Christian Coelho
email: christian.coelho@u-bourgogne.fr

Charles Brottier

Florent Beuchet

Peio Elichiry-Ortiz

Benoit Bach

Céline Lafarge

Raphaëlle Tourdot-Maréchal

EFFECT OF AGEING ON LEES DISTILLATION PROCESS ON FERMENTED SUGARCANE MOLASSES FOR THE PRODUCTION OF RUM

Keywords: sugarcane molasses, ageing on lees, distillation process, aroma analysis, chemical diversity

The study aimed at evaluating the influence of fermented sugarcane molasses ageing on lees and the distillation process used for the production of rums.

Molasses were freshly fermented or 3-months lees aged. Batch (PS: Pot Still) or continuous (CS: Coffey Still) distillation was carried out resulting in four different rum distillates. Gas chromatography and 3D-fluorescence enabled to differentiate rum distillates chemical composition according to the distillation process, regardless of the ageing on lees of fermented molasses. Differences in fluorescent PARAFAC components and volatile acids, acetals and carbonyls contents revealed the predominance of the physicochemical processes driven at the liquid-vapor interface of fermented molasses, generated by the distillation systems. Notwithstanding the distilling conditions, the long chain fatty ester content was significantly higher in the 3-months lees aged condition.

Multivariate analysis highlighted that CS rum distillates were chemically more homogeneous than those obtained by PS that preserved the lees effect.

Introduction :

Sugarcane molasses are the viscous end product of sugar companies which is mostly valued as raw material prior to fermentation and distillation for rum production. The choice of yeasts and the conditions of fermentation differentiate molasses wort chemical composition which are revealed later in the characteristics of volatile composition of distillates [START_REF] Medeiros | 16 -Cachaça and Rum[END_REF]. During the elaboration of fruit, cereal or plant fermented beverages, a great diversity of microorganisms can be used but the yeast Saccharomyces cerevisiae remains the main species generally used [START_REF] Campos | Features of Saccharomyces cerevisiae as a culture starter for the production of the distilled sugar cane beverage, cachaça in Brazil[END_REF][START_REF] Walker | Saccharomyces cerevisiae in the Production of Fermented Beverages[END_REF]. Additionally in the area of distilled beverages, particularly in whisky production, specific strains of Saccharomyces cerevisiae have been selected for their high alcohol content tolerance and their capacities to convert mash sugars into ethanol, carbon dioxide and numerous flavor congeners [START_REF] Stewart | 125th Anniversary Review: Developments in brewing and distilling yeast strains[END_REF]. In the area of rum production, the inoculation of selected yeasts strains for sugar cane fermentation can be excluded, n favor of the expression of indigenous microbial flora, often associated with rums richer in aromas.For example, the "Rhum Agricole" involves ai complex indigenous microbiota made of mixes of yeasts and bacteria, already present in the sugarcane juices.

Lactobacillus and Propionibacterium species have also been shown to remain in sugarcane molasses used for "Rhum Grand Arôme" production [START_REF] Fahrasmane | Microbial flora of rum fermentation media[END_REF]. Another practice used for producing heavy rums consists of adding the "dunder" in the fermenting molasses wort. The "dunder" is the residual creamy vinasse from the previous distillation, made of sugars and dead yeast cells [START_REF] Fahrasmane | RUM[END_REF][START_REF] Medeiros | 16 -Cachaça and Rum[END_REF]. Such ancestral practice could be hazardous with the risk of low alcoholic fermentation yields, unachieved fermentations and the development of spoilage microorganisms. The control of fermentation can be improved by direct inoculation of pure cultures of microorganisms or inoculation of a mother yeasting pre-cultured in a fermenter. In some cases, dried yeasts can be directly added in the washing media [START_REF] Fahrasmane | Microbial flora of rum fermentation media[END_REF][START_REF] Murtagh | The Alcohol Textbook: A Reference for the Beverages, Fuel and Industrial Alcohol Industries[END_REF]. The choice of strains impacts the quality of rums. The distinction between the different types of rums, light or heavy rums for instance, can be designed by the choice of inoculated yeast strains belonging to Saccharomyces cerevisiae, Saccharomyces bayanus or Schizosaccharomyces pombe [START_REF] Fahrasmane | Microbial flora of rum fermentation media[END_REF][START_REF] Medeiros | 16 -Cachaça and Rum[END_REF]. The quality of the final spirit can be also modulated with a sugarcane fermentation obtained by co-inoculation of a consortium of microorganisms [START_REF] Duarte | Effect of Co-Inoculation of Saccharomyces cerevisiae and Lactobacillus fermentum on the Quality of the Distilled Sugar Cane Beverage Cachaça[END_REF]. Moreover, the presence of yeast lees in the mash could positively impact the spirit's quality, especially for heavy rums [START_REF] Medeiros | 16 -Cachaça and Rum[END_REF][START_REF] Murtagh | The Alcohol Textbook: A Reference for the Beverages, Fuel and Industrial Alcohol Industries[END_REF]. The presence of yeast lees during distillation has been shown to promote different releases in ethyl esters, ethyl hexanoate and octanoate in particular, leading to differences in rum styles [START_REF] Suomalainen | Yeast esterases and aroma esters in alcoholic beverages[END_REF].

Rum technology involves two distillation techniques used all around the world of distilled beverages: the ancestral one with the pot still and the industrial one with coffey still (L. [START_REF] Fahrasmane | RUM[END_REF]. In both cases, odorous volatile compounds, concentrated in the final spirit, enabled a classification of the different types of rums according to their level of concentration. Traditional agricultural rums produced from raw sugar cane differ from sugar refinery molasses rums in composition and concentration, generally due to differences in the distillation process [START_REF] Pigott | The alcohol textbook 4th Edition. Recueil des méthodes internationalesd'analyse des boissons spiritueuses des alcools et de la fraction aromatique des boissons[END_REF]. [START_REF] Liebich | Analysis of the Flavor of Rum by Gas-Liquid Chromatography and Mass Spectrometry[END_REF] identified more than 200 flavor compounds in a Jamaican rum using liquid extraction of rum prior to rum analysis by gas chromatography coupled to mass spectrometry, with concentrations reaching 800 ppm, particularly for fused alcohols [START_REF] Liebich | Analysis of the Flavor of Rum by Gas-Liquid Chromatography and Mass Spectrometry[END_REF]. According to Marse et al. (2004) rum is one of the distilled beverages that has the most of volatile compounds, reaching 550 different aromas [START_REF] Maarse | Flavour of distilled beverages[END_REF]. Some Grand Arôme and heavy rums, often appreciated from rum tasters due to their elevated esters content, can reach concentrations of more than 500 g/hL of pure alcohol (L. [START_REF] Fahrasmane | De la canne au rhum[END_REF]. According to [START_REF] Fahrasmane | Trente ans de travaux en technologie rhumière à l'Inra-Antilles-Guyane : Trente ans de recherche en technologie des rhums[END_REF], the control of the organoleptic quality of heavy rums production remains a big challenge for rum producers and scientists due to the variability in microbiota and the impact of distillation processes. This study presents a quantification of the effect of ageing on lees and the distillation process based on the quantification of chemical differences in the composition of major volatile compounds families and fluorescent components. The discrimination potential of each fermenting and distilling practices in sugarcane molasses rums was evaluated by multivariate statistical analysis.

Materials and methods

Wort samples and fermentations

2.1.1. Sugarcane molasses characteristics and wort preparations Sugarcane molasses were supplied by a French rum company (Compagnie des Indes, Beaune, France). Prior to fermentation, the molasses were diluted with distilled water, in order to obtain 50 kg of diluted molasses characterized by a density of 1.090 at 20°C with a DMA 35 densimeter (Anton Paar, Graz, Austria).

The diluted molasses presented a Brix degree of 16 and an initial pH of 4.9. Then 16 kg of diluted molasses were poured into three 20 L glass demijohns and supplemented with 30 g/hL of diammonium phosphate (Sigma), 30 g/hL of yeast assimilable nitrogen (Mauriferm Gold, AB Maury, Peterborough, UK). The strain of Saccharomyces cerevisiae was Pinnacle MG+ (AB Mauri, Peterborough, UK), packaged in active dry form. The yeast inoculation was applied at the dose of 40 g/hL, according to the manufacturer's recommendations.

Fermentation processes

The fermentations were conducted in demijohns without stirring at room temperature (18-25°C) and monitored in terms of density and temperature.

Measures were realized twice per day with a DMA 35 densimeter (Anton Paar, Graz, Austria). Demijohns were weighed with a numeric analytical scale of 35 kg (Mettler Toledo, Greifensee, Switzerland).

Two series of fermentations were carried out in biological triplicates at three months of interval. After fermentation, the first mashes were left at 4°C in contact with the yeast lees (L: Lees) to age during three months (L1, L2, L3).

The second fermentations (F: Fresh) were carried out in triplicates (F1, F2, F3), with the same protocol as previously described, just prior to the distillation. In all cases, yeast lees (fresh or aged) were removed from mashes before distillation.

S. cerevisiae strain implantations were controlled at the middle of alcoholic fermentation using a PCR interdelta analysis according to a previously published procedure [START_REF] Legras | Optimisation of interdelta analysis for Saccharomyces cerevisiae strain characterisation[END_REF]. As illustrated in Fig. S.I.1 all sugarcane molasses were fermented with the same yeast strain.

