An overview of peroxidation reactions using liposomes as model systems and analytical methods as monitoring tools.

Phospholipids are building blocks of biological membranes having a key role in cellular functionality. The presence of unsaturated fatty acids in their conformation makes them prompt to oxidation reactions, leading to dysfunctions of living cells or to instability of lipid containing food products. The aim of this review is to gather together the latest advances on the understanding on lipids' peroxidation, using liposomes as model systems, including the main available analytical methods to monitor peroxidation reactions, with special emphasis on Fourier Transform Infrared (FTIR) and Raman spectroscopies. Lipid peroxidation is the most widely studied free radical chain reaction, which occurs in three steps: initiation, propagation and termination, making difficult to determine peroxidation products. Using liposomes as model membrane systems provides a useful tool to investigate the effects of free radicals. Different analytical methods enable the determination of peroxidation primary or secondary products. In particular, FTIR and Raman spectroscopies allow the simultaneous determination of peroxidation products in a non-destructive and easy-to-use manner. A quick monitoring of both reagents and products provides a reliable method for the quality control of industrial products or even for diagnostics, thus underlying the strong potential of vibrational spectroscopic based techniques.

Study of surface damage on cell envelope assessed by AFM and flow cytometry of Lactobacillus plantarum exposed to ethanol and dehydration.

AIMS: In this work, we evaluated freeze-drying damage at the surface level of oenological strain Lactobacillus plantarum UNQLp155, as well as its ability to grow in a synthetic wine with and without pre-acclimation. METHODS AND RESULTS: Damage on cell surface was studied by flow cytometry, zeta potential and atomic force microscopy, and cell survival was analysed by plate count. Results showed that beside cells acclimated at lower ethanol concentration (6% v/v) became more susceptible to drying than nonacclimated ones, after rehydration they maintain their increased ability to grow in a synthetic wine. Acclimation at a higher ethanol concentration (10% v/v) produces several damages on the cell surface losing its ability to grow in a synthetic wine. CONCLUSIONS: In this work, we showed for the first time that sublethal alterations on bacterial surface induced by a pre-acclimation with a low ethanol concentration (6%), upon a freeze-drying process, result in a better bacterial adaptation to the stress conditions of wine-like medium, as well as to the preservation process. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding the adaptation to ethanol of oenological strains and their effects on the preservation process has a strong impact on winemaking process and allows to define the most appropriate conditions to obtain malolactic starters cultures.

Effect of the fatty acid composition of acclimated oenological Lactobacillus plantarum on the resistance to ethanol.

The aim of this work was to evaluate the changes due to acclimation to ethanol on the fatty acid composition of three oenological Lactobacillus plantarum strains and their effect on the resistance to ethanol and malic acid consumption (MAC). Lactobacillus plantarum UNQLp 133, UNQLp 65.3 and UNQLp 155 were acclimated in the presence of 6 or 10% v/v ethanol, for 48 h at 28°C. Lipids were extracted to obtain fatty acid methyl esters and analysed by gas chromatography interfaced with mass spectroscopy. The influence of change in fatty acid composition on the viability and MAC in synthetic wine was analysed by determining the Pearson correlation coefficient. Acclimated strains showed a significant change in the fatty composition with regard to the nonacclimated strains. Adaptation to ethanol led to a decrease in the unsaturated/saturated ratio, mainly resulting from an increase in the contribution of short-length fatty acid C12:0 and a decrease of C18:1. The content of C12:0 was related to a higher viability after inoculation of synthetic wine. The MAC increased at higher contents in saturated fatty acid, but its efficiency was strain dependent.

Stabilization of polymer lipid complexes prepared with lipids of lactic acid bacteria upon preservation and internalization into eukaryotic cells.

