Deciphering the Mechanism of Action of the Antimicrobial Peptide BP100.

The linear undecapeptide KKLFKKILKYL-NH2 (BP100) highlights for its antibacterial activity against Gram-negative bacteria and its low toxicity. These excellent biological properties prompted the investigation of its mechanism of action, which were undertaken using spectroscopic techniques, biophysical analysis, microscopy, and molecular dynamic simulations. Studies were conducted in different membrane environments, such as anionic, zwitterionic, and mixed membranes, as well as in vesicles (LUVs and GUVs) and bacteria. The findings suggest that BP100 exhibits a preference for anionic membranes, and its mechanism of action involves charge neutralization and membrane permeabilization. In these membranes, BP100 transitions from an unstructured state in water to an α-helix with the axis parallel to the surface. MD simulations suggest that after electrostatic interaction with the membrane, BP100 flips, facilitating the insertion of its hydrophobic face into the membrane bilayer. Thus, BP100 adopts an almost vertical transmembrane orientation with lysine side chains snorkelling on both sides of the membrane. As a result of the rotation, BP100 induces membrane thinning and slow lipid diffusion and promotes water penetration, particularly in anionic lipid membranes. These investigations pointed towards a carpet-like mechanism and are aligned with the biological activity profile described for BP100. This review covers all the studies carried out on the mechanism of action of BP100 published between 2009 and 2023.

Immobilisation of yeasts on oak chips or cellulose powder for use in bottle-fermented sparkling wine.

Sparkling wine production comprises two successive fermentations performed by Sacharomyces cerevisiae strains. This research aimed to: develop yeast immobilisation processes on two wine-compatible supports; study the effects of yeast type (IOC 18-2007 and 55A) and the immobilisation support type (oak chips and cellulose powder) on the fermentation kinetics, the deposition rate of lees and the volatile composition of the finished sparkling wine; compare the fermentation parameters of the wines inoculated with immobilised or non-immobilised cells. Proper immobilisation of yeast on oak chips and cellulose powder was demonstrated by electron microscopy. Total sugar consumption occurred in under 60 days in all bottles, regardless of the strain used and the way they were inoculated in wine. Deposition of lees was 3-fold faster in the bottles containing immobilised cells than in those with free cells; no addition of adjuvants was necessary. The analysis of the volatile compounds of the finished sparkling wines showed significant differences in the formation of esters, acids, alcohols, aldehydes and lactones according to the yeast and the immobilisation support used. Oak chips were the more appropriate support for yeast immobilisation. No significant differences in the sensorial analysis of the sparkling wines produced by the different strategies were found.

Acetobacter musti sp. nov., isolated from Bobal grape must.

An acetic acid bacterium (strain Bo7T), obtained during a study of the microbial diversity of spontaneous fermentations of Bobal grape must, was subjected to a taxonomic study using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences allocated strain Bo7T to the genus Acetobacter, and revealed Acetobacter aceti and Acetobacter oeni to be nearest neighbours (99.57 % 16S rRNA gene sequence similarity between strain Bo7T and A. oeni CECT 5830T, and 98.76 % between strain Bo7T and A. aceti CECT 298T). Cells of strain Bo7T are Gram-negative, motile rods, catalase-positive and oxidase-negative. The DNA G+C content of strain Bo7T was 58.0 mol%. DNA-DNA hybridizations demonstrated that strain Bo7T belongs to a single novel genospecies that can be differentiated from its nearest phylogenetic neighbours by the following phenotypic characteristics: no production of 5-keto-d-gluconic acid from d-glucose, growth with glycerol but not with methanol or maltose as sole carbon sources, and growth on yeast extract with 30 % d-glucose. The major fatty acid was C18 : 1ω7c/C18 : 1ω6c (summed feature 8; approx. 56 %); other fatty acids in significant amounts (>5 %) were C16 : 0 2-OH (11 %), C16 : 0 (7 %), C14 : 0 2-OH (7 %) and C14 : 0 3-OH/iso-C16 : 1 I (summed feature 2; 6 %). The results obtained indicate that strain Bo7T represents a novel species of the genus Acetobacter, for which the name Acetobacter musti sp. nov. is proposed. The type strain is Bo7T ( = DSM 23824T = CECT 7722T).

