2.6. Cell surface hydrophobicity test
The hydrophobicity of LAB strains was assessed by the test for
microbial adhesion to hydrocarbons (MATH) [30,31]. Three
different solvents: n-hexadecane (apolar solvents), chloroform (a
monopolar and acidic solvent) and ethyl acetate (a monopolar and
basic solvent) were used in this test. Briefly, LAB was grown in MRS
broth at 37 C for 18 h, centrifuged at 10.000 g for 10 min, and the
pellet was washed three times and re-suspended in quarterstrength
Ringer’s solution (1.5 g NaCl, 0.02 g KCl, 0.03 g CaCl2 and
0.03 g NaHCO3). The optical density at 580 nm of the suspension
was measured using UVevis spectrophotometer and recorded as
reading 1. Then, 1.5 mL of cell suspension was mixed for 2 min with
equal volume of each solvents. The mixture was left to stand for
20 min to ensure the complete separation of the two phases after
which 1 mL of the top (water) phase was carefully collected, and the
optical density was measured at 580 nm and recorded (reading 2).
The experiment was repeated three times.
The percentage of cell-surface hydrophobicity (% H) of the strain
adhering to solvent was expressed as: % Hydrophobicity ¼ [(OD580
reading 1 - OD580 reading 2)/OD580 reading 1] 100. According to
their hydrophobicity, isolates were classified into: isolates with
high (71e100%), medium (36e70%) and low (0e35%) hydrophobicity
[32].