Analytical combinations to evaluate the macromolecular composition of extracellular substances (ECS) from Lactobacillus plantarum cell culture media.

Sugar-enriched media are used to produce extracellular substances (ECS) by Lactobacillus plantarum WCSF1, with a focus on growing stages and carbon source substrates. Combination of size exclusion chromatography and ATR-FTIR spectroscopy provides physicochemical patterns of bulk ECS produced along culture growing time. Secreted biopolymers present polydisperse and high molecular weight distributions, with significant amounts of carbohydrates and proteins. Results, supported by a multivariate statistical analysis, enable to differentiate the macromolecular content of bacterial ECS along the growing stages regardless of the growing media, highlighting a higher production of proteinaceous materials compared to polysaccharides. At the end of the exponential phase, common exoproteins were present in all the tested sugar-enriched media such as transglycosylases between 20 and 35 kDa, a muropeptidase at 36.9 kDa and a cell wall hydrolase. Additionally, L. plantarum WCFS1 secretes ECS with a greater diversity of proteins, when growing in the sucrose-enriched media. Graphical abstract.

Measurement of white wines resistance against oxidation by Electron Paramagnetic Resonance spectroscopy.

Free radical theory of aging hypothesizes that oxygen-derived radicals are responsible for the storage-related flavor instability in wine. In an optimal situation, a balanced-distribution exists between oxidants and antioxidants among wines intrinsic/extrinsic metabolites. Based on the kinetic study of POBN-1-hydroxyethyl spin adduct (POBN-1-HER) formation in wines initiated via the Fenton reaction, a novel tool based on EPR spin trapping methodology was developed to quantify wines resistance against oxidation. Antioxidant capacities of wines were evaluated according to POBN-1-HER maximum signal intensity (Imax POBN-1-HER) and rate formation (rPOBN-1-HER) kinetic parameters. Low Imax POBN-1-HER and rPOBN-1-HER values suggest that endogenous antioxidants in wine are able to quench a substantial amount of radicals capable to take part in deleterious oxidative reactions. This will be very valuable in understanding aging potential and will provide an avenue to better control the process by knowing how it might be possible to change wines resistance against oxidation.

Direct investigation of viscosity of an atypical inner membrane of Bacillus spores: a molecular rotor/FLIM study.

We utilize the fluorescent molecular rotor Bodipy-C12 to investigate the viscoelastic properties of hydrophobic layers of bacterial spores Bacillus subtilis. The molecular rotor shows a marked increase in fluorescence lifetime, from 0.3 to 4ns, upon viscosity increase from 1 to 1500cP and can be incorporated into the hydrophobic layers within the spores from dormant state through to germination. We use fluorescence lifetime imaging microscopy to visualize the viscosity inside different compartments of the bacterial spore in order to investigate the inner membrane and relate its compaction to the extreme resistance observed during exposure of spores to toxic chemicals. We demonstrate that the bacterial spores possess an inner membrane that is characterized by a very high viscosity, exceeding 1000cP, where the lipid bilayer is likely in a gel state. We also show that this membrane evolves during germination to reach a viscosity value close to that of a vegetative cell membrane, ca. 600cP. The present study demonstrates quantitative imaging of the microscopic viscosity in hydrophobic layers of bacterial spores Bacillus subtilis and shows the potential for further investigation of spore membranes under environmental stress.

Effect of plasticizers (water and glycerol) on the diffusion of a small molecule in iota-carrageenan biopolymer films for edible coating application.

Translational diffusion of a fluorescein probe has been measured in iota-carrageenan edible films containing different amounts of glycerol (0, 15, 30, and 45%), using fluorescence recovery after photobleaching (FRAP) experiments. The effects of this plasticizer as well as the plasticizing effect of water on the diffusion of fluorescein have been studied in this edible coating mainly composed of natural biopolymer. Diffusion coefficients of about 10(-13) m2 s(-1) have been measured in these films for water activity (aw) lower than 0.7. Above this water content threshold, fluorescein translational diffusion coefficient increases up to 10(-12) m2 s(-1). Another interesting information obtained from FRAP experiments on this system is the ratio of the diffusing molecules which are immobilized in the carrageenan matrix at aw lower than 0.98. Moreover, films containing more than 30% glycerol (w/w carrageenan) present a huge increase of the diffusion coefficient of fluorescein at high water activity (about 2 orders of magnitude), this effect being less pronounced at low water activity. The increase of diffusion seems to be only related to the water content, and glycerol only acts through the enhancement of water adsorption. Therefore, in biopolymer films containing polyol plasticizers, the gain in mobility could be devoted to the effect of the ubiquitous plasticizing molecule, water, whose adsorption is increased by the plasticizer.

Influence of sucrose and water content on molecular mobility in starch-based glasses as assessed through structure and secondary relaxation.