Technological properties of Lactobacillus plantarum strains isolated from grape must fermentation.

Malolactic fermentation (MLF) is a secondary fermentation in wine that usually takes place during or at the end of alcoholic fermentation. Lactobacillus plantarum is able to conduct MLF (particularly under high pH conditions and in co-inoculation with yeasts), and some strains are commercially used as MLF starter cultures. Recent evidences suggest a further use of selected L. plantarum strains for the pre-alcoholic acidification of grape must. In this study, we have carried out an integrated (molecular, technological, and biotechnological) characterization of L. plantarum strains isolated from Apulian wines in order to combine the two protechnological features (MLF performances and must acidification aptitudes). Several parameters such as sugar, pH and ethanol tolerance, resistance to lyophilisation and behaviour in grape must were evaluated. Moreover, the expression of stress gene markers was investigated and was linked to the ability of L. plantarum strains to grow and perform MLF. Co-inoculation of Saccharomyces cerevisiae and L. plantarum in grape must improves the bacterial adaptation to harsh conditions of wine and reduced total fermentation time. For the first time, we applied a polyphasic approach for the characterization of L. plantarum in reason of the MLF performances. The proposed procedure can be generalized as a standard method for the selection of bacterial resources for the design of MLF starter cultures tailored for high pH must.

The use of core-shell high-performance liquid chromatography column technology to improve biogenic amine quantification in wine.

BACKGROUND: HPLC column technology has been improved, providing better resolution of closely eluting compounds, better analyte sensitivity, and shorter analysis times. The core-shell technology columns offer a faster analysis through the use of shorter columns without compromising resolution. The aim of this work was to improve the methods for determination of biogenic amines (BAs) in wine using the new HPLC PFP core-shell column technology. RESULTS: Two different elution programs were designed to quantify BAs with the core-shell PFP column. Program I flow rate was 2 mL min(-1). The total elution time was 10 min. In elution program II, the flow rate was 0.8 mL min(-1) and the total elution time was 25 min. The two elution programs used with the core-shell PFP HPLC column showed differences related mainly to the histamine peak. The chromatograms showed that when a temporary isocratic elution was added in the gradient (program II), the histamine peak was eluted later, causing its isolation, and therefore its quantification was easier. CONCLUSIONS: Compared to the previous C18 HPLC column for the BAs determination in wine, the main advantage of the presented technique is the reduction of the run times and solvent volumes, and has a better sensitivity and selectivity as peaks are higher and sharper.

Malic enzyme and malolactic enzyme pathways are functionally linked but independently regulated in Lactobacillus casei BL23.

Lactobacillus casei is the only lactic acid bacterium in which two pathways for l-malate degradation have been described: the malolactic enzyme (MLE) and the malic enzyme (ME) pathways. Whereas the ME pathway enables L. casei to grow on l-malate, MLE does not support growth. The mle gene cluster consists of three genes encoding MLE (mleS), the putative l-malate transporter MleT, and the putative regulator MleR. The mae gene cluster consists of four genes encoding ME (maeE), the putative transporter MaeP, and the two-component system MaeKR. Since both pathways compete for the same substrate, we sought to determine whether they are coordinately regulated and their role in l-malate utilization as a carbon source. Transcriptional analyses revealed that the mle and mae genes are independently regulated and showed that MleR acts as an activator and requires internalization of l-malate to induce the expression of mle genes. Notwithstanding, both l-malate transporters were required for maximal l-malate uptake, although only an mleT mutation caused a growth defect on l-malate, indicating its crucial role in l-malate metabolism. However, inactivation of MLE resulted in higher growth rates and higher final optical densities on l-malate. The limited growth on l-malate of the wild-type strain was correlated to a rapid degradation of the available l-malate to l-lactate, which cannot be further metabolized. Taken together, our results indicate that L. casei l-malate metabolism is not optimized for utilization of l-malate as a carbon source but for deacidification of the medium by conversion of l-malate into l-lactate via MLE.

Assessment of trace elements and stable isotopes of three Ardeid species at Birama Swamp, Cuba.

