Faustine Gomand

Frédéric Borges

Justine Guerin

Sofiane El-Kirat-Chatel

Gregory Francius

Dominique Dumas

Jennifer Burgain

Claire Gaiani
email: claire.gaiani@univ-lorraine.fr

Adhesive interactions between lactic acid bacteria and β-lactoglobulin: specificity and impact on bacterial location in whey protein isolate

Keywords: adhesion, lactic acid bacteria, dairy, β-lactoglobulin, high-throughput screening, bacterial distribution, Atomic Force Microscopy (AFM), Confocal Laser Scanning Microscopy (CLSM)

In the last decade, there has been an increasing interest in the potential health effects associated with the consumption of Lactic Acid Bacteria (LAB) in foods. Some of these bacteria such as Lactobacillus rhamnosus GG (LGG) are known to adhere to milk components, which may impact their distribution and protection within dairy matrices and therefore is likely to modulate the efficiency of their delivery. However, the adhesive behavior of most LAB, as well as its effect on food structuration and on the final bacterial distribution within the food matrix remain very poorly studied. Using a recently developed high-throughput approach, we have screened a collection of 73 LAB strains for their adhesive behavior towards the major whey protein β-lactoglobulin. Adhesion was then studied by genomics in relation to common bacterial surface characteristics such as pili and adhesion-related domain containing proteins. Representative adhesive and non-adhesive strains have been studied in further depth through biophysical measurement using Atomic Force Microscopy (AFM) and a relation with bacterial distribution in Whey Protein Isolate (WPI) solution has been established. AFM measurements have revealed that bacterial adhesion to β-lactoglobulin is highly specific and cannot be predicted accurately using only genomic information. Non-adhesive strains were found to remain homogeneously distributed in solution whereas adhesive strains gathered in flocs. These findings show that several LAB strains are able to adhere to β-lactoglobulin, whereas this had only been previously observed on LGG. We also show that these adhesive interactions present similar characteristics and are likely to impact bacterial location and distribution in dairy matrices containing β-lactoglobulin. This may help with designing more efficient dairy food matrices for optimized LAB delivery.

Introduction

Adhesion is a major property of microorganisms which effectively impacts microorganism activities as well as human health, and has been identified as a key factor involved in microorganism ecology.

Adhesion enables bacteria to stick to both biotic and abiotic surfaces. Adhesion to abiotic surfaces leads to biofilm formation, which has been widely studied in relation to the food industry [START_REF] Barnes | Correlated XPS, AFM and bacterial adhesion studies on milk and milk proteins adherent to stainless steel[END_REF][START_REF] Garrett | Bacterial adhesion and biofilms on surfaces[END_REF][START_REF] Notermans | Contribution of surface attachment to the establishment of micro-organisms in food processing plants: A review[END_REF][START_REF] Pontefract | Bacterial Adherence: Its Consequences in Food Processing[END_REF]. Adhesion to biotic surfaces enables bacteria to establish direct contact with mucous membranes, and especially the intestinal epithelium, to colonize a host [START_REF] Conway | Survival of Lactic Acid Bacteria in the Human Stomach and Adhesion to Intestinal Cells[END_REF][START_REF] Pizarro-Cerdá | Bacterial Adhesion and Entry into Host Cells[END_REF][START_REF] Servin | Adhesion of probiotic strains to the intestinal mucosa and interaction with pathogens[END_REF]. Adhesion of pathogens is therefore considered to be a virulence factor as it facilitates host invasion [START_REF] Pizarro-Cerdá | Bacterial Adhesion and Entry into Host Cells[END_REF][START_REF] Proft | Pili in Gram-negative and Gram-positive bacteria -structure, assembly and their role in disease[END_REF]. Amongst non-pathogenic bacteria, adhesion is considered essential in order for probiotic bacteria to remain functional and therefore provide health benefits to the host [START_REF] Ouwehand | Assessment of adhesion properties of novel probiotic strains to human intestinal mucus[END_REF][START_REF] Quinto | Probiotic Lactic Acid Bacteria: A Review[END_REF][START_REF] Servin | Adhesion of probiotic strains to the intestinal mucosa and interaction with pathogens[END_REF]. In the case of Gram-positive bacteria, bacteria-environment interactions such as bacterial adhesion are mediated by sortase-dependent proteins [START_REF] Comfort | A Comparative Genome Analysis Identifies Distinct Sorting Pathways in Gram-Positive Bacteria[END_REF][START_REF] Maresso | Sortase as a Target of Anti-Infective Therapy[END_REF], which are covalently anchored to the cell wall and possess an LPxTG like motif at their C-terminal end [START_REF] Schneewind | Sec-secretion and sortase-mediated anchoring of proteins in Gram-positive bacteria[END_REF].

Bacteria have also been shown to be able to adhere to food components, especially to meat [START_REF] Firstenberg-Eden | Attachment of Bacteria to Meat Surfaces: A Review[END_REF][START_REF] Piette | New method to study bacterial adhesion to meat[END_REF]) and more recently to dairy components (Burgain et al., 2014a;[START_REF] Gomand | High-throughput screening approach to evaluate the adhesive properties of bacteria to milk biomolecules[END_REF][START_REF] Guerin | Pili of Lactobacillus rhamnosus GG mediate interaction with beta-lactoglobulin[END_REF]. Bacterial adhesive interactions to food components can compete with bacterial adhesion to the host [START_REF] Sun | Food derived anti-adhesive components against bacterial adhesion: Current progresses and future perspectives[END_REF]. Therefore food components such as milk fat globule membrane [START_REF] Douëllou | Invited review: Anti-adhesive properties of bovine oligosaccharides and bovine milk fat globule membrane-associated glycoconjugates against bacterial food enteropathogens[END_REF]Guerin et al., 2018b), milk proteins [START_REF] Halpin | Inhibition of adhesion of Streptococcus mutans to hydroxylapatite by commercial dairy powders and individual milk proteins[END_REF], and milk oligosaccharides [START_REF] Lane | Methodologies for screening of bacteria-carbohydrate interactions: Anti-adhesive milk oligosaccharides as a case study[END_REF] can play an anti-adhesive role by decreasing bacterial adhesion to the intestine (Guerin et al., 2018b). Some food additives including stabilizers (such as sucrose fatty acid esters) and colors (gardenia yellow, monascus pigment, etc.) have also been found to feature similar effects [START_REF] Islam | Combined effects of selected food additives on adhesion of various foodborne pathogens onto microtiter plate and cabbage leaves[END_REF].

In food matrices, adhesive interactions are likely to play an important part in bacterial spatial distribution and viability during the structuration of the food matrix [START_REF] Gomand | Food Matrix Design for Effective Lactic Acid Bacteria Delivery[END_REF]. Adhesive interactions occurring between the model strain Lactobacillus rhamnosus GG (LGG) and βlactoglobulin is mediated by the pili produced by LGG cells on their surface [START_REF] Guerin | Pili of Lactobacillus rhamnosus GG mediate interaction with beta-lactoglobulin[END_REF]. These interactions result in an increased encapsulation efficiency when using dairy components as well as a higher resistance to gastric digestion for this strain (Burgain et al., 2013a(Burgain et al., , 2014b;;[START_REF] Guerin | Use of imaging techniques to identify efficient controlled release systems of Lactobacillus rhamnosus GG during in vitro digestion[END_REF]. Adhesive interactions between genetically engineered Lactococcus lactis producing pili and dairy components result in texture alteration in fermented milk (Tarazanova et al., 2018a) and can modulate this strain distribution in cheese curd [START_REF] Tarazanova | Influence of lactococcal surface properties on cell retention and distribution in cheese curd[END_REF]. Similarly, during curdling and cheese ripening, bacterial cells mostly co-localize with fat globules or at the casein-fat interface, which suggest adhesive interactions between fat and LAB strains [START_REF] Laloy | Influence of the fat content of Cheddar cheese on retention and localization of starters[END_REF][START_REF] Lopez | Lipolysis during Ripening of Emmental Cheese Considering Organization of Fat and Preferential Localization of Bacteria[END_REF]. This is likely to play a role in lipolysis thus affecting the development of characteristic flavors and textures during ripening [START_REF] Laloy | Influence of the fat content of Cheddar cheese on retention and localization of starters[END_REF][START_REF] Lopez | Lipolysis during Ripening of Emmental Cheese Considering Organization of Fat and Preferential Localization of Bacteria[END_REF].

However, the impact and technological interest of adhesive interactions is yet poorly documented and largely remains to be investigated [START_REF] Hickey | Growth and location of bacterial colonies within dairy foods using microscopy techniques: a review[END_REF]. Adhesive interactions between bacterial surface components and dairy components have only been studied for very few wild type strains, namely LGG [START_REF] Guerin | Pili of Lactobacillus rhamnosus GG mediate interaction with beta-lactoglobulin[END_REF], Lactobacillus amylovorus [START_REF] Chumphon | Development of rice as potential carriers for probiotic Lactobacillus amylovorus[END_REF], and Lactobacillus paracasei [START_REF] De Bellis | Probiotic table olives: Microbial populations adhering on olive surface in fermentation sets inoculated with the probiotic strain Lactobacillus paracasei IMPC2.1 in an industrial plant[END_REF]. This article goes one step forward in that direction by applying the high-throughput screening method recently developed by [START_REF] Gomand | High-throughput screening approach to evaluate the adhesive properties of bacteria to milk biomolecules[END_REF] to a collection of 73 LAB strains (for which genome sequence is available) in order to characterize their potential adhesive behavior towards the major dairy protein β-lactoglobulin, to which the adhesive behavior of the model strain LGG is already well-known (Burgain et al., 2013b(Burgain et al., , 2014b[START_REF] Burgain | Impacts of pH-mediated EPS structure on probiotic bacterial pili-whey proteins interactions[END_REF][START_REF] Guerin | Pili of Lactobacillus rhamnosus GG mediate interaction with beta-lactoglobulin[END_REF]Guerin et al., , 2018a)). Two strains featuring extreme adhesive and non-adhesive behaviors have then been studied in further depth through AFM. The AFM results were then studied in relation to Confocal Laser Scanning Microscopy (CLSM) experiments, allowing to observe the spatial distribution of these strains in Whey Protein Isolate (WPI) solution.

