Lactiplantibacillus plantarum strain UNQLp 11 is a lactic acid bacterium with the potential to carry out malolactic fermentation (MLF) in red wines. Recently, the complete genome of UNQLp 11 was ...sequenced and this strain possesses four loci of the enzyme β-glucosidase. In order to demonstrate that these glucosidase enzymes could be functional under harsh wine conditions, we evaluated the hydrolysis of p-nitrophenyl-β-D-glucopyranoside (p-NPG) in synthetic wine with different ethanol contents (0%, 12%, and 14% v/v) and at different pH values (3.2, 3.5, and 3.8). Then, the hydrolysis of precursor n-octyl β-D-glucopyranoside was analyzed in sterile Pinot Noir wine (containing 14.5% v/v of ethanol, at different pH values) by headspace sorptive extraction gas chromatography-mass spectrometry (HSSE-GC/MS). The hydrolysis of p-NPG showed that β-glucosidase activity is very susceptible to low pH but induced in the presence of high ethanol content. Furthermore, UNQLp 11 was able to release the glycosilated precursor n-octyl, during MLF to a greater extent than a commercial enzyme. In conclusion, UNQLp 11 could improve the aromatic profile of the wine by the release of volatile precursors during MLF.
The aim of this study was to characterize the microbial communities of Malbec vineyards recently established in a re-emerging wine region of Argentina. We studied the wine microbiota at different ...fermentation stages and the soil and rhizosphere microbial communities of two vineyards. A next-generation sequencing (NGS) approach was used to identify bacterial and fungal communities. The soil and rhizosphere samples showed a predominance of the phyla Proteobacteria and Actinobacteria. The order Rhizobiales stood out in the soil and rhizosphere of the two vineyards analyzed. Members of this order are recognized for their plant-growth promotion properties. Regarding fungal communities, Ascomycota and Basidiomycota were the most abundant phyla. The high abundance of the genus Ilyonectria in one of the vineyards may have impact on the health of vines. In wine samples, we detected low levels of lactic acid bacteria and the persistence of acetic acid bacteria (AAB) throughout the fermentation process, although there were no discernible effects on the acidity of wine. The results achieved could allow winemakers to improve the vineyard management practices and the fermentation process to favor the growth of microorganisms potentially beneficial for the health of the vines and the wine quality, while maintaining the regional microbial biodiversity.
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•Alpha & Beta Diversity suggested differences in the bacterial and fungal community composition.•Bacterial communities were largely dominated by Proteobacteria spp.•Acetic Acid bacteria were detected in all the wine fermentation stages of Malbec wine.•Control of excess volatile acidity and use of autochthonous LAB should be considered.•The high abundance of Ilyonectria could have an impact on the health of the vines.
The spontaneous malolactic fermentation (MLF) in a centenary winery from Patagonia, Argentina, is conducted by predominantly mesophilic Oenococcus oeni and Lactiplantibacillus plantarum. In this ...region, MLF takes place from 14 to 4 °C, leading to heat cellars incurring in higher costs and non-sustainable practices. Previously, psychrotrophic strains of O. oeni had been obtained from a Patagonian wine. The goal of this work was to identify the Lactobacillaceae microbiota related to low-temperature MLF and assess their contribution. Nine sychrotrophicc Lentilactibacillus hilgardii strains were identified by sequencing the 16S rRNA gene, and the strains typified by RAPD-PCR. All strains consumed L-malic acid at 4 and 10 °C in sterile wine. The selected UNQLh1.1 strain revealed implantation capacity and L-malic acid consumption at 4 and 10 °C in the presence of the native microbial consortium. Furthermore, the histidine decarboxylase (hdc) gene was not detected in any of the Len. hilgardii strains. The prevalence of Len. hilgardii under low-temperature conditions represents a novelty compared to previous findings of LAB diversity in the MLF of Patagonian wines. The native Patagonian psychrotrophic Len. hilgardii strains are a new player in fermentations conducted at low temperatures with the potential to be used as a sustainable MLF starter.
The Lacticaseibacillus paracasei UNQLpc 10 strain was isolated from a Malbec wine produced in North Patagonia, Argentina, and identified by 16S rRNA gene sequencing. The aim of this work was to ...obtain the fully assembled genome of the UNQLpc 10 strain, analyze its structure, and evaluate the possible functions of the predicted genes with regard to its oenological potential as a malolactic starter. UNQLpc10 is the first whole assembled genome of an oenological strain of Lcb. paracasei reported in databases. This information is of great interest inexpanding the knowledge of diversity of oenological lactic acid bacteria and in searching for new candidate species/strains to design starter cultures. The in silico genome-wide analysis of UNQLpc 10 confirms the existence of genes encoding enzymes involved in the synthesis of several metabolites of oenological interest, and proteins related to stress responses. Furthermore, when UNQLpc 10 was incubated in synthetic wine, it exhibited a very good survival and L-malic acid consumption ability.
