F Fonseca

C Pénicaud

E E Tymczyszyn

A Gomez-Zavaglia

S Passot

Factors influencing the membrane fluidity and the impact on production of lactic acid bacteria starters

Keywords: Process conditions, membrane fluidity and bacterial resistance fluorescence anisotropy, lipid phase transition, preservation processes, environmental stress

Production of lactic acid bacteria starters for manufacturing food, probiotic and chemical products, requires the application of successive steps: fermentation, concentration, stabilisation and storage. Despite process optimization, losses of bacterial viability and functional activities are observed after stabilisation and storage steps due to cell exposure to environmental stresses (thermal, osmotic, mechanical, and oxidative). Bacterial membrane is the primary target for injury and its damage is highly dependent on its physical properties and lipid organization. Membrane fluidity is a key property for maintaining cell functionality, and depends on lipid composition and cell environment. Extensive evidence has been reported on changes in membrane fatty acyl chains when modifying fermentation conditions. However, a deep characterization of membrane physical properties and their evolution following production processes is scarcely reported.

Therefore, the aims of this mini-review are: i) to define the membrane fluidity and the methods used to assess it; and ii) to summarize the effect of environmental conditions on membrane fluidity and the resulting impact on the resistance of lactic acid bacteria to the stabilisation processes. This will make it possible to highlight existing gaps of knowledge and opens up novel approaches for future investigations.

Introduction

Lactic acid bacteria (LAB) are of great importance for the food industry because of their role in the manufacture of fermented meat, vegetables, fruit and dairy products. The market of concentrated LAB cultures (starters) is continuously growing due to the development of health benefit products and green chemistry applications.

However, the most promising bacteria will have no commercial value if the long term stability of the target functional properties (acidifying activity, production of aroma compounds and texturizing agents, probiotic activity,...) is not ensured up to their final use (fermentation of food, direct ingestion of probiotics). The commercialization of LAB requires the application of successive processes including fermentation, concentration, stabilisation and storage, for delivering LAB under the form of ready-to-use, highly concentrated and stable starters to food companies or to consumers. Stabilisation strategies are based on the decrease of water activity to inhibit or strongly slow down degradation reactions. Freezing and freeze-drying are the stabilisation processes most commonly used since they allow to maximize the technological properties and shelf-life of LAB cells [START_REF] Fonseca | Freeze-Drying of Lactic Acid Bacteria[END_REF][START_REF] Béal | Freezing of Probiotic Bacteria[END_REF].

During the stabilisation process, bacteria are exposed to several environmental changes, such as change in temperature, solute concentration, and hydration level [START_REF] Fonseca | Stabilization of Frozen Lactobacillus delbrueckii subsp. bulgaricus in Glycerol Suspensions: Freezing Kinetics and Storage Temperature Effects[END_REF][START_REF] Santivarangkna | Inactivation mechanisms of lactic acid starter cultures preserved by drying processes[END_REF][START_REF] Fonseca | Freeze-Drying of Lactic Acid Bacteria[END_REF]. Cells will thus exhibit passive responses to these environmental changes and membrane fluidity will play a key role in the cellular response [START_REF] Beney | Influence of the fluidity of the membrane on the response of microorganisms to environmental stresses[END_REF][START_REF] Santivarangkna | Inactivation mechanisms of lactic acid starter cultures preserved by drying processes[END_REF]. Membrane of LAB are mainly composed of phospholipids forming a lamellar lipid bilayer with embedded proteins. Membrane fluidity characterizes the dynamics of lipids within the bilayer, and is thus the inverse of membrane viscosity [START_REF] Denich | Effect of selected environmental and physico-chemical factors on bacterial cytoplasmic membranes[END_REF]). To maintain cell integrity while ensuring the main functions of cell membrane (regulating the transport and diffusion of biological substances), an optimal value of cell membrane viscosity of 0.1 Pa.s (100 times higher than water viscosity) is reported in literature. This value corresponds to a 90% glycerol solution [START_REF] Schechter | Biochimie et biophysique des membranes -Aspects structuraux et fonctionnels. Paris Schneiter R, Toulmay A[END_REF]). Membrane fluidity is thus dependent on temperature, decreasing temperature results in increasing viscosity and in a more rigid membrane. During freezing, bacterial cells are exposed to cold and osmotic stresses, resulting in membrane stiffening, and cell dehydration and volume reduction, due to the cryoconcentration of the extracellular medium [START_REF] Dumont | Cell Size and Water Permeability as Determining Factors for Cell Viability after Freezing at Different Cooling Rates[END_REF][START_REF] Gautier | A low membrane lipid phase transition temperature is associated with a high cryotolerance of Lactobacillus delbrueckii lssp. <i>bulgaricus<i> CFL1[END_REF][START_REF] Fonseca | Determination of Intracellular Vitrification Temperatures for Unicellular Micro Organisms under Conditions Relevant for Cryopreservation[END_REF]). These events can induce membrane damage, such as membrane leakage or loss of integrity, whose degree will be dependent on membrane fluidity. The fermentation conditions and the resulting membrane lipid composition are commonly related to the resistance of LAB to stabilisation processes. Even, it is well admitted that membrane fluidity is governed by the fatty acid composition of the membrane, few works report direct assessment of membrane fluidity after fermentation [START_REF] Velly | Cyclopropanation of unsaturated fatty acids and membrane rigidification improve the freeze-drying resistance of Lactococcus lactis subsp. lactis TOMSC161[END_REF][START_REF] Bouix | Assessment of bacterial membrane fluidity by flow cytometry[END_REF] and following stabilisation process [START_REF] Schwab | Influence of oligosaccharides on the viability and membrane properties of Lactobacillus reuteri TMW1.106 during freeze-drying[END_REF][START_REF] Passot | Synchrotron UV fluorescence microscopy for determining membrane fluidity modification of single bacteria with temperatures[END_REF]Meneghel et al. 2017b).

This review is structured in four sections aiming at: i) summarizing the lipid membrane composition of LAB and its contribution to membrane fluidity; ii) reviewing the progress on methods for characterizing membrane fluidity;

iii) overviewing the influence of environmental conditions during production and stabilisation processes on membrane fluidity and the consequences on LAB resistance; iv) sharing future prospects for LAB research.

Contribution of lipid composition to LAB's membrane organization and fluidity

The principal type of lipids involved in LAB membrane are polar phospholipids, although other polar glycolipids and neutral lipids can contribute to membrane organization [START_REF] Drucker | Phospholipids of Lactobacillus spp[END_REF][START_REF] Gómez-Zavaglia | Fatty acid composition and freeze-thaw resistance in lactobacilli[END_REF].

Phospholipids (PLs) contain both a hydrophilic region including a phosphate group, and a hydrophobic region including two acyl chains. The acyl chains of fatty acids are mainly composed of an even number of carbons, from 12 to 22, involving no, one or two unsaturations (C12:0, C14:0, C16:0, C16:1, C18:0, C18:1, C18:2, C20:0, C22:0). Fatty acids (FA) of 16 and 18 carbons account for more than 60% of total FA of LAB membranes [START_REF] Johnsson | Cellular fatty acid profiles of Lactobacillus and Lactococcus strains in relation to the oleic acid content of the cultivation medium[END_REF][START_REF] Gómez-Zavaglia | Fatty acid composition and freeze-thaw resistance in lactobacilli[END_REF][START_REF] Wang | Influence of cooling temperature and duration on cold adaptation of Lactobacillus acidophilus RD758[END_REF]Li et al. 2009a;[START_REF] Broadbent | Physiological and Transcriptional Response of Lactobacillus casei ATCC 334 to Acid Stress[END_REF][START_REF] Gautier | A low membrane lipid phase transition temperature is associated with a high cryotolerance of Lactobacillus delbrueckii lssp. <i>bulgaricus<i> CFL1[END_REF][START_REF] Velly | Cyclopropanation of unsaturated fatty acids and membrane rigidification improve the freeze-drying resistance of Lactococcus lactis subsp. lactis TOMSC161[END_REF]. Furthermore, cyclic fatty acids (CFA, such as cycC19:0) are widely found in LAB and are formed by the addition of a methylene group to the carbon-carbon double bond of unsaturated fatty acids (UFA, mainly C18:1).

Under physiological conditions, membrane lipid bilayers are in a lamellar liquid crystalline state (L), as illustrated in Fig. 1a. In this state, lipids have considerable motional freedom (rotation, rocking and lateral diffusion), that allows the embedded proteins to work properly and ensure the main functions of bacterial membrane (fluid-mosaic model). Upon decreasing temperature (or removing water), the fatty acid chains and head-groups (or the PLs) start packing into a crystal-like structure, forming a more stable and ordered gel phase (L), where lipid movements are highly reduced.

