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International audience; Fourier transform infrared (FTIR) spectroscopy has proven to be a non-invasive tool to analyse cells without the hurdle of employing exogenous dyes or probes. Nevertheless, the study of single live bacteria in their aqueous environment has long remained a big challenge, due to the strong infrared absorption of water and the small size of bacteria compared to the micron-range infrared wavelengths of the probing photons. To record infrared spectra of bacteria in an aqueous environment, at different spatial resolutions, two setups were developed. A custom-built attenuated total reflection inverted microscope was coupled to a synchrotron-based FTIR spectrometer, using a germanium hemisphere. With such a setup, a projected spot size of 1 × 1 μm2 was achieved, which allowed spectral acquisition at the single-cell level in the 1800–1300 cm−1 region. The second setup used a demountable liquid micro-chamber with a thermal source-powered FTIR microscope, in transmission geometry, for probing clusters of a few thousands of live cells in the mid-IR region (4000–975 cm−1). Both setups were applied for studying two strains of a model lactic acid bacterium exhibiting different cryo-resistances. The two approaches allowed the discrimination of both strains and revealed population heterogeneity among bacteria at different spatial resolutions. The multivariate analysis of spectra indicated that the cryo-sensitive cells presented the highest cell heterogeneity and the highest content of proteins with the α-helix structure. Furthermore, the results from clusters of bacterial cells evidenced phosphate and peptidoglycan vibrational bands associated with the cell envelope, as potential markers of resistance to environmental conditions.

Freezing is widely used for bacterial cell preservation. However, resistance to freezing can greatly vary depending on bacterial species or growth conditions. Our study aims at identifying cellular markers of cryoresistance based on the comparison of three lactic acid bacteria (LAB) exhibiting different tolerance to freezing: Carnobacterium maltaromaticum CNCM I-3298, Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842, and Lactobacillus delbrueckii subsp. bulgaricus CFL1. A thorough characterization of their cytoplasmic membrane properties was carried out by measuring their fatty acid composition, membrane fluidity, and lipid phase transition upon cooling from 50 to -50 °C. Vitrification temperatures of the intra- and extra-cellular compartments were also quantified by differential scanning calorimetry. Additionally, the cell biochemical characterization was carried out using a recently developed Fourier transform infrared (FTIR) micro-spectroscopic approach allowing the analysis of live bacteria in an aqueous environment. The multivariate analysis of the FTIR spectra of fresh and thawed cells enabled the discrimination of the three bacteria according to their lipid, protein, and cell wall peptidoglycan components. It also revealed freezing-induced modifications of these three cellular components and an increase in bacteria heterogeneity for the two strains of L. bulgaricus, the freeze-sensitive bacteria. No cellular damage was observed for C. maltaromaticum, the freeze-resistant bacteria. Comparison of the results obtained from the different analytical methods confirmed previously reported cryoresistance markers and suggested new ones, such as changes in the absorbance of specific infrared spectral bands. FTIR microspectroscopy could be used as a rapid and non-invasive technique to evaluate the freeze-sensitivity of LAB.

Environmental emission of pharmaceutical pollutants notably causes the contamination of aquatic ecosystems and drinking water. Typically, reduction of these pollutants in the environment is mostly managed by ameliorated wastewater treatments. Here, we report a method for the eco-design of drugs through the introduction within the molecular structure of a sensitive chemical group responsive to water treatments. The new drugs are thus programmed to fragment more easily and quickly than the original drugs. In this "retro catabolic drug design" strategy, methotrexate was used as drug model and an ether analog displaying a similar pharmacological profile was selected. Using photo-irradiation experiments at 254 nm, a representative drinking water treatment process, the identified transformation products were predominantly obtained from the expected molecular scission. Moreover, a faster kinetics of degradation was measured for the ether analog as compared to methotrexate and its transformation products were far less cytotoxic.

