Inhibition of the Aspergillus flavus Growth and Aflatoxin B1 Contamination on Pistachio Nut by Fengycin and Surfactin-Producing Bacillus subtilis UTBSP1

Abstract

In this study, the treatment of pistachio nuts by Bacillus subtilis UTBSP1, a promising isolate to degrade aflatoxin B1 (AFB1), caused to reduce the growth of Aspergillus flavus R5 and AFB1 content on pistachio nuts. Fluorescence probes revealed that the cell free supernatant fluid from UTBSP1 affects spore viability considerably. Using high-performance liquid chromatographic (HPLC) method, 10 fractions were separated and collected from methanol extract of cell free supernatant fluid. Two fractions showed inhibition zones against A. flavus. Mass spectrometric analysis of the both antifungal fractions revealed a high similarity between these anti-A. flavus compounds and cyclic-lipopeptides of surfactin, and fengycin families. Coproduction of surfactin and fengycin acted in a synergistic manner and consequently caused a strong antifungal activity against A. flavus R5. There was a positive significant correlation between the reduction of A. flavus growth and the reduction of AFB1 contamination on pistachio nut by UTBSP1. The results indicated that fengycin and surfactin-producing B. subtilis UTBSP1 can potentially reduce A. flavus growth and AFB1 content in pistachio nut.

Keywords: Bacillus subtilis; aflatoxin B1; antifungal activity; fengycin; surfactin.

Fig. 1

Reduction of Aspergillus flavus growth (spore population) and aflatoxin B1 (AFB1) content in pistachio nuts by using different concentrations of Bacillus subtilis UTBSP1, including 10⁵, 10⁶, and 10⁷ cfu/ml saline solution. In the infected control (inoculated nut with saline solution and A. flavus R5), 1.3 × 10⁷ spores and 12,465.1 ng AFB1 per g nut were detected. The treatments with same letter were not significantly different (P ≤ 0.05). Error bars indicate standard error of four replications.

Fig. 2

Fluorescence microscopy pictures of Aspergillus flavus spores that were incubated for 3 hours in the HCl precipitate of Bacillus subtilis UTBSP1 (500 μg·ml⁻¹) and then stained with PI (A) or cFDA (F). The fluorescence microscopy pictures of PI-stained (C) or cFDA-stained (H) spores in control (water:methanol [50:50 v/v]) are presented. In addition, the light microscopy pictures of HCl precipitate-treated spores (B, E) and untreated (control) spores (D, G) are shown. PI, propidium iodide; cFDA, carboxyfluorescein diacetate.

Fig. 3

Purification chromatogram of the methanol extraction of the HCl precipitate of Bacillus subtilis UTBSP1 by using reverse phase high-performance liquid chromatography (HPLC) with C18 column. Mobile phase: water (0.1% trifluroacetic acid) and methanol; detector: 214 and 280 nm. All the distinct fractions (10 peaks) were collected manually. Peaks ‘A’ and ‘B’ showed inhibition zones against Aspergillus flavus R5.

Fig. 4

The inhibition zones against Aspergillus flavus growth caused by the fraction A (right) and mixture of fractions A + B (left) after 4 days incubation at 30°C in darkness.

Fig. 5

Full mass spectrometric spectra of surfactin (A), and fengycin (B), produced by Bacillus subtilis UTBSP1, and obtained from electrospray ionization triple-quadrupole mass spectrometer. The peaks belong to the HCl precipitate of B. subtilis UTBSP1.