the Junta de Andalucía. A.M.-B. was the recipient of a grant from MCYT, Madrid, Spain. ; Peer reviewed

27 páginas, 5 figuras ; The present work studies the in vitro interactions between Lactobacillus pentosus and yeast strains isolated from table olive processing to form mixed biofilms. Among the different pairs assayed, the strongest biofilms were obtained from L. pentosus and Candida boidinii strain co-cultures. However, biofilm formation was inhibited in the presence of D(+)-mannose. In addition, biofilm formation by C. boidinii mono-cultures was stimulated in the absence of cell-cell contact with L. pentosus. Scanning electron microscopy revealed that a sort of "sticky" material formed by the yeasts contributed to its substrate adherence. Hence, the data obtained in this work suggests that yeast-lactobacilli biofilm may be favored by the presence of a specific mate of yeast-L. pentosus, and that more than one mechanism could be implicated in the biofilm formation. This knowledge will help for designing appropriate mixed starter cultures of pairs of L. pentosus-yeast species able to improve the quality and safety of Spanish-style green table olive processing. ; The Spanish Government supported this work through MICIIN projects AGL2009- 08016/ALI, AGL2010-15529/ALI and AGL2013-48300-R/ALI (project OliFilm: www.olifilm.science.com.es) and the Junta de Andalucía (through financial support to Project P11-AGR-7051). F.N.A-L was the recipient of a Ramón y Cajal post-doctoral research contract (Spanish Government) while A.L.-R and J.D.-M. were the beneficiaries of an F.P.I grant from the MICIIN and of a JAE-predoc grant from the C.S.I.C., respectively. ; Peer reviewed

36 Páginas.-- 8 Figuras.-- 1 Tabla ; This work relates native lactic acid bacteria (LAB) (Lactobacillus pentosus LPG1, L. pentosus Lp13, and Lactobacillus plantarum Lpl15) and yeast (Wickerhamomyces anomalus Y12) starters to the volatile components (VOCs) produced in green Spanish-style table olives. For this aim, the VOC profile was considered as compositional data (CoDa). The CoDa analysis generated new information on the relationship among inocula and VOCs through the tetrahedral plot, CoDa-biplot, variation array matrix, and CoDa dendrogram. The ilr (which includes pivot) coordinates (Euclidean space) from VOCs produced more reliable starters' clustering than the original data. The potential VOC markers, identified by a test based on the pairwise comparison of the logratio variation arrays from the whole data set and the individual groups, were (starters in the parenthesis): 2-phenylethyl acetate (LPG1, Y12, Y12 + LAB), methanol (Lpl15), cis-2-penten-1-ol (LPG1, Y12, Y12 + LAB), 2-methyl-3-hexanol (LPG1, Y12), U (non-identified) C (m/z 83-112-97) (Y12) and UF (m/z 95-154-110) (LPG1, Y12 + LAB). Besides, some VOCs were partial/totally inhibited by specific starters: 2-methyl-1-propanol (Lp13, Y12 + LAB), 2-phenyl ethanol (Lp13), furfuryl methyl ether (Y12 + LAB), purpurocatechol (Y12, Y12 + LAB), 4-ethyl guaiacol (Lp13, Lpl15), 4-ethyl phenol (Lpl15), 5-tert-butylpyrogallol (Lp13, Lpl15), and UE (m/z 111–198) (Lp13). A better understanding of the relationship between starters and their VOC may facilitate modelling the flavour and quality of Spanish-style green table olive fermentations. ; The research was funded by the Spanish Government (Project OliFilm AGL-2013-48300-R: www.olifilm.science.com.es) A-BC thanks the Spanish Ministry of Economy and Competitiveness for their FPI grant. ; Peer reviewed