Methotrexate is an antineoplastic folate analog of high environmental concern, due to its low biodegradability and toxicological properties. This study focused on its photodegradation under two irradiation conditions, aiming to be representative of environment (300–450 nm) and drinking water treatment (254 nm). The photodegradation experiments were conducted at two pH, to vary the methotrexate ionization state and to produce a large variety of transformation products (TPs). The degradation kinetics determined through LC-UV monitoring were contrasted according to pH and irradiation wavelength. However, the quantum yields were independent of ionization state at 254 nm and the changes in kinetics at higher wavelengths were attributed to a change in the degradation mechanism. The TPs formed during the reactions were identified by UHPLC-MS/MS, using both the positive and negative modes. Among the eleven proposed structures, five were described as methotrexate TPs for the first time. The TPs result from N-demethylation, glutamic acid oxidation and C-N cleavage, all of them leading to further degraded photoproducts presenting modified or lost glutamic acid part. This was made possible thanks to the negative mode, which allowed the exploration of the glutamic acid moiety modifications. Cytotoxicity assessment on A549 cancer cells demonstrated that all photoproducts formed at pH 7 were less toxic than the parent compound.

The study of food microorganism domestication can bring important insights on adaptation mechanisms and have industrial applications. The Penicillium roqueforti mold is divided into four main populations, with two populations domesticated for blue-cheese making and two populations thriving in other environments. While most blue cheeses worldwide are made with the same P. roqueforti clonal lineage, the emblematic Roquefort cheeses are inoculated with a specific population. To study the differences among P. roqueforti populations in the context of domestication for cheesemaking, we compared blue cheeses made with the four fungal populations following Roquefort-type production specifications. We found that the P. roqueforti populations had a minor impact on the cheese bacterial diversity and none on the main microorganism abundance. The cheese P. roqueforti populations produced cheeses with higher percentages of blue area and with different sets and higher quantities of desired volatile compounds. The Roquefort P. roqueforti population in particular produced higher quantities of positive aromatic compounds in cheeses, which was related due to its most efficient proteolysis and lipolysis, and also produced cheeses with lower water activity, thus restricting spoiler microorganisms. Our results show the strong influence of P. roqueforti populations on several important aspects of cheese safety, appearance and flavour. The typical appearance and flavours of blue cheeses are therefore the result of human selection on P. roqueforti , thus constituting domestication, and the two cheese populations have acquired specificities. This has important implications for our understanding of adaptation and domestication processes as well as for improving cheese production. Importance Several fungi have been domesticated for food fermentation, with selection for traits beneficial for food production. The mold used for making blue cheeses, Penicillium roqueforti , is subdivided into four genetically different populations, two being found in cheese, one being specific of the Roquefort protected designation of origin, and two in other environments. The cheese P. roqueforti populations produced bluer cheeses with higher quantities of desired volatile compounds. The Roquefort P. roqueforti population in particular produced higher quantities of positive aromatic compounds in cheeses, in relation to its most efficient proteolysis and lipolysis, and also produced cheeses with lower water activity, thus restricting spoiler microorganisms. Our results support that the blue cheese typical aspect and flavors are the result of a selection by humans and show the strong influence of P. roqueforti populations for several important aspects of cheese safety, aspect and flavor, paving the way for improving cheese production.

Geotrichum candidum is a fungus-like yeast widely used as a starter culture for cheese ripening for its proteolytic and lipolytic activities and its contribution to the cheese flavours. The sequenced strain G. candidum CLIB 918 was isolated from cheese Pont-L’Evêque. This strain's ability to produce volatile compounds was compared to the ability of a known strong sulphur compound producer G. candidum strain (Gc203). The aminotransferase-coding genes BAT2 and ARO8 were identified to be involved in methionine catabolism. The production of volatile compounds indicated that the sequenced strain was a moderate producer compared to the strong producer strain. The major volatile compounds were produced from sulphur amino acid, branched-chain amino acid and fatty acid metabolisms. Metabolite content of the cells showed that the ability of the strain to produce volatile compounds was inversely proportional to its ability to store amino acids inside the cells. Reduced glutathione, hypotaurine and taurine intracellular concentrations and volatile fatty aldehyde production indicated the role of oxidative stress sensitivity in flavour production. The increase in expression of several genes in a Reblochon-type cheese at the end of ripening confirmed that oxygen and iron were key factors regulating cheese flavour production., The ability of Geotrichum candidum to produce volatile compounds is strain dependent and inversely proportional to its ability to store metabolites inside the cells