2 Material and methods

High-throughput screening

Adhesive interactions between bacteria and β-lactoglobulin were screened using the method recently developed by [START_REF] Gomand | High-throughput screening approach to evaluate the adhesive properties of bacteria to milk biomolecules[END_REF] using an automated liquid handling system for 96-well microplates.

Briefly, this method consists in immobilizing the biomolecules of interest on the surface of 96 well adherent microplates. Microplates are then washed with a blocking agent in order to remove all unbound molecules and to block the remaining empty sites. The bacterial suspension is then added into the wells and incubated for 1 h at 37 °C in order to allow bacterial adhesion to the immobilized biomolecules. Non-adherent bacteria are removed by successive washes using the same blocking agent. The amount of immobilized bacteria is measured through bacterial growth monitoring (turbidity measurements at 595 nm) after the addition of MRS culture growing medium [START_REF] De Man | A Medium for the Cultivation of Lactobacilli[END_REF]) in the wells. The higher the initial quantity of bound bacteria, the earlier the growth starts. Adjustments made to this protocol are listed below.

Bacterial strains and cultures

A list of the 73 screened LAB strains is given in Supplementary data 1 (S1). This collection of strains has previously been studied for their genomics and surface properties [START_REF] Sun | Expanding the biotechnology potential of lactobacilli through comparative genomics of 213 strains and associated genera[END_REF]. The model strain Lactobacillus rhamnosus GG ATCC53103 (LGG wild type, "WT") and the mutant strain LGG spaCBA CMPG 5357 impaired in pili synthesis, which adhesive properties of both are well-known [START_REF] Guerin | Pili of Lactobacillus rhamnosus GG mediate interaction with beta-lactoglobulin[END_REF][START_REF] Lebeer | Functional Analysis of Lactobacillus rhamnosus GG Pili in Relation to Adhesion and Immunomodulatory Interactions with Intestinal Epithelial Cells[END_REF][START_REF] Tripathi | Deciphering the Nanometer-Scale Organization and Assembly of Lactobacillus rhamnosus GG Pili Using Atomic Force Microscopy[END_REF][START_REF] Tripathi | Adhesion and Nanomechanics of Pili from the Probiotic Lactobacillus rhamnosus GG[END_REF] were respectively used as positive (adherent) and negative (non-adherent) control strains.

For each series of experiments, a 96-well microplate previously stored at -80 °C was thawed and replicated on working microplates using 50 µL of bacterial suspension to inoculate 150 µL of MRS by well. The working microplates were incubated at 30 °C two days before the adhesion assay. During the adhesion assay, microplates were only centrifuged once at 1,642 g for 20 min, emptied and the resulting cell pellets were resuspended in 200 µL of PBS adjusted at pH 6.8. Triplicates on independent cultures were performed as well as duplicates by strain on each plate (6 repetitions for control strains).

Preparation of the β-lactoglobulin solution and microplate coating

Beta-lactoglobulin (Sigma-Aldrich Co. LLC, St Louis, MO, USA) was prepared in solution (1% w/w) as described by [START_REF] Gomand | High-throughput screening approach to evaluate the adhesive properties of bacteria to milk biomolecules[END_REF].

Bacterial growth monitoring

Adhesion and growth monitoring were done according to [START_REF] Gomand | High-throughput screening approach to evaluate the adhesive properties of bacteria to milk biomolecules[END_REF]. The incubation temperature was changed to 30 °C in order to match the diversity of the growing conditions for all strains [START_REF] Gomand | High-throughput screening approach to evaluate the adhesive properties of bacteria to milk biomolecules[END_REF]. Bacterial growth was monitored through OD595nm measurements over 48 h.

Data processing

Strain growth comparison

The times at which the apparent bacterial growth starts (tstart) were monitored such as described by [START_REF] Gomand | High-throughput screening approach to evaluate the adhesive properties of bacteria to milk biomolecules[END_REF]. The higher these time values are, the later the growth starts i.e. the fewer bacteria have adhered i.e. the lower the affinity. These values were averaged on all series of experiments and standard deviations are computed. Strains were compared to one another based on their Minimum Adhesion Value (MAV) corresponding to the difference between the smallest tstart (highest adhesion) obtained on a control without β-lactoglobulin and the highest tstart (lowest adhesion) obtained on βlactoglobulin:

𝑴𝒊𝒏𝒊𝒎𝒖𝒎 𝑨𝒅𝒉𝒆𝒔𝒊𝒐𝒏 𝑽𝒂𝒍𝒖𝒆 (𝑴𝑨𝑽) = (𝒕 𝒔𝒕𝒂𝒓𝒕 ) 𝒂𝒗𝒆𝒓𝒂𝒈𝒆 -𝝈 𝑪𝑶𝑵𝑻𝑹𝑶𝑳 -(𝒕 𝒔𝒕𝒂𝒓𝒕 ) 𝒂𝒗𝒆𝒓𝒂𝒈𝒆 -𝝈 𝛃 𝐋𝐀𝐂
Where σ stands for standard deviation. A strain is considered to adhere to β-lactoglobulin if its MAV is significantly superior to zero for all three series of experiments.

Functional domain prediction for the bacterial surface proteome

Bacterial surface proteins featuring LPxTG motif were predicted using the InterPro resource, that provides functional analysis of protein sequences by classifying them into families and predicting the presence of domains and important sites [START_REF] Finn | InterPro in 2017-beyond protein family and domain annotations[END_REF]. Protein sequences were obtained from [START_REF] Sun | Expanding the biotechnology potential of lactobacilli through comparative genomics of 213 strains and associated genera[END_REF] and were scanned against InterPro's signatures using the software package InterProScan [START_REF] Jones | InterProScan 5: genome-scale protein function classification[END_REF]. Gene sequence resemblance with known domains was performed using the Basic Local Alignment Search Tool resource (BLAST), according to [START_REF] Altschul | Basic local alignment search tool[END_REF].

Statistical analysis

Statistical analysis were performed via t-tests and Tukey tests (parametric) for normal data and Wilcoxon-Mann Whitney and Steel-Dwass tests (non-parametric) for data that did not fit normal distribution using Kyplot software (Kyens Lab Inc.).

Adhesive interactions between bacteria and β-lactoglobulin characterized through Atomic Force Microscopy

Protocols used in this part have been adapted from Guerin et al. (2018a). Briefly, this method consists in immobilizing the bacterial strains of interest on functionalized gold-coated mica by deposing the bacterial suspension during 15 h at 4 °C (pH 6.8). The mica is rinsed with PBS (pH 6.8) before use. Milk proteins are prepared in distilled water (1% w/w) and adsorbed on modified AFM probes (gold coated and with NH2-terminated PEG linker) by immersion for 15 h at 4 °C and then rinsed with milli-Q-grade water before use. Force measurements are performed at room temperature in PBS buffer (pH 6.8). AFM force distance curves are obtained by following the cantilever deflection as a function of the vertical displacement of the piezoelectric scanner with a scan speed of 400 mm/s. Adjustments to this protocol are listed below.

Bacterial cultures

Cultures were prepared according to Guerin et al. (2018a). Precultures of Lactobacillus aquaticus DSM 21051 and Lactobacillus sharpeae DSM 20505 were prepared by inoculating 9 mL of MRS broth with 100 µL of bacterial stock and grown overnight at 37 °C. These precultures were used to inoculate 9 mL of fresh MRS broth the next day and the growth was performed at 37 °C until an optical density of 1.2 was reached at 660 nm (for about 8 h). Cultures were then centrifuged at 3,000 g for 10 min at room temperature. Pellets were suspended in 1 mL of PBS (pH 6.8).

Preparation of bacteria-coated mica and protein-coated tips

According to Guerin et al. (2018a), a mica coated with a gold layer functionalized with a NH2terminated PEG-linker (Novascan, Ames, Iowa, USA) was used, as well as AFM probes with borosilicate glass particle (2 µm), coated with gold and modified with NH2 terminated PEG linker (Novascan, Ames, Iowa, USA). The bacterial suspension is deposed on mica at 4 °C and left overnight (pH 6.8). Preparation of the β-lactoglobulin and Bovine Serum Albumine (BSA) 1% (w/w) solutions (Sigma-Aldrich Co. LLC, St Louis, MO, USA) was done according to Guerin et al. (2018a). Probes tips were left to incubate overnight at 4°C in wells containing 1 mL of the β-lactoglobulin or BSA solutions to maximize protein adsorption. β-lactoglobulin was the candidate protein tested and BSA was the negative control.