The fatty acyl-chains structure and geometry govern the lipid's shape, the degree of lipid packing within the bilayer (Fig. 1b). Any conformation of the acyl-chain that will make it more difficult to pack densely and regularly the chains will contribute to increase membrane fluidity. For instance, compared to straight chain of saturated fatty acid, the presence of unsaturation in cis conformation within the chain will clearly limit the chains packing [START_REF] Loffhagen | Suitability of the trans/cis ratio of unsaturated fatty acids in Pseudomonas putida NCTC 10936 as an indicator of the acute toxicity of chemicals[END_REF]. As a consequence, the unsaturated/saturated fatty acids' ratio (UFA/SFA) is widely related to bacterial membrane fluidity [START_REF] Denich | Effect of selected environmental and physico-chemical factors on bacterial cytoplasmic membranes[END_REF]. Furthermore, short length chains and ante-isobranched chains of fatty acids have also been reported to increase membrane fluidity [START_REF] Kaneda | Iso-and anteiso-fatty acids in bacteria: biosynthesis, function, and taxonomic significance[END_REF][START_REF] Denich | Effect of selected environmental and physico-chemical factors on bacterial cytoplasmic membranes[END_REF][START_REF] Wang | Starvation induces physiological changes that act on the cryotolerance of Lactobacillus acidophilus RD758[END_REF].

The presence of cyclic fatty acids (CFA) also modulates membrane fluidity but their effect remains unclear and contradictory. For some authors, CFAs should have the same physical properties as UFA and would contribute to increase membrane fluidity [START_REF] Machado | Osmotic response in Lactobacillus casei ATCC 393: biochemical and biophysical characteristics of membrane[END_REF][START_REF] Zhang | Membrane lipid homeostasis in bacteria[END_REF]. Other researchers reported a decrease of membrane fluidity with increasing content of CFA (Li et al. 2009a;[START_REF] Velly | Cyclopropanation of unsaturated fatty acids and membrane rigidification improve the freeze-drying resistance of Lactococcus lactis subsp. lactis TOMSC161[END_REF], or no modification of membrane fluidity [START_REF] To | Cyyclopropanation of Membrane Unsaturated Fatty Acids Is Not Essential to the Acid Stress Response of Lactococcus lactis subsp. cremoris[END_REF].

The fatty acid composition of LAB membrane determines the temperature of phase transition from the liquid crystalline (L) to the gel (L) phases. The longer and more saturated the chains of FA are, the higher the transition temperature is. For instance, for a diacylphosphatidylethanolamine bilayer, the temperature of lipid phase transition (Tm) change from 30 °C to 90 °C when increasing the number of carbons of the acyl chains from 12 to 22 [START_REF] Koynova | Recent Patents on Nonlamellar Liquid Crystalline Lipid Phases in Drug Delivery[END_REF]. When considering lipids with unsaturated chains, the position and type of the double bond substantially modulate the membrane lipid phase transition. For a dioctadecenoyl phosphatidylcholine bilayer, the Tm vary from 20 °C, -20 °C and 0 °C when the position of the double bond changes from 4, 9 or 13 position in carbon chains [START_REF] Koynova | Recent Patents on Nonlamellar Liquid Crystalline Lipid Phases in Drug Delivery[END_REF].

Although it is generally accepted that modification of FA composition is an effective way for modulating membrane fluidity and phase transition [START_REF] Denich | Effect of selected environmental and physico-chemical factors on bacterial cytoplasmic membranes[END_REF], little is known about the effect of polar head-group composition on membrane properties. Phospholipids heads are composed of phosphate groups covalently bound to glycerol, leading to phosphatidylglycerol (PG), or diphosphatydilglycerol (DPG) also called cardiolipin (CL), [START_REF] Gómez-Zavaglia | Fatty acid composition and freeze-thaw resistance in lactobacilli[END_REF][START_REF] Machado | Osmotic response in Lactobacillus casei ATCC 393: biochemical and biophysical characteristics of membrane[END_REF][START_REF] Tymczyszyn | Volume recovery, surface properties and membrane integrity of Lactobacillus delbrueckii subsp. bulgaricus dehydrated in the presence of trehalose or sucrose: Volume recovery, surface properties and membrane integrity of dehydrated L. bulgaricus[END_REF], or lysylphosphatidylglycerol (LPG) [START_REF] Russell | Membranes as a target for stress adaptation[END_REF][START_REF] Machado | Osmotic response in Lactobacillus casei ATCC 393: biochemical and biophysical characteristics of membrane[END_REF]), all anionic phospholipids. LAB phospholipid head can also be bounded to ethanolamine [START_REF] Teixeira | Lactobacillic Acid Accumulation in the Plasma Membrane of Oenococcus oeni: A Response to Ethanol Stress?[END_REF], thus leading to phosphatidylethanolamine (PE), a zwitterionic PL. Some phospholipids located at the outer surfaces of the LAB membranes are associated with a sugar group (mono and oligosaccharides) attached by a glycosidic bond to the polar head-group (glycolipids). Each type of polar head is associated to a given number of water molecules, tightly bound through hydrogen bonds [START_REF] Luzardo | Effect of Trehalose and Sucrose on the Hydration and Dipole Potential of Lipid Bilayers[END_REF], determining the radius of the polar head and the geometry of the lipid. PLs thus present different sizes and shapes that will affect the extent of interfacial area between head-groups as well as the PLs distribution and packing in the membrane, eventually modulating membrane fluidity.

Membrane fluidity appears thus a complex property that depends on several factors, membrane FA composition, organization and temperature. A reliable measurement of this property is thus mandatory in order to well understand the relationship between membrane fluidity and resistance of LAB to production processes.

Methods for evaluating membrane fluidity

Three main ways for assessing membrane fluidity of LAB are commonly reported:

-the measurement of the UFA/SFA ratio by identifying and quantifying the fatty acid methyl esters (FAME) by gaseous chromatography coupled to mass spectrometry (GC-MS); -the direct measurement of membrane fluidity by quantifying the fluorescence anisotropy of a probe inserted in the lipid bilayer; -the characterization of membrane lipid phase transition from the liquid crystalline to the gel phases (L<=> L) by FTIR (Fourier Transform InfraRed) spectroscopy, fluorescence spectroscopy using Laurdan probe or differential scanning calorimetry (DSC).

Assessment of membrane fluidity and lipid phase transition can also be measured by nuclear magnetic resonance, electron spin resonance and X-ray diffraction [START_REF] Denich | Effect of selected environmental and physico-chemical factors on bacterial cytoplasmic membranes[END_REF][START_REF] Silveira | Membrane Fluidity Adjustments in Ethanol-Stressed Oenococcus oeni Cells[END_REF][START_REF] Mykytczuk | Fluorescence polarization in studies of bacterial cytoplasmic membrane fluidity under environmental stress[END_REF].

Table 1 summarizes the works characterizing the membrane physical properties of LAB. Some details concerning the sample preparation, the conditions of measurement are also reported.

Fluorescence anisotropy

This the most common method applied to measure the relative changes in fluidity of bacterial membranes under environmental conditions. Fluorescent lipid soluble membrane probes are used as biomarkers of membrane lipids structure and motion. The degree of polarization of the fluorescent probe is generally characterized by the anisotropy (r), which decreases when cell membrane fluidity increases. Most reported works concern steady state anisotropy measurements by using spectrofluorometer with probes exhibiting fluorescence lifetimes (of 10 -8 and 10 -9 s) corresponding to the rate of lipid movement. In the last 15 years, this technique has been increasingly employed to study intact membranes of whole LAB to better understand the role of membrane fluidity on the physiological responses to different environmental conditions (Table 1). The most commonly used probe is 1,6diphenyl-1,3,5-hexatriene (DPH), an extremely hydrophobic and symmetrical probe that penetrates into the hydrophobic core orientating itself parallel to the fatty acid side chains. Another DPH-analogue (1-[4 (trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH)) has also been used on LAB. TMA-DPH anchors at the aqueous membrane interface because the side chains contribute to amphipathic behaviour [START_REF] Trevors | Fluorescent probes for bacterial cytoplasmic membrane research[END_REF].

Recently, fluorescence anisotropy measurements were performed on LAB using flow cytometry. A three costaining method, involving DPH, propidium iodide (PI) and carboxyfluorescein diacetate (cFDA) was developed to assess membrane fluidity of viable, injured and dead cells of Lb. bulgaricus and S. thermophilus [START_REF] Bouix | Assessment of bacterial membrane fluidity by flow cytometry[END_REF].

By replacing fluorescence spectroscopy with fluorescence microscopy to measure the emitted fluorescence of the probe, it is possible to obtain subcellular mapping of membrane fluidity and to detect any membrane heterogeneity. For instance, [START_REF] Passot | Synchrotron UV fluorescence microscopy for determining membrane fluidity modification of single bacteria with temperatures[END_REF], evidenced the formation of rigid domains within the membrane of Lb. bulgaricus cells when submitting to cold stress. Meneghel et al. (2017aMeneghel et al. ( , 2017b) ) investigated the evolution of membrane fluidity of two strains of Lb. bulgaricus (ATCC11842, resistant / CFL1, sensitive) submitted to cold and osmotic stresses. The measurements of membrane fluidity were carried out at population (Fig. 2a) and subcellular scales (Fig. 2b). Similar values of fluorescence anisotropy were observed at the population level, regardless the stress conditions applied, whereas significant differences were observed at the cellular level when quantifying the number of rigid domains within the bacterial membrane.

