Insaf Nachi

Imene Fhoula

Imene Smida

Hadda-Imene Ouzari

Mnasser Hassouna

Microbiological analysis and Assessment of biotechnological potential of lactic acid bacteria isolated from Tunisian flours

Keywords: Baking flour, lactic acid bacteria, acidification, proteolytic, amylolytic, antimicrobial activity

The present work was carried out to identify lactic acid bacteria (LAB) from wheat flours and to evaluate their technological capabilities for potential incorporation in sourdough process. Six samples of baking wheat flours were collected from mills situated in different geographical regions of Tunisia and used for microbiological analysis of total mesophilic aerobic microorganisms, yeasts, moulds and LAB. A total of 45 autochthonous LAB were isolated and identified by genetic analysis of 16S-23S rRNA intergenic transcribed spacer ITS generated patterns ITS-PCR. One representative strain of each ITS-PCR pattern was subjected to partial sequencing of the 16S rRNA. Strains were identified as Weissella cibaria, Lactobacillus plantarum, Lactobacillus brevis, Pediococcus pentosaceus, Pediococcus pentoseus, Pediococcus acidilactici, Enterococcus faecium, Enterococcus casseliflavus and Enterococcus faecalis. Technological features including acidification, antimicrobial, amylolytic, proteolytic and antioxidant activities of six selected LAB strains were investigated for future in situ applications. Moreover, LAB were investigated for their ability to produce exopolysaccharides. All tested LAB showed good acidifying ability by decreasing significantly (p<0.05) the pH of flour extract below 4.0 after 24 h and below 3.0 after 72 h. P. pentoseus and P. acidilactici presented fermentation quotient FQ (ratio of lactic and acetic acids) close to the optimal range. All flour LAB isolates, demonstrated extracellular proteolytic activity. W. cibaria S25 had the highest radical-scavenging activity with a rate of 25.57%. L. plantarum S28 demonstrated the highest amylolytic activity (1386 U/mL) followed by P. acidilactici S16 (1086 U/mL). Although, L. plantarum S28 showed the highest production of exopolysaccharides (0.97 g/L). Moreover, different halo of inhibition were detected against E. coli, Staphylococcus aureus, Aspergillus niger and Penicillium expansum. This study revealed that autochthonous flour LAB exhibited interesting technological features and thus could be used in sourdough production.

Introduction

Cereals are one of the most important food sources worldwide for both humans and animals. They supplies a considerable portion of the nutrients required for growth, well-being and maintenance of health (FAO 2002;FAO 2014). In the past, many populations of several cultures are directly linked to cereals and, thus are recognized by the cereals they eat. This is the case of Mediterranean people which are recognized to be "wheat people" [START_REF] Alfonzo | Diversity and technological potential of lactic acid bacteria of wheat flours[END_REF]. Tunisia is placed among the largest wheat per capita consumers in the world with a total of 2.8 million metric tons MT per year between 2016/2017 (USDA 2016). Moreover, wheat bread is the most popular staple food consumed in Tunisia under different forms such as backer's yeast bread, Tabouna, Mlawi, Mtabga [START_REF] Mamhoud | Selection of lactic acid bacteria isolated from Tunisian cereals and exploitation of the use as starters for sourdough fermentation[END_REF].

However, cereal grains, including wheat, are naturally contaminated by microorganisms (yeast, moulds and bacteria) which can occur in the field during growth, harvest, post-harvest and storage [START_REF] Eglezos | Microbiological Quality of Wheat Grain and Flour from Two Mills in Queensland, Australia[END_REF].

During milling, a small fraction of microorganisms initially concentrated in the outer layers of kernels, could contaminate the flour, leading to microbial rate increase during storage [START_REF] Berghofer | Microbiology of wheat and flour milling in Australia[END_REF]. The microflora composition and the relative species ratio on wheat grains are influenced by several factors including temperature, moisture, physical damage caused by molds and application of pesticides [START_REF] Karlsson | Fungicide Effects on Fungal Community Composition in the Wheat Phyllosphere[END_REF][START_REF] Fleurat-Lessard | Integrated management of the risks of stored grain spoilage by seedborne fungi and contamination by storage mould mycotoxins -An update[END_REF]. Several works highlights that flour's wheat are dominated by fungi. Food spoilage by moulds and the occurrence of their mycotoxins constitute a potential health hazard (Čonková et al. 2006).

Among the microorganisms associated with flours, lactic acid bacteria (LAB) play an important role in preserving the balance of microbial flora and enhancing the shelf life of final products by controlling and inhibiting spoilage organisms during fermentation, due to their antimicrobial properties [START_REF] Dalié | Lactic acid bacteria -Potential for control of mould growth and mycotoxins[END_REF][START_REF] Cizeikiene | Antimicrobial activity of lactic acid bacteria against pathogenic and spoilage microorganism isolated from food and their control in wheat bread[END_REF].

LAB are commonly present in various habitats, including milk and dairy products, vegetables, meat and cereal products, where fermentation can take place [START_REF] Giraffa | Overview of the ecology and biodiversity of the LAB[END_REF]) and they constitute the majority of the rate of the commercial starter cultures (Corsetti and Settanni 2007;[START_REF] Vuyst | Biodiversity, ecological determinants, and metabolic exploitation of sourdough microbiota[END_REF][START_REF] Guyot | Fermented cereal products[END_REF]. LAB have been isolated, identified and characterized from cereals [START_REF] Mamhoud | Selection of lactic acid bacteria isolated from Tunisian cereals and exploitation of the use as starters for sourdough fermentation[END_REF] or mature sourdoughs [START_REF] Digaitiene | Lactic acid bacteria isolated from rye sourdoughs produce bacteriocin-like inhibitory substances active against Bacillus subtilis and fungi[END_REF] or fermented dough [START_REF] Mhir | Microflora distribution and species ratio of Tunisian fermented doughs for bakery industry[END_REF].

However, few studies were focused on the characterization of LAB isolated from wheat flours (Corsetti et al. 2007;[START_REF] Alfonzo | Diversity and technological potential of lactic acid bacteria of wheat flours[END_REF]). To our knowledge, there are no previous studies focused on the characterization of lactic acid bacteria isolated from Tunisian wheat flours.

Despite the availability of essential nutrients for LAB growth, the investigation of flour's LAB content remains difficult and critique because of the low water content [START_REF] Alfonzo | Diversity and technological potential of lactic acid bacteria of wheat flours[END_REF]. Indeed, within flours, several LAB species are found in a dormant state (Corsetti et al. 2007). Some studies showed that Lactobacillus and Enterococcus were the most abundant in Italian flours (Corsetti et al. 2007;[START_REF] Robert | Biodiversity of lactic acid bacteria in French wheat sourdough as determined by molecular characterization using species-specific PCR[END_REF]. The aims of this study were to: (i) enumerate total mesophilic aerobic microorganisms, yeasts, fungi and LAB in flour samples;

(ii) isolate, identify and characterize LAB naturally occurring on Tunisian wheat flour; (iii) and evaluate the technological capabilities of LAB by examining their acidifying ability in a flour extract system, proteolytic, amylolytic, antibacterial and antifungal activities for a further incorporation as starter in sourdough formulation.

