黄曲霉菌株的筛选和产毒能力鉴定

本研究从发霉粮食中分离出数株黄曲霉菌菌株,并进行了形态学和分子生物学鉴定。为了探究分离菌株与黄曲霉标准菌株之间产毒能力的差异,通过对分离菌株和黄曲霉标准菌株进行发酵培养和HPLC测定,分析确定产毒能力。结果表明黄曲霉菌株之间产毒能力差异巨大：黄曲霉菌株3.4408产毒量很高,黄曲霉菌株HDWH产毒量很低,黄曲霉菌株3.2572甚至不产生黄曲霉毒素;产生黄曲霉毒素菌株中部分黄曲霉菌株产生四种黄曲霉毒素AFB1、AFB2、AFG1、AFG2,黄曲霉菌株HDWS只产生黄曲霉毒素AFB1、AFB2。     

黄曲霉菌株的分离、鉴定及产毒能力分析

对几株从发霉粮食中分离出的黄曲霉菌菌株进行形态学和分子生物学鉴定,并进行发酵培养和产毒能力的HPLC测定。结果表明:试验分离菌株均为黄曲霉菌株且含有黄曲霉毒素产生的关键基因aflR;黄曲霉菌株之间产毒能力差异巨大:黄曲霉菌株3.4408产毒量最高,黄曲霉菌株HDWS产毒量最低,黄曲霉菌株3.2572甚至不产生黄曲霉毒素;产生黄曲霉毒素菌株中部分黄曲霉菌株产生4种黄曲霉毒素AFB1、AFB2、AFG1、AFG2,黄曲霉菌株HDWH只产生黄曲霉毒素AFB1、AFB2。     

不产毒黄曲霉菌对产毒黄曲霉菌产毒抑制效果分析

本实验6株菌分离自广东、山东、辽宁和湖北四省的花生土壤中,通过形态学和分子生物学鉴定均为黄曲霉菌,HPLC测定其产毒能力,其中GZ-6为产毒菌,GZ-15、WF-5、WF-20、JZ-2和YC-8为不产毒菌。分别以花生和玉米为培养基,将不产毒黄曲霉菌和产毒菌(孢子浓度:104:105或105:105)进行混合培养,测定不产毒菌对产毒黄曲霉产毒的抑制效果。结果显示:不产毒菌对产毒菌产毒的抑制率随着其孢子浓度的增加而明显加强,当孢子浓度比为105:105(不产毒菌:产毒菌)时,5株不产毒菌在玉米培养基上对产毒菌产毒的抑制率为34.55%~75.94%,在花生培养基上对产毒菌产毒的抑制率为38.03%~83.03%,其中WF-5、WF-20和GZ-15这三株不产毒菌对产毒黄曲霉产毒的抑制效果均达到75.00%以上,可以作为田间防治黄曲霉毒素污染的候选菌株。     

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。     

薄荷精油对黄曲霉生长的抑制作用研究

目的 研究薄荷精油对黄曲霉生长及其毒素B1合成的抑制作用。方法 在培养基中加入不同体积浓度的薄荷精油, 以不加薄荷精油作为对照, 通过测定其对产毒黄曲霉的最小抑菌浓度(minimum inhibitory concentration, MIC)、孢子萌发、菌丝干重、菌落直径、微观结构及黄曲霉毒素B1合成量的影响, 探究薄荷精油对黄曲霉的抑制效果。结果 薄荷精油对产毒黄曲霉的MIC为1.6 μL/mL; 2MIC薄荷精油处理对黄曲霉孢子萌发抑制率、黄曲霉菌丝生物量抑制率及黄曲霉毒素B1合成抑制率达到了99%以上，且扫描电镜结果表明，薄荷精油会影响黄曲霉菌丝形态; 同时, 通过熏蒸处理, 薄荷精油能够有效抑制花生中的霉菌增长, 防止霉菌侵染花生。结论 薄荷精油对黄曲霉具有良好的抑制作用, 在粮食储藏、食品防霉等方面具有进一步开发成安全防腐抑菌剂的潜力。     