The Birama Swamp is the second largest wetland in the Caribbean region and it is inhabited by large populations of waterbirds. Here we report, for the first time, the foraging ecology and pollutant levels of three Ardeidae species: Cattle egret (Bubulcus ibis), Snowy egret (Egretta thula), and Tricolored heron (E. tricolor) breeding in this wetland using stable-isotope (δ (15)N and δ (13)C) and trace elements [mercury (Hg), lead (Pb), and selenium (Se)] analysis of chick feathers. Our results showed that individuals from all species occupied similar trophic levels. However, we found significant differences for δ (13)C, with the highest values in cattle egret indicating its use of terrestrial habitats and a generalist and opportunist behavior. No significant differences were found for Pb among species. Yet, Hg levels were greater and similar in tricolored heron and snowy egret than in cattle egret, which was associated with their greater use of aquatic environments. Snowy egret had the lowest values of Se differing significantly with the other two species suggesting a different relative use of prey type. Modeling log-Hg concentration in relation to δ (15)N and δ (13)C showed an independent and significant relationship among species but without interaction with species level indicating that within a particular species, higher Hg levels were associated with higher δ (15)N values. There was no interaction between δ (15)N and δ (13)C in the general linear models for Se and Pb in all species. We found an association between δ (15)N and species in Pb for snowy egret. The foraging habitat use of these species and the low levels of pollutants, which are lower than in other similar habitats in other areas of the world, indicated that there is not risk of negative effects in juvenile birds of the Birama Swamp colony that may impair their survival. Our results can be used as a baseline to achieve management regulations.

Erwinia piriflorinigrans sp. nov., a novel pathogen that causes necrosis of pear blossoms.

Eight Erwinia strains, isolated from necrotic pear blossoms in València, Spain, were compared with reference strains of Erwinia amylovora and Erwinia pyrifoliae, both of which are pathogenic to species of pear tree, and to other species of the family Enterobacteriaceae using a polyphasic approach. Phenotypic analyses clustered the novel isolates into one phenon, distinct from other species of the genus Erwinia, showing that the novel isolates constituted a homogeneous phenotypic group. Rep-PCR profiles, PCR products obtained with different pairs of primers and plasmid contents determined by restriction analysis showed differences between the novel strains and reference strains of E. amylovora and E. pyrifoliae. Phylogenetic analysis of 16S rRNA, gpd and recA gene sequences showed that the eight novel strains could not be assigned to any recognized species. On the basis of DNA-DNA hybridization studies, the novel isolates constituted a single group with relatedness values of 87-100  % to the designated type strain of the group, CFBP 5888(T). Depending on the method used, strain CFBP 5888(T) showed DNA-DNA relatedness values of between 22.7 and 50  % to strains of the closely related species E. amylovora and E. tasmaniensis. The DNA G+C contents of two of the novel strains, CFBP 5888(T) and CFBP 5883, were 51.1 and 50.5 mol%, respectively. On the basis of these and previous results, the novel isolates represent a novel species of the genus Erwinia, for which the name Erwinia piriflorinigrans sp. nov. is proposed. The type strain is CFBP 5888(T) (=CECT 7348(T)).

Characterization of Lactobacillus isolates from fermented olives and their bacteriocin gene profiles.

Near one hundred isolates of Lactobacillus paraplantarum, Lactobacillus pentosus and Lactobacillus plantarum from table olives were studied. Strains were genotyped by rep-PCR. Although the technique failed to differentiate some isolates at the species level, it proved a robust and easy procedure that could be useful for distinguishing between related strains of L. paraplantarum, L. pentosus and L. plantarum from a large pool of unrelated strains of these species. A PCR-based screening revealed the presence of the plantaricin encoding genes plnA, plnB, plnC, plnD, plnE/F, plnF, plnI, plnJ, plnK, plnG and plnN in most isolates of the three species. Sequences of bacteriocin genes present in L. paraplantarum and L. pentosus were homologous to L. plantarum genes. Through a discriminating analysis of the bacteriocin gene profiles, it was possible to establish a relationship between the origin of isolation and the LAB isolates, regardless of species.

Structural analysis and biochemical properties of laccase enzymes from two Pediococcus species.

