| dc.description.abstracteng | Summary: Mycotoxins are secondary metabolites produced by fungi. Enniatins are a group of cyclohexadepsipeptides made of three hydroxyisovaleric acid molecules and alternating amino acids mostly valine, leucine and isoleucine. Enniatins have been associated with mainly cereal and cereal-based products, but also with nuts and some dried fruits. There is no evidence that high concentrations of enniatins in food commodities are not harmful for human and animal health in the long term. Therefore, methods to reduce enniatin content in food by transforming them into less toxic compounds are of crucial importance. Decontamination strategies for mycotoxin contaminated food or feed involve physical and chemical treatments which are to some extent successful but also pose some disadvantages like limited efficacy and possible losses of the sensorial and nutritive value. Therefore, intensive work has been done in the field of biodegradation of mycotoxins in food. Within that framework, the main objective of this study was to find microorganisms (bacteria, yeast or fungi) able to degrade enniatins into less toxic compounds. The strategy used involved a random search of such microorganisms followed by their classification.
Due to the high quantity of mycotoxins that are involved in such studies resulting in great expense, the first objective of this study was to purify enniatins from fungal extract. Therefore two strains of F. tricinctum (DSM 23357 and O32) were tested for enniatin production in solid white bean medium. Enniatins were extracted every 3 days. A short method for the detection and quantification of enniatins A, A1, B and B1 in the fungal crude extracts was established and validated using HPLC-DAD. For both studied strains, enniatin concentrations increased until 24 days with 614 g/kg and 479g/kg for F. tricinctum O32 and DSM 23357 respectively. Throughout the 30 cultures days F. tricinctum O32 produced about two times more enniatins than the strain DSM 23357. As a result F. tricinctum O32 was used for the large scale production of enniatins over 30 days on solid white bean medium. Enniatin in the fungal crude extract were separated using flash chromatography and recrystallization. 3.18 g enniatins mixture/kg of white bean medium with a purity of 96.25% and containing about 77% enniatin B, 17% enniatin B1, 6% enniatin A1 and 0.3% enniatin A was obtained.
The second part of this study involved the search for microorganisms having the ability to degrade enniatins. For this purpose, microorganisms were first isolated from several sources (grains, nuts, water, soil, cereal based-products, juice) based on their ability to grow on a minimal medium (MM) containing enniatin as the unique carbon source. Wash fluid from several materials were mixed with MM containing enniatins (final concentration 1 mg/ml) and incubated for 7 days at 20°C. The cultures were diluted 25-times in MM with enniatins (final concentration 1 mg/ml) and incubated for further 21 days at 20°C. In total 114 bacteria or yeast-like microorganisms and 34 fungi were isolated. The major sources of bacteria were water, grains and nuts from which 38, 35, 20 and 7 bacteria were isolated respectively. Whereas the majority of fungi originated from nuts (16), grains (10) and soil (3) samples. 20 bacteria and 10 fungi were not able to be cultivated and after a second selection using single culture in MM containing (final concentration 1 mg/ml), 6 bacteria and 1 fungus did not grow anymore. Then 88 bacteria (and yeast-like microorganisms) and 23 fungi were screened for their property to break down enniatins into new compounds using HPLC-DAD (as described previously) in MM and in a minimal medium containing a limited amount of glucose and tryptone (MMGP). The results showed that enniatin concentrations were reduced in all the tested samples (enniatin A was mostly reduced than enniatin B). The HPLC-UV chromatograms of a bacterium (K4) and two fungi (F18 and F20) produced new signals compared to those of the controls. K4 and F18 originated from wheat field soil sample, whereas F20 was isolated form hazelnuts sample. These samples were further analyzed using LC-MS, differential metabolic profiling (Noise reduction, chromatogram alignment, normalization) and the mass of enniatins products were deduced and confirmed using extracted ions chromatograms and the corresponding mass spectra. Four enniatin products were identified using this technique; they were probably the products of hydrolysis of either an ester or an amide bond in the ring structure of enniatins, leading to the opening of the ring and therefore probably producing less toxic compounds. The molecular masses of the protonated ions of enniatins degradation products were 658, 672, 686 and 700 for enniatin B, B1, A1 and A respectively.
Finally the identification of the pure cultures responsible for enniatin degradation was carried out using phenotypic (visual and microscopic), biochemical (for the bacterium only, gram test, catalase, sulfide-indole-motility, starch hydrolysis, citrate, urease tests…) and molecular characteristics of the microorganisms. The bacterium was preliminary identified as Bacillus sp. and the sequence of the 16S rRNA gene analysis enabled the species identification of the bacterium K4 as Bacillus licheniformis. Based on morphological characteristics (visual and microscopic observations, spore size, conidia, conidiophores, presence or absence of septae…), fungi F18 and F20 were identified as Acremonium sp. and Clonostachys sp. respectively. The sequence of the ITS1, 5.8S RNA subunit and ITS2 of the DNA revealed that F18 and F20 were Acremonium strictum and Clonostachys rosea. | de |
| dc.subject.eng | Enniatins, F. tricinctum, production, HPLC-DAD, flash chromatography, degradation, degradation products, HPLC-MS, Bacillus licheniformis, Clonostachys rosea, Acremonium strictum, morphological and molecular characterization, ITS1, ITS2, 16S rRNA | de |