Nutritional requirements for the production of dithiolopyrrolone antibiotics by Saccharothrix algeriensis NRRL B-24137

The amino acid and humic acid requirements of Saccharothrix algeriensis NRRL B-24137 for growth and production of the dithiolopyrrolone antibiotics were studied in a semi-synthetic medium (SSM). Nature and concentration of amino acids and humic acid strongly influenced the growth and dithiolopyrrolone specific production. The highest value of thiolutin (acetyl-pyrrothine) specific production was obtained in the presence of 1 g/l humic acid (336 mg/g DCW), and in the presence of 5mM l-cystine (309 mg/g DCW) as compared to 19 mg/g DCW obtained with the control. Furthermore, thiolutin production was increased about six-fold, four-fold and three-fold in the presence of l-proline, l-glutamic acid and dl-histidine, respectively. In contrast,the production of thiolutin was reduced by addition of other amino acids such as l-glutamine, dl-ethionine, l-methionine and l-arginine. The highest value of isobutyryl-pyrrothine production was obtained in the presence of 2,6-diaminopimelic acid and l-lysine (7.8 and 1.0 mg/g DCW, respectively). However, the highest value of butanoyl-pyrrothine production was obtained in the presence of humic acid (6.6 mg/g DCW), followed by l-cysteine and l-proline (3.6 and 3.2 mg/g DCW, respectively). In addition, the maximum specific production of senecioyl-pyrrothine (29 mg/g DCW) and tigloyl-pyrrothine (21 mg/g DCW) was obtained in the presence of humic acid. We found that, except for isobutyryl-pyrrothine, production of all dithiolopyrrolones was favoured by addition of l-proline. The maximum specific production was obtained with l-proline at concentrations of 2.50mM for thiolutin (133mg/gDCW),1.25mMfor senecioyl-pyrrothine, tigloyl-pyrrothine and butanoyl-pyrrothine production (29, 23 and 3.9 mg/gDCW, respectively). Production of all dithiolopyrrolones strongly decreased as the l-methionine or dl-ethionine concentration was increased in the culture medium

Quantification of Fusarium graminearum and Fusarium culmorum by real-time PCR system and zearalenone assessment in maize

Zearalenone (ZEA) is a mycotoxin produced by some species of Fusarium, especially by Fusarium graminearum and F. culmorum. ZEA induces hyperoestrogenic responses in mammals and can result in reproductive disorders in farm animals. In the present study, a real-time PCR (qPCR) assay has been successfully developed for the detection and quantification of Fusarium graminearum based on primers targeting the gene PKS13 involved in ZEA biosynthesis. A standard curve was developed by plotting the logarithm of known concentrations of F. graminearum DNA against the cycle threshold (Ct) value. The developed real time PCR system was also used to analyze the occurrence of zearalenone producing F. graminearum strains on maize. In this context, DNA extractions were performed from thirty-two maize samples, and subjected to real time PCR. Maize samples also were analyzed for zearalenone content by HPLC. F. graminearum DNA content (pg DNA/ mg of maize) was then plotted against ZEA content (ppb) in maize samples. The regression curve showed a positive and good correlation (R2=0.760) allowing for the estimation of the potential risk from ZEA contamination. Consequently, this work offers a quick alternative to conventional methods of ZEA quantification and mycological detection and quantification of F. graminearum in maize

A stoichiometric reaction scheme for Saccharothrix algeriensis growth and thiolutin production

A new bacterial species, Saccharothrix algeriensis NRRL B-24137, was isolated in 1992 in the Sahara desert. This filamentous bacterium is able to produce dithiolopyrrolones, molecules presenting antibacterial, antifungal, and anticancer properties. In this study, a “reaction engineering” approach was adopted to gain more knowledge on the growth of Sa. algeriensis and its dithiolopyrrolone production on a semi-synthetic liquid medium. The objective is to establish a reaction scheme of the bacterium metabolism from extracellular experimental information, relatively easy to obtain. The approach enabled us to show that Sa. algeriensis could grow using several substrates that were sequentially consumed and that substrate limitation may induce a secondary metabolism in antibiotic production. From these qualitative data, a general reaction scheme was extracted consisting of four reactions: growth via amino acids, glucose consumption for maintenance, growth using glucose, and thiolutin production. The stoichiometric coefficients and the reaction extends were identified using a factorial analysis based on the bilinear structure of the component mass balances in a batch reactor. The analysis of the reaction stoichiometry enabled us to draw some conclusions concerning the substrate consumption pathway

Influence of barley malting operating parameters on T-2 and HT-2 toxinogenesis of Fusarium langsethiae, a worrying contaminant of malting barley in Europe.

