RESUMEN
Propylthiol functionalized SBA-15 silica was investigated to detoxify aqueous solutions contaminated with the regulated mycotoxin patulin. Micelle templated silicas with a specific pore size were synthetically modified to possess propylthiol groups, a functional group known to form Michael reaction products with the conjugated double bond system of patulin. BET surface area analysis indicated the propylthiol functionalized SBA-15 possesses channels with the pore size of 5.4 nm and a surface area of 345 m(2)g(-1). Elemental analysis indicates the silicon/sulfur ratio to be 10:1, inferring one propylthiol substituent for every ten silica residues. The propylthiol modified SBA-15 was effective at significantly reducing high levels of patulin from aqueous solutions (pH 7.0) in batch sorption assays at room temperature. The material was less effective at lower pH; however heating low pH solutions and apple juice to 60 °C in the presence of propylthiol functionalized SBA-15 significantly reduced the levels of patulin in contaminated samples. Composite molecular models developed by semi-empirical PM3 and empirical force field methods support patulin permeation through the mesoporous channels of propylthiol functionalized SBA-15. Density functional study at the B3LYP/6-31G(d,p) level predicts the proposed patulin adducts formed by reaction with the thiol residues exhibit less electrophilic properties than patulin. It is demonstrated the use of propylthiol functionalized SBA-15 is a viable approach to reduce patulin levels in aqueous solutions, including contaminated apple juice.
Asunto(s)
Patulina/aislamiento & purificación , Dióxido de Silicio/química , Compuestos de Sulfhidrilo/química , Contaminantes Químicos del Agua/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Modelos Moleculares , Patulina/toxicidad , Contaminantes Químicos del Agua/toxicidadRESUMEN
The presence of deoxynivalenol (DON) in cereal-based baby food, a primary source of the first solid food for infants, was studied in order to develop a method to detect its presence at low concentrations. DON, produced primarily by Fusarium graminearum, is commonly isolated from grains and feed around the world and affects both animal and human health, producing diarrhea, vomiting, gastrointestinal inflammation, and immunomodulation. An aqueous extract of infant cereal was cleaned by means of an immunoaffinity chromatography column. After the eluate was evaporated and redissolved, DON was determined by high-pressure liquid chromatography-UV. The level of quantification for DON was 10 ppb for three types of infant cereal (mixed, barley, and oatmeal); the level of detection was 5 ppb. The protocol we have developed can measure DON between 10 to 500 ppb. An advisory level of 1 ppm for wheat products has been established by the U.S. Food and Drug Administration; however, the European Communities (EC) regulations have been set at 200 ppb for cereal-based foods for infants. Only 1 of 52 samples of barley-, mixed-, or oat-based infant cereal purchased in 2008 and 2009 in the United States exceeded the European standard.
Asunto(s)
Grano Comestible/química , Contaminación de Alimentos/análisis , Alimentos Infantiles/análisis , Tricotecenos/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Seguridad de Productos para el Consumidor , Grano Comestible/microbiología , Microbiología de Alimentos , Humanos , Lactante , Medición de Riesgo , Tricotecenos/análisisRESUMEN
The genes for the patulin biosynthetic pathway are most likely arranged in a cluster, as is often the case for other mycotoxins. With this in mind, GeneWalking has been performed to identify genes both upstream and downstream of the isoepoxydon dehydrogenase (idh) gene. A gene present in Penicillium griseofulvum NRRL 2159A had high sequence homology to the isoamyl alcohol oxidase (iao) gene and was detected downstream of the idh gene and in the same orientation. By virtue of the presence of a signal peptide sequence, the newly identified gene coded for a secreted protein with an FAD-binding domain and potential for N-glycosylation. An open reading frame consisted of 1946 nucleotides, containing four putative introns and encoding a 22 amino acid signal peptide. The 571 amino acid mature protein contained nine cysteine residues and had 11 potential N-linked glycosylation sites. Searches using GenBank indicated that Aspergillus terreus, A. oryzae, A. fumigatus, and Gibberella zeae contain genes coding for a putative isoamyl alcohol oxidase. When the translated query was compared with the translated database, the highest scores were seen with A. clavatus (E value of 0.00), A. fumigatus (E value of 8e(-142)), and A. oryzae and A. terreus (each having an E value of 2e(-141)). Reverse transcription-polymerase chain reaction analysis confirmed that the iao gene was transcribed. The amplified products were sequenced for confirmation of their identities. This is the first report of an isoamyl alcohol oxidase gene in a species of the genus Penicillium.