Distillates samples

Two types of distillation: the pot still (PS) and the column still (CS) were carried on the six samples of fermented sugarcane molasses. Distillation systems used in this study can be viewed in Fig. S.I.2. For that, half of the demijohn content, corresponding to 8 kg was poured into the pot still and 8kg was poured into the column still generating twelve distillates that were used for chemical analyses.

Pot Still distillation

Pot still distillation was heated directly by flame contact with the copper surface of the 25 L copper still. Two distillations were carried out, the first one leading to the "low wines" and the second one leading to the final white distillated spirit.

Volumes and ethanol content of these final distillates were analyzed. For this second pot still distillation, we decided to cut at 50 % of alcohol content for the six wort batches (PS-F1, PS-F2, PS-F3, PS-L1, PS-L2, PS-L3) in order to keep an optimized control of pot still distillation process. The foreshots were removed and corresponded in each case to an approximated volume of 100 mL characterized by an intense solvent olfactive character.

Coffey Still distillation

Column still distillation was carried out on a 25 L Holstein column (Markdorf, Germany). Temperatures in the boiler, heater, column and deflegmator were automatically measured, with a control of the boiler temperature. Heat was generated by a steam flow in direct contact with the copper still and controlled by a pressure of 150 mbars, enabling to keep a constant temperature of 90 °C inside the still. Temperature, alcohol content and distillate flow rate were automatically monitored online thanks to an infrared detector for the six wort batches (CS-F1, CS-F2, CS-F3, CS-L1, CS-L2, CS-L3). The control of the cooling system was adjusted with an automatic valve. The distillate flow rate was kept constant between 15-20 mL/min and collected as the hearts of the distillation and once passing below 10 mL/min the hearts were separated from the tails. The foreshots were removed the same way as described in the pot still distillation Ethanol content was determined in the mashes at the end of fermentation by an enzymatic method following the manufacturer's instructions (BioSentec®, France).

Distillate volatile composition

The distillates were also submitted to a targeted analysis of the volatile chemical composition. The liquid extracts (990 mL of distillate sample and 10 µL of octan-3-ol at 1 g/L) were analyzed with a Agilent Technology 5975C spectrometry (Shimadzu QP2010+, electronic impact at 70 eV) paired with a Agilent Technology 7890 A gas chromatograph fitted with a split/splitless injector (250°C). The chromatograph was equipped with a capillary column PEG of 30 m × 0.32 mm (J&W Scientific). Film thickness was 0.50 µm. Helium was used as vector gas at a rate of 1.5 mL/min (average velocity of 44 cm/sec). The temperature of the oven was increased from 50°C to 240°C at 5°C/min, and finally held at 240°C for 5 minutes. The injection mode was splitless. The analyses were done in triplicate. Spectrometry Selected Ion Monitoring method (SIM method) was used for molecules detection. The mass spectrometer scanned from m/z 29 to 500. The volatile compounds were identified by matching their spectral fragmentation with those provided by the mass spectral library of the National Institute of Standards and Technology (NIST) and the Wiley Registry (WILEY) and by validating with pure chemical standards. Quantification was carried out via an internal standard method by the addition of octan-3-ol to distillates reduced to 50 % ethanol (v/v) with ultrapure water prior to injection. Response factors were calculated for volatile compounds from calibration curves obtained by analyzing hydroalcoholic solutions (ethanol 50 %, v/v) made from pure analytical grade standards (SigmaAldrich, Saint Louis, MO) in the ranges 0.05-10 mg/L for phenylethanol, eugenol, ethyl acetate, isoamyl acetate, ethyl lactate, ethyl butanoate, ethyl octanoate, ethyl decanoate, ethyl dodecanoate, 1,1-diethoxy ethane, diacetyle, 2-methyltetrahydrofuran-3-one, furfural, propanoic acid, ndecanoic acid, propanoic acid, 2-methyl and octanoic acid and 1-200 mg/L for propanol, 2-methyl-propanol, butanol, 3-methyl-butanol, 2 methyl-butanol. The concentrations of volatile compounds were converted in grams per hectoliter of pure alcohol following CE regulation 2870/2000.

Excitation Emission Matrices of Fluorescence (EEMF) of rum distillates

All rum distillates were analyzed with an untargeted approach consisting of measuring Excitation Emission Matrices of Fluorescence (EEMF). For that, rum distillates were diluted twenty times with ultrapure water and put in 1 cm pathlength quartz cuvette and EEMFs were recorded in a Horiba Aqualog unit, enabling to automatically correct the Rayleigh and Raman scattering and the inner filtering effect and to normalize EEMFs to a quinine sulfate 1 ppm solution.

Statistical analysis

Aroma concentrations were statistically analyzed by multivariate analysis using Origin Lab software. PARAFAC model of rum distillates EEMFs was built on home made Matlab software, previously used for wine PARAFAC modeling [START_REF] Coelho | Fluorescence Fingerprinting of Bottled White Wines Can Reveal Memories Related to Sulfur Dioxide Treatments of the Must[END_REF]. Mean Fmax values of PARAFAC components and mean volatile compounds concentrations were statistically compared with an ANOVA test with an interval of confidence of 95 %, followed by a Tukey's HSD post hoc test to evaluate the impact of yeast lees ageing and distillation practice.

Results and discussion

Fermentation monitoring

The evolutions of the molasses wort density, weight and temperature upon the fermentation stage for the fresh (F) and yeast lees (L) modalities are presented in Fig. 1A and 1B, respectively. Fermentations started at a density of 1.086 and reached a final density of 1.030 for each modality. Molasses weight decreased from 16.1 kg to 15.3 kg for F modalities and from 16.0 kg to 15.1-15.2 kg for L modalities. For F modalities, fermentation started just after the yeast strain inoculation and finished within 48 hours for the three biological replicates (F1, F2 and F3). For L modalities, we observed a lag phase of 24 hours following yeast strain inoculation for L1 and L2. This lag phase was around 40 hours for L3. These delays were probably due to lower non-controlled fermentation temperatures of 20 °C compared to 26 °C for the fresh modality. Fermentation at lower temperatures values affected yeast metabolism by slowing their proliferation in the molasses wort. Nevertheless, the real duration of the alcoholic fermentation for L modalities was comparable to that obtained with F modalities, ie 48 hours. For all modalities, the average ethanol contents presented no statistical differences (p=0.05) and were comprised between 6.45 % and 6.80 %, for L and F modalities, respectively. Final pH was measured at 4.5 and 4.6 for (F) and (L) conditions, respectively.

A) B)

Rum distillates chemical analysis

Aromatic Volatile congeners composition

Major volatile congeners concentrations were quantified in each rum distillate.

Distillation process differentiation

First of all, CS and PS rum distillates generated by the two distillation systems presented different normalized concentrations of volatile congeners, particularly for chemical families like acetals, carbonyls and acids and to a lesser extent alcohols (Fig. 2A). Statistical differences were found in PS distillates with higher concentrations in acetals (1,1-diethoxyethane), carbonyls (furfural, diacetyl, 2methyloxolan-3-one) and acids (propanoic, isobutyric, octanoic, decanoic) compared to CS distillates. No statistical differences were found for alcohols and esters (Fig. 2B). Were also more detected in PS rum distillate, some individual volatile compounds such as 3-methyl-propanol phenylethanol, eugenol, ethyl acetate and ethyl lactate (Fig. S.I.4). Such results have already been pointed out in brandy, cachaça and whisky production [START_REF] Maarse | Flavour of distilled beverages[END_REF][START_REF] Nascimento | Quantitative Ester Analysis in Cachaça and Distilled Spirits by Gas Chromatography-Mass Spectrometry (GC-MS)[END_REF][START_REF] Piggott | Understanding Natural Flavors[END_REF][START_REF] Simpson | Manufacture of brandy[END_REF]. Furfural, already present in sugarcane molasses, is formed by Maillard reaction when using direct heating pot still units [START_REF] Simpson | Manufacture of brandy[END_REF]. To our knowledge methyloxolan-3-one, a Maillard reaction product already found in rum [START_REF] Nykänen | Aroma of beer, wine and Distilled alcoholic beverages[END_REF] which has a pleasant coffee note has never been shown to depend on the type of distillation. 1,1-diethoxyethane, conferring a fruity note to the distillate was only present on pot still batches and was not detected in the CS rum distillates, meaning the continuous distillation reduced acetals formation [START_REF] Piggott | Understanding Natural Flavors[END_REF]. Organic acids were not detected in CS distillates, revealing they were eliminated due to different partitioning of these compounds in the CS column plates, particularly due to the elevated amount of reflux [START_REF] Maarse | Flavour of distilled beverages[END_REF]. Another plausible reason is these organic acids were more prone to esterification with ethanol leading to higher concentrations of their esterified forms, particularly ethyl hexanoate, ethyl octanoate, ethyl decanoate and ethyl dodecanoate.

