Yeast nitrogen base without aminoacids, bacto casitone peptone and soy peptone were purchased from Difco (Becton Dickinson, Le Pont De Claix, France). De Man, Rogosa and Sharpe Medium (MRS), medium M17, bacteriological agar and the AnaeroGen Compact atmosphere generation system for solid state incubation on petri dishes were from Oxoid (Basingstoke, England). All other chemicals used to prepare the semi-defined medium and the buffers were purchased from Sigma-Aldrich (Milan, Italy). A kit containing acetic acid, lactic acid, citric acid, butyrric acid, iso-butyrric acid, succinic acid, oxalic acid, maleic acid was obtained by Supelco (Milan, Italy) for the analytical quantification of organic acids.
Microorganism and media
Vaginal fluids collected from healthy women (after informed consent) were plated onto lactobacilli selective medium, namely MRS-agar (Oxoid) and incubated in anaerobic conditions (Gas-Pak System; BBL, Becton Dickinson Biosciences) for 48 h at 37°C.
Microorganisms were maintained in MRS-broth as suspended culture (stabs) at −80°C using glycerol (20% w/v) as cryoprotectant. These stabs were used to inoculate pyrex bottles (250 ml) completely filled with culture media to study cell growth and lactic acid production under microaerofilic conditions over a period of 24–30 h at 37°C, in a rotary shaker (HT Aquatron, Infors, Switzerland) at 160 rpm. Experiments were performed by adding different carbon sources (20 g∙l−1) to the semi-defined medium, SDM : in particular fructose, sucrose, lactose, trehalose and dextrins were used alternatively to analyze how microbial growth and organic acids production were affected. Shake flask experiments were also performed adding sodium lactate (0–60 g∙l−1) at increasing concentrations in the SDM, to evaluate strain growth inhibition.
Single colonies were collected from MRS plates and characterized with the API 50 CHL system (BioMérieux) according to the manufacturer’s instructions. In order to correctly identify the Lactobacillus at species level, 16S ribosomal DNA (rDNA) was sequenced . The sequences of the selected Lactobacillus-specific primers LcrisF (AGCGAGCGGAACTAACAGATTTAC) and LcrisR (AGCTGATCATGCGATCTGCTT) confirmed the amplification of a 154-bp fragment of 16S rRNA from the reference strain L. crispatus ATCC33820 . Briefly genomic DNA was extracted from pure cultures using a QIAamp DNA mini kit (Qiagen) according to the manufacturer’s instructions. 4 μl of DNA (≈40 ng), in 50 μl reaction mixtures containing 1× Fast Start High Fidelity PCR system mix (Roche), and 100nM (each) primer were amplified. PCR was performed with the GeneAmp PCR System 9700 (Perkin Elmer, Wellesley, Mass.) with an initial denaturation step of 95°C for 15 min, followed by 40 cycles of 95°C for 15 s and 62°C for 1 min.
Determination of H2O2 production
Since is reported that the lack of vaginal H2O2 producing lactobacilli is associated with bacterial vaginosis, the Lactobacillus isolates were also characterised for their production of H2O2. The capacity of L. crispatus L1 to produce H2O2 was tested with a semiquantitative assay on tetramethylbenzidine agar plates  using Brucella agar (Difco) containing 0.001% (w/v) horseradish peroxidase (Sigma), 0.023% (w/v) tetramethylbenzidine (Sigma) and 1% (w/v) starch. This medium was supplemented with 0.5 mg of bovine haemin (Sigma) and 0.1 mg of vitamin K1 (Sigma) in 100 ml of final volume. Serial dilutions of lactobacilli were inoculated in the medium and incubated in anaerobic conditions at 37°C for 72 h. Plates were then exposed to ambient air and H2O2-producing colonies were revealed by the appearance of a blue colour. According to the colour intensity, the strains were classified as strong, medium, weak or negative (white colonies) producers .
