Tannase produced optimally on an agroresidue by an Aspergillus niger isolate under submerged fermentation immobilized on sodium alginate beads with 93.6% efficiency was applied for tannin removal from myrobalan/aonla (Phyllanthus emblica) juice. The pH and temperature optima of the immobilized enzyme were found to be 5.4 and 40°C while the corresponding values of the soluble enzyme were 5.8 and 35°C. Maximum tannin removal of 73.6% was obtained at 40°C and 150 rpm in 180 min with 36.6 U/ml of immobilized enzyme while the same amount of the soluble enzyme removed 45.2% of tannin at 37°C and 150 rpm in the same time period. The immobilized beads could be used repeatedly till 7th cycle with 77% efficiency. When preserved at 6°C the beads retained 71.7% of enzyme activity after 60 days. Reduction in vitamin C content, which is responsible for antioxidant property of the fruit, was minimum at only 2% during the treatment.
Aspergillus niger; Immobilized tannase; Myrobalan; Submerged fermentation; Sodium alginate
Individually Andrographis paniculata Nees. (Acanthaceae), Phyllanthus niruri Linn.(Euphorbiaceae) and Phyllanthus emblica Linn. single plant extracts have been reported to have hepatoprotective activity. However, literature survey shows that no sufficient scientific data has been publish on pharmacological evaluation of these plants in combined form.
Hepatoprotective activity of the polyherbal hepatoprotaective formulation (PHF)-containing spray-dried aqueous extracts of Andrographis paniculata Nees. (Acanthaceae), Phyllanthus niruri Linn. (Euphorbiaceae) and Phyllanthus emblica Linn. (Euphorbiaceae), was screened against paracetamol, carbon tetrachloride (CCl4), and ethanol-induced hepatic damage in rats. PHF was evaluated by measuring levels of serum marker enzymes like SGOT, SGPT, ALP, direct bilirubin (DB), and lactate dehydrogenase (LDH). The histological studies were also studied support the biochemical parameters. Silymarin was used as standard drug.
Administration of PHF (100 and 200 mg/kg p.o.) significantly inhibited paracetamol, CCl4 and ethanol-induced elevation levels of SGPT, SGOT, ALP, DB and LDH. A comparative histopathological study of liver exhibited almost normal architecture as compared to toxicant group.
Results suggests that the hepatoprotective effects of PHF might be useful for liver protection due to combined action of all plant extracts along with their phytoconstituents.
Andrographis paniculata Nees; carbon tetrachloride; hepatoprotective activity; Marker enzymes; paracetamol; Phyllanthus niruri Linn
Post-treatment of the indomethacin induced ulcerated rats at the optimal dose of 100 mg/kg body-wt. orally for 7 consecutive days with the lyophilized aqueous extract of the fruits ofPhyllanthus emblica L. syn.Emblica officinalis Gaertn. (Euphorbiaceae) exhibited highly significant (p<0.001) enhancement of secretion of catalase, reduced glutathione and decrease in malonyldialdehyde (MDA). Furthermore, the gross morphological observation and highly significant (p<0.001) decrease of ulcer index (81.43%) indicated healing effect of the extract on gastric ulcer.
Antioxidant; antiulcer; healing; Phyllanthus emblica
Emblica (Phyllanthus emblica L.), an euphorbiaceous plant, is widely distributed in subtropical and tropical areas of India, China and Indonesia. The fruits possess antimicrobial, antioxidant, anti-inflammatory, analgesic and antipyretic properties. In the current article a new, simple, sensitive, selective, precise, and robust high-performance thin-layer chromatographic (HPTLC) method was developed and validated for the determination of gallic acid in dried fruit powder of Phyllanthus emblica.
Materials and Methods:
The quantitative determination of gallic acid was performed on TLC aluminium plates pre-coated with silica gel 60F-254 as the stationary phase. The linear ascending development was carried out in a twin trough glass chamber saturated with a mobile phase consisting of toluene: ethyl acetate: formic acid: methanol (3:3:0.8:0.2) at room temperature (25 ± 2°C). Camag TLC scanner III was used for spectrodensitometric scanning and analysis, in the absorbance mode, at 278 nm.