Materials and Methods

Sampling and microbiological analyses

Six samples of baking wheat flours were collected from mills situated in different geographical regions of Tunisia (Nabeul, Tunis, Sousse, Djerba and Sfax), and used for microbiological analysis of total mesophilic aerobic microorganisms, yeasts, moulds and Lactic acid bacteria (LAB) (Table 1). Ten grams of flour were homogenized with 90 mL of sterile peptone water solution (peptone 0.1% and NaCl 0.85%) [START_REF] Minervini | Lactic Acid Bacteria in durum wheat flour are endophytic components of the plant during its entire life cycle[END_REF]. Total mesophilic aerobic microorganisms, yeasts and moulds were enumerated using the PCA (Plate Count Agar) and SD (Sabouraud Dextrose) agar media with chloramphenicol 5%, respectively [START_REF] Minervini | Lactic Acid Bacteria in durum wheat flour are endophytic components of the plant during its entire life cycle[END_REF]. Lactic acid bacteria (LAB) were enumerated using MRS medium (Man, Rogosa, Sharpe agar) supplemented with 0.01% of cycloheximide (MP Biomedicals) to avoid fungal growth and 0.0025% of bromocresol green (MP Biomedicals) to indicate acidification of the medium (yellow turn) by LAB.

Isolation of lactic acid bacteria

The isolation of lactic acid bacteria was carried by enriching 10 g of each sample into 90 mL of modified-MRS (mMRS) broth (1% maltose, 1% lactose and 10% yeast extract). Cultures were incubated at 30°C for 3 days.

Then 1 mL of each resulting culture was diluted using decimal dilutions and plated in MRS agar supplemented with 0.0025% of bromocresol green (MP Biomedicals) and 0.01% cycloheximide (MP Biomedicals) and incubated under anaerobic conditions at 30 °C for 48-72 h. Acid-producing bacteria characterized by a yellow zone around each colony were picked and purified on MRS agar. Gram-positive and catalase negative isolates were restreaked and cultivated into the same agar medium. The pure cultures were maintained in 20% of glycerol (v/v) and conserved at -80 °C.

DNA extraction, characterization and molecular identification of lactic acid bacteria

Genomic DNA was extracted according to the method described by [START_REF] Wilson | Preparation of genomic DNA from bacteria[END_REF] using CTAB/NaCl method.

Bacterial strains were characterized genotypically by profile analysis of the 16S-23S rDNA Internal Transcribed Spacer (ITS) region using universal primers (s-d-bact-1494a-20 and s-d-bact-0035-a-15) [START_REF] Daffonchio | PCR fingerprinting of whole genomes: the spacers between the 16S and 23S rRNA genes and of intergenic tRNA gene regions reveal a different intraspecific genomic variability of Bacillus cereus and Bacillus licheniformis[END_REF]. Bacteria strains presenting the same band patterns were clustered in the same ITS-haplotype. One or two representative strains from each cluster have been selected for 16S rDNA gene PCR amplification which was performed using universal primers (s-d-bact-0008-a-S-20 and s-d-bact-1495-a-A20) according to [START_REF] Daffonchio | PCR fingerprinting of whole genomes: the spacers between the 16S and 23S rRNA genes and of intergenic tRNA gene regions reveal a different intraspecific genomic variability of Bacillus cereus and Bacillus licheniformis[END_REF]. The two PCR were performed on a thermocycler (BioRad) using this program: 94 °C for 3 min, 35 cycles of 94 °C (45s), 55 °C (1 min), 72°C for 2 min and a final cycle at 72 °C for 10 min. PCR products were separated by electrophoresis through 1.5% (w/v) agarose gel containing ethidium bromide (0.5 mg/mL).

The 16S rDNA PCR amplicons were purified with Exonuclease-I and Shrimp Alkaline Phosphatase (Exo-Sap, Fermentas, Life Sciences) based on the manufacturer's standard protocol. DNA sequencing was performed in an automated capillary DNA sequencer (Applied Biosystems 3130XL) using a Big Dye Terminator cycle sequencing Kit V3.1 (Applied Biosystems). Sequence identification to the closet relative taxa of the strains was achieved using BLAST analysis tool [START_REF] Tamura | MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[END_REF] in the GenBank DNA database (www.ncbi.nih.gov).

Phylogenetic analysis of 16S rRNA gene sequences was conducted with MEGA-6 software [START_REF] Tamura | MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[END_REF].

Phylogenetic tree was constructed by using neighbor-joining method [START_REF] Saitou | The neighbor-joining method: a new method for reconstructing phylogenetic trees[END_REF].

Technological characterization of LAB

Acidification activity

To evaluate the acidification activity of isolated LAB, a sterile flour extract (SFE) was prepared according to the method described by [START_REF] Alfonzo | Diversity and technological potential of lactic acid bacteria of wheat flours[END_REF]: 200 g of flour was suspended in distilled water (1 L) and sterilized at 121 °C for 20 min. After precipitation, the flour was removed. The supernatant was used as liquid broth in subsequent experiments. Overnight LAB cultures on MRS broth were centrifuged (5000 x g for 5 min and washed 3 times with sterile distilled water. LAB cells were inoculated in 50 mL of the solution SFE with 1% (v/v) of bacterial suspension at 10 9 CFU/mL and incubated at 30 °C. The pH measurements were taken initially, after 24 h, 48 h and 72 h of inoculation.

Strains that rapidly and greatly decreased SFE pH, were analyzed for their ability to produce lactic and acetic acids, following 24, 48 and 72h of fermentation. Each resultant sample of acidified SFE (aSFE), was mixed with 1/10 mixture of ethyl-butyric acid 2 mg mL (internal standard), filtered through 0.2-µm filters and stored at -80 °C until analysis. A system composed of a 7020A gas chromatograph (Agilent Technologies Inc., Palo Alto, CA) connected to a mass spectrometry (MS) 5975N detector (Agilent) was used to quantify the short chain fatty acids SCFA. Data were collected with Enhanced ChemStation G1701DA software (Agilent). 1 µl were directly injected into the gas chromatograph equipped with a ZB-WAX capillary column (30-mm length by 250 µm internal diameter, with a 0.25 µm film thickness; Agilent) using H 2 as the gas carrier, with a constant flow rate of 1.5 mL/min. The temperature of the injector was kept at 220 °C, and the injection was performed in split less mode. Chromatographic conditions were as follows: an initial oven temperature of 50 °C, 5 °C/min up to 180 °C, 1 min at 180 °C, and 20 °C/min up to 220 °C for cleaning the column. The column was directly connected to the MS detector, and the electron impact energy was set to 70 eV. The data collected were in the range of 25 to 250 atomic mass units (3.25 scans/s). The organic acids were identified by comparing their mass spectra with those held in the NSIT MS Data Center and 2HP-Wiley 138 library (Agilent) and by comparing their retention times with those of the corresponding standards (Volatile Free fatty acid mixture CRM46975) purchased from Sigma.

