Evaluation of mycotoxins,mycobiota, and toxigenic fungi in selected medicinal plants of Khyber Pakhtunkhwa,Pakistan

Medicinal plants are used worldwide to treat a variety of ailments. Due to the provenance of medicinal plants, they are subjected to contamination by moulds, which may be responsible for spoilage and production of mycotoxins. The investigation was designed to throw light on mycological and mycotoxicological status of some medicinal plants from Pakistan and the result showed 30 % and 26.7 % samples were contaminated with aflatoxins and ochratoxin A, respectively. Mould contamination was present in 90 % samples, of which 70 % exceeded the permissible limits. Opium poppy, licorice root, and Indian rennet were most contaminated samples. The predominant moulds found were Aspergillus flavus, Aspergillus niger, Aspergillus parasiticus, and Penicillium spp. and 31 % of the 47 isolates tested were found to be toxigenic. The findings indicate that the contamination in the medicinal plants may contribute to adverse human health problems. This information would prove helpful for regulatory agencies to establish limits for these contaminants in medicinal plants and will explore ways for export of herbal products to countries where more stringent permissible limits of mycotoxins exist. The study is first of its kind in the country reporting natural occurrence of mycotoxins in medicinal plants in Pakistan.     

Fungi associated with black tea and tea quality in the Sultanate of Oman

Forty-eight samples of four popular commercial brands of black tea ( Camellia sinensis L.) were purchased from the local markets in Muscat area, Sultanate of Oman. Tea leaves were surveyed for mycoflora. Five fungal species were isolated with A. niger as the most dominant in all the brands having percentage contamination ranging between 0.66% and 30.34%. Other fungi isolated were Aspergillus flavus, Penicillium spp. and Pacelomyces spp. but having average percentages of 0.6%, 0.84% and 0.21% respectively. Significant differences were found among the batches contaminated by A. niger. None of the 25 A. flavus strains screened for aflatoxins were found aflatoxigenic. The total ash, water-soluble ash, and mineral concentration of the samples were within the British standards and were not affected by fungal contamination. The results showed that black tea is contaminated by fungi that might constitute health hazards for humans. The post harvest contamination of tea could be eliminated or reduced if processing is conducted under more hygienic conditions. This revised version was published online in June 2006 with corrections to the Cover Date.     

The experimental contamination of foodstuffs with the spores of toxigenic micromycetes and the production of mycotoxins

A study of experimental contamination of foods with spores of toxigenic micromycetes (Aspergillus flavus andPenicillium verrucosum) was realized. Samples of bread, apricot jam, Edam cheese, processed cheese and half-fat dried milk purchased on the market were used for experimental contamination. Aflatoxins and ochratoxin A were determinated in experimental contaminated foodstuffs.     

Assessment of toxigenic fungi on Argentinean medicinal herbs

This work was performed to determine the incidence of toxigenic fungi and their mycotoxins on 152 dried medicinal and aromatic herbs, belonging to 56 species, which are used as raw material for drugs. International methodologies for fungal enumeration and identification were applied as well as TLC and HPLC techniques for toxins detection. The 52% out of 152 samples were contaminated with species from Aspergillus genus, 27% belonging to the Flavi section and 25% to the Circumdati section. The 16% of the total samples was contaminated with species from Fusarium genus. Aspergillus flavus and A. parasiticus (Flavi section), were the predominant species isolated, 50% out of 40 isolates were toxigenic. Aflatoxin concentrations ranged from 10 to 2000 ng/g. Only 26% of isolates from the Circumdati section (A. alliaceus, A. ochraceus and A. sclerotiorum) produced ochratoxin A in low concentrations between 0.12 and 9 ng/g. From a total of 29 strains of Fusarium spp., 27.5% were Fusarium verticillioides and F. proliferatum, which produced fumonisin Bland fumonisin B2 ranged from 20 to 22000 microg/g and from 5 to 3000 microg/g respectively. The remaining species, F. equiseti, F. oxysporum, F. semitectum, F. compactum, F. sombucinum and F. solani were able to produce neither group A and B trichothecenes nor zearalenone. The incidence of A. ochraceus and Fusarium spp. and their toxigenic capacities on medicinal herbs were studied for the first time in Argentina. It would be important to look for natural contamination to define acceptability Limits which allow the control of sanitary quality of medicinal herbs used as phytotherapic medicines in several countries.     