This paper describes the dominant bacterial species metabolically active through the industrial production of Spanish-style Manzanilla and Gordal olives. For this purpose, samples (brines and fruits) obtained at 0, 15, and 90 fermentation days were analyzed by a culture-independent approach to determine viable cells by reverse transcription of RNA and further PCR-DGGE analysis, detecting at least 7 different species. Vibrio vulnificus, Lactobacillus plantarum group, and Lactobacillus parafarraginis were present in samples from both cultivars; Lactobacillus sanfranciscensis and Halolactobacillus halophilus were detected only in Gordal samples, while Staphylococcus sp. was exclusively found at the onset of Manzanilla fermentations. Physicochemical data showed a typical fermentation profile while scanning electron microscopy confirmed the in situ biofilm formation on the olive epidermis. Different Bacillus, Staphylococcus, and Enterococcus species, not detected during the fermentation process, were also found in the solid marine salt used by the industry for preparation of brines. Elucidation of these non-lactic acid bacteria species role during fermentation is then an appealingly challenge, particularly regarding safety issues. ; The research has received funding from the Spanish Government (Project OliFilm AGL-2013-48300-R: www.olifilm.science.com.es). AB-C and FA-L wish to express thanks to the Spanish Government for their pre-doctoral fellowship and postdoctoral research contract (Ramón y Cajal), respectively. ; Peer Reviewed

© 2015, Springer International Publishing Switzerland. This work examines the formation of poly-microbial communities adhered to the epidermis of natural green Gordal olives and the application of different methodologies for recovery and counting of the microorganisms embedded in olive biofilms. The fermentation process was physicochemical and microbiologically monitored for 90 days, at which, formation of true biofilms on the skin of fermented fruits was confirmed by scanning electron microscopy. Then, samples of olives were taken and treated with sonication, enzymes, mechanical homogenization with stomacher and ultrasonic bath for biofilm disaggregation. The use of the stomacher for 1 min was the most effective treatment to release the lactic acid bacteria (6.6 log 10 cfu g −1 ), whereas sonication for 5 min was the most efficient method for quantification of yeasts (up to 3.5 log 10 cfu g −1 ). Molecular identification of isolates obtained from natural Gordal olive biofilms revealed that Lactobacillus pentosus was the only species found among lactic acid bacteria, while Pichia membranifaciens was the dominant yeast species, with higher counts obtained for the bacteria. ; The research leading to these results has received funding from the Spanish Government (project OliFilm AGL-2013-48300-R: www.olifilm.science.com.es), and the Junta de Andalucía (through financial support to project P11-AGR-7051 and group AGR-125). A. Benítez and F.N. Arroyo-López wish to express thanks to the Spanish Government for their pre-doctoral fellowship and postdoctoral research contract (Ramón y Cajal), respectively. V. Romero-Gil would like also to thank the AgriFood Campus of International Excellence (ceiA3), Bank of Santander, Spanish Government and 'Aloreña de Málaga' Olive Manufacturing Association for her pre-doctoral fellowship (training program of Ph.D. in companies). ; Peer Reviewed

The bacteriocin producer Lactobacillus plantarum LPCO10, a strain originally isolated from an olive fermentation, was used as a starter culture for traditional Spanish-style green olive fermentation. By means of a 23 (7 4) fractional factorial design, the salt concentration in brines was found to be the most important factor for producing the highest acidity and the highest initial population of the starter culture. To a lesser extent, other factors such as a high inoculum size, use of MRS as carrier for the starter culture, inoculation time, and type of acid to correct the pH had also an overall significant effect on the final acidity or total corrected acidity. For all initial conditions investigated, Lb. plantarum LPCO10 was able to dominate over the natural population of lactic acid bacteria and led to a faster decrease of pH and a faster acidification than the spontaneous process during the first 25 days after brining. Then, wild lactobacilli grew slowly and the population of lactic cocci rose, while LPCO10 decreased. Inoculation also stimulated the growth of yeasts. Thus, the traditional fermentation of Spanish green table olives could be improved by using Lb. plantarum LPCO10 (suspended in MRS) as a starter culture at X107 cfu/ml of brine and well defined starting brine parameters, i.e. p4% w/v of NaCl, and initial pH correction (from 4.5 to 6.5) with acetic acid. Inoculation can take place in 1–4 days after brining. r 2002 Elsevier Science Ltd. All rights reserved. ; This work was supported in part by contracts from the Spanish Government (CICYT projects ALI97-0658-C03-01, ALI97-0646, and AGL2000-1539-CO2-01) and the EU (FAIR-97-9526). ; Peer reviewed