The linear regression analysis data for the calibration plots showed good linear relationship with r2 = 0.99977 in the concentration range of 40 – 240 ng spot—1, with respect to the peak area. According to the guidelines of the International Conference on Harmonization (ICH), the method was validated for precision, accuracy, and recovery.
Statistical analysis of the data showed that the method was reproducible and selective for the estimation of gallic acid.
Phyllanthus emblica; HPTLC fingerprinting; gallic acid
Kaumarbhritya a branch of Asthanga Ayurveda deals with neonatal, infant and child health care. Multicentric studies conducted in various developed and developing countries have indicated that Infant Mortality Rate (I.M.R.) is very high in developing countries, and infection has been observed as the major cause. Immune system in neonates is not yet fully functional. Bala compound having the ingredients of
Atibala (Abutilon indicum Linn), Amalaki (Emblica officinalis Linn), Vidanga (Emblica ribes burn), Guduchi (Tinospora cordifolia Welld Miers), Pippali (Piperlongum linn), Yashtimadhu (Glycyrrhiza glabra Linn), Shankhapuspi (Convolvulus pluricaulis Chois ), Vacha (Acorus calamus Linn), Musta (Cyperus rotundus Linn) and Ativisha (Aconitum heterophyllum wall) are Medhya as well as Rasayana drugs mention in Ayurvedic classics. ‘Bala compound” was tried in infants in the form of oral drops for a period of six months and result was assessed for serum immuoglobulins IgG, IgM, IgA for three months of interval of two follow ups (i.e., third and six month of infant). There is significant increase of immunoglobulins observed after six months administration of ‘Bala compoumd”
In this study, the RAPD (Random Amplified Polymorphic DNA) technique was employed for determination of the components in an Ayurvedic herbal prescription, Rasayana Churna. One-hundred-and-twenty decamer oligonucleotide primers were screened in the RAPD analysis to identify three Ayurvedic medicines, dried stem of Tinospora cordifolia, dried fruit of Emblica officinalis and dried fruit of Tribulus terestris, the Ayurvedic prescription. Primer OPC-6 simultaneously generated three distinct amplicons, each specific to one component. The marker with 600 bp is specific to Tinospora cordifolia; the marker 500 bp is specific to Emblica officinalis and the remaining marker >1000 bp was present in Tribulus terestris. Presence of three herbal medicines was determined when RAPD reaction with OPC-6 was performed. The technique was proved to contribute to the identification of components in Ayurvedic herbal preparation and thus helping to serve as a complementary tool for quality control.
DNA fingerprinting; herbal medicine; RAPD; standardization
The aim of this study was to evaluate fungi and contamination levels of aflatoxin B1, ochratoxin A, fumonisin B1, and zearalenone in raw materials and finished feed intended for sows at different reproductive stages. Total fungi, Aspergillus, Penicillium, and Fusarium species occurrence, were examined. Aspergillus flavus, A. niger aggregate spp., and F. verticillioides were the prevalent species. Fungal counts exceeded the levels proposed as feed hygienic quality limits (1 × 104 colony forming units) at all reproductive stages. Aflatoxin B1, ochratoxin A, fumonisin B1, and zearalenone were detected by high-pressure liquid chromatography. Aflatoxin levels in 80% samples of finished sow feeds were over the permitted levels of 0.02 μg g−1
(mean 228.2 ± 95 μg Kg−1). Fumonisin B1 was detected in all tested raw materials at levels that varied from 50.3 to 1137.64 μg Kg−1 and finished feed samples at levels that ranged from 99.8 to 512.4 μg Kg−1. Aflatoxin B1, zearalenone, and ochratoxin A were not detected in raw materials. All finished feeds were negative for zearalenone contamination whereas all nonpregnant gilt samples were contaminated with low OTA levels (mean 0.259 ± 0.123). This fact requires periodic monitoring to prevent the occurrence of mycotoxicosis in animal production, to reduce the economic losses, and to minimize hazards to human health.