玉米黄曲霉毒素污染生防菌筛选及菌株B42-3抗菌活性研究

从采自河北、山东、山西及吉林玉米产区的健康玉米籽粒内分离到350株内生菌。拮抗实验发现有250株菌对黄曲霉菌具有明显的抑制作用,其中有10菌株抑菌带宽在8 mm以上;玉米籽粒活体抑毒实验发现8株内生细菌对黄曲霉菌生长及毒素产生有较强的抑制作用,拮抗菌对玉米黄曲霉菌侵染的防治效果从42.5%到75.0%,对黄曲霉毒素污染的抑制率分别从45.0%到70.5%,其中,菌株B42-3、B96-1及B101-2对玉米黄曲霉防治效果分别达到75.0%、71.5%和68.5%,菌株B42-3、B12-3及B88-5抑毒率分别达到70.5%、68.5%和62.7%;田间实验结果显示,菌株B42-3促生率及抑毒率最高,分别达到4.5%及75.5%,生防潜力巨大。实验结果显示,菌株B42-3的发酵液、上清液、滤液及粗蛋白提取液对黄曲霉菌生长有较强的抑制作用。经生理生化特征和16SrDNA序列分析,菌株B42-3初步鉴定为解淀粉芽孢杆菌。     

抗菌肽AMP-17对黄曲霉抑制作用的初步研究

探讨抗菌肽AMP-17对食源性微生物黄曲霉孢子萌发、菌丝生长、产毒等的影响。通过微量液体稀释法测定AMP-17对黄曲霉最低抑菌浓度(minimum inhibitory concentration, MIC)并绘制生长曲线。血球计数板及干重法计算经AMP-17作用后,黄曲霉的孢子萌发率、菌丝生长抑制率。酶联免疫吸附法分析AMP-17对黄曲霉毒素产量的影响。扫描电镜观察AMP-17作用后黄曲霉孢子及菌丝体的形态结构变化。结果显示,AMP-17对黄曲霉的MIC为50μg/mL;生长曲线显示其能显著抑制黄曲霉的生长;50μg/mL AMP-17作用后黄曲霉孢子萌发率为8%,菌丝抑制率可达72.5%;低浓度的AMP-17即可显著降低黄曲霉毒素的合成;经AMP-17干预后,黄曲霉孢子及菌丝形态结构均遭到明显破坏。因此AMP-17对黄曲霉的生长及产毒具有显著抑制作用。该研究结果为AMP-17应用于食品防腐提供了实验依据,可进一步丰富食物防腐剂种类。     

植物提取物抑制玉米中黄曲霉生长及产毒研究

通过液体培养 直接接触法比较植物提取物肉桂醛、柠檬醛和丁香酚对黄曲霉生长及产毒的抑制作用,选取抑制作用最强的肉桂醛应用到玉米中,研究了添加量、玉米水分含量和储藏温度对肉桂醛抑制黄曲霉生长及产毒的影响。结果表明：液体培养基中,肉桂醛、柠檬醛和丁香酚的最低杀菌浓度与各自完全抑制产毒浓度一致,分别为 100、500和500 μl/L。随着添加量的加大,肉桂醛对黄曲霉生长和产毒的抑制率逐渐升高,挥发浓度为96 μl/L时完全抑制黄曲霉生长,对黄曲霉毒素B1的抑制率为97.98％。在玉米水分为14%~40％时,肉桂醛对玉米黄曲霉污染的抑制率随水分含量的升高显著下降,14％时抑制率最高,为88.24%;对黄曲霉毒素B1始终保持较强的抑制作用,14％时产毒抑制率最高,为97.51％。储藏温度在20~37℃时,肉桂醛对黄曲霉污染的抑制率随温度的升高呈现先升高后降低的趋势,28℃黄曲霉污染抑制率最高,为85.67%;对黄曲霉毒素B1抑制率随温度的升高呈现先降低后升高又降低的趋势,20℃时抑制率最高,达到91.00%。     

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。     

香菇纤维素衍生物抑制黄曲霉菌产毒的研究

黄曲霉毒素具有诱导突变、抑制免疫和致癌作用。控制黄曲霉毒素污染一直是世界性难题,也是近年来的研究热点之一。实验研究了香菇纤维素衍生物GPX抑制黄曲霉菌产毒活性,初步探究了GPX抑制黄曲霉菌产毒的作用机制。结果表明,10、50μg/mL的GPX对黄曲霉菌产毒的抑制率分别达到70%、95%,而100、250、500、750、1000μg/mL的GPX对黄曲霉菌产毒的抑制率均可达到100%,抑制活性极其明显。GPX离体抗氧化能力很低,但是能够缓解黄曲霉菌丝的氧化胁迫。此外GPX促进了黄曲霉细胞内囊泡过早与大液泡的融合,该现象能够抑制黄曲霉毒素的产生。     