31 Páginas; 4 Figuras ; Directly brined black table olives of Bosana variety are a traditional food product of Sardinia island (Italy), spontaneously fermented by yeasts among other microorganisms. However, as far as we know, the identification, biotechnological and probiotic potential of this yeast community has not been investigated yet. In this work, a total of 72 yeast isolates previously obtained from Bosana olive brines were first genotyped by Random Amplified Polymorphic DNA (RAPD-PCR) analysis with primer M13, and then identified by sequencing of D1/D2 domains of rDNA 26S gene. The dominant species were Wickerhamomyces anomalus and Nakazawaea molendini-olei, albeit Candida diddensiae, Candida boidinii, Zygotorulaspora mrakii, and Saccharomyces cerevisiae were also present in lower proportions. For the different biotypes of yeasts obtained, the multivariate analysis of their technological (esterase, lipase and β-glucosidase activities, growth in presence of oleuropein, resistance and susceptibility to NaCl) and probiotic (removal of cholesterol, gastric and pancreatic digestions, biofilms assays alone and in co-culture with Lactobacillus pentosus) features, showed that W. anomalus Wa1 exhibited the best technological characteristics, while S. cerevisiae Sc24 and C. boidinii Cb60 showed promising probiotic features. Therefore, they may have potential application as multifunctional starters, alone or in combination with lactic acid bacteria, during olive processing, albeit further studies are necessary to validate these results. ; The research leading to these results has received funding from the Spanish Government (Project OliFilm AGL-2013-48300-R: www.olifilm.science.com.es), and Junta de Andalucía (through financial support to projects P11-AGR-7051, P11-AGR-7755, and groups AGR-125 and BIO-160). CP gratefully acknowledges Sardinia regional Government for the financial support of her PhD scholarship (POR Sardegna FSE OP European Social Fund2007/2013 – Axis IV Human Resources, Objective 1.3, Line of activity 1.3.1) and the Foundation of Sardinia (Prat. 2013.1364) for financial support. FNAL wish to express thanks to the Spanish Government for his postdoctoral research contract (Ramón y Cajal). Authors express their gratitude to Dr. Antonio Garrido Fernández for his constructive comments and suggestions with drafting of manuscript. CP gratetfully acknowledges Dr Ivo Pinna for kindly provided support of her thesis manuscript. ; Peer reviewed

4 Páginas; 1 Figura ; easts play an important role in the food and beverage industry, especially in products such as bread, wine, and beer, among many others. However, their use as a starter in table olive processing has not yet been studied in detail. The candidate yeast strains should be able to dominate fermentation, together with lactic acid bacteria, but should also provide a number of beneficial advantages. Technologically, yeasts should resist low pH and high salt concentrations, produce desirable aromas, improve lactic acid bacteria growth, and inhibit spoilage microorganisms. Nowadays, they are being considered as probiotic agents because many species are able to resist the passage through the gastrointestinal tract and show favorable effects on the host. In this way, yeasts may improve the health of consumers by means of the degradation of non-assimilated compounds (such as phytate complexes), a decrease in cholesterol levels, the production of vitamins and antioxidants, the inhibition of pathogens, an adhesion to intestinal cell line Caco-2, and the maintenance of epithelial barrier integrity. Many yeast species, usually found in table olive processing (Wickerhamomyces anomalus, Saccharomyces cerevisiae, Pichia membranifaciens, and Kluyveromyces lactis, among others), have exhibited some of these properties. Thus, the selection of the most appropriate strains to be used as starters in this fermented vegetable, alone or in combination with lactic acid bacteria, is a promising research line to develop in the near future. ; This research line has received funding from the EU's Seventh Framework Programme [FP7/2007-2013; under grant agreement n° 243471 (PROBIOLIVES)]. We also thank the Spanish Government for financial support to projects AGL2009-07436/ALI and AGL2010-15529/ALI, and Junta de Andalucía through financial support to group AGR-125. Thanks to JOLCA and ASEMESA for their invaluable collaboration. Joaquín Bautista-Gallego and Francisco N. Arroyo-López would also like to thank CSIC and Spanish Government for their JAE-PRE fellowship and Ramón y Cajal postdoctoral research contract, respectively. ; Peer reviewed

