Use of UV photography to identify aflatoxin-producing strains ofAspergillus flavus andA. parasiticus

UV photography in glucose, yeast extract (GY) agar medium was tested as a simple and rapid method for the distinction of afiatoxin-positive from aflatoxin-negative strains ofAspergillus flavus andA. parasiticus. In the UV photographs aflatoxin-producing moulds were identified as grey or black colonies, whereas aflatoxin-non-producing moulds appeared as white colonies. Of the afiatoxin-positive strains detected by the UV photographic method, 10% was confirmed by extraction of the GY agar medium and mould mycelium in chloroform, extracts which were analysed subsequently using thinlayer chromatography. Confirmation of aflatoxigenic strains was achieved by biosynthesis on liquid medium yeast extract sucrose (YES) broth.     

Mature seeds of Sorghum contain proteins toxic to aflatoxin-producing Aspergillus flavus

Crude acid extract of mature Sorghum seeds showed partial inhibition of spore germination and growth of aflatoxin-producing Aspergillus flavus strains. To find the protein components involved in this mechanism of inhibition, the crude extract was fractionated by differential precipitation followed by ion exchange chromatography in carboxymethyl cellulose. Five proteins of low molecular weight were isolated. The ability of each of the five proteins to inhibit the spore germination and hyphal extension of aflatoxin-producing Aspergillus flavus strains was tested. Protein 5 did not show any inhibition. Proteins 1, 2 and 4 showed complete inhibition of spore germination (1500–2000 spores/100 μl of medium) at a concentration of 15 μg/100 μl. Protein 3 showed partial inhibition only.     

Spectral analyses of fluorescences in dent maize infected with Aspergillus flavus and Aspergillus parasiticus

Bright greenish yellow (BGYF) and blue white (BWF) fluorescences were associated with Aspergillus flavus and A. parasiticus infected maize. The fluorescences were studied spectrofluorometrically, the BGYF exhibiting a peak wave length between 480–485 nm and the BWF between 440–445 nm. Neither fluorescence varied in maize stored under different moistures and temperatures.

BWF was similar spectrally to the fluorescence of the endosperm of sound kernels but × 5 20 more intense. The spectrum of BWF was similar to Aflatoxin G₁ or a mixture of aflatoxin B₁, B₂, G₁ and G₂ when they were spotted on endosperm tissue. A color reference for BGYF was similar in peak wave length to BGYF. Amsoy soybeans without the seed coat fluoresced with a peak 470–475 nm and the intensity was low compared to BGYF in maize. A fluorescence of maize kernels visually similar to BGYF but not associated with Aspergillus infection or aflatoxin contamination was also investigated. This “false BGY” fluorescence was spectrally similar to the BGYF in infected kernels.     

Effect of application of nontoxigenic strains of Aspergillus flavus and A. parasiticus on subsequent aflatoxin contamination of peanuts in storage

Experiments were conducted to determine the potential for biological control of aflatoxin contamination of peanuts during storage. Florunner peanuts were treated in field plots by applying competitive, nontoxigenic strains of Aspergillus flavus and A. parasiticus, at 76 and 67 days after planting in 1998 and 1999, respectively. After harvest, half the peanuts from both treated and control plots were sprayed with an aqueous conidial suspension containing the nontoxigenic strains; the other half of the peanuts from each group were not sprayed. The peanuts were then placed in separate compartments of a miniature warehouse. Therefore, storage treatments consisted of peanuts that were (1) not treated at all; (2) treated prior to storage only; (3) field-treated only; (4) treated both in the field and prior to storage. Peanuts were stored for 3-5 months under high temperature and relative humidity conditions designed to promote aflatoxin contamination. In 1998, peanuts were not contaminated with aflatoxins prior to storage. After storage, peanuts that were never treated with the competitive fungi contained an average of 78.0 ppb of aflatoxins. Peanuts not treated in the field but receiving the spray treatment before storage contained 48.8 ppb. Peanuts treated in the field only averaged 1.4 ppb, and peanuts treated both in the field and prior to storage contained 0.8 ppb. In 1999, peanuts suffered from late-season drought and were contaminated with aflatoxins at harvest, with controls averaging 516.8 ppb compared with 54.1 ppb in treated peanuts. After storage, non-field-treated peanuts averaged 9145.1 ppb compared with 374.2 ppb for peanuts that had been field-treated, a 95.9% reduction. Spraying of pods with the nontoxigenic strains postharvest but prior to storage provided no additional protection against aflatoxin contamination. Results demonstrated that field application of the nontoxigenic strains had a carry-over effect and reduced aflatoxin contamination that occurred in storage.     