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     

Wachstum vonAspergillus flavus und Aflatoxinbildung in geschnittenem und verpacktem Weizenvollkornbrot

Zusammenfassung In weitgehend wasserdampf- und sauerstoffdichter Folie (MXT 450) verpacktes, geschnittenes Weizenvollkornbrot wurde mit einem Aflatoxin-bildenden Stamm vonAspergillus flavus beimpft und Mycelwachstum sowie Aflatoxin (mit Hilfe der DSC) bestimmt. Im Mycel konnten keine Aflatoxine nachgewiesen werden, während in Proben, die zusätzlich zum Mycel auch Sporen enthielten, these Mycotoxine vorhanden waren. Ursache für these Erscheinung ist vermutlich der rasch abnehmende Sauerstoffgehalt im engen Raum zwischen Brot und Verpackung, der bei zunächst noch nahezu normalem Mycelwachstum eine Aflatoxin-Synthese nicht mehr ermöglicht.

---

Growth ofAspergillus flaves (aflatoxin producing strain) and production of aflatoxin in sliced and packed whole wheat bread

Summary Whole wheat bread being packed in almost oxygen-proof foil material (MXT 450; Kalle, Wiesbaden, Germany) was inoculated with an aflatoxin producing strain ofAspergillus flaves. Mycelial growth as well as aflatoxin production (by TLC) were determined. Whereas no aflatoxins could be detected in bread with mycelium alone, bread with mycelium and spores contained these mycotoxins. This phenomenon is probably due to the rapidly decreasing oxygen content between bread and packaging foil which inhibits aflatoxin synthesis while mycelial growth is still normal.

     

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。     

EFFECT OF GAMMA IRRADIATION ON AFLATOXIN B1 PRODUCTION BY ASPERGILLUS FLAVUS IN GROUND BEEF STORED AT 5C

The effect of γ-irradiation for controlling the production of aflatoxin B₁ by Aspergillus flavus in ground beef stored at 5C for 2 weeks was investigated. Aspergillus, Penicillium, Cladosporium, Mucor, Scopulariopsis, Candida and Rhodotorula were the most common fungal genera contaminating ground beef. A. flavus and A. niger were the most common Aspergillus spp. Aspergillus flavus isolates were able to produce aflatoxin B₁ in ground beef. Only 3 (20%) samples of ground beef were contaminated with aflatoxin B₁ (25–45 μg/Kg). Gamma irradiation dose levels resulted in an immediate reduction in the total numbers of A. flavus. No growth or aflatoxin B₁ production occurred at 1.50 kGy during storage.     

Growth and aflatoxin production by Aspergillus parasiticus in a medium at different pH values and with or without pimaricin

A glucose-yeast extract-salt medium containing 0, 5, 7.5, 10, 15 or 20 micrograms pimaricin/ml with an initial pH of 3.5 or 5.5 was inoculated with Aspergillus parasiticus WB 108 and incubated at 15 degrees or 28 degrees C. The pH, weight of mycelium and amount of aflatoxin produced were determined after 3, 7, and 10 days and after 14, 21, and 30 days when incubation was at 28 degrees or 15 degrees C, respectively. Increasing the concentration of pimaricin in the medium with an initial pH of 5.5 decreased the amounts of aflatoxin B1 and G1 produced after 3 days of incubation. When the initial pH of the medium was 3.5, no growth or toxin production occurred after 3 days of incubation in the medium containing 7.5 micrograms or more of pimaricin/ml. The presence of 20 micrograms of pimaricin/ml inhibited growth and toxin production after 7 days of incubation. When cultures were incubated at 15 degrees C, there was a lag phase which extended from 9 to 16 days, and the amounts of aflatoxin produced decreased with an increasing concentration of pimaricin. Pimaricin did not completely inhibit the growth and aflatoxin production by A. parasiticus. Pimaricin, in combination with a low pH, low temperature or 4% or 6% NaCl, initially caused slow mycelial growth and low toxin production, but the mold overcame the inhibitory effects and produced substantial amounts of mycelium and toxin.     