Pharmacognostical and preliminary phytochemical studies of Triphala churnam were carried out. The churnam of triphala consists of equal quantities of deseeded fruits of Terminalia chebula, Terminalia bellerica and Emblica officinalis. Triphala is exclusively used in more than 200 drug formulations in Indian system of Medicine. The present study involved the pharmacognostical evaluation of Triphala, in which morphological and powder microscopical characters were established. In addition, physico-chemical parameters such as ash values viz, total ash (10.21± 0.42), acid insoluble ash (2.54 ± 0.06), water-soluble ash (5.46±0.24) and sulphated ash (13.12 ± 0.63), extractive values viz, alcohol soluble extractive (11.20±0.18)) and water-soluble extractive (52.56±2.04), fluorescent analysis and microchmical tests were determined. The preliminary phytochemical study revealed the presence of carbohydrates, reducing sugar and tannins in aqueous extract and carbohydrates, flavonoids and tannins in alcoholic extract. This standardization would be very much helpful for the identification of Triphala churnam to differentiate from other powdered sources.
Production of the harmful carcinogenic aflatoxins by Aspergillus parasiticus and Aspergillus flavus has been postulated to be a mechanism to relieve oxidative stress. The msnA gene of A. parasiticus and A. flavus is the ortholog of Saccharomyces cerevisiae MSN2 that is associated with multi-stress response. Compared to wild type strains, the msnA deletion (∆msnA) strains of A. parasiticus and A. flavus exhibited retarded colony growth with increased conidiation. The ∆msnA strains also produced slightly higher amounts of aflatoxins and elevated amounts of kojic acid on mixed cereal medium. Microarray assays showed that expression of genes encoding oxidative stress defense enzymes, i.e., superoxide dismutase, catalase, and cytochrome c peroxidase in A. parasiticus ∆msnA, and the catalase A gene in A. flavus ∆msnA, was up-regulated. Both A. parasiticus and A. flavus ∆msnA strains produced higher levels of reactive oxygen species (ROS), and ROS production of A. flavus msnA addback strains was decreased to levels comparable to that of the wild type A. flavus. The msnA gene appears to be required for the maintenance of the normal oxidative state. The impairment of msnA resulted in the aforementioned changes, which might be used to combat the increased oxidative stress in the cells.
Aspergillus; aflatoxin; kojic acid; oxidative stress; development
With an initial microbial level of ca. 107 microorganisms per g of Ivory Coast cacao beans, 5 kGy of gamma radiation under an atmosphere of air reduced the microflora per g by 2.49 and 3.03 logs at temperatures of 35 and 50°C, respectively. Bahia cacao beans were artificially contaminated with dried spores of Aspergillus flavus and Penicillium citrinum, giving initial fungal levels of 1.9 × 104 and 1.4 × 103 spores per g of whole Bahia cacao beans, respectively. The average D10 values for A. flavus and P. citrinum spores on Bahia cacao beans were 0.66 and 0.88 kGy, respectively.
Single-spore colonies of Aspergillus flavus and Aspergillus parasiticus, grown for 4 to 5 days at 25 degrees C on a coconut extract agar containing sodium desoxycholate as a growth inhibitor, produced aflatoxin, readily detectable as blue fluorescent zones under long-wave (365 nm) UV light. Over 100 colonies per standard petri dish were scored for aflatoxin production by this procedure. Progeny from some strains remained consistently stable for toxin production after repeated subculture, whereas instability for toxin synthesis was revealed among progeny from other strains. Spore color markers were used to rule out cross-contamination in monitoring strains. A yellow-spored and nontoxigenic strain of A. flavus, reported previously to produce aflatoxin in response to cycloheximide treatment, proved to be toxin negative even after repeated exposure to cycloheximide. Extended series of progeny from another strain of A. flavus and from a strain of A. parasiticus were each compared by this plating procedure and by fluorometric analysis for aflatoxin when grown in a coconut extract broth. Both of these strains showed variation for toxin synthesis among their respective progeny, and specific progeny showed a good correlation for aflatoxin synthesis when examined by the two procedures.