The peaks were quantified as the relative total ionic count abundance with respect to the internal standard. The concentration estimated in mM of each acid was calculated using linear regression equations (R 2 ≥ 0.99) from the corresponding curves of standards obtained with eight different concentrations.

Proteolytic activity

Extracellular proteolytic activity of LAB strains inoculated for 72 h in SFE, was measured according to the method described by [START_REF] Miralles | Biochemical tests for the selection of Staphylococcus strains as potential meat starter cultures[END_REF] using Azocasein as substrate. Absorbance was measured at 440 nm against a blank containing only the Azocasein. Proteolytic activity was expressed as ΔDO 440 × h -1 × mg -1 dry weight.

Antioxidant activity

The antioxidant activity was measured using DPPH according to the method described by [START_REF] Lin | Antioxidative Effect of Intestinal Bacteria Bifidobacterium longum ATCC 15708 and Lactobacillus acidophilus ATCC 4356[END_REF].

LAB were harvested by centrifugation at 4400 x g for 10 min after culture of 18 h incubation at 37 °C. The test was performed on the intact bacterial cells. Cells were washed three times with phosphate buffer solution (PBS: 0.85% NaCl, 2.68 mM KCl, 10 mM Na 2 HPO 4 , and 1.76 mM KH 2 PO 4 , pH 7.4) and resuspended in the same buffer. The sample (0.8 mL) was reacted with 1 mL DPPH solution (0.2 mM in 0.5% ethanol). The control was prepared by using ultra-pure water. The tubes were incubated for 30 min in the dark. Absorbance was measured by spectrophotometer (Thermo Scientific Multiskan GO) at 517 nm in triplicate. The results are expressed as a percentage of the anti-radical activity:

Antioxidant activity % = [ A 517c -A 517 a e A 517 c
] x

Amylolytic activity

The amylolytic activity of LAB was measured using the starch-iodine method, described by [START_REF] Bartkiene | Study on the reduction of acrylamide in mixed rye bread by fermentation with bacteriocin-like inhibitory substances producing lactic acid bacteria in combination with Aspergillus niger glucoamylase[END_REF].To evaluate this activity, the sterile flour extract SFE was enriched with glucose 0.15% in order de enhance the bacterial growth. The absorbance was measured at 670 nm using a spectrophotometer (Thermo Scientific Multiskan GO). One unit of -amylase activity (1 AU) was defined as an amount of enzyme that catalyzes 1 g soluble starch hydrolysis to dextrins in 10 min at 30 °C temperature.

Antimicrobial activity

The antimicrobial activities of LAB were determined against pathogenic bacterial and fungal strains.

Enterococcus faecalis ATCC 29212, Escherichia coli DH5α, Pseudomonas aeruginosa ATCC 27853 and

Staphylococcus aureus ATCC 25923 were used as indicator strains. These pathogens were grown in Brain heart infusion (BHI) broth at 37 °C. The inhibitory activity was evaluated with spots method. The colonies of LAB were grown in MRS medium incubated for 24 h at 30 °C. The indicator strains were inoculated at 10 5 CFU/mL in 5 mL of soft agar (containing 0.75% agar) and poured into petri dishes which were then incubated anaerobically at 37 °C for 24 h. Antimicrobial activity was expressed as inhibition diameter zones in millimeter (mm) against the pathological strains. The zone of inhibition was divided as follows: strong inhibitor (d ≥ 15 mm), medium inhibitor (11 ≤ d <15 mm), no significant inhibitory effect (d < 11 mm).

The antifungal activity of LAB was evaluated against two fungi Aspergillus niger and Penicillium expansum according to the method described by [START_REF] Whipps | Effect of Media on Growth and Interactions between a Range of Soil-Borne Glasshouse Pathogens and Antagonistic Fungi[END_REF]. LAB were inoculated on the modified MRS agar (without ammonium citrate and sodium acetate), 2 cm from the edge line of the petri dish and then grown at 30 °C for 48 h. A piece of fungus was placed in the center of the petri dish and then incubated at 25 °C for 2 to 3 days.

Control plates were inoculated with fungi only. The antifungal activity of LAB was expressed according to the following formula:

Antifungal activity % = [ R -R ] *
R1 is the radial distance developed by fungi in the direction of antagonism; R2 is the radial distance developed by the fungi.

Production of exopolysaccharides

The measurement of the amount of exopolysaccharides was done by the colorimetric test developed by [START_REF] Dubois | Colorimetric method for determination of sugars and related substances[END_REF].The supernatant (0.4 mL) of an overnight culture of LAB strain was mixed with 0.2 mL of phenol solution (5%) and 0.5 mL of concentrated sulfuric acid. After incubation in the dark for 30 minutes, the absorbance was measured at 490 nm against a blank without LAB strain.

Statistical analysis

All experiments were repeated three times and illustrated as the mean values ± standard deviations. Statistical analyses were performed using the IBM SPSS Statistics software version 23.0. The data were analyzed using one-way analysis of variance (ANOVA), followed by the Duncan's test with the significance level set at p<0.05

to establish the significance of differences between the samples.

Results and discussion

Microflora of wheat flour

Examination of the microbiological quality of wheat flour samples was performed in order to gain a common view about the hygienic quality and microbiological load. The microbial content of flours is composed of mesophilic aerobic bacteria, yeasts, moulds, and lactic acid bacteria. The results of microbiological analysis showed that yeasts constituted the major microbiota of the flour samples (Table 1). Yeast counts ranged from 0.30 to 2.57 Log CFU/g, whereas presumptive LAB counts varied from 0.17 to 1.68 Log CFU/g. The mean mesophilic aerobic bacteria counts in all flour samples studied were 1.53 to 2.11 Log CFU/g. All samples were below the maximum acceptable limits of the Codex Alimentarius (FAO 1995b). According to [START_REF] Minervini | Lactic Acid Bacteria in durum wheat flour are endophytic components of the plant during its entire life cycle[END_REF], LAB contaminating flour is strongly affected by the endophytic microbiota of cereals, mainly by the plant organs, the cultivars, and the phenological growth stages. A number of plant-associated microorganisms infect grains, and so flours, touching the whole quality of leavened baked goods [START_REF] Vuyst | Biodiversity, ecological determinants, and metabolic exploitation of sourdough microbiota[END_REF][START_REF] Gobbetti | How the sourdough may affect the functional features of leavened baked goods[END_REF].

