Inhibition of growth and mycotoxin production of Aspergillus flavus and Aspergillus parasiticus by extracts of Agave species

In this work, the effect of ethanolic, methanolic and aqueous extracts of Agave asperrima and Agave striata on growth and production of aflatoxin (in A&M medium) and cyclopiazonic acid (CPA; in Czpaek-Dox medium) and on growth in corn under storage conditions was determined. Aspergillus strains were inoculated (10(6) conidia per ml of medium or per 6 g of corn), then plant extracts were added and incubated without shaking at 28 degrees C for 8 days (for aflatoxin-producing analysis) or for 12 days (for CPA-producing analysis). Aflatoxin was assayed by HPLC and cyclopiazonic acid by absorbance at 580 nm using the Erlich reagent. The extracts that most effectively inhibited growth were those from the flowers of both plants. These exhibited an MIC from 0.5 to 2 mg/ml in culture media. Extracts from scape showed an MIC from 15 to 30 mg/ml in culture media. The MIC of the flower extracts was higher (>30 mg/g) when examined in corn. However, concentrations lower than the MIC drastically inhibited production of aflatoxins in culture medium or in corn. Half of the MIC inhibited 99% of the production of aflatoxins and 85% of cyclopiazonic acid.     

Aspergillus flavus population isolated from soil of Argentina's peanut‐growing region. Sclerotia production and toxigenic profile

The Aspergillus flavus population was evaluated in the period 1998–2001 in soil samples from the peanut‐growing region in Argentina. A total of 369 A flavus isolates were examined for sclerotia, aflatoxin and cyclopiazonic acid production. The L phenotype was isolated in a higher percentage than the S phenotype and represented 59% of the total isolates. Statistical analysis showed significant differences between L, S and non‐sclerotial strains with regard to aflatoxin and cyclopiazonic acid production (p < 0.05). The S strains produced higher mycotoxin levels than the L and non‐sclerotial strains. About 10% of the S strains had an unusual pattern of mycotoxin production because they simultaneously produce aflatoxins B and G and CPA. The S_BG strains isolated in the present study have all morphological and microscopic characteristics of A flavus. These strains are of concern in food safety, as there is a higher probability of aflatoxin contamination in peanuts. Copyright © 2005 Society of Chemical Industry     

一株防治花生采后黄曲霉污染的拮抗菌的筛选及其防控效果

为预防花生储藏过程中的黄曲霉污染,从农田土中分离出2株拮抗芽孢杆菌,通过平板对峙法优选出1株作为研究对象,研究其对黄曲霉的抑制效果及对花生采后黄曲霉污染的防控效果。采用平板对峙法检测拮抗菌的抑菌广谱性并绘制生长曲线,并通过生理生化实验、形态特征观察及16S rDNA序列分析对该菌株进行鉴定。结果表明：优选出拮抗菌B419作为研究对象,其可有效抑制黄曲霉的生长,在拮抗菌浓度为1.0×10⁶ CFU/mL,黄曲霉孢子浓度为1.0×10³ CFU/mL时,黄曲霉孢子萌发抑制率达到98%;相比拮抗菌无菌发酵滤液和拮抗菌细胞悬浮液,拮抗菌发酵液防治花生黄曲霉污染效果最好;该菌株对互隔交链孢霉、枝孢霉、青霉、拟盘多毛孢等多种霉菌都有抑制作用,对大肠杆菌、酵母菌也有抑制作用;该菌株在培养4 h后进入对数期,14 h达到最大,26 h后进入衰亡期;该菌株被鉴定为贝莱斯芽孢杆菌(Bacillus velezensis)。综上,分离的拮抗菌B419对多种微生物都有抑制作用,不仅可以用来预防花生储藏过程中的真菌污染,在防治植物微生物病害方面也具有良好的应用潜力。     

Phenolic compounds in peanut seeds: Enhanced elicitation by chitosan and effects on growth and aflatoxin B1 production by Aspergillus flavus