Lees ageing effect after distillation Interestingly, ester compounds were more present in rum distillates generated from yeast lees aged mashes compared to the fresh mashes, independently of the distillation process (Fig. 2A and Fig. 2C). This increase in ester content in rum distillate had already been described when lees were directly incorporated into the still with a progressive release of their lipophilic content in the wort with the temperature increase during the distillation [START_REF] Suomalainen | Yeast esterases and aroma esters in alcoholic beverages[END_REF]. This abundance in ester compounds was never previously attributed to the lees ageing process on fermented sugarcane molasses. Only the 3-months yeast lees aged rum distillates showed higher amounts of 1,1-diethoxyethane, diacetyl, octanoic acid and decanoic acid compared to the fresh rum distillates (Fig. S.I.4). Such fatty acids increase after 3-months lees aging has been proposed by Troton et al. (1999) as degradation of membrane compounds from cells [START_REF] Troton | Evolution of the Lipid Contents of Champagne Wine During the Second Fermentation of Saccharomyces cerevisiae[END_REF]. Nevertheless, propanoic and isobutyric acids were found in higher amounts in fresh fermented rum distillates traducing their preferential accumulation in the distillate after a pot still distillation. With The same tendency is observed with compounds like eugenol or furfural, which are more present in fresh fermented rum distillates. As previously mentioned in wine medium, these woody-flavored compounds tend to bind to yeast lees and be less detected in the resulting wines [START_REF] Chatonnet | Incidence des conditions de fermentations et d'élevage des vins blancs secs en barriques sur leur composition en substances cedeés par le bois de chene[END_REF][START_REF] Moreno | Binding of oak volatile compounds by wine lees during simulation of wine ageing[END_REF]. This phenomenon could also explain the reduced concentration of eugenol and furfural in the rum distillates from 3 months lees aged mashes.

Rum distillates EEMF Analysis

The chemical composition of rum distillate was assessed by means of 3D fluorescence spectroscopy in order to strengthen the previous volatile congeners differentiations between the fermentation and distillation modalities. Excitation-Emission Matrices of Fluorescence of rum distillates elaborated from fresh and 3-months yeast lees aged sugarcane molasses in pot still and coffey still are shown in Fig. 3A. All rum distillates present two typical emission areas centered at 340 nm for 250 and 300 nm of excitation wavelengths. These emissions have been attributed in other food systems to a great variety of compounds such as phenolics, furfurals, NADH and Maillard reaction products [START_REF] Coelho | Fluorescence Fingerprinting of Bottled White Wines Can Reveal Memories Related to Sulfur Dioxide Treatments of the Must[END_REF][START_REF] Elcoroaristizabal | Fluorescence excitation-emission matrix spectroscopy as a tool for determining quality of sparkling wines[END_REF][START_REF] Ghosh | A fluorescence spectroscopic study of honey and cane sugar syrup[END_REF][START_REF] Markechova | Fluorescence spectroscopy and multivariate methods for the determination of brandy adulteration with mixed wine spirit[END_REF][START_REF] Matiacevich | A critical evaluation of fluorescence as a potential marker for the Maillard reaction[END_REF]. The intensity of each emission area was higher in rum A) B)

Impact of the lees ageing and distillation practices

As rum distillates were differentiated by means of their volatile congener composition and their fluorescence fingerprinting, prediction statistic models were built using multivariate approaches by partial least squares discrimination similarities once they were elaborated from fresh fermented sugarcane molasses whereas the 3-months yeast lees aged one led to a higher discrepancy between the triplicates of rum distillates. This statistical approach permitted a better evaluation of the variability of the distillation process taking into account the heterogeneity of fermented sugarcane molasses. Continuous distillation enabled a better homogenization of rum distillates whereas batch distillation preserved the yeast lees ageing practice on mashes that could be applied or desired by some rum producers.

Conclusion

Sugarcane molasses were fermented freshly or yeast lees aged during three months prior to distillation in order to obtain different styles of rum distillates.

Regardless of the nature of the distillation process, yeast lees ageing led to higher amounts of ester contents, particularly long chain fatty esters and some of their precursors like fatty acid in C8 and C10. Once distillation is carried out, pot still rum distillates differ from coffey still distillates by presenting specific fluorescence fingerprinting related to their chemical volatile composition. This study also highlights for the first time that yeast lees ageing practice on sugarcane molasses coupled to batch distillation could confer a differentiated rum style whereas continuous distillation tends to minimize its impact.

  2.3. Chemical analysis2.3.1. Wort and distillate characterization Wort and distillate classical parameters such as ethanol, pH and total acidity and ethanol (only for distillates) were determined according to OIV standardized methods (Recueil des méthodes internationales d'analyse des boissons spiritueuses des alcools et de la fraction aromatique des boissons. OIV 1994).

  PARAFAC model was validated by core consistency and split half validation of the dataset. PARAFAC model described each PARAFAC components by their fluorescence intensites at their maximum, represented as Fmax values. Fmax values were used to statistically interpret the distillate fluorescent composition and classify the different rum distillates in function of their elaboration processes.

  Ethanol contents measured at the end of the alcoholic fermentation are specified in Fig. S.I.3.

Fig. 1 .

 1 Fig. 1. Fermentation monitoring of sugar cane molasses wort density (filled symbols), weight (emptied symbols) and temperature (red cross) for the three biological replicates for (A) fresh (F1, F2 and F3) and (B) 3-months yeast lees aged (L1, L2 and L3) modalities.

Fig.

  Fig. 2A illustrates a heatmap representation of volatile compounds normalized by the maximum concentration found among the twelve samples per volatile compound and grouped by chemical families (alcohols, esters, acetals, carbonyls and acids). The mean concentrations for CS and PS rum distillates, regardless of the presence/absence of lees on fermented sugarcane molasses are presented in Fig. 2B. The mean concentrations for L and F rum distillates, regardless of the distillation process are indicated in Fig. 2C. Raw concentrations values of individual volatile congeners found in rum distillates are indicated in additional information (Fig. S.I.4).

Fig. 2 .

 2 Fig. 2. (A) Heatmap of aromatic compounds quantified in the rum distillates after Coffey still (CS) and Pot still (PS) distillations from fresh (F) and 3-months yeast lees aged (L) sugarcane molasses fermentations in triplicates. Concentrations are normalized by the maximum concentration per volatile congeners and represented by the color scale from black (O) to red (1). Mean aroma concentrations grouped by chemical families by comparing PS and CS distillation regardless of the type of fermentation (B) and by comparing L and F fermentation regardless of the type of distillation (C). Letters a and b indicate the results of the variance analysis realized performed for each chemical family.

  distillates from pot still compared to coffey still, regardless of the lees ageing on mashes. For finest discriminations and statistical validation, a PARAFAC model was built based on the analysis of twelve rum distillates samples analyzed in triplicates. The model generated three PARAFAC components (F1, F2 and F3), shown in Fig. S.I.5, enabled to statistically differentiate the effect of distillation process used in the elaboration of rum distillates. Fig. 3B illustrates this differentiation obtained by analyzing each Fmax values of the model. PS rum distillates present higher mean Fmax values of PARAFAC component F1 from 12.34 (PS F) to 16.25 (PS L) compared to CS rum distillates (Fmax mean values of 7.07 and 7.59, for CS F and CS L, respectively). CS rum distillates present higher mean Fmax values of PARAFAC component F3 from 9.99 (CS F) to 8.67 (CS L) compared to PS rum distillates (Fmax 3 mean values of 3.95 and 5.54 for PS F and PS L, respectively. No statistical differences were found for Fmax values of PARAFAC component F2 for the four rum distillates. This spectral discrimination between batch and continuous distilled liquids by means of PARAFAC components F1 and F3 could be attributed to the influence of volatile compounds mainly present in distillates such as alcohols, esters and acids that affect the chemical environment of intrinsic fluorophores[START_REF] Sadecka | Fluorescence spectroscopy for the analysis of spirit drinks[END_REF]. Longer wavelength emissive compounds, associated to the statistical PARAFAC component F1, could also be attributed to volatile carbonyls such as furfural, that were analytically measured at higher levels in PS distillates (previously shown in Fig.2B), coinciding with the observed higher Fmax values of this component. Nevertheless, chemical assignments should be performed carefully due to several overlapping bands originating from different volatile fluorophores present in the total fluorescence spectra of rum distillates.

Fig. 3 .

 3 Fig. 3. Excitation Emission Matrices of Fluorescence of the four rum distillates PS-F, CS-F, PS-L and CS-L (A) and mean Fmax values of PARAFAC components F1, F2, and F3 of the same four rum distillates analyzed in biological triplicates (B).

  analysis and hierarchical clustering analysis. Results are shown in Fig. 4 where volatile congeners concentrations and PARAFAC components were used as predicable variables and the distillation type (PS: group 1 or CS: group 2) as dependent variables. Fig. 4A illustrates statistically the clear discrimination found between the two types of distillation along the first component t[1] regardless of the treatment of mashes after fermentation. This PS/CS distinction is essentially driven by higher Fmax values of PARAFAC component F3 and some long chain fatty esters in C8, C10 and C12 for CS rum distillates and by higher values in Fmax 1, volatile acids, furfural and phenylethanol in PS rum distillates. Fig. 4B shows the number of clusters and the level of cluster similarity represented by the Y-axis. It is interesting to notice that CS distillates presented closest similarities compared to the PS rum distillates independently of fermented mashes. In the same way, PS rum distillates presented close

Fig. 4 .