Isolation and identification of lactic acid bacteria

LAB were isolated from Tunisian wheat flours collected from six different regions (Table 1). Upon enrichment in MRS broth 113 LAB isolates were initially selected based on their ability to produce acid by the presence of yellow halo surrounding the colonies on MRS-bromocresol green plates and purified. A total of 45 isolates

Gram-positive and catalase-negative rods and cocci were kept on MRS agar for further identification.

The isolates were subjected to ITS-PCR amplification analysis and 16S-PCR sequencing. In fact, diverse studies have previously described the efficiency of ITS for inter-and intra-differentiation at the genus/ species level [START_REF] Gürtler | New approaches to typing and identification of bacteria using the 16S-23S rDNA spacer region[END_REF] due to the high variability of these spacers. The ITS-PCR amplification generated different patterns (bands ranging from 50 to 1000 base pairs). Comparing the generated patterns of PCR products obtained from the studied isolates, we distinguished 09 ITS fingerprints. One representative of each ITS-PCR pattern were subjected to partial sequencing of the 16S rRNA. LAB isolates were identified at species level by 16S rRNA gene sequencing which is generally regarded as a more reliable solution for the classification and identification of LAB [START_REF] Ehrmann | Molecular taxonomy and genetics of sourdough lactic acid bacteria[END_REF]. According to the comparison of 16S rDNA sequences with those available in GenBank, all isolates were related to LAB species with sequence homology >97%.

Phylogenetic tree of LAB isolated from wheat flours was constructed based on the 16S rDNA sequences from evolutionary distances by the neighbor-joining method (Figure 1). LAB identification showed several consistent recognized species that are affiliated to four lactic genotypic groups (Figure 1). LAB strains were identified to:

Weissella cibaria (05 strains), Lactobacillus plantarum (08 strains), Lactobacillus brevis (04 strains),

Pediococcus pentosaceus (10 strains), Pediococcus pentoseus (04 strains), Pediococcus acidilactici (04 strains),

Enterococcus faecium (05 strains), Enterococcus casseliflavus (03 strains) and Enterococcus faecalis (02 strains).

In our studies, several LAB species found were reported to be associated with bread production, such as wheat grains and flours (Corsetti et al. 2007). In addition, some of LAB species isolated in our wheat flours are naturally found in mature sourdoughs such as L. plantarum (Corsetti and Settanni 2007;[START_REF] Alfonzo | Diversity and technological potential of lactic acid bacteria of wheat flours[END_REF]) and W. cibaria [START_REF] Alfonzo | Diversity and technological potential of lactic acid bacteria of wheat flours[END_REF]. We noted that Pediococcus genus was presented by P. pentosaceus (followed by P. pentoseus and P. acidilactici) as the most commonly isolated bacterium from wheat flours. P. acidilactici is emerging as a potential probiotic in animal and human [START_REF] Guerra | Dynamic mathematical models to describe the growth and nisin production by Lactococcus lactis subsp. lactis CECT 539 in both batch and re-alkalized fed-batch cultures[END_REF]).

The dominant species of Lactobacillus found from wheat flours was L. plantarum followed by L. brevis. This finding reinforced the concept that L. plantarum has a strong ecological or metabolic adaptability to different habitats [START_REF] Alfonzo | Diversity and technological potential of lactic acid bacteria of wheat flours[END_REF][START_REF] Minervini | Lactic Acid Bacteria in durum wheat flour are endophytic components of the plant during its entire life cycle[END_REF]. However, these two Lactobacillus species were reported to dominate raw and fermented vegetables [START_REF] Rhee | Importance of lactic acid bacteria in Asian fermented foods[END_REF]). Similar findings were noted for P. pentosaceus [START_REF] Yang | Purification antifungal compound of a new produced by Lactobacillus plantarum AF1 isolated from kimchi[END_REF][START_REF] Swain | Fermented Fruits and Vegetables of Asia: A Potential Source of Probiotics[END_REF]). Together with L. plantarum and W. cibaria are generally isolated from plants, wheat grains and fermented wheat products (sourdough) (Corsetti et al. 2007;[START_REF] Trias | Lactic acid bacteria from fresh fruit and vegetables as biocontrol agents of phytopathogenic bacteria and fungi[END_REF][START_REF] Alfonzo | Diversity and technological potential of lactic acid bacteria of wheat flours[END_REF]. For Enterococcus genus, En. faecium was the most commonly species presented followed by En. casseliflavus and En. faecalis. In fact, enterococci are normal inhabitants of gastrointestinal tract [START_REF] Giraffa | Enterococci from foods[END_REF]. The isolated Enterococcus strains were discarded from further studies to avoid risks for antibiotic resistance and virulence gene dissemination which contribute to the pathogenesis of virulent bacteria [START_REF] Bortolaia | Human health risks associated with antimicrobialresistant enterococci and Staphylococcus aureus on poultry meat[END_REF]. Weissella genus was represented at low occurrence by only W. cibaria species. Recording to our findings, there was no correlation between LAB species and the geographical location of the wheat flour samples.

Assessment of the technological properties of LAB isolates

Acidification activity

In order to select lactic bacteria with technological characteristics relevant for sourdough process, 06 selected representative LAB species from wheat flour samples were investigated for the acidification capacity to decrease pH of sterile flour extract SFE. The results of the acidification activity are shown in Figure 2. We note that all tested LAB strains presented good acidifying abilities. They were able to decrease the SFE pH significantly (p<0.05) below 4.0 after 24 h. At 72 h, almost all tested LAB strains acidified the medium below pH 3.0. Except P. acidilactici S16, the remained strains were fast acidifiers revealing a ∆pH (difference between pH before and after inoculation of SFE by strains) around 2.8 after 24h and a final pH (after 72h) ranging between 2.0 and 3.0.

The acidifying ability of the majority of the identified LAB strains after 24 h, 48 h and 72 h incubation were similar. Compared to initial pH of incubated culture, Lactobacillus plantarum S28 showed the rapid acidification ability ∆pH to decrease pH significantly (p<0.05) of the flour extract broth at 24 h (4.84 ± 0.2). This finding result concurred with previous studies of [START_REF] Ventimiglia | Codominance of Lactobacillus plantarum and obligate heterofermentative lactic acid bacteria during sourdough fermentation[END_REF] which reported that L. plantarum is the highest acid-producing species of LAB group. It causes rapid acidification of the raw material through the production of organic acids, mostly lactic acid. Besides, it is an abundant food related species reported to be common as well for sourdough environments (Corsetti and Settanni 2007). Accordingly, their fast lowering of the pH ability improves safety and organoleptic properties of fermented food products [START_REF] Hansen | Commercial bacterial starter cultures for fermented foods of the future[END_REF].

