Niger andA Research Articles

Assessment of toxin-producing genes in Aspergillus species from traditional herbal products in Khon Kaen province, Thailand using molecular method

A large number of traditional herbal products from medicinal plants are available in local markets. However, the risk of fungal contamination especially toxigenic Aspergillus species, Aspergillus niger and Aspergillus flavus was potentially possible. Thus, this study targeted to investigate the prevalence of A. niger and A. flavus and the occurrence of genes encoding toxins, aflatoxins and ochratoxin A among the fungi using the molecular method in 33 herbal products randomly collected from local markets in Khon Kaen province, Thailand. Based on the identification using DNA sequences of the internal transcribed spacer region (ITS), there were two Aspergillus species, A. niger and A. flavus, with prevalence rates of 73.6% and 0.1% respectively. The genes related to aflatoxins and ochratoxin A were undetectable in all isolates. This study suggested the products were naturally contaminated with the fungi, but they were safe from the toxigenic A. niger and A. flavus.

Molecular strategy to discriminate between two ochratoxin A producingAspergillus niger aggregate species isolated from fresh and dried grapes

Aspergillus genus is an ubiquitous fungal group that colonizes a wide range of substrates. A total of 100 Tunisian fungal strains isolated at harvest time from fresh and dried grapes were identified within the sectionNigri and tested for their ochratoxin A (OTA) producing abilities. Of the isolates, 45% were identified asAspergillus tubingensis, 34% asAspergillus niger, 12% asAspergillus japonicus and 9% asAspergillus carbonarius. The OTA production was assessed using Czapeck yeast extract agar which revealed that 25% of the isolates belonging to theAspergillus niger aggregate were OTA producers, while 97% were identified as belonging to theA. Carbonarius species. To distinguish betweenA. Niger andA. Tubingensis, PCR was used to amplify the ITS-5.8S rDNA for these two strains. The sequence analysis of the PCR products revealed a full similarity between the two species. Thus, a new molecular strategy has been developed based on the targeting of the gene from nitrate reductase (niiA) and nitrite reductase gene (niaD), as well as the intergenic region of the gene (niiA-niaD). The subsequent screening revealed that two new specific primer pairs can be used to identify specificallyA. Niger andA. Tubingensis.

Production, purification and characterisation of proteases from whey by some fungi

The results regarding the production of proteases fromAspergillus niger andAspergillus terreus revealed that different nitrogen complements induced different growth and enzyme productivity depending on the individual organism, nature of cosupplement and/or concentration. The two steps fermentation process increased the yield of fungal biomass and proteases. Proteases were isolated and purified fromA. niger andA. terreus with specific activity of 179.0 and 294.7 U mg−1 protein, respectively. The purification fold values were 28.9 and 81.9 from the two organisms in the same order. The Vmax values were 56 and 166 U ml−1 for acid and alkaline proteases fromA. niger. However, Vmax for alkaline protease fromA. terreus was 29 U ml−1. The three proteases expressed the same value of Hill coefficient. Abscisic acid inhibited activities of the three proteases particularly those produced by A.terreus, a phenomenon that completely reversed with gibberellic acid (GA3), more permanently with acid protease fromA. niger. The purified protease was hardly affected with chelating agents and SH-reagents whereas inhibition was dependent on the nature of agent concentration and/or the source of enzyme. Proteases from the testedAspergillus strains exhibited promising hydrolytic activities towards fibrinogen, fibrin and blood clot. Thrombolytic activity varied among organisms and/or substrates.

Chemical composition and antifungal effect of anise (Pimpinella anisum L.) fruit oil at ripening stage

The composition of the essential oil ofPimpinella anisum L fruit is determined by GC and GC-MS. The volatile oil content obtained by hydrodistillation was 1.91%. Ten compounds representing 98.3% of the oil was identified. The main constituents of he oil obtained from dried fruits were trans-a nethole (93.9%) and estragole (2.4%). The olfactorially valuable constituents that were found with concentration higher than 0.06% were (E)-methyeugenol, α-cuparene, α-himachalene, β-bisabolene, p-anisaldehyde and cis-anethole. Also, the different concentrations of anise oil exerted varying levels of inhibitory effects on the mycelial growth off/ternaria alternata, Aspergillus niger andAspergillus parasiticus used in experimental. The results showed that the most effected fungus from anise oil wasA. parasiticus, which is followed byA. niger andA. alternata. Individual of this plant oil may provide a useful to achive adequate shelf-life of foods.