The consumption protocol used during alcoholic beverage tasting may affect aroma perception. We used an integrated approach combining sensory analysis and physicochemistry to investigate the impact of swallowing on aroma release and perception. A panel of 10 persons evaluated the dynamics of aroma perception during the consumption of a commercial flavored vodka, using the method of temporal dominance of sensations. Two protocols (spitting out or swallowing of the product) were tested. Nosespace analysis was simultaneously carried out by proton transfer reaction mass spectrometry to evaluate aroma release in the nasal cavity. Comparison of the results obtained with the 2 protocols highlighted significant differences in both the perception and the release of aroma: the swallowing of the product resulted in more complex perceptions but decreased the dominance rates of aromatic attributes. Ethanol perception also had an impact when the product was swallowed. Aroma release data partly accounted for the differences in perception, particularly as concerned ethanol release. The time at which dominance appears as well as the dominance duration of some attributes can be related to some temporal parameters of release data. But the lack of knowledge concerning the variety and complexity of mechanisms continues to limit our understanding of relationship between aroma release and perception.

Communication orale sur invitation; Strategies for improving freeze-drying resistance of bacteria: Modulating membrane properties during fermentation. 52. Annual Meeting of the Society for Cryobiology

This work aimed at characterizing the biochemical and biophysical properties of the membrane of Lactococcus lactis TOMSC161 cells during fermentation at different temperatures, in relation to their freeze-drying and storage resistance. Cells were cultivated at two different temperatures (22 and 30 A degrees C) and were harvested at different growth phases (from the middle exponential phase to the late stationary phase). Bacterial membranes were characterized by determining the fatty acid composition, the lipid phase transition, and the membrane fluidity. Cultivability and acidification activity losses of L. lactis were quantified after freezing, drying, and 3 months of storage. The direct measurement of membrane fluidity by fluorescence anisotropy was linked to lipid composition, and it was established that the cyclopropanation of unsaturated fatty acids with concomitant membrane rigidification during growth led to an increase in the freeze-drying and storage resistance of L. lactis. As expected, cultivating cells at a lower fermentation temperature than the optimum growth temperature induced a homeoviscous adaptation that was demonstrated by a lowered lipid phase transition temperature but that was not related to any improvement in freeze-drying resistance. L. lactis TOMSC161 was therefore able to develop a combined biochemical and biophysical response at the membrane level during fermentation. The ratio of cyclic fatty acids to unsaturated fatty acids (CFA/UFA) appeared to be the most relevant parameter associated with membrane rigidification and cell resistance to freeze-drying and storage. This study increased our knowledge about the physiological mechanisms that explain the resistance of lactic acid bacteria (LAB) to freeze-drying and storage stresses and demonstrated the relevance of complementary methods of membrane characterization.

A GC/MS method previously described for diacetyl was developed for the quantification of 2,3-pentanedione, and the derivatization procedure was modified for the determination of α-acetohydroxy acid. The reaction of 2,3-pentanedione with 4,5-dichloro-1,2-diaminobenzene produced 6,7-dichloro-2-methyl-3-ethylquinoxaline (DCMEQ), which was extracted with toluene and determined by gas chromatography using a mass selective detector. The formation of DCMEQ was linearly correlated with the 2,3-pentanedione concentration. The method was very simple and sensitive. The detection limit of the 2,3-pentanedione derivative and diacetyl derivative was 0.0007 mg/litre and 0.0002 mg/litre of the toluene extract respectively, and the determination limit was 0.001 mg/litre and 0.0007 mg/litre, respectively. Cautious sample treatment led to a low (10%) and controlled conversion of α-acetohydroxy acids to vicinal diketones. This reproducible percentage of conversion made it possible to determine precisely free vicinal diketones and α-acetohydroxy acids. The method was applied to the determination of precursors and vicinal diketones concentrations during beer fermentation.

An integrated approach combining sensory analysis and physicochemistry was used to investigate the impact of swallowing on aroma release and perception. A panel of 10 people evaluated the dynamics of aroma perception during the consumption of a commercial flavored vodka, using two protocols (spitting out or swallowing the product) and the temporal dominance of sensations method. Aroma release in their nasal cavity was simultaneously measured by proton transfer reaction mass spectrometry. The swallowing of the product resulted in more complex perceptions, but decreased the dominance rates of aromatic attributes. Ethanol perception also had a high impact when the product was swallowed.