The fungus Fusarium langsethiae, exclusively described in Europe at present, seems to have taken the place of other Fusarium species in barley fields over the last 5 years. It has proved to be a highly toxic type-A trichothecene producer (T-2 and HT-2 toxins). The aim of this work was to study the ecotoxinogenesis of this fungus the better to identify and manage the health risk it may pose during the beer manufacturing process. The influence of temperature and water activity on its growth rate and production of toxins are particularly assessed from a macroscopic point of view. Different cultures were grown on sterilized rehydrated barley with a water activity between 0.630 and 0.997 and a temperature ranging from 5 to 35°C. Biomass specific to F. langsethiae and T-2 and HT-2 toxins were quantified by real-time polymerase chain reaction and liquid chromatography-mass spectrometry, respectively. It appears that the optimal temperature and water activity for F. langsethiae toxinogenesis are 28°C and 0.997. This fungus was able to produce 2.22 g kg−1 of these toxins in 16 days on barley in optimal production conditions. The malting process seems to be a critical step because, in its temperature range, specific production was six times higher than under optimal temperatures for fungus growth. In the short-term, this work will help redefine the process conditions for malting. In the medium-term, the results will contribute to the development of a molecular tool to diagnose the presence of this contaminant and the detection of the toxins in barley, from fields to the end product

Review of mycotoxin reduction in food and feed: from prevention in the field to detoxification by adsorption or transformation

Mycotoxins are secondary metabolites present worldwide in agricultural commodities and produced by filamentous fungi that cause a toxic response (mycotoxicosis) when ingested by animals. Prevention of mycotoxicoses includes pre- and post-harvest strategies. The best way to reduce the mycotoxin content in food and feed is the prevention of mycotoxin formation in the field, but this is often not sufficient, so other methods are needed. To decontaminate and/or detoxify mycotoxin-contaminated food and feed, the most prevalent approach in the feed industry is the inclusion of sorbent materials in the feed thus obtaining more or less selective removal of toxins by adsorption during passage through the gastrointestinal tract. Another reliable approach is to add enzymes or microorganisms capable of detoxifying some mycotoxins. Through a comprehensive review of published reports on the strategies for mycotoxin removal, this present work aims to update our understanding of mycotoxin removal. It provides an insight into the detoxification of mycotoxin present in food and feed. In the future, more emphasis needs to be placed on adsorption of mycotoxins in the gastrointestinal tract. Concerning the enzymatic transformation of mycotoxins, further efforts are required in understanding detoxification reactions, the toxicity of transformation products and in the characterization of enzymes responsible for transformations

Identification of a new strain of Actinomadura isolated from Saharan soil and partial characterization of its antifungal compounds

One promising strain Actinomadura sp. AC170, isolated from Algerian Saharan soil, with strong antifungal activity against pathogenic and toxinogenic fungi, was selected for further studies. The 16S rRNA results showed a distinct phylogenetic lineage from the other species within the Actinomadura genus. The production of antibiotic substances was investigated using GYEA solid medium. The butanolic extract contained four bioactive spots detected on thin layer chromatography plates. Among these antibiotics, a complex called 170A, which showed the more interesting antifungal activity, was selected and purified by reverse-phase HPLC. This complex is composed of four compounds. Ultraviolet-visible, infrared, mass and H nuclear magnetic resonance spectroscopy studies showed that these molecules contain an aromatic ring substituted by aliphatic chains. These compounds differ from the known antibiotics produced by Actinomadura species

Taxonomic study and partial characterization of antimicrobial compounds from a moderately halophilic strain of the genus Actinoalloteichus