AFM measurements

Protocol followed is described by Guerin et al. (2018a). Force-volume measurements are performed at room temperature in PBS buffer (pH 6.8) using a Bruker Bioscope Resolve atomic force microscope (Bruker corporation, Santa Barbara, CA) mounted on an inverted microscope (DMi8, Leica microsystems). The spring constants of the cantilevers was measured using the thermal noise method and found to be 0.01 N m -1 . Force distance curves were recorded between the bacteria deposited on functionalized mica and the probe coated with β-lactoglobulin or BSA. Three adhesion force maps (20 µm × 20 µm, 256 force curves) were recorded for each protein-bacteria interaction analysis. Data analysis was performed using the Nanoscope Analysis software from Bruker (Santa Barbara, CA, USA) and the last peak was calculated for each curve before plotting adhesion forces and last rupture length histograms. The last peak is used for analysis instead of the maximum peak in order to characterize the last interacting point between the β-lactoglobulin and the cell receptor and not the unfolding of a biomolecular domain.

Adhesive interactions imaged by confocal microscopy

The cultures were prepared as described in 2.2.1., then centrifuged at 3,000 g for 10 min at room temperature. Pellets were suspended in 10 mL of WPI solution (15 %, w/w). The WPI solution was prepared using PRODIET 90 S (Ingredia, Arras, France) that is a soluble milk protein isolate containing native whey proteins including β-lactoglobulin. One milliliter of resuspended cells was stained with the LIVE/DEAD BacLight viability kit (1:200 v/v; LIVE/DEAD BacLight viability kit was prepared according to the procedure described for the kit L13152 by ThermoFisher Scientific). Two hundred microliters of LAB suspension (same conditions as 2.2.1) were introduced on chambered glass slides (Nunc Lab-Tek, ThermoFisher Scientific). Confocal Laser Scanning Microscopy (CLSM) images were taken using a Leica TCS SP5-X-AOBS confocal laser scanning microscope (Leica Microsystems CMS GmbH, Mannheim, Germany) equipped with WLL lasers. The objective lens used was a HCX PL APO CS 100 × 1.40 (oil immersion). The excitation wavelength was 488 nm and emission bandwidth was of 495-510 nm for SYTO 9 and 600-620 nm for propidium iodide. Two independent repetitions were performed and approximately 20 representative images were acquired for each sample.

3 Results

Identification of strains adhesive to β-lactoglobulin

Most strains were found not to be adhesive to β-lactoglobulin as the average MAV calculated on the 73 strains was negative (-180 ± 22) although higher than the MAV of the negative control LGG spaCBA (-386), known to be non-adhesive to β-lactoglobulin [START_REF] Guerin | Pili of Lactobacillus rhamnosus GG mediate interaction with beta-lactoglobulin[END_REF]. The microplate adhesive assays revealed four adhesive candidates to β-lactoglobulin amongst the 73 strains tested: The MAV for all strains are listed as supplementary material (S1).

Lactobacillus

Biophysical deciphering of bacterial adhesive interaction with β-lactoglobulin through AFM

The adhesive interactions between β-lactoglobulin and the strains at the extremes of the adhesion spectrum, L. aquaticus DSM 21051 (the most adhesive strain) and L. sharpeae DSM 20505 (the least adhesive strain) were studied through AFM, in order to characterize them in further depth. Only two strains were chosen to precise our understanding of the interaction mechanism of the LAB surface with β-lactoglobulin since AFM is not a suitable method for screening of large populations. This is why we decided to select only the two strains at the extreme of the adhesion spectrum for this analysis. BSA was used as a negative control as LAB strains have previously been found to feature low adhesion to it [START_REF] Gomand | High-throughput screening approach to evaluate the adhesive properties of bacteria to milk biomolecules[END_REF][START_REF] Guerin | Pili of Lactobacillus rhamnosus GG mediate interaction with beta-lactoglobulin[END_REF]. The percentages of adhesive events (frequencies) observed between L. aquaticus DSM 21051 and the two proteins, β-lactoglobulin and BSA, were respectively of 82.6 ± 7.1% and 27.6 ± 10.4% (Figure 1A1). The frequencies of adhesive events observed between L. sharpeae DSM 20505 and the same two proteins were respectively of 3.4 ± 1.5% for β-lactoglobulin and 2.5 ± 0.6 % for BSA (Figure 1B1). Typical force-distance curves obtained for the interactions occurring between the two strains and the AFM probes functionalized with the two proteins are presented, i.e. L. aquaticus DSM 21051 and -lactoglobulin (Figure 1A2), L. aquaticus DSM 21051 and BSA (Figure 1A3), L. sharpeae DSM 20505 and -lactoglobulin (Figure 1B2), and L. sharpeae DSM 20505 and BSA (Figure 1B3). During the withdrawal of functionalized β-lactoglobulin-coated probe from the surface of L. aquaticus DSM 21051 several specific adhesive events occur (Figure 1A2), whereas more than 70% of the curves observed for BSA-coated probes did not feature any adhesive event (Figure 1A3). Moreover, the few adhesive events observed between BSA and L. aquaticus DSM 21051 appeared to be random and therefore could not be associated to any specific interaction (Figure 1A3). Almost no adhesive event was observed for both BSA-and β-lactoglobulincoated probes on L. sharpeae DSM 20505 cells (Figures 1B1,B2,B3). These results are consistent with those obtained using the screening method: L. aquaticus DSM 21051 significantly adheres to -lac whereas poor adhesion was observed for L. sharpeae DSM 20505. Retraction curves recorded between L. aquaticus DSM 21051 and β-lactoglobulin attest the specificity of occurring adhesive interactions, which would happen according to a lock and key mechanism (Figure 2A). 3D-AFM images recorded on mica attest of the good coverage of L. aquaticus DSM 21051 and therefore that adhesive events recorded did occur between L. aquaticus DSM 21051 cells and β-lactoglobulin-coated probes (Figure 2B). The biophysical properties of the adhesion between L. aquaticus DSM 21051 and -lac were analyzed using additional force parameters including adhesion forces (Figure 2C) and final rupture length (Figure 2D). Retraction curves exhibited adhesion forces averaging around 1.43 ± 0.03 nN.

Final rupture length averaged around 0.90 ± 0.03 µm. These results will be compared with those of LGG WT and the mutant strains LGG spaCBA and welE in the discussion section.

Impact of adhesive interactions on bacterial distribution in whey protein isolate probed by confocal microscopy

L. aquaticus DSM 21051, L. sharpeae DSM 20505, LGG WT and LGG spaCBA were first imaged in MRS to make sure that they were originally homogeneously distributed (Figure 3A1, 3B1 and 4A1, 4B1). Live cells of L. aquaticus DSM 21051 were found to aggregate in the WPI solution whereas L. sharpeae DSM 20505 live cells remained homogeneously distributed (Figure 3A2 and 3B2). This is consistent with the adhesive properties of the control strains: LGG WT (positive control) aggregate in the WPI solution whereas LGG spaCBA remained homogeneously distributed (Figure 4A2 and4B2).

Dead bacterial cells or cells with a damaged membrane gathered in flocs for all 4 strain types (data not shown).

Relation between bacterial adhesion to β-lactoglobulin and predicted bacterial surface characteristics

Presence of pili genes clusters (PGCs)

Predicted bacterial surface characteristics were analyzed in relation to the results of the adhesive assays in order to delineate gene candidates predicted to encode surface proteins that could be involved in bacterial adhesion to β-lactoglobulin. Amongst the 73 strains tested, 32 of them possessed at least one sortase-dependent pilus gene cluster (PGC) and therefore were predicted to express pili on their surface [START_REF] Sun | Expanding the biotechnology potential of lactobacilli through comparative genomics of 213 strains and associated genera[END_REF]. The average MAV of these 32 strains was -163 ± 33.2 whereas the average MAV of the 41 non-piliated strains was -194 ± 30.1. Amongst the 32 strains presenting PGCs, 16 possessed PGCs similar to LGG pilus clusters in terms of gene order, that is, a cluster of three pilin genes and one pilin-specific sortase gene [START_REF] Sun | Expanding the biotechnology potential of lactobacilli through comparative genomics of 213 strains and associated genera[END_REF]. The MAV of these 16 strains was -165 ± 53.8 whereas the MAV of the 16 strains with PGCs different from LGG was -160 ± 38.8. Although a mean comparison of the MAV for strains featuring PGCs compared to non-piliated strains would suggest that the presence of PGCs fosters adhesion to β-lactoglobulin, this was not supported statistically. No difference could be observed between strains featuring PGCs similar to LGG WT's and PGCs different from LGG WT's. The number of PGCs, sortase enzymes or proteins with LPxTG motif (listed for all strains in S1) were not found either to impact strain adhesion to β-lactoglobulin (data not shown).

Predicted protein domains candidates for mediating bacterial adhesion to β-lactoglobulin

More predicted surface characteristics were analyzed for the four strains found to be adhesive to βlactoglobulin. Predicted protein domains featuring LPxTG motif found for each strain are listed in Table 1. Strains were analyzed for gene sequence resemblance with the spaCBA domain, known to be responsible for adhesion to β-lactoglobulin for LGG WT [START_REF] Guerin | Pili of Lactobacillus rhamnosus GG mediate interaction with beta-lactoglobulin[END_REF] but no homologue sequence could be identified for any of the four adhesive strains. All strains are predicted to feature immunoglobulin-like (Ig-like) fold domains, which are usually involved in binding or molecular recognition processes [START_REF] Bodelón | Immunoglobulin domains in Escherichia coli and other enterobacteria: from pathogenesis to applications in antibody technologies[END_REF]. Other and more specific adhesion-related domains present on the four adhesive strains studied as well as on LGG WT include MucBP (mucin-binding), CBME/CBM3 (carbohydrate-binding), figbrinogen-and collagen-binding domains, cysteine-and leucine-rich domains, and SD-repeat B-domain. Most of these domains are present once in the genome of the adhesive strains (L. plantarum DSM 13273 is the only adhesive strain presenting 3 MucBP domains) and are not repeated within a given protein.