The physicochemical characterization of polymer liposome complexes (PLCs) prepared with lipids of lactic acid bacteria and poly(N,N-dimethylaminoethyl methacrylate) covalently bound to cholesterol (CHO-PDMAEMA) was carried out in an integrated approach, including their stability upon preservation and incorporation into eukaryotic cells. PLCs were prepared with different polymer:lipid molar ratios (0, 0.05 and 0.10). Zeta potential, particle size distribution and polydispersity index were determined. The optimal polymer:lipid ratio and the stability of both bare liposomes and PLCs were evaluated at 37 °C and at different pHs, as well as after storage at 4 °C, -80 °C and freeze-drying in the presence or absence of trehalose 250 mM. Internalization of PLCs by eukaryotic cells was assessed to give a complete picture of the system. Incorporation of CHO-PDMAEMA onto bacterial lipids (ratio 0.05 and 0.10) led to stabilization at 37 °C and pH 7. A slight decrease of pH led to their strong destabilization. Bacteria PLCs showed to be more stable than lecithin (LEC) PLCs (used for comparison) upon preservation at 4 and -80 °C. The harmful nature of the preservation processes led to a strong decrease in the stability of PLCs, bacterial formulations being more stable than LEC PLCs. The addition of trehalose to the suspension of liposomes stabilized LEC PLC and did not have effect on bacterial PLCs. In vitro studies on Raw 264.7 and Caco-2/TC7 cells demonstrated an efficient incorporation of PLCs into the cells. Preparations with higher stability were the ones that showed a better cell-uptake. The nature of the lipid composition is determinant for the stability of PLCs. Lipids from lactic acid bacteria are composed of glycolipids and phospholipids like cardiolipin and phosphatidylglycerol. The presence of negatively charged lipids strongly improves the interaction with the positively charged CHO-PDMAEMA, thus stabilizing liposomes. In addition, glycolipids and phosphatidylglycerol act as intrinsic protectants of PLCs upon preservation. This particular lipid composition of lactic acid bacteria makes them natural formulations potentially useful as drug delivery systems.

Effect of acclimation medium on cell viability, membrane integrity and ability to consume malic acid in synthetic wine by oenological Lactobacillus plantarum strains.

AIMS: The aim of this work was to evaluate the effect of acclimation on the viability, membrane integrity and the ability to consume malic acid of three oenological strains of Lactobacillus plantarum. METHODS AND RESULTS: Cultures in the stationary phase were inoculated in an acclimation medium (Accl.) containing 0, 6 or 10% v/v ethanol and incubated 48 h at 28°C. After incubation, cells were harvested by centrifugation and inoculated in a synthetic wine, containing 14% v/v ethanol and pH 3.5 at 28°C. Viability and membrane integrity were determined by flow cytometry (FC) using carboxyfluorescein diacetate (cFDA) and propidium iodide. Bacterial growth and malic acid consumption were monitored in a synthetic wine during 15 days. In nonacclimated strains, the damage of bacterial membranes produced a dramatic decrease in microbial viability in synthetic wine. In contrast, survival of strains previously acclimated in Accl. with 6 and 10% v/v ethanol was noticeable higher. Therefore, acclimation with ethanol increased the cultivability in synthetic wine and consequently, the consumption of l-malic acid after 15 days of growth. CONCLUSION: Acclimation of oenological strains in media containing ethanol prior to wine inoculation significantly decreases the membrane damage and improves viability in the harsh wine conditions. The role of membrane integrity is crucial to warrant the degradation of l-malic acid. SIGNIFICANCE AND IMPACT OF THE STUDY: The efficiency of multiparametric FC in monitoring viability and membrane damage along with the malic acid consumption has a strong impact on winemaking because it represents a useful tool for a quick and highly reliable evaluation of oenological parameters.

Effect of cholesterol-poly(N,N-dimethylaminoethyl methacrylate) on the properties of stimuli-responsive polymer liposome complexes.

The development of new polymer-liposome complexes (PLCs) as delivery systems is the key issue of this work. Three main areas are dealt with: polymer synthesis/characterization, liposome formulation/characterization and evaluation of the PLCs uptake by eukaryotic cells. Poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA) with low molecular weight and narrow polydispersity was synthesized by Atom Transfer Radical Polymerization (ATRP). The polymers were synthesized using two different bromide initiators (cholesteryl-2-bromoisobutyrate and ethyl 2-bromoisobutyrate) as a route to afford PDMAEMA and CHO-PDMAEMA. Both synthesized polymers (PDMAEMA and CHO-PDMAEMA) were incorporated in the preparation of lecithin liposomes (LEC) to obtain PLCs. Three polymer/lipid ratios were investigated: 5, 10 and 20%. Physicochemical characterization of PLCs was carried out by determining the zeta potential, particle size distribution, and the release of fluorescent dyes (carboxyfluorescein CF and calcein) at different temperatures and pHs. The leakage experiments showed that CHO covalently bound to PDMAEMA strongly stabilizes PLCs. The incorporation of 5% CHO-PDMAEMA to LEC (LEC_CHO-PD5) appeared to be the stablest preparation at pH 7.0 and at 37°C. LEC_CHO-PD5 destabilized upon slight changes in pH and temperature, supporting the potential use of CHO-PDMAEMA incorporated to lecithin liposomes (LEC_CHO-PDs) as stimuli-responsive systems. In vitro studies on Raw 264.7 and Caco-2/TC7 cells demonstrated an efficient incorporation of PLCs into the cells. No toxicity of the prepared PLCs was observed according to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. These results substantiate the efficiency of CHO-PDMAEMA incorporated onto LEC to assist for the release of the liposome content in mildly acidic environments, like those found in early endosomes where pH is slightly lower than the physiologic. In summary, the main achievements of this work are: (a) novel synthesis of CHO-PDMAEMA by ATRP, (b) stabilization of LEC by incorporation of CHO-PDMAEMA at neutral pH and destabilization upon slight changes of pH, (c) efficient uptake of LEC_CHO-PDs by phagocytic and non-phagocytic eukaryotic cells.

