flavus RC2053, RC2054, RC2055, RC2056, RC2057, RC2058, RC2059, RC2060, RC2061, A. parasiticus RC2062). All isolates were used in qualitative
experiments and only the most 3-MA in vivo potent AFB1 producers were used in growth studies (A. flavus RC2053, RC2054, RC2055, RC2056). The isolates were maintained at 4 °C on malt extract agar (MEA) slants and at −80 °C in 15% glycerol. The effect of lactobacilli strains on A. flavus strains was detected by two qualitative methods: Lactobacillus rhamnosus L60 and L. fermentum L23 strains were assayed for inhibition of 10 A. flavus strains. The agar overlay method was used with some modifications (Magnusson & Schnürer, 2001). MRS agar plates on which L. rhamnosus L60 and L. fermentum L23 were inoculated in 2-cm-wide lines each and incubated at 37 °C under a 5% CO2 atmosphere for 48 h. After the incubation period, the plates were overlaid with a soft agar (75% by weight agar) preparation of MEA containing 9.5 × 102 fungal spores mL−1, determined by counting on a Neubauer haemocytometer. The plates were incubated this website aerobically at 25 °C for 5 days. The zones of inhibition of Aspergillus were estimated using a semiquantitative scale: (−), lack of Aspergillus growth inhibition over Lactobacillus culture; (+/−),
minimal inhibition of Aspergillus growth over Lactobacillus culture; (+), partial inhibition of Aspergillus growth over Lactobacillus culture; (++), total inhibition of Aspergillus growth over Lactobacillus culture. Plates containing only the fungal spore inoculums (without Lactobacillus strains) were used as a control. Lactobacillus L60 and L23 strains were seeded until covering one-third of the surface of MRS agar plates and incubated in optimal conditions at 37 °C for 48 h. An MEA agar plug with A. flavus was placed on the centre of the free surface of these MRS agar plates and incubated aerobically at
25 °C for 5 days in the dark. Lactobacillus rhamnosus L60 and L. fermentum L23 suspensions Liothyronine Sodium were prepared in MRS broth (bioMérieux) (Rogosa & Sharpe, 1963) and adjusted to 0.5 of the McFarland scale, corresponding to final concentration of 1.5 × 108 CFU mL−1. An aliquot of 1 mL from each lactobacillus suspension was placed into sterile Petri dishes. MRS agar (bioMérieux) (Rogosa & Sharpe, 1963) was poured into Petri dishes and stirred to homogenize the content. The plates were inoculated in the centre with a suspension of fungal spores from 7-day-old cultures on MEA in semisolid agar. The plates were incubated at 25 °C and the colony radius was measured daily. For each colony, two radii, measured at right angles to one another, were averaged to find the mean radius for that colony. All colony radii were determined by using three replicates for each tested fungus. The radial growth rate (mm day−1) was subsequently calculated by linear regression of the linear phase of growth and the time at which the line intercepted the x-axis was used to calculate the lag phase.