Shiga toxin (Stx) is the principal virulence factor during Shiga toxin-producing
(STEC) infections. We have previously reported the inactivation of bacteriophage encoding Stx after treatment with ...chitosan, a linear polysaccharide polymer with cationic properties. Cationic antimicrobial peptides (cAMPs) are short linear aminoacidic sequences, with a positive net charge, which display bactericidal or bacteriostatic activity against a wide range of bacterial species. They are promising novel antibiotics since they have shown bactericidal effects against multiresistant bacteria. To evaluate whether cationic properties are responsible for bacteriophage inactivation, we tested seven cationic peptides with proven antimicrobial activity as anti-bacteriophage agents, and one random sequence cationic peptide with no antimicrobial activity as a control. We observed bacteriophage inactivation after incubation with five cAMPs, but no inactivating activity was observed with the random sequence cationic peptide or with the non-alpha helical cAMP Omiganan. Finally, to confirm peptide-bacteriophage interaction, zeta potential was analyzed by following changes on bacteriophage surface charges after peptide incubation. According to our results we could propose that: (1) direct interaction of peptides with phage is a necessary step for bacteriophage inactivation, (2) cationic properties are necessary but not sufficient for bacteriophage inactivation, and (3) inactivation by cationic peptides could be sequence (or structure) specific. Overall our data suggest that these peptides could be considered a new family of molecules potentially useful to decrease bacteriophage replication and Stx expression.
In the search for new antimicrobial molecules, antimicrobial peptides (AMPs) offer a viable alternative to conventional antibiotics, as they physically disrupt the bacterial membranes, leading to ...membrane disruption and eventually cell death. In particular, the group of linear α-helical cationic peptides has attracted increasing research and clinical interest. The AMP P5 has been previously designed as a cationic linear α-helical sequence, being its antimicrobial and hemolytic properties also evaluated. In this work, we analyzed the feasibility of using P5 against a carbapenem-resistant clinical isolate of Pseudomonas aeruginosa, one of the most common and risky pathogens in clinical practice. After antimicrobial activity confirmation in in vitro studies, synergistic activity of P5 with meropenem was evaluated, showing that P5 displayed significant synergistic activity in a time kill curve assay. The ability of P5 to permeabilize the outer membrane of P. aeruginosa can explain the obtained results. Finally, the antibiofilm activity was investigated by viability analysis (MTT assay), crystal violet and confocal imaging, with P5 displaying mild biofilm inhibition in the range of concentrations tested. Regarding biofilm disruption activity, P5 showed a higher efficacy, interfering with biofilm structure and promoting bacterial cell death. Atomic force microscope images further demonstrated the peptide potential in P. aeruginosa biofilm eradication, confirming the promising application of P5 in multi-resistant infections therapeutics.
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•The antimicrobial peptide P5 displays synergistic activity with meropenem in a carbapenem-resistant strain of P. aeruginosa.•P5 permeablizes the membrane of P. aeruginosa.•P5 eradicates P.aeruginosa biofilms and displays bactericidal activity against biofilm associated bacteria.
•The biophysical and microbiological characteristics of 3 new designed cationic antimicrobial peptides (CAMPs) were studied.•A CAMP sequence was modified in order to increase or diminish the ...hydrophobic face when structured as alpha helix.•Importance of the hydrophobic moment in the peptide-membrane interaction was analyzed through extensive biophysical studies.•A mode of action of these amphipathic alpha helical peptides is also proposed.
Cationic antimicrobial peptides (CAMPs) represent important self defense molecules in many organisms, including humans. These peptides have a broad spectrum of activities, killing or neutralizing many Gram-negative and Gram-positive bacteria. The emergence of multidrug resistant microbes has stimulated research on the development of alternative antibiotics. In the search for new antibiotics, cationic antimicrobial peptides (CAMPs) offer a viable alternative to conventional antibiotics, as they physically disrupt the bacterial membranes, leading to lysis of microbial membranes and eventually cell death. In particular, the group of linear α-helical cationic peptides has attracted increasing interest from clinical as well as basic research during the last decade.
In this work, we studied the biophysical and microbiological characteristics of three new designed CAMPs. We modified a previously studied CAMP sequence, in order to increase or diminish the hydrophobic face, changing the position of two lysines or replacing three leucines, respectively. These mutations modified the hydrophobic moment of the resulting peptides and allowed us to study the importance of this parameter in the membrane interactions of the peptides. The structural properties of the peptides were also correlated with their membrane-disruptive abilities, antimicrobial activities and hemolysis of human red blood cells.