Effects of chitosan and Aspergillus flavus to enhance elicitation of phenolic compounds in viable peanut seeds were conducted at two water activity levels. In vitro effects of phenolic acids on A. flavus growth and aflatoxin B₁ production were also studied. Chitosan enhanced elicitation of free phenolic compounds (FPC) at Aw .85 and .95 levels. A. flavus initially decreased and subsequently increased FPC content, but bound phenolic compounds (BPC) decreased during incubation. Chitosan + A. flavus treatment caused an increase in FPC reaching a plateau between 24–48 h at Aw .85 while BPC levels increased over the same period at both Aw levels. Major free and bound phenolic acids detected were p‐coumaric, ferulic and an unknown phenolic acid eluting at a retention time of 22 min. Generally, chitosan significantly enhanced elicitation of free ferulic and p‐coumaric acids and bound p‐coumaric acid at Aw .95. Free unknown phenolic and bound ferulic acids at Aw .85 were enhanced by chitosan. A. flavus caused significant induction of bound p‐coumaric and ferulic acids and free unknown phenol at Aw .85. Chitosan + A. flavus enhanced free p‐coumaric (3 h) and unknown phenolic acids and bound p‐coumaric acid at Aw .95 while bound ferulic acid was enhanced at Aw .85. Chitosan limited A. flavus growth and subsequent aflatoxin production by inducing susceptible tissues to produce more preformed phenolic compounds.

Analysis of liquid cultures of A. flavus revealed that p‐coumaric, ferulic, and vanillic acids and a mixture of these phenolic acids slightly inhibited mycelial growth. Production of aflatoxin B₁ by A. flavus was completely inhibited at 1 mM and 10 mM concentrations of the phenolic acids and their mixture on four days of incubation. Mode of action of phenolic acids is likely on the secondary pathway for aflatoxin B₁ production and not on the primary metabolism for fungal growth.     

Potential use of phenolic antioxidants on peanut to control growth and aflatoxin B1 accumulation by Aspergillus flavus and Aspergillus parasiticus

Food‐grade antioxidants: butylated hydroxyanisole (BHA), propyl paraben (PP) and butylated hydroxytoluene (BHT) (10 and 20 mmol g^?1) and all the mixtures of these chemicals were tested for inhibitory activity on the growth of and aflatoxin B₁ (AFB₁) accumulation by Aspergillus parasiticus and A. flavus on irradiated (7 kGy) peanut grains. Also, the influence of these treatments was evaluated in different water conditions (0.982, 0.955, 0.937a_w) at 11 and 35 days of incubation at 28 °C. Water activity (a_w) affected the fungal growth, no fungal development was observed at the highest stress water condition (0.937a_w). Butylated hydroxyanisole at 10 mmol g^?1 level and all the mixtures with PP and/or BHT were significantly effective (P = 0.05) in increasing lag phase and reducing growth rate and colony forming units per gram of peanut of both Aspergillus section Flavi strains and AFB₁ accumulation. The application of BHA at concentrations of 20 mmol g^?1 alone or with PP and/or BHT totally inhibited fungal growth at 11 and 35 days of incubation. The results suggest that the addition of these chemical mixtures on peanut grains at low levels has potential to impact synergically on the control of Aspergillus section Flavi. Copyright © 2007 Society of Chemical Industry     

茶多酚对黄曲霉生长及产毒能力的影响

基于氧化应激与黄曲霉毒素生物合成的持续相关性,研究了天然抗氧化剂茶多酚对黄曲霉生长及产毒的影响,并通过扫描电镜观察茶多酚对黄曲霉菌丝体形态的影响,旨在寻找一种安全、高效、环境友好型的抑菌剂来控制食品中黄曲霉毒素的污染。结果表明:茶多酚对黄曲霉菌落生长有一定抑制作用,在10 mg/m L浓度下,抑制率达到48.41%,使黄曲霉菌落生长速率为(5.57±0.16)mm/d,延滞期为(1.32±0.13)d;而茶多酚对黄曲霉产毒抑制效果显著,在1,5和10 mg/m L浓度下,茶多酚对AFB1生物合成的抑制率分别达到了60.15%,87.12%和97.48%;且茶多酚可严重破坏黄曲霉菌丝体的超微结构。因此,茶多酚可作为天然抑菌剂应用于食品与饲料中,控制黄曲霉毒素的污染。     

Effect of Equisetum arvense and Stevia rebaudiana extracts on growth and mycotoxin production by Aspergillus flavus and Fusarium verticillioides in maize seeds as affected by water activity