Prokaryotic laccases are emergent biocatalysts. However, they have not been broadly found and characterized in bacterial organisms, especially in lactic acid bacteria. Recently, a prokaryotic laccase from the lactic acid bacterium Pediococcus acidilactici 5930, which can degrade biogenic amines, was discovered. Thus, our study aimed to shed light on laccases from lactic acid bacteria focusing on two Pediococcus laccases, P. acidilactici 5930 and Pediococcus pentosaceus 4816, which have provided valuable information on their biochemical activities on redox mediators and biogenic amines. Both laccases are able to oxidize canonical substrates as ABTS, ferrocyanide and 2,6-DMP, and non-conventional substrates as biogenic amines. With ABTS as a substrate, they prefer an acidic environment and show sigmoidal kinetic activity, and are rather thermostable. Moreover, this study has provided the first structural view of two lactic acid bacteria laccases, revealing new structural features not seen before in other well-studied laccases, but which seem characteristic for this group of bacteria. We believe that understanding the role of laccases in lactic acid bacteria will have an impact on their biotechnological applications and provide a framework for the development of engineered lactic acid bacteria with enhanced properties.

The role of two families of bacterial enzymes in putrescine synthesis from agmatine via agmatine deiminase.

Putrescine, one of the main biogenic amines associated to microbial food spoilage, can be formed by bacteria from arginine via ornithine decarboxylase (ODC), or from agmatine via agmatine deiminase (AgDI). This study aims to correlate putrescine production from agmatine to the pathway involving N-carbamoylputrescine formation via AdDI (the aguA product) and N-carbamoylputrescine amidohydrolase (the aguB product), or putrescine carbamoyltransferase (the ptcA product) in bacteria. PCR methods were developed to detect the two genes involved in putrescine production from agmatine. Putrescine production from agmatine could be linked to the aguA and ptcA genes in Lactobacillus hilgardii X1B, Enterococcus faecalis ATCC 11700, and Bacillus cereus ATCC 14579. By contrast Lactobacillus sakei 23K was unable to produce putrescine, and although a fragment of DNA corresponding to the gene aguA was amplified, no amplification was observed for the ptcA gene. Pseudomonas aeruginosa PAO1 produces putrescine and is reported to harbour aguA and aguB genes, responsible for agmatine deiminase and N-carbamoylputrescine amidohydrolase activities. The enzyme from P. aeruginosa PAO1 that converts N-carbamoylputrescine to putrescine (the aguB product) is different from other microorganisms studied (the ptcA product). Therefore, the aguB gene from P. aeruginosa PAO1 could not be amplified with ptcA-specific primers. The aguB and ptcA genes have frequently been erroneously annotated in the past, as in fact these two enzymes are neither homologous nor analogous. Furthermore, the aguA, aguB and ptcA sequences available from GenBank were subjected to phylogenetic analysis, revealing that gram-positive bacteria harboured ptcA, whereas gram-negative bacteria harbour aguB. This paper also discusses the role of the agmatine deiminase system (AgDS) in acid stress resistance.

Lactobacillus uvarum sp. nov.--a new lactic acid bacterium isolated from Spanish Bobal grape must.

Five strains isolated from grape musts in Spain in 1997, have been characterized by several molecular techniques, and three of them have been identified as pertaining to a new species. All strains are Gram-positive rods, aerotolerant and homofermentative bacteria that do not exhibit catalase activity. Phylogenetic analysis based on 16S rRNA gene sequences placed these strains within the genus Lactobacillus, closely related to Lactobacillus mali. DNA-DNA hybridization experiments confirmed that strain 71 belongs to the lately described species L. satsumensis, strain 88 belongs to L. mali and the other three isolates have an independent status at species level. Restriction analysis of the amplified 16S rRNA gene (16S-ARDRA), internal spacer region (ISR) analysis, random amplified polymorphism DNA (RAPD) and ribotyping were performed in order to establish genotypic similarities and differences between the new species and their closest species. The three isolates can be genetically differentiated from their closest relatives by RAPD analysis and ribotyping. Phenotypically, they can be distinguished by several traits such as their ability to grow at pH 3.3 and NaCl 5% (w/v) and by certain carbohydrate fermentations. The name L. uvarum sp. nov. is proposed. The type strain is 8T (=DSM 19971T = colección española de cultivos tipo (CECT) 7335T).

Direct and Rapid Detection and Quantification of Oenococcus oeni Cells in Wine by Cells-LAMP and Cells-qLAMP.

