Penicillium expansum, one of the patulin producing fungi that causes decay on apple, is recognized as the main source of patulin contamination on apple and apple products. The widely used method for patulin analysis in apple juice is liquid-liquid extraction with ethyl acetate followed by HPLC-UV or LC-MS detection. Previous studies have shown cyclodextrin polymers to exhibit favorable adsorption properties for several classes of small organic molecules, including patulin in apple juice. In this study, an insoluble polymer composed of cyclodextrin crosslinked with 4, 4'-methylenebis(phenyl isocyanate) was synthesized for use in the solid phase extraction of patulin from apple juice. Conditions investigated for this method were solvent for column conditioning, sample volume to load patulin on the column, solvent for washing, and solvent and volume for patulin elution and optimized recovery of patulin from the column. At the optimized conditions, the recovery and relative standard deviation (RSD) of patulin from apple juice spiked at 10, 20, 50, 80 and 100 ng mL-1 were 78 and 20%, 71 and 13%, 78 and 17%, 71 and 7.1%, 67 and 2.9%, respectively. Limit of quantitation (LOQ) of patulin in apple juice by this method was 10 ng mL-1.
Trichothecenes are mycotoxins produced by various species of fungi, including Fusarium species. They contaminate important crops such as wheat and corn, and cause toxicity in animals and humans mainly by inhibition of protein synthesis. In this study, we performed genome-wide screening of genes that play roles in protection of Saccharomyces cerevisiae against A-type trichothecene, T-2 toxin, and D-type trichothecene, verrucarin A. Although a distinct difference exists in the structures of these two trichothecenes, these toxins inhibited the growth of the mutant strains of S. cerevisiae with common disrupted genes. The genes whose deletion conferred high sensitivity to these trichothecenes were considered as trichothecene resistance genes, and they included the ones encoding ABC transporter protein, enzymes for ergosterol biosynthesis, vacuolar H+-ATPase, and others. These results possibly lead to the construction of a yeast detection system with improved sensitivity to trichothecenes.
The central region of the trichothecene gene cluster contains two pathway and two regulatory genes (four Tri genes) that are necessary for forming the trichothecene skeleton. One of these genes, Tri4, encodes a multifunctional cytochrome P 450 monooxygenase, and the expression of this gene is highly induced under trichothecene-inducing conditions. Apart from this gene cluster, Tri101 occurs as a single non-cluster gene, and the product of this gene contributes to the function of self-protection in Fusarium graminearum. The promoter activity of these Tri genes was compared with that of TEF1α (a highly expressed translation elongation factor 1-alpha) gene under toxin-inducing conditions by using the β-glucuronidase (GUS) gene as a reporter. The Tri101 promoter- and TEF1α promoter-reporter fusions integrated at the end of the gene cluster showed activities that were similar to those of the original loci with respect to the timing and the level of GUS protein accumulation. However, as opposed to the normal pattern of Tri4 transcription from the original locus, no GUS activity was detected when the Tri4 promoter was placed at the end of the gene cluster. Thus, we can conclude that the position of the promoter in the gene cluster is important for native transcriptional regulation of Tri4 in trichothecene biosynthesis.
Seeds of two okra accessions and 17 samples of sesame seeds were examined for contamination by Fusarium species. Altogether, 37 Fusarium isolates were obtained from the two crops: 6 isolates from two okra accessions and 31 isolates from 10 sesame samples. The isolates belonged to three species: F. oxysporum, F. semitectum and F. verticillioides. All isolates from okra were identified as F. semitectum while the three species of Fusarium occurred in sesame. Six randomly selected isolates from the two crops were screened for their ability to produce mycotoxins in ofada rice culture and the crude extracts of the mycotoxins were tested on week-old Clarias gariepinus fingerlings. Six metabolites were produced by the isolates in ofada rice: equisetin (EQUS), fumonisin B1 (FB1), FB2, methyl-equisetin (M-EQUS), moniliformin (MON) and zearalenone (ZEA). All isolates produced EQUS at concentrations ranging 454-29,983 μg/kg. All isolates except F. semitectum BUFC 041 and F. oxysporum BUFC 024 produced MON and ZEA, while three F. semitectum isolates produced M-EQUS. Only F. verticillioides isolates produced fumonisins. There was evidence of variation within species obtained from both crops based on toxin profile and level of toxin produced. The culture extracts of all isolates induced 100% lethality to C. gariepinus fingerlings except for extracts of F. semitectum BUFC 041 which killed only 62.2% of the fingerlings, possibly due to the absence of MON. Our data suggest that okra and sesame may be potential sources of toxigenic Fusarium.