A note on the screening of dried shrimps, shrimp paste and raw groundnut kernels for aflatoxin-producing Aspergillus flavus

All of 33 samples of dried shrimps, shrimp paste, peanut butter and raw groundnut kernels were contaminated with fungi. Aspergillus and Penicillium spp. were the predominant types in dried shrimps and raw groundnut kernels but no Aspergillus spp. were present in peanut butter or shrimp paste samples. Among 81 Aspergillus isolates obtained from dried shrimps and raw groundnut kernels, 10 were A. flavus/A. parasiticus, of which five were potential aflatoxin-producing A. flavus strains. No aflatoxins were detected in the food samples although some were visibly mouldy and some had high mould counts. The occurrence of aflatoxin-producing strains of A. flavus in dried shrimps and raw groundnut kernels warrants further investigation of these foods and their products as potentially significant sources of aflatoxins.     

Toxigenic Fungi in Food

Forty-five fungal isolates from moldy supermarket foods were tested for toxicity to brine shrimp, and twenty-two of these isolates were subsequently tested for toxicity to chicken embryos. Highly toxigenic fungi were Cladosporium sphaerospermum from a bakery product, Fusarium oxysporum from carrots, F. solani from cabbage, Aspergillus niger and Penicillium corylophilum from bread, P. cyclopium and P. herguei from corn meal, P. lanosum from onions,P. steckii from chocolate syrup, Penicillium sp. from jelly, and Rhizopus nigricans isolates from sweet potato, applesauce, and strawberries. Approximately one-third of the fungal cultures were moderately to highly toxigenic to brine shrimp and chicken embryos, while several additional cultures were slightly toxigenic.     

Cyclopiazonic acid production by Penicillium camemberti Thom and natural occurrence of this mycotoxin in cheese.

Every Penicillium camemberti strain freshly isolated from 20 commercial cheese brands produced cyclopiazonic acid in two culture media at 25, 13, and 4 degrees C; the toxin yield was greatly dependent on the strain and environmental parameters (medium, temperature, and incubation time). The toxigenic ability appeared as a log-normal distribution. This mycotoxin was found in the crust (0.05 to 0.1 microgram/g in three samples, 0.1 to 0.2 microgram/g in five samples, and 0.4, 1, and 1.5 microgram/g in three other samples) but not in the inner part. When its acute toxicity is considered, doses eventually ingested by consumers are very low (lower than 4 microgram). Means for prevention are discussed. A highly toxigenic strength and rate appear to be necessary features leading to natural contamination in cheeses. The distribution of toxigenic ability makes possible without delay a choice of weakly toxic strains.     

Cyclopiazonic acid production by Penicillium camemberti Thom and natural occurrence of this mycotoxin in cheese

Every Penicillium camemberti strain freshly isolated from 20 commercial cheese brands produced cyclopiazonic acid in two culture media at 25, 13, and 4 degrees C; the toxin yield was greatly dependent on the strain and environmental parameters (medium, temperature, and incubation time). The toxigenic ability appeared as a log-normal distribution. This mycotoxin was found in the crust (0.05 to 0.1 microgram/g in three samples, 0.1 to 0.2 microgram/g in five samples, and 0.4, 1, and 1.5 microgram/g in three other samples) but not in the inner part. When its acute toxicity is considered, doses eventually ingested by consumers are very low (lower than 4 microgram). Means for prevention are discussed. A highly toxigenic strength and rate appear to be necessary features leading to natural contamination in cheeses. The distribution of toxigenic ability makes possible without delay a choice of weakly toxic strains.     

Fungal Contamination of Raw Materials of Some Herbal Drugs and Recommendation of <Emphasis Type="Italic">Cinnamomum camphora</Emphasis> Oil as Herbal Fungitoxicant

The paper explores fungal infection and aflatoxin B(1) contamination of six medicinal plant samples viz. Adhatoda vasica Nees, Asparagus racemosus Linn., Evolvulus alsinoides Linn., Glycyrrhiza glabra Linn., Plumbago zeylanica Linn. and Terminalia chebula Retz. A total of 858 fungal isolates were detected from the raw materials. Maximum number of fungal isolates was detected from A. racemosus (228). The genus Aspergillus was found to be the most dominant genus causing infection to most of the raw materials. Among the 32 isolates of A. flavus tested, 13 isolates were found to be toxigenic elaborating aflatoxin B(1). The highest elaboration of aflatoxin B(1) was 394.95 ppb by the isolates of A. flavus from G. glabra. The essential oil of Cinnamomum camphora (L.) Presl showed efficacy in arresting aflatoxin B(1) by the toxigenic strain. The growth of a toxigenic strain of A. flavus decreased progressively with increasing concentration of essential oil from leaves of C. camphora. The oil completely inhibited aflatoxin B(1) production even at 750 ppm. Hence, the oil of C. camphora is recommended as herbal fungitoxicant against the fungal contamination of the raw materials.     

Experimental aflatoxin production in Manchego-type cheese

Manchego-type cheese, a typical Spanish cheese, was inoculated in various ways with an aflatoxigenic organism, Aspergillus parasiticus NRRL 2999, to study the production of aflatoxin. When the original milk was contaminated with a spore suspension, aflatoxin was not detected in paraffin-covered cheeses although it was present in the top layer of non-paraffin-covered cheeses after ripening at 15 degrees C for 60 d. When the cheese surface was inoculated, no aflatoxins were detected in paraffin-covered cheeses after ripening for 60 d although they were found when the cheeses were ripened for 30 d. In non-paraffin-covered cheeses aflatoxins were detected only in the top layer and in the second 10 mm layer when cheeses were incubated after the normal ripening at 28 degrees C for 30 d. When the centre of the cheese was inoculated, no aflatoxins were detected although Aspergillus grew slightly along the inoculation area. When cheese portions were inoculated, fungal growth was evident after incubation at 28 degrees and 15 degrees C for 6 d but there was no growth at 10 degrees C after 50 d. At 28 degrees C aflatoxins were detected at a concentration of 132 micrograms/g after 13 d, the highest level obtained. In cheese paste at 28 degrees and 15 degrees C, growth was intense, but the level of aflatoxins detected was lower than in cheese portions. At 10 degrees C the growth was heavy, but aflatoxins were not detected.     

Experimental aflatoxin production in Manchego-type cheese

Manchego-type cheese, a typical Spanish cheese, was inoculated in various ways with an aflatoxigenic organism, Aspergillus parasiticus NRRL 2999, to study the production of aflatoxin. When the original milk was contaminated with a spore suspension, aflatoxin was not detected in paraffin-covered cheeses although it was present in the top layer of non-paraffin-covered cheeses after ripening at 15°C for 60 d. When the cheese surface was inoculated, no aflatoxins were detected in paraffin-covered cheeses after ripening for 60 d although they were found when the cheeses were ripened for 30 d. In non-paraffin-covered cheeses aflatoxins were detected only in the top layer and in the second 10 mm layer when cheeses were incubated after the normal ripening at 28°C for 30 d. When the centre of the cheese was inoculated, no aflatoxins were detected although Aspergillus grew slightly along the inoculation area. When cheese portions were inoculated, fungal growth was evident after incubation at 28° and 15°C for 6 d but there was no growth at 10°C after 50 d. At 28°C aflatoxins were detected at a concentration of 132 μg/g after 13 d, the highest level obtained. In cheese paste at 28° and 15°C, growth was intense, but the level of aflatoxins detected was lower than in cheese portions. At 10°C the growth was heavy, but aflatoxins were not detected.     

Nematophagous fungi of Toxocara canis eggs in a public park of La Plata, Argentina

Fungi have showed a great potential for the biological control of nematodes. However, they have not been evaluated for the control of animal and/or human parasites transmitted by egg contaminated soils. Environmental contamination with Toxocara spp. eggs is a public health problem. Accidental swallowing of Toxocara canis eggs (a nematode of dogs) usually results on a zoonotic infection (toxocarosis). The objectives of this research were: 1) To test the presence of antagonistic fungi against T. canis in the soil in public places of La Plata city, Argentina, infected with eggs of this parasite, 2) To determine the possible association between biotic and abiotic factors of the soil with the presence of fungal parasites of egg nematodes. Soil samples were tested for: textural type, organic matter (%), pH, presence of egg-parasite fungi, of larvae and of nematode eggs, in particular of Toxocara spp. The studied area showed the following characteristics: pH: 6.6-8.0, organic matter: 1.2-70%, with a predominantly loam texture. The following antagonistic fungal genera were identified: Acremonium, Aspergillus, Chrysosporium, Fusarium, Humicola, Mortierella, Paecilomyces and Penicillium. A prevalence of 70% was detected for nematode eggs, of 33% for Toxocara spp. eggs and of 90% for larvae. No association between the presence of egg-parasite fungi and the considered factors was found. More studies are necessary to know the natural antagonism factors to T. canis eggs for its in situ biological control.     

Microbial contamination of cell cultures: a 2 years study.

Cell line contamination is a major drawback of main cell banks of the world and it has cost of losing important biological products or valuable research. The causative agents are different chemicals, invertebrates, bacteria, fungi, parasites, viral species and even other cell lines. In this retrospective study, cell lines from various species such as human, fish, insect, animals either offered or accessed through usual official accession in CGBRI were studied during 2 years (2002-2004) to detect their microbial contaminations and the causative organisms. Samples were taken for sterility test upon cell lines receipt and upon each cell line sub-culture. Samples were examined for bacterial (including mycoplasmas) and fungal contamination using conventional microbiological techniques. The study excluded parasites, viruses and other contaminating agents. This study revealed 39% of specimens were contaminated. The major contaminating agents were mycoplasmas (19%) followed by mixed infection (8%), fungi (8%) and bacteria (4%). Among various bacterial species (except mycoplasmas) Bacillus sp., Enterococcus sp. and Staphylococcus sp. are main bacterial agents and among various fungi Aspergillus sp. followed by Penicillium sp., Sepedonium sp. and Botrytis sp. were main fungal causative agents of CGBRI cell line contamination. Our study also delineates each cell line contamination rate and its causative agents. This is the first report of cell culture contamination from cell banks of Middle-East countries like Iran.     

Nickel Tolerance and Accumulation by Filamentous Fungi From Sludge of Metal Finishing Industry

Industrialization, urbanization and increased vehicular traffic have resulted in increased contamination of our environment by heavy metals. The long persistence of heavy metals in nature has in turn resulted in development of metal resistant microbial strains. These strains are minimizing heavy metals toxicity, either by metal complexation or precipitation and other mechanisms. Characterization of fungal diversity was done in contaminated soil of the Wazirpur industrial area throughout the year. In this area highly acidic hazardous solid waste produced high concentration of heavy metals (Ni, Cu, Cr, Fe, Mn). Nickel toxicity is a major environmental concern. Due to long persistence of this waste in the environment without any treatment, many fungal isolates from the surrounding environment settle on the upper surface of waste. Few of them are capable of growing in the toxic conditions. More than 20 strains were isolated, most of them belonging to species of Aspergillus, Penicillium, Fusarium and Mucor genera. Seasonal variation in fungal diversity was significant. Four filamentous fungal isolates were found to be resistant for nickel (II) and a strain of Papulaspora sepedonoides reported first time for bioremediation of Ni (II) in this investigation, which is absorbing 62.33 μmol Ni gr^?1. These fungal isolates showed a high level (100–10000 mg kg^?1) of resistance for Ni (II) salt and removing Ni (II) from solution. Metal uptake varied with fungi. The toxicity also was influenced by different factors like pH and composition of growth medium.     

The mycoflora of gari

Mycological studies have been carried out on market samples of gari, a ready-to-use staple fermentation product of cassava ( Manihot esculenta cranz ). Commercial and industrial samples were analysed. The moisture contents of samples varied from 7% to 17% while the water activity ( a _w) varied from 0·52 to 0·85 and the pH of samples ranged from 3·9 to 7·26. Several fungi were isolated from the commercial samples while the industrial packaged samples were relatively mould-free. Moulds isolated were mainly Aspergillus spp., Penicillium spp., and several Mucorales, e.g. Rhizopus sp., Syncephalastrum sp., Mucor sp. and Circinella sp.     

A note on the screening of dried shrimps, shrimp paste and raw groundnut kernels for aflatoxin-producing Aspergillus flavus

S im , T.S., T eo , T heresa & S im , T.F. 1985. A note on the screening of dried shrimps, shrimp paste and raw groundnut kernels for aflatoxin-producing Aspergillus flavus. Journal of Applied Bacteriology 59 , 29–34.
All of 33 samples of dried shrimps, shrimp paste, peanut butter and raw groundnut kernels were contaminated with fungi. Aspergillus and Penicillium spp. were the predominant types in dried shrimps and raw groundnut kernels but no Aspergillus spp. were present in peanut butter or shrimp paste samples. Among 81 Aspergillus isolates obtained from dried shrimps and raw groundnut kernels, 10 were A. flavus/ A. parasiticus , of which five were potential aflatoxin-producing A. flavus strains. No aflatoxins were detected in the food samples although some were visibly mouldy and some had high mould counts. The occurrence of aflatoxin-producing strains of A. flavus in dried shrimps and raw groundnut kernels warrants further investigation of these foods and their products as potentially significant sources of aflatoxins.     