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.     

Environmental factors modify carbon nutritional patterns and niche overlap between Aspergillus flavus and Fusarium verticillioides strains from maize

This study examined the utilization patterns of key carbon sources (CS, 24: including key sugars, amino acids and fatty acids) in maize by strains of Aspergillus flavus and Fusarium verticillioides under different water activity (a_w, 0.87–0.98 a_w) and temperature (20–35 °C) values and compared the niche overlap indices (NOI) that estimate the in vitro CS utilization profiles [Wilson, M., Lindow, S.E., 1994. Coexistence among epiphytic bacterial populations mediated through nutritional resource partitioning. Applied and Environmental Microbiology 60, 4468–4477.]. The ability to grow in these key CS in minimal media was studied for 120 h in 12 h steps. The NOI was calculated for inter-species (F. verticillioides–A. flavus) and for intra-species (A. flavus–A. flavus) using CS utilization patterns over the range of interacting environmental conditions. 30 °C, over the whole a_w range examined, was found to be optimal for utilization of the maximum number of CS by A. flavus. In contrast, for F. verticillioides this was more so at 20 °C; 25 °C allowed a suboptimal usage of CS for both species. NOIs confirmed the nutritional dominance of A. flavus at 30 °C, especially at lower a_w levels and that of F. verticillioides at 20 °C, mainly at 0.95 a_w. In other conditions of a_w, based on CS utilization patterns, the data indicated that A. flavus and F. verticillioides occupied different ecological niches. The variability in nutritional sources utilization between A. flavus strains was not related to their ability to produce aflatoxins (AFs). This type of data helps to explain the nutritional dominance of fungal species and strains under different environmental conditions. This could be useful in trying to find appropriate natural biocontrol microorganisms to compete with these mycotoxigenic species.     

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.     

Kinetic and mutation analyses of aspartate kinase from thermus flavus

To reveal the catalytic mechanism of Thermus aspartate kinase, each of 29 amino acid residues that were highly conserved in the sequenced aspartate kinases, was replaced with alanine or leucine by PCR site-directed mutagenesis. Comparison of the kinetic parameters of these mutants with those of the wild-type aspartate kinase suggested that Thr47 was involved in binding aspartate and that Lys7 and Glu74 were involved in catalysis. Analysis of the effective concentrations of magnesium ion on the activity showed that the mutants with replacements at Ser41, Thr47, Asp154 and Asp182 required higher concentrations of magnesium ion. This suggests that these four residues play important roles in the binding of magnesium ions which are required for enzymatic activity.     

山苍子精油抑制黄曲霉菌的生长和产毒作用研究

山苍子精油是一种纯天然植物精油,本文研究了其对黄曲霉生长、代谢和毒素产生的抑制作用,探讨了山苍子精油对黄曲霉菌的抑菌能力和作用机理。本研究将花生放置于自然环境染菌并分离纯化目标菌,采用形态学并结合ITS序列法进行菌株分类鉴定;结合抑菌圈、抑菌率和最低抑菌浓度(MIC)的测定探讨山苍子精油对黄曲霉菌的抑制能力;进行了山苍子精油影响黄曲霉孢子萌发率、生长曲线和黄曲霉毒素B1产生的实验研究;从细胞膜渗透性、细胞酶活性的变化探讨了山苍子精油抑制黄曲霉的作用机理。实验结果表明:从腐败花生中分离筛选出菌株HB₂,经ITS序列法鉴定为黄曲霉(Aspergillus flavus);黄曲霉素测定结果显示其含有黄曲霉素B1(AFB1),质量浓度为3.4×10³μg·kg^-1(纯湿菌体);抑菌圈随精油浓度的增大明显变大,对黄曲霉的最低抑菌体积分数(MIC)为0.800μL·mL^-1;孢子萌发率、牙管长度、黄曲霉菌体的生长量和AFB1的浓度随培养液中精油浓度的增大呈显著下降趋势,当山苍子精油浓度为0.100μL·mL     