Molecular diagnosis of ochratoxinogenicAspergillus species

Toxigenic and non-toxigenic black aspergilli belonging to theAspergillus niger aggregate and toA. carbonarius were compared to each other and to strains of other species by DNA fingerprinting. AFLPs showed a clear separation ofA. niger andA. carbonarius. However, no clear correlation between the genetic similarity of the strains and the ability to produce ochratoxin A (OTA) was detected. Based on AFLP, marker sequences were chosen for the construction of SCAR-PCR primers for the detection ofA. carbonarius. A similar approach was used forA. ochraceus, another fungus of concern regarding ochratoxin A contamination of coffee. Cluster analysis ofA. ochraceus isolates mainly from Brazilian coffee showed a very close genetic similarity. Three species specific primer pairs were developed and one of these was used for the PCR and realtime PCR (RT-PCR) based detection of the mould in green coffee.A. ochraceus was specifically and rapidly detected and quantified in green coffee. A positive correlation between the amount of DNA and OTA content was established.

Natural occurrence of citrinin in rice grains and its biocontrol byTrichoderma hamatum

Paddy rice was sampled from El-Sharkia, El-Gharbia, El-Dakahlia and Kafr El-Shekh governorates, Egypt. Of the 30 samples taken, ten were contaminated with the mycotoxin citrinin. An average of 6.79 × 104 fungal spores per gram rice was found. The isolated fungi represented 47 species and 28 genera. The predominant genera wereAspergillus, Cladosporium andPenicillium. Aspergilli were represented by 22 species;Aspergillus niger andA. flavus had the highest occurrence.Penicillium viridicatum produced the highest amount of citrinin on glucose ammonium nitrate salts broth and rice grains, and hence this isolate was selected as a good producer of citrinin in this study. The presence ofTrichoderma hamatum reduced the amount of citrinin produced byP. viridicatum compared with its respective control. The excessive growth ofT. hamatum onP. viridicatum was increased with time. Viability ofP. viridicatum conidia decreased byT. hamatum with an increase in the incubation period. Chitinases and 1,3-β-glucanase enzyme activity ofT. hamatum increased with extending the incubation period onP. viridicatum mycelia up to maximum values at 72 and 84 h, respectively.T. hamatum led to a decrease in the production of citrinin byP. viridicatum on rice grains compared with the respective control values.

Enhancement of faba bean nodulation, nitrogen fixation and growth by different microorganisms

The effect of four bacterial and six fungal species on nodulation and growth ofVicia faba cv. Giza 3 inoculated withRhizobium leguminosarum biovarviceae strain RCR 1001 were assessed in a pot experiment.Bacillus cereus, Pseudomonas fluorescens, Aspergillus niger andA. quadriliniatus either alive cells or sterile filtrate significantly promoted nodulation, growth and nitrogen accumulation. Heat-killed cells had no effect.

Studies on the fungi associated with tomato fruit rots and effects of environment on storage

Seven fungi associated with fruit rot of tomato were isolated includingFusarium equiseti, F. chlamydosporum, Alternaria solani, Geotrichum candidum, Acremonium recifei, Aspergillus flavus andA. niger. They were all pathogenic on tomato fruits, most pathogenic beingGeotrichum candidum followed byA. niger. Least rot was caused byAlternaria solani. The optimum temperature for maximum rotting caused byG. candidum, A. niger andA. flavus was 30°C. The relative humidity for maximum rot ranged from 70–90%. Tomato fruits stored well at 0–10°C and rather poorly at 20–30°C. Fruits stored at 35°C showed blemishes. The best RH for storage ranged between 60 and 90%.