The main advantage of fluorescence anisotropy measurements is that it directly measures membrane fluidity through the mobility of a fluorescent probe within the membrane, while requiring low volumes of dyes. Adequate co-staining, makes it possible the membrane fluidity quantification of subpopulations (viable, injured and dead cells) or subcellular mapping if coupled to flow cytometry or fluorescence microscopy, respectively. The main disadvantage is that cells must be in suspension and it is not possible to study the membrane fluidity in dried matrices. Moreover, as each fluorescent probe has a well-defined target (i.e., interface -carbonyls-, polar headsphosphates-, hydrophobic region -acyl chains-), to have a full landscape of lipid membranes different probes have to be used.

Membrane lipid phase transition (L L)

FTIR spectroscopy is a non-invasive technique for studying in situ the membrane lipid phase transition of LAB, the change from liquid crystalline to gel phases when decreasing temperature (Table 1). It is a particularly useful technique because of its large flexibility, making it possible the study of both liquid and dried samples with no need of reagents. Despite FTIR spectroscopy does not provide direct measurement of membrane fluidity, the spectra can provide complete information about the different membrane regions, namely the interface, the polar heads, and the hydrophobic region, which is of great advantage over other techniques used to this aim (i.e., fluorescence anisotropy, DSC, X-rays diffraction, electron spin resonance spectroscopy -ESR-). Furthermore, FTIR spectroscopy can be used to characterise the membrane lipid behaviour during stabilization processes (i.e., freezing, drying).

Membrane phase behaviour is commonly monitored by observing the evolution of the position of the symmetric CH 2 stretching band at approximately 2850 cm -1 (vCH 2 symmetric) with cooling and subsequent heating (Fig. 3) [START_REF] Crowe | Effects of Water on the Stability of Phospholipid Bilayers: The Problem of Imbibition Damage in Dry Organisms[END_REF]. Figure 3 shows the membrane phase behaviour for two population of Lb. bulgaricus CFL1 exhibiting different cryotolerance (black diamond symbols: bacteria resistant to freezing when cultured in MRS medium/ grey triangle symbols: bacteria sensitive to freezing when cultured in whey medium). A shift in vCH 2 symmetric from 2854 cm -1 to lower wavenumbers (2851 cm -1 for the resistant condition and 2850 cm -1 for the sensitive one) was observed with decreasing temperature. Resistant bacteria were characterized by a lower lipid phase transition (-8 °C) than sensitive bacteria (22 °C) during cooling. The evolution of fluorescence anisotropy with temperature was also reported in Fig. 3 for both culture conditions (black circles for the freeze-resistant bacteria and grey circles for the freeze-sensitive bacteria). When considering the sensitive condition, the shift of vCH 2 symmetric to lower wavenumbers, is associated with an increase of fluorescence anisotropy (i.e. a decrease in membrane fluidity).

A native membrane has different types of lipids (acyl chains and head-groups) with different melting temperatures and capacities to bind water. As a result, during freezing or drying, the various phospholipids enter their respective gel phases at different temperatures, and the gel and liquid-crystal phases transiently coexist. The gel-phase domains would exclude more fluid domains, and in such two-phase systems, membranes are expected to leak during thawing or rehydration with potentially negative consequences for cells survival [START_REF] Crowe | Effects of Water on the Stability of Phospholipid Bilayers: The Problem of Imbibition Damage in Dry Organisms[END_REF]. Consequently, in addition to the determination of the lipid phase transition temperatures (Ts following cooling and Tm following heating), other useful parameters can be obtained from the membrane lipid transition curves: i) the broadness of the transition indicates lipid heterogeneity [START_REF] Oldenhof | Effect of Sucrose and Maltodextrin on the Physical Properties and Survival of Air-Dried Lactobacillus bulgaricus: An in Situ Fourier Transform Infrared Spectroscopy Study[END_REF][START_REF] Gautier | A low membrane lipid phase transition temperature is associated with a high cryotolerance of Lactobacillus delbrueckii lssp. <i>bulgaricus<i> CFL1[END_REF] and possible phase separation due to the coexistence of rigid an fluid domains [START_REF] Hazel | The role of alterations in membrane lipid composition in enabling physiological adaptation of organisms to their physical environnement[END_REF][START_REF] Hazel | Thermal Adaptation in Biological Membranes: Is Homeoviscous Adaptation the Explanation?[END_REF]; ii) the wavenumber increase at high and/or low temperatures denotes high disorder and fluidity [START_REF] Gautier | A low membrane lipid phase transition temperature is associated with a high cryotolerance of Lactobacillus delbrueckii lssp. <i>bulgaricus<i> CFL1[END_REF]; and iii) hysteresis between cooling and heating, has been ascribed to irreversible phenomena occurring during freezing, probably lateral phase separation [START_REF] Gautier | A low membrane lipid phase transition temperature is associated with a high cryotolerance of Lactobacillus delbrueckii lssp. <i>bulgaricus<i> CFL1[END_REF].

Besides, the phosphate symmetric stretching vibration band (vPO 2 -asym, around 1220 cm -1 ) has been employed

for studying the interaction between phospholipids head-groups and the extracellular environment of LAB cells (Meneghel et al. 2017b).

The liquid crystalline to gel phase transition in LAB membrane was also detected (Table 1) by using Laurdan (an amphiphilic fluorescence probe) fluorescence spectroscopy [START_REF] Harris | Use of laurdan fluorescence intensity and polarization to distinguish between changes in membrane fluidity and phospholipid order[END_REF]. [START_REF] Molina-Höppner | Protective Effect of Sucrose and Sodium Chloride for Lactococcus lactis during Sublethal and Lethal High-Pressure Treatments[END_REF] thus investigated the effect of milk buffer with 0.5 M sucrose and milk buffer with 4 M sodium chloride on the membrane phase behaviour of Lc. lactis by applying FTIR spectroscopy and Laurdan fluorescence spectroscopy.

Although only FTIR lipid phase transition is presented, the authors declared a similar temperature-dependent phase behaviour with Laurdan approach.

Membrane-fluidity related responses to environmental conditions occurring during the production process of LAB concentrates and their impact on bacterial resistance

Changes of environmental conditions that take place during the production process of LAB starters, generate stresses that induce, in turn, different LAB responses. Stress are mainly caused by modifications in temperature, pH, medium composition, solute concentration, water activity, atmosphere composition, etc. According to the process step (fermentation, cooling, concentration, stabilisation, and storage), the kinetics of the stressful event (sudden or gradual), its intensity and duration can vary. Consequently, different cellular responses can be promoted:

• The active responses that generally help bacteria to withstand the stress taking place during first steps of production (fermentation and cooling). They lead to modifications of membrane lipid composition, changes in protein contents and regulation of some genes' expression. They could also result in cell adaptation to the stresses taking place during the stabilisation processes.

• The passive responses that are related to cell's physico-chemical modifications taking place during downstream processes of stabilisation (membrane phase change, lipids oxidation, protein denaturation, modification of ionic force and viscosity). They are influenced by the cellular active responses and lead to various degrees of deterioration of cell biological properties and functionalities.

Table 2 summarises the studies reported on the impact of environmental factors on LAB resistance to the stabilisation processes (freezing, freeze-drying, and drying), that have reported either modification of lipid composition and/or quantification of membrane physical properties (lipid phase transition or direct assessment of membrane fluidity by fluorescence anisotropy).

Changes in fatty acyl chain composition are more largely investigated and quantified than changes in phospholipid classes, e.g. head-group composition. Moreover, research work on changes of membrane physical properties due to process conditions are also scarcely reported. To our knowledge, only two works have investigated simultaneously fatty acid composition, membrane phase transition and fluidity in relation with the LAB resistance to process [START_REF] Velly | Cyclopropanation of unsaturated fatty acids and membrane rigidification improve the freeze-drying resistance of Lactococcus lactis subsp. lactis TOMSC161[END_REF]Meneghel et al. 2017b).

Fermentation (LAB active responses)

Temperature

Applying temperature lower than the optimal one for cell's growth, improved the resistance of LAB to freezing [START_REF] Fernández-Murga | Influence of growth temperature on cryotolerance and lipid composition of Lactobacillus acidophilus[END_REF], to frozen storage [START_REF] Wang | Influence of cooling temperature and duration on cold adaptation of Lactobacillus acidophilus RD758[END_REF] and to freeze-drying (Li et al. 2009a). [START_REF] Velly | Cyclopropanation of unsaturated fatty acids and membrane rigidification improve the freeze-drying resistance of Lactococcus lactis subsp. lactis TOMSC161[END_REF] reported no improvement of freeze-drying resistance of Lc. lactis. Cold adaptation resulted in a modification of fatty acid membrane with an increase of the UFA/SFA ratio, which could be related to an increase of membrane fluidity. Only [START_REF] Velly | Cyclopropanation of unsaturated fatty acids and membrane rigidification improve the freeze-drying resistance of Lactococcus lactis subsp. lactis TOMSC161[END_REF] characterized the physical properties of Lc. lactis and reported an increase of membrane fluidity and a decrease of the lipid phase transition temperature when cells were grown at low temperature. When cells are grown at non-optimal temperatures, they adapt their membrane fatty acid composition in order to maintain membrane fluidity (i.e. liquid crystalline phase) [START_REF] Hazel | The role of alterations in membrane lipid composition in enabling physiological adaptation of organisms to their physical environnement[END_REF][START_REF] Hazel | Thermal Adaptation in Biological Membranes: Is Homeoviscous Adaptation the Explanation?[END_REF].

