Aspergillus flavus Growth Inhibition and Aflatoxin B1 Decontamination by Streptomyces Isolates and Their Metabolites

Abstract

Aflatoxin B₁ is a potent carcinogen produced by Aspergillus flavus, mainly during grain storage. As pre-harvest methods are insufficient to avoid mycotoxin presence during storage, diverse curative techniques are being investigated for the inhibition of fungal growth and aflatoxin detoxification. Streptomyces spp. represent an alternative as they are a promising source of detoxifying enzymes. Fifty-nine Streptomyces isolates and a Streptomyces griseoviridis strain from the commercial product Mycostop^®, evaluated against Penicillium verrucosum and ochratoxin A during previous work, were screened for their ability to inhibit Aspergillus flavus growth and decrease the aflatoxin amount. The activities of bacterial cells and cell-free extracts (CFEs) from liquid cultures were also evaluated. Fifty-eight isolates were able to inhibit fungal growth during dual culture assays, with a maximal reduction going down to 13% of the control. Aflatoxin-specific production was decreased by all isolates to at least 54% of the control. CFEs were less effective in decreasing fungal growth (down to 40% and 55% for unheated and heated CFEs, respectively) and aflatoxin-specific production, with a few CFEs causing an overproduction of mycotoxins. Nearly all Streptomyces isolates were able to degrade AFB₁ when growing in solid and liquid media. A total degradation of AFB₁ was achieved by Mycostop^® on solid medium, as well as an almost complete degradation by IX20 in liquid medium (6% of the control). CFE maximal degradation went down to 37% of the control for isolate IX09. The search for degradation by-products indicated the presence of a few unknown molecules. The evaluation of residual toxicity of the tested isolates by the SOS chromotest indicated a detoxification of at least 68% of AFB₁'s genotoxicity.

Keywords: actinobacteria; biodegradation; detoxification; enzymes; fungi; mycotoxins.

Figure 1

Effect of 59 Streptomyces isolates and Mycostop^® (MYC) on Aspergillus flavus growth and aflatoxin B₁- and B₂-specific production (accumulation) during dual culture on CYA medium at 25 °C for 8 days. The boxplot represents the distribution of the data expressed as a % of control (fungal growth and aflatoxin-specific production without bacteria).

Figure 2

Growth profiles of Aspergillus flavus alone and during dual culture assay against Streptomyces isolates IX14 and IX50. Cultures were performed on CYA medium for 8 days at 25 °C.

Figure 3

Effect of cell-free extracts (CFEs) of 59 Streptomyces isolates and Mycostop^® strain (MYC), added at 10% in the solid culture medium (CYA) of Aspergillus flavus, on fungal growth and aflatoxin-specific production (accumulation) after 8 days at 25 °C. The boxplot represents the distribution of the data expressed as a % of control (fungal growth and specific mycotoxin production without CFEs). Thermal treatment for heated CFEs: 100 °C for 10 min.

Figure 4

Degradation of aflatoxin B₁ by Streptomyces isolates and Mycostop^® (MYC) cells in solid (CYA) and liquid (CYB) media after 10 and 5 days of culture, respectively, and by their unheated CFEs after 48 h at 25 °C and 180 rpm. The boxplot represents the distribution of the mycotoxin decrease as a % of control. Controls were included by adding AFB₁ to CYA or CYB media, incubated under the same conditions.

Figure 5

Boxplots of the effect of Streptomyces isolates and CFEs on Aspergillus flavus growth and AFB₁-specific production (AFB₁sp) (accumulation). Each sub-figure represents a subcluster of the heatmap from Figure S1. GDC = A. flavus growth in dual culture, GCFE = A. flavus growth vs. cell-free extracts (CFEs), GHCFE = A. flavus growth vs. heated CFEs, AFBDC = AFB₁sp in dual culture, AFBCFE = AFB₁sp vs. CFEs, AFBHCFE = AFB₁sp vs. heated CFEs, AFBDS = AFB₁ degradation by cells in solid medium, AFBDL = AFB₁ degradation by cells in liquid medium, AFBDCFE = AFB₁ degradation by CFEs.

Figure 5

Boxplots of the effect of Streptomyces isolates and CFEs on Aspergillus flavus growth and AFB₁-specific production (AFB₁sp) (accumulation). Each sub-figure represents a subcluster of the heatmap from Figure S1. GDC = A. flavus growth in dual culture, GCFE = A. flavus growth vs. cell-free extracts (CFEs), GHCFE = A. flavus growth vs. heated CFEs, AFBDC = AFB₁sp in dual culture, AFBCFE = AFB₁sp vs. CFEs, AFBHCFE = AFB₁sp vs. heated CFEs, AFBDS = AFB₁ degradation by cells in solid medium, AFBDL = AFB₁ degradation by cells in liquid medium, AFBDCFE = AFB₁ degradation by CFEs.

Figure 6

Mass spectrometry spectral data for aflatoxin B₁ remaining after 12 days of incubation at 25 °C and 180 rpm with Streptomyces isolates IX20 and IX45 and MYC strain in CYB medium. Controls consisted of CYB with AFB₁, incubated without bacteria, under the same conditions. Spectra were obtained for the extraction with two solvents: chloroform (top) and ethyl acetate (bottom). The peaks of the medium for the control and samples without AFB₁ were subtracted as blanks.

Figure 7

Plot of the SOS induction factors (IF) as a function of the initial concentration of AFB₁ during degradation, and residual toxicity assays of Streptomyces isolates IX20 and IX45 and MYC strain after 12 days at 25 °C and 180 rpm. Control consisted of CYB medium without bacteria incubated under the same conditions. Results are compared to the plot of the IF values of the AFB₁ standard at the initial concentrations.

Figure 8

Workflow of the presented study for the preliminary identification of hypothetical antagonistic mechanisms of Streptomyces isolates against Aspergillus flavus development and AFB₁ accumulation. Blue-colored rectangles indicate the different experimental stages, and filled rectangles (green, orange) indicate putative mechanisms. Examples of subclusters exhibiting the described mechanism are indicated in purple.