Cereals are a very important part of the human and animal diets. However, agricultural products can be contaminated by moulds and their mycotoxins. Plant extracts, particularly those of Equisetum arvense and Stevia rebaudiana have been reported previously to contain antioxidant compounds which may have antifungal properties. In this study, E. arvense and S. rebaudiana extracts were tested for their control of mycotoxigenic fungi in maize. The extracts were tested separately and as a mixture for their effect on growth of Aspergillus flavus and Fusarium verticillioides. Extracts were added to unsterilised inoculated maize at different water activity (a(w)) levels (0.85-0.95). Moulds were inoculated and incubated for 30 days. Results confirmed that the extract of E. arvense and a mixture 1:1 of Equisetum-Stevia may be effective for the inhibition of both growth of A. flavus and aflatoxin production at high water activity levels (pre-harvest conditions). In general, growth of the F. verticillioides was reduced by the use of plant extracts, especially at 0.95 a(w). However, fumonisin presence was not significantly affected. E. arvense and S. rebaudiana extracts could be developed as an alternative treatment to control aflatoxigenic mycobiota in moist maize.     

Cyclopiazonic acid and aflatoxin production by cultures of Aspergillus flavus isolated from dried beans and maize

From the storerooms of individual households 150 samples of dried beans and 90 samples of stored maize were collected for mycological analyses. Two of 27 isolates of A. flavus grown on malt extract agar (MEA) were found to produce the mycotoxin cyclopiazonic acid (25–36 μg/g). Three of the A. flavus isolates grown on crushed moist wheat produced aflatoxin B₁ (0.72–1.6 μg/g) and 6 of 26 A. ochraceus isolates were OA positive (0.5–10.4 μg/g). None of 25 bean samples were contaminated with CPA, AF or OA, while 4 samples of 30 tested maize samples were OA positive with level of OA 0.4–400 μg/g. Toxins were determined by thin layer chromatography and colorimetric method was used for quantitations of CPA.     

不同植物抑制剂对黄曲霉菌生长和产毒的影响

目的研究不同植物抑制剂对黄曲霉菌生长和产毒的影响。方法将产毒的黄曲霉菌接种在EMA培养基上,活化后分别培养在含不同浓度抑制剂的培养基中,在30℃的温度下恒温培养,通过观察抑菌圈直径的大小判断不同抑制剂对黄曲霉生长的影响,并将菌丝抽提使用酶标仪测定不同抑制条件下黄曲霉毒素含量。结果 0.2mg/mL的紫苏醛、烟草萜和香芹醇在第2d对黄曲霉菌生长抑制率分别为100%、67%和79%,第5d时分别降低到46%、63%和55%。柠檬醛、紫苏醛、香茅醇和芳樟醇处理组培养基中第5d时黄曲霉毒素含量很低,对照组中黄曲霉毒素含量是这4个处理组的2000倍以上,第6d时,6种植物抑制剂的抑制黄曲霉菌产毒能力都有所降低,但是0.2mg/mL的柠檬醛、紫苏醛、香茅醇、烟草萜和香芹醇仍具有较高的抑制黄曲霉菌产毒能力。结论柠檬醛、紫苏醛、烟草萜和香芹醇对黄曲霉菌的生长和产毒有着明显的抑制作用。     

Effects of smoke produced from smoldering plants on the Aspergillus flavus growth and production of aflatoxin in pistachio

Aflatoxin (AF) contamination of the Iranian exporting pistachio has become a major problem in the last decades. In this study, the antifungal effects of smoke produced from the smoldering of several herbal plants were investigated. Four different ratios of plant weight/exposure time (5/5, 10/15, 15/30, and 20 g/45 min) were used from each plant material to smoke two isolates of Aspergillus flavus (A47 and A3), grown in potato dextrose agar (PDA). The results showed that the 20 g/45 min treatment using smoldering cinnamon bark, neem leaves, and clove flowers had efficient inhibitions of 100, 85, and 75%, respectively. Furthermore, the smoking of pistachio inoculated with a spore concentration of 1 × 10⁶ / ml using 30 g of cinnamon bark, neem leaves, and clove flowers for 75 min was capable of preventing the production of different types of AF, that is, B1, B2, G1, and G2 in the treated products. There is a good potential to smoke pistachio with these three herbal plants and prevent the production of AF in pistachio during the handling, storage, and transportation.     