Modulation of cyclic fatty acid (cycC19:0) membrane content following cold treatment remains unclear and varies according to the LAB strains. When decreasing culture temperature, the cycC19:0 content decreased for Lb. acidophilus CRL 640 [START_REF] Fernández-Murga | Influence of growth temperature on cryotolerance and lipid composition of Lactobacillus acidophilus[END_REF], Lactobacillus delbrueckii ssp. lactis [START_REF] Veerkamp | Fatty Acid Composition of Bifidobacterium and Lactobacillus Strains[END_REF] and Lactobacillus plantarum [START_REF] Russell | Membranes as a target for stress adaptation[END_REF] or increased for Lb. acidophilus RD758 [START_REF] Wang | Influence of cooling temperature and duration on cold adaptation of Lactobacillus acidophilus RD758[END_REF] and Lc. Lactis ssp. cremoris [START_REF] Guillot | Fatty acid membrane composition and activation of glycine-betaine transport in Lactococcus lactis subjected to osmotic stress[END_REF], whereas a bell shape dependence with temperature was observed for Lb. fermentum [START_REF] Suutari | Temperature adaptation in Lactobacillus fermentum: interconversions of oleic, vaccenic and dihydrosterulic acids[END_REF].

Furthermore, when increasing the growth temperature (from 42 °C to 60 °C), a decrease of phospholipids content compared to proteins was reported for O. oeni [START_REF] Garbay | Response of Leuconostoc oenos to environmental changes[END_REF].

The application of a moderate cold stress after cells growth at optimal temperature could afford resistance to upcoming stabilisation process. The production of cold shock proteins and the modulation of membrane FA composition are proposed as the main adaptive responses to suboptimal temperatures [START_REF] Papadimitriou | Stress Physiology of Lactic Acid Bacteria[END_REF]. [START_REF] Wang | Influence of cooling temperature and duration on cold adaptation of Lactobacillus acidophilus RD758[END_REF] and [START_REF] Schoug | Impact of fermentation pH and temperature on freeze-drying survival and membrane lipid composition of Lactobacillus coryniformis Si3[END_REF] reported increase in the UFA/SFA ratio, similarly to the low temperature effect observed during fermentation, but leading to opposite effects on the resistance to stabilisation process. The better resistance to frozen storage of "cold adapted" Lb. acidophilus cells was related to the increase in cycC19:0 membrane fatty acid content [START_REF] Wang | Influence of cooling temperature and duration on cold adaptation of Lactobacillus acidophilus RD758[END_REF]. In turn, the cold step induced a lower resistance of Lb. coryniformis to freeze-drying accompanied of a decrease in the content of cycC19:0 when compared to optimal growth conditions [START_REF] Schoug | Impact of fermentation pH and temperature on freeze-drying survival and membrane lipid composition of Lactobacillus coryniformis Si3[END_REF].

pH

Exposing LAB to pH values lower than the optimal one during growth resulted in improvement of cell survival either after the stabilisation processes (freezing or freeze-drying) or the application of acidic treatment. Only [START_REF] Gilliland | Relationship of cellular components to the stability of concentrated lactic streptococcus cultures at -17°C[END_REF] and Li et al. (2009a) observed a decrease of survival of Lc lactis and Lb. bulgaricus with decreasing pH when bacteria were grown at uncontrolled pH and 39 °C, respectively. However, opposite results were obtained when growth of Lb. bulgaricus was carried out at 30 °C, suggesting that these two factors, pH and temperature, have important and combined impact on LAB survival after stabilisation processes. The effect of low pH on the modulation of membrane fatty acids composition appeared to be dependent on the LAB considered: 60 % of the works reported a decrease of the UFA/SFA ratio, whereas 66 % of the studies observed an increase of the CFA content. The scarce works performing direct measurements of membrane fluidity evidenced a membrane stiffness when bacterial growth was carried out at low pH.

This might be ascribed either to a decrease of UFA/SFA combined with an increase of CFA, or to the increase of CFA and long chain FA (higher mean acyl chain length, MCL) content. The increase in the proportion of long length chains FA, is often associated to an increase in UFA/SFA, thus probably counterbalancing the effect of FA unsaturation on membrane fluidity.

Growth phase (or age of the culture)

During growth, LAB encounter various environmental changes: decrease of pH (for uncontrolled pH culture condition), production of metabolites (lactate for controlled pH condition, reactive oxygen species) and eventual nutrient depletion.

An increasing content of CFA was systematically evidenced with increasing culture time associated with a decrease of the UFA/SFA ratio for 60 % of them. This modulation of membrane fatty acid composition during growth seems to result in a decrease of membrane fluidity (similar to the pH effect) and for most of the reported cases, in an improvement of LAB resistance to stabilisation processes. [START_REF] Velly | Cyclopropanation of unsaturated fatty acids and membrane rigidification improve the freeze-drying resistance of Lactococcus lactis subsp. lactis TOMSC161[END_REF] correlated the membrane stiffness to an increase if the CFA/UFA ratio by means of C18:1 cyclopropanation.

When examining the effect of pH and growth phase parameters, LAB growth at uncontrolled pH conditions up to low pH values or at controlled pH up to stationary phase induces similar effects on membrane FA composition (increase CFA, decrease UFA/SFA) and membrane stiffness when measured.

Cultures performed at controlled and low pH values (acid adaptation) and at standard pH but followed by acid shock/challenge, induce similar bacterial responses than growth under lactose depletion [START_REF] Wang | Starvation induces physiological changes that act on the cryotolerance of Lactobacillus acidophilus RD758[END_REF]):

increase CFA, UFA/SFA and MCL and decrease of membrane fluidity.

Composition of culture medium and growth in hyperosmotic condition

Modifying the composition of the culture medium is often an efficient way for modulating membrane fatty acid profile (Table 2). Culture medium promoting the synthesis of UFA (increase UFA/SFA ratio), CFA and long chain FAs made it possible to improve the LAB resistance to freezing and freeze-drying processes. These membrane composition modifications seem to be correlated with an increase of membrane fluidity, in particular at low temperatures [START_REF] Passot | Synchrotron UV fluorescence microscopy for determining membrane fluidity modification of single bacteria with temperatures[END_REF][START_REF] Gautier | A low membrane lipid phase transition temperature is associated with a high cryotolerance of Lactobacillus delbrueckii lssp. <i>bulgaricus<i> CFL1[END_REF]). An increase of membrane fluidity can be expected when increasing CFA content due to its decreasing effect on the lipid phase transition temperature [START_REF] Perly | Effects of the Replacement of a Double Bond by a Cyclopropane Ring in Phosphatidylethanolamines: A 2H NMR Study of Phase Transitions and Molecular Organizationt[END_REF] and from recent molecular dynamic simulations of model membranes containing CFA [START_REF] Poger | A Ring to Rule Them All: The Effect of Cyclopropane Fatty Acids on the Fluidity of Lipid Bilayers[END_REF]. However, the reported studies on LAB did not make it possible to clarify the effect of CFA on membrane fluidity. Addition of lipids with high content of UFA, such as oleic derivatives (Tween 80, sodium oleate) or using MRS (containing Tween 80) medium appears as efficient ways to increase membrane content of UFA and CFA. By varying pH and culture age in MRS medium, accumulation levels of CFA up to 33% and 42% were reported for Lb. fermentum and Lb. buchneri, respectively [START_REF] Nikkilä | Effect of pH on growth and fatty acid composition of Lactobacillus fermentum[END_REF].

Some authors have investigated the effect of the osmolarity of the culture medium (addition of salts and solute to decrease water activity) on the membrane properties. Unfortunately, no data on LAB survival after stabilisation processes were reported. Increasing osmolarity (decreasing water activity) resulted in modification not only of the FA composition but also on the lipids polar head-groups. A decrease of the UFA/SFA ratio and an increase of CFA was evidenced by several authors for Lb. pentosus [START_REF] Gilarová | Cellular fatty acids analysis in the identification of lactic acid bacteria[END_REF], Lc. lactis ssp. cremoris [START_REF] Guillot | Fatty acid membrane composition and activation of glycine-betaine transport in Lactococcus lactis subjected to osmotic stress[END_REF], Lb. helveticus [START_REF] Guerzoni | Alteration in cellular fatty acid composition as a response to salt, acid, oxidative and thermal stresses in Lactobacillus helveticus[END_REF] and Lb. casei [START_REF] Machado | Osmotic response in Lactobacillus casei ATCC 393: biochemical and biophysical characteristics of membrane[END_REF]). However, other LAB exhibited different behaviors: increase of UFA/SFA ratio and decrease of CFA in Lb. acidophilus [START_REF] Murga | Permeability and Stability Properties of Membranes Formed by Lipids Extracted from Lactobacillus acidophilus Grown at Different Temperatures[END_REF]; decrease of UFA/SFA ratio and CFA in Lb. bulgaricus [START_REF] Tymczyszyn | Influence of the growth at high osmolality on the lipid composition, water permeability and osmotic response of Lactobacillus bulgaricus[END_REF] and no modification Lb. plantarum [START_REF] Russell | Membranes as a target for stress adaptation[END_REF].

