Carol Verheecke

Microbial degradation of aflatoxin B1: Current status and future advances

Aflatoxin B1 (AFB1) is a natural toxin produced by many food-contaminant fungi and is a threat to human and animal health. This review summarizes current knowledge of the different ways to limit AFB1 in the food chain. We start by introducing current data and reviews available on the prevention of AFB1 occurrence, on AFB1 non-biological decontamination and biological adsorption. We then focus on microbial AFB1-degradation. The latter has already been well studied using living organisms, supernatants or purified enzymes. This review compiles information on the variety of protocols and the efficacy of the different sub-kingdoms or classes of microorganisms or their enzymes. We pay particular attention to publications closest to in vivo applications of microbial AFB1-degradation. In addition, this review also provides a summary of the currently known microbial degradation metabolites of AFB1 and their levels of toxicity, and provides recommendations on the most promising techniques to pursue the aim of minimizing ABF1 in the food supply.

In vitro interaction of actinomycetes isolates with Aspergillus flavus: impact on aflatoxins B1 and B2 production

This work aimed to study the interaction between Actinomycetal isolates and Aspergillus flavus to promote mutual antagonism in contact. Thirty‐seven soilborn Streptomyces spp. isolates were chosen as potential candidates. After a 10‐day in vitro co‐incubation period, 27 isolates respond to the criteria, that is, mutual antagonism in contact. Further aflatoxins B1 and B2 analysis revealed that those 27 isolates reduced aflatoxin B1 residual concentration from 38·6 to 4·4%, depending on the isolate. We selected 12 isolates and tested their capacity to reduce AFB1 in pure culture to start identifying the mechanisms involved in its reduction. AFB1 was reduced by eight isolates. The remaining AFB1 concentration varied between 82·2 and 15·6%. These findings led us to suggest that these eight isolates could be used as biocontrol agents against AFB1 and B2 with low risk of impacting the natural microbial equilibrium.

Reduction of aflatoxin production by Aspergillus flavus and Aspergillus parasiticus in interaction with Streptomyces

The aim of this study is to investigate aflatoxin gene expression during Streptomyces-Aspergillus interaction. Aflatoxins are carcinogenic compounds produced mainly by Aspergillus flavus and Aspergillus parasiticus. A previous study has shown that Streptomyces-A. flavus interaction can reduce aflatoxin content in vitro. Here, we first validated this same effect in the interaction with A. parasiticus. Moreover, we showed that growth reduction and aflatoxin content were correlated in A. parasiticus but not in A. flavus. Secondly, we investigated the mechanisms of action by reverse-transcriptase quantitative PCR. As microbial interaction can lead to variations in expression of household genes, the most stable [act1, βtub (and cox5 for A. parasiticus)] were chosen using geNorm software. To shed light on the mechanisms involved, we studied during the interaction the expression of five genes (aflD, aflM, aflP, aflR and aflS). Overall, the results of aflatoxin gene expression showed that Streptomyces repressed gene expression to a greater level in A. parasiticus than in A. flavus. Expression of aflR and aflS was generally repressed in both Aspergillus species. Expression of aflM was repressed and was correlated with aflatoxin B1 content. The results suggest that aflM expression could be a potential aflatoxin indicator in Streptomyces species interactions. Therefore, we demonstrate that Streptomyces can reduce aflatoxin production by both Aspergillus species and that this effect can be correlated with the repression of aflM expression.

Contamination of common spices by aflatoxigenic fungi and aflatoxin B1 in Algeria

Spices are usually produced in areas where the climatic conditions are favourable to growth of toxigenic fungi and production of mycotoxins. This study assesses the occurrence of aflatoxigenic fungi and aflatoxin B1 (AFB1) in spices marketed in Algeria. A total of 44 spice samples (4 for each type of spice) composed of aniseed, black pepper, caraway, cinnamon, coriander, cumin, ginger, red pepper, saffron, sweet cumin, and sweet pepper were collected from four popular markets located in Algeria. Mycological analysis of the spice was by dilution plating while AFB1 contamination levels were determined by high-performance liquid chromatography coupled with fluorescence detection (HPLC-FLD) after post-column derivatisation. The commonly isolated fungi were species of Aspergillus (56.4%), Penicillium (25.1%), Mucor (12.8%) and Eurotium (5.7%). Species belonging to Aspergillus section Flavi represented 28.9% of the total Aspergilli. The aflatoxin producing ability of isolates belonging to Aspergillus section Flavi was determined on coconut agar medium and confirmed by thin layer chromatography and HPLC-FLD. Ninety-four isolates (38.4%) of the 245 Aspergillus section Flavi examined produced aflatoxins. The most frequent chemotypes (84%) correspond to isolates able to produce both aflatoxin B and cyclopiazonic acid followed by the producers of only aflatoxin B. Twenty-three (63.9%) of the 36 spices contained AFB1 at levels ranging from 0.10 to 26.50 μg/kg. Two saffron (24.34 and 26.50 μg/kg) and two sweet cumin (14.65 and 19.07 μg/kg) samples were above the Algerian regulatory limit of 10 μg/kg. This work represents the first report about the occurrence of aflatoxigenic fungi and AFB1 in the common spices in Algeria.