The concentration of lactic and acetic acids in SFE was carried out on four strains (P. acidilactici, P. pentoseus, L. plantarum and L. brevis), that displayed interesting technological properties, after 24, 48 and 72 h of fermentation (Figure 3). Lactic acid concentration increased after 48 h for all LAB strains. It reached 5.73 -8.36 mg/g. After 72 h, the content increased significantly (p<0.05) and reached 7.77 mg/g for P. acidilactici and 7.06 mg/g for P. pentoseus. However, we noted a significant (p<0.05) decline of lactic acid concentration after 72h for L. plantarum (6.18 mg/g) and L. brevis (6.79 mg/g). Acetic acid production ranged from 0 to 3.87 mg/g after 24 h, and 3.74 to 5.95 mg/g after 72 h. In fact, acetic acid can occur via the citrate metabolism [START_REF] Zalán | Production of organic acids by Lactobacillus strains in three different media[END_REF] or the degradation of lactic acid produced (Oude [START_REF] Elferink | Anaerobic conversion of lactic acid to acetic acid and 1, 2-propanediol by Lactobacillus buchneri[END_REF]) which may justify the reduction of lactic acid concentration in SFE extract of L. plantarum and L. brevis

The strains displayed a fermentation quotient (FQ: molar ratio between lactic and acetic acids) varied from 0.84 to 2.38, which is considered to affect the organoleptic features (aroma and texture) and to prevent ripeness and fungi spoilage of final products and so to guarantee longer shelf life [START_REF] Gobbetti | Arabinose fermentation by Lactobacillus plantarum in sourdough with added pentosans and alphaalpha-L-arabinofuranosidase: a tool to increase the production of acetic acid[END_REF][START_REF] Datta | Lactic acid: recent advances in products, processes and technologiesa review[END_REF]. P. pentoseus S14 and P. acidilactici S16 presented FQ close to the optimal range of 2.0-2.7 suggested by [START_REF] Hammes | Sourdough breads and related products[END_REF].

Proteolytic activity

The proteolytic activity of tested LAB was determined by their inoculation in sterile flour extract SFE. The extracellular proteolytic activity assessed by azocasein degradation was detected in all flour LAB strains (Figure 4). P. pentoseus S14 and L. brevis S12 exhibited significantly (p<0.05) highest proteolytic activity (1.51 ± 0.18 and 1.41 ± 0.14, respectively). The extracellular protease activity is known to improve organoleptic features of leavened baked goods by generating small peptides and free amino acids as precursors for flavor development [START_REF] Cagno | Proteolysis by Sourdough Lactic Acid Bacteria: Effects on Wheat Flour Protein Fractions and Gliadin Peptides Involved in Human Cereal Intolerance[END_REF][START_REF] Rizzello | Use of fungal proteases and selected sourdough lactic acid bacteria for making wheat bread with an intermediate content of gluten[END_REF]. Moreover, the extracellular protease activity is important for the rheology and staleness of breads [START_REF] Corsetti | Combined Effect of Sourdough Lactic Acid Bacteria and Additives on Bread Firmness and Staling[END_REF]. Moreover, proteolysis generated small peptides which are important for rapid microbial growth and acidification during fermentation [START_REF] Cagno | Proteolysis by Sourdough Lactic Acid Bacteria: Effects on Wheat Flour Protein Fractions and Gliadin Peptides Involved in Human Cereal Intolerance[END_REF]. In addition, certain LAB strains are further known to be able to release bioactive peptides from proteins, which are thought to have a role in promoting health [START_REF] Leroy | Functional meat starter cultures for improved sausage fermentation[END_REF]. This finding revealed the potential use of these LAB in bread making.

Antioxidant activity

The DPPH scavenging activity of the six LAB strains from flour samples are shown in Figure 5. The results

showed DPPH scavenging activity ranging between 4.39 to 25.57%. Among the tested strains, W. cibaria S25 had significantly (p<0.05) the highest radical-scavenging activity with a rate of 25.57%, followed by L. brevis S12 with 24.81% and P. pentosaceus S30 with 21.49%. Several authors have reported that the fermentation by lactic acid bacteria with antioxidant activity is considered as one of the most important tool suitable to enhance the functional and the organoleptic potential of several fermented cereal flours [START_REF] Coda | Selected Lactic Acid Bacteria Synthesize Antioxidant Peptides during Sourdough Fermentation of Cereal Flours[END_REF][START_REF] Rizzello | Lactic acid fermentation as a tool to enhance the functional features of Echinacea spp[END_REF][START_REF] Curiel | Lactic acid fermentation as a tool to enhance the antioxidant properties of Myrtus communis berries[END_REF].

Amylolytic activity

The biodiversity of amylolytic lactobacilli, ubiquitously used in cereals processing, is quite limited [START_REF] Reddy | Amylolytic bacterial lactic acid fermentation: A review[END_REF]. In this work, the in-vitro amylolytic activity of the tested LAB was evaluated by starch-iodine method.

According to the results presented in Figure 4, all tested LAB are characterized by starch hydrolysis. They showed amylase activity ranging from 658± 13 to 1386± 4 U/mL (for 1 g of starch hydrolyzed (SH)) which are equivalent to 6.58 to 13.86 U/ml (for 0.01 g of SH). L. plantarum S28 demonstrated significantly (p<0.05) the highest amylolytic activity (1386 ± 4 U/mL) followed by P. acidilactici S16 (1086 ± 6 U/mL) and P. pentoseus S14 (1046 ± 9 U/mL). These results obtained for the amylolytic activity are in line with previous reports [START_REF] Petrova | Amylolytic Lactobacillus strains from Bulgarian fermented beverage boza[END_REF][START_REF] Amapu | Amylolytic Potential of Lactic Acid Bacteria Isolated from Wet Milled Cereals, Cassava Flour and Fruits[END_REF]). Production of amylase has been described primordially for L.