Furthermore, the effect of hyperosmotic condition on the composition of the membrane phospholipids headgroups remains difficult to generalize and appears to be dependent on the bacteria and on the solutes. Increasing proportions of anionic PLs (PG, CL and LPG) were reported in Lb. plantarum [START_REF] Russell | Membranes as a target for stress adaptation[END_REF] and Lb. casei [START_REF] Machado | Osmotic response in Lactobacillus casei ATCC 393: biochemical and biophysical characteristics of membrane[END_REF]) when subjected to KCl 0.8 M and NaCl 1M respectively. Osmotic stress adaptation conducts to a high overall negative charges of bacterial membrane lipids for acting as a binding site for cations [START_REF] Mykytczuk | Fluorescence polarization in studies of bacterial cytoplasmic membrane fluidity under environmental stress[END_REF]). The relative proportions of anionic phospholipids however varied according to microorganisms.

After fermentation / Stabilisation processes (LAB passive responses)

The main strategy to stabilise and increase the shelf-life of LAB starters is to reduce the availability of water by freezing or drying. Due to the heat sensitivity of LAB, freeze-drying is often the drying method of choice.

However, stabilisation processes generate some undesirable side effects that induce decreased cell activity and death. Freezing process induces mainly cold and osmotic stresses whereas mechanical, osmotic, oxidative and heat stresses characterize the drying processes. Two main kinds of cellular damage are reported: i) changes in the physical state of cytoplasmic membrane, resulting in loss of membrane integrity [START_REF] Linders | Effect of Added Carbohydrates on Membrane Phase Behavior and Survival of Dried Lactobacillus plantarum[END_REF][START_REF] Schwab | Influence of oligosaccharides on the viability and membrane properties of Lactobacillus reuteri TMW1.106 during freeze-drying[END_REF]) and ii) modifications in the secondary structure of proteins [START_REF] Carpenter | The mechanism of cryoprotection of proteins by solutes[END_REF][START_REF] Oldenhof | Effect of Sucrose and Maltodextrin on the Physical Properties and Survival of Air-Dried Lactobacillus bulgaricus: An in Situ Fourier Transform Infrared Spectroscopy Study[END_REF].

Modification of membrane properties following stabilisation processes

Few studies have investigated the modification of membrane physical properties following stabilisation During drying processes, removal of unfrozen water results in profound changes in the physical properties of biomolecules, particularly phospholipids and proteins [START_REF] Crowe | Effects of Water on the Stability of Phospholipid Bilayers: The Problem of Imbibition Damage in Dry Organisms[END_REF]. A decrease of the lateral spacing of the polar head-groups and the subsequent packing of the hydrocarbons chains leads to a considerable increase of the membrane lipid phase transition after drying [START_REF] Potts | Desiccation Tolerance of Prokaryotes[END_REF]. In LAB, the membrane lipid phase transition (Tm)

of Lb. plantarum was reported to shift from 4 °C in hydrated cells to 20 °C in dried cells [START_REF] Linders | Effect of Added Carbohydrates on Membrane Phase Behavior and Survival of Dried Lactobacillus plantarum[END_REF] and from 35 °C to 40 °C in Lb. bulgaricus [START_REF] Oldenhof | Effect of Sucrose and Maltodextrin on the Physical Properties and Survival of Air-Dried Lactobacillus bulgaricus: An in Situ Fourier Transform Infrared Spectroscopy Study[END_REF].

No significant modification of fatty acid composition was observed upon freeze-drying of Lb. acidophilus while a decrease of CFA was observed following vacuum drying (Brennan et al. 1986). However, the fatty acid composition of freeze-dried and spray-dried Lb. bulgaricus was reported to evolve upon storage [START_REF] Castro | Storage of lyophilized cultures of Lactobacillus bulgaricus under different relative humidities and atmospheres[END_REF][START_REF] Teixeira | Evidence of membrane lipid oxidation of spray-dried Lactobacillus bulgaricus during storage[END_REF]. [START_REF] Teixeira | Evidence of membrane lipid oxidation of spray-dried Lactobacillus bulgaricus during storage[END_REF] showed that UFA/SFA ratio was stable within 49 days of storage and then decreased, while CFA content decreased from 32 days of storage after spray drying. Similarly, the low survival of Lb. plantarum and Leuconostoc mesenteroïdes to 90 days storage at 20 °C was associated to the decrease in C18:2/C16:0 and C18:3/C16:0 ratios [START_REF] Coulibaly | Survival of Freeze-dried Leuconostoc mesenteroides and Lactobacillus plantarum Related to Their Cellular Fatty Acids Composition during Storage[END_REF]). These decreases were ascribed to the oxidation of UFA and CFA that are sensitive to oxygen [START_REF] Castro | Changes in the cell membrane of Lactobacillus bulgaricus during storage following freeze-drying[END_REF]) and accentuated by an increase in the residual relative humidity that probably activates the oxidation processes [START_REF] Castro | Storage of lyophilized cultures of Lactobacillus bulgaricus under different relative humidities and atmospheres[END_REF]. Damage through reactive oxygen species is indeed recognized as one of the main stress that face micro-organisms during dehydration process [START_REF] Potts | Desiccation Tolerance of Prokaryotes[END_REF]. Oxidation of cell components upon drying and storage have been confirmed by the improvement of survival when adding antioxidants to starters before stabilisation [START_REF] Andersen | Storage stability of freeze-dried starter cultures (Streptococcus thermophilus) as related to physical state of freezing matrix[END_REF][START_REF] Kurtmann | Water activity-temperature state diagrams of freeze-dried Lactobacillus acidophilus (La-5): Influence of physical state on bacterial survival during storage[END_REF]) and by storage in nitrogen atmosphere [START_REF] Castro | Storage of lyophilized cultures of Lactobacillus bulgaricus under different relative humidities and atmospheres[END_REF][START_REF] Andersen | Storage stability of freeze-dried starter cultures (Streptococcus thermophilus) as related to physical state of freezing matrix[END_REF].

Furthermore, no direct measurements of membrane fluidity are reported upon drying and rehydration of LAB.

Interaction of protective molecules with membrane and effect on LAB resistance

The production and cellular accumulation of sugars (i.e. trehalose, sucrose, fructo-oligosaccharides (FOS)) is one of the most studied phenomena in organisms resistant to anhydrobiosis-involving processes [START_REF] García | Anhydrobiosis in bacteria: From physiology to applications[END_REF]. In order to mimic the processes naturally occurring in cells, protective molecules such as sugars, amino acids, polyols, polysaccharides, antioxidants, are currently added, after fermentation, to cell concentrates [START_REF] Santivarangkna | Inactivation mechanisms of lactic acid starter cultures preserved by drying processes[END_REF]. The protective mechanisms of these molecules, in particular their potential interaction with membranes are still controversial and have been studied mainly on model lipid systems (liposomes, monolayers) [START_REF] Crowe | Anhydrobiosis: An Unsolved Problem with Applications in Human Welfare[END_REF].

The interaction of the polar head-groups of cell membranes with water molecules present in the environment is crucial to maintain membrane in functional state. During dehydration, sugars like trehalose or sucrose are reported to directly interact with the polar head-groups by establishing hydrogen bonds, and replacing water molecules. The consequence is the decrease of the lipid membrane phase transition temperature after dehydration.

Membranes dehydrated in the presence of sugars remain in the liquid crystalline phase as if they were hydrated, thus preserving their biological function [START_REF] Crowe | Interactions of sugars with membranes[END_REF][START_REF] Milhaud | New insights into water-phospholipid model membrane interactions[END_REF]).

Works on LAB have not confirmed Tm depression of whole cells of model micro-organisms stabilised with sugars [START_REF] Leslie | Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying[END_REF]. No significant effect of maltose, trehalose, sorbitol was observed on Tm of Lb.

plantarum dried cells [START_REF] Linders | Effect of Added Carbohydrates on Membrane Phase Behavior and Survival of Dried Lactobacillus plantarum[END_REF]). These results were explained by an already low modification of Tm on drying without protectant and the authors ascribed the protective effect of carbohydrates to their free radical scavenging activity and not to the direct interaction with the polar lipid head-groups. Similarly, sucrose, matodextrin and skim milk had also minor effects on membrane phase behavior and the overall protein secondary structure of Lb. bulgaricus dried cells [START_REF] Oldenhof | Effect of Sucrose and Maltodextrin on the Physical Properties and Survival of Air-Dried Lactobacillus bulgaricus: An in Situ Fourier Transform Infrared Spectroscopy Study[END_REF]. Furthermore, an increased stability upon freezedrying of stationary phase cells of Lb. reuteri in the presence of FOS was ascribed to direct interaction of FOS with membranes [START_REF] Schwab | Influence of oligosaccharides on the viability and membrane properties of Lactobacillus reuteri TMW1.106 during freeze-drying[END_REF]), but no lipid phase transition was assessed. In the presence of FOS, the authors evidenced a decreased generalized polarization that they interpreted as an increased membrane fluidity of Lb. reuteri. However, Molina-Hoppner et al. ( 2004) reported a decrease of Tm of Lc. lactis suspended in milk buffer in presence of sucrose or NaCl, from 21.4 °C to 16.8 °C or 16.6 °C, respectively. In this study, accumulation of sugars within the intracellular medium was observed.