The mechanisms of cellular damage that lactic acid bacteria incur during freeze-thaw processes have not been elucidated to date. Fourier transform infrared spectroscopy was used to investigate in situ the lipid phase transition behavior of the membrane of [i]Lactobacillus delbrueckii[/i] ssp. [i]bulgaricus[/i] CFL1 cells during the freeze-thaw process. Our objective was to relate the lipid membrane behavior to membrane integrity losses during freezing and to cell-freezing resistance. Cells were produced by using 2 different culture media: de Man, Rogosa, and Sharpe (MRS) broth (complex medium) or mild whey-based medium (minimal medium commonly used in the dairy industry), to obtain different membrane lipid compositions corresponding to different recovery rates of cell viability and functionality after freezing. The lipid membrane behavior studied by Fourier transform infrared spectroscopy was found to be different according to the cell lipid composition and cryotolerance. Freeze-resistant cells, exhibiting a higher content of unsaturated and cyclic fatty acids, presented a lower lipid phase transition temperature (Ts) during freezing (Ts = -8 degrees C), occurring within the same temperature range as the ice nucleation, than freeze-sensitive cells (Ts = +22 degrees C). A subzero value of lipid phase transition allowed the maintenance of the cell membrane in a relatively fluid state during freezing, thus facilitating water flux from the cell and the concomitant volume reduction following ice formation in the extracellular medium. In addition, the lipid phase transition of freeze-resistant cells occurred within a short temperature range, which could be ascribed to a reduced number of fatty acids, representing more than 80% of the total. This short lipid phase transition could be associated with a limited phenomenon of lateral phase separation and membrane permeabilization. This work highlights that membrane phase transitions occurring during freeze-thawing play a fundamental role in the cryotolerance of [i]Lb. delbrueckii[/i] ssp. [i]bulgaricus[/i] CFL1 cells.

Towards a better understanding of the freezing resistance of lactic acid bacteria. International Conference on Membrane Domains

International audience; Mid-infrared spectroscopy was applied to the analysis and discrimination of Cognacs and other distilled drinks (Armagnacs, whiskies, brandies, bourbons, rums, and counterfeit products). Strong correlations were found between dry extract spectra, polyphenolic dry extract spectra, and the total polyphenol concentration of samples, notably of Cognacs. Principal component analysis applied to spectral data made it possible to emphasize the importance of dry extract data when a distinction is made between Cognacs and Armagnacs, whiskies, bourbons, and rums, and of polyphenol concentration when Cognacs, brandies, and counterfeit products are separated. Ninety-six percent of samples in the test set were correctly assigned to Cognacs and non-Cognacs by partial least-squares discriminant analysis.

article scientifique The following values have no corresponding Zotero field: number: 6; International audience; The characterisation of the physical state of frozen and freeze dried biological products delivers powerful information for freeze-drying process optimisation. The influence of lactic acid bacterial cell size, shape and concentration on collapse temperature of concentrated bacterial suspensions was investigated. Lactobacillus bulgaricus (long rods), and Streptococcus thermophilus (small spherical cells) were used as cellular models for this study. Whatever the strain, when lactic acid bacterial cells were added to protective solutions, the collapse temperature increased, thus allowing the use of higher sublimation temperatures during primary drying than expected from the protective medium alone. Moreover, the higher the cell concentration, the greater the effect, linear relationships existing between the collapse temperatures and the total dried matter. Cells of both strains gave a kind of robustness to the freeze-dried product, but the increase observed in collapse temperature was considerably higher (3-5degreesC) for L. bulgaricus compared to S. thermophilus. This result was ascribed to the different size and shape of the strains.