Actinomadura adrarensis sp. nov., an actinobacterium isolated from Saharan soil.

A novel actinobacterial strain, designated ACD12T, was isolated from a Saharan soil sample collected from Adrar province, southern Algeria. A polyphasic study was carried out to establish the taxonomic position of this strain. Strain ACD12T was observed to form extensively branched substrate mycelia. Aerial mycelium was absent or was weakly produced on all media tested, while spore chains were short with a hooked and irregular spiral form (2-3 turns). The dominant diaminopimelic acid isomer in the cell wall was meso-diaminopimelic acid. Glucose, ribose, galactose, mannose and madurose occured in whole-cell hydrolysates. The major phospholipid was diphosphatidylglycerol and phosphatidylinositol. The predominant menaquinone was MK-9(H6). The fatty acid profile was characterized by the presence of C16 : 0, C17 : 0, C15 : 0, C18 : 0, C18 : 1 cis9 and iso-C16 : 0. Results of 16S rRNA gene sequence comparisons revealed that strain ACD12T shared the highest degree of 16S rRNA gene sequence similarity with Actinomadura sputi DSM 45233T (98.3 %) and Actinomadura hallensis DSM 45043T (97.8 %). All tree-making algorithms used also supported strain ACD12T forming a distinct clade with its most closely related species. In addition, DNA-DNA hybridization indicated only 39.8 % relatedness with A. sputi DSM 45233T and 18.7 % relatedness with A. hallensis DSM 45043T. The combined phenotypic and genotypic data show that the novel isolate represents a novel species of the genus Actinomadura, for which the name Actinomadura adrarensis sp. nov., is proposed, with the type strain ACD12T (=DSM 46745T =CECT 8842T).

Streptomyces–Aspergillus flavus interactions: impact on aflatoxin B accumulation

The aim of this work was to investigate the potential of Streptomyces sp. as biocontrol agents against aflatoxins in maize. As such, we assumed that Streptomyces sp. could provide a complementary approach to current biocontrol systems such as Afla-guard® and we focused on biocontrol that was able to have an antagonistic contact with A. flavus. A previous study showed that 27 (out of 38) Streptomyces sp. had mutual antagonism in contact with A. flavus. Among these, 16 Streptomyces sp. were able to reduce aflatoxin content to below 17% of the residual concentration. We selected six strains to understand the mechanisms involved in the prevention of aflatoxin accumulation. Thus, in interaction with A. flavus, we monitored by RT-qPCR the gene expression of aflD, aflM, aflP, aflR and aflS. All the Streptomyces sp. were able to reduce aflatoxin concentration (24.0–0.2% residual aflatoxin B1). They all impacted on gene expression, but only S35 and S38 were able to repress expression significantly. Indeed, S35 significantly repressed aflM expression and S38 significantly repressed aflR, aflM and aflP. S6 reduced aflatoxin concentrations (2.3% residual aflatoxin B1) and repressed aflS, aflM and enhanced aflR expression. In addition, the S6 strain (previously identified as the most reducing pure aflatoxin B1) was further tested to determine a potential adsorption mechanism. We did not observe any adsorption phenomenon. In conclusion, this study showed that Streptomyces sp. prevent the production of (aflatoxin gene expression) and decontamination of (aflatoxin B1 reduction) aflatoxins in vitro.

Essential Oils Modulate Gene Expression and Ochratoxin A Production in Aspergillus carbonarius

Ochratoxin A (OTA) is a mycotoxin, mainly produced on grapes by Aspergillus carbonarius, that causes massive health problems for humans. This study aims to reduce the occurrence of OTA by using the ten following essential oils (E.Os): fennel, cardamom, anise, chamomile, celery, cinnamon, thyme, taramira, oregano and rosemary at 1 µL/mL and 5 µL/mL for each E.O.As a matter of fact, their effects on the OTA production and the growth of A. carbonarius S402 cultures were evaluated, after four days at 28 °C on a Synthetic Grape Medium (SGM). Results showed that A. carbonarius growth was reduced up to 100%, when cultured with the E.Os of cinnamon, taramira, and oregano at both concentrations and the thyme at 5 µL/mL. As for the other six E.Os, their effect on A. carbonarius growth was insignificant, but highly important on the OTA production. Interestingly, the fennel E.O at 5 µL/mL reduced the OTA production up to 88.9% compared to the control, with only 13.8% of fungal growth reduction. We further investigated the effect of these E.Os on the expression levels of the genes responsible for the OTA biosynthesis (acOTApks and acOTAnrps along with the acpks gene) as well as the two regulatory genes laeA and vea, using the quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) method. The results revealed that these six E.Os reduced the expression of the five studied genes, where the ackps was downregulated by 99.2% (the highest downregulation in this study) with 5 µL/mL of fennel E.O.As for the acOTApks, acOTAnrps, veA and laeA, their reduction levels ranged between 10% and 96% depending on the nature of the E.O and its concentration in the medium.