Effect of human defensins on lactobacilli and liposomes.

AIMS: To study the effect of human ß-defensins (HBD-1 and HBD-2) on lactobacilli membranes as well as on liposomes prepared from purified bacterial lipids. METHODS AND RESULTS: Lactobacillus delbrueckii subsp. bulgaricus CIDCA 331 and Lact. delbrueckii subsp. lactis CIDCA 133 were grown in Man, Rogosa, Sharpe broth for 16 h at 37 °C. After being washed, micro-organisms were treated with 0.1-10 µg ml(-1) of HBD-1 and HBD-2 (30 min, 37 °C). Bacterial damage was determined by flow cytometry after propidium iodide staining. In parallel experiments, release of carboxyfluorescein from liposomes prepared from bacterial lipids was determined fluorometrically (excitation 485/20 nm, emission 528/20 nm) in the presence of HBD-1, HBD-2 or Nisin. Exposure of lactobacilli to HBD-2 resulted in a significant membrane permeabilization being Lact. delbrueckii subsp. bulgaricus CIDCA 331 the most susceptible strain. Liposomes prepared with lipids from strain CIDCA 133 were destabilized neither by HBD-1 nor by HBD-2, whereas liposomes derived from strain CIDCA 331 were susceptible to HBD-2 but not to HBD-1. Effect of defensins was strongly inhibited in the presence of NaCl, and the activity increased in water. CONCLUSIONS: Results reported in the presented work indicate that lipid composition of bacterial membranes lead to a different interaction with cationic peptides such as defensins. SIGNIFICANCE AND IMPACT OF THE STUDY: The results represent an advance in the understanding of the differential effect of HBDs on micro-organisms. Differences in susceptibility to anti-microbial peptides could modify the fate of micro-organisms after the interaction with host's cells.

Use of Raman spectroscopy and chemometrics for the quantification of metal ions attached to Lactobacillus kefir.

AIMS: To set-up an experimental and analytical methodology to evaluate the feasibility of developing simple, accurate and quantitative models based on Raman spectroscopy and multivariate analysis for the quantification of metal ions adsorbed to the bacterial surface of Lactobacillus kefir. METHODS AND RESULTS: One millilitre cultures from two strains of Lact. kefir in the stationary phase were harvested and washed twice with ultra pure water. The bacterial pellets were resuspended into 1 ml solutions of Pb(+2), Cd(+2) or Ni(+2) ranging from 0 to 0·9 mmol l(-1). The suspensions were further incubated for 1 h at 30°C at pH 5·5. After centrifugation, the pellets were kept to register the Raman spectra and the supernatants were used for the analytical determination of Pb(+2) , Cd(+2) and Ni(+2). Micro-organisms nontreated with metal ions were used as controls. Principal component analysis (PCA) was performed over the preprocessed Raman spectra to evaluate whether the clusters obtained could be correlated with the concentration of metal ions attached to the bacterial biomass. After that, partial least squares (PLS) models were calibrated with the aim of quantifying the metal ions adsorbed to the bacterial surface. According to the analytical determinations, the maximum binding capacity of all the metals (q(max)) attained values that are comparable with those observed for other lactic acid bacteria (ca. 0·200 mmol g(-1)). The spectral analysis revealed that the main functional groups involved in the bacteria/metal interaction are carboxylates, phosphates and polysaccharides. In PCA, the first two principal components explain more than 72% variance of the spectral data set contained in the data structure, allowing a clear discrimination among samples of different concentrations. Based on this information and using as reference the results obtained by analytical methods, PLS prediction models were successfully defined for the quantification of Pb(+2), Cd(+2) and Ni(+2) attached to the bacterial surface. CONCLUSIONS: The calibration and validation of methods based on multivariate analysis allowed the definition of models for the quantification of Pb(+2), Cd(+2) and Ni(+2) attached to bacterial surfaces. The high percentages of explained variances in PCA gave a strong support to calibrate the prediction models, depicting very good correlations with the reference method (correlations ∼0·90 in all cases). SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacillus kefir CIDCA 8348 and JCM 5818 bind Pb(+2), Cd(+2) and Ni(+2) in an efficient way. This fact gives support for their potential use as sequestrants of traces of these metals in products addressed to human and animal consume. The prediction models developed would be useful for the determination of the investigated metal ions in unknown samples giving at the same time, structural information about this interaction. This is certainly the most important contribution of this work.