By describing the cooling process as a combination of cold and osmotic stresses, Meneghel et al. 2017a and2017b, proposed a complete characterization of the membrane physical behaviour following freezing in presence of sucrose, using FTIR spectroscopy and fluorescence of anisotropy at the subcellular level. The organization of membrane phospholipids head-groups, and its modification with osmotic stress (the most cell damaging stress of freezing process) monitored by FTIR spectroscopy (PO 2 -(+)) suggested preferential exclusion of sucrose from the LAB membrane as the preservation mechanism of the freeze-resistant cell. When considering freeze-sensitive cells, direct interaction between sucrose and membrane was proposed to explain loss of biological activity following freezing. Furthermore, occurrence of rigid domains within the membrane was more important in the freeze-sensitive bacterial population following cold and/or osmotic stresses. The broadening of existing highly rigid lipid domains in freeze-sensitive cells when applying osmotic stress is proposed to be caused by the interaction of sucrose with membrane phospholipids, leading to membrane disorganization and cell degradation.

The visual observation of rigid lipid domains within the membrane of LAB and the identification of FTIR markers of phospholipids organization requires further investigation, in particular to identify the precise composition of lipid domains and their mechanisms of formation in LAB.

Future prospects for research on LAB membranes Existence of Lipids domains within bacterial membrane

Domains of specific lipid composition have recently been evidenced within bacterial membranes and the characterization of mechanisms underlying the local enrichment of PLs has become an active research area [START_REF] Romantsov | Cardiolipin and the osmotic stress responses of bacteria[END_REF][START_REF] Passot | Synchrotron UV fluorescence microscopy for determining membrane fluidity modification of single bacteria with temperatures[END_REF][START_REF] Lin | Organization and function of anionic phospholipids in bacteria[END_REF]. Membrane poles and septa of bacilli including Gram-positive bacteria were reported to be enriched in anionic phospholipids, especially cardiolipin (CL) [START_REF] Kawai | Cardiolipin Domains in Bacillus subtilis Marburg Membranes[END_REF][START_REF] Lopez | Role of anionic phospholipids in the adaptation of Bacillus subtilis to high salinity[END_REF][START_REF] Bernal | Compensatory role of the cis -trans -isomerase and cardiolipin synthase in the membrane fluidity of Pseudomonas putida DOT-T1E: Cis -trans -isomerase and cardiolipin synthase[END_REF][START_REF] Seydlová | Surfactin production enhances the level of cardiolipin in the cytoplasmic membrane of Bacillus subtilis[END_REF]. CL content varies from 5 to 30% in bacteria, and has been associated to the membrane rigidification of B. subtilis [START_REF] Seydlová | Surfactin production enhances the level of cardiolipin in the cytoplasmic membrane of Bacillus subtilis[END_REF]) and P. aeruginosa [START_REF] Khoury | Targeting Bacterial Cardiolipin Enriched Microdomains: An Antimicrobial Strategy Used by Amphiphilic Aminoglycoside Antibiotics[END_REF]. A phosphatidylglycerol (PG) domain spiral-shaped that extends along the long axis of B subtilis was also evidenced [START_REF] Barák | Lipid spirals in Bacillus subtilis and their role in cell division[END_REF][START_REF] Hachmann | Genetic Analysis of Factors Affecting Susceptibility of Bacillus subtilis to Daptomycin[END_REF][START_REF] Muchová | Lipid domains in Bacillus subtilis anucleate cells[END_REF]. Moreover, the existence of domains similar to lipid rafts in eukaryotic cells was suggested [START_REF] Donovan | Characterization and subcellular localization of a bacterial flotillin homologue[END_REF][START_REF] Lopez | Functional microdomains in bacterial membranes[END_REF]. Lipid domains, lipid rafts and membrane physical properties have been mainly studied in model organisms (B. subtilis and E. coli) and evidenced their relevance for several important cellular processes [START_REF] Barák | The Role of Lipid Domains in Bacterial Cell Processes[END_REF]. Many interesting findings are thus expected to come to shed light on other bacteria, with new markers and imaging technologies continuously been developed. The recent visualization of lipid domains of high rigidity within the membrane of freeze-sensitive Lb. bulgaricus (Meneghel et al. 2017a) raises scientific questions concerning the composition of LAB lipid domains (head-groups and acyl-chains), their mechanisms of formation and their role on LAB resistance to stress.

(El

Lipids-proteins interactions and the modulation of membrane fluidity by proteins

Bacterial membrane proteins, accounting for about 20-30% of the cell proteome, are inserted in the membrane lipid bilayer through protein-aqueous channels. The lipid composition of the membrane can affect the biogenesis, activity and function of integral membrane proteins [START_REF] Lee | How lipids affect the activities of integral membrane proteins[END_REF]Schneiter and Toulmay 2007).

The coordination of lipid and proteins position in the membrane is however still poorly understood.

Lipid-protein interactions are controlled by several factors: the thickness, curvature and fluidity of membrane, composition of lipid head-group (charge, size, hydration) and fatty acid (chain length, transition temperature) [START_REF] Denich | Effect of selected environmental and physico-chemical factors on bacterial cytoplasmic membranes[END_REF][START_REF] Lee | How lipids affect the activities of integral membrane proteins[END_REF]. For example, non-bilayer forming lipid assemblies will occupy spaces in the protein surface to ensure a good contact between the protein and the lipid bilayer. Proteins also contribute to the stability of the membrane by limiting the flexibility of acyl chains, and decreasing their motion [START_REF] Heipieper | Mechanisms of resistance of whole cells to toxic organic solvents[END_REF][START_REF] Epand | Lipid polymorphism and protein-lipid interactions[END_REF]), particularly in the stationary growth phase [START_REF] Souzu | Fluorescence polarization studies on Escherichia coli membrane stability and its relation to the resistance of the cell to freeze-thawing. I. Membrane stability in cells of differing growth phase[END_REF]). The insertion of large proteins in the lipid bilayers results in ordering the lipid acyl chains, which in turn causes an increase of the membrane lipid phase transition temperature.

Some proteins can modify the membrane fluidity when cells are exposed to short-term changes in environmental conditions, such as heat-shock. In this case, the bacterial metabolic pathways would not make it possible a rapid change in membrane lipid composition. Heat shock proteins (such as GroEL, a soluble chaperonin from E. coli) can associate with lipids, leading to an increasing molecular order in the lipid bilayer, thus counterbalancing the increased membrane fluidity induced by high temperature [START_REF] Torok | Evidence for a lipochaperonin: Association of active proteinfolding GroESL oligomers with lipids can stabilize membranes under heat shock conditions[END_REF].

Furthermore, the membrane lipid phase transition from the liquid crystalline to the gel phases occurring following a decrease of temperature or hydration level, can induce protein segregation and thus the formation of concentrated domains of lipids within bacterial membranes [START_REF] Letellier | Lipid and protein segregation in Escherichia coli membrane: morphological and structural study of different cytoplasmic membrane fractions[END_REF][START_REF] Sperotto | Theory of protein-induced lateral phase separation in lilpid membranes[END_REF]). This phenomenon called lateral phase separation can perturb the membrane biological function, inactivating some proteins. Better knowledge about lipid-protein interactions and lipid domains formation in LAB would help to improve the maintenance of membrane properties following stabilisation processes.

The "sensing" role of membrane fluidity

Membrane fluidity modifications also contributes to the perception of environmental changes (cold, hyperosmotic, etc.) by bacterial cells and to the subsequent expression of genes that ensures acclimation to a new set of environmental conditions. Putative sensors that perceive changes in membrane fluidity have been reviewed

for bacteria and plants [START_REF] Los | Membrane fluidity and its roles in the perception of environmental signals[END_REF]. A cytosolic thermosensor governs the temperature-dependent adjustment of membrane fluidity (associated to production of unsaturated fatty acid) in E. coli while in B. subtilis and cyanobacteria this control is exerted by a membrane-associated thermosensor [START_REF] Mansilla | Control of Membrane Lipid Fluidity by Molecular Thermosensors[END_REF]).

Membrane integrated osmosensors have also been proposed for E. coli and Lc. lactis and it has been suggested that the changes in fluidity and in physical state of membrane lipids regulate the activity of osmosensors [START_REF] Los | Membrane fluidity and its roles in the perception of environmental signals[END_REF]. The identification of lipids or lipid domains that interact with these environmental sensors can provide clues for understanding how bacteria and in particular LAB respond to environmental stresses.