 4 Fig. 4. Statistical discrimination of rum distillates based on their chemical analysis and the way fermentation and distillation was carried measured by a partial least squares discrimination analysis (A) and a hierarchical clustering analysis (B)

Acknowledgements

This work was partly supported by Regional Council of Bourgogne-Franche Comté, and the Fonds Européen de Developpement Régional (FEDER). We also thank the French spirit company Compagnie des Indes for its financial support for this study and Changins, Viticulture and Oenology, University of Applied Sciences and Arts Western Switzerland for the column distillation facilities. We also thank Horiba Scientific Company for the equipment support. A) B)


Aurore Palud
email: palud.aurore@gmail.com

Karima Salem

Jean-François Cavin

Laurent Beney

Hélène Licandro
email: helene.licandro@u-bourgogne.fr

Identification and transcriptional profile of Lactobacillus paracasei genes involved in the response to desiccation and rehydration

Keywords: Lactobacillus paracasei, transposon mutants, desiccation, rehydration, gene expression

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Water is essential for all living organisms as it contributes to the structure of cells, stabilizes proteins, lipids and nucleic acids, and maintains vital metabolic systems and chemical reactions [START_REF] Potts | Desiccation tolerance of prokaryotes[END_REF]. Desiccation leads to water exit from cells, induces structural modifications, and causes osmotic and oxidative stresses [START_REF] Dupont | Survival kit of Saccharomyces cerevisiae for anhydrobiosis[END_REF][START_REF] França | Oxidative stress and its effects during dehydration[END_REF][START_REF] Potts | Desiccation tolerance of prokaryotes[END_REF]. In addition, the rehydration phase could lead to membrane alterations [START_REF] Mille | Magnitude and kinetics of rehydration influence the viability of dehydrated E. coli K-12[END_REF]. The capacity to return to life after hydric fluctuations is not only crucial for bacteria as a function of diurnal and seasonal cycles in natural environments but also during food industry processes such as conventional drying, freeze drying and spray drying [START_REF] Billi | Life and death of dried prokaryotes[END_REF][START_REF] Esbelin | Desiccation: An environmental and food industry stress that bacteria commonly face[END_REF][START_REF] Pointing | Microbial colonization and controls in dryland systems[END_REF][START_REF] Potts | Desiccation tolerance of prokaryotes[END_REF]. In many organisms, desiccation survival is correlated with the accumulation of protective molecules, in particular trehalose [START_REF] Crowe | The Role of Vitrification in Anhydrobiosis[END_REF]. However, desiccation tolerance involves other mechanisms that are mostly unknown except for extreme desiccation tolerant bacteria (anhydrobiotes) such as such as cyanobacteria [START_REF] Potts | Mechanisms of desiccation tolerance in cyanobacteria[END_REF], yeast [START_REF] Dupont | Survival kit of Saccharomyces cerevisiae for anhydrobiosis[END_REF], resurrection plants [START_REF] Crowe | Anhydrobiosis: an unsolved problem[END_REF] and microscopic animals [START_REF] Wełnicz | Anhydrobiosis in tardigrades-The last decade[END_REF].

In the last few years, progress has been made in understanding the mechanisms of bacteria tolerance to partial desiccation using transcriptome analysis, in particular for Salmonella, one of the most common foodborne pathogens able to survive for extended periods after desiccation [START_REF] Chen | Thermal Resistance and Gene Expression of both Desiccation-Adapted and Rehydrated Salmonella enterica Serovar Typhimurium Cells in Aged Broiler Litter[END_REF][START_REF] Deng | Transcriptome sequencing of Salmonella enterica serovar Enteritidis under desiccation and starvation stress in peanut oil[END_REF][START_REF] Finn | ProP Is Required for the Survival of Desiccated Salmonella enterica Serovar Typhimurium Cells on a Stainless Steel Surface[END_REF][START_REF] Gruzdev | Global Transcriptional Analysis of Dehydrated Salmonella enterica Serovar Typhimurium[END_REF][START_REF] Li | Transcriptomic Analysis of Salmonella Desiccation Resistance[END_REF][START_REF] Maserati | General response of Salmonella enterica serovar Typhimurium to desiccation: A new role for the virulence factors sopD and sseD in survival[END_REF]. The comparison of these studies has revealed differences in identified genes that could be explained by the procedures used to dry bacteria (surface, drying medium, RH, desiccation periods). Several common genes were linked to amino acid transport and metabolism functions. Global transcriptional analyses were also performed for soil-residing bacteria [START_REF] Cytryn | Transcriptional and Physiological Responses of Bradyrhizobium japonicum to Desiccation-Induced Stress[END_REF][START_REF] Higo | Dynamic transcriptional changes in response to rehydration in Anabaena sp[END_REF][START_REF] Leblanc | Global Response to Desiccation Stress in the Soil Actinomycete Rhodococcus jostii RHA1[END_REF] and similar responses were identified such as compatible solute and heat shock protein accumulation, reactive oxygen species neutralization, and DNA modification and repair.

However, transcriptomics has limitations since genes essential for a function can be constitutively expressed and even inducible genes can be overexpressed during a very short period that does not overlap with that of the experiment. Recently, random transposon mutagenesis approaches were developed to identify genes for resistance to desiccation in Listeria monocytogenes [START_REF] Hingston | Insertional mutagenesis reveals genes associated with desiccation and osmotic stress in Listeria monocytogenes[END_REF] and Salmonella enterica [START_REF] Mandal | Global Screening of Salmonella enterica Serovar Typhimurium Genes for Desiccation Survival[END_REF]. For L. monocytogenes screening, genes were involved in energy production, membrane transport, amino acid metabolism, fatty acid metabolism and oxidative damage control. In S. enterica, in comparison to previous transcriptional studies, several genes were related to amino acid metabolism and more than 20% encoded hypothetical proteins. Recently, changes in proteomic expression were investigated for S. enterica during desiccation and rehydration [START_REF] Maserati | iTRAQ-Based Global Proteomic Analysis of Salmonella enterica Serovar Typhimurium in Response to Desiccation, Low Water Activity, and Thermal Treatment[END_REF]. The proteins with higher expression levels in dried samples were mainly ribosomal proteins whereas flagellar proteins, membrane proteins, and export systems as well as stress response proteins were identified in rehydrated samples.

Although L. casei/paracasei is one of the most emblematic groups of lactic acid bacteria (LAB), the genetic mechanisms involved in desiccation resistance and adaptation are not fully understood, limiting prospects for improving and developing preservation processes.

Comparative genomics has demonstrated that LAB are highly adaptable to various niches such as soil, foods (dairy, meat, and vegetable), as well as oral, vaginal and gastrointestinal cavities [START_REF] Cai | Genome Sequence and Comparative Genome Analysis of Lactobacillus casei: Insights into Their Niche-Associated Evolution[END_REF][START_REF] Douillard | Comparative Genomic and Functional Analysis of Lactobacillus casei and Lactobacillus rhamnosus Strains Marketed as Probiotics[END_REF][START_REF] Duar | Lifestyles in transition: evolution and natural history of the genus Lactobacillus[END_REF]. This is correlated with an ability to adapt and persist under diverse environmental stresses [START_REF] Hosseini Nezhad | Stress Responses in Probiotic Lactobacillus casei[END_REF]. Thanks to this capacity, humans have succeeded in selecting and producing certain LAB strains as efficient starters for fermented foods (particularly hard cheese) and as probiotics. The adequate preservation of starter cultures is necessary though it is currently only possible using advanced desiccation technologies and storage in frozen state [START_REF] Santivarangkna | Alternative Drying Processes for the Industrial Preservation of Lactic Acid Starter Cultures[END_REF] Considering the importance of the L.casei/paracasei group for health and industrial applications, we compiled a non-redundant, annotated transposon mutant library of L. paracasei [START_REF] Licandro-Seraut | Functional genomics of Lactobacillus casei establishment in the gut[END_REF][START_REF] Scornec | Rapid 96-well plates DNA extraction and sequencing procedures to identify genome-wide transposon insertion sites in a difficult to lyse bacterium: Lactobacillus casei[END_REF] based on the Pjunc-TpaseIS1223 system, a random mutagenesis tool specifically designed for the Lactobacillus genus [START_REF] Licandro-Seraut | Development of an Efficient In Vivo System (Pjunc-TpaseIS1223) for Random Transposon Mutagenesis of Lactobacillus casei[END_REF]. Recently, using this approach we determined five Lactobacillus pentosus mutants sensitive to olive brine due to multifactorial stress [START_REF] Perpetuini | Identification of Critical Genes for Growth in Olive Brine by Transposon Mutagenesis of Lactobacillus pentosus C11[END_REF]. In addition, new genetic determinants were identified during the early stage of L. paracasei establishment in the gut [START_REF] Licandro-Seraut | Functional genomics of Lactobacillus casei establishment in the gut[END_REF] and during monofactorial perturbations of mild intensities [START_REF] Palud | New Genes Involved in Mild Stress Response Identified by Transposon Mutagenesis in Lactobacillus paracasei[END_REF]. In the present work, the L. paracasei transposon mutant library was screened to identify genes involved in the adaptation of this LAB to desiccation and rehydration. The expression of corresponding genes was studied by RT-qPCR during the two-step process to draw a chronological transcriptional profile during hydric fluctuations.

