Microbial detoxification of eleven food and feed contaminating trichothecene mycotoxins

Abstract

Background: Contamination of agricultural commodities with multiple trichothecene mycotoxins, produced by toxigenic Fusarium species, is a food safety issue, which greatly affects grain production and marketing worldwide. Importantly, exposure to multiple trichothecenes may increase toxicity in animals due to their synergistic and/or additive effects. To address the problem this study aimed to achieve a novel biological trait capable of detoxifying various food and feed contaminating trichothecenes under aerobic and anaerobic conditions and wide range of temperatures.

Results: A highly enriched microbial consortium (called DX100) capable of transforming eleven trichothecenes to significantly less toxic de-epoxy forms was achieved after prolonged incubation of soil microbial culture with 200 μg/mL deoxynivalenol (DON). DX100 demonstrated de-epoxidation activity under aerobic and anaerobic conditions, a greater range of temperatures and around neutral pH. The consortium contains 70% known and 30% unknown bacterial species, dominated by Stenotrophomonas species. Probably novel bacteria including strains of Stenotrophomonas and Alkaliphilus-Blautia species complex could be involved in aerobic and anaerobic de-epoxidation of trichothecenes, respectively. DX100 showed rapid and stable activity by de-epoxidizing 100% of 50 μg/mL deoxynivalenol at 48 h of incubation and retaining de-epoxidation ability after 100 subcultures in mineral salts broth (MSB). It was able to de-epoxidize high concentration of DON (500 μg/mL), and transformed ten more food contaminating trichothecenes into de-epoxy forms and/or other known/unknown compounds. Microbial de-epoxidation rate increased with increasing trichothecene concentrations in the broth media, suggesting that DX100 maintains a robust trichothecene detoxifying mechanism. Furthermore, the nature of microbial de-epoxidation reaction and inhibition of the reaction by sodium azide and the finding that bacterial cell culture lysate retained activity suggests that certain cytoplasmic reductases may be responsible for the de-epoxidation activity.

Conclusions: This study reports the enrichment procedure for obtaining an effective and stable microbial consortium DX100 capable of de-epoxidizing several food contaminating trichothecene mycotoxins. DX100, which has de-epoxidation ability under wide range of conditions, represents a unique enzymatic source which has great industrial potential for reducing contamination of foods/feeds with multiple trichothecenes, and minimizing their synergistic/additive cytotoxic effects on consumer health.

Keywords: Biodetoxification; Food contamination; Fusarium mycotoxins; Microbial de-epoxidation; Trichothecenes.

Fig. 1

Identification and abundance of bacterial genera and species in trichothecene de-epoxidizing DX100 microbial consortium. a: Relative abundance of known and unknown bacterial genera in DX100. b: Relative abundance of known and unknown bacterial species in DX100. The areas of different color represent abundance of different bacterial genera or species present in DX100. The initials of bacterial species A, B and S indicate genera Alkaliphilus, Blautia and Stenotrophomonas, respectively

Fig. 2

Mass spectrometric evidence of microbial de-epoxidation and de-acetylation of HT-2 trichothecene mycotoxin. The upper panel illustrates the mass spectrometer chromatograph of the de-acetylated and de-epoxidized products. The middle and lower panels show the mass spectra of the transformed compounds. Arrows on the upper panel indicate the targeted acetyl (red) and epoxy (blue) functional groups of HT-2 toxin. Red and blue arrows on the middle and lower panels identify the degradation sites of HT-2 toxin. dA.HT-2 = de-acetylated HT-2, and dE.HT-2 = de-epoxidized HT-2 toxin

Fig. 3

Effect of deoxynivalenol concentrations on the growth and de-epoxidation activity of DX100 microbial consortium. a: Microbial cell density (OD_600nm) in MSB with different DON concentrations measured at 24, 36, 48, 60, 72 and 96 h. b: Microbial cell growth (OD_600nm) in NB with different DON concentrations, measured at 24, 48, 72 and 96 h. c: Temporal DON de-epoxidation activity of DX100 in MSB containing 20, 200 and 500 μg/mL DON. The relative activity was measured by quantifying de-epoxidation activity divided by microbial growth (OD_600nm value) at different time points. Results are the mean of three replicate observations, and bars shown are ± standard errors of the means. The averages ± standard errors for three treatments and the control are presented in the graph. MSB = broth containing six mineral salts + 0.5% Bacto Peptone, DON = deoxynivalenol, and NB = nutrient broth

Fig. 4

Relative growth and de-epoxidation capabilities of DX100 microbial consortium under aerobic and anaerobic conditions at moderate temperature. a: Microbial growth (determined by OD₆₀₀ value) under aerobic conditions in MSB and NB medium and anaerobic conditions in MSB medium. b: Microbial DON de-epoxidation ability under aerobic conditions in MSB and NB media and anaerobic conditions in MSB medium. Tubes containing MSB or NB were supplemented with 50 μg/mL DON. The activity was measured by quantifying de-epoxidation activity divided by microbial growth (OD₆₀₀ value) at different time points. Values are means of triplicate experiments, and bars shown are ± standard errors of the means. The average ± standard errors for the treatments are indicated in the graph. MSB = broth containing six mineral salts + 0.5% Bacto Peptone, NB = nutrient broth, DON = deoxynivalenol

Fig. 5

Effects of temperature, substrate and pH on growth and de-epoxidation activities of DX100 under aerobic conditions. a: OD_600nm values show relative growth performance of DX100 in different broth media. b: Bacterial de-epoxidation activity (DON to dE-DON transformation) in different broth media. c: OD_600nm values demonstrate the effects of temperatures on the growth capacity of DX100 in MSB. d: Bacterial DON de-epoxidation in μg/mL under different temperatures in MSB. e: OD_600nm values show growth behavior of DX100 at different pHs in MSB. f: Bacterial de-epoxidation (DON to dE-DON transformation) in μg/mL in different pHs. The activity was measured by quantifying de-epoxidation activity divided by microbial growth (OD₆₀₀ value) under different growth conditions. Results are the mean of five replicate observations, and bars shown are ± standard errors of the means. The averages of ± standard errors for different growth conditions are presented in the graph. Treatments labeled with the same letter are not significantly different, according to Tukey’s HSD test (p < 0.05). Media: NB = nutrient broth, 1/NB = 1/10 strength NB, MM = Minimal medium, MS-DON = six mineral salts having 200 μg/mL DON. dE-DON = de-epoxy deoxynivalenol, MSB = six mineral salts + 0.5% Bacto Peptone, MSC = six mineral salts broth + 0.3% Bacto Peptone, MSY = six mineral salts with 0.5% yeast extract, S.E = standard error, SE = soil extract, PDB = potato dextrose broth and LB = Luria Bertani