The MucBP domain is the only domain with a known adhesive-related function (apart from the Ig-like fold domain) which could be identified on L. aquaticus DSM 21051, the most adhesive strain to βlactoglobulin. MucBP domains have been found predominantly in lactobacilli found naturally in intestinal niches, which suggests that they play an important role in establishing host-microbial interactions in the gut by binding mucus [START_REF] Roos | A high-molecular-mass cell-surface protein from Lactobacillus reuteri 1063 adheres to mucus components[END_REF][START_REF] Tassell | Lactobacillus Adhesion to Mucus[END_REF]. L. plantarum DSM 13273 is the strain featuring the highest number of adhesion-related domains in its genome (Table 1). This is also the only strain out of the four presenting fibrinogen-and collagenbinding domains. The fibrinogen-binding domain has been found to accommodate linear peptides with a certain degree of ligand sequence variability [START_REF] Ponnuraj | A "dock, lock, and latch" structural model for a staphylococcal adhesin binding to fibrinogen[END_REF] and therefore might be able to interact with β-lactoglobulin. L. brantae DSM 23927 features leucine-rich repeats (LRRs) and SDrepeat (Sdr) domains (Table 1), both of them susceptible to play a role in adhesive interactions to βlactoglobulin. LRRs have been found to provide a structural framework for the formation of proteinprotein binding and interactions [START_REF] Gay | A leucine-rich repeat peptide derived from the Drosophila Toll receptor forms extended filaments with a β-sheet structure[END_REF][START_REF] Kobe | The leucine-rich repeat as a protein recognition motif[END_REF] and are likely to allow a broad range of ligands [START_REF] Kobe | The leucine-rich repeat as a protein recognition motif[END_REF]. Sdr-repeat domains are surface proteins that play an important role in Staphylococcus aureus adhesion and pathogenesis [START_REF] Mccrea | The serineaspartate repeat (Sdr) protein family in Staphylococcus epidermidis[END_REF][START_REF] Wang | Structures of SdrD from Staphylococcus aureus reveal the molecular mechanism of how the cell surface receptors recognize their ligands[END_REF]. The protein containing Sdr-repeat domains may therefore be a good candidate for mediating adhesion to β-lactoglobulin for the strain L. brantae DSM 23927. No other adhesion-related domain than the Ig-like fold domain was identified on L. murinus DSM 20452 (Table 1), which would suggest that the protein containing this domain would likely be the one involved in adhesive interactions with β-lactoglobulin.

Discussion

The aim of this study was to evaluate and characterize adhesive interactions occurring between lactic acid bacteria and β-lactoglobulin. A collection of 73 LAB strains was screened for their adhesive behavior towards β-lactoglobulin and strains at the extreme of the adhesion spectrum i.e. a highly adhesive and a poorly adhesive strains were studied in further depth.

Only four strains out of 73 were found to present adhesive affinities towards β-lactoglobulin. Therefore, adhesion to β-lactoglobulin appears not to be a common characteristic of the LAB group. The consequences of these adhesive interactions, when they occur, are not fully understood. However, it could be hypothesized that strains featuring adhesive affinities towards whey proteins would be lost during the drainage step of cheese manufacturing processes, alongside with whey expulsion from the cheese network. It would be interesting to test the affinity of this same strain collection to other food components in future work, in order to dispose of more comparison points to our study and to get a better understanding of the importance of adhesion to β-lactoglobulin compared to adhesion to other food components. Currently, the rare existing studies discussing bacterial adhesion to food components other than β-lactoglobulin concern up to four strains at most at a time [START_REF] Chumphon | Development of rice as potential carriers for probiotic Lactobacillus amylovorus[END_REF][START_REF] De Bellis | Probiotic table olives: Microbial populations adhering on olive surface in fermentation sets inoculated with the probiotic strain Lactobacillus paracasei IMPC2.1 in an industrial plant[END_REF][START_REF] Tarazanova | Cell Surface Properties of Lactococcus lactis Reveal Milk Protein Binding Specifically Evolved in Dairy Isolates[END_REF][START_REF] Tarazanova | Influence of lactococcal surface properties on cell retention and distribution in cheese curd[END_REF]Tarazanova et al., , 2018a;;[START_REF] Utratna | Rapid screening for specific glycosylation and pathogen interactions on a 78 species avian egg white glycoprotein microarray[END_REF], therefore failing to provide an overview of adhesion to food components amongst wide bacterial groups such as the LAB group.

The study performed by [START_REF] Tarazanova | Cell Surface Properties of Lactococcus lactis Reveal Milk Protein Binding Specifically Evolved in Dairy Isolates[END_REF] is the only one to our knowledge that compares the adhesion level of a wide number of strains (55) to food (casein-derived) components, however these strains are all of the same species, Lactoccocus lactis. Out of 55, 30 to 40 strains presented adhesive affinities towards casein-derived components, depending on their growth phase, and strains isolated from a dairy environment presented much stronger binding of milk proteins versus strains isolated from plants, suggesting a selective advantage [START_REF] Tarazanova | Cell Surface Properties of Lactococcus lactis Reveal Milk Protein Binding Specifically Evolved in Dairy Isolates[END_REF]. However, this was not confirmed in our case, as the four strains out of 73 that were originally isolated from dairy products, i.e. Lactobacillus casei DSM 20011, Lactobacillus paracasei subsp. tolerans DSM 20258, Lactobacillus bifermentans DSM 20003, and Lactobacillus kefiri DSM 20587, did not present more adhesive affinities towards βlactoglobulin in average than the strains isolated from nondairy sources (data not shown).

The strain found to be the most adhesive to β-lactoglobulin, L. aquaticus DSM 21051, exhibited a specific adhesive behavior when studied by AFM. The signature of the observed retraction curves was identified as specific of biomolecules stretching, suggesting that the surface of L. aquaticus DSM 21051 features a strong affinity towards -lac. This has also been shown previously for the model strain LGG WT by our team as well as for the mutant strain LGG welE, expolysaccharide-depleted and known to adhere more to β-lactoglobulin than LGG WT due to its increased pili exposure [START_REF] Guerin | Pili of Lactobacillus rhamnosus GG mediate interaction with beta-lactoglobulin[END_REF](Guerin et al., , 2018a)). A contrario, L. sharpeae DSM 20505 which screening results show not to adhere to β-lactoglobulin presented retraction curves characteristic of a lack of adhesion to -lac when studied by AFM (frequency of adhesive events was inferior to 5 %). Similarly, our team demonstrated previously this same fact for the model strain non-adhesive to β-lactoglobulin, LGG spaCBA [START_REF] Guerin | Pili of Lactobacillus rhamnosus GG mediate interaction with beta-lactoglobulin[END_REF]. Comparative results are presented in Table 2.

The adhesive behavior of L. aquaticus DSM 21051 towards -lactoglobulin appears relatively close to the one of LGG welE in terms of frequency of adhesive events. The high specificity of the adhesion phenomenon occurring between L. aquaticus DSM 21051 and β-lactoglobulin is highlighted by the fact that the frequency of adhesion is almost twice as high as the one characterizing adhesive interactions between LGG WT and β-lactoglobulin, whereas the frequency of adhesion of L. aquaticus DSM 21051 on BSA is almost 4 times lower than the one occurring between LGG welE and BSA. The mean adhesion force recorded on the last peak is also 3 times higher than the mean adhesion force recorded for LGG WT and β-lactoglobulin, and higher than the highest adhesion force recorded on the last peak for LGG welE and β-lactoglobulin, reaffirming the idea of a very strong specificity and adhesion strength. When comparing the length of biomolecules stretched by adhesive interactions with β-lactoglobulin, L. aquaticus DSM 21051 and LGG welE both exhibit molecules stretched up to 1µm i.e. 3 times longer than the molecule stretched in the case of LGG WT (Table 2). The molecule mediating adhesive interactions with β-lactoglobulin in the case of L. aquaticus DSM 21051 is therefore comparable in length to LGG pili when stretched, which may explain the higher specificity and adhesion strength found for L. aquaticus DSM 21051 compared to LGG WT, which pili are partially hidden within the expolysaccharides layer [START_REF] Guerin | Pili of Lactobacillus rhamnosus GG mediate interaction with beta-lactoglobulin[END_REF].

On the other hand, the frequency of adhesive events observed between L. sharpeae DSM 20505 and β-lactoglobulin is inferior to 5 % and similar to the frequency of adhesive events observed on BSA for both this strain and L. aquaticus DSM 21051. The frequency of adhesive events recorded when using BSA-coated probes is also 4 times lower for L. sharpeae DSM 20505 than for LGG spaCBA (negative control). Overall, L. sharpeae DSM 20505 has demonstrated very poor adhesive capacities towards βlactoglobulin. However, when analyzed for predicted adhesion-related protein domains, this strain revealed a total of 23 adhesion-related domains, 8 of which being different, including MucBP and Gram positive pilin subunit D1 N-terminal, although no sequence homologue to the spaCBA domain was found (data not shown). The spaCBA domain is known to mediate adhesion to β-lactoglobulin for the piliated strain LGG WT [START_REF] Guerin | Pili of Lactobacillus rhamnosus GG mediate interaction with beta-lactoglobulin[END_REF]. This confirms that adhesive interactions with βlactoglobulin are specific, and cannot be predicted accurately using only genomic predictions (the functions of these domains may not be accurately predicted or they may not be expressed).