Associative effect of cellulolytic fungi andAzospirillum lipoferum on yield and nitrogen uptake by wheat

The associative effect of cellulolytic fungi, such asAspergillus awamori andA. niger, with the nitrogen fixer,Azospirillum lipoferum was studied in a soil amended with rice straw. All the inoculants gave significantly higher grain and straw yield and nitrogen uptake by wheat crop than did the uninoculated treatment. The doubling of chemical nitrogen dose significantly increased the yield and nitrogen uptake. It was observed thatA awamori performed significantly better followed byA. niger andA. lipoferum. The maximum benefit was obtained with combined inoculation ofA. awamori andA. lipoferum.

Comparative studies on glucoamylases from three fungal sources

Five commercial preparations of glucoamylases (three fromAspergillus niger, one each fromAspergillus foetidus andAspergillus candidus) were purified by ultrafiltration, Sepharose-gel filtration and DEAE-sephadex chromatography. Two forms of the enzyme, namely glucoamylase I and glucoamylase II were obtained from the fungi except from one strain ofA. Niger. All the enzymes appeared homogeneous by electrophoresis and ultracentrifugation. The specific activities varied between 85 and 142 units. The pH and temperature optima were between 4 and 5, and 60‡C respectively. The molecular weight as determined by the sodium dodecyl sulphate-polyacrylamide gel electrophoresis ranged from 75,000 to 79,000 for glucoamylase I and 60,000 to 72,000 for glucoamylase II. OnlyA. niger glucoamylases contained phenylalanine at the N-terminal end. The amino acid composition of the enzymes was generally similar. However,A. niger andA. foetidus glucoamylases, in contrast toA. candidus enzymes, contained greater percentage of acidic than of basic amino acids. The enzymes contained 15 to 30% carbohydrate and 49 to 57 residues of monosaccharides per mol.A. niger enzymes contained mannose, glucose, galactose, xylose and glucosamine but theA. candidus enzyme lacked xylose and glucose and only xylose was absent inA, foetidus enzymes. Majority of the carbohydrate moieties were O-glycosidically linked through mannose to the hydroxyl groups of seline and threonine of the polypeptide chain.

Survey of Aspergillus species associated with the human respiratory tract.

A comprehensive survey has been carried out on the occurrence ofAspergillus species in the respiratory tract of patients of bronchopulmonary diseases in Delhi. In all, 1238 clinical specimens, which included 1082 sputa, 143 bronchial aspirates and 13 throat swabs obtained from 812 patients, were examined. Of these 61.7 per cent patients were culturally positive yielding 29 different species ofAspergillus. The prevalence of aspergilli in sputa was significantly higher than in the bronchial aspirates.Aspergillus niger was the commonest species isolated showing a prevalence of 36.7 per cent. It was followed byA. flavus, A. nidulans, A. terreus, A. versicolor, A. sydowi, A. japonicus andA. oryzae. None of theAspergillus species showed a significant correlation with any of the diseases, or the type of treatment the patients had received. Of the 8 broad occupational groups investigated farmers and labourers showed higher prevalence ofA. niger andA. flavus. The prevalence ofAspergillus species in the throats of healthy persons was 16 per cent withA. versicolor being the commonest species followed byA. flavus, A. amstelodami, A. sydowi andA. terreus. A comparison of the prevalence ofAspergillus species in the patients, healthy individuals and atmosphere of Delhi appears to support the view that the aspergilli are transient residents in the human respiratory tract following their inhalation from the environment.

Deep mycoses in India. A critical review.