plantarum [START_REF] Giraud | Degradation of raw starch by a wild amylolytic strain of Lactobacillus plantarum[END_REF]. Besides, the amylolytic activity recorded for tested LAB strains was equivalent to 8 -11.5 U/ml reported for amylolytic strains [START_REF] Petrova | Amylolytic Lactobacillus strains from Bulgarian fermented beverage boza[END_REF]. In fact, high amylolytic activity was reported for L. plantarum, P. acidilactici and L. brevis which are predominant amylase producing LAB in wet milled cereals [START_REF] Amapu | Amylolytic Potential of Lactic Acid Bacteria Isolated from Wet Milled Cereals, Cassava Flour and Fruits[END_REF]. Besides, [START_REF] Sanni | New efficient amylase-producing strains of Lactobacillus plantarum and L fermentum isolated from different Nigerian traditional fermented foods[END_REF] described amylolytic strains of L. plantarum in several traditional amylaceous fermented foods. The potential LAB strain with amylolytic activity could be used as one of the factors to improve the fermentation rate, reduction of dough viscosity with resultant improvements in the volume and texture of the bread for food industries (making of high density gruels, baking, brewing) [START_REF] Fossi | Application of Amylolytic Lactobacillus fermentum 04BBA19 in Fermentation for Simultaneous Production of Thermostable Alpha-Amylase and Lactic Acid. R and D for food[END_REF]. [START_REF] Ray | Amylolytic Lactic Acid Bacteria: Microbiology and Technological Interventions in Food Fermentations[END_REF] reported that they are also employed in preparing high energy density (ED) cereal-based foods for improving dietary starch utilization in infants and small children. On the basis of their technological potentials, the strains L. plantarum S28, P. acidilactici S16, P. pentoseus S14, W. cibaria S25 and L. brevis S12 isolated from wheat flours retains better starch degradation ability. It may developed into starter cultures (sourdoughs) in the making of fermented cereal foods, including bread, to contribute to the enzymatic pool and yield products with a higher fermentable sugar content [START_REF] Amapu | Amylolytic Potential of Lactic Acid Bacteria Isolated from Wet Milled Cereals, Cassava Flour and Fruits[END_REF][START_REF] Hattingh | Amylolytic strains of Lactobacillus plantarum isolated from barley[END_REF]. The hypothesis that the amylolytic activity of strains studied are cell wall bound or/and produced in cell-free supernatants needs to be verified.

Exopolysaccharide production

The cell-free supernatants of selected LAB strains were examined for the amounts of exopolysaccharide (EPS) yields using phenol-sulfuric acid method. EPS production of each strain was presented in Figure 6. EPS amount produced by selected LAB isolates ranged from 0.172 to 0.970 g/L. Figure 6 obviously demonstrates that the strains P. acidilactici S16 and L. plantarum S28 showed significantly (p<0.05) highest EPS production (0.97 and 0.636 g/L, respectively). Several health benefits have been attributed to exopolysaccharides EPS from LAB which are used to improve the textural properties of fermented foods [START_REF] Crescenzi | Microbial polysaccharides of applied interest: ongoing research activities in Europe[END_REF][START_REF] Ruas-Madiedo | Molecular aspects of lactic acid bacteria for traditional and new applications[END_REF]. These strains can be used in sourdough and may have applications in bread making.

Antibacterial activity

In order to select bacterial strains with antibacterial activity, selected LAB were assessed against four pathogens (Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus) as indicator strains. En.faecalis with an average of 11 mm. In our case, we observe that the inhibition of microbial growth by the six LAB is due to pH variation on culture media and might be a result of fermentative compounds accumulation.

LAB are known to produce a range of metabolic end compounds that are able of interfering with the growth of certain undesirable microbes in food systems [START_REF] Vandenbergh | Lactic acid bacteria, their metabolic products and interference with microbial growth[END_REF][START_REF] Alvarado | Food-associated lactic acid bacteria with antimicrobial potential from traditional Mexican foods[END_REF]. In fact, most of the inhibitory activity exhibited by LAB strains was attributed to pH reduction by organic acids. Besides, it is suggested that acid products interfere with permeability of plasmic membrane and raise its diffusion leading to stop metabolic activities and so to the inhibition effect of sensitive microorganisms [START_REF] Piard | Inhibiting factors produced by lactic acid bacteria. 1. Oxygen metabolites and catabolism end-products[END_REF]. So, these findings led to suggest that sourdough and bread produced with these tested LAB strains,

showing consistent ability to retard the growth of both pathogen moulds and bacteria species, thus have the potential to improve the shelf-life of wheat bread.

Antifungal activity

All the studied lactic acid bacteria strains, isolated from different wheat flour samples, were assessed for inhibitory activity against two common post-harvest fungus Aspergillus niger and Penicillium expansum using a dual culture method. Varying degrees of inhibition were detected against the two moulds in-vitro (Figure 7). All LAB strains exhibited significant (p<0.05) inhibitory effects towards the tested fungi. The growth of Penicillium expansum was strongly inhibited by P. pentoseus S14 with an inhibitory rate of 84%. While growth of Aspergillus niger was moderately (p<0.05) inhibited by all the tested LAB isolates from 16.7% to 36.1% of inhibition. Almost all strains, especially L. plantarum S28 and P. acidilactici S16, showed radial (p<0.05) growth reductions against P. expansum and A. niger by an average of 36 % and 25%, respectively. Penicillium expansum was the most sensitive strain in dual-culture method. Dal [START_REF] Bello | Improvement of the quality and shelf life of wheat bread by fermentation with the antifungal strain Lactobacillus plantarum FST 1.7[END_REF] and [START_REF] Djossou | Robusta coffee beans post-harvest microflora: Lactobacillus plantarum sp. as potential antagonist of Aspergillus carbonarius[END_REF] reported that L. plantarum isolated from sourdough and plant materials is known by its antifungal activity. This study revealed that Tunisian wheat flours included autochthonous and selected LAB of interesting technological features relevant to sourdough production. In fact, the obtained results might be helpful to use a mixed starter culture including L. plantarum, L. brevis, P. acidilactici and P. pentoseus for producing sourdough bread. Each value represents the mean value standard deviation (SD) from two trials; a, b and c, represent significant (p<0.05) differences in the same column; *: Counts<1.

Table 2. Inhibitory diameter (mm) of neutralized cell-free supernatant of six selected LAB flour isolates.

Numbers indicated the diameter of the inhibition zone in mm; Each value represents the mean value standard deviation (SD) from two trials; a, b and c, represent significant (p<0.05) differences in the same column.

Enterococcus faecalis ATCC 29212

Pseudomonas aeruginosa ATCC 27853

Escherichia coli DH5α

Staphylococcus aureus ATCC 25923

Pediococcus acidilactici S16 8.5 ± 0.71 a 11.5 ± 0.71 b 8 ± 0 a 10.75 ± 0.

Fig. 1

 1 Figure captionsFig.1Dendrogram obtained by comparing 16S rDNA sequences of the isolates from Tunisian wheat flours, based on 16S rDNA partial sequences, using the neighbor-joining method. Bootstrap values for a total of 1000 replicates are shown at the nodes of the tree, using MEGA-6. The scale bar corresponds to 0.01 units of the number of base substitutions per site.