Gastrointestinal survival: simulated gastric and pancreatic juices
Shake flask experiments were performed to evaluate the capability of L. crispatus L1 to survive the gastrointestinal tract. Simulated gastric and pancreatic juices were prepared by slightly modifying the protocols reported by Kos and colleagues . Briefly, gastric juices were simulated with a solution of NaCl, 125 mM, KCl 7 mM, NaHCO3, 45 mM and pepsine (Sigma Aldrich) 0.3% (w/v), with a final pH equal to 2 obtained by HCl addition. Either 6.0∙108 cells · ml−1 (low dose, minimal starting density for shake flasks experiments necessary to avoid the lag phase) or 1.8·109 cells · ml−1 (high dose, typical amount delivered in probiotic commercial products) were inoculated into the medium and incubated 2–3 h in shaker at 37°C and 110 rpm to simulate physiological conditions. This step was followed by centrifugation (15 min at 1200 × g) and re-suspension of the cells in a solution containing pancreatine (Sigma Aldrich) 0.1% (w/v), Oxgall bile (Sigma Aldrich) 0.15% (w/v) with a final pH equal to 4, to simulate pancreatic juices. The suspension was incubated for 3 h, after which cells were centrifuged and re-suspended in fresh MRS medium to evaluate bacterial growth. At the end of each step cell viability was measured by plating aliquotes and counting colony forming units (cfu).
The fermenter used was a Biostat CT, Braun Biotech International (Melsungen, Germany), 2 l working volume, equipped with a digital control unit and connected to a PC for remote control via MFCS-win software. L. crispatus L1 was grown at T = 37°C, pH = 6.5. The stirring velocity was initially set to 100–200 rpm and increased up to 300 rpm during the experiment. The medium was sparged with nitrogen after sterilization prior to inoculation for at least 30 min. Experiments in batch mode were carried out using the SDM medium, controlling the pH by automatic addition of NH4OH (2.5 M).
MF experiments were performed using SDM, starting in batch mode, and switching after 8–10 h to fed-batch and approximately 4 h later to MF mode. The duration of each phase was set based on lactate formation, carbon source consumption rate and their influence on growth rates. Filtered exhaust medium was replaced with a fresh salt solution with a level controller, to maintain a constant fermentation volume. Microorganisms were therefore held in the vessel and fed with appropriate profiles generally ranging from 1 to 5 g · l−1 · h−1. However, differently from previous data , the C/N ratio in the nutrient solution was lowered from 1/4 to 1/16 during the MF phase to further decrease the impact of raw materials on process costs.
A Biostat C Braun Biotech International (Melsungen,Germany) bioreactor with a 15 l working volume was used for the production of exopolysaccharides. Two repeated batch experiments were carried out using SDM medium as previously described, in order to purify higher amounts of EPS to allow extensive structural characterization.
Cell growth was followed during experiments by measuring absorbance at 600 nm on a Beckman DU 640 Spectrophotometer (Milan, Italy). Samples collected every hour were spinned down in an ALC PK 131R centrifuge at 2000×g, and the wet weight was measured after centrifugation and washing in saline solution (0.9% NaCl w/v). The washed pellet was dried overnight (16–18 h) at 85°C and a calibration curve relating the absorbance value to the cell dry weight was generated. One gram per litre of dry cell weight corresponded to 1.9 OD600. This correlation was extrapolated on many different fermentation experiments. Cell number was also measured by direct counts at the optical microscope and plating for viability determination (cfu). The supernatant (1 ml) was ultrafiltered on a centricon tube (10 KDa Mw cut–off, Millipore) at 5000×g to prepare the samples for analytical quantification. The concentration of glucose, or other carbon sources, was measured through HPAEC-PAD analysis performed with a Dionex chromatographer (model DX 500); the organic acids from the culture broth and the permeate solutions were analysed by HPLC as previously described . A quick off-line determination was obtained for glucose by using the Haemo-Glukotest 20–800 stripes (Boehringer-Manheim, In vitro diagnosticum).