Materials and Methods

Strains and growth conditions

Wild-type (WT) L. paracasei ATCC 334 (CIP 107868, Institut Pasteur Collection) and its corresponding mutants obtained by random transposon insertion mutagenesis [START_REF] Licandro-Seraut | Development of an Efficient In Vivo System (Pjunc-TpaseIS1223) for Random Transposon Mutagenesis of Lactobacillus casei[END_REF] were grown statically at 37°C in MRS (Difco). 5 µg/mL erythromycin (Em) was used to select mutants. The mutants correspond to the 1287 genic and intergenic mutants already described [START_REF] Palud | New Genes Involved in Mild Stress Response Identified by Transposon Mutagenesis in Lactobacillus paracasei[END_REF]) (Licandro-Seraut et al., 2014). For each mutant, the putative inactivated function was assigned thanks to the genome annotation of this strain [START_REF] Cai | Genome Sequence and Comparative Genome Analysis of Lactobacillus casei: Insights into Their Niche-Associated Evolution[END_REF]. The mutant library was grown in 96-well plates (200 µl) for 48 h. Plates were mixed (30 s, 700 rpm, Eppendorf MixMate) and 10 µL of each well was used to inoculate 190 µL of MRS in new 96-well plates. Individual mutants were grown in tubes for 48h (2 ml) and vortexed to inoculate at a dilution of 1/100 in new tubes. Plates and tubes were incubated for 24h at 37°C to obtain cells in stationary phase (concentration between 1.0x10 9 and 2.0x10 9 UFC/mL) because it was the most convenient to obtain the same growth phase for the 1287 mutants, and thus to compare viability after drying.

Drying and rehydration conditions

Convective drying in a ventilated chamber at room temperature with a relative humidity (RH) of 25% was selected to mimic the severe desiccation conditions that can occur in natural environments during drought in soil, on plants, and on animal skin, for example. This process consisted in passing a flow of dry air over the cell suspension approximately simulating the desiccation conditions that organisms can undergo in the natural environment. The drying chambers were hermetic plastic boxes (20 cm × 13 cm × 6 cm) containing 100 mL saturated potassium acetate (Sigma-Aldrich) solution to obtain 25% RH at 25°C. Samples were placed on a rack in the drying chamber to keep them above the salt solution and the atmosphere was maintained using a ventilator (Sunon, Radiospare, France). Temperature and RH were controlled using an EASY Log USB tool (Lascar Electronics). Cells were rehydrated with bromocresol purple (BCP) medium which contains 5 g/L of tryptone, pepsic peptone, yeast extract and sodium acetate, 2 g/L of ammonium citrate and dipotassium phosphate, 1g/L of glucose, 1 ml of tween 80, 0.20 g/L of magnesium sulfate, 0.17 g/L of bromocresol purple and 0.05 g/L of manganese sulfate.

Effect of several sugar solutions on the viability of L. paracasei after desiccation

We selected three disaccharides related in the literature to lactic acid bacteria preservation, in particular during freeze drying [START_REF] Carvalho | Relevant factors for the preparation of freeze-dried lactic acid bacteria[END_REF][START_REF] Costa | Effect of protective agents, rehydration media and initial cell concentration on viability of Pantoea agglomerans strain CPA-2 subjected to freeze-drying[END_REF][START_REF] Jofré | Impact of different cryoprotectants on the survival of freeze-dried Lactobacillus rhamnosus and Lactobacillus casei/paracasei during long-term storage[END_REF], and their corresponding monosaccharides. L. paracasei cultures in stationary phase (1 mL) were centrifuged (5 min, 4000 g, 25°C) and pellets were suspended by vortexing with one volume of sugar solution diluted in water. After incubation for 15 min, 10 µL of cell suspensions were placed onto a sterile polypropylene (PP) coupon of 15 mm × 10 mm × 2 mm (Scientix, Fougères, France). The inoculated coupons were placed in the ventilated chamber in plastic petri dishes. For rehydration, 110 µL of BCP medium was deposited on the dried cells and the latter were resuspended by 15 successive cycles using a micropipette.

Colony enumeration by plate counts was averaged and the viability percentage was obtained with the ratio of colony enumeration (in CFU/mL) before desiccation to that after desiccation. Lactose 50 g/L was selected because it corresponds to the composition of milk, thus a condition often encountered by L. paracasei in the natural and technological environments of cheese-making, for example.

Screening of the mutant library for viability after desiccation

A screening method composed of easy and rapid steps to save time and reduce the risk of bias was developed. The whole strategy followed in this work is illustrated in figure 1. The mutant library was subjected to 24-h desiccation (pronounced desiccation) followed by rapid rehydration. Stationary growth phase cultures were mixed (1 min, 1000 rpm) and 50 µL of cells were spotted in a U bottom 96-well microplate and centrifuged (5 min, 4000g, 25°C).

Pellets were suspended with 50 µL of 50 g/L lactose diluted in water (1 min, 1700 rpm) and incubated 15 min at 20°C. Then, only 10 µL of suspension were placed in each well and the plates were placed in the ventilated chamber. After 24 h, each well was rehydrated rapidly with 110 µL of BCP medium at 37°C. This specifically designed culture medium with pH indicator turns to yellow when bacteria metabolize sugars after rehydration. Thus, the viability of the 1287 mutants after 24-h desiccation followed by rapid rehydration was evaluated in 96-well microplates by monitoring the acidification kinetics (due to sugar metabolism). Absorbance at 420 nm (A420nm) was measured in a plate reader for 7h at 37°C (Paradigm, Beckman Coulter) and was correlated to the quantity of metabolically active (or viable) bacteria. The viability of the mutants could be compared to that of the WT (A420nm (7 h) = 1.91 ± 0.08). Mutants were considered as potentially sensitive when absorbance at 7 h was lower than 1.75, which is the mean value of the WT minus twice the standard derivation, for the two biological replicates. Mutant phenotypes were subsequently validated by individual drying on PP coupons and by determining viability by the plate count method in order to remove possible false positive mutants (for instance, metabolic deficiency mutants).

Colony enumeration by plate counts was averaged and the viability percentage was obtained with the ratio of colony enumeration (in CFU/mL) before desiccation to that after desiccation (3 biological replicates). Mutants were classified into three categories according to their viability after rehydration: + (mean viability at least 1.5-fold less than WT), ++ (mean viability at least 2-fold less than WT) and +++ (mean viability at least 3-fold less than WT).

Determination of the viability of selected mutants after drying with phosphate buffer

L. paracasei cultures in stationary phase (1 mL) were centrifuged (5 min, 4000 g, 25°C) and pellets were suspended by vortexing with one volume of drying solution composed of phosphate buffer (10 mM pH 6,5). After incubation for 15 min, 10 µL of cell suspensions were placed onto a sterile PP coupon. Three coupons were prepared for each mutant. The inoculated coupons were placed in the ventilated chamber in plastic petri dishes. For rapid rehydration, 110 µL of BCP medium at 37°C was deposited on the dried cells and the latter were resuspended by 15 successive cycles using a micropipette. Colony enumeration by plate counts was averaged and the viability percentage was obtained with the ratio of colony enumeration (in CFU/mL) before desiccation to that after desiccation. Mutants exhibiting a viability percentage significantly lower than the WT (Student test, p < 0.05) and at least 1.5fold less were determined as sensitive.

2.6. Determination of the viability of selected mutants after progressive and rapid rehydration L. paracasei cultures in stationary phase (1 mL) were centrifuged (5 min, 4000 g, 25°C) and pellets were suspended by vortexing with one volume of drying solution composed of lactose 50 g/L. After incubation for 15 min, 10 µL of cell suspensions were placed onto a sterile PP coupon. Three coupons were prepared for each mutant. The inoculated coupons were placed in the ventilated chamber in plastic petri dishes. For rapid rehydration, 110 µL of BCP medium was deposited on the dried cells and the latter were resuspended by 15 successive cycles using a micropipette. For progressive rehydration, dried cells were introduced into a hermetic chamber at 99% RH for 2 h at 25°C. Then, 110 µL of BCP medium was deposited on the wet bacteria cells that were recovered by 15 successive cycles using a micropipette.

Colony enumeration by plate counts was averaged and the viability percentage was obtained with the ratio of colony enumeration (in CFU/mL) before desiccation to that after desiccation.

Mutants exhibiting a viability percentage significantly lower than the WT (Student test, p < 0.05) and at least 1.5-fold less were determined as sensitive.

2.7.Bioinformatics

The putative operon organization of corresponding genes was established using the Biocyc website. Then, genes were aligned using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) against all bacteria to determine their specificity.

2.8.RT-qPCR

The transcriptional analysis was performed using mid-exponential growth phase cells (OD600 between 0.5 and 0.6) which are the most metabolically active and which generally present the strongest and the fastest gene regulation. Cells were centrifuged and concentrated to an OD600 of 20. Cell pellets were suspended with phosphate buffer (10 mM pH 6.5) supplemented with 50 g/L lactose except for control. The suspension was incubated for 15 min at 25°C and 1 mL was deposited on a 0.22 µm polyvinylidene membrane in a glass Petri dish to prevent cell adhesion. Cells were dried in ventilated chambers and RNA extractions were performed at two different stages of desiccation: partial desiccation D1 (76% of water evaporated after 1 h) and pronounced desiccation D2 (96% of water evaporated after 2 h) which corresponded to completely dried cells (Figure 2). As rehydration kinetics is decisive for bacterial survival, rapid and progressive rehydration were applied for RNA extractions after 2 h. Rehydrated cells were subsequently detached from the membrane using a cell lifter. Total RNA isolation, cDNA synthesis and qPCR were performed as previously described [START_REF] Licandro-Seraut | Kinetics and Intensity of the Expression of Genes Involved in the Stress Response Tightly Induced by Phenolic Acids in Lactobacillus plantarum[END_REF] using TRI Reagent (Sigma Aldrich), DNase I (Roche), iScript™ Reverse Transcription Supermix (Bio-Rad) and SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad).

