Kultur vonAspergillus parasiticus im Apfelsaft

Zusammenfassung Kulturen vonAspergillus parasiticus NRRL 2999 in Apfelsaft (aus Sirup) wurden Natriumbenzoat bzw. Kaliumsorbat (100, 200, 300 und 400 mg/l zugesetzt, danach wurden diese bei 25 °C, 3, 6, 9, 12, oder 15 Tage bebrütet und Myceltrokkengewicht, pH und Konzentration der Aflatoxine B₁ und G₁ bestimmt. Der pH-Anfangswert von 2,5 blieb in allen Fällen während der ganzen Inkubationsdauer unverändert. Natriumbenzoat unterdrückte bei allen getesteten Konzentrationen das Wachstum und förderte die Biosynthese von Aflatoxin G₁, während es die Anhäufung von B₁ wenig beeinflußte. Kaliumsorbat förderte das Wachstum bei 100 mg/kg, doch alle getesteten Konzentrationen inhibierten die Produktion der Toxine erheblich (keine nachweisbaren Mengen von B₁ und 3- bis 5 mal weniger G₁ als in der Kontrollreihe). Ausnahmslos wurde Aflatoxin G₁ stärker als B₁ angehäuft.

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The cultivation ofAspergillus parasiticus on apple juiceI. Influence of sodium benzoate and potassium sorbate on fungal growth and aflatoxin biosynthesis

Summary Sodium benzoate or potassium sorbate (100, 200, 300 and 400 mg/l) were added to cultures ofAspergillus parasiticus NRRL 2999 on apple juice (from syrup) and incubated quiescently at 25 °C for 3, 6, 9, 12 or 15 days. The cultures were analyzed for pH, mycelial dry weight and accumulation of aflatoxin B₁ and G₁. The initial pH of 2.5 remained constant in all instances throughout the incubation period. Sodium benzoate, at all concentrations, supressed fungal growth and stimulated the biosynthesis of G₁, whereas little influence was exerted upon the accumulation of B₁. Potassium sorbate stimulated fungal growth at 100 mg/l, while at all concentrations it considerably inhibited toxin production (no detectable amounts of B₁ and 3 to 5 times less G₁ than in controls). The concentration of G₁ surpassed that of B₁ without exception.

     

Aflatoxinbildung in butter und margarine

Zusammenfassung Drei Stämme vonAspergillus flavus entwickelten sich — wenn auch sehr langsam — auf Butter, zwei dieser Stämme bildeten geringe Mengen an Aflatoxinen. Auf Margarine konnte sich nur einer von drei untersuchtenAspergillus flavus-Stämmen entwickeln und dabei Spuren an Aflatoxinen bilden.

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Production of aflatoxins in butter and margarine

Summary Three strains ofAspergillus flavus developed very slowly on butter. Two of these strains produced small amounts of aflatoxins. On margarine only one of the three tested strains ofAspergillus flavus was able to grow. This strain produced traces of aflatoxins on margarine.

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Die Arbeit wurde mit Unterstützung der DFG durchgeführt. FrauK. Blase danken wir für die sorgfältige Unterstützung bei der technischen Durchführung der Versuche.     

Aflatoxin production by Aspergillus flavus in Brazil nuts

Experiments were conducted to evaluate the effects of relative humidity (r.h.; 75%, 80%, 85%, 97%) and temperature (10, 13, 15, 25, 30 °C) on aflatoxin production in previously dried (3.5% moisture content; m.c.) Brazil nuts. Initially Aspergillus spp. were isolated from the surfaces of whole in-shell (WIS) Brazil nuts imported from Peru using A. flavus and A. parasiticus agar (AFPA). Isolates were subsequently screened for aflatoxin production using yeast extract sucrose medium. Total aflatoxin (B₁+B₂+G₁+G₂+M₁) was analyzed using an immunoassay technique while the presence of aflatoxin was confirmed using thin-layer chromatography. The surface of shelled half-nuts (simulating damaged or trimmed nuts), shelled whole (SW) nuts, and WIS nuts following a chlorine wash and water rinse, served as sites for inoculation (10 μl; 10⁵/ml) using an aflatoxigenic isolate. Maximum concentrations of total aflatoxin and B₁ were detected in nuts stored at 97% r.h. and at temperatures of 25–30 °C. Shelled half-nuts contained the highest total (6817 ng/g) and B₁ (4483 ng/g) aflatoxin. WIS nuts contained the least total and B₁ toxin with maximum concentrations of 93 and 49 ng/g, respectively. Aflatoxin was not detected (detection limit of 1.75 ng/g) in nuts maintained at either 10 °C (97% r.h.) or at 30 °C (75% r.h.) for up to 60 d. Maximal moisture contents (%) and water activity values (a_W) for nuts stored at these conditions were 4.50 and 0.78, and 9.14 and 0.92, respectively. Results of this study indicate that the limiting moisture content and a_W values required to control aflatoxin production () in SW and WIS stored at 30 °C for up to 60 d are 4.5, 0.68, 5.0, and 0.75, respectively. Overall, increasing the relative humidity and temperature during storage resulted in an increase in aflatoxin and these were shown to be the most significant variables influencing toxin production in Brazil nuts.     