Fast detection and enumeration of Oenococcus oeni in winemaking are necessary to determine whether malolactic fermentation (MLF) is likely to be performed or not and to decide if the use of a commercial starter is needed. In other wines, however, performing MLF can be detrimental for wine and should be avoided. The traditional identification and quantification of this bacteria using culture-dependent techniques in wine-related matrices require up to 14 days to yield results, which can be a very long time to perform possible enological operations. Loop-mediated isothermal amplification (LAMP) is a novel culture-independent technique that amplifies nucleic acid sequences under isothermal conditions with high specificity and efficiency in less than 1 h with inexpensive equipment. We designed LAMP primers for the specific detection and quantification of O. oeni cells. The developed LAMP method allows O. oeni to be detected directly from both grape musts and wines within 1 h from the time that the LAMP reaction begins, and without DNA extraction and purification requirements. The high sensitivity of LAMP methodology is achieved by previous mechanical cells lysis with no further purification by detecting one single cell per reaction in culture media, and in white/red grape musts and wines by avoiding reaction inhibition by ethanol, polyphenols, and other wine inhibitors. Cells can be concentrated prior to the LAMP reaction to further increase this sensitivity. Moreover, the LAMP method does not require expensive equipment and can be easily operated. The developed method is both economic and fast and offers high sensitivity and specificity.

Tyramine and phenylethylamine production among lactic acid bacteria isolated from wine.

The ability of wine lactic acid bacteria to produce tyramine and phenylethylamine was investigated by biochemical and genetic methods. An easy and accurate plate medium was developed to detect tyramine-producer strains, and a specific PCR assay that detects the presence of tdc gene was employed. All strains possessing the tdc gene were shown to produce tyramine and phenylethylamine. Wines containing high quantities of tyramine and phenylethylamine were found to contain Lactobacillus brevis or Lactobacillus hilgardii. The main tyramine producer was L. brevis. The ability to produce tyramine was absent or infrequent in the rest of the analysed wine species.

Cells-qPCR as a direct quantitative PCR method to avoid microbial DNA extractions in grape musts and wines.

A novel quantitative PCR assay called Cells-qPCR has been developed for the rapid detection and quantification of yeasts, lactic acid bacteria (LAB) and acetic acid bacteria (AAB) directly from grape must and wine that does not require DNA extraction. The assay was tested on Brettanomyces bruxellensis, Saccharomyces cerevisiae, Lactobacillus plantarum, Oenococcus oeni, Acetobacter aceti and Gluconobacter oxydans in culture media, and in white and red grape musts and wines. Standard curves were constructed from DNA and cells for the six target species in all the matrices. Good efficiencies were obtained for both when comparing DNA and cells standard curves. No reaction inhibition was observed between matrices for each species. Cells quantification was linear over a range of cell concentrations (7, 5 or 4 orders of magnitude) and detected as few as one cell per reaction in all the matrices. The developed Cells-qPCR assay is a robust, reliable, fast and specific method to detect and quantify different yeasts, LAB and AAB species in grape must and wine that avoids DNA extraction and overcomes the presence of inhibitors like polyphenols and ethanol.

Recombinant laccase from Pediococcus acidilactici CECT 5930 with ability to degrade tyramine.

Biogenic amines degradation by bacterial laccases is little known, so we have cloned and heterologously expressed, in E. coli, a new laccase from Pediococcus acidilactici CECT 5930 (Lpa5930), a lactic acid bacterium commonly found in foods able to degrade tyramine. The recombinant enzyme has been characterized by physical and biochemical assays. Here we report the optimization of expression and purification procedures of this laccase. DNA encoding sequence of laccase from P. acidilactici was amplified by PCR and cloned into the expression plasmid pET28a for induction by isopropyl-ß-D-thiogalactoipyranoside. Protein expression was performed in E. coli BL21(DE3) harboring pGro7 plasmid expressing a chaperone folding assistant induced by arabinose. Purification was performed by column metal-chelating chromatography on Ni-NTA-agarose. The laccase enzyme obtained has an apparent molecular mass of ∼60 kDa, an optimum temperature activity toward 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) of 28°C, and was quickly inactivated at temperatures higher than 70°C. The apparent Km value for ABTS was 1.7 mM and the Vmax obtained was 24 U/mg. In addition to ABTS, recombinant Lpa5930 laccase degraded the biogenic amine tyramine at pH 9.5 and pH 4.0 with or without ABTS as a mediator. Tyramine degradation by laccases could solve the problems generated in food due to the presence of this toxic compound.