植物精油对黄曲霉的抑制机理及应用方式研究进展

黄曲霉是一种常见的真菌,其产生的黄曲霉毒素会对人和动物的生命健康造成威胁,近年来黄曲霉毒素中毒事件频繁发生。植物精油作为一种天然的活性物质,对黄曲霉有较强的抑制作用,其抑菌机理和应用方式依旧受到各国科研工作者广泛关注。本文主要概述了黄曲霉的危害,植物精油对黄曲霉的抑制效果及作用机理,总结了近几年植物精油抑制黄曲霉的应用方式,以期为植物精油在抑制黄曲霉中的合理应用提供理论依据。     

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.     

非脱羧勒克菌wt16对黄曲霉菌生长与产毒的抑制作用

黄曲霉菌及其毒素严重威胁农产品质量安全,本文旨在探寻非脱羧勒克菌对黄曲霉菌及其毒素污染防控效果。从湖北黄陂分离筛选出一株非脱梭勒克菌wt16,将其与黄曲霉菌在液体培养基中共培养后测定非脱羧勒克菌wt16菌株对黄曲霉菌生长及产毒的抑制率。结果表明,在沙氏液体培养基中,非脱羧勒克菌wt16能明显抑制黄曲霉菌的生长及产毒,对其菌丝生长的抑制率为77%~92%,对其产毒的抑制率为90%~96%。通过扫描电子显微镜观察发现,非脱羧勒克菌wt16能改变黄曲霉菌丝的形态,使得黄曲霉菌丝体由规则的球体聚集成不规则形状,单个菌丝会由细长型断裂成小截形态,菌丝表面也变得更为光滑;并且发现wt16在花生粉及未受机械损伤的花生颗粒上对黄曲霉菌的生长及产毒均表现出很强的抑制作用。进一步研究发现,非脱羧勒克菌wt16菌株发酵上清液中含有能抑制黄曲霉毒素合成的有效成分,且该发酵上清液的制备以培养4 d以上为最佳,培养温度为15~40 ℃。     

Combined effect of temperature and propionic acid concentration on the growth of Aspergillus parasiticus

Effects of temperature (25, 30 and 36°C) and propionic acid concentration (129, 258 and 516 ppm) on the growth of Aspergillus parasiticus in solid media were analyzed. Growth rate, expressed as the increment of colony diameter per unit of time, was studied as a function of storage time employing the Arrhenius model. The inverse of the lag phase was fitted to an Arrhenius-type equation and the inhibition index was also calculated. A linear relationship between the lag phase and the reciprocal growth rate at different propionic acid concentrations was assessed by linear regression analysis. Fungi behavior was modeled considering the main controlling factors and a response surface methodology was established in terms of propionic acid and temperature. The proposed model could be used in food microbiology to predict the growth of toxicogenic fungi growth.     

钾离子通道在黄曲霉菌孢子萌发中的作用

黄曲霉毒素严重污染农产品,威胁人畜生命健康,而孢子萌发是黄曲霉菌生存及侵染过程中的决定性事件,从最上游阻断孢子萌发可能是有效控制黄曲霉污染、开发新型抗菌药物的突破口之一。研究发现一定浓度的胞外KCl能够影响黄曲霉孢子的萌发。通过使用K+特异性荧光探针PBFI-AM,发现适合孢子萌发的特定浓度K+能够激发黄曲霉孢子瞬间产生较高的钾离子流,说明K+响应可能是黄曲霉孢子萌发的早期信号。4-AP作为特异性电压门控钾离子通道阻滞剂,能够明显抑制并延迟孢子的萌发。通过生物信息学预测,发现黄曲霉菌KCNA具有钾离子通道选择器序列,具备电压门控钾离子通道的α亚基属性。进一步构建KCNA非洲爪蟾卵母细胞表达体系,用双电极电压钳系统测得KCNA外向型电流,一定程度上证明了KCNA电压门控钾离子通道蛋白质属性,也说明电压门控钾离子通道参与了黄曲霉孢子萌发。     

