L. brevis IOEB 9809, isolated from Bordeaux red wine, was obtained from the IOEB strain collection (Institute of Oenology of Bordeaux, ISVV, Villenave d’Ornon, France). The probiotic bacteria Lactobacillus acidophilus LA-5 and Bifidobacterium animalis subps. lactis BB-12 (Chr. Hansen A/S., Hørsholm, Denmark) were also used. All strains were maintained at −80°C in de Man Rogosa Sharpe (MRS)  broth (Pronadisa, Madrid, Spain) supplemented with 20% (vol/vol) glycerol.
Analysis of cell survival under upper digestive tract stress
Induction of BA production
Four cultures of L. brevis IOEB 9809 were grown at 30°C in MRS initial pH 6.2. One culture was unsupplemented (uninduced), and the other three were supplemented with 10 mM tyrosine (Sigma-Aldrich, St Louis, MO), or 4.38 mM agmatine sulphate (Sigma-Aldrich, St Louis, MO) or both. These concentrations of BA precursors were optimal for production of BA during bacterial growth (results not shown). Pyridoxal phosphate 0.005% (wt/vol) final concentration (Sigma-Aldrich, St Louis, MO) was added to all cultures as coenzyme for decarboxylation reactions. All of the above was performed in triplicate (12 cultures in total). Cells were harvested in the mid-exponential phase (OD620 = 0.8, approximately 8 × 108 CFU mL-1) by centrifugation, and resuspended in the same volume of the corresponding fresh MRS medium.
Digestive tract simulation
To determine the tolerance to saliva and gastric stresses, we modified a previous method . Each of the 12 resuspended cell samples (above) was dispensed in 7 groups of 2.5 ml aliquots. Group 1 (control) was untreated. Group 2 (saliva simulation) 10% (vol/vol) of a sterile electrolyte solution  pH 6.5 supplemented with 1% (wt/vol) lysozyme (Sigma-Aldrich, St Louis, MO) was added to each aliquot, and they were incubated for 5 min at 37°C with shaking. Groups 3–7 (gastric environment simulation) 0.3% (wt/vol) pepsin (Sigma-Aldrich, St Louis, MO) was added to saliva simulation followed by acidification with 1 M HCl to pH 5.0, 4.1, 3.0, 2.1 or 1.8 respectively. All aliquots subjected to gastric stress were independently incubated for 20 min, at 37°C with shaking. After the treatments, the bacteria were collected by centrifugation (8.000 × g, 8 min) and cell survival was determined by plate counting on MRS agar. Supernatants were filtered (0.2 μm filters, VWR international, West Chester, PA) and analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC) (see below) for tyramine and putrescine.
Cell culture and in vitro adhesion assay
The Caco-2 cell line was obtained from the cell bank of the Centro de Investigaciones Biológicas (Madrid, Spain), and was grown and differentiated as previously described . For the adhesion assay, Dulbecco’s Modified Eagle Medium (DMEM) with L-glutamine (580 mg L-1), D-glucose (4500 mg L-1) and sodium pyruvate (110 mg L-1) pH 8.2 was prepared without L-tyrosine, according to the Invitrogen formulation. DMEM medium was supplemented with the same concentration of L-tyrosine and agmatine sulphate as used for the gastrointestinal experiments. In the adhesion assay experiments, bacteria grown in MRS to the mid-exponential phase (OD620 = 0.8) as for BA induction, were centrifuged (10.000 x g, 10 min), washed once with cold phosphate-buffered saline (PBS) pH 7.1 (10 mM Na2HPO4, 1 mM KH2PO4, 140 mM NaCl, 3 mM KCl, all purchased from Merck, Darmstadt, Germany) and resuspended in the same DMEM medium supplemented, or not, with tyrosine, agmatine or both. Bacterial suspensions were added to Caco-2 intestinal cells in a final volume of 0.1 mL and a final concentration of 1.25 x 107 CFU mL-1 (ratio 1:100, Caco-2 cells to bacteria) and incubated at 37°C for 1 h. Unbound bacteria were then removed by washing three times with 0.2 mL of PBS at pH 7.1. Some wells, unwashed, were used as control. Cell cultures were then resuspended in 0.1 mL of PBS and detached by adding 0.1 ml of 0.05% trypsin-EDTA (Gibco, Carlsbad, CA). After incubation at 37°C for 10 min, the detachment reaction was interrupted by adding 0.1 mL of cold PBS. The number of total and adhered bacteria was determined by serial dilution and quantitation on agar plates as for viable counts. The adhesion percentage was calculated by comparing the number of CFU from three washed wells with those from control wells. Every experiment was performed in triplicate.