Exploring the biodiversity of two groups of Oenococcus oeni isolated from grape musts and wines: Are they equally diverse?

One hundred and four Oenococcus oeni isolates were characterised by the carbohydrate fermentation (CH) profile and DNA fingerprinting. Forty-four isolates came from grape must, and 60 from wines sampled at the end of alcoholic fermentation or during malolactic fermentation. The grape must isolates fermented more CH than the wine isolates. In genotypical terms, no clear boundary between grape must and wine isolates was found. Diversities were deduced by considering the isolates of grape must and of wine separately and jointly. By considering only CH fermentation abilities, the group of grape must isolates gave higher diversity index (DICH) values than those isolated from wine; i.e., these isolates were metabolically more diverse. The contrary occurred when the DNA fingerprints were used to calculate DIRAPD-VNTR: wine isolates were genotypically more diverse than grape must ones. With a polyphasic approach, which considered metabolic and genotypic data, the diversity index of both isolate groups (from grape must and wine) was the same, 0.993, which was slightly lower than that calculated from all the isolates (0.997).

Lowering histamine formation in a red Ribera del Duero wine (Spain) by using an indigenous O. oeni strain as a malolactic starter.

This study demonstrates for the first time that a non-commercial selected autochthonous O. oeni strain has been used to conduct malolactic fermentation (MLF) while lowering histamine formation in the same winery. Lactic acid bacteria (LAB) were isolated from 13 vats before and after spontaneous MLF at the Pago de Carraovejas winery from the Ribera del Duero region (Spain). Only O. oeni were present, typed and characterized, and both histamine producer and non-producers existed. From the non-producers, one strain was selected to become a starter according to its genetic profile, prevalence in the different wines in the winery, resistance to alcoholic degree, resistance to high polyphenolic content, inability to synthesise histamine, growth kinetics and malolactic activity. This starter was produced at semi-industrial levels to inoculate 20,000L of Tempranillo red wine. The inoculated vat showed 5-fold less histamine than the non-inoculated control vat. After 1year, the barrel-ageing histamine concentrations were 3-fold lower in the inoculated vat than in the non-inoculated vat.

Histamine, histidine, and growth-phase mediated regulation of the histidine decarboxylase gene in lactic acid bacteria isolated from wine.

Fermented foods are frequently contaminated by histamine that is generated by microorganisms with histidine decarboxylase activity. The ingestion of large amounts of histamine can cause serious toxicological problems in humans. A study of the effects of histamine, histidine, and growth phase on histamine production by lactic acid bacteria isolated from wine is reported here. With northern blots and specific activity analysis, we observed that histidine induces the expression of the histidine decarboxylase gene (hdc) and that histamine causes a decrease in the expression of this gene. The expression of hdc is also mediated by the bacterial growth phase. Histidine and histamine do not affect histidine decarboxylase activity, whereas pyridoxal 5'-phosphate does. Data on histamine-producing lactic acid bacteria isolated from wine should contribute to the prevention of histamine formation during winemaking and storage.

Biogenic amines in wines from three Spanish regions.

One hundred and sixty-three wines from La Rioja, Utiel-Requena, and Tarragona were analyzed to determine if there were any differences in the concentrations of six biogenic amines that are found in these three regions. The influence of grape variety, type of vinification, wine pH, malolactic fermentation, and storage in bottle on biogenic amine concentrations was studied. Results show important differences in putrescine and histamine concentrations among regions, varieties of grape, and type of wine; differences were less appreciable for the remaining biogenic amines studied. Low pH prevented biogenic amine formation. Malolactic fermentation and short storage periods in bottle (3-6 months) showed increases in histamine concentration, whereas longer periods of storage led to a general decrease in histamine. Several strains of lactic acid bacteria were isolated in this work, and their ability to form biogenic amines was assayed in synthetic media, grape must, and wine. Grape varieties, different types of winemaking, pH, and lactic acid bacteria may be responsible for the differences observed in the biogenic amine concentrations of the wines analyzed.