α d glucose

TPC was determined spectrophotometrically at 760 nm with the Folin–Ciocalteu reagent (Sigma Aldrich), using gallic acid as standard (Sigma Aldrich) [14 (link)]. The phenolic content is expressed as gallic acid equivalents (GAE). Dissolved sugars were also determined spectrophotometrically with a reagent containing l-tryptophan and boric acid (both from Sigma Aldrich) dissolved in concentrated sulfuric acid (Sigma Aldrich) [15 (link)]. This method can quantify sugars that can act as reducing agents and since most monosaccharides and oligosaccharides can be mild reductants, it was chosen as a suitable method for this analysis. The calibration standard for this method was α-d-glucose (Sigma Aldrich) and detection was performed at 525 nm. Both spectrophotometric methods gave concentrations of extracted compounds expressed as grams per liter of solvent (g/L). The data presented in this work are expressed as grams of extracted compounds per kilograms of olive leaf powder (g/kg) simply by dividing their concentration in the liquid phase by the solid–liquid ratio. This gives us an overestimate of the extracted material because there is a considerable amount of retention in the solid phase (approximately 30%) but is presented in this way to express the whole amount of extractable material. Error bars correspond to the standard deviation of measurements carried out in triplicate.

Potassium chloride, α-(d)-glucose and l-glutamine were purchased from Sigma-Aldrich; osmolyte solutions were prepared using deionised water and were stored overnight at 4 °C before use. The solution pH was measured by means of a pH meter (Eutech Instruments, WP 600 series meter, model PC 650) in order to detect potential variations with respect to the neutral value due to the presence of glutamine (because of its ionisation in water, as amino acid).

LPC 18:1 (Avanti Polar Lipids) in chloroform was aliquoted under argon, evaporated under nitrogen until dry and stored at −20°C under argon until use. LPC aliquots were dissolved in PBS to yield a stock solution (3 mM) and used fresh for every experiment. NaCl, KCl and CaCl₂ were from Roth, KH₂PO₄ and NaHCO₃ from Merck (Darmstadt, Germany), MgSO₄ was from Fluka and α-D-Glucose was from Sigma-Aldrich.

G. vaginalis ATCC 14018^T, G. leopoldii UGent 06.41^T, G. piotii UGent 18.01^T and G. swidsinskii GS 10234^T were grown on chocolate (Choc) agar plates (Becton Dickinson, Franklin Lakes, NJ, USA) at 37 °C under anaerobic conditions (10% CO₂, 10% H₂, 80%N₂, Concept 400, Anaerobic Workstation). Liquid cultures were prepared in New York City Broth III (10 mM HEPES (Sigma Aldrich, Burlington, MA, USA), 15 g/L Proteose Peptone No. 3 (Becton Dickinson), 3.8 g/L yeast extract (Thermo Fisher Scientific, Waltham, MA, USA), 86 mM sodium chloride (Carl Roth, Karlsruhe, Germany), 28 mM α-D-glucose (Sigma–Aldrich)), at pH 5 (NYB5), supplemented with 10% horse serum (HS, Thermo Fisher Scientific) (NYB5 + HS).

α-d-Glucose, α,α′-trehalose, allyl bromide, Amberlite IR-120H resin, hydrochloric acid, ethanol, methanol, vinyl acetate, D₂O, CD₃OD and CDCl₃ were purchased from Sigma Aldrich. Potassium hydroxide was purchased from Carlo Erba. Azobisisobutyronitrile (AIBN) was purchased from Fluka. Acetone was purchased from VWR International. All the chemicals were reagent grade and were used without further purification.
Amberlite IR-120H resin (250 mg) was activated by washing with methanol (3 × 10 min, 1.25 ml each time) and standing overnight in methanol (1.25 ml).

Campylobacter jejuni strain 81116 (NCTC11828), C. coli NCTC 11366 and three C. hepaticus isolates, C. hepticus HV10, C. hepaticus 19L and C. hepaticus 44L were used in glucose utilization studies. After cultures were grown in HBA for 3 days, cells were collected and resuspended in physiological saline (0.9% NaCl) to an OD₆₀₀ of 1.0. The medium used to test the ability of C. hepaticus to utilize glucose consisted of inorganic salts (IS) as described previously (Alazzam et al., 2011 (link)). L-cysteine (0.2 mM) was used as a nitrogen source, and α-D-glucose (10 mM) (Sigma) was used as the sole carbon source. The experiment was carried out in 24-well plates. Each well-contained 100 μl of culture (OD₆₀₀ = 1). Controls included culture in IS plus L-cysteine only and IS plus α-D-glucose only. 2,3,5 tetrazolium chloride (TTC) (Sigma) (0.0665 g/L) was used as an indicator in all wells (Menolasino, 1959 (link)). A color change is observed in growing cultures, indicating utilization. The color change results were read after 36 h of incubation at 37°C. C. hepaticus, C. jejuni, and C. coli were also grown in Brucella broth to confirm their viability. The experiment was repeated twice, each time in biological triplicate.