EPSs purification and quantification
EPSs were collected and isolated from fermentation supernatants of L. crispatus L1. To quantify EPSs during growth, opportunely diafiltered supernatants were assayed using the anthrone/H2SO4 method , using a glucose solution as standard. After harvesting (e.g. 24 h) removal of cells was obtained by centrifugation (2000 × g 30 min) and the supernatants were recovered to purify EPSs.
The developed downstream procedure consisted in a pre-treatment of the fermentation supernatant with 4U per litre of protease (Aspergillus oryzae 3.2 U⋅mg−1, Sigma) for 60 min at room temperature followed by membrane-based UF and DF steps. The procedure was carried on Uniflux-10, (GE Healthcare, USA) an automated tangential flow filtration pilot system also equipped with a sensor level to perform fed-batch concentration or constant volume DF; temperature probe on the retentate line; pressure sensors and flow meters on feed, retentate and permeate lines; pH meter, conductivity meter and UV (with λ = 280 nm) detector on the permeate line. It is connected to a PC and a UNICORN TM software, that allows to control, manage and monitor the process and its parameters. The supernatant was ultrafiltered on 5KDa membranes with a filtering area of 0.1 m2 and diafiltered with 5 volumes of distilled water. After addition of 0.08 M NaCl the recovered retentate was precipitated with 6 volumes of acetone and ethanol (1:1 v/v). The precipitate was dried, resuspended in sterile water and treated with active charcoal to decolorization and purification from accidental endotoxin contamination. Finally the concentrated EPS solution was microfiltered on 0.22 μm membranes and lyophilized. The powder obtained was used for further characterization.
General analytical and spectroscopic methods
Determination of sugars residues and of their absolute configuration, GLC and GLC-MS were all carried out as described. 1D 2D NMR experiments were carried out as described [44, 45].
Culturing of Vk2/E6E7cells
Vk2/E6E7, immortalized human vaginal epithelial cell line (American Type Culture Collection), were grown in 75-cm2 flasks (Falcon, Becton Dickinson Biosciences, Milan, Italy) at 37°C (5% CO2) in Keratinocyte-Serum Free medium (GIBCO-BRL San Giuliano Milanese, Milan, Italy) with 0.1 ng∙ml−1 human recombinant EGF, 0.05 mg∙ml−1 bovine pituitary extract, and additional calcium to a final concentration of 0.4 mM. The medium was changed every 2 days. Confluent monolayers (2.5 × 105 cells) were grown in six-well tissue culture plates (Falcon, Becton Dickinson Biosciences, Milan, Italy) in Dulbecco’s modified Eagle’s medium and Ham’s F12 medium (D-MEM) (GIBCO-BRL San Giuliano Milanese, Milan, Italy), antibiotic-free and FCS-free, for 24 h, before starting experiments. One million Vk2/E6E7 cells/well were used for the adhesion assay.
Adhesion of L. crispatus L1 to Vk2/E6E7 cells and competition with C. albicans for adherence
Cell suspensions of L. crispatus L1 were grown in MRS broth at 37°C in anaerobic conditions.
C. albicans was identified on the basis of growth characteristics, colony morphology, cellular appearance, and carbohydrate assimilation patterns using commercially available ATB ID 32 C test kit (bioMérieux, Marcy/Etoile, France) at the Operative Unit of Microbiology, Second University of Naples, Italy. Yeast cells were prepared by inoculating four colonies isolated from Saburaud agar (Oxoid, Milan, Italy) plates in 6 ml Brain Heart infusion broth (BHI broth) (Oxoid, Milan, Italy), and incubating the suspension at 30°C for 18 h under constant shaking. These conditions yield cultures composed primarily of blastospores at the late exponential growth phase.