14 Páginas, 14 Tablas, 5 Figuras ; Candida boidinii is an Ascomycota yeast with important biotechnological applications. In this paper we present the genome sequencing and annotation of eight strains of this species isolated from human activities and wild environments. The produced assemblies revealed several strain specific features in terms of genomic GC content (ranging from 30.9 to 32.7%), genome size (comprised between 18,791,129 and 19,169,086 bp) and total number of protein coding genes (ranging from 5819 to 5998), with putative assignation to their general KOG functional categories. The obtained data underlined the presence of two different groups for this species. The results reported herein provide new insights into the plasticity of the genome of this yeast species and represent a starting point for further studies in view of its biotechnological applications ; The research leading to these results has received funding from the Spanish Government (project OliFilm AGL-2013-48300-R: www.olifilm.science.com.es), and Junta de Andalucía (through financial support to project P11-AGR-7051 and groups AGR-125 and BIO-160). ABC and FNAL wish to express thanks to the Spanish Government for their pre-doctoral fellowship and postdoctoral research contract (Ramón y Cajal), respectively, while BCD is a recipient of a pre-doctoral grant from Junta de Andalucía. CP gratefully acknowledges Sardinia regional Government for the financial supporter of her PhD scholarship (POR Sardegna FSE OP European Social Fund 2007/2013 – Axis IV Human Resources, Objective 1.3, Line of activity 1.3.1) and the Foundation of Sardinia (Prat. 2013.1364) for financial support. ; Peer reviewed

The present survey uses a dynamic gastric and small intestinal model to assess the survival of one pathogenic (Escherichia coli O157:H7 EDL 933) and three lactobacilli bacteria with probiotic potential (Lactobacillus rhamnosus GG, L. pentosus TOMC-LAB2, and L. pentosus TOMC-LAB4) during their passage through the human gastrointestinal tract using fermented olives as the food matrix. The data showed that the survival of the E. coli strain in the stomach and duodenum was very low, while its transit through the distal parts (jejunum and ileum) resulted in an increase in the pathogen population. The production of Shiga toxins by this enterohemorrhagic microorganism in the ileal effluents of the in vitro system was too low to be detected by ELISA assays. On the contrary, the three lactobacilli species assayed showed a considerable resistance to the gastric digestion, but not to the intestinal one, which affected their survival, and was especially evident in the case of both L. pentosus strains. In spite of this, high population levels for all assayed microorganisms were recovered at the end of the gastrointestinal passage. The results obtained in the present study show the potential use of table olives as a vehicle of beneficial microorganisms to the human body, as well as the need for good hygienic practices on the part of olive manufacturers in order to avoid the possibility of contamination by food-borne pathogens. ; The research leading to these results has received funding from the EU's Seventh Framework Program (FP7/2007-2013) under grant agreement n° 243471 (PROBIOLIVES). Francisco N. Arroyo-López wish to express his gratitude to the Spanish Government for his Ramón y Cajal postdoctoral research contract, as well as CSIC for financial support of the research mobility program in foreign countries (A12012PM2-PA1003309). Verónica Romero-Gil thanks to the Agrifood Campus of International Excellence (ceiA3) for her predoctoral fellowship (program of formation of Ph.D. in companies). ; Peer Reviewed