Primers were designed by using Primer3Plus [START_REF] Untergasser | Primer3Plus, an enhanced web interface to Primer3[END_REF] (Table 1). Quantitative PCR were performed using a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad) in triplicate, in a 20 µL-reaction mixture. Cq (threshold value) calculation was determined by a regression model of the CFX Manager™ Software. The relative transcript levels of genes were calculated using the 2 -ΔΔCT method [START_REF] Livak | Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2-ΔΔCT Method[END_REF]. In order to select appropriate reference genes, 10 potential housekeeping genes (fusA, ileS, lepA, leuS, mutL, pcrA, pyrG, recA, recG and rpoB) [START_REF] Landete | Requirement of the Lactobacillus casei MaeKR Two-Component System for L-Malic Acid Utilization via a Malic Enzyme Pathway[END_REF] were tested with all the experimental conditions and analyzed using the CFX Manager™ Software. The genes fusA, lepA and rpoB were selected as the references because they displayed the lowest M values (0.22) and coefficients of variation (0.09), meaning that they have the most stable expression in the tested conditions.

Results

Selection of a protective drying solution for the screening

A library containing a total number of 1287 L. paracasei mutants was screened for their capacity to resist a 24-h desiccation period followed by rapid rehydration in comparison with their parental strain (L. paracasei ATCC 334, named WT). The viability of L. paracasei WT after 24-h desiccation at 25% RH was determined with and without the protectors (Table 2).

Viability was very low in the absence of sugar and with monosaccharides. On the contrary, with disaccharides, viability was between 19 and 45%, which was an adequate range for screening sensitive mutants.

Identification of genes involved in resistance to pronounced desiccation followed by rapid rehydration.

Screening of the 1287 transposon mutants resulted in the detection of 47 sensitive mutants (A420nm at 7 h < 1.75 for 2 biological replicates). After the validation step by individual drying of the 47 mutants, 16 mutants displayed a significant decrease of survival at least 1.5-fold less compared to the WT (p<0.05) (Table 3). Identified genes were mostly involved in metabolism and transport, cell wall structure and function, regulation of stress response and DNA related enzymes. Moreover, three sensitive mutants containing transposon in a gene encoding a hypothetical protein were identified (LSEI_0040, LSEI_1045 and LSEI_1316). The majority of mutants (12) were categorized as +. The two most sensitive mutants (+++) were disrupted for a putative ribonucleotide diphosphate reductase (LSEI_1468) and for a hypothetical protein (LSEI_ 1316). Two mutants disrupted for a putative PTS (LSEI_0178) and for a putative alpha/beta fold family hydrolase (LSEI_0756) were categorized as ++. Nine genes out of the 16 belonged to putative operons (Table 4). Seven genes were specific to L. paracasei or related species, four to Lactobacillus genus and five were well conserved among Gram (+) bacteria. Two genes among the three encoding hypothetical proteins presented a transmembrane domain (LSEI_0040 and LSEI_1045). All hypothetical protein genes were specific to the L. paracasei group.

Analysis of selected mutant after pronounced desiccation with phosphate buffer

We chose to omit the protector in order to strictly analyze the impact of the drying process.

The WT and the 16 sensitive mutants identified previously (viability reduction > 1.5 in comparison of the WT) were subjected to a desiccation for 24 h without protector. In these conditions, the viability of the WT was 0.059 ± 0.006 %, which is considerably lower than that obtained for the pronounced desiccation with lactose 50 g/L. Among the 16 sensitive mutants identified, nine were also more sensitive to a drying without protector than the WT (Table 5). They are disrupted for the following genes: a putative diadenosine tetraphosphatase (LSEI_0167), a PTS system (LSEI_0178), a Pyridoxine 5'-phosphate oxidase (LSEI_0363), an NAD (FAD)-dependent dehydrogenase (LSEI_0397), a ribonucleotide reductase (LSEI_1468), a sorbitol operon transcription regulator (LSEI_2725) and hypothetical protein (LSEI_0040, LSEI_1045 and LSEI_1316). As observed for drying with lactose, the most sensitive mutant was disrupted for LSEI_1468.

Analysis of selected mutant after pronounced desiccation followed by progressive rehydration

To assess if the identified genes were involved during dehydration whatever the rehydration process, the WT and the 16 sensitive mutants identified previously (viability reduction > 1.5 in comparison of the WT) were subjected to desiccation for 24-h followed by progressive rehydration (for 2 h) instead of rapid rehydration. In these conditions, the viability of the WT was 98 ± 3%, which is considerably higher than obtained with a rapid rehydration. Among the 16 sensitive mutants identified previously, only four were also sensitive after desiccation and progressive rehydration (Table 6). They encode a putative diadenosine tetraphosphatase (LSEI_0167), an NAD (FAD)-dependent dehydrogenase (LSEI_0397), a cation transport ATPase (LSEI_0749) and a ribonucleotide reductase (LSEI_1468). As observed for desiccation and rapid rehydration, the most sensitive mutant was ribonucleotide diphosphate reductase.

Transcriptional analysis of the identified genes during a desiccationrehydration cycle

Mutant library screening allowed the identification of 16 genes involved in the global perturbation which consisted in desiccation followed by rehydration. Thus, it was not possible to determine during which phase these genes were involved since rehydration was necessary to analyze mutant phenotypes. To determine the chronology of the genetic mechanisms during humidity fluctuations, transcriptional analysis of the identified genes was carried out on the WT.

As lactose was required to ensure good survival of bacteria during the desiccation/rehydration process it was reasonable to expect that some candidate genes could be differentially expressed in the presence of lactose. However, for the 16 genes, expressions after 15-min incubation in lactose were comparable to that after a 15-min phosphate buffer incubation (Figure 3). Differentially expressed genes were classified to obtain the most upregulated (Table 7, values in dark red) and downregulated (Table 7, values in dark blue) genes, with a mean expression value higher or lower than 2.0. Among the 16 genes studied, 13 were differentially expressed (p < 0.05) for at least one condition (Table 7). The three genes involved in hydric changes but not differentially expressed encoded a putative PTS transporter (LSEI_0178), a reductase (LSEI_0758), and hypothetical proteins (LSEI_1045).

Four genes were upregulated for all the hydric fluctuations tested. These genes encoded a putative diadenosine tetraphosphatase (LSEI_0167), a polysaccharide transporter (LSEI_0238), a pyridoxine 5'-phosphate oxidase (LSEI_0363), and hypothetical proteins (LSEI_0040). Also, LSEI_0397 encoding a putative NAD dependent dehydrogenase was upregulated during desiccation and progressive rehydration. Conversely, LSEI_0749 encoding a putative cation transport ATPase was upregulated during desiccation and rapid rehydration. Interestingly, these genes were the most upregulated (> 2-fold change) for at least one condition. One gene, LSEI_1754, encoding a putative SAICAR synthase was the most downregulated gene in these experiments (mean expression value < 2.0 for all the drying and rehydration conditions).

One gene, LSEI_2725 (a putative sorbitol operon transcription regulator) was upregulated after only 2 h of desiccation and represent potential dry state biomarkers. LSEI_1316 encoding a putative hypothetical protein was upregulated after 1 h and 2 h of desiccation.

Interestingly, no gene was specifically upregulated during subsequent rehydration.

Conversely, four genes were downregulated specifically during rehydration: LSEI_0756 (a putative hydrolase) LSEI_1009 (a putative spermidine/putrescine-binding protein), LSEI_1468 (a putative ribonucleotide reductase) and LSEI_1659 (a putative glucokinase).

Discussion

Since water is the fundamental component of all living organisms, desiccation and rehydration alternation is one of the most prevalent and severe stress for most microorganisms. Adaptation to this stress occurs via a combination of mechanisms which depend on the genetic background of the microorganism. In L. paracasei, we developed a strategy to identify genes involved in the adaptation to hydric fluctuations using random transposon mutagenesis and targeted transcriptional analysis.

Sixteen desiccation-sensitive mutants were identified from the L. paracasei mutant library screening after desiccation followed by rapid rehydration. Contrary to our recent screening of this library for mild stress sensitivity [START_REF] Palud | New Genes Involved in Mild Stress Response Identified by Transposon Mutagenesis in Lactobacillus paracasei[END_REF], no mutants for putative promoters were identified as sensitive to desiccation. Seven of the identified genes were specific of the L. casei/paracasei/rhamnosus group. On the contrary, five genes were well conserved among Gram-positive bacteria. Interestingly, these genes, although present in Listeria monocytogenes genome, were not identified by the screening of L. monocytogenes random mutants for desiccation resistance [START_REF] Hingston | Insertional mutagenesis reveals genes associated with desiccation and osmotic stress in Listeria monocytogenes[END_REF]. Otherwise, this study of L. monocytogenes desiccation resistance shows the involvement of a gene encoding a putative glutathione peroxidase, known to prevent oxidative damages. This gene is present in the L. paracasei mutant library but was not identified as sensitive. These differences may result from the drying conditions applied such as the surface used for desiccation, the relative humidity, the drying medium or the rehydration conditions.