The gathering behavior observed by CLSM for the adhesive strains in the WPI solution also pledges in favor of a specific bacterial adhesion to β-lactoglobulin for L. aquaticus DSM 21051. CLSM results indicate that the location of bacteria in a dairy matrix strongly depends on bacterial surface properties.

These observations are important as it was evidenced recently that physical properties of dairy products, such as viscosity and gel hardness, are affected by bacterial surface properties in the case of surface-engineered strains (Tarazanova et al., 2018a). In light of our results, it would be interesting to see if that is also the case for wild strains presenting different surface properties inducing different adhesive behaviors. Some peptides shown to be linked to bacterial aggregation were also recently evidenced to be able to promote bacterial adhesion to functionalized surfaces and Caco-2-cells [START_REF] Okochi | Screening of peptides associated with adhesion and aggregation of Lactobacillus rhamnosus GG in vitro[END_REF]. This typical behavior was responsible for observed enhanced interactions between LAB and the host intestinal mucosa [START_REF] Okochi | Screening of peptides associated with adhesion and aggregation of Lactobacillus rhamnosus GG in vitro[END_REF]. Adhesive interactions with β-lactoglobulin leading to the aggregation of L. aquaticus DSM 21051 and LGG WT cells might therefore be considered for further study in order to determine whether they would promote such kind of behavior as well.

This work was performed in the continuity of previous studies, in which a method was developed allowing screening a wide number of strains for their adhesive affinities towards biomolecules such as dairy food components [START_REF] Gomand | High-throughput screening approach to evaluate the adhesive properties of bacteria to milk biomolecules[END_REF], and which identified the bacterial surface molecules (pili) involved in the adhesion of LGG to dairy components using AFM [START_REF] Guerin | Pili of Lactobacillus rhamnosus GG mediate interaction with beta-lactoglobulin[END_REF]. The present study sought to go beyond bacterial species differences in revealing common adhesive characteristics of Lactic Acid Bacteria in relation to dairy food components such as β-lactoglobulin. We first looked for LAB species featuring adhesive affinities for β-lactoglobulin, then focused on the molecular characteristics of this adhesion. We observed adhesion to β-lactoglobulin for few LAB (less than 6% of our collection). However, for those which did feature adhesive affinities, some common characteristics were pointed out that matched the characteristics previously identified on the model strain LGG. These characteristics include the specificity of the affinity, as well as the impact on bacterial spatial distribution in the matrix. The major findings of the present paper are that (i) Adhesion to whey proteins is apparently not a common characteristic to the LAB group (few strains presented adhesive affinities towards β-lactoglobulin), (ii) Strains featuring adhesive affinities towards βlactoglobulin present common adhesive characteristics (specific β-lactoglobulin-adhesion domains related to the specificity of the AFM signature), and (iii) Adhesion to β-lactoglobulin was shown to strongly influence bacterial distribution in dairy matrices featuring this component (adhesive bacteria gathered in flocs in whey matrices whereas non-adhesive bacteria distribute more homogeneously), and could therefore modulate their accessibility and later delivery when designing functional foods containing LAB with potential associated health effects.

According to these findings, food matrices could play a protective role on bacteria by influencing their spatial distribution, which may prove especially useful for probiotic bacteria. Indeed, as bacteria adhering to a component have been found to flocculate in the food matrix containing this component, this could result in later heterogeneous delivery in the gastro-intestinal tract (GIT) which would impact host colonization, but may also better protect bacterial survival until they reach the GIT. These findings also pave the road to future experiments aiming generalizing bacterial adhesion characteristics to broad bacterial groups, thus helping with practical food matrix design. It would therefore be interesting to study the potential protective effect of components to which bacteria are adherent during critical steps of the food manufacturing process, such as spray-drying during probiotic milk powder production.

Author contributions FG, JG, JB, FB, CG conceived the research. FG, JG, JB, SEKC, DD, GF carried out the experiments. FG, JB, JG, SEKC, DD, GF analyzed the data. FG, JG, JB wrote the manuscript. All authors commented on the manuscript.

Funding

This article has been written as part of a LUE project (Lorraine Université d'Excellence). The authors would like to thank the LUE initiative for having provided fundings and supported this work.

The authors declare no competing interest. *Frequency of adhesive events as found to be inferior to 5%.

  aquaticus DSM 21051 (MAV = 61.5), Lactobacillus murinus DSM 20452 (MAV = 12.8), Lactobacillus plantarum DSM 13273 (MAV = 12.6), Lactobacillus brantae DSM 23927 (MAV = 6.97), although these strains were still less adhesive than the positive control LGG WT (MAV = 104). Nine strains were also found to have a MAV inferior to the one of the negative control LGG spaCBA: Lactobacillus sharpeae DSM 20505 (MAV = -857), Lactobacillus kefiri DSM 20587 (MAV = -787), Lactobacillus similis DSM 23365 (MAV = -780), Lactobacillus pobuzihii DSM 28122 (MAV = -617), Lactobacillus namurensis DSM 19117 (MAV = 516), Lactobacillus satsumensis DSM 16230 (MAV = -490), Pediococcus parvulus DSM 20332 (MAV = -477), Lactobacillus senmazukei DSM 21775 (MAV = -404), Lactobacillus lindneri DSM 20690 (MAV = -387).

Figure 1 Figure 2

 12 Figure 1

  Figure 3

Table 1 :

 1 proteins containing-domains written in bold characters for the four adhesive strains are likely to mediate adhesive interactions with β-lactoglobulin Predicted proteins domains with LPxTG motif which may play a role in bacterial adhesion to β-lactoglobulin. Domains present on L. rhamnosus GG (known to be adhesive to β-lactoglobulin) are included as a reference. Proteins sequences used were those provided by[START_REF] Sun | Expanding the biotechnology potential of lactobacilli through comparative genomics of 213 strains and associated genera[END_REF].

	Strain		MAV	Predicted adhesion-related protein domains*
	Lactobacillus aquaticus DSM 21051 61.5 Immunoglobulin-like fold, MucBP
	Lactobacillus murinus	DSM 20452 12.8 Immunoglobulin-like fold
				Immunoglobulin-like fold, MucBP, CMBE-
	Lactobacillus plantarum DSM 13273 12.6	CBM3 (carbohydrate-binding), fibrinogen-
				binding, collagen-binding
				Immunoglobulin-like fold, Collagen-binding
	Lactobacillus brantae	DSM 23927 6.97	surface protein Cna-like (type B), Leucin-rich
				repeat, SD-repeat (type B)
				Immunoglobulin-like fold, MucBP, Leucin-rich
				repeat, Fn3-like (frequently found in the
	Lactobacillus rhamnosus GG WT	104	adhesin/invasin streptococcal C5a), Gram positive pilin subunit D1 N-terminal (containing
				spaCBA domain, responsible of adhesion to β-
				lactoglobulin)

*

Table 2 :

 2 Comparison of the adhesive capabilities of five strains to β-lactoglobulin when studied by Atomic Force Microscopy: L. aquaticus DSM 21051, L. sharpeae DSM 20505, and the model strains LGG WT, LGG spaCBA (pili-depleted) and LGG welE (expolysaccharides-depleted).

Acknowledgment

The wild type strain LGG ATCC53103 (WT) and the derivative mutant strains spaCBA CMPG 5357 (impaired in pili synthesis) were kindly provided by Dr Sarah Lebeer (Centre of Microbial and Plant Genetics, K.U. Leuven, Leuven, Belgium, and Department of Bioscience Engineering, University of Antwerp, Antwerp, Belgium).

Supplementary material

The Supplementary Material for this article can be found online at: … Bacterial concentration is 10 7 u.f.c./ mL. Bacteria cells are represented in green on this figure whether they are viable or damaged (no difference is made here that would depend on bacterial status).


Justine Guerin

Jennifer Burgain

Gregory Francius

Sofiane El-Kirat-Chatel

Audrey Beaussart

Joël Scher

Claire Gaiani
email: claire.gaiani@univ-lorraine.fr

Adhesion of Lactobacillus rhamnosus GG surface biomolecules to milk proteins

Keywords: Milk proteins, Lactobacillus rhamnosus GG, Bacterial surface biomolecules, Pili, Adhesion, Atomic force microscopy

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

Lactic acid bacteria (LAB) are incorporated in many dairy products due to their fermentative properties and their probiotic characteristics. LAB are the most important starter cultures used in all area of dairy and food fermentation [START_REF] Leroy | Lactic acid bacteria as functional starter cultures for the food fermentation industry[END_REF]. By causing a rapid acidification of the raw material through the conversion of lactose into lactic acid, LAB play a central role in fermentation processes. LAB also produce several compounds (as acetic acid, ethanol, aromatic compounds, bacteriocins, exopolysaccharides or enzymes) often involved in biochemical reactions during food processing which influence its texture and flavor. This is particularly the case during cheese ripening (Hickey, Sheehan, Wilkinson, & Auty, 2015;[START_REF] Leroy | Lactic acid bacteria as functional starter cultures for the food fermentation industry[END_REF]. Many LAB are also incorporated into food products for their health benefits to the consumer. The strains Lactobacillus rhamnosus GG (LGG) (Valio), Lactobacillus paracasei Shirota (Yakult) or Bifidobacterium lactis BB12 (Chr. Hansen) are the most documented LAB used in food industries [START_REF] Linares | Beneficial Microbes: The pharmacy in the gut: Bioengineered[END_REF]. Among these strains, LGG is also an interesting LAB model in food research because of its wide use, its available genome sequence [START_REF] Kankainen | Comparative genomic analysis of Lactobacillus rhamnosus GG reveals pili containing a human-mucus binding protein[END_REF] and the availability of numerous surface mutants [START_REF] Lebeer | Functional analysis of Lactobacillus rhamnosus GG pili in relation to adhesion and immunomodulatory interactions with intestinal epithelial cells[END_REF]. The surface composition of LGG is well-known and contains numerous SpaCBA pili distributed all around the bacterial cells [START_REF] Reunanen | Characterization of the SpaCBA pilus fibers in the probiotic Lactobacillus rhamnosus GG[END_REF][START_REF] Tripathi | Deciphering the Nanometer-Scale Organization and Assembly of Lactobacillus rhamnosus GG Pili Using Atomic Force Microscopy[END_REF], long galactose-rich exopolysaccharides (EPS), small glucose-rich EPS [START_REF] Francius | Stretching polysaccharides on live cells using single molecule force spectroscopy[END_REF] and other proteins as MBF [START_REF] Von Ossowski | Functional characterization of a mucus-specific LPXTG surface adhesin from probiotic Lactobacillus rhamnosus GG[END_REF] or MabA [START_REF] Perea Vélez | Characterization of MabA, a modulator of Lactobacillus rhamnosus GG adhesion and biofilm formation[END_REF].