Mycotoxins are secondary metabolites produced by several fungal species and represent a great concern for the economical and healthy implications on food and feed chain. Cereals are the primary source of human diet, wheat being the third most produced grain worldwide. Although Fusarium still represents the main source of mycotoxin contamination of wheat, in recent years, due also to evident climate changes that influence agricultural environment, other mycotoxingenic fungi have been pointed out as important wheat contaminants. Among these a disease called “Black Point”, caused by Alternaria spp., is increasing it importance as re-emerging risk. Diseases and mycotoxins (alternariol, altenuene, alternariol methyl-ether and tenuazolic acid) associated with Alternaria infection have been reported in several countries suggesting to deepen the knowledge about this genus. This paper summarizes the recent .ndings on wheat contamination by Alternaria spp and their related toxins.
Okinawa prefecture is located south of the Japanese Islands, and causes of natural toxin food poisoning are different from other parts of our country due to its subtropical climate and natural environments. During ten years from 2001 to 2010, 24% of food poisoning incidences occurring in Okinawa were caused by natural toxins, which comprises of animal origins in as much as 91% of the cases. Most of the animal toxins are ciguatera toxins produced by seafood that inhabit the coral reef. In addition to ciguatera, incidences of food poisoning are also attributed to cucurbitacin-containing plants in the family Cucurbitaceae, tropical mushroom Chlorophyllum molybdites, and hallucinogenic mushrooms.
Tofuyo is an indigenous fermented tofu in Okinawa, Japan. Red furu was brought to Okinawa from China, and was introduced during the period of the Ryukyu dynasty, around the 18th century. Because the product was brined and had strong taste, it was initially unpalatable to the Okinawan people in its original form. Therefore, it was re-created using awamori instead of salt by cooks in the dynasty for a more palatable taste that was milder in .avor. Microorganisms commonly used to the manufacturing of tofuyo belong to the genus Monascus and Aspergillus oryzae. The main components forming the body of tofuyo consist of basic subunit of glycinin and other polypeptides (Mr. 10,500-15,000). They are related to the textures of tofuyo. The soybean proteins were digested into amino acids and peptides during maturation. The amount of free glutamic acid and aspartic acid greatly related to good taste of the product. Some of the peptides (IFL and WL) liberated from tofuyo inhibited the angiotensin I-converting enzyme activity that produces the vasopressor peptide. Both IFL and WL were highly preserved after treatment with gastrointestinal proteases. Furthermore, the systolic blood pressure of rats in the tofuyo group was significantly lower than that in control group. Therefore, tofuyo was expected to have an antihypertensive effect.
Monitoring of marine toxins in shellfish and fin fish are carried out mainly by mouse bioassays in Japan (MBA). To achieve higher specificity, sensitivity, accuracy, and rapidity, however, the world is shifting to alternative methods, as exemplified by the EU decision to abolish MBA by the end of 2014. Lipophilic shellfish toxins would be determined by LC-MS and paralytic shellfish toxins by HPLC methods. Ciguatera toxins in the Pacific could be monitored by the LC-MS method we are developing. A promising aspect of receptor binding assays is shown for Caribbean type ciguatoxins. The severe poisoning due to ingestion of Scarus ovifrons does not involve palytoxin and should be regarded as rhabdomyolysis known to occur worldwide after eating various aquatic animals.
Marker genes are required for the genetic manipulation of living organisms. For selecting transformants after the process of transformation, two types of marker genes are employed: one for the positive selection of the transformants carrying the positive selection marker gene and another for the negative selection of the transformants lacking the negative selection marker gene. Here we have summarized the current advancements in genome engineering of Fusarium species. We have focused on introducing arbitrary mutations by applying both positive and negative selection approaches that result in the exclusion of the marker genes from the target genome. We have discussed the advantages and necessity of using this technique in studies on the regulation of biosynthetic gene expression in mycotoxin production.
An international symposium on mycotoxin entitled “WMF meets IUPAC 2012” (7th Conference of the World Mycotoxin Forum and 13th IUPAC International Symposium on Mycotoxins and Phycotoxins) was held in Rotterdam (the Netherlands) in November 2012. Many people from all over the world participated to this symposium, and lectures of more than 100 speakers and posters of more than 210 researchers were presented throughout the conference, resulting in active and fruitful discussions among the participants. This report shows a summary of the symposium with citation of several interesting topics provided there.