Molecular mobility is known to be a key parameter in controlling the physical properties of materials and thus their quality and performance. Beyond glass transition related changes, attention should be called to the impact of local motions remaining in the glassy state. Gelatinized waxy maize starch at different sucrose contents (0-20% solids) was equilibrated between 0 and 14% water and sorption isotherms determined at 25 degrees C. The effect of water and sucrose content on the molecular mobility of glassy starch was investigated by differential scanning calorimetry through enthalpy relaxation studies and dynamical mechanical thermal analysis. The existence of sucrose-starch interactions was suggested by the sorption isotherms not following the expected additivity of the single component sorption curves. Contrary to the glass transition or associated alpha relaxation, water and sucrose affected differently the secondary relaxations. Indeed, the beta relaxation observed around -15 degrees C was shifted to lower temperature upon increasing hydration, and to higher temperature when sucrose content increased, suggesting a hindering of these local motions. Enthalpy relaxation of the ternary mixtures was studied following aging up to 668 h at Tg -15 degrees C. Ternary mixtures exhibited an enthalpy relaxation upon aging lower than starch alone as a sign of lower polymer mobility in the presence of small molecules, contrary to the free volume theory. Relaxation kinetics were characterized with the Cowie-Ferguson model and compared to literature data. The extent of the enthalpy relaxation appeared to be controlled by the distance between the aging temperature and the beta relaxation temperature.

Wetting properties at the surface of iota-carrageenan-based edible films.

Surface properties of edible films composed of a polymeric matrix of carrageenan in association with hydrophobic material were studied by contact angle measurements. The use of this technique not only in a static mode but also in a dynamic way enables investigation of surface hydrophobicity as well as surface wettability. The absorption flux inside the material can be estimated from the wetting kinetic, which can be very useful to quickly compare water barrier efficiency of the tested films. Comparison of carrageenan films with films containing known amounts of additives enables understanding and correlation of changes of the surface properties with the nature of used additives (glycerol used as a plasticizer, glycerol monostearate used as a surfactant, and fat) and their influence on the orientation of polymer chains at the surface during film formation. Very different responses were observed from one surface of the film (film-casting-support interface) to the other (film-air interface), which could be also attributed to the influence of the support on the polymer and to macromolecular orientation during drying after casting.

Translational diffusion coefficients of volatile compounds in various aqueous solutions at low and subzero temperatures.

Translational diffusion coefficients (D(12)) of volatile compounds were measured in model media with the profile concentration method. The influence of sample temperature (from 25 to -10 degrees C) was studied on translational diffusion in sucrose or maltodextrin solutions at various concentrations. Results show that diffusivity of volatile compounds in sucrose solutions is controlled by temperature, molecule size, and the viscosity of the liquid phase as expected with the Stokes-Einstein equation; moreover, physicochemical interactions between volatile compounds and the medium are determinant for diffusion estimation. At negative temperature, the winding path induced by an ice crystal content of >70% lowered volatile compound diffusion. On the contrary, no influence on translational diffusion coefficients was observed for lower ice content.

Aroma volatility from aqueous sucrose solutions at low and subzero temperatures.

The gas-liquid partition coefficients of ethyl acetate and ethyl hexanoate have been measured in water and aqueous sucrose solutions from 25 to -10 degrees C by dynamic headspace. Experiments were carried out on sucrose solutions at temperatures where no ice formation was possible. Results showed that when sucrose concentration increased, aroma volatility increased except for ethyl hexanoate and in the highest sucrose concentration solution (57.5%). A quasi-linear temperature decrease on aroma volatility was observed in sucrose solutions from 25 to around 4 and 0 degrees C. Then, from 0 to -10 degrees C, aroma volatility did not decrease: ethyl acetate volatility remained constant but that of ethyl hexanoate increased. Enthalpy of vaporization and activity coefficients of the aroma compounds were calculated.

Ascorbic acid oxidation in sucrose aqueous model systems at subzero temperatures.

The reduction of Tempol by ascorbic acid in concentrated sucrose solutions was measured by electron paramagnetic resonance (EPR) at temperatures ranging from 16 to -16 degrees C. This method allowed the determination of the rate constants (k) of this fast reaction, by recording the Tempol reduction as a function of time. The two reactants were initially separated and had to migrate for the reaction to occur. The experimental findings were compared with predicted values according to the equation for diffusion-controlled reaction proposed by Atkins. The experimental reaction rate constants were observed to be lower than the calculated ones. However, the experimental values were found to be controlled by the temperature and viscosity changes of the reaction media, as expected for a diffusion-controlled reaction.

Study of release kinetics of small and high molecular weight substances dispersed into spray-dried ethylcellulose microspheres.

Spray-dried ethylcellulose microspheres were used as matrices for the encapsulation of a fungal lactase and/or small paramagnetic probes (Tempol or Tempo). Their dissolution in water was studied. Kinetics fitted with the model Q = kt(n) of Korsemeyer et al. [Int. J. Pharm. 15 (1983) 25] exhibited a non-Fickian diffusion. The calculated diffusional exponent (n) values were near 0.26 whatever the encapsulated probes. The release rates (k) were only slightly different for paramagnetic probes and lactase. This result indicated that the probes' release mechanisms are not diffusion controlled. Other factors such as matrix porosity and probe solubility in the matrix and in water could influence the probes' release rate.