柠檬醛缓释制剂的稳定性及其对黄曲霉菌的抑制作用

本文旨在研究柠檬醛脂质体（Citral Liposome,CL）和柠檬醛-壳聚糖复合脂质体(Citral Liposome-Chitosan,CL-CS)这两种柠檬醛缓释制剂的稳定性及对黄曲霉菌（Aspergillus flavus）的抑菌效果、抑菌机理,为后期开发绿色、高效、安全的柠檬醛缓释抑菌剂提供数据支撑。通过设定不同的温度及pH条件,以外观、粒径、Zeta电位和保留率为指标探究储藏稳定性;用孢子计数法测定柠檬醛缓释制剂的瞬时抑菌率,并分析抑菌长效性;根据细胞通透性变化揭示柠檬醛缓释制剂的抑菌机理。结果表明,两种柠檬醛缓释制剂均具有纳米级粒径且粒径均一,经壳聚糖修饰后,体系Zeta电位由?16.63±1.67变为35.72±3.29。4 ℃条件下储藏28 d后粒径和Zeta电位变化较小,保留率更高,且CL-CS比CL更稳定;pH为4~6时,两种柠檬醛缓释制剂更稳定,CL和CL-CS的粒径别在150和200 nm左右。CL、CL-CS在48 h内对黄曲霉菌的瞬时抑菌EC₅₀分别为77.88和68.20 mg·L?1,继续培养48 h后CL和CL-CS的抑制率分别为67.69%和82.89%,而柠檬醛只有30.26%,表明CL-CS具有良好的瞬时抑菌效果和长效抑菌效果。这两种柠檬醛缓释制剂处理后黄曲霉菌的胞外电导率和核酸含量均升高,说明细胞膜通透性增加,且所需要的作用时间比游离柠檬醛更短。综上,两种柠檬醛缓释制剂具有良好的稳定性,能通过改变细胞通透性、使内溶物渗出,从而抑制黄曲霉菌的生长,且具有长效抑菌能力,CL-CS比CL性能更好,具有开发成植物源防霉剂的潜能。     

Physiological properties of Lactobacillus paracasei, L. danicus and L. curvatus strains isolated from Estonian semi-hard cheese

Growth and survival of Lactobacillus paracasei (six strains), L. danicus sp. nov. (four isolates, two strains) and L. curvatus (two strains) from semi-hard Estonian cheeses were comparatively studied in different environmental conditions of relevance for their growth in cheese and survival in gastric environment. Maximum specific growth rates for L. paracasei strains varied between 0.40 and 0.57 h⁻¹, and all strains were tolerant to low water activities, heating at 60 °C for 30 min and pH 3. The newly discovered genetically distinct species L. danicus was characterized by low maximum growth rates (0.26–0.38 h⁻¹) and low temperature optimum (<30 °C). It was acid and heat sensitive and inhibited at salt concentrations from 4% and water activities below 0.93. L. curvatus was characterized by the highest growth rates (0.65–0.70 h⁻¹), tolerance to high NaCl concentrations, but sensitivity to heating, bile salts and low pH. The study showed that genetically different LAB species isolated from cheese could be distinguished by simple cultivation experiments.     

丝状真菌Talaromyces flavus S1改善污泥脱水性能及机理研究

从城市剩余污泥中分离出一株丝状真菌Talaromyces flavus S1,将预培养的菌丝体接种到污泥后,能够有效地改善污泥脱水性能。通过分离菌丝体培养液,发现并非真菌分泌物(微生物絮凝剂)而是菌丝体本身改善了污泥脱水性能;菌丝体对高浓度污泥的脱水性能影响显著,最多可提高40.58%,污泥浓度较小时,脱水性能不但没有改善反而变差;与聚丙烯酰胺(PAM)和聚合氯化铝铁(PAFC)相比,菌丝体调理对毛细吸水时间的降低更显著,而添加PAM对污泥比阻效果更好,单独添加PAFC效果最差,在PAM与菌丝体协同调理下,污泥的脱水性能可提高60%左右。通过污泥粒径分布与SEM电镜扫描的结果发现:菌丝体通过缠绕包裹小颗粒的污泥,使污泥絮体和污泥颗粒增大从而改善了污泥脱水性能。菌丝体接种的接种方式比孢子直接接种更加实际有效,利用生物法改善了污泥脱水性能,对以该菌为基础开发生物脱水工艺有重要意义。     