Volume recovery, surface properties and membrane integrity of Lactobacillus delbrueckii subsp. bulgaricus dehydrated in the presence of trehalose or sucrose.

AIMS: Although the practical importance of adding sugars before drying is well known, the mechanism of protection of bacteria by sugars is not clear. The response of the dehydrated micro-organisms to rehydration is analysed in terms of structural and functional changes, and correlated with their potentiality to grow in rich media. These aspects are related with the membrane integrity and the metabolic state of the rehydrated bacteria, measured by means of surface properties and permeability. To attain this objective, Lactobacillus delbrueckii subsp. bulgaricus was dehydrated in the presence and in the absence of sucrose and trehalose. The bacterial response upon rehydration was investigated by determining: (i) the lag time of the bacterial growing in rich media, (ii) the restoration of the surface properties and the cellular volume and (iii) the membrane integrity. METHODS AND RESULTS: Lactobacillus delbrueckii subsp. bulgaricus was grown in MRS at 37 degrees C overnight [De Man et al. (1960)J Appl Bacteriol 23, 130] and then dehydrated for 10, 20 and 30 min at 70 degrees C in a vacuum centrifuge. The lag time of micro-organisms was determined by optical density changes after rehydration. The surface properties were determined by measuring the zeta potential of the bacteria suspended in aqueous solution. The cellular volume recovery was measured, after stabilization in saline solution, by light scattering and by the haematocrit method [Alemohammad and Knowles (1974)J Gen Microbiol 82, 125]. Finally, the membrane integrity has been determined by using specific fluorescent probes [SYTO 9 and propidium iodide, (PI)] that bind differentially depending on the integrity of the bacterial membrane. The lag time of Lact. delbrueckii subsp bulgaricus, dehydrated by heat in the presence of sucrose or trehalose and after that rehydrated, was significantly shortened, when compared with that obtained for bacteria dried in the absence of sugars. In these conditions, trehalose and sucrose maintained the zeta potential and the cell volume close to the control (nondried) cells. However, the membrane integrity, measured with fluorescent probes, was maintained only when cells were dehydrated for 10 min in the presence of sugars. For larger times of dehydration, the membrane integrity was not preserved, even in the presence of sugars. CONCLUSIONS: When the micro-organisms are dehydrated in the absence of protectants, the membrane damage occurs with a decrease in the absolute value of the zeta potential and a decrease in the cellular volume recovered after rehydration. In contrast, when the zeta potential and the cellular volume are restored after rehydration to that corresponding to nondried cells, the micro-organisms are able to recover and grow with a reduced lag time. This can only be achieved when the dehydration is carried out in the presence of sugars. At short dehydration times, the response is associated with the preservation of the membrane integrity. However, for longer times of dehydration the zeta potential and volume recovery occurs in the presence of sugars in spite of a severe damage at membrane level. In this condition, cells are also recovered. In conclusion, to predict the ability of growing after dehydration, other bacterial structural parameters besides membrane integrity, such as zeta potential and cellular volume, should be taken into account. SIGNIFICANCE AND IMPACT OF THE STUDY: The correlation of the lag time with the surface and permeability properties is of practical importance because the correlation of these two parameters with cell viability, allow to determine the potential bacterial capacity to grow in a rich medium after the preservation procedure, without necessity of performing a kinetic curve of growth, which is certainly time-consuming.

Effect of sugars and growth media on the dehydration of Lactobacillus delbrueckii ssp. bulgaricus.

AIMS: The efficiency of trehalose, sucrose and maltose to protect Lactobacillus bulgaricus during drying has been evaluated in bacteria grown at low water activity. METHODS AND RESULTS: Bacteria were grown in MRS (control), and in MRS supplemented with sucrose (MRS-sucrose) or with polyethyleneglycol (PEG) (MRS-PEG) as low water activity media. The growth in low water activity media (MRS-sucrose and MRS-PEG) prior to drying enhanced the effectiveness of trehalose as thermoprotectant during drying. The efficiency of sucrose was improved when bacteria were grown in MRS-sucrose. On the other hand, the growth in both low water activity media did not affect the efficiency of maltose. The damage produced during dehydration has been evaluated by means of growth kinetics in milk. The preservation of bacteria dehydrated with sucrose, after growing them in MRS-sucrose, appears to be as efficient as the dehydration with trehalose. CONCLUSIONS: The growth of L. bulgaricus in low water activity media enhances the protective action of trehalose and sucrose. SIGNIFICANCE AND IMPACT OF THE THE STUDY: These results may aid the dairy industry to improve the recovery of the starters at low costs after preservation processes.