Fig. 2

 2 Fig. 2 Kinetics of acidification of wheat flour LAB. Histograms are in the order of increasing pH after 24h, 48h and 72 h.

Fig. 3

 3 Fig.3Organic acids content of wheat flour LAB (mg/g) after 24h, 48h and 72 h.

Fig. 4

 4 Fig. 4 Proteolytic (grey) and amylolytic (black) activities of wheat flour LAB.

Fig. 5

 5 Fig. 5 Antioxidant activity (%) of wheat flour LAB.

Fig. 6

 6 Fig. 6 Exopolysaccharide production (g/L) of wheat flour LAB.

Fig. 7

 7 Fig. 7 Antifungal inhibition (%) of wheat flour LAB against Aspergillus niger and Penicillium expansum..

  Table2gives the results for antibacterial activity of the LAB isolates in terms of inhibition diameter around the spot. All of LAB strains presented aptitude to inhibit the growth of tested pathogens with different percentages. The antibacterial activity was recorded as strong growth inhibition against Pseudomonas aeruginosa for L. brevis S12 (15 mm), P. pentoseus S14 (16 mm), W. cibaria S25 (17 mm) and mostly for P. pentosaceus S30 (18 mm). Except P. acidilactici S16, the entire tested LAB showed a significant (p<0.05) moderate antagonistic activity towards E. coli and St. aureus. L. brevis S12 and P. pentoseus S14 presented high values of diameter of growth inhibition against St. aureus (14.5 mm) and E. coli (12 mm), respectively. Only L. brevis S12 and P. pentosaceus S30 presented significantly (p<0.05) a medium growth inhibition activity against

Table 1 .

 1 Microflora in flour samples collected from mills located throughout Tunisia (Log CFU/g).

	Sample	Company	Geographical origin	Mesophilic aerobic bacteria	Yeasts	Moulds	Lactic acid bacteria
	E1	Grands Moulins de Nabeul	Nabeul	1.89 ± 0.17 b	2.51 ± 0.11 a	<1 *	1.68 ± 0.03 a
	E5	Epis de Carthage	Sousse	1.72 ± 0.11 b	2.27 ± 0.20 a	<1 *	1.16 ± 0.15 b
	E6	RANDA	Tunis	1.89 ± 0.14 b	2.57 ± 0.17 a	<1 *	1.67 ± 0.04 a
	E7	Couscousseries du sud	Sfax	2.11 ± 0.24 a	2.41 ± 0.21 a	<1 *	1.21 ± 0.07 b
	E8	Epi d'or	Sousse	1.53 ± 0.12 c	0.30 ± 0.04 c	1.35 ± 0.12 a 0.17 ± 0.04 c
	E11	AZIZA	Djerba	2.04 ± 0.21 a	1.27 ± 0.12 b	0.39 ± 0.07 b 0.30 ± 0.05 c

Acknowledgments

This work was supported by the Tunisian Ministry of Higher Education and Scientific Research (LR10CBBC02). Aurelie sauvager (CORINT, UMR CNRS ISCR 6226, UFR Sciences Pharmaceutiques et Biologiques, Université Rennes 1, France) is acknowledged for her technical help.


Marc Aberle

Olivier Christin

Fabrice Flückiger

Raphaële Rasina

Vincent Alamercery

Francesco Beretta

Bernard Hours

Voting on Faith

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

The commune has spoken! Mass shall not be celebrated anymore, and all sacred images shall be instantly removed! A similar proclamation may have been pronounced by city heralds after the communities had voted on their faith in many Swiss villages and towns. Our project focuses on ballots held in cities and villages under supervision of the cantons in order to choose between either the reformed ideas or the teachings of the Roman Church. We want to understand why a vote was considered by parishioners and magistrates as the right path to lead to a wise religious choice and under which circumstances ballots were organized.

Équipe de recherche

UniNE/FNS

LARHRA

Pôle histoire Numérique

We have noticed that a lot of ballots were held during the 1523-1550 period and that the process differed in various ways between each ballot. Therefore, we thought that the best way to understand the process is to analyse its whereabouts with tools provided by Digital Humanities. The «Reformation and Ballots» project works therefore in close partnership with the LARHRA-Pôle histoire numérique in Lyon, where the symogih.org project is hosted. symogih.org is an open modular platform that stores geohistorical information. The platform allows researchers to share their knowledge in a collaborative and interlinked environment and to produce structured data.

symogih.org allows us to produce and store structured data about ballots in Reformation times and to make the data available to researchers in compliance with FAIR principles. The information is organised around a CIDOC CRM compatible and an event-centered model: classes (persistent items -objets -and temporal entities -informations) interact with each other as well as with temporal entities through what the platform calls properties (or roles). The principal benefits of working within this frame rather than to build a specific database for our project are interoperability and evolutive perspectives. As new information will be found, it will be added in the database and connected to the existing information, enhancing existing knowledge and producing new results. symogih.org also allows future research projects to re-use our data in order to develop their own analyses.

The patterns of the vote are shown through the reconstruction of complex processes: -What was the object of the vote? People did not vote "yes" or "no" to "the Reformation" in these days; they pronounced themselves on the mass, on devotional images or on financial matters like the very much hated tithe. Who could vote, and which authority would supervise the process? The communes and parishes which choose their faith in the 1520-1530s were entangled in complex networks of alliances and tied by overlapping jurisdictions. They often found themselves in the middle of political and religious conflicts, the most important one being the growing disagreement over the true faith. Using qualitative analysis, symogih.org allows us to recreate these relations and leads to a better understanding of the reasons why a commune could vote on its faith, and why the same possibility was denied to another.

VOTING ON FAITH

symogih

  , L'Unanimité, étude, 1912Unanimité, étude, /1913, Huile sur toile © Musée d'art et d'histoire de Genève, photo : Y. Siza , Inv. 1939-0044 DARIAH-CH 2018, Neuchâtel CC BY-SA 4.0 also allows the use of data visualization processes. Results can be shown in form of diagrams, maps, tag clouds... The diagram on the left shows a part of the complex network of temporal and spiritual jurisdictions which connected communities in the Moutier-Grandval bailiwick: it reveals the link between parishes and communes, which is crucial to determine how many people would be affected by a vote, often hold on parishional level. The map on the right shows which villages underwent a vote to determine the choice between the reformed and Roman interpretations of the Church.