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.     

Zur Aflatoxinbildung in Milch und Milchprodukten IV. Modellversuche mit Milchpulver

Zusammenfassung In Modellversuchen mit Magermilchpulver konnte nach Beimpfung mit den 3 SchimmelpilzenAspergillus flavus, Aspergillus parasiticus undPenicillium puberulum eine Bildung von Aflatoxin B₁ und G₁ beobachtet werden. Je nach dem Wassergehalt und Pilzart schwankte die Aflatoxinmenge zwischen 0,06–19,2 mg je 100 g Milchpulver (jeweils auf Trockenmasse bezogen).

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IV. Model experiments with milk powder

Summary In model experiments with skim milk powder, a formation of aflatoxin B₁ and G₁ could be observed after inoculation with the following moulds:Aspergillus flavus, Aspergillus parasiticus andPenicillium. puberulum. The amount of aflatoxin found varied between 0.06–19.2 mg/100 g milk powder (related to dry matter), depending on the water content and on the mould strain.

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Als Mitteilungen I–III gelten die bereits in dieser Z. veröfentlichten Publikationen (vgl. 1–3).

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Frau Gertrud Behringer sei auch an dieser Stelle für ihre experimentelle Mitarbeit gedankt.

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Die Deutsche Forschungsgemeinschaft, Godesberg, hat diese Arbeit durch eine Sachbeihilfe in dankenswerter Weise nachhaltig unterstützt.

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Die Deutsche Forschungsgemeinschaft, Godesberg, hat diese Arbeit durch eine Sacbeihilfe in dankenswerter Weise nachhaltig unterstützt.     

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.     

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.     

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.     

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     

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。     

Wirkung von Sorbins?ure und Sorboylpalmitat auf die Aflatoxinbildung im Brot

Zusammenfassung Unter Bedingungen, die für das Wachstum von Schimmelpilzen optimal sind, verlängert ein Zusatz von 0,13% Sorbinsäure die Haltbarkeit von Weizenbrot um 100%, der Zusatz einer entsprechenden Menge an Sorboylpalmitat um 180%, bezogen auf die Haltbarkeit unkonservierter Kontrollproben. Beide Konservierungsstoffe verzögern die Aflatoxinbildung erheblich, Sorboylpalmitat stärker als Sorbinsäure.

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Effect of sorbic acid and sorboyl-palmitate on aflatoxine-formation in bread

Summary Under conditions which are optimal for the growth of moulds, addition of 0.13% sorbic acid increased the keeping quality of wheat bread for 100%, addition of a corresponding amount of sorboyl-palmitate for 180%, as compared to untreated control samples. Both preservatives considerably retard the aflatoxine formation, sorboyl-palmitate stronger, however, than sorbic acid.

     

Zur Aflatoxinbildung in Milch und Milchprodukten VII. Zur Problematik der Probenahme in aflatoxinhaltigen Lebensmitteln

Zusammenfassung Von einem Käselaib wurde bei mehr als 100 Proben — über die ganze Oberfläche verteilt — der Gehalt an Aflatoxin B₁, B₂, G₁ und G₂ halbquantitativ bestimmt. Erwartungsgemäß traten regional sehr unterschiedliche Konzentrationen an Aflatoxin auf. Daraus ergeben sich Schwierigkeiten für die Probenahme and für die rechtliche Beurteilung.

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On aflatoxin formation in milk and milk products VII. Difficulties in sampling of foods containing aflatoxin

Summary In more than 100 samples distributed over the whole surface of one cheese loaf, the content of aflatoxine B₁, B₂, G₁ and G₂ was determined half-quantitatively. As expected, there were very different concentrations of aflatoxines in different sections. From there, problems of sampling and legal evaluation arise.

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Vgl. von den vorausgegangenen besonders Mitteilung III. (1)     

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 ℃。     

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。     

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.     

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.     

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。