大米中黄曲霉的微波杀菌工艺优化

目的探索微波处理对大米中黄曲霉的杀菌工艺条件。方法以黄曲霉孢子减少对数周期为检测指标,考察微波功率、微波时间和装载量对黄曲霉孢子减少对数周期的影响,在单因素试验的基础上,采用3因素3水平响应面法设计并优化微波杀菌工艺,建立相应的回归方程。结果微波杀菌最佳工艺条件为:微波功率231 W,微波时间32 s,装载量34 g,在此条件下黄曲霉孢子减少对数周期为3.496±0.069,实际值与理论值的相对误差为-9.97%。结论本研究获得了微波杀灭大米中黄曲霉的最佳工艺条件,应用微波技术可以作为粮食杀菌的一种有效手段。     

Inhibitory effect of Aspergillus niger on the growth of Aspergillus ochraceus and Aspergillus flavus,and on aflatoxin formation

Aspergillus ochraceus and A flavus were grown on synthetic media (SM) supplemented with 50 or 200 ml litre^?1 SM on which A niger had been grown previously ( ‘A niger medium’ = ANM). Controls included SM acidified to pH 6.0 or 4.4, SM diluted with 50 or 200 ml litre^?1 water, and diluted-acidified SM. For both fungi, higher growth inhibition was recorded on ANM-containing SM than in the controls. Aflatoxin formation was markedly inhibited on SM to which 20 ml litre^?1 ANM extract (in methanol/chloroform, 2:1 v) had been added, although the growth of A flavus on that medium was almost the same as that in the control. It is concluded that the inhibitory effect of A niger on the growth of fungi should not be attributed merely to pH reduction, but also, mainly, to metabolites produced by the fungus in the growth medium, even at early stages of its growth.     

Aspergillus flavus infection and aflatoxin production in mixtures of high-moisture and dry maize

High-moisture (26·6–27·9% m.c.) and dry (9·8% m.c.) fractions of white and yellow maize were examined for fungal development and aflatoxin production during an 8-week incubation at 25°C. Treatment procedures included blending of either high-moisture white with dry yellow or high-moisture yellow with dry white maize fractions (average moisture in blend, 14%) and inoculation of some test maizes with A. flavus spores. At sampling time white and yellow components of maize blends were manually separated and all of the maize samples were analyzed for levels of moisture, fungal infection and aflatoxin. Moisture levels in maize blends equilibrated rapidly during the initial 2–4 days of incubation; neither dry yellow nor dry white exceeded 13% moisture during the trial period. Only a limited incidence of A. flavus was observed on uninoculated maize. but in samples treated with A. flavus spores a high infection rate developed; from 58 to 98% of the kernels in dry fractions of inoculated blends were infected with A. flavus during the trial. Aflatoxin was detected in high-moisture maize and in both high-moisture and dry fractions of inoculated maize blends. Up to 500 μg aflatoxin B₁/kg of corn was found after the 8-week incubation in a dry fraction of inoculated maize blends.     

Aflatoxin-producing potential of Aspergillus flavus and Aspergillus parasiticus isolated from samples of smoked-dried meat

Over a period of three years 420 samples of various smoke-dried meat products, collected from individual households in different region of Croatia were analysed for the presence of aflatoxigenic strains of the Aspergillus flavus group. Strains of A. flavus and A. parasiticus were present in 17,8% of the samples, and aflatoxin-producing ability was tested in 75 strains. In relation to sequential method of aflatoxin detection, 5 of 8 isolates were found in the first step (fluorescence in aflatoxin-producing ability medium - APA) and all of them in the second step (extraction method from syntheses on moist shredded wheat - SW). A. flavus strains produced mainly aflatoxin B₁, and had various levels of toxigenicity (1.4–3.12 mg/kg). Some strains of A. parasiticus produced all four aflatoxins B₁ B₂ G₁ G₂, while the other ones produced AF B₁ + G₁ only, with concentrations of aflatoxins from 0.1 to 450 mg/kg.     