RP-HPLC determination of BA
Pre-column dabsyl chloride manual derivatisation was performed for BA detection. The derivatisation reaction was carried out as described by Krause et al. . 10 μl of the dabsylated supernatants were used for injection. HPLC analysis was performed using an Alliance 2795 system (Waters, Milford, MA) equipped with a Waters Nova-Pack C18 column (150 × 3.9 mm 4 μm particle size). Dabsylated amino acids and amines were eluted using the gradient described by Krause et al. . Detection was carried out by a Waters 2996 Photodiode array detector at 436 nm.
RNA extraction and Real Time PCR analysis
Transcriptional analysis was performed after 20 min gastric stress simulation. Control and samples mimicking gastric stress at pH 5.0, were analyzed in the presence or absence of biogenic amine precursors. Total RNAs were extracted from 2 × 109 cells using the FastRNA pro blue kit (Qbiogene, Montreal, QC) following the manufacturer’s instructions. Cells were lysed mechanically with a Hybaid Ribolyser for 30 s. The RNAs' quantity and quality was determined by spectrophotometry, and their integrity was assessed by visualization of the rRNA bands on 1.2% agarose gels. Absence of chromosomal DNA was confirmed by quantitative real-time PCR.
cDNAs were synthesized using 0.8 μg of total RNA and Quantitect Reverse Transcription (Qiagen, Hilden, Germany) which included a DNase treatment and reverse transcription.
Primers for real time PCR were designed to have a length around 20 bases, a GC content of approximately 50% and a Tm around 60°C. OligoPerfect Designer software (Invitrogen, Carlsbad, CA) was used to select primers sequences. Secondary structures and dimer formation were predicted using Oligo Analyzer 3.0 software (Integrated DNA Technologies, Coralville, IA). Primers were purchased from Sigma-Aldrich (St Louis, MO).
Real time PCR was performed using an Applied Biosystems 7300 Real-Time PCR System. The tuf gene of L. brevis, encoding elongation factor Tu, was used as internal control for the analysis of tyrDC and aguA1 genes expression, as previously described for Streptococcus thermophilus. Standard curves for both the internal-control and target genes were obtained by amplifying serial dilutions (ratio, 1:10) of the target sequences. Additionally, data were normalized in function of the amount of total RNA, according to Torriani et al. .
The amplifications were carried out in 20 μl reactions, by adding 5 μl of 1:20 diluted cDNA, to a real-time PCR mix containing Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA), according to the manufacturer’s instructions, and 100 nM of each primer. The tyrDC (EMBL accession number LVIS_2213) specific cDNA was amplified with the TDC_F (5′-TGAGAAGGGTGCCGATATTC-3′) forward and the TDC_R (5′-GCACCTTCCAACTTCCCATA-3′) reverse primers. The aguA1 (EMBL accession number LVIS_2208) specific cDNA was amplified with the AGUA1_F (5′-TCTTGAAAATGCGACAGACG-3′) forward and the AGUA1_R (5′-TCCAACGTAGCCTGAGCTTT-3′) reverse primers. The TUF_F (5′-AGGCGACGAAGAACAAGAAA-3′) forward and the TUF_R (5′-CGATACGACCAGAAGCAACA-3′) reverse primers were used to amplify the tuf (EMBL accession number LVIS_1389) specific cDNA.
Thermal cycling was as follows: initial denaturing at 95°C for 5 min followed by 35 cycles at 95°C for 15 s and 60°C for 35 s. The amplicons' lengths were 141 bp, 240 bp and 159 bp for the tyrDC, aguA1 and tuf genes respectively and their specificity was checked by melting curve analysis.
A threshold cycle value (CT) was determined with a base line settled automatically. The relative expression level of genes was calculated by the 2-∆∆ct method, using unstressed, and unsupplemented with BA precursors, total RNA as calibrator. The relative expression of tyrDC and aguA1 during the other experimental conditions was quantified as n-fold differences with respect to the calibrator.
Real-time PCRs were performed in duplicate for each sample of cDNA, including a negative control in each run. Data were expressed as the mean of three independent experiments.
Confocal laser scanning microscope
Samples from each gastric stress condition were analyzed by confocal laser scanning microscopy (model TCS-SP2-AOBS, Leica Microsystems GmbH, Wetzlar, Germany), after staining with SYTO9 and propidium iodide (LIVE/DEAD® BacLight™ bacterial viability kit, Molecular Probes, Inc. AA Leiden, The Netherlands) to differentiate the cells as a function of compromised membranes. Confocal illumination was provided with a X63 magnification objective and numerical aperture of 1.40–0.60 and by Argon laser (488 nm laser excitation) with a long pass 520–565 nm filter (for green emission) and long pass 630–685 nm filter (for red emission). Image analysis was performed using FRET and FRAP software (Leica Microsystems GmbH, Wetzlar, Germany).
Anova statistical tests were used to evaluate the consistency of the data.