Metabolism and transport

Two transporters are required for survival to hydric fluctuation including a putative phosphotransferase system (PTS, LSEI_0178) and one cation transporter (LSEI_0749).

Several authors have reported a link between PTS systems and stress response for

Lactobacillus genus. L. plantarum mutants with an impaired expression of the mannose PTS operon exhibited increased sensitivity to peroxide, probably due to a diminution of glucose capture and energy production [START_REF] Stevens | Involvement of the Mannose Phosphotransferase System of Lactobacillus plantarum WCFS1 in Peroxide Stress Tolerance[END_REF].

Next, we identified a membrane associated protein (LSEI_1009) predicted to bind polyamines before their transport into the cell by the ABC transporters encoded by other genes of the LSEI_1005-1009 operon. Polyamines have various important physiological roles during stress response, including the modulation of gene expression, signal transduction, oxidative defense mechanism, and cell-to-cell communication [START_REF] Miller-Fleming | Remaining Mysteries of Molecular Biology: The Role of Polyamines in the Cell[END_REF][START_REF] Shah | A multifaceted role for polyamines in bacterial pathogens[END_REF].

One identified gene encoded a glucokinase (LSEI_1659), a key enzyme of glucose metabolism. In S. aureus, glucokinase is also involved in pathogenicity (biofilm formation, virulence factors, cell wall synthesis) [START_REF] Kumar | Comparative Structural and Functional Analysis of Staphylococcus aureus Glucokinase with other Bacterial Glucokinases[END_REF]. In our case, glucokinase as well as PTS interruption could lead to energy diminution and then sensitivity to hydric fluctuations.

Another gene, LSEI_0363, encodes a putative enzyme that catalyzes the oxidation of pyridoxamine-5-P (PMP) and pyridoxine-5-P (PNP) to pyridoxal-5-P (PLP) in the vitamin B6 pathway. Vitamin B6, an antioxidant molecule, has been implicated in defense against cellular oxidative stress in Saccharomyces cerevisiae [START_REF] Chumnantana | Vitamin B6 compounds prevent the death of yeast cells due to menadione, a reactive oxygen generator[END_REF].

Cell wall function and structure

Modification at the cell wall level appears to be essential for surviving hydric fluctuation, considering the three genes related to the cell wall and identified as essential for hydric fluctuation survival. The gene LSEI_0238 encodes a polysaccharide transporter involved in the export of lipoteichoic acid (LTA), a major constituent of the Gram-positive cell wall [START_REF] Cai | Genome Sequence and Comparative Genome Analysis of Lactobacillus casei: Insights into Their Niche-Associated Evolution[END_REF]. Interestingly, this gene was also needed for L. paracasei gut establishment [START_REF] Licandro-Seraut | Functional genomics of Lactobacillus casei establishment in the gut[END_REF]. The mutant for LSEI_2546, another polysaccharide transporter involved in LTA export, was not identified as sensitive.

Finally, two hypothetical proteins exhibited a transmembrane domain (LSEI_0040 and LSEI_1045). Interestingly LSEI_0040 was involved in the adaptation of L. paracasei to thermal, ethanol and oxidative stresses [START_REF] Palud | New Genes Involved in Mild Stress Response Identified by Transposon Mutagenesis in Lactobacillus paracasei[END_REF]. The present and previous results

show that membrane proteins are important for L. paracasei adaptation to various environments and involved in general stress response.

Regulation of stress response

One gene involved in alarmone degradation is needed for L. paracasei survival during hydric fluctuations. Alarmones are putative chemical messengers produced during environmental changes. LSEI_0167 encodes a putative diadenosine tetraphosphatase, an enzyme that hydrolyzes diadenosine tetraphosphate (Ap4A) into two molecules of adenosine diphosphate (ADP). Ap4A has been reported to be involved in heat stress response in E.coli [START_REF] Lee | AppppA, heat-shock stress, and cell oxidation[END_REF]. Moreover apaH (diadenosine tetraphosphatase) mutation causes Ap4A accumulation and sensitivity to thermal stress [START_REF] Johnstone | AppppA binds to several proteins in Escherichia coli, including the heat shock and oxidative stress proteins DnaK, GroEL, E89, C45 and C40[END_REF]. Nevertheless, Despotović and collaborators wondered whether Ap4A was an alarmone or a damage metabolite because they observed that no signaling cascade was triggered by Ap4A and that its accumulation at high level was toxic for E. coli [START_REF] Despotović | Diadenosine tetraphosphate (Ap4A) -an E. coli alarmone or a damage metabolite?[END_REF]. LSEI_2725 (gutR) has been identified as a determinant for surviving hydric fluctuations.

Although it is annotated as a sorbitol operon transcription regulator, its predicted function should be reconsidered. [START_REF] Alcantara | Regulation of Lactobacillus casei Sorbitol Utilization Genes Requires DNA-Binding Transcriptional Activator GutR and the Conserved Protein GutM[END_REF] reported that in L. paracasei ATCC334, gutR is split into 3 ORFs (LSEI_2728, LSEI_2726 and LSEI_2725) as the result of transposition events, and consequently this strain is unable to use sorbitol, contrary to L. casei BL23. In addition, the LSEI_ 2724 (gutM) gene, encoding a sorbitol operon activator with gutR, is dispensable for desiccation resistance. In conclusion, we assume that LSEI_2725 functions as a regulator but independently of the sorbitol operon.

DNA related enzymes

It was widely reported that desiccation induces DNA damage [START_REF] Fredrickson | Protein oxidation: key to bacterial desiccation resistance?[END_REF][START_REF] Potts | Desiccation tolerance of prokaryotes[END_REF]. Indeed, the mutant library screening identified two genes involved in nucleotide metabolism, LSEI_1468 (a ribonucleotide reductase, RNR) and LSEI_1754 (a SAICAR synthase). The RNR mutant was particularly sensitive (mean viability 10-fold less than WT).

RNR is an essential enzyme since its mediates the synthesis of deoxyribonucleotides, the precursors of DNA synthesis [START_REF] Torrents | Ribonucleotide reductases: essential enzymes for bacterial life[END_REF], and we have previously reported that the RNR gene was involved in general response to mild stresses in L. paracasei [START_REF] Palud | New Genes Involved in Mild Stress Response Identified by Transposon Mutagenesis in Lactobacillus paracasei[END_REF]. SAICAR synthase is involved in de novo purine biosynthesis [START_REF] Nelson | Mechanism of Action of Escherichia coli Phosphoribosylaminoimidazolesuccinocarboxamide Synthetase[END_REF]. These two genes could be involved in DNA repair. Recently, [START_REF] García-Fontana | A New Physiological Role for the DNA Molecule as a Protector against Drying Stress in Desiccation-Tolerant Microorganisms[END_REF] reported that the DNA molecule was overproduced and acted as a protein protector in desiccation tolerant bacteria. This phenomenon could occur in L. paracasei.

Taking into account desiccation and rehydration kinetics

The results indicate that rapid rehydration of WT strain cells was more detrimental than progressive rehydration. It has been reported that progressive rehydration could reduce the stress applied to bacteria as it promotes membrane integrity recovery [START_REF] Lang | Drying parameters greatly affect the destruction of Cronobacter sakazakii and Salmonella Typhimurium in standard buffer and milk[END_REF][START_REF] Zoz | Control of Relative Air Humidity as a Potential Means to Improve Hygiene on Surfaces: A Preliminary Approach with Listeria monocytogenes[END_REF]. We found nine common sensitive mutants after drying with or without protector and only four sensitive mutants whatever their rehydration kinetics. Identified genes were generally specific to the screening strategy applied for the mutant library. It is very likely that the modifications of drying conditions lead to the identification of different genes. Studies on Salmonella species exposed to desiccation stress showed a response specificity in function of methodologies used [START_REF] Finn | ProP Is Required for the Survival of Desiccated Salmonella enterica Serovar Typhimurium Cells on a Stainless Steel Surface[END_REF]

Transcriptional profiles

Our findings are schematized in Figure 3. Among the genes essential for survival, three genes were expressed at the same level as the control whatever the hydration phase. This result again shows the strength of global reverse genetics, since some genes may be essential for a function, although expressed constitutively. Four transcriptional profiles were observed during hydric fluctuations: six genes were upregulated during both desiccation and rehydration, two genes were upregulated only during the desiccation stage, one gene was downregulated during both desiccation and rehydration, four genes were downregulated only during the rehydration stage. We assume that adaption to hydric fluctuations occurs during dehydration and continues or not during rehydration. Up regulation of genes was particularly observed after 2 hours of drying (96% of water evaporated). These results suggest that this step could be the most stressful for bacteria. Our data highlighted the importance of cell wall structure as well as diadenosine tetraphosphate degradation since LSEI_0238 and LSEI_0167 were up regulated for all dehydration and rehydration phases.