LAB biomimetic membranes as research tools

The complexity of bacterial membrane composition and organization, as well as, the asymmetric characteristic of lipid bilayers make it highly difficult the identification of key components that governs the membrane properties and bacterial resistance to environmental changes induced by stabilisation processes. The use of model membranes with well-defined lipid composition is a promising way to circumvent this high complexity and to identify the role of specific lipids (polar head-groups as well as fatty acyl chains).

Methodological breakthroughs

Complementary physical methods are continuously improved to investigate interactions between molecules and lipid bilayers [START_REF] Deleu | Complementary biophysical tools to investigate lipid specificity in the interaction between bioactive molecules and the plasma membrane: A review[END_REF][START_REF] Kent | Direct Comparison of Disaccharide Interaction with Lipid Membranes at Reduced Hydrations[END_REF].

As mentioned before in this mini-review, FTIR spectroscopy is a powerful tool for studying the structure and organization of membrane lipid bilayers (whole cells or vesicles) in physiological conditions and following exposure to environmental stresses without introducing extrinsic probes. However, since the lipid phase transition does not give a direct measurement of membrane fluidity it still needs to be combined to fluorescence anisotropy.

Besides, FTIR spectroscopy remains under-exploited and could provide relevant information on lipid domains formation [START_REF] Mendelsohn | Vibrational spectroscopic studies of lipid domains in biomembranes and model systems[END_REF], lipid-protein interactions in model membranes [START_REF] Silvestro | Infrared spectroscopy of supported lipid monolayer, bilayer, and multibilayer membranes[END_REF], and interactions between lipid polar head-groups [START_REF] Lewis | Membrane lipid phase transitions and phase organization studied by Fourier transform infrared spectroscopy[END_REF]. The maximal extraction of structural information encoded in the IR spectra will certainly require input from complementary approaches (isotopic labelling, recently developed sub-micron IR micro-spectroscopy, fluorescence microscopy, light scattering, neutron scattering). Polarized FTIR ATR measurements can also deliver valuable information on the preferential orientation of functional groups on membrane's surface [START_REF] Silvestro | Infrared spectroscopy of supported lipid monolayer, bilayer, and multibilayer membranes[END_REF][START_REF] Hutter | Fourier Transform Infrared Spectroscopy Using Polarization Modulation and Polarization Selective Techniques for Internal and External Reflection Geometries: Investigation of Self-Assembled Octadecylmercaptan on a Thin Gold Film[END_REF].

A summary of the main factors identified as having an influence on membrane fluidity is proposed in Figure 4.

The factors relate to membrane composition and process parameters, and are classified according to their ability to induce increasing, decreasing, or uncertain effect on membrane fluidity and also.

Conclusion

The prediction of membrane fluidity from the membrane fatty acid composition has shown strong limitations.

Despite all reported work on LAB lipid membranes, research is still needed to understand lipids organization and their interaction with proteins, to elucidate the factors governing membrane fluidity.

The systematic assessment of complete lipid composition (e.g. lipid classes, fatty acids and head-groups quantification) and physical properties of LAB membranes (membrane fluidity and lipid phase transition) becomes essential for understanding the role of different fatty acids (i.e. CFA), lipid classes (phospholipids and glycolipids), and lipid head-groups (i.e. PG, PE, etc.) in membrane fluidity.

For fully understanding the causes of bacterial membranes modulation and injury by environmental stress, it appears mandatory to assess membrane physical properties, through going forth and back model membranes and real cells, and to combine complementary methods: covering different observation scales (from cell population to molecules), involving in real time measurements, close to environmental conditions: if possible in situ or mimicking temperature and water activity process changes.

The systematic deep characterization of lipid composition and physical properties of membranes at different steps of the production of stabilised LAB would make it possible to relate process environmental conditions, membrane fluidity and LAB active or passive responses. This knowledge is of paramount importance for the optimization of industrial fermentations and stabilisation processes of LAB starters. It will also allow by reverse engineering, to select, produce and deliver populations of LAB with preferred characteristics in terms of membrane fluidity and physiological state. Legend:

FCP: fresh cell pellet; FT: freeze-thawing; PLs: phospholipids; FOS (fructo-oligosaccharides); IMO (iso-malto oligosaccharides); DPH (1,6-diphenyl-1,3,5-hexatriene) / Hydrophobic Probe located at the hydrophobic core of lipid bilayer; TMA-DPH (1-[4 (trimethylamino) phenyl]-6-phenyl-1,3,5-hexatriene) / Amphiphilic probe located at the aqueous interface between lipid bilayer and extracellular environment (PLs head-groups); Laurdan: 6-Dodecanoyl-N,N-dimethyl-2-naphthylamine. / Amphiphilic fluorescence probe located at the hydrophobic core of lipid bilayer, Ts: temperature of lipid phase transition during cooling (s, solidification); Tm: temperature of lipid phase transition during heating (m, melting); PLs head-groups: PO 2 -band by 1215 cm -1 associated to PLs head-groups hydration. The modifications on membrane fatty acid composition, fluidity as well as resistance to stabilisation processes are indicated as follows: increase (green and "+"), decrease (red and "-") and no significant variation (black and "~"). NR: not reported; FOS (fructo-oligosaccharides); IMO (iso-malto oligosaccharides) PLs: phospholipids; GLs: glycolipids; PG: Phosphatidylglycerol; CL: cardiolipin; LPG: Lysylphosphatidylglycerol; PGL: phosphoglicolipid FA: fatty acids; UFA: unsaturated FA; SFA: saturated FA; CFA: cyclic FA (CycC19:0); BFA: branched FA; MCL: mean acyl chain length; Ts: lipid transition during cooling (s, solidification); Tm: lipid transition during heating (m, melting); PO 2 -: band by 1215 cm -1 associated to PLs head-groups hydration; FT: freeze-thawing; FS: freezing storage; FD: freeze-drying; AD: air drying; VD: vacuum drying; SD: spraydrying; Resistant and sensitive cells were obtained when culturing in MRS broth and mild whey based culture medium, respectively (Adapted from [START_REF] Gautier | A low membrane lipid phase transition temperature is associated with a high cryotolerance of Lactobacillus delbrueckii lssp. <i>bulgaricus<i> CFL1[END_REF].

  processes. Following cooling, LAB membrane fluidity decreases with the transition from the disordered liquid crystalline (L) to the ordered gel phase (L) of the lipid bilayer.[START_REF] Gautier | A low membrane lipid phase transition temperature is associated with a high cryotolerance of Lactobacillus delbrueckii lssp. <i>bulgaricus<i> CFL1[END_REF] and[START_REF] Passot | Synchrotron UV fluorescence microscopy for determining membrane fluidity modification of single bacteria with temperatures[END_REF] studied the evolution of membrane properties of two populations of Lb. bulgaricus CFL1 exhibiting different freezing-resistance following cooling by FTIR spectroscopy and anisotropy of fluorescence (Fig.3). Freezeresistant cells exhibited a lower lipid phase transition (Ts) during freezing (Ts = -8 °C) and a higher membrane fluidity (r = 0.240) at the ice nucleation temperature range, than the freeze-sensitive cells (Ts = +22 °C and r = 0.388, respectively). A sub-zero value of lipid phase transition, associated to high membrane fluidity allowed the maintenance of the cell membrane in a relatively fluid state during freezing. Therefore, water flux from the cell and the concomitant volume reduction following ice formation in the extracellular medium (and associated solute cryoconcentration) was facilitated.

Figure 1 .Figure 2 .

 12 Figure 1. Lipid phase transition between a lamellar liquid crystalline structure (L) and a gel state (L) during cooling or dehydration (A). Schema of the membrane fluidity changes expected according to acyl chains structure and geometry (B). FA: fatty acids; UFA: unsaturated FA; SFA: saturated FA; BFA: branched FA; CFA: cyclic FA (CycC19:0).

Figure 3 .

 3 Figure 3. Membrane lipid phase behavior of two populations of Lb. bulgaricus CFL1 exhibiting different resistance to freeze-thawing. Resistant (black diamonds) and sensitive (grey triangles) cells' lipid transitions (liquid-crystalline  gel phase) were obtained by FTIR following cooling (close symbols) and heating (open symbols) by FTIR spectroscopy. Lipid transition temperatures during cooling (Ts, solidification) and during heating (Tm, melting) are indicated. Anisotropy values are also presented at three temperatures for both resistant (black circles) and sensitive cells (grey circles).

Figure 4 :

 4 Figure 4: Summary of main factors affecting LAB membrane fluidity FA: fatty acids; UFA: unsaturated FA; SFA: saturated FA; CFA: cyclic FA (CycC19:0).

  To our knowledge, no work has been reported on the development of model membranes that are good representations of LAB. Most of the reported studies have focused on single-component model lipid membrane. Considerable work is needed to generate data on more complex model lipid membranes and real biological membranes.