The spatial distribution of LAB within the dairy matrix plays an essential role during cheese processing, ripening and storage. This parameter may affect enzymes release and solutes diffusion in the matrix, which would influences cheese texture, flavor and aroma development (Hickey, Auty, Wilkinson, & Sheehan, 2015;Hickey et al., 2015). Numerous studies reported that LAB are not evenly distributed in the dairy matrix. During cheese ripening, various microscopy methods reported the preferential bacterial location at the fat-protein interface or in direct contact with the milk fat globule membrane [START_REF] Laloy | Influence of the fat content of Cheddar cheese on retention and localization of starters[END_REF][START_REF] Lopez | Lipolysis during ripening of Emmental cheese considering organization of fat and preferential localization of bacteria[END_REF][START_REF] Oberg | Microstructure of Mozzarella Cheese During Manufacture[END_REF][START_REF] Tunick | Rheology and microstructure of low-fat Mozzarella cheese[END_REF].

Previously published work from the authors demonstrated that bacteria surface biomolecules can play a key role in bacteria distribution within the food matrix. The presence of the SpaCBA pili, a proteinaceous surface appendages, at the LGG surface leads to a homogeneous location of the bacteria in the matrix. In absence of pili on their surface, LGG bacteria are closely spaced and aggregated at the same place in the matrix (Figure S1). Therefore, bacteria location in the dairy matrix seems to be governed both by the matrix composition and the surface composition of the bacteria.

Deciphering adhesive interactions between LAB surface biomolecules and dairy compounds would allow to better understand -and eventually predict-bacteria location in a food product.

Nevertheless, very few studies focused on this concern. In a recent work, atomic force microscopy (AFM) was used to reveal specific adhesive interactions between LGG and whey proteins (J. [START_REF] Burgain | In vitro interactions between probiotic bacteria and milk proteins probed by atomic force microscopy[END_REF], and more particularly with the -lactoglobulin (-LG) [START_REF] Guerin | Pili of Lactobacillus rhamnosus GG mediate interaction with β-lactoglobulin[END_REF].

Different LGG surface mutants were compared to understand the mechanism occurring during adhesive interactions and the key role of the SpaCBA pili was underlined [START_REF] Burgain | In vitro interactions between probiotic bacteria and milk proteins probed by atomic force microscopy[END_REF][START_REF] Guerin | Pili of Lactobacillus rhamnosus GG mediate interaction with β-lactoglobulin[END_REF]. However, the role of the other surface biomolecules remains, to the best of our knowledge, unknown.

The aim of this study was to decipher, at the nanoscale, the interactions involved in the adhesion of LGG to different pure milk proteins: micellar caseins, -LG, -lactalbumin (-LA) and bovine serum albumin (BSA). By comparing the adhesive forces of various LGG cell-wall mutants with these proteins, the role of different LGG surface biomolecules in the binding mechanisms was interpreted.

Material and methods

Material

Micellar caseins (Promilk 872B) are obtained from Ingredia IDI (Arras, France). The -LG, -LA and BSA are purchased from Sigma-Aldrich (France).

L. rhamnosus GG (ATCC 53103) (LGG WT) and three of its surface mutants are used. The surface mutants are: the pilus spaCBA knockout mutant CMPG5357 (LGG spaCBA) [START_REF] Lebeer | Functional analysis of Lactobacillus rhamnosus GG pili in relation to adhesion and immunomodulatory interactions with intestinal epithelial cells[END_REF], the welE knockout EPS-deficient mutant CMPG5351 (LGG welE) [START_REF] Lebeer | Identification of a gene cluster for the biosynthesis of a long galactose-rich exopolysaccharide in Lactobacillus rhamnosus GG and functional analysis of the priming glycosyltransferase[END_REF][START_REF] Lebeer | Exopolysaccharides of Lactobacillus rhamnosus GG form a protective shield against innate immune factors in the intestine[END_REF], the double knockout of the spaCBA operon and the welE gene CMPG5365 (LGG welEspaCBA) [START_REF] Lebeer | Functional analysis of Lactobacillus rhamnosus GG pili in relation to adhesion and immunomodulatory interactions with intestinal epithelial cells[END_REF].

Microbial Adhesion To Solvent (MATS)

The four strains of LGG are cultivated as described by [START_REF] Guerin | Pili of Lactobacillus rhamnosus GG mediate interaction with β-lactoglobulin[END_REF]. Briefly, the growth of the four strains is performed at 37 °C in MRS broth until an optical density at 600 nm around 1.2 is reached. The culture is centrifuged at 3,000 g for 10 min at room temperature. The pellet is washed with PBS and re-suspended in PBS to obtain an optical density of 0.5 at 600 nm. The ability of LGG cell surface to adhere to solvent was evaluated as described by Salotti de Souza et al.

(2018), with some modifications. For this determination, 1.2 ml of bacterial suspension in PBS is mixed for 90 s with 0.2 ml of different solvents. The bacterial affinity to a polar solvent (chloroform) and an apolar solvent (hexadecane) is studied. The mixtures are allowed to stand for 15 min to ensure the good separation of the two phases. Then, the optical density of the aqueous phase is measured at 600 nm. The percentage of bound cells is calculated with:

% 𝑎𝑑ℎ𝑒𝑠𝑖𝑜𝑛 = 𝐴 0 -𝐴 𝐴 0 × 100
Where A0 is the absorbance of the bacteria suspension measured at 600 nm before mixing and A is the absorbance of the bacteria suspension measured at 600 nm after mixing.

Atomic force microscopy

Preparation of bacteria-coated mica

A mica coated with a gold layer functionalized with a NH2 terminated PEG linker (Novascan, Ames, Iowa, USA) is used in this study. Culture of LGG is performed as describe by Guerin et al. [START_REF] Guerin | Pili of Lactobacillus rhamnosus GG mediate interaction with β-lactoglobulin[END_REF]. The bacterial suspension is deposed on mica during 15 h at 4 °C (pH 6.8).

The mica is rinsed with PBS (pH 6.8) before their use. AFM topographic images confirmed the presence and the good coverage of LGG on the mica surface (Figure S2).

Preparation of proteins-coated tips

AFM probes with borosilicate glass particle (2 µm), coated with gold and modified with NH2 terminated PEG linker are used (Novascan, Ames, Iowa, USA). The nominal spring constant is 0.01 N/m. Milk proteins (micellar caseins, -LG, -LA, BSA) are prepared in distilled water at a concentration of 1 % (w/w). The rehydration is done under stirring for 2 h at room temperature and overnight at 4 °C. Proteins are adsorbed on the probe by immersion for 15 h at 4 °C. The time left for adsorption is much higher than time necessary for the proteins to absorb on the tip. Probes are rinsed with milli-Q-grade water before use.

Atomic force microscopy measurements

Force measurements are performed at room temperature in PBS buffer (pH 6.8) using an Asylum MFP-3D atomic force microscope (Santa Barbara, CA, USA) controlled by the operation software IGOR Pro 6.04 (Wavemetrics, Lake Osewego, OR, USA) as described by [START_REF] Guerin | Pili of Lactobacillus rhamnosus GG mediate interaction with β-lactoglobulin[END_REF]. The tip coating can slightly modify the cantilever spring constant but the modification is taken into account by readjusting the value via the thermal noise method. For each experiment, the force map is obtained on a 10 × 10 µm² surface corresponding to 32 × 32 points, i.e. 1024 force curves. AFM force distance curves are obtained by following the cantilever deflection as a function of the vertical displacement of the piezoelectric scanner with a scan speed of 400 mm/s.

Curve processing

Determination of the percentage of adhesive events. For each AFM force measurement, a force map containing 1024 force curves is recorded. The presence of specific adhesion between bacteria and milk proteins are determined by analyzing the retraction curves. The force profiles often show multipeaks, corresponding to the stretching of biomolecules. For each experiment, 100 force curves are selected and the corresponding retraction curves are considered to determine the percentage of adhesive events with specific biomolecule stretching signatures.

Retraction curves analysis.