To evaluate the potential for mycotoxin production by molds in dried beans, the mold flora of 114 samples was determined both before and after surface disinfection of the beans with 5% NaOCl. Surface disinfection substantially reduced mold incidence, indicating that contamination was mainly on the surface. The flora, both before and after disinfection, was dominated by species of the Aspergillus glaucus group, the toxicogenic species A. ochraceus, Penicillium cyclopium, and P. viridicatum, and species of Alternaria, Cladosporium, and Fusarium. The toxicogenic species Aspergillus flavis, A. versicolor, Penicillium citrinum, P. expansum, P. islandicum, and P. urticae were encountered less frequently. Of 209 species of Aspergillus and Penicillium screened for mycotoxin production on sterile rice substrate, 114 produced one or more of the following mycotoxins: A. flavus, aflatoxins; A. ochraceus, ochratoxins; A. nidulans, A. unguis, and A. versicolor, sterigmatocystin; P. cyclopium, penicillic acid; P. citrinum and P. viridicatum, citrinin; P. urticae, patulin and griseofulvin. Sterigmatocystin production by A. unguis is reported for the first time.
Diabetes prevalence is increasing worldwide. More than 800,000 Australians live with diabetes, and there are stark inequities in prevalence and clinical outcomes among Indigenous people and low socio-economic groups.
This paper focuses on food security issues experienced by low-income earners living with type 2 diabetes in Perth, Western Australia. The results presented here are part of a broader qualitative study exploring the impact of socioeconomic disadvantage on diabetes.
Data was collected through focus groups and semistructured interviews conducted from October 2008 to November 2009. The sample, comprising 38 participants ( Indigenous and non-Indigenous), was recruited from areas with high indices of socio-economic disadvantage in Perth. Deductive data analysis identified categories from an existing conceptual framework for the relationship between socio-economic position and diabetes health outcomes, while an inductive approach was adopted to identify new themes.
Participants had a good understanding of their dietary requirements. However, access to healthy food was not always realised, as many participants depended on others for food provision and meal preparation and had little control over their diets. Furthermore, the majority struggled to accommodate the price of healthy food within a limited budget.
In this study, low-income earners living with diabetes faced food security issues. Participants reported cost barriers, but also physical barriers relating to functional limitations and lack of transport. This study highlights that the socioeconomic circumstances in which vulnerable populations experience their disease need to be understood and addressed in order to reduce the inequities surrounding diabetes outcomes.
Diabetes; food security; access; disadvantage; low income; Indigenous health
The effect of 12 surfactants on aflatoxin production, growth, and conidial germination by the fungus Aspergillus flavus is reported. Five nonionic surfactants, Triton X-100, Tergitol NP-7, Tergitol NP-10, polyoxyethylene (POE) 10 lauryl ether, and Latron AG-98, reduced aflatoxin production by 96 to 99% at 1% (wt/vol). Colony growth was restricted by the five nonionic surfactants at this concentration. Aflatoxin production was inhibited 31 to 53% by lower concentrations of Triton X-100 (0.001 to 0.0001%) at which colony growth was not affected. Triton X-301, a POE-derived anionic surfactant, had an effect on colony growth and aflatoxin production similar to that of the five POE-derived nonionic surfactants. Sodium dodecyl sulfate (SDS), an anionic surfactant, and dodecyltrimethylammonium bromide, a cationic surfactant, suppressed conidial germination at 1% (wt/vol). SDS had no effect on aflatoxin production or colony growth at 0.001%. The degree of aflatoxin inhibition by a surfactant appears to be a function of the length of the hydrophobic and hydrophilic chains of POE-derived surfactants.