Table 1 :

 1 Reported work on the characterization of LAB cells and LAB lipids, and the associated methods

	Method (scale)	Micro-organism	Sample type	Measurement conditions	Membrane properties physical	References
	FLUORESCENCE ANISOTROPY
		O. oeni Lo84.13	FCP	At 42°C following heat; at 30°Cfollowing acid and ethanol shocks		(Tourdot-Maréchal et al. 2000)
		Lb. casei ATCC 393 liposomes	From 10°C to 55°C, at 5°C intervals, hyperosmotic conditions		(Machado et al. 2004)
		O. oeni ATCC BAA-1163	FCP	At 30°C, following cold, acid and ethanol shocks		(Chu-Ky et al. 2005)
		Lb. bulgaricus	FCP,	From 15°C to 50°C, at 5°C intervals,		(Tymczyszyn et al.
		CIDCA 333	liposomes	following osmotic stress.		2005)
	Fluorescence spectroscopy	Lb. bulgaricusL2	FCP	At 30°C, following various fermentation pH and temperatures	DPH	(Li et al. 2009a)
	(population scale)	Lc. cremorisMG1363 FCP	At 30°C, following ethanol stress or acid shock		(To et al. 2011)
		Lb. casei Zhang and acid-resistant mutant Lbz-2	FCP	At 37°C, following lactic acid and gastric juice stress in chemostat		(Wu et al. 2012)
		Lb. buchneri R1102, B. longum R0175	FCP	From 37°C to 0°C and back 0°C to 37°C, stationary growth phases following harvest at exponential and		(Louesdon et al. 2015)
		Lc. lactis ML3 Lb. bulgaricus CFL1 FCP PLs	At 25°C At 0°C and 25°C, following osmotic stress (sucrose)	DPH and TMA-DPH	(In't Veld et al. 1992) (Meneghel et al. 2017a)
		Lb. bulgaricusCFL1		At 42, 25 and 5°C following growth in MRS and whey medium	TMA-DPH (Passot et al. 2014)
	Flow cytometry (cellular scale)	Lc. lactis TOMSC161 Lb. bulgaricus CFL1 S. thermophilus CFS2	Fresh cells	At 20°C, fermentation (22°C, 30°C), different growth phases At 42°C, following growth in (MRS or whey) and harvested at different growth phases	DPH DPH and TMA-DPH	(Velly et al. 2015) (Bouix and Ghorbal 2017)
	Fluorescence microscopy (subcellular scale)	Lb. bulgaricusCFL1	Fresh single cell	At 0°C and 25°C, following osmotic stress (sucrose) From 0°C to 37°C at 5°C intervals, following growth in MRS and whey	TMA-DPH	(Meneghel et al. 2017a) (Passot et al. 2014)
				medium
	MEMBRANE LIPID PHASE TRANSITION
		Lb. plantarum P743	FCP, liposomes	FT: -50°C/+80°C; protectants: sorbitol, maltose, trehalose	Tm, PLs head-groups	(Linders et al. 1997)
			FCP, dried cells	FT: -50°C/+80°C; protectants: sucrose, maltodextrin, skim milk	Tm	(Oldenhof et al. 2005)
	FTIR spectroscopy (population scale)	Lb. bulgaricus CFL1	FCP	FT: -50°C/+80°C, MRS and whey growth media FT: -50°C/+80°C; protectants: glycerol, DMSO, sucrose	Ts, Tm Ts, Tm	(Gautier et al. 2013) (Fonseca et al. 2016)
				FT: -50°C/+80°C; osmotic stress (sucrose)	Ts, , Tm, PLs head-groups	(Meneghel et al. 2017b)
		Lc. lactis TOMSC161	FCP	FT: growth phases -50°C/+80°C; fermentation (22°C, 30°C), different following	Ts and Tm (Velly et al. 2015)
		Lc. cremoris MG1363	FCP	Heating: 0°C to 40°C; addition of sucrose, NaCl, pressure	Tm	(Molina-Höppner et al. 2004)
		Lc. cremoris MG1363	FCP	Heating: 0°C to 40°C; addition of sucrose, NaCl, pressure		(Molina-Höppner et al. 2004)
	Fluoresence spectroscopy (population scale)	Lb. acidophilus CRL 640 Lb. casei ATCC393 liposomes liposomes	At 5, 15, 25, 37, 50°C, following salt and bile stress hyperosmotic conditions At 20°C and 37°C, following	Laurdan Tm	(Fernández Murga et al. 1999) (Machado et al. 2004)
		Lb. reuteri TMW1.106	FCP	From 20°C to 50°C at 10°C intervals; skim milk protectants: Inulin, FOS, IMO, sucrose,		(Schwab et al. 2007)

Table 2 :

 2 Impact of environmental factors on lipid composition, membrane physical properties and LAB's resistance to stabilisation processes (freezing, freeze-drying, and drying).

	Factor	LAB strain	Stress conditions	Lipid composition	Physical	Resistance to
					properties	stabilisation	Reference
						process
	Fermentation (LAB active response)
		Lb. acidophilus	Cold: growth at 30°C UFA/SFA (+), CFA (+)	NR	(+) to FT and FS	(Wang et al.
		RD758		C18:0 (-), C16:0 (+)		at -20 °C	2005)
		Lb. acidophilus	Cold: growth at 25,	UFA/SFA (~), CFA (-)	NR	(+) to FT	(Fernández-
		CRL 640	30°C	MCL (-)			Murga et al.
				C18:2 (+), GLs/PLs (+)			2000)
		Lc. lactis	Cold: growth at 22°C UFA/SFA (+), CFA (~)	Tm (-), Fluidity	(~) to FD and	(Velly et al.
	Temperature	TOMSC161 Lb. bulgaricus L2 Cold: growth at 30°C, 35°C, 37°C, 39°C (at pH 5)	UFA/SFA (+), CFA (~)	(+) at 22 °C NR	storage at 25°C (+) to FD	2015) (Li et al. 2009a)
		Lb. acidophilus	Cold step after culture	UFA/SFA (+), CFA (+)	NR	(+) to FS	(Wang et al.
		RD 758	(8h at 15°C)				2005)
		Lb. coryniformis	Cold step after culture	UFA/SFA (+),CFA (-)	NR	(-) to FD	(Schoug et al.
			(26 °C, 6-8h)				2008)

UFA/SFA (~), CFA (~) (+) LPG (-) PG, CL NR NR After fermentation / Stabilisation processes (Passive responses of LAB)

	Factor	LAB strain	Stress conditions	Lipid composition		Physical	Resistance to
					properties	stabilisation	Reference
							process
		Lb. reuteri	Freeze-drying	NR	Fluidity (+) with	(+) to FD with
		TMW1.106	Protective solutes:		FOS	FOS	(Schwab et al.
			FOS, sucrose, inulin,					2007)
			IMO
		Lb. bulgaricus	Air Drying	NR	Tm (+) when	(~) AD
		CFL1	Protective solutes:		drying.		(Oldenhof et
			sucrose, maltodextrin,		Tm (~) with		al. 2005)
			S/MD mixture		protectants
	Drying	Lb. plantarum CWBI-B534, L. mesenteroïdes Kenya MRog2	Freeze-drying Protective solute: maltodextrin+glycerol Storage conditions	C18:2/C16:0 (-) and C18:3/C16:0 (-) following storage in presence of air, at high moisture content	NR		(-) Storage in presence of air, at high temperature and moisture	(Coulibaly et al. 2009)
				and high temperature			content
		Lb. acidophilus W Freeze-drying (FD)	UFA/SFA (~)	NR		(~) FD, VD	(Brennan et al.
			Vacuum drying (VD)	CFA (-) for VD				1986)
		Lb. bulgaricus NCFB 1489	Spray-drying	UFA/SFA (-), CFA (-) after spray-drying and storage	NR		(-) SD and storage (Teixeira et al. 1996)
		O. oeni ATCC	Cold stress	NR	Fluidity (-) at	NR after
		BAA-1163	cold shocks: 5 and 30		8°C		stabilisation	(Chu-Ky et al.
			min, at 8 °C or 14 °C				No effect on	2005)
							survival
					Ts / Tm (+) in		Gautier et al.
			Culture medium		whey	(-) FT when LAB	(2013)
		Lb. bulgaricus	(MRS vs. Whey)	UFA/SFA (-), CFA (-),	Fluidity (-) and	grown in whey
		CFL1	Cooling from 42 °C to	MCL (-) with whey	lipid rigid	medium	(Passot et al.
			0°C		domains at low		2014)
					T° in whey
	Freezing	Lb. bulgaricus CFL1 and ATCC 11842	Exposure to cold (5°C) and/or osmotic stress (50% of sucrose) Culture in whey	UFA/SFA (-), MCL (-) for CFL1	Ts (+), Fluidity (-) PO 2 -(-), in CFL1 Broad rigid lipid	(-) to FT for CFL1	(Meneghel et al. 2017b) (Meneghel et
			medium		domains in CFL1		al. 2017a)
		Lb. plantarum	Air Drying	NR	Tm (+) dried,	(+) to AD with	(Linders et al.
		P743	Protective solutes:		Tm (~) with	sorbitol	1997)
			Maltose, sorbitol,		protectants.
			trehalose		PO 2	-(-) with
					sorbitol

Funding: This work has received funding from the European Union's Horizon 2020 Marie Skłodowska-Curie research and innovation program under grant agreement N° 777657.

Compliance with Ethical Standards

Conflict of interest:

The authors declare that they have no conflict of interest.

Ethical approval: This article does not contain any studies with human participants or animals performed by any of the authors.