黑曲霉抗产毒黄曲霉作用的初步研究

采用90 × 2.6 × 1013N+/cm2 注入黑曲霉筛选能抗黄曲霉(Aflavus)生长的突变菌株,以利发酵中控制被黄曲霉污染的原材料的再污染。进行产毒黄曲霉与被离子注入的黑曲霉混合对峙、原黑曲霉菌株与黄曲霉单独培养生长及混合对峙培养实验。结果显示经离子注入的菌株及未注入菌株均对黄曲霉产生抑制作用,但后者仅有微弱抑制,前者不仅表现出几乎不能使黄曲霉生长,且已长出的黄曲霉菌丝体较瘦小,并呈灰白色。从培养基中提取物检验结果显示,黄曲霉组表现出有较明显的荧光反应,而黑曲霉菌株对峙培养物提取物中有微弱的荧光反应,其黑曲霉突变株对峙培养物未见荧光反应检出。这表明黑曲霉原菌株虽然能对黄曲霉只有微弱抑制,但表现出黄曲霉产毒和合成色素能力下降。与对照组相比,突变株有较强抑制黄曲霉生长能力。     

复配精油对粮油中霉菌防治的增效作用及机理研究

开发新型天然防霉剂控制粮食霉变是保障粮食质量安全的重要途径之一。为研究复配植物精油对粮油储藏过程中常见霉菌赭曲霉(Aspergillus ochrator)、黄曲霉(Aspergillus flavus)和黑曲霉(Aspergillus niger)的防霉效果,挑选活性较强的植物精油进行复配并对联合防霉效果进行评价。通过复配植物精油对霉菌孢子萌发、菌丝干重和细胞完整性的影响,对其防霉机理进行初步探究。结果显示,牛至精油对赭曲霉和黑曲霉的防霉效果最好,抑菌圈直径分别为（27.83±0.58 ）、（15.33±0.29）mm,肉桂醛对黄曲霉的防霉效果最好,抑菌圈直径为（18.50±0.87 ）mm;山苍子精油与牛至精油、肉桂醛与牛至精油复配体积比为2：8时对3种霉菌的防霉效果较优;通过对部分抑菌浓度指数判读,两组植物精油复配对黑曲霉和赭曲霉的防治效果为协同作用,对黄曲霉的防治效果为相加作用;山苍子精油与牛至精油按体积比2：8复配可抑制霉菌孢子萌发和菌丝生长、破坏细胞的完整性、改变孢子和菌丝结构;山苍子精油和牛至精油按体积比2：8复配施用于含黄曲霉的玉米上,可有效降低玉米中黄曲霉毒素B1和赭曲霉毒素的含量。本研究为山苍子精油与牛至精油复配作为防霉剂提供理论支持。     

Correlation of growth and aflatoxin production by Aspergillus flavus with some essential metals in gamma irradiated crushed corn

The effects of gamma irradiation and some essential metals on growth and aflatoxin B1 production by Aspergillus flavus in crushed corn were investigated. The production of aflatoxin by A. flavus was influenced by the addition of zinc, copper or iron and the effect gradually decreased with increasing metal concentration from 0 to 300 ppm. A. flavus grew and depleted zinc, copper and iron at initial concentration of 100, 200 or 300 ppm. Presence of 100 ppm zinc, copper or iron plus gamma irradiation (0.5, 1.0, 2.0 kGy) enhanced the growth of A. flavus and the production of aflatoxin in contrast with irradiated samples alone. A. flavus was able to metabolize and deplete elements in all gamma-irradiated samples. These results suggest that stricter control of element levels in gamma irradiated grains could control aflatoxin contamination.     

Effect of inoculum level on aflatoxin production by Aspergillus flavus under modified atmosphere packaging (MAP) conditions

The combined effect of inoculum level and other environmental factors (water activity, storage temperature and level of headspace oxygen) on the growth and aflatoxin production by Aspergillus flavus under MAP conditions on a synthetic medium was studied using a full factorial experimental design. Regression analysis of the data showed that inoculum level, water activity (a_w), storage temperature and headspace O₂ all had a significant effect on both growth of, and aflatoxin production by, A. flavus. Maximum growth (4·17 mg g^-1) occurred at an inoculum level of 10⁴ spores per plate, an a_w of 0·95, storage temperature of 20°C and headspace O₂ of 15% (balance CO₂:N₂ (60%:40%)). However, maximum aflatoxin level of 3·45 μg g^-1 (3450 ng g^-1) occurred at an inoculum level of 10³ spores per plate, an a_w of 0·96, storage temperature of 25°C and a reduced level of headspace O₂ (10%). Packing under almost completely anaerobic conditions using Ageless oxygen absorbents inhibited both growth and aflatoxin production by A. flavus, irrespective of the inoculum levels and storage temperature used.