A moderately halophilic actinomycete strain designated AH97 was isolated from a saline Saharan soil, and selected for its antimicrobial activities against bacteria and fungi. The AH97 strain was identified by morphological, chemotaxonomic and phylogenetic analyses to the genus Actinoalloteichus. Analysis of the 16S rDNA sequence of strain AH97 showed a similarity level ranging between 95.8% and 98.4% within Actinoalloteichus species, with A. hymeniacidonis the most closely related. The comparison of the physiological characteristics of AH97 with those of known species of Actinoalloteichus showed significant differences. Strain AH97 showed an antibacterial and antifungal activity against broad spectrum of microorganisms known to be human and plant pathogens. The bioactive compounds were extracted from the filtrate culture with n-butanol and purified using thin layer chromatography and high pressure liquid chromatography procedures. Two active products were isolated, one hydrophilic fraction (F1) and another hydrophobic (F2). Ultraviolet-visible, infrared, mass and 1H and 13C nuclear magnetic resonance spectroscopy studies suggested that these molecules were the dioctyl phthalate (F2) and an aminoglycosidic compound (F1)

Aspergillus westerdijkiae polyketide synthase gene “aoks1” is involved in the biosynthesis of ochratoxin A

OchratoxinA (OTA) is a potential nephrotoxic, teratogenic, immunogenic, hepatotoxic and carcinogenic mycotoxin, produced by Aspergillus westerdijkiae NRRL 3174. Herein we describe the characterization of a putative OTA-polyketide synthasegene “aoks1”, cloned by using gene walking approach. The predicted amino acid sequence of the 2 kb clone display 34–60% similarities to different polyketide synthasegenes including lovastatine biosynthesis gene “lovb” in A. terreus, compactin biosynthesis gene “mlcA” in Penicillium citrinum and OTA biosynthesis gene “otapksPN” in P. nordicum. Based on the reverse transcription PCR and kinetic secondary metabolites production studies, aoks1 expression was found to be associated with OTA biosynthesis. Further a mutant, in which the aoks1gene was inactivated by Escherichia coli hygromycin B phosphotransferase gene, lost the capacity to produce OTA, but still producing mellein. To our knowledge this report describes for the first time characterization of a gene involved in OTA biosynthesis, with the information about mellein which was proposed in the literature to be an intermediate OTA. This study also suggests that aoks1 may be the second polyketide synthase gene required for OTA biosynthesis in A. westerdijkiae NRRL 3174

Identification and partial characterization of antifungal and antibacterial activities of two Bacillus sp. strains isolated from salt soil in Tunisia

Two Bacillus sp. strains (B29 and B27) isolated from soil in the South of Tunisia were tested for their abilities to produce antimicrobial compounds. Both strains showed antimicrobial activity against Gram-positive and Gram-negative bacteria, yeasts and fungi. The produced compounds were extracted by using four different solvents. The hexane solvent allowed to obtain maximum of activity of the strain B29. The activity of the strain B27 was not elucidated by the four solvents used. Bio-autography results of B29 hexane extract revealed presence of different antibiotics and antifungal compounds with different Rf values of 0.3 and 0.76 for antifungal compounds and of 0.12, 0.14, 0.19 and 0.3 for antibacterial ones. Two active fractions were isolated from the culture broth of the strain B29 by semi-preparative high performance liquid chromatography (HPLC). The partial sequencing of the 16S rDNA gene was used to identify the two Bacillus strains. They may be assigned to new Bacillus specie

Aspergillus section Flavi and aflatoxins in Algerian wheat and derived products

Wheat and its derivatives are a very important staple food for North African populations. The aim of this study was to analyze populations of Aspergillus section Flavi from local wheat based on aflatoxins (AFs),cyclopiazonic acid (CPA) and sclerotia production, and also to evaluate AFs-contaminated wheat collected from two different climatic regions in Algeria. A total of 108 samples of wheat were collected during the following phases: pre-harvest, storage in silos and after processing. The results revealed that among the Aspergillus species isolated, those belonging to section Flavi were predominant. Of the 150 strains of Aspergillus section Flavi isolated, 144 were identified as Aspergillus flavus and 6 as Aspergillus tamarii. We showed that 72% and 10% of the A. flavus strains produced AFs and CPA, respectively. Among the 150 strains tested, 60 produced amounts of AFB1 ranging from 12.1 to 234.6 lg/g of CYA medium. Also, we showed that most strains produced large sclerotia. AFB1was detected by HPLC in 56.6% of the wheat samples and derived products (flour, semolina and bran) with contamination levels ranging from 0.13 to 37.42 lg/kg