The aqueous and successive extracts of the fruit pulp of Emblica officinalis and fresh leaves and stems of Ocimum sanctum were prepared and evaluated for antimicrobial activity. The successive extracts such as petroleum ether,chloroform, ethyl acetate and methanol were prepared by successive solvent extraction method and aqueous extract by maceration process and screened for antimicrobial activity against gram positive bacteria Staphylococcus aureus, gram negative bacteria E.coli and fungal strains of Candida species by using agar cup plate method. The extracts showed different degree of activity against pathogenic microbes. The results obtained were compared with standard drugs Amoxicillin (10μg) and Amphotericin B(10μg). The methanolic extract of Emblica officinalis was found to be more effective than the leaf and stem extracts of Ocimum sanctum in inhibiting all the microbial strains.
Samples of freshly harvested and remoistened corn, of various moisture contents, were stored at different temperatures; analyses for aflatoxin content were made periodically. At moisture levels above 17.5% and at temperatures of 24 C or warmer, aflatoxins were formed by Aspergillus flavus present in the original epiphytic mycoflora. Remoistened dried corn was subject to more rapid fungal deterioration and aflatoxin formation than freshly harvested corn. Screening of the fungi present in the corn revealed aflatoxin production only by A. flavus. The toxigenic strains produced only aflatoxins B1 and B2.
AIM: To examine the growth inhibitory effects of Phyllanthus emblica (P. emblica) and Terminalia bellerica (T. bellerica) extracts on human hepatocellular carcinoma (HepG2), and lung carcinoma (A549) cells and their synergistic effect with doxorubicin or cisplatin.
METHODS: HepG2 and A549 cells were treated with P. emblica and T. bellerica extracts either alone or in combination with doxorubicin or cisplatin and effects on cell growth were determined using the sulforhodamine B (SRB) assay. The isobologram and combination index (CI) method of Chou-Talalay were used to evaluate interactions between plant extracts and drugs.
RESULTS: P. emblica and T. bellerica extracts demonstrated growth inhibitory activity, with a certain degree of selectivity against the two cancer cell lines tested. Synergistic effects (CI < 1) for P. emblica/doxorubicin or cisplatin at different dose levels were demonstrated in A549 and HepG2 cells. The T. bellerica/cisplatin or doxorubicin also showed synergistic effects in A549 and HepG2 cells. In some instances, the combinations resulted in antagonistic effects. The dose reduction level was different and specific to each combination and cell line.
CONCLUSION: The growth inhibitory activity of doxorubicin or cisplatin, as a single agent, may be modified by combinations of P. emblica or T. bellerica extracts and be synergistically enhanced in some cases. Depending on the combination ratio, the doses for each drug for a given degree of effect in the combination may be reduced. The mechanisms involved in this interaction between chemotherapeutic drugs and plant extracts remain unclear and should be further evaluated.
Cisplatin; Doxorubicin; Liver cancer; Phyllanthus emblica; Synergistic effect; Terminalia bellerica
Aspergillus flavus first gained scientific attention for its production of aflatoxin. The underlying regulation of aflatoxin biosynthesis has been serving as a theoretical model for biosynthesis of other microbial secondary metabolites. Nevertheless, for several decades, the DNA methylation status, one of the important epigenomic modifications involved in gene regulation, in A. flavus remains to be controversial. Here, we applied bisulfite sequencing in conjunction with a biological replicate strategy to investigate the DNA methylation profiling of A. flavus genome. Both the bisulfite sequencing data and the methylome comparisons with other fungi confirm that the DNA methylation level of this fungus is negligible. Further investigation into the DNA methyltransferase of Aspergillus uncovers its close relationship with RID-like enzymes as well as its divergence with the methyltransferase of species with validated DNA methylation. The lack of repeat contents of the A. flavus' genome and the high RIP-index of the small amount of remanent repeat potentially support our speculation that DNA methylation may be absent in A. flavus or that it may possess de novo DNA methylation which occurs very transiently during the obscure sexual stage of this fungal species. This work contributes to our understanding on the DNA methylation status of A. flavus, as well as reinforces our views on the DNA methylation in fungal species. In addition, our strategy of applying bisulfite sequencing to DNA methylation detection in species with low DNA methylation may serve as a reference for later scientific investigations in other hypomethylated species.