Activity of essential oil and its major compound, 1,8-cineole, from Eucalyptus globulus Labill., against the storage fungi Aspergillus flavus Link and Aspergillus parasiticus Speare

The essential oil from leaves of Eucalyptus globulus obtained by hydrodistillation, as well as its major compound 1,8-cineole, identified by gas chromatography coupled with a mass selective detector, were evaluated for their effectiveness against the storage fungi Aspergillus flavus and Aspergillus parasiticus. The evaluation was performed by compound dissolution in yeast extract sucrose (YES) medium and exposure to headspace volatiles. Complete fungal growth inhibition of both species was achieved with the essential oil by contact and volatile assays. Volatile exposure showed total inhibition at the lower level tested of 500 μL. The 1,8-cineole tested alone showed partial inhibition only at the highest level of 1.3492 μL. Aflatoxin B₁ production was reduced in headspace volatile assays and partial inhibition was observed at the 200 μL dose of the essential oil.     

Complex regulation of the aflatoxin biosynthesis gene cluster of Aspergillus flavus in relation to various combinations of water activity and temperature

A microarray analysis was performed to study the effect of varying combinations of water activity and temperature on the activation of aflatoxin biosynthesis genes in Aspergillus flavus grown on YES medium. Generally A. flavus showed expression of the aflatoxin biosynthetic genes at all parameter combinations tested. Certain combinations of a_w and temperature, especially combinations which imposed stress on the fungus resulted in a significant reduction of the growth rate. At these conditions induction of the whole aflatoxin biosynthesis gene cluster occurred, however the produced aflatoxin B₁ was low. At all other combinations (25 °C/0.95 and 0.99; 30 °C/0.95 and 0.99; 35 °C/0.95 and 0.99) a reduced basal level of cluster gene expression occurred. At these combinations a high growth rate was obtained as well as high aflatoxin production. When single genes were compared, two groups with different expression profiles in relation to water activity/temperature combinations occurred. These two groups were co-ordinately localized within the aflatoxin gene cluster. The ratio of aflR/aflJ expression was correlated with increased aflatoxin biosynthesis.     

The influence of oxygen on the differentiation of temperature-sensitive and temperature-insensitive Lactococcus lactis subsp. cremoris starter strains

The differentiation of temperature-insensitive and temperature-sensitive strains of Lactococcus lactis subsp. cremoris by using a modified sodium-β-glycerophosphate/milk medium is described (temperature-insensitive strains are defined as those that continue to grow at 38°C and temperature-sensitive strains as those that do not grow, or grow poorly, at 38°C). The physiological basis for the differentiation assay was examined by using L. lactis subsp. cremoris strains 2146 (temperature-insensitive) and 2182 (temperature-sensitive) as test strains. After aerobic incubation on the medium, strain 2146 formed uniform colonies, 0·5 mm in diameter, while strain 2182 formed larger colonies, 1·0–1·5 mm in diameter. The differential was dependent on the medium constituents, on an aerobic gas phase, and on the effects of H₂O₂ generated within the colonies. The addition of 0·5% (w/v) pyruvate to the medium facilitated the growth of colonies of strain 2146 to 3-mm diameter, while the colony size of strain 2182 remained at 1-mm diameter, and thus the colony-size differential between strains was reversed. The growth of both strains was inhibited at 0·4–0·8% air saturation during suspension culture in sodium-β-glycerophosphate/milk medium. The inclusion of catalase in the cultures overcame the growth inhibition. There was no observable difference between the two strains in their oxygen sensitivity or NADH oxidase/peroxidase enzymology.