In conclusion, this work identified 16 genetic determinants for the resistance of L. paracasei to desiccation. Transcriptional analysis of the corresponding genes highlighted that six genes were upregulated during both desiccation and rehydration and two during desiccation only. Thus, our results show that desiccation is a critical phase for inducing stress response in L.

paracasei. This analysis provides clues for developing genetic biomarkers to monitor the intensity of desiccation during industrial processes since we identified two genes overexpressed only when the cells are dried during 2 h at 25% RH (dry state). However, it was reported that drying conditions (surface used for desiccation, the drying medium, the rehydration conditions) may modify the genes involved in the desiccation stress. Indeed, gene expression of these two genes should be studied during industrial drying process before stating that they constitute genetic biomarkers. Some genes identified in this study have higher potential since they are not limited to L. paracasei genomes. From an applied point of view, these genes will be preferred targets for obtaining more efficient Lactobacillus starters.

Transcriptional studies during freeze drying could bring complemental information. In a fundamental point of view, these findings provide novel insights into the genetic mechanisms involved in desiccation and rehydration adaptation in L. paracasei. Table 1. Primers designed for RT-qPCR.

Tables

Primers

Sequence (5' 3') Primers Sequence (5' 3')

0040-F CCACAAATGAGTCAAACCATCT 0040-R GGTCGTTATCGCACCTAATGA 0167-F ATGCCGACTGCTGTTGAGTT 0167-R CGGACCCACATTCGATTAAA 0178-F TAGGCAACATTCGCAGTCAA 0178-R ATAATCGGCAACGGCTTTC 0238-F CGGATCGCTTCCCTTATTCT 0238-R CCAACATCTGTGAGCCAGAC 0363-F GGTCCCAAGGGTTCACTACA 0363-R GCCAAATGGTTGTTCGAGAG 0397-F GGCACGACGCAGGATTAT 0397-R CCACGCATCAAGAAGACATT 0749-F ATGACTGGTGACGGTGTGAA 0749-R AACGAAATCCGCAATAGAGC 0756-F CACTGACAGCCACGATCAAA 0756-R CTTGCTGCCGTGAATAAACA 0758-F TGATTCAGGCACAACGATTT 0758-R GCGTCACGTCGGTTTCTTTA 1009-F CAGGTGAAGCCAGTGAGATG 1009-R CCGTCTTAGGGATGACCAAGT 1045-F CAATACACAGACCGCCAGTG 1045-R ATCGTTGGCTTTGTCGTCTT 1316-F CGCCGCATGAAGTATGTTTA 1316-R GAGGGCTCAACACGTTTAACA 1468-F CAATGGTTCGTGCTTTGACTT 1468-R TGCTGTGAGCCTGGTGATTA 1659-F TGGATGAAGGCAGTCACATC 1659-R CCCATACCGATACCGACAAA 1754-F CCGCCAATCCACGAATACTA 1754-R GCACTTGCTTGAGTTGTTCAG 2725-F CAGATCGACCATTGACACACA 2725-R GCCGATGCCCTTACCTTAGT fusA-F CCTGAAACTGGCGAAACATT fusA-R CCAACCTTAGCAGCAACCTT ileS-F GCAACGGTTGACTCTTCCTC ileS-R GCTACCATAAACCGCATCGT lepA-F AAGAGCAGCACGAAGGGATA lepA-R AGGATCGCCATTAAGCAAGA leuS-F GCTGGATGCTGGTATTGCTT leuS-R ATCACGCAGTTTGCCTTCAT mutL-F CCTGCCAGTGTTGTCAAAGA mutL-R CGTTATCGCTAACCCGAATC pcrA-F GCAACACAAGCGTTGAGAAG pcrA-R ACCAGCAATCGGACTGAGTT pyrG-F GAAGCGTGATGTTGGTTCG pyrG-R TGTGCTGAGTTGGTTTCGTC recA-F TTGGCAACCGATAAAGACAA recA-R TGAGATGCGTTGACAAGTCC recG-F CGTAGCGTGATTCTGGTGAC recG-R TTCCAAAGATTGCTGCTTCA rpoB-F AATACAAGGCAGCCCATGAC rpoB-R ATCCAAGGCACCATCTTCAC

  Mutants were classified into three categories according to their viability: + (mean viability at least 1.5-fold less), ++ (mean viability at least 2-fold less) and +++ (mean viability at least 3fold less than WT). *, significant changes in viability (p<0.05) compared to the WT strain (3 biological replicates). **, significant changes in viability (p<0.01) compared to the WT strain (3 biological replicates).

Figure legends Figure 1 .

 legends1 Figure legends

Figure 2 .

 2 Figure 2. Evolution of the cell suspension mass during desiccation (orange curve) and

Figure 3 .

 3 Figure 3. Schematic representation of genes differentially transcribed in L. paracasei

Table 2 . L. paracasei ATCC 334 viability after a pronounced desiccation period followed by rapid rehydration with or without saccharides used as protectors.

 2 Bacterial cells in stationary growth phase suspended in various protective solutions were air dried for 24 h at 25% RH and 25°C on PP coupons. Survival was determined after suspending dried cells in BCP medium. Viability was measured by plate count (CFU/ml).

	Condition of	Concentration	Viability percentage
	drying	(g/L)	after desiccation/rehydration
	No protector (distilled water)		0.001 ± 0.000
	Monosaccharides
	Glucose	25	0.13 ± 0.09
		50	0.01 ± 0.01
	Fructose	25	0.01 ± 0.01
		50	0.04 ± 0.02
	Galactose	25	0.05 ± 0.02
		50	0.09 ± 0.04
	Disaccharides
	Lactose	25	21 ± 2
		50	35 ± 7
	Trehalose	25	41± 9
		50	19 ± 7
	Sucrose	25	27 ± 9
		50	45 ± 0

Table 3 . Viability of L. paracasei sensitive mutants after a pronounced desiccation period followed by rapid rehydration

 3 Cells in stationary growth phase were air dried for 24 h at 25% RH and 25°C with 50 g/L lactose solution in microplates or on PP coupons. Sensitive mutants were determined by monitoring the A420nm of dried cells suspended in BCP medium (2 biological replicates) and confirmed by individual drying on PP coupons and plate count (3 biological replicates).

	Disrupted	Predictive function	A420nm at 7 h	Viability %	Sensitivity
	gene				level
	WT		1.91 ± 0.08	41 ± 2
	LSEI_0040 Hypothetical protein	1.56 / 1.69	23 ± 4**	+
	LSEI_0167 Diadenosine tetraphosphatase	1.71 / 1.64	22 ± 2**	+
	LSEI_0178 PTS	1.73 / 1.59	18 ± 9*	++
	LSEI_0238 PST transporter	1.63 / 1.66	23 ± 7**	+
	LSEI_0363 Pyridoxine 5'-phosphate oxidase	1.74 / 1.75	26 ± 6*	+
	LSEI_0397 NAD (FAD)-dependent dehydrogenase	1.34 / 1.40	25 ± 5**	+
	LSEI_0749 Cation transport ATPase	1.74 / 1.75	23 ± 3**	+
	LSEI_0756 Alpha/beta fold family hydrolase	1.62 / 1.75	18 ± 1**	++
	LSEI_0758 Aldo/keto reductase related enzyme	1.45 / 1.48	22 ± 3**	+
	LSEI_1009 Spermidine/putrescine-binding protein	1.63 / 1.45	26 ± 1**	+
	LSEI_1045 Hypothetical protein	1.72 / 1.69	23 ± 4**	+
	LSEI_1316 Hypothetical protein	1.30 / 1.27	14 ± 5**	+++
	LSEI_1468 Ribonucleotide diphosphate reductase	1.10 / 1.11	4 ± 1**	+++
	LSEI_1659 Glucokinase	1.65 / 1.66	25 ± 8*	+
	LSEI_1754 SAICAR synthase	1.75 / 1.46	23 ± 4**	+
	LSEI_2725 Sorbitol transcription regulator	1.74 / 1.70	27 ± 5*	+

Acknowledgments

This work was funded by the Regional Council of Bourgogne Franche-Comté and the Fonds Européen de DEveloppement Régional (FEDER). AP was supported by a PhD fellowship from the Ministère de l'Enseignement Supérieur, de la Recherche et de l'Innovation (MESRI). We thank the other members of the laboratory for their assistance, including, in particular, Christine Rojas for her skillful technical assistance. We thank the Pasteur Institute Microorganism Collection for the L. paracasei strain (CIP 107868).

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Author Contributions

Study conception and design: AP, KS, HL, LB and JFC. Acquisition of data: AP, KS.

Analysis and interpretation of data: AP, HL, LB and JFC. Drafting of manuscript: AP. Critical revision: AP, HL, LB and JFC. All authors read and approved the final manuscript. LAC (cells incubated 15 min with lactose), D1 (cells dehydrated for 1 h), D2 (cells dehydrated for 2 h), RR (rapid rehydration with 1 mL of distilled water), PR (progressive rehydration in a closed chamber with RH adjusted at 99% for 2 h). RTL were calculated using 2 -ΔΔCt method. For the phosphate buffer control condition, a gene expression value of 1.0 was attributed and RTL of genes in stress condition were calculated as a function of this value.

Positive values (> 1.0) represent upregulation and negative values (< 1.0) represent downregulation. *, significant changes in gene expression (p<0.05) compared to the phosphate buffer condition (4 biological replicates). Values in light red correspond to