Cultures of microorganisms were collected by centrifugation from the broth cultures, washed three times and finally suspended in phosphate-buffered saline (PBS; pH 7.1). The working dilution of the microorganism suspensions was determined by performing sequential measurements of optical densities of cultures at 600 nm and quantification of viable microorganisms by colony counts. For each strain, the correlation between the OD600 and cfu was established. The microorganism cells suspended in DMEM were used for the adhesion and interference assays.
Adherence of L. crispatus L1 to Vk2/E6E7 cells was assayed by a method described previously with slight modifications . Preliminary experiments using 10:1, 100:1, and 1000:1 multiplicities of infection (MOI) were conducted to determine the optimal bacterial-to-epithelial cell ratio in our adhesion model. These pilot investigations demonstrated a saturation of adhesion of L. crispatus L1 to Vk2/E6E7 cells at a MOI of 10:1. Therefore, for all subsequent adhesion experiments described in this study a MOI of 10:1 was utilized.
Interference experiments were performed with C. albicans, a potential vaginal pathogen, that showed a significant capacity to adhere to host cells. The procedures described by Osset et al.  were used, with some modifications. For exclusion tests, 1×107 lactobacilli and vaginal epithelial cells were incubated together for 1 h at 37°C in microaerophilic conditions; afterwards, C. albicans cells were added, and incubation was further continued for 1 h. During competition tests, 1×107 lactobacilli and 1×107
C. albicans were mixed and Vk2/E6E7 cell monolayers then inoculated and incubated for 1 h at 37°C in microaerophilic conditions. For displacement tests, 1×107
C. albicans and epithelial cells were incubated together for 1 h at 37°C in microaerophilic conditions. Successively, 1×107 lactobacilli were added and incubation was prolonged for 1 h. Vk2/E6E7 cells were scored for the presence and number of bacteria and C. albicans attached, and cell observation was performed as indicated above.
For exopolysaccharide-interference experiments, Vk2/E6E7 cell monolayers were treated with EPS as follows: for competition tests, exopolysaccharide (0.01-0.1-1.0 mg∙ml−1) and 1×107
C. albicans were mixed and, successively, Vk2/E6E7 cell monolayers were inoculated and incubated for 1 h at 37°C in microaerophilic conditions. For exclusion tests, vaginal epithelial cells were pre-treated with EPS (0.01-0.1-1.0 mg∙ml−1), before addition of the C. albicans suspension for 1 h at 37°C in microaerophilic conditions. At the concentrations used, the EPS did not affect epithelial cell viability. In preliminary experiments monolayers were pre-treated with EPS for 1, 4, 6 and 18 h at 37°C in microaerophilic conditions.
Microorganism adhesion to Vk2/E6E7 cells was assessed by microscopy (×100) after Gram’s stain by counting the number of micro-organisms attached to 30 consecutive cells. The results of the three conditions (i.e. exclusion, competition and displacement) were expressed as the average number of C. albicans per Vk2/E6E7 cells and compared with adhesion without lactobacilli or EPS (control value). The control values were taken as 100% of adhesion and the inhibition of C. albicans adherence was calculated by subtracting each adhesion percentage from its corresponding control value.
Adhesion experiments were conducted three times with at least three replicates per group. A difference in mean values was deemed significant if the P values were <0.05 or highly significant if the P values were <0.01. The three experimental groups were compared using a one-way analysis of variance. Post hoc group comparisons were conducted using the Student-Newman-Keuls test.
HBD- 2 ELISA
Semi-confluent Vk2/E6E7 were grown in six-well tissue culture plates and were treated with EPS (0.01-0.1-1.0 -5.0 mg∙ml−1) for 18 h. Cell-free supernatants were recovered by centrifugation and assayed to establish the concentration of Human beta-defensin 2 (HBD-2) by an enzyme-linked immunosorbent assay (Phoenix Pharmaceuticals, Inc.).
The data were presented as means ± standard errors. All pair wise comparisons were examined using unpaired Student’s two-tailed t-test. Differences were considered significant when P ≤ 0.05.