Stored and cooked samples of pearl millet (Pennesetum typhoides), which is regularly consumed as food by the Paharia tribe in the hilly regions of Santhal Pargana, Bihar State, India, that were harvested in January 1989 were analyzed for mold flora, natural occurrence of Aspergillus flavus and A. parasiticus, and incidence and levels of aflatoxin B1. Of the 22 fungal species isolated, A. flavus and A. parasiticus were the predominant species (63.8%) during the rainy season, followed by other species of Aspergillus, Penicillium, Fusarium, Rhizopus, Helminthosporium, and Curvularia. Screening of 169 A. flavus and A. parasiticus strains showed that 59 of them were toxigenic, producing various combinations of aflatoxins B1, B2, G1, and G2. The amounts of aflatoxin B1 ranged between 4 and 30 mg/100 ml of liquid medium. Analysis of stored and cooked samples also revealed a high incidence and alarming levels of naturally produced aflatoxin B1. Forty-nine of 75 stored and 16 of 38 cooked samples contained various combinations of aflatoxins. The levels of aflatoxin B1 ranged between 17 and 2,110 ppb in stored samples and 18 and 549 ppb in cooked samples. The correlation of insect damage with A. flavus and A. parasiticus incidence and quantity of aflatoxin B1 was found to be insignificant.
Triphala (TP) is composed of Emblica officinalis, Terminalia chebula, and Terminalia belerica. The present study was undertaken to evaluate its anticataract potential in vitro and in vivo in a selenite-induced experimental model of cataract. In vitro enucleated rat lenses were maintained in organ culture containing Dulbecco’s Modified Eagles Medium alone or with the addition of 100μM selenite. These served as the normal and control groups, respectively. In the test group, the medium was supplemented with selenite and different concentrations of TP aqueous extract. The lenses were incubated for 24 h at 37°C. After incubation, the lenses were processed to estimate reduced glutathione (GSH), lipid peroxidation product, and antioxidant enzymes. In vivo selenite cataract was induced in 9-day-old rat pups by subcutaneous injection of sodium selenite (25 μmole/kg body weight). The test groups received 25, 50, and 75 mg/kg of TP intraperitoneally 4 h before the selenite challenge. At the end of the study period, the rats’ eyes were examined by slit-lamp. TP significantly (P < 0.01) restored GSH and decreased malondialdehyde levels. A significant restoration in the activities of antioxidant enzymes such as superoxide dismutase (P < 0.05), catalase (P < 0.05), glutathione peroxidase (P < 0.05), and glutathione-s-transferase (P < 0.005) was observed in the TP-supplemented group compared to controls. In vivo TF 25mg/kg developed only 20% nuclear cataract as compared to 100% in control. TP prevents or retards experimental selenite-induced cataract. This effect may be due to antioxidant activity. Further studies are warranted to explore its role in human cataract.
Anticataract; antioxidant; glutathione; malondialdehyde; selenite; superoxide dismutase; triphala
The present study was carried out to investigate the protective role of Triphala (a combination in equal proportions by weight of fruit powder of Terminalia belerica, Terminalia chebula and Emblica officinalis) against 1,2-dimethylhydrazinedihydrochloride (DMH) induced Endoplasmic reticulum stress (ER stress) in mouse liver. An oral dose of 3 mg/kg body wt in drinking water for 5 weeks significantly (P < 0.001) increased the levels of serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), serum Alkaline phosphatase (ALP) and total bilirubin thus suggesting damage to mouse liver and biliary dysfunction. The DMH administration invariably led to increase in the liver microsomal proteins of molecular weight of about 29 (ERp29) and 53 kDa (ERp53) and decrease in the protein of molecular weight of 36 kDa (ERp36) thereby suggesting the interference of DMH and its metabolites with normal protein biosynthesis and folding, in the reticular membranes of the liver cells thus developing ER stress. Histological studies show necrosis, large sized hepatocytes with increased N:C ratio, aberrant mitotic figures and prominent nucleoli in the liver of DMH treated mice. In animals fed 5% Triphala in diet (w/w) during DMH administration, there was significant decrease in the above changes in the liver suggesting the suppression of DMH induced ER stress in liver. Triphala significantly (P < 0.05) decreased lipid peroxidation and also the activity of lactate dehydrogenase (LDH) in mouse liver. It simultaneously increased the level of reduced glutathione (GSH) and the activity of glutathione-S-transferase (GST) thereby suggesting that it prevents peroxidative damage and also diverts the active metabolites (electrophiles) of DMH from their interactions with critical cellular bio-molecules which could be responsible for its protective action against DMH.