In the last years, the decreasing effectiveness of conventional antimicrobial-drugs has caused serious problems due to the rapid emergence of multidrug-resistant pathogens. This situation has brought ...attention to other antimicrobial agents like antimicrobial peptides (AMPs), for being considered an alternative to conventional drugs. These compounds target bacterial membranes for their activity, which gives them a broad spectrum of action and less probable resistance development. That is why the peptide-membrane interaction is a crucial aspect to consider in the study of AMPs. The aim of this work was the characterization of the “de novo” designed peptide P1, studying its interactions with model membranes (i.e. liposomes of DMPC:DMPG 5:1) in order to evaluate the final position of the peptide upon interacting with the membrane. Also, we tested the effects of the peptide in gram-positive and gram-negative bacteria. Later, by spectroscopic methods, the ability of the peptide to permeabilize the inner and outer membrane of E. coli and plasmatic membrane of S. aureus was assessed. The results obtained confirmed that P1 can disrupt both membranes, showing some difference in its activity as a function of the nature of each bacterial cell wall, confirming higher effects on gram-positive S. aureus. Finally, we also showed the ability of P1 to inhibit biofilms of that gram-positive bacterium.
All data obtained in this work allowed us to propose a model, where the first interactions of the peptide with the bacterial envelope, seem to depend on the gram-negative and gram-positive cell wall structure. After that first interaction, the peptide is stabilized by Trp residues depth inserted into the hydrocarbon region, promoting several changes in the organization of the lipid bilayer, following a carpet-like mechanism, which results in permeabilization of the membrane, triggering the antimicrobial activity.
•The difference in gram-negative and gram-positive envelop play a key role in the interaction of P1.•P1 acts through the carpet-like model, covering the surface in parallel alignments to the membrane.•Cooperativity and kinetics of membrane permeabilization are key factors in antimicrobial activity.•P1 was able to inhibit the biofilm formation of S. aureus at sub-MIC concentrations.
The ability of Patagonian L. plantarum and O. oeni strains to change the volatile profile of a sterile Pinot noir wine was studied through fermentation assays, at laboratory scale. Two strains of ...each LAB species were selected based on data regarding to their ability to survive in wine and to consume l-malic acid. Both O. oeni strains but only one L. plantarum (UNQLp 11) strain were able to remain viable, consuming l-malic acid through the fermentation assay with a concomitant increase of l-lactic acid. The volatile profile of Pinot noir wine, before and after LAB inoculation, was measured by using HS-SPME gas chromatography technique. This analysis showed that alcohols were the main volatile compounds after alcoholic fermentation and that after fermentation with the selected LAB strains, a decrease in the volatile alcohols concentration and an increase in the volatile esters concentration could be observed. The O. oeni UNQOe 73.2 strain produced the most notable change in the volatile profile, with the production of some important odorant esters at higher concentration, compared to O. oeni UNQOe 31b strain. Although, L. plantarum UNQLp 11 strain showed a better performance in the consumption of l-malic acid, this strain had a low capacity to modify the volatile compounds profile after incubation in red wine. The results found in the present work showed that different strains selected as potential malolactic starters could have different behavior when are incubated in real wine. Although L. plantarum UNQLp 11 strain showed a good consumption of l-malic acid, the O. oeni UNQOe 73.2 strain exhibited superior capacity to improve the flavor of wine due to its esterase activity that produce an increase of fruity and creamy odorants.
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•Changes in volatile profile of inoculated Pinot noir wine was studied.•Selected L. plantarum and O. oeni strains were used in the vinification assays.•Inoculation diminished alcohols concentration and increased esters concentration.•All strains assayed were able to modify the wine volatile profile.•O. oeni strain UNQOe 73.2 produced the most notable change in wine volatile profile.
Antimicrobial peptides are small molecules that display antimicrobial activity against a wide range of pathogens. In a previous work, by using model membranes we studied P6, a peptide that shows no ...antimicrobial activity, and P6.2, which exhibits antibacterial activity. In the present work we aimed to unravel the mode of action of these peptides by studying their interaction in vivo with Escherichia coli and Staphylococcus aureus. In this sense, to study the interactions with bacterial cells and their effect on the bacterial surface, zeta potential, spectroscopic, and microscopic methodologies were applied. P6.2 exhibits a higher affinity toward both bacterial envelopes. The ability of both peptides to disrupt afterwards the bacterial membrane was also studied. Both peptides were able to induce bacterial membrane damage, but higher concentrations of P6 were needed to obtain results comparable to those obtained for P6.2. Additionally, P6.2 exhibited faster damage kinetics. Altogether, these data allow postulating, in a physiologic model, that the lower affinity of P6 for bacterial envelope results in a minor final concentration of the peptide in the bacterial membrane unable to trigger the antimicrobial activity. Finally, the fact that the active P6.2 has the same MIC value for the Gram-positive and Gram-negative bacteria tested, but not the same profile in the permeabilization assays, reinforces the question of whether cell wall components act as electrostatic barriers preventing or minimizing membrane-active AMPs lethal action at the membrane level.
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•Achieving the permeabilization of the inner membrane seems to be the critical step in the P6.2 killing mechanism in E. coli.•In S. aureus faster full permeabilization takes place probably as they lack outer membrane.•Fast membrane disruption kinetics is a key feature to avoid bacteria to overcome the damage.•AFM data revealed that P6.2 induces leakage of bacterial content and bubbles on the bacterial membrane.
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