Asunto(s)
Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Patulina/biosíntesis , Penicillium/enzimología , Penicillium/genética , Pentanoles/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Paseo de Cromosoma/métodos , Cartilla de ADN , Datos de Secuencia Molecular , Oxidorreductasas/química , Penicillium/clasificación , Reacción en Cadena de la Polimerasa/métodos , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de SecuenciaRESUMEN
Certain species of Penicillium have been reported to produce the mycotoxin patulin, and research was undertaken to identify these with the use of oligonucleotide primer pairs. Species examined were found in food, plants, and soil and were reported to produce patulin. Penicillium expansum is the most commonly detected species linked to the presence of patulin in apple juice. At least 10 different enzymes are involved in the patulin biosynthetic pathway, including the isoepoxydon dehydrogenase (idh) gene. Based on nucleotide sequences previously determined for the idh gene in Penicillium species, PCR primers were designed for the species-specific detection of patulin-producing species. The 5' primers were based on differences in the second intron of the idh gene. To ensure that the primer pairs produced a PCR product restricted to the species for which it was designed, and not to unrelated species, all of the primer pairs were tested against all of the Penicillium species. With one exception, it was possible to detect a reaction only with the organism of interest. The primer pair for Penicillium griseofulvum also amplified DNA from Penicillium dipodomyicola, a closely related species; however, it was possible to distinguish between these two species by doing a second amplification, with a different primer pair specific only for P. dipodomyicola. Consequently, with different primer sets, it was possible to identify individual patulin-producing species of Penicillium.
Asunto(s)
Bebidas , ADN de Hongos/análisis , Contaminación de Alimentos/análisis , Patulina/biosíntesis , Penicillium/metabolismo , Bebidas/análisis , Bebidas/microbiología , Microbiología de Alimentos , Amplificación de Genes , Humanos , Malus , Patulina/clasificación , Patulina/aislamiento & purificación , Penicillium/clasificación , Alineación de Secuencia , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
Interest in species of the genus Penicillium is related to their ability to produce the mycotoxin patulin and to cause spoilage of fruit products worldwide. The sequence of the isoepoxydon dehydrogenase (idh) gene, a gene in the patulin biosynthetic pathway, was determined for 28 strains representing 12 different Penicillium species known to produce the mycotoxin patulin. Isolates of Penicillium carneum, Penicillium clavigerum, Penicillium concentricum, Penicillium coprobium, Penicillium dipodomyicola, Penicillium expansum, Penicillium gladioli, Penicillium glandicola, Penicillium griseofulvum, Penicillium paneum, Penicillium sclerotigenum and Penicillium vulpinum were compared. Primer pairs for DNA amplification and sequencing were designed from the P. griseofulvum idh gene (GenBank AF006680). The two introns present were removed from the nucleotide sequences, which were translated to produce the IDH sequences of the 12 species for comparison. Phylogenetic relationships among the species were determined from rDNA (ITS1, 5.8 S, ITS2 and partial sequence of 28S rDNA) and from the idh nucleotide sequences minus the two introns. Maximum parsimony analysis showed trees based on rDNA and idh sequences to be congruent. It is anticipated that the genetic information obtained in the present study will aid in the design of probes, specific for patulin biosynthetic pathway genes, to identify the presence of these mycotoxigenic fungi.
Asunto(s)
Proteínas Fúngicas/genética , Oxidorreductasas/genética , Patulina/biosíntesis , Penicillium/enzimología , Penicillium/genética , Secuencia de Aminoácidos , Vías Biosintéticas/genética , ADN de Hongos/química , ADN de Hongos/genética , ADN de Hongos/aislamiento & purificación , ADN Ribosómico/química , ADN Espaciador Ribosómico/química , ADN Espaciador Ribosómico/genética , Genes de ARNr/genética , Intrones/genética , Datos de Secuencia Molecular , Filogenia , ARN de Hongos/genética , ARN Ribosómico 28S/genética , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
Nucleotide sequences of the isoepoxydon dehydrogenase gene (idh) for eight strains of Byssochlamys nivea were determined by constructing GenomeWalker libraries. A striking finding was that all eight strains of B. nivea examined had identical nucleotide sequences, including those of the two introns present. The length of intron 2 was nearly three times the size of introns in strains of Penicillium expansum and P. griseofulvum, but intron 1 was comparable in size to the number of nucleotides present in introns 1 and 2 of P. expansum and P. griseofulvum. A high degree of amino acid homology (88%) existed for the idh genes of the strains of B. nivea when compared with sequences of P. expansum and P. griseofulvum. There were many nucleotide differences present, but they did not affect the amino acid sequence because they were present in the third position. The identity of the B. nivea isolates was confirmed by sequencing the ITS/partial LSU (28 S) rDNA genes. Four B. nivea strains were analysed for production of patulin, a mycotoxin found primarily in apple juice and other fruit products. The B. nivea strains produced patulin in amounts comparable to P. expansum strains. Interest in the genus Byssochlamys is related to the ability of its ascospores to survive pasteurization and cause spoilage of heat-processed fruit products worldwide.