All retraction curves presenting specific biomolecule stretching signatures are fitted with two predictive models: FJC (Freely-Jointed Chain) model and WLC (Worm-Like Chain) model. These two models are used to describe the elongation by statistical mechanics of ideal chains [START_REF] Guerin | Pili of Lactobacillus rhamnosus GG mediate interaction with β-lactoglobulin[END_REF]. These predictive models permit to access parameters such as: number of rupture, maximal rupture force needed to detach the biomolecule, the maximum extension length of the biomolecule before rupture and the contour length of the biomolecule. The maximal adhesion force needed to break the contact between bacteria and milk proteins is calculated through the analysis of the retraction curve. All the information are illustrated in Figure 1.

SpaCBA pili mechanical behavior. To quantify the mechanical behavior of SpaCBA pili, spring constant in different loading regimes is estimated as described by [START_REF] Tripathi | Adhesion and Nanomechanics of Pili from the Probiotic Lactobacillus rhamnosus GG[END_REF]. The pili spring constant (kp) is determined using the slope (s) of the linear portion of the curve and the following equation:

𝑘 𝑝 = (𝑘 𝑐 × 𝑠) ÷ (1 -𝑠)
with kc (pN/µm) the spring constant of AFM cantilever. The constant force step values (Fp, pN) and the constant length values (Lp, nm) are determined by measuring the force and the length of the constant force plateaus on the retraction curve.

Results and Discussion

Bacterial surface characterization

To better clarify and explain the adhesive interactions occurring between bacteria and milk proteins, surface properties of the different LGG mutant strains were investigated. Bacterial surface charge and hydrophobicity may be very important to explain the nature of the interactions. In this study, adhesion force measurements are performed in PBS. PBS possesses a high molarity sufficient to mask the charges of both LGG surface and proteins [START_REF] Burgain | Impacts of pH-mediated EPS structure on probiotic bacterial pili-whey proteins interactions[END_REF]. Therefore, the charge of bacteria should not influence the adhesive interactions with proteins. On the contrary, the surface hydrophobicity of the four different strains seems important to better highlight the nature of the interactions between bacteria and milk proteins. With the MATS method, LGG WT,

LGG welE and LGG welEspaCBA showed a more hydrophilic surface character with a best affinity to chloroform compared to hexadecane. On the contrary, LGG SpaCBA presented a more hydrophobic surface character with a poorer affinity to chloroform (Figure 2).

Key role of SpaCBA pili in milk proteins adhesion

Some of the biomolecules present at the surface of LGG WT comprise the SpaCBA pili, the long galactose-rich EPS, the small glucose-rich EPS and other proteins as MBF (Mucus Binding Factor)

or MabA (Figure 3A). AFM-based force spectroscopy is used with milk proteins coated probes to decipher how LGG WT cells interact with these individual proteins. The percentage of adhesive events presented in Figure 3B reveals major differences between the four milk proteins tested.

The percentage of specific adhesive events is of 66.5 ± 0.5, 3.5 ± 1.5 and 9.0 ± 0.0 % for -LG, -LA and BSA, respectively. No adhesive event is observed with caseins. The percentage of adhesive events is very high for -LG. The force signatures observed with -LG are very repeatable and correspond to specific events (Figure 3C). For -LA and BSA, the percentage of adhesive events is very low (Figure 3B). The force signatures corresponding to these low adhesive interactions with -LA and BSA (Figures 3E and3F) are poorly repeatable which suggest that they are unspecific events.

The specific adhesion between LGG WT and -LG reinforces previous hypothesis suggested by [START_REF] Guerin | Pili of Lactobacillus rhamnosus GG mediate interaction with β-lactoglobulin[END_REF] who proposed that the adhesion between LGG WT and -LG is mediated by SpaCBA pili. To support this result, the retraction curves recorded between these two entities are analyzed in details (Figure 3C and3D). Remarkable adhesion signatures are detected on retraction curves between LGG WT and-LG. Large adhesion force peaks with linear shape and characteristic horizontal force steps are observed (Figure 3C). These adhesion signatures are comparable to those observed by [START_REF] Tripathi | Adhesion and Nanomechanics of Pili from the Probiotic Lactobacillus rhamnosus GG[END_REF], who demonstrate, using AFM tips decorated with SpaC proteins to pull on pili at the surface of LGG cells, that the appendages behave like a nanospring under mechanical stretching. Superimposition of multiple forces profiles reveals a high reproducibility of the events (Figure 3D). As described by [START_REF] Tripathi | Adhesion and Nanomechanics of Pili from the Probiotic Lactobacillus rhamnosus GG[END_REF], the specific signatures repetition are most-likely due to the intrinsic mechanical properties of SpaCBA pili. The different linear slopes and plateau values observed on the 'step curves' were then analyzed. The slopes of the linear segments are found to increase with increasing the applied pulling force. This behavior describes the stiffening of the stretched pilus. The spring constant of pili (kp) in the different loading rate is then calculated and found to be kp1 = 5.6 ± 1.0, kp2 = 11.7 ± 0. Caseins and whey proteins structures differ. Caseins are organized in spherical and voluminous superstructures called micelles and present a lack of secondary organization [START_REF] Fox | Milk: an overview[END_REF]. On the contrary, whey proteins (including -LG) possess a rich secondary organization with -sheet and -helix structures and the presence of disulfide bonds. Contrary to -LA and BSA, -LG monomers are folded into eight stranded antiparallel-sheets that form a hydrophobic pocket, called calix, with a 3-turn -helix on the outer surface [START_REF] Monaco | Crystal structure of the trigonal form of bovine beta-lactoglobulin and of its complex with retinol at 2.5 A resolution[END_REF], able to bind multiple hydrophobic molecules (Çelebioğlu et al., 2015; Dominguez-Ramirez, Del Moral-Ramirez, Cortes-Hernandez, Garcia-Garibay, & Jimenez-Guzman, 2013; [START_REF] Kontopidis | The ligand-binding site of bovine betalactoglobulin: evidence for a function?[END_REF], 2004).

The difference observed in the percentage of adhesive events between LGG WT and the proteins may be due to a difference of structure. It worth consider that LGG WT interact preferentially with -LG due to the presence of a hydrophobic calix. However the calix is probably not able to interact with a larger molecule such as the LGG pili SpaCBA protein. Moreover the SpaCBA pili is a glycosylated proteins [START_REF] Tytgat | Probiotic Gut Microbiota Isolate Interacts with Dendritic Cells via Glycosylated Heterotrimeric Pili[END_REF], so the interaction between LGG WT and the -LG would be more hydrophilic rather than hydrophobic. MATS method confirms the hydrophilic character of the LGG WT surface (Figure 2). Therefore, the hydrophilic nature of the interaction occurring between the LGG WT and the -LG is confirmed.

To confirm the role of SpaCBA pili in adhesion to -LG, the LGG welE strain, deprived of long rich-galactose EPS layer, is used. This mutant is known to overexpose SpaCBA pili, due to the EPS removal (Figure 4A) [START_REF] Lebeer | Functional analysis of Lactobacillus rhamnosus GG pili in relation to adhesion and immunomodulatory interactions with intestinal epithelial cells[END_REF]. The percentages of adhesive events between LGG welE and-LG (96.5 ± 1.5 %) are higher than LGG WT (66.5 ± 0.5 %) (Figure 4B). In LGG welE, pili located on bacterial surface are not embedded within the EPS layer and are entirely free to establish contact points with -LG. A recent work has demonstrated the glycosylated behavior of the stretched biomolecule during interaction between -LG and LGG WT and LGG welE [START_REF] Guerin | Pili of Lactobacillus rhamnosus GG mediate interaction with β-lactoglobulin[END_REF]). It appears that the SpaC pilins of the SpaCBA pili are the pilins carrying the glycosylation [START_REF] Tytgat | Probiotic Gut Microbiota Isolate Interacts with Dendritic Cells via Glycosylated Heterotrimeric Pili[END_REF], suggesting that SpaC subunits is implicated in -LG interaction.

In LGG welE, the increased exposure of SpaC pilins at the basal part of each pilus could allow SpaCBA pili to establish more contact points with -LG resulting in an increased number of rupture events in the retraction curve [START_REF] Lebeer | Functional analysis of Lactobacillus rhamnosus GG pili in relation to adhesion and immunomodulatory interactions with intestinal epithelial cells[END_REF]. The specific biomolecules stretching signatures between -LG and LGG welE differed from LGG WT with the apparition of more irregular and jerky signatures and a more important maximal rupture distance (2.8 ± 0.5 and 1.1 ± 0.1 µm for

LGG welE and LGG WT, respectively) (Figure 4C). As the size of a SpaCBA pilus is around 1 µm [START_REF] Guerin | Pili of Lactobacillus rhamnosus GG mediate interaction with β-lactoglobulin[END_REF][START_REF] Tripathi | Adhesion and Nanomechanics of Pili from the Probiotic Lactobacillus rhamnosus GG[END_REF], the biomolecule stretched at a distance of around 2.8 µm with LGG welE might not be an individual pilus. In fact, the pilus SpaCBA is able to interact each other with a zipper-like adhesion mechanism involving multiple SpaC molecules distributed along the pilus length. In LGG welE, SpaCBA pili are not embedded in EPS layer and are totally free to interact with each other. SpaCBA pili may form a network entirely engaged in the interaction and leading to the observed numerous ruptures and the presence of jerky signatures during the retraction of the tip containing -LG which leads to a stretched distance of 2.8 µm with

LGG welE.