The cell membrane is the primary site for freezing injury. During cooling, ice forms outside the cell resulting in an increase of the solutes concentration in the extracellular medium. This cryo-concentration of solutes induces water transport from the intracellular medium of cell to the extracellular medium and thus cell dehydration and cell injury. Cell membrane permeability to water is influenced by its fluidity property which is governed by the composition and the structure of the lipid bilayer. By changing the composition of the medium, the composition and organisation of the lipid bilayer of the cell membrane will be modified resulting in increasing or decreasing freezing resistance. Furthermore, another important phenomenon occurs during cooling of cell suspensions leading to the modification of membrane fluidity: the lipid phase transition from a liquid crystalline phase to a gel phase. The liquid crystalline state is characterized by increased lipid head group spacing, increased disorder in the lipid acyl chains, and a small bilayer thickness. In the gel phase the lipid head groups are very tightly packed, the lipid acyl chains become straighter and ordered, and the bilayer thickness increases. Our objective is to gain a better understanding of the relationships between membrane fluidity, lipid phase transition, membrane water permeability and freezing resistance of lactic acid bacteria. Two suspensions of Lactobacillus bulgaricus CFL1 were produced using either MRS broth or mild whey based culture media. The freezing resistance of both bacterial populations protected with sucrose was evaluated by measuring the cultivability and acidification activity of bacteria before and after freezing at −80 °C. The membrane lipid phase behaviour was studied in situ using Fourier transform infrared spectroscopy during freezing and thawing. Membrane fluidity was evaluated with fluorescence polarization using a lipophilic fluorescent probe embedded in the membrane, namely the DPH. The fluorescence measurements were performed on a flow cytometer at various temperatures. Bacterial cell dehydration was investigated by submitting cells to osmotic stress by increasing the concentration on a non penetrating solute (sucrose). The changes in cell volume with time were measured by Coulter techniques and the biophysical parameters of water transport were estimated. Bacterial population grown in MRS broth exhibited a better resistance to freezing than bacteria grown in mild whey. This better resistance can be ascribed to a lower lipid phase transition temperature and higher membrane fluidity. The detailed analysis of the results is in progress and will enable clarification of the relationships between freezing resistance, membrane fluidity and water permeability.

Influence of the membrane phase behaviour and lipid composition on the freezing-resistance of lactic acid bacteria. Faraday Discussion 161: Lipids and Membrane Biophysics

International audience; The characterisation of the physical state of frozen and freeze dried biological products delivers powerful information for freeze-drying process optimisation. The influence of lactic acid bacterial cell size, shape and concentration on collapse temperature of concentrated bacterial suspensions was investigated. Lactobacillus bulgaricus (long rods), and Streptococcus thermophilus (small spherical cells) were used as cellular models for this study. Whatever the strain, when lactic acid bacterial cells were added to protective solutions, the collapse temperature increased, thus allowing the use of higher sublimation temperatures during primary drying than expected from the protective medium alone. Moreover, the higher the cell concentration, the greater the effect, linear relationships existing between the collapse temperatures and the total dried matter. Cells of both strains gave a kind of robustness to the freeze-dried product, but the increase observed in collapse temperature was considerably higher (3 - 5 degree C) for L. bulgaricus compared to S. thermophilus. This result was ascribed to the different size and shape of the strains.

The European PREMIUM project aims at proposing new eco-friendly strategies to preserve lactic acid bacteria (LAB). One approach considered is the development of new protective agents in order to allow the application of more aggressive drying processes than freeze-drying. Recently, oligosaccharides such as fructo-oligosaccharides and galacto-oligosaccharides have been reported as promising molecules for protecting LAB. It is generally assumed in the literature that direct interaction between membrane and protective agent is important to stabilize membrane during water removal. The ability of oligosaccharides to interact with bacterial membrane will be dependent not only on their sugar composition but also on the lipid membrane composition. The objective of this study is to develop a simplified lipid model representative of bacterial membrane to investigate the specific interaction between oligosaccharides and lipids. The membrane of two strains of lactic acid bacteria exhibiting different freezing resistances and extremely low survival to freeze-drying were fully characterized to determine lipid composition and lipid phase transition. Lipid membrane models of various level of complexity were subsequently designed: i) liposomes and monolayers from bacterial lipid extracts; ii) liposomes from lipid extracts enriched with standard lipids, and ii) liposomes and monolayers made from two standard lipids. The standard lipids were chosen in accordance with the predominant lipids present within the bacterial membrane. The biophysical properties of these models were determined using Fourier Transform Infrared spectroscopy (lipid phase transition) and Langmuir isotherms (surface pressure area isotherms). Two binary lipid models satisfactorily representing the two bacterial strains according to their biophysical properties were set up and could thus be further used to investigate specific interaction between lipid and oligosaccharides.