  Understanding the Swiss Reformation through Digital Humanities Marc Aberle (UNINE/FNS), Olivier Christin (UNINE/CEDRE PSL) Fabrice Flückiger (UNINE/FNS/CEDRE PSL), Raphaële Rasina (UNINE/FNS) Vincent Alamercery (Université de Lyon/LARHRA) Francesco Beretta (CNRS/Université de Lyon/LARHRA), Bernard Hours (Université de Lyon/LARHRA)

											AbOb 1875 Combourgeoisie	SoCh1683 Evêque de Bâle	AbOb1948 Juridiction spirituelle		CoAc20448 Gouv. de la principauté	AbOb1940 Suzeraineté
																Typer
											Typer		Exercer un pouvoir			Exercer un pouvoir	Typer
									CoAc 18413	Exercer un pouvoir	TyIn99	Etre soumis à un pouvoir	CoAc 19422			TyIn99		CoAc 19396
									Conseil de		Relation de pouvoir		Prévôté de			Relation		Chapitre collégial de
									Berne		entre acteurs collectifs	Moutier-Grandval		pouvoir	Etre soumis	Moutier-Grandval
									Exercer un					Etre soumis à un pouvoir		à un pouvoir
									pouvoir								Etre soumis à un pouvoir		Etre concerné
				CoAc 19418 Conseil de Soleure	AbOb1972 Envoi d'un prédicateur Typer		SoCh1960 Abbé de Bellelay Etre concerné	AbOb1985 Protection d'un acteur	TyIn66 Evènement	TyIn66 Evènement Exercer un pouvoir concerné Etre	Actr47248 Guillaume Farel	TyIn99 Relation de pouvoir entre acteurs collectifs Exercer un pouvoir	Etre soumis à un pouvoir	CoAc 19966 Communauté de Tavannes	Etre concerné	TyIn66 Evènement	Etre concerné	CoAc19958 Communauté de Sornetan	Typer	AbOb1988 Conflit sur bénéfices	AbOb2133 Injonction à remplir ses devoirs	AbOb1970 Refus de payer les dîmes
																			Etre
				Etre concerné				Typer			Etre concerné Etre soumis à un pouvoir					localisé			localisé Etre
																								Etre soumis
												Typer					Etre soumis à un pouvoir						à un pouvoir	Typer
														Exercer un pouvoir
	SoCh717 Pasteur	Actr60045 Jacques Moeschler	AbOb1878 Droit de collation	AbOb1973 Installation d'un pasteur	TyIn7 Exercice d'une fonction	TyIn99 Relation pouvoir		Etre soumis à un pouvoir		AbOb1948 Juridiction spirituelle	CoAc 19968 Paroisse de Tavannes	TyIn66 Evènement	TyIn99 Relation pouvoir	TyIn36 Localisation	Localiser	NaPl 127423 Tavannes	TyIn36 Localisation	Localiser	NaPl127519 Sornetan	TyIn99 Relation pouvoir	Exercer un pouvoir	CoAc19959 Paroisse de Sapran	Etre concerné	TyIn66 Evènement	AbOb1948 Juridiction spirituelle
															Occasionner								Typer	Occasionner
					SoCh121 Curé	AbOb1955 Abolition de la messe							AbOb1952 Adoption de la Réforme	AbOb1949 Votation								AbOb1952 Adoption de la Réforme	AbOb1949 Votation

Projet FNS Réformation et Votations


Claire Thérial

Francoise S Lucas

Adélaïde Roguet

Saad Mohamed

Laurent Moulin

Laure Huguenard

Microbial sources tracking in recreational water in a leisure center

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

Introduction

Materials and methods

Results

Conclusions

Microbial sources tracking in recreational water in a leisure center.

Claire Thérial 1 , Françoise Lucas 1 , Adélaïde Roguet 1 , Mohamed Saad 1 , Laurent Moulin 2 , Laure Huguenard 3 .

Claire Thérial : claire.therial@u-pec.fr Recreational activities on the Champs-sur-Marne leisure area are regularly affected by water contamination issues associated with pathogens of fecal origin. The objective was to identify the origin of these microbes to provide technical development and management solutions favorable to the improvement of the water quality and compatible with bathing activity.

A part of this project was to test the hypothesis of bacteriological contamination by aquatic birds massively occupying the leisure center (fig. 1) using a microbial source tracking technique. This approach relies on the quantification by real-time PCR (qPCR) in water, sand, and sediment of bacterial specific markers of the digestive tract of different animal species (geese, seagulls, dogs) and humans.

No contamination by human enteric viruses was found. This suggests that the contamination was not from human origin.

The detection of animal markers in all matrices before and after the swimming season indicates that dogs and geese contribute to the most to the fecal contamination of the water, sand and sediment. The contamination could be originated from two watchdogs roaming around the lake during closing hours, and several geese settled around the large bathing area or on the dock. The fences around the beaches do not stop geese and dogs. Thus, fecal contamination may occur by direct defecation of the birds in the water or by runoff during rain events from the grass, sand and docks.

These results are reinforced with a microbial community analysis where sequencing patterns from the lake samples (water and sediment) and different fecal sources were compared (animal feces, urban waste water, and urban runoff). Virus: • Influenza virus A and B were not detected (qPCR was not inhibited).

• Human enterovirus and adenovirus were not detected in 10L, but some samples were inconclusive (qPCR amplification inhibition).

[

Sampling strategy

Bacterial sources:

• Gull marker was detected only in the water from the large bathing area (A) in September, and in low quantity (less than 3 genome copies per ml).

• C. jejuni was detected in just one sample of lake water with a quantity of 0.2 genome copies per ml.

• Dog bacterial marker was detected in most samples with a large quantity range (0.4 to 1.6 x 10 7 genome copies per ml or g). Dog marker is present in both sediment and sand of the bathing areas, and less in the water. The patterns differ between June (B is more contaminated) and September (A is more contaminated).

• Goose bacterial marker was detected in all types of samples ranging from 1 to 9.4 x 10 6 genome copies per ml or g. Goose marker was predominant in the sand and the sediment of the large bathing area (A). The pollution was higher in September than in June.

• Dog and goose markers were more present in the lake sediment than the lake water in September.

Analysis

Two sampling campaigns took place in late June and late August 2015 at the bathing areas (A and B) and in the lake (C and D) (fig. 2) in 4 replicates:

-2L water for qPCR analysis -10L water for virus analysis -2x50mL tubes of sediment

In duplicates :

-2 tubes 50mL of mixed sand from quadrat 50x50x1cm on beach For each bacterial target, a plasmid vector was built for the standard curve. Additionally, another plasmid was used as an internal inhibition control. For virus analysis, a control DNA was added to the samples.

Figure

  Figure 1: Scarecrow (Eagle) to scare off birds.

  1. Laboratoire Eau Environnement Systèmes Urbains (LEESU), Université Paris-Est Créteil, 2. Laboratoire Eau de Paris, 3. Conseil départemental de Seine-Saint-Denis.

Figure 2 :

 2 Figure 2: Aerial view of the Champs-sur-Marne leisure lake.

  Figure 3: number of genome copies of dog and goose markers (average Log+1). Lake values are averages of C and D values.