Surprisingly, a lot of adhesive events are observed between LGG welE and other milk proteins (caseins, -LA and BSA). The percentages of adhesive events are of 56.0 ± 6.5, 98.5 ± 1.5 and 88.5 ± 2.5 % for caseins, -LA and BSA, respectively (Figure 4B). It was previously shown that these three proteins poorly interact with LGG WT. In contrast, a lot of specific signatures, similar to those observed with -LG, are observed with LGG welE (Figure 3C, E andF). In LGG welE, the over-exposition of the SpaCBA pili in absence of long EPS layer could allowed caseins, -LA and BSA to interact with SpaCBA pili. An explanation would be that, for LGG welE, the absence of long EPS lead to uncover pili subunits initially buried in the EPS structure. It can be the case of the SpaB pilins, which are located at the pilus base. In the absence of long-EPS layer, these SpaB subunits would be accessible to promote interaction between LGG welE and these three milk proteins. To verify this hypothesis, adhesion force measurements should be made between milk proteins and purified SpaCBA pili or purified subunits SpaB of the pilus. The rupture distance observed on retraction curve was too long to believe that other surface biomolecules such as MabA and MBF proteins or small EPS governed interaction between LGG welE and milk proteins.

In conclusion, the SpaCBA pili environment seems to determine LGG ability to interact with milk proteins. In presence of long-EPS layer, the SpaCBA pili interact preferably with -LG. In absence of long-EPS layer, SpaCBA pili possess the ability to adhere with all the studied milk proteins.

Role of EPS and other surface proteins in adhesion to milk proteins in absence of SpaCBA

pili. To study the role of others LGG surface biomolecules (i.e. other than SpaCBA pili), two LGG mutants depleted in SpaCBA pili are compared: LGG spaCBA and LGG welEspaCBA.

LGG spaCBA surface is composed of a long rich-galactose EPS layer, small glucose-rich EPS and proteins as MBF or MabA (Figure 5A) [START_REF] Francius | Stretching polysaccharides on live cells using single molecule force spectroscopy[END_REF][START_REF] Perea Vélez | Characterization of MabA, a modulator of Lactobacillus rhamnosus GG adhesion and biofilm formation[END_REF][START_REF] Von Ossowski | Functional characterization of a mucus-specific LPXTG surface adhesin from probiotic Lactobacillus rhamnosus GG[END_REF]. The percentages of adhesive events between LGG spaCBA and the different milk proteins are of 13.5 ± 5.5, 13.5 ± 0.5 and 11.0 ± 3.0 % for -LG, -LA and BSA, respectively (Figure 5B) and are not significantly different from each other. First, results observed with -LG demonstrated a dramatic decrease of adhesive events in absence of SpaCBA pili on LGG surface, which reinforce the key role of pili in adhesion between LGG and -LG. In contrast, specific biomolecules stretching signatures recorded on retraction curves are rare but highly reproducible for all whey proteins (Figure 5C,D,E). The rupture number is ranging from 1 and 2, the maximal force varies between 100 and 200 pN and the maximal stretching distance is ranging from 0.6 and 1.4 µm. These signatures are similar to those observed by [START_REF] Francius | Stretching polysaccharides on live cells using single molecule force spectroscopy[END_REF] when they used lectin-functionalized AFM tips. [START_REF] Francius | Stretching polysaccharides on live cells using single molecule force spectroscopy[END_REF] obtained the same multiple force peak with a magnitude ranging from 50 to 100 pN and up to 1000 nm length during the stretching of EPS. In another work, similar results are obtained during the LGG spaCBA adhesion to hydrophobic surfaces. The observed forces profiles resemble to those obtained from stretching of EPS on LGG cells [START_REF] Sullan | Single-cell force spectroscopy of pili-mediated adhesion[END_REF]. Signatures recorded with LGG spaCBA during biomolecules stretching seem to be those of sugars which should correspond to the stretching behavior of EPS (Francius et al., 2009). Thus, in the absence of SpaCBA pilus, all whey proteins may be able to interact with

LGG via EPS. With the casein-modified tip, interaction with bacterial surface reveals specific signatures of biomolecules stretching (Figure 5F). The rupture number is of 2.8 ± 0.2, the maximal force is of 1.8 ± 0.1 pN and the maximal stretching distance is of 1.1 ± 0.1 µm (Figure 5F). With caseins, the particular shape of the retraction curves does not allow the determination of the biomolecule involved in the interaction with LGG SpaCBA.

In conclusion, these results demonstrated the ability of EPS to interact with milk proteins, in a less pronounced way than pili and only when pili are absent. These results suppose that the presence of pili masked the other biomolecules and inhibit their ability to mediate adhesion to milk proteins.

The surface of LGG welEspaCBA mutant is composed of different biomolecules such as small glucose-rich EPS, other proteins as MBF or MabA, lipoteichoïc acids or peptidoglycans (Figure 6A) [START_REF] Lebeer | Functional analysis of Lactobacillus rhamnosus GG pili in relation to adhesion and immunomodulatory interactions with intestinal epithelial cells[END_REF]. The percentages of adhesive events measured with LGG welEspaCBA are of 44.0 ± 6.7, 17.5 ± 0.5 and 14.0 ± 2.0 % for -LG, -LA and BSA, respectively (Figure 6B).

No specific adhesive events are observed between LGG welEspaCBA and caseins. The specific signatures recorded for whey proteins on retraction curves are presented on Figures 6C, D andE.

These specific signatures revealed the presence of surface biomolecules, other than SpaCBA pili and long EPS, implicated in adhesion with whey proteins. The observed signatures may recall the small EPS stretching [START_REF] Francius | Stretching polysaccharides on live cells using single molecule force spectroscopy[END_REF]. These signatures may also be due to the detection of peptidoglycan, accessible in the absence of both long-EPS and SpaCBA pili, with the milk proteins [START_REF] Beaussart | Binding Mechanism of the Peptidoglycan Hydrolase Acm2: Low Affinity, Broad Specificity[END_REF] and the observed peak will be due to the stretching of milk proteins.

Nevertheless, other surface mutants will be required to validate these hypotheses.

Conclusion

AFM in force mode was used in this work to underline the role of LGG surface biomolecules in adhesion to milk proteins. The high frequency of adhesive events demonstrated that the SpaCBA pili and other LGG surface biomolecules such as EPS and proteins are involved in the adhesion.

However, the ability of each biomolecule to interact with milk proteins depend on their environment. In LGG WT, only the pili are implicated in adhesive interactions with the -LG as other surface biomolecules are masked. When pili are absents, the results demonstrated that EPS are also able to interact with all milk proteins, in a less pronounced way than pili. This work revealed the ability of LGG surface biomolecules to interact to different milk proteins. Depending on the surface biomolecules composition, LAB present different affinity with milk components. In that respect, it is expected that the media and the growth phase may have an impact on the preferential location of bacteria in the dairy matrix, and could be extended to other LAB strains.

Schema of the principle of AFM-based force spectroscopy to measure the interaction between milk proteins coated probes and the surface of LGG WT (A). Frequency of adhesive events between LGG and individual milk proteins (B). Representative retraction curves recorded between LGG and -LG (C). Superimposition of retraction curves and determination of spring-like properties of pili SpaCBA (D). Representative retraction curves recorded between

LGG and -LA (E) or BSA (F).

Schema of the principle of AFM-based force spectroscopy to measure the interaction between milk proteins coated probes and the surface of LGG welEspaCBA (A). Frequency of adhesive events between LGG and individual milk proteins (B). Representative retraction curves recorded between

LGG and -LG (C), -LA (D) or BSA (E).

  8 and kp3 = 19.1 ± 1.1 pN/µm. Besides, the constant force steps values (Fp1 = 129 ± 8 and Fp2 = 310 ± 8 pN) and the constant step length values (Lp1 = 91 ± 6 and Lp2 = 109 ± 10 nm) are highly reproducible. The values variation follow the same trend that those measured by Tripathi et al. (2013) and Sullan et al. (2014). These characteristics are reproducible and confirm the structural changes of the SpaCBA pilus induced during the retraction of the -LG tip, leading to a more rigid conformation of the pilus. These results demonstrated the nanospring-like behavior of pilus SpaCBA during adhesion with the -LG.

  Figure 1

Figure 1 :

 1 Figure 1: Illustration of a biomolecule displaying force-induced structural modification (A). Example of data obtained by adjustment of the retraction curve with the FJC or WLC models (red lines) (B).

Figure 2 :

 2 Figure 2: Microbial adhesion to solvents test to determine the surface hydrophobicity of LGG WT and the three mutants: LGG spaCBA, LGG welE and LGG welEspaCBA.

Figure 3 :

 3 Figure 3: Adhesive interactions between LGG WT and milk proteins (caseins, β-LG, α-LA and BSA).

Figure 4 :

 4 Figure 4: Adhesion between LGG welE and milk proteins.Schema of the principle of AFM-based force spectroscopy to measure the interaction between milk proteins coated probes and the surface of LGG welE (A). Frequency of adhesive events between LGG and individual milk proteins (B). Representative retraction curves recorded betweenLGG and -LG (C), caseins (D), -LA (E) or BSA (F).

Figure 5 :

 5 Figure 5: Adhesion between LGG spaCBA and milk proteins.Schema of the principle of AFM-based force spectroscopy to measure the interaction between milk proteins coated probes and the surface of LGG spaCBA (A). Frequency of adhesive events between LGG and individual milk proteins (B). Representative retraction curves recorded betweenLGG and -LG (C), -LA (D), BSA (E) or caseins (F).

Figure 6 :

 6 Figure 6: Adhesion between LGG welEspaCBA and milk proteins.

Acknowledgement

The authors thank the Centre of Microbial and Plant Genetics, KU Leuven and the Department of Bioscience Engineering, University of Antwerp for providing the bacterial strains.