Temperature,water activity and gas composition effects on the growth and aflatoxin production by Aspergillus flavus on paddy

The main aim of this study was to evaluate the combined effects of temperature with water activity (a_w) and CO₂ with a_w on the growth and aflatoxin production by Aspergillus flavus Link on paddy. The effects of temperature (20–30 °C) and a_w (0.92–0.98) on the relationship between colony diameter and aflatoxin production, and the influence of a_w (0.92–0.98) and CO₂ (20–80%) on the growth and toxin production were studied using full factorial design. Colony diameters were regularly measured and aflatoxins were periodically analyzed using HPLC with fluorescence detector. The growth and aflatoxin formation increased with a_w at the temperatures studied, and toxin production was positively correlated with the incubation time and colony diameter. Except at 0.92 a_w, as much as 80% CO₂ failed to inhibit the growth of fungi completely. However, at all a_w levels studied the growth parameters as estimated by Baranyi function and aflatoxin were affected by the increment in CO₂ where growth rates and aflatoxin were negatively correlated with CO₂ while the lag phase durations were positively correlated with CO₂. Under 0.98 a_w, the atmosphere enriched with 20% and 80% CO₂ lead to at least 59% and 88% reduction in growth and 47% and 97% in the toxin production, respectively. At 0.95 a_w, the lag phases of both isolates in average increased by a factor of 1.7–12 when the CO₂ levels in the headspace were between 20 and 80% compared to the control. The growth rate and lag phase durations under the modified atmospheres were successfully described using a polynomial equation (R² > 0.97). The results of the study could form a basis of indicative guidelines on the possible control of A. flavus and aflatoxin in paddy during temporary storage prior to drying.     

Influence of gamma-irradiation and maize lipids on the production of aflatoxin B1 by Aspergillus flavus

The effect of gamma-irradiation and maize lipids on aflatoxin B1 production by Aspergillus flavus artificially inoculated into sterilized maize at reduced water activity (aw 0.84) was investigated. By increasing the irradiation doses the total viable population of A. flavus decreased and the fungus was completely inhibited at 3.0 kGy. The amounts of aflatoxin B1 were enhanced at irradiation dose levels 1.0 and 1.5 kGy in both full-fat maize (FM) and defatted maize (DM) media and no aflatoxin B1 production at 3.0 kGy gamma-irradiation over 45 days of storage was observed. The level in free lipids of FM decreased gradually, whereas free fatty acid values and fungal lipase activity increased markedly by increasing the storage periods. The free fatty acid values decreased by increasing the irradiation dose levels and there was a significant enhancement of fungal lipase activity at doses of 1.0 and 1.50 kGy. The ability of A. flavus to grow at aw 0.84 and produce aflatoxin B1 is related to the lipid composition of maize. The enhancement of aflatoxin B1 at low doses was correlated to the enhancement of fungal lipase activity.     

Mould infection and aflatoxin contamination of the peanut kernels harvested from spring and fall crops as affected by artificial inoculation of the seeded kernels with Aspergillus flavus and Aspergillus niger

Spring and fall crops of peanut are grown each year in Taiwan. Mould infection and aflatoxin contamination of crops as affected by artificial inoculation of the seeded kernels with conidia of Aspergillus flavus, A niger and a combination of A flavus and A niger (inocula > 10⁵ CFU kernel⁻¹) were determined. Three cultivars, ie Tainan 9, Tainan 11 and Tainan 12, were consecutively grown for fall 1996, spring 1997, fall 1997 and spring 1998 crops with green vegetable pea for rotation. In crops from uninoculated kernels (control), percentages of germination and harvested plants were higher in spring crops than in fall crops. Inoculation with A niger alone or with A flavus resulted in various levels of seed and seedling mortality and lower yields of peanut pods than yields of the other inoculation treatments. When harvested and sized (US No 1), kernels were subjected to examination for mould colonisation and analysis of aflatoxin content. Fairly low percentages were colonised and aflatoxin contents were low or non-detectable. Colonisation and aflatoxin content were independent of artificial inoculation. Average aflatoxin contents in the kernels harvested from all cultivars and crops ranged from 0 to 6.1 µg kg⁻¹. However, the highest levels of aflatoxin content among samples of the four crops were 4.0, 18.2, 9.6 and 36.7 µg kg⁻¹, respectively. © 1999 Society of Chemical Industry