2.2.1. Study of the Effect of Bacterial Count on Mycotoxin Binding of *Lactobacillus* Strains

According to the method described in Section 4.4.1, the effect of bacterial concentration on the mycotoxin binding capacity of *Lactobacillus* strains was determined for AFB1 and ST. The result shown in Figure 1 indicates that detectable mycotoxin binding could only be found above 10<sup>7</sup> cells/mL for both AFB1 and ST.

This result is in agreement with the findings of Ma et al. [26], who only found one strain that was able to bind mycotoxin at 10<sup>6</sup> cell/mL concentration (4.27%), and they obtained cut-off values at 10<sup>9</sup> cell/mL.

**Figure 1.** The effect of bacterial concentration on the aflatoxin B1 (continuous line) and sterigmatocystin (dashed line) binding of *L. pentosus* TV3 (black), *L. paracasei* MA2 (red), and *L. plantarum* TS23 (green). (logN means the logarithm of the number of colony-forming units per mL of bacterial cell suspension).

2.2.2. Study of the Effect of Incubation Time on Mycotoxin Binding of *Lactobacillus* Strains

The effect of incubation time on the AFB1 mycotoxin binding capacity of *Lactobacillus* strains was also examined. For this experiment, five strains of different genera were selected. The strains were incubated with AFB1 mycotoxin for 10 min or 48 h, according to the method described in Section 4.4.2. The two-time values were selected according to the literature data available on AFB1 binding (see at the end of the paragraph), 10 min was the lowest with satisfactory results and 48 h is the incubation period in which lactobacilli reached the highest cell count under the study parameters. Our aim was to determine whether it is necessary to add the mycotoxin at the beginning of culturing or it is enough to add it after the bacteria reached their final cell concentrations. On the studied strains, very diverse results were obtained (Figure 2). For strain TV3, a significantly (*p* < 0.005) higher mycotoxin binding was found for the shorter incubation time, on the other hand, for TS23, significantly (*p* < 0.00005) better binding could be observed for the longer incubation in the presence of the mycotoxin. For strains MA2, TV24, and SK63 the incubation times had no significant (*p* > 0.4, 0.6 and 0.5, respectively) effect on the mycotoxin binding capacity. Regarding data in the literature, contradictory results can also be found. Studying 1, 10, 30 and 60 min, no effect on the incubation time on mycotoxin binding was found by Bueno et al. [22]. In another paper of Kasmani et al. [27], however, AFB1 binding was assayed at 0, 0.5, 4, 12, 24, and 72 h, with the lowest mycotoxin binding obtained at 0.5 h and the highest at 12 h, with a twofold difference. El-Nezami et al. and Peltonen et al. studied the binding of AFB1 by different species for 0, 4, 24, 48 and 72 h, and suggested that mycotoxin elimination is a rapid process [24,28]. As no satisfactory conclusions could be drawn regarding the optimal incubation time with the mycotoxin, a practical decision was made, the mycotoxin binding experiments were performed with 10 min incubation with the mycotoxin for our experiments.

**Figure 2.** The effect of incubation time of 10 min (blue) and 48 h (white) on the aflatoxin B1 binding of *L. pentosus*TV3, *L. paracasei*MA2, *L. plantarum*TS23, *L. graminis*TV24, and*L. salivarius* SK63. (means±standard deviation, *N* = 5).