Available published reports on deep mycoses in India have been critically and exhaustively reviewed. So far there seem to be only 9 cases of actinomycosis reported, mostly of thoracic type and diagnosed on the basis of the presence of “sulphur granules” in the lesions. Nocardiosis and its chief causal agentNocardia asteroides have received particular attention in recent studies. To-date there are 18 authentic cases reported from India and significantly 12 of these have been diagnosed by applying the paraffin bait technique to the isolation ofN. asteroides from sputa and other clinical specimens. In most of these 12 cases timely diagnosis allowed for the successful treatment of the disease with heavy doses of sulphadiazine. Case reports on cryptococcosis which include 26 adequately documented cases, have been published from various parts of the country. Occurrence ofCryptococcus neoformans in soil and its association with old pigeon excreta have also been confirmed by studies done in some northern and western regions of this country. The status of histoplasmosis in India still remains a debatable subject although there is a suggestive evidence that the disease may be endemic in the northeastern parts. There are 9 case reports in which diagnosis has been supported by histopathologic findings and in 3 cultures have also been positive. However, attempts to isolateHistoplasma capsulatum from soil or any other extra-human source have remained futile and the limited surveys have revealed only low skin sensitivity to histoplasmin and none to blastomycin and coccidioidin. As yet there is no authentic case of blastomycosis, coccidioidomycosis or paracoccidioidomycosis reported from India. Two cases of invasive aspergillosis and 6 of bronchopulmonary aspergillomas have been published. In the latterAspergillus fumigatus, A. niger andA. flavus have been found to be the aetiologic agents. In addition, a recent report on a series of 8 patients recognises for the first time the occurrence of allergic aspergillosis in this country. Two cases of phycomycoses, involving the lungs in one and brain in the other case have been described. Diagnosis of bronchopulmonary candidiasis has been claimed in as many as 16 patients by several authors but in none the evidence is unequivocal. The isolation ofCandida viswanathii from the cerebrospinal fluid of two fatal cases is suggestive of the possible aetiologic role of this new yeast in human meningitis. Besides, there are 3 cases of brain mycoses described in Indian literature, two due toCladosporium trichoides while in the third caseUstilago maydis, the causal agent of maize smut, has been implicated.

Studies on Aspergilli

The effect of foliar sprays of urea on the Aspergilli of the rhizosphere ofTriticum vulgare L was studied in detail. In all 16 species ofAspergillus were isolated from the rhizosphere region of wheat. The urea-treated plants (five sprayings) registered largest number ofAspergillus species. Out of 16 species the percentage occurrence of the following species,A. nidulans, A. rugulosus, A. sydowi A. fumigatus, A. niger andA. flavus, increased with the sprayings of urea. There was a general increase in the concentration of amino acids in urea-treated plants. But fourfold increase in the concentration was noted in the case of γ-aminobutyric acid and glutamine. In contrast to this the concentration of organic acids decreased as the sprayings increased.

Ферментация лимонн ой кислоты из тростн ика, м елассу

The authors describe experiments with the fermentation of citric acid from cane molasses byAspergillus niger. The yield on the original medium, improved only by phosphoric acid and potassium ferrocyanide, was low (20–23%) and was not greatly increased by the various clarification methods tested. Citric acid formation was improved by the addition of ammonium nitrate (32–35%) and still more by the addition of malt (37–41%). Dilution of the cane molasses, with replenishment of sugars, did not eliminate the influence of inhibitors, which in fermentation with a mixture of cane and beet molasses was much stronger than that of stimulators. The most marked increase in the yield, particularly in the presence of organic and inorganic nitrogen, was observed on adding 3–3.5% ethyl alcohol, which gave a citric acid yield of about 50%. The simultaneous addition of iodine up to 0.05% did not affect the yield and had an antiseptic effect. Old, unsuitable molasses can be neutralized and give comparatively good yields if nitrogen and ethyl alcohol are added. Foreign strains ofA. niger andA. luchuensis were not more effective than the production strain ofA. niger, the activity of which was not increased by passage on a cane molasses medium.

Studies on lipolytic molds. Comparative study of lipases obtained from molds grown on oil seeds

SummaryA. niger andA. flavus isolated from oil seed source show an appreciable amount of lipolytic activity.The optimum pH of the mold lipase varies to a certain extent with the nature of the substrate and buffer used.In the experiments carried out the optimum buffer concentration is 2 cc., the optimum substrate concentration is 1–2 cc., and the optimum temperature is 35–37°C. for hydrolysis of peanut oil by mold lipases.Sodium taurocholate, managanese sulphate, KH2PO4, sodium acetate, etc., accelerate the activity of the mold lipase.The change in activity of acetone‐dried sample of mold lipase with time is negligible.The synthetic activity of the mold lipase is higher than that of seed and cake lipases.A definite relation between the vitamin contents and the lipolytic activity of the molds could not be found.Peanut cake medium appears to be a good source to grow the lipolytic molds on a large scale.