真菌及昆虫在储藏小麦中生长产生CO_2气体的特点

在各种温、湿度条件下对小麦储藏期间真菌和昆虫活动产生二氧化碳气体(CO2)进行监测,研究可用于判断小麦储藏安全性的方法。试验结果表明,储粮温度对处于生长状态的真菌产CO2速率影响最大,从20℃升高到25℃,产CO2速率可提高10倍。在安全水分小麦中,昆虫是产CO2的主要类群,对高水分小麦,真菌是产CO2的主体。在产CO2特性方面,昆虫密度与产CO2量具线性关系(r0.99),而真菌产CO2呈明显的加速现象,在储藏初期带菌量没有显著增加(p0.05)时,产气速率已提高5.96倍。在大型粮仓储藏中的试验也表明,昆虫的产气量恒定,粮堆4 m深度昆虫活动部位25 d的CO2浓度变化幅度为13%,而离霉变点0.5 m的CO2浓度变化高达37倍。因此,利用检测CO2气体浓度变化的方法可以灵敏地发现小麦储藏中昆虫和真菌的危害活动。     

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。     

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。     

我国部分省区2008年产小麦玉米中真菌污染状况研究

目的对安徽、江苏、河北、河南四省部分地区2008年产小麦、玉米真菌污染情况和菌相分布进行调查,为进行粮食中主要污染真菌产毒预测微生物学研究提供菌株,为粮食防霉提供依据。方法将采集的小麦、玉米样品消毒后点种于PDA培养基上,培养后进行菌落计数并鉴定菌相。结果四省小麦样品真菌污染率几乎100%,优势菌是交链孢霉;玉米全部样品均被真菌污染,优势菌是镰刀菌。结论四省的小麦、玉米样品真菌污染严重,粮食储藏过程中应采取必要的措施防止粮食霉变。     

基于HPLC检测方法的真菌毒素代谢规律研究

玉米是我国主要的储备粮种之一,营养丰富且带菌量大,在收获、储藏等过程中,易发生霉变,产生大量真菌毒素,引发储粮安全问题。本文通过一次前处理实现对玉米样品中多种毒素的提取与检测,确定了AFTB₁、AFTB₂、AFTG₁、AFTG₂、ZEN、DON共6种玉米中常见真菌毒素的高效液相色谱检测方法。为了明确玉米储藏过程中真菌毒素代谢和温度的关系,我们将前期获得的两株产毒霉菌:黄曲霉和禾谷镰刀菌,接种于无菌玉米粉中,在22、28、38℃以及常温下储藏40 d后,监测其毒素含量变化。结果表明:温度对两种菌的真菌毒素代谢能力有较大的影响,较高的温度虽然会有利于霉菌的生长繁殖,但是其代谢产毒会受到抑制,较低的温度下,虽然霉菌的生长受到抑制,但也可能产生了大量的毒素。为更好的解析霉菌在玉米储藏过程中的产毒规律,确保储粮安全提供了数据支撑。     

小麦中致病真菌的分离鉴定及熏蒸对其毒力的影响

小麦受镰刀菌的感染会导致赤霉病、真菌毒素污染和减产,镰刀菌对粮食安全有着严重威胁。通过对小麦中镰刀菌株进行分离鉴定,分离出的三种菌株N1、N2、N3分别属于禾谷镰刀菌属、亚洲镰刀菌属和高秆镰刀菌属。利用产毒基因检测和真菌毒素检测对三株菌株玉米赤霉烯酮、呕吐毒素和伏马毒素的产毒情况进行分析,结果一致显示N1和N3能产生玉米赤霉烯酮和呕吐毒素,均含有PSK和Tri5两种产毒基因,而N2不产生这三种毒素。进一步通过臭氧和二氧化氯进行气体熏蒸,探究气体熏蒸对分离出的三株菌株的影响,通过观察孢子形态、菌丝体长度、菌丝形态,结果表明二氧化氯熏蒸能有效抑制菌丝生长和孢子萌发,低浓度二氧化氯（300 mg/L）处理0.5 h,可明显抑制菌丝的生长。而臭氧只能抑制孢子萌发,对菌丝的生长没有明显的抑制作用。     

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。     

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 ℃。     

不同水分活度下稻谷和糙米黄曲毒素累积风险的比较

谷物水分活度及加工过程对于储藏期谷物的微生物安全性具有重要作用。本研究的目的是对稻谷和糙米接种高产毒黄曲霉菌孢子来模拟黄曲霉菌污染过程,比较不同储存水分活度（0.85、0.90、0.95）稻谷和糙米霉变过程中的呼吸速率（R）、干物质损失（DML）和黄曲霉毒素B1（AFB1）累积风险的差异。研究结果表明,霉变过程中稻谷和糙米的干物质损失和AFB1含量都随水分活度的增加而增加,但糙米中的干物质损失水平更大。所有接种过黄曲霉菌的样本中检测到的AFB1累积水平都高于欧盟立法限制的谷物及其制品中的AFB1水平（2 μg/kg）,但在稻谷中只检测到了少量的AFB1累积,而糙米中检测到大量的AFB1累积污染。这些研究结果表明谷物等大宗商品中的霉菌毒素污染风险受到谷物是否加工的影响,另谷物呼吸速率（R）用于预测谷物霉变过程中真菌毒素累积的风险具有一定的可行性。     

槲皮素抑制黄曲霉毒素产生的机制初探

研究发现茶叶中的茶多酚单体普遍具有抑制黄曲霉毒素B1(AFB1)产生的活性,而槲皮素的抑毒活性要高于等浓度下儿茶素类茶多酚。为了解槲皮素抑制黄曲霉毒素产生的分子机制,对黄曲霉菌的抗氧化系统、毒素产生的相关基因进行了分析。试验结果显示槲皮素处理能后降低黄曲霉菌内的ROS水平,降低MDA含量。RT-PCR结果证实槲皮素能够激活抗氧化系统转录因子Yap1,导致黄曲霉体内的抗氧化酶系统活性的增加,POD、CAT、SOD都得到了显著的提高,这很可能是槲皮素抑制AFB1产生的关键因素;槲皮素能同时下调AflR与AflS的表达,而AflS能够通过结合AflR调控产毒基因的表达,这很可能是槲皮素抑制AFB1产生的核心分子机制,这种机制也与其激活抗氧化系统缓解菌体内氧化胁迫的作用相对应。以上结果表明槲皮素作为一种高效的黄曲霉毒素合成抑制剂,将对提高食品安全保障具有较高的应用价值。     

Effect of Soybean Volatile Compounds on Aspergillus flavus Growth and Aflatoxin Production

ABSTRACT:  Soybean homogenates produced volatile compounds upon exposure to lipase. These induced volatiles were identified by SPME. Seventeen volatile compounds identified by SPME were chosen for determination of their ability to inhibit Aspergillus flavus growth and aflatoxin B₁ (AFB1) production in a solid media assay. These volatiles included aldehydes, alcohols, ketones, and furans. Of the tested compounds, the aldehydes showed the greatest inhibition of fungal growth and AFB1 production. These compounds inhibited up to 100% of the observed growth and AFB1 production as compared to the controls. The greatest activity by the aldehydes to disrupt growth was ranked as follows: 2,4 hexadienal > benzaldehyde > 2-octenal > ( E )-2-heptenal > octanal > ( E )-2-hexenal > nonanal > hexanal. The greatest activity by the aldehydes to reduce AFB1 was ranked as follows: ( E )-2-hexenal > 2,4 hexadienal > ( E )-2-heptenal > hