Fungal colonists of maize grain conditioned at constant temperatures and humidities

Fungal colonization of shelled maize (Pioneer 3320) harvested from a field near Furman, South Carolina, in 1992 was determined after 348 and 751 days of continuous storage at each of seven temperatures (10, 15, 20, 25, 30, 35, or 40°C) and four constant relative humidities, giving equilibrium grain moisture contents ranging from 9.4% to 17.5% m.c. in 28 grain conditioning environments. Twenty fungal species infected surface sterilized seeds and were recorded from these conditioned grain treatments, including species commonly found in preharvest maize [e.g. Acremonium zeae, Aspergillus flavus, Fusarium moniliforme (syn. F. verticillioides), Penicillium pinophilum (syn. P. funiculosum), etc.]. Eupenicillium cinnamopurpureum and Monascus ruber were recorded only from conditioned grain treatments. Eurotium chevalieri colonized 50–96% of the kernels from grain conditioning treatments with the highest moisture content for each incubation temperature. Grain samples with>33% E. chevalieri infection had a decreased occurrence of F. moniliforme and A. zeae, and no kernels from these samples germinated. No fungi colonized more than 50% of the kernels conditioned at 30–40°C and 9.4–14.2% m.c. The results of this study indicate that individual patterns of fungal colonization during grain conditioning were a function of the survival rates for preharvest fungal colonists and their potential replacement by E. chevalieri.     

六堡茶的水溶性成分分析与研究进展

六堡茶独特的感官品质与保健功效与其后发酵过程微生物的参与具有密切联系。为探究六堡茶渥堆与蒸压工艺过程中真菌的多样性及分布规律,利用传统分离培养法与分子生物学鉴定技术对六堡茶毛茶、渥堆各个阶段以及蒸压后共14个样品中的真菌进行研究。结果表明：从研究样品中分离到13个属,39种真菌。其中,22种真菌为首次在六堡茶中报道,包括3种金花菌和6种酵母菌。研究发现六堡毛茶及渥堆茶样中Aspergillus（曲霉属）和Penicillium（青霉属）是主要的优势菌属,蒸压后,样品中只能分离出Aspergillus niger（黑曲霉）、Aspergillus sydowii（聚多曲霉）、Penicillium laeve、Penicillium gerundense四种真菌,其他曲霉属和青霉属真菌几乎无法检测到。蒸压后的样品中以Talaromyces（篮状菌属）为主要的优势菌属。在种水平上,所有茶样中,Aspergillus niger和Arxula adeninivorans是主要的优势菌种。研究结果丰富了六堡茶加工过程真菌多样性的认知,并为进一步探究不同茶源真菌对六堡茶品质影响提供了材料基础和理论依据。     

Putranjiva roxburghii oil—A potential herbal preservative for peanuts during storage

Samples of peanut seeds were collected from stores in eastern Uttar Pradesh, India and examined for their associated mycoflora and insects. Fifteen species of fungi and one insect, Trogoderma granarium, were isolated from the samples. Volatile constituents extracted in the form of essential oils from 32 plant species were evaluated against the dominant fungi, Aspergillus flavus and Aspergillus niger, as well as Trogoderma granarium. The oil of Putranjiva roxburghii exhibited the greatest toxicity. The oil was found to be fungicidal and thermostable at its minimum inhibitory concentration (MIC) of 400 ppm. The oil was characterized by the determination of its various physico-chemical properties.

The oil protected the peanut seeds completely for 6 months at 0.25 and 0.38 mL in containers of 250 mL capacity holding 200 g seeds. It did not exhibit any adverse effect on seed germination, seedling growth and general health and morphology of plants. Thus, the oil of Putranjiva roxburghii showed potential as a preservative for peanut seeds against spoilage by fungi and insects during storage.     

Kinetic Properties and Enantioselectivity of The Lipases Produced by Four Aspergillus Species

Four high lipase-producing Aspergillus species, selected in our laboratory, were compared in terms of their stability and reactivity for enantioselective esterification between (R, S)-2-octanol with octanoic acid in n-hexane. We determined the pH and temperature reactions dependences of lipases activities, and we found that these enzymes exhibited various pH sensitivities. The optimum pH observed for Aspergillus terreus lipase was 5.5, for A. niger and A. oryzae lipases in the range of 6.0 to 6.5 and pH 7.0 for A. flavus lipase. Good stability was observed at pH ranging from 5.0 to 8.5 after 24 hours at 40° C, and the optimum activity was observed at 35-40° C for all lipases tested. The lipases from A. terreus and A. niger were highly thermostable, retaining 60% and 50% activity at 60° C after 1 hour, respectively. The lipases from A. niger and A. terreus lipases provided the best results in terms of enantioselectivity (E) in the esterification of (R, S)-2-octanol with octanoic acid in n-hexane (E = 4.9 and E = 4.5, respectively). These properties make these lipases good candidates for biocatalysis in organic media.     

Aspergillus flavus link and its occurrence in relation to other mycoflora on stored spices

Spices are used sparingly to flavour foods; their mycoflora and its possible role in food infection has not been thoroughly investigated. In an examination of 6 spices, Aspergillus flavus, other Aspergillus species, and species of Rhizopus, Mucor, Penicillium, Tricothecium, and Fusarium were found on seed surfaces. A. flavus which is implicated in the production of carcinogenic aflatoxin, was mainly found on ginger, mustard, garlic, and pepper. The highest fungal counts (10.6 × 10⁴/g) occurred in stored pepper (Piper nigrum L.) and the lowest in curry leaves (Murraya koenigii L.). Contamination of pepper and mustard with A. flavus seems to be related to the processing and storage of these spices.     

Incidence of toxigenic fungi and ochratoxin A in dried fruits sold in Brazil

A total of 117 dried fruit samples (black sultanas, white sultanas, dates, dried plums, dried figs and apricots) from different origins were analysed both for toxigenic fungi and for the presence of ochratoxin A. Amongst the fungi found, Aspergillus niger was predominant, with 406 isolates, of which 15% were ochratoxin A producers. They were followed by A. ochraceus, with 15 isolates and 87% ochratoxigenics, and A. carbonarius, with only five isolates of which 60% were ochratoxin A producers. The average infection rates for A. niger in black sultanas, plums, figs, dates and white sultanas were 22.0, 8.0, 4.0, 1.5 and 0.5%, respectively. The apricot samples were not contaminated by any fungi or ochratoxin A. Black sultana and dried figs contained the highest contamination with ochratoxin A, with 33 and 26.3% of the samples containing more than 5 µg kg^-1 respectively, while all the white sultanas, dates and plums had no sample that exceeded this limit.     

On water dynamics and germination time of mold spores in concentrated sugar and polyol solutions

The relative effect of concentrated solutions of selected glass formers on germination time at 28 °C of mold spores of Aspergillus flavus, A. niger and Eurotium herbariorum was determined. Solutes included glucose, fructose, mannose, glycerol, sucrose, maltose and propylene glycol. The effect of fructose concentration on the germination time of A. parasitions was also studied. The results did not agree with previous literature experimental data used to support a ‘water dynamics’ approach of microbial stabilization in concentrated sugar and polyol solutions.     

Impact of habitats on toxigenic potential of Aspergillus flavus

Toxigenic potentials of Aspergillus flavus strains isolated from different organic substrates, air and soils were evaluated. The frequency of occurrence of A. flavus ranged from 76% in maize to 87% (coconut and groundnut) and 92% in makhana (Euryale ferox indica.) Incidence was lowest in green gram (42%). Analysis of variance showed that the percentage incidence of A. flavus in the aerosphere of maize fields was significantly affected by season × location. In soil samples the frequency of occurrence of A. flavus was high during the monsoons (76% in non-diara land soils, 69% in diara land soil). Among 1706 isolates of A. flavus obtained from different sources, 826 (48.4%) were found to be toxigenic. The frequency of non-toxigenic strains of A. flavus was comparatively higher (ratio = 1.07:1) than the toxigenic strains. Percentage incidence of the toxigenic strains of A. flavus was the highest (73.3%; ratio = 0.36:1) in the soil samples. No attempt was made to differentiate A. flavus and A. parasiticus and therefore all references to A. flavus include A. parasiticus.     

Population ecology of Aspergillus flavus associated with Mississippi Delta soils

Understanding the source of Aspergillus flavus is required to manage aflatoxin contamination of maize (Zea mays L.). Studies assessed A. flavus propagules, Fusarium spp., and total fungi associated with Mississippi Delta soils. Soils from 12 and 15 sites were collected in 2000 and 2001, respectively. The propagule density of A. flavus ranged from log(10) 2.0 to 4.3 colony-forming units (cfu) g^-1 soil, while total fusaria ranged from log(10) 3.0 to 5.4 cfu g^-1 soil. The highest populations of A. flavus were associated with soils containing higher organic matter, especially in sites under a no-tillage management. The frequency of aflatoxin production in isolates ranged from 13 to 81% depending on soil. In 2001, there was a highly significant correlation between A. flavus and the history of maize cultivation. Soil fertility factors such as organic matter content, nitrate and extractable phosphorus correlated with the density of Aspergillus, Fusarium spp., and total fungi. The relationship between soil parameters and Aspergillus populations may be useful in predicting the contribution of soil microflora to aflatoxin contamination.     

Effect of pH on ochratoxin A production by Aspergillus niger aggregate species

The effect of pH (2-10) on growth and ochratoxin A (OTA) production by 12 Aspergillus niger aggregate strains was studied in two culture media: Czapek yeast autolysate agar (CYA) and yeast extract sucrose agar (YES), over 30 days. The strains were selected to include different sources, different reported abilities to produce OTA and different ITS-5.8S rDNA RFLP patterns. YES was a better culture medium than CYA for OTA production. In this medium, OTA was produced from pH 2 or 3 to 10 depending on the strain. The results show the ability of A. niger aggregate strains not only to grow, but also to produce OTA over a wide pH range. The results will lead to a better understanding of the role of A. niger aggregate strains in the OTA contamination of several food commodities.     

Insect pests, Aspergillus flavus and aflatoxin contamination in stored wheat: A survey at North Bihar (India)

Fifty stored wheat samples (25 each of insect pest free and infested) were collected from North Bihar (India). Of the four major pests recorded, Sitophilus oryzae was the dominant insect, followed by Tribolium castaneum, Rhizopertha dominica and Trogoderma granarium. Aspergillus flavus was recorded in all samples: infection in insect-damaged samples was 87% and in insect-free samples 25%. Aflatoxin contamination was found in 19 and 2 of the insect-damaged and insect-free lots respectively. The role of insect pests in making wheat grains vulnerable to A. flavus infection and subsequent aflatoxin production is discussed.     

Aflatoxin formation in hay treated with formic acid and in isolated strains of Aspergillus flavus

The possibility of improving the keeping qualities of field-dried and hayloft-dried hay, with dry matter contents of 55 and 73% by adding sodium chloride, formic acid and propionic acid was investigated. Field-dried, sodium-chloride-treated hay was stored in containers, while controls and acid-treated hay were pressed into high-density bales. All hayloft-dried hay was stored in containers. Fungal attacks were very heavy, especially in the baled hay. The contaminating flora was examined at the temperature maximum and after 40 and 32 days storage for field-dried and hayloft-dried hay respectively. The genera Penicillium, Cladosporium and Trichothecium were predominant in hayloft-dried hay, and the differences between varying dry matter contents were insignificant. Mostly Aspergillus spp., but also Penicillium spp., occurred in the field-dried hay at 73% dry matter content but at 55% the flora consisted mostly of Aspergillus flavus, and the formic acid-treated hay contained almost a pure culture of this fungus. In addition, aflatoxin B₁ and G₁ (635–1000 μg/l) were demonstrated in this formic acid-treated hay.

Toxin formation in isolates of A. flavus from differently treated hay specimens was also investigated on various substrates.     

Comparison of surface sampling methods and cleanability assessment of stainless steel surfaces subjected or not to shot peening

The cleanability of AISI 304 stainless steel surfaces, indicated by the removal of Escherichia coli cells or Aspergillus niger spores was assessed by controlled inoculation and washing treatment of samples in standardised conditions. Two systems of recapture (Rodac plate technique and swabbing technique) were compared. Four industrial finishes, subjected or not to shot peening, contaminated at low concentration (1–10 cfu/cm²), and then washed with distilled water or alkaline detergent, were examined. The Rodac plate technique detected most of microorganisms inoculated (80% for E. coli cells and 67% for A. niger spores), whereas the swabbing technique recovered only 1% of the E. coli cells and 26% of the A. niger spores. Using the Rodac plate technique E. coli cells proved to be easily detachable from samples either with distilled water (98%) or alkaline detergent (>99%). For the surfaces contaminated with A. niger spores, the cleanability increased from 34% with distilled water to 77% with alkaline detergent. In these working conditions type of finish (shot treated or not) had no significant effect on cleanability of stainless steel.     

Inhibition of fungal growth on bread by volatile components from spices and herbs, and the possible application in active packaging, with special emphasis on mustard essential oil

The effect of modified atmosphere packaging (MAP) on the most important spoilage fungi of bread was investigated. Penicillium commune, P. roqueforti, Aspergillus flavus and Endomyces fibuliger were able to grow at oxygen levels down to 0.03%, while the chalk mould E. fibuliger was capable of growing even in the presence of an oxygen absorber. High levels of carbon dioxide retarded growth but not completely. As an alternative to MAP active packaging (AP) using volatile essential oils (EO) and oleoresins (OL) from spices and herbs were tested against a range of fungi commonly found on bread. Concentrations of 1, 10 or 100 μl EO or OL were added to a filter paper placed in the lid of a Petri dish inoculated with one of the test fungi. The Petri dish was sealed hermetically to avoid the exchange of gases. Mustard essential oil showed the strongest effect. Cinnamon, garlic and clove also had high activity, while oregano oleoresin only inhibited growth weakly. Vanilla showed no inhibitory effect towards the tested microorganisms at the applied concentrations. A. flavus was more resistant than the other microorganisms while P. roqueforti was the most sensitive. Mustard essential oil was investigated in greater detail. The minimal inhibitory concentration (MIC) for the active component, allyl isothiocyanate (AITC), was determined for the same species and an additional three moulds and one yeast. MIC values ranged from 1.8 to 3.5 μg/ml gas phase. Results showed that whether AITC was fungistatic or fungicidal depended on its concentration, and the concentration of spores. When the gas phase contained at least 3.5 μg/ml, AITC was fungicidal to all tested fungi. Results of sensory evaluation showed, that hot-dog bread was more sensitive to AITC than rye bread. The minimal recognisable concentration of AITC was 2.4 μg/ml gas phase for rye bread and between 1.8 and 3.5 μg/ml gas phase for hot-dog bread. These findings showed that the required shelf-life of rye bread could be achieved by active packaging with AITC. Active packaging of hot-dog bread, may nevertheless require the additional effect of other preserving factors to avoid off-flavour formation     

Management and prevention of mycotoxins in peanuts

Contamination of peanuts with mycotoxins, particularly aflatoxins, is a worldwide problem that affects both food safety and agricultural economies. Most countries have adopted regulations that limit the quantity of aflatoxins in food and feed to 20 µg kg^-1 or less; however, environmental conditions in most of the world where peanuts are produced and stored often make it difficult or impossible to attain such low concentrations. In addition to aflatoxins, peanuts are often contaminated with cyclopiazonic acid (CPA). Both mycotoxins are produced by Aspergillus flavus, a ubiquitous fungus that can infect and grow in peanuts under both pre- and post-harvest conditions. Management of mycotoxin contamination in peanuts generally involves removal of high-risk components from shelled lots or the removal of individual, highly contaminated nuts. This is accomplished by various processes such as screening, kernel sizing, electronic colour sorting, hand sorting, and blanching followed by electronic colour sorting. Recently, biological control technology has been developed that prevents much of the contamination that might otherwise occur. Biocontrol is based on competitive exclusion whereby a dominant population of a non-toxigenic strain of A. flavus is established in the soil before peanuts are subjected to conditions favouring contamination. The applied strain competes with toxigenic strains for infection sites, resulting in significantly reduced concentrations of aflatoxins in peanuts. Monitoring of the first commercial use of the technology showed that aflatoxins were reduced by an average of 85% in farmers' stock peanuts and by as much as 98% in shelled, edible grade peanuts.     

Effect of gas barrier characteristics of films on aflatoxin production by Aspergillus flavus in peanuts packaged under modified atmosphere packaging (MAP) conditions

The effects of the gas barrier characteristics of three films (ASI, ASII and ASIII) and storage temperature on the growth of, and aflatoxin production by, Aspergillus flavus in peanuts packaged in air and under a modified gas atmosphere of CO₂:N₂ (65:35) were investigated. Mold growth was barely visible in air packaged peanuts using high-medium barrier films (ASI and ASII) and stored at 20°C with more extensive growth occurring in air packaged peanuts stored at 25 and 30°C. Extensive mycelial growth and sporulation occurred in all air packaged peanuts in a low barrier film (ASIII), especially at 30°C. Gas packaging inhibited mold growth in peanuts packaged in a high gas barrier film at 20°C. However, mold growth occurred in gas packaged peanuts packaged in film ASII at higher storage temperatures while extensive mycelial growth was observed in all peanuts packaged in film ASIII irrespective of storage temperature. Levels of aflatoxin greater than the regulatory limit of 20 ng/g were detected in all air packaged peanuts with the highest level of aflatoxin (76 ng/g) being detected in peanuts packaged in a high gas barrier film ASI. Aflatoxin production was inhibited in gas packaged peanuts using a high barrier film. However, higher levels of aflatoxin were detected in all gas packaged peanuts in medium-low gas barrier films (ASII and ASIII), particularly at higher storage temperatures. This study has shown that MAP using a CO₂:N₂ (65:35) gas mixture was effective in controlling aflatoxin production by A. flavus in peanuts to levels less than the regulatory limit of 20 ng/g. However, the antimycotic effect of low O₂-high CO₂ atmospheres is dependent on the gas barrier characteristics of the packaging films, especially at higher storage temperatures.     

Mycotoxin occurrence and Aspergillus flavus soil propagules in a corn and cotton glyphosate-resistant cropping systems

The effects of cotton-corn rotation and glyphosate use on levels of soil-borne Aspergillus flavus, aflatoxin and fumonisin contamination in corn and cotton seed were determined during 2002-2005 in Stoneville, Mississippi (USA). There were four rotation systems (continuous cotton, continuous corn, cotton-corn and corn-cotton) for both glyphosate-resistant (GR) and non-GR cultivars-herbicide system arranged in a randomized complete block design with four replications. Aspergillus flavus populations in surface (5-cm depth) soil, sampled before planting (March/April), mid-season (June) and after harvest (September), ranged from 1.47 to 2.99 log (10) cfu g^-1 soil in the four rotation systems. Propagules of A. flavus were higher in the continuous corn system compared to the continuous cotton system on three sample dates, and cotton rotated with corn decreased A. flavus propagules in three of nine sample dates. Propagules of A. flavus were significantly greater in plots with GR cultivars compared to non-GR cultivars in three samples. In cotton seed, aflatoxin and fumonisin levels were similar (≤4 µg kg^-1 and non-detectable, respectively) regardless of rotation and glyphosate. In corn grain, aflatoxin was above the regulatory level (≥20 µg kg^-1) only in GR cultivar in 2004 and 2005. Fumonisin was higher in non-GR cultivar (4 mg kg^-1) regardless of rotation in 2004; however, in 2002, 2003 and 2005, aflatoxin and fumonisin levels were similar regardless of rotation and glyphosate. These results indicate the potential for increased aflatoxin and fumonisin levels (1 of 4 years) in corn; however, climatic conditions encountered during this study did not allow for mycotoxin production. In laboratory incubation studies, fairly high concentrations of glyphosate were required to inhibit A. flavus growth; however no short-term effect of soil treatment with glyphosate on A. flavus populations were observed. These data suggest that altered populations of A. flavus or higher aflatoxin concentrations in corn grain were due to indirect effects of the GR cropping system.     

The effect of storage time and agroecological zone on mould incidence and aflatoxin contamination of maize from traders in Uganda

A study to determine mould incidence and aflatoxin contamination of maize kernels was carried out among dealers (traders) in the three agroecological zones of Uganda. The maize kernels were categorized into those stored for two to six months or for more than six months to one year. Results indicate that the mean moisture content of the kernels was within the recommended safe storage levels of ≤ 15% but was significantly lower in the Highland maize kernels followed by the Mid-Altitude (dry) kernels while the Mid-Altitude (moist) kernels had the highest levels. Across the agroecological zones, Aspergillus, Fusarium, Penicillium and Rhizopus were the most predominant fungal genera identified and, among their species, A. niger had the highest incidence, followed by A. flavus, F. verticillioides, A. wentii, A. penicillioides and Rhizopus stolonifer. There were more aflatoxin positive samples from the Mid-Altitude (moist) zone (88%) followed by those samples from the Mid-Altitude (dry) zone (78%) while samples from the Highland zone (69%) were least contaminated. Aflatoxin levels increased with storage time such that maize samples from the Mid-Altitude (dry and moist) stored for more than six months had mean levels greater than the 20 ppb FDA/WHO regulatory limits. Aflatoxin B₁ was the most predominant type and was found to contaminate maize kernels from all the three agroecological zones. These results indicate that maize consumers in Uganda are exposed to the danger of aflatoxin poisoning. Thus, there is the need for policy makers to establish and enforce maize quality standards and regulations related to moulds and aflatoxins across the agroecological zones to minimize health hazards related to consumption of contaminated kernels.     

单宁酶的研究与应用

运用基因工程手段,构建高效表达单宁酶的黑曲霉重组菌株,并对重组酶活性及酶学性质进行研究,以期提高单宁酶的表达,更好地实现单宁酶在茶饮料、饲料等行业的重要作用。以内源高表达的glaA基因位点为整合靶位点,集成glaA多拷贝强启动子PglaA6R和信号肽SglaA,构建单宁酶基因AnTan黑曲霉重组表达载体pSZHG6R-AnTan,采用农杆菌介导法转化黑曲霉,获得单宁酶黑曲霉纯合重组菌株A1。聚丙烯酰胺凝胶电泳（SDS-PAGE）检测结果表明,目的蛋白大小约为76 kDa;以10%糊精为碳源摇瓶发酵至11 d时,酶活最高达到134.36 U·mL⁻¹,约为宿主菌株的192倍。酶学性质研究表明,最适温度为50 ℃,高温时热稳定性较差;最适pH为7.0。K⁺和Ca²⁺对重组酶没有影响;Mg²⁺对重组酶有微弱的促进作用;Zn²⁺对重组酶有明显的促进作用;Cu²⁺和Fe²⁺对重组酶有微弱的抑制作用;Mn²⁺和EDTA对重组酶有明显的抑制作用。研究结果为进一步优化单宁酶的分泌表达提供了有价值的参考。     

Some biological and chemical characteristics of damaged corn

The predominant fungi isolated from damaged kernels of corn (maize) plated on agar media were species of Aspergillus (mainly A. glaucus) and Penicillium. Masses of spores and mycelium of Aspergillus, Penicillium, Cladosporium and Fusarium were present in the cavities of the embryo of many kernels from which no fungi grew. Some samples had a high percentage of blue-eye kernels, evidently caused mainly by A. restrictus and A. glaucus. The embryos of all damaged kernels were decayed to some extent by fungi. Weight averaged 51 · 7 lb/bu, not much below that of sound corn. Fat acidity values ranged from 90–324. No aflatoxin was found in any of the 24 samples tested.