1,2-Dimethylhydrazinedihydrochloride; Neoplastic lesions; Triphala; ER stress; ERp29; ERp53; Antioxidant status; Chemoprotective effect
A heterologous transformation system was developed for Aspergillus flavus with efficiencies greater than 20 stable transformants per micrograms of DNA. Protoplasts of uracil-requiring strains of the fungus were transformed with plasmid and cosmid vectors containing the pyr-4 gene of Neurospora crassa. Transformants were selected for their ability to grow and sporulate on medium lacking uracil. Vector DNA appeared to integrate randomly into the genome of A. flavus with a tendency for multiple, tandem insertion. Transformants with single or multiple insertions were stable after five consecutive transfers on medium containing uracil. Uracil-requiring recipient strains were obtained either by UV-irradiating conidia and selecting colonies resistant to 5-fluoroorotic acid or by transferring the mutated pyr locus to strains by parasexual recombination. This is the first report of a transformation system for an aflatoxin-producing fungus. The transformation system and the availability of aflatoxin-negative mutants provide a new approach to studying the biosynthesis and regulation of aflatoxin.
Nine isolates of Aspergillus flavus and Aspergillus parasiticus were screened for aflatoxin production on a coconut extract agar medium. Aflatoxin-producing colonies were detected under long-wave UV light (365 nm) by blue fluorescence on the reverse side after 2 to 5 days of growth. Aflatoxin production was verified by chemical analysis. Several types of shredded coconut available in the United States were tested and found to be satisfactory. No additives were required. Various parameters affecting the test were investigated.
Aflatoxins are carcinogenic secondary metabolites produced by the fungi Aspergillus flavus and Aspergillus parasiticus. Previous studies found that repeated serial mycelial transfer or treatment of A. parasiticus with 5-azacytidine produced colonies with a fluffy phenotype and inability to produce aflatoxins. To understand how these treatments affect expression of genes involved in aflatoxin production and development, we carried out expressed sequence tag (EST)-based microarray assays to identify genes in treated clones that are differentially expressed compared to the wild-type. Expression of 183 genes was significantly dysregulated. Of these, 38 had at least two-fold or lower expression compared to the untreated control and only two had two-fold or higher expression. The most frequent change was downregulation of genes predicted to encode membrane-bound proteins. Based on this result we hypothesize that the treatments cause changes in the structure of cellular and organelle membranes that prevent normal development and aflatoxin biosynthesis.
aflatoxin; microarray; 5-azacytidine; Aspergillus parasiticus; secondary metabolism; fluffy phenotype
Peanuts grown under dryland conditions where drought stress occurred accumulated more aflatoxin before digging than peanuts grown under irrigation. Kernels became more susceptible to Aspergillus flavus and A. parasiticus invasion when the soil moisture in the pod zone approached levels at which moisture moved from the pod into the soil and the kernel moisture dropped below 31%. Isolation frequencies of these aspergilli from fresh-dug kernels were lowest in 1968 (maximum of 3%). In 1967 and 1969, maximum percentages of 100 and 74, respectively, were noted. Kernel infestation was correlated with degree of aflatoxin contamination. Dryland fresh-dug kernels contained a maximum of 35,800 parts per billion aflatoxin while a maximum of 50 parts per billion was detected in kernels from irrigated plots. In 1969 A. flavus infestation was as high as 59% in peanuts from irrigated plots; however, no aflatoxin was detected. Absence of aflatoxin in these samples is attributed to the higher kernel moisture content which reduced the aflatoxin-producing potential of A. flavus. Statistical analysis of the data revealed no significant differences in degree of fungal infestation, production levels, and grade factors between any fungicide treatments.