Asunto(s)
Eurotiales/enzimología , Oxidorreductasas/química , Oxidorreductasas/genética , Patulina/biosíntesis , Penicillium/enzimología , Homología de Secuencia de Aminoácido , Secuencia de Aminoácidos , ADN Ribosómico , Eurotiales/genética , Eurotiales/metabolismo , Datos de Secuencia Molecular , Penicillium/genética , ARN Ribosómico 28S/genética , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
Purified DNA from isolates of Penicillium griseofulvum and P. expansum was used as a template to amplify a 600-bp fragment of the isoepoxydon dehydrogenase (idh) gene of the patulin biosynthetic pathway. Primer pairs designed from the P. griseofulvum gene to amplify specific regions of the idh gene yielded similar-sized bands for all strains. Asymmetrical amplification produced DNA products for sequencing and DNA sequences were translated to produce the corresponding amino acid sequences. After removal of two introns present in the region sequenced, amino acid sequences were compared. There were 12 amino acid differences between P. expansum and P. griseofulvum in the coding region. The differences correlated with the amount of patulin previously produced in culture, with strains of P. griseofulvum producing the greatest amounts of patulin.
Asunto(s)
Genes Fúngicos , Oxidorreductasas/genética , Patulina/biosíntesis , Penicillium/genética , Penicillium/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , ADN de Hongos/genética , ADN de Hongos/aislamiento & purificación , Intrones , Datos de Secuencia Molecular , Oxidorreductasas/metabolismo , Penicillium/enzimología , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Especificidad de la EspecieRESUMEN
The aim of this study was to evaluate different species of Penicillium to identify those which have the potential to produce the greatest amount of the mycotoxin, patulin. Additionally, six different culture media were compared to determine maximum patulin production. Eleven different strains of Penicillium species were selected because they had previously been reported to be producers of patulin. The strains included Penicillium expansum, Penicillium griseofulvum (formerly Penicillium urticae), Penicillium clavigerum, and Penicillium coprobium and a recent Penicillium sp. isolated from an apple. Cultures were grown in duplicate in three different liquid media: potato dextrose, malt extract, and glucose/yeast extract/peptone, both with and without manganese supplementation. Patulin production was compared at 24, 48, 72, and 96 h. Variability in patulin production occurred among the different species, growth media used, and time of incubation. All three of the P. griseofulvum isolates were the highest producers of patulin at 96 h. For most of the strains, potato dextrose broth supplemented with manganese was optimal for maximum production of patulin. Although P. expansum is frequently cited as the most likely source of patulin in apple juice, certain other Penicillium species are capable of producing more patulin than strains of P. expansum. The apple juice industry should be alert to the possibility that Penicillium species other than P. expansum can be responsible for the occurrence of patulin.
Asunto(s)
Medios de Cultivo/química , Frutas , Patulina/biosíntesis , Penicillium/metabolismo , Contaminación de Alimentos/análisis , Contaminación de Alimentos/prevención & control , Microbiología de Alimentos , Frutas/química , Frutas/microbiología , Patulina/análisis , Especificidad de la Especie , Factores de TiempoRESUMEN
Fumonisins, a family of mycotoxins produced by Fusarium verticillioides (synonym Fusarium moniliforme Sheldon) and F. proliferatum, have been associated with various deleterious effects in different animal species. Serological, hematological and pathological effects and mortality have previously been observed in broiler chicks fed F. proliferatum culture material containing known concentrations of fumonisin, moniliformin and beauvericin. Turkey peripheral blood lymphocytes were exposed in vitro for 72 hours to fumonisin B1 (FB1), fumonisin B2 (FB2), hydrolyzed fumonisin B1 (HFB1), moniliformin and tricarballylic acid (TCA) (0.01-25 microg/ml). A decrease in cell proliferation, as determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] bioassay, occurred in the order: FB2 > FB1 > HFB1, with IC50 = 0.6 microM, 1 microM and 10 microM, respectively. Internucleosomal DNA fragmentation and morphological features characteristic of apoptosis were observed following exposure to fumonisin B1 and beauvericin; cytoplasmic condensation and membrane blebbing were seen by light microscopy. Tricarballylic acid and moniliformin did not interfere with cell proliferation. Results suggested that fumonisin B1 and beauvericin may affect immune functions by suppressing proliferation and inducing apoptosis of lymphocytes.