Growth reduction of Listeria spp. caused by undefined industrial red smear cheese cultures and bacteriocin-producing Brevibacterium lines as evaluated in situ on soft cheese.

The undefined microbial floras derived from the surface of ripe cheese which are used for the ripening of commercial red smear cheeses have a strong impact on the growth of Listeria spp. In some cases, these microbial consortia inhibit Listeria almost completely. From such undefined industrial cheese-ripening floras, linocin M18-producing (lin+) (N. Valdés-Stauber and S. Scherer, Appl. Environ. Microbiol. 60:3809-3814, 1994) and -nonproducing Brevibacterium linens strains were isolated and used as single-strain starter cultures on model red smear cheeses to evaluate their potential inhibitory effects on Listeria strains in situ. On cheeses ripened with lin+ strains, a growth reduction of L. ivanovii and L. monocytogenes of 1 to 2 log units was observed compared to cheeses ripened with lin strains. Linocin M18 activity was detected in cheeses ripened with lin+ strains but was not found in those ripened with lin strains. We suggest that production of linocin M18 contributes to the growth reduction of Listeria observed on model red smear cheeses but is unsufficient to explain the almost complete inhibition of Listeria caused by some undefined microbial floras derived from the surface of ripe cheeses.     

Filamentous Fungi Isolated from Estuarine Sediments Contaminated with Industrial Discharges

Filamentous fungi were isolated from estuarine sediments collected from two contaminated sites. One site was contaminated mainly by polycyclic aromatic hydrocarbon (PAH) with a concentration around 407?µg g^?1 of different PAHs, and the other by different sources of industrial pollutants; both were compared to a pristine site. All three sites were located in the Baixada Santista, São Paulo State, Brazil. The aim of the present investigation was the isolation of filamentous fungi from pristine and industrially polluted sediments in order to assess the mycobiota present in those sites and to evaluate their tolerance to phenanthrene and pyrene. Most of the isolated fungi were mitosporic ascomycetes, including Aspergillus sp., Chrysosporium sp., Cyclothyrium spp., Gliocladium sp., Penicillium spp., Phoma spp., and Trichoderma spp. There were also representatives of sexual Ascomycetes, Basidiomycetes, and Zygomycetes. The results showed that 59% of the evaluated fungi were tolerant to pyrene and 30% to phenanthrene. Representatives of Trichoderma were the most tolerant among the filamentous fungi investigated. A representative of Penicillium simplicissimum was the only isolate tested that had a better growth in the presence of pyrene than in its absence.     

Mycotoxin Producing Potential of Fungi Associated with Dry Shrimps

Among 114 fungi isolated from 20 dry shrimp samples, 27 isolates were capable of producing mycotoxins. Aspergillus and Penicillium were-the-predominant fungi. Two of 3 Aspergillus flavus could produce aflatoxin B₁ and G₁ on yeast extract sucrose medium and on surface sterilized dry shrimps. For other toxigenic species, toxicity was found mostly in the chloroform extracts of cultures. Visibly mouldy dry shrimps had significantly higher moisture content than the non-mouldy ones. Most of the fungi were isolated from mouldy samples.     

Fungi associated with dried fish in Sri Lanka

The mycoflora of dried-salted fish from markets in Kandy, Sri Lanka was studied with emphasis on visibly spoiled dried fish. A total of 61 fungal isolates from 25 dried-fish were isolated and identified. The most prevalent fungus wasAspergillus niger. Species ofAspergillus flavus, A. fumigatus, A. glaucus, A. restrictus, Aureobasidium spp.Basipetospora halophila (a genuinely halophilic fungus)Cladosporium herbarum, Gliomastix, spp.,Penicillium chalybeum andPenicillium expansum were present. The isolated fungi did not grow in synthetic media containing more than 30% sodium chloride.Aureobasidium spp. andGliomastix spp. did not grow on dried-fish under laboratory conditions. The protective exoskeleton appeared to prevent fungal growth on dried shrimp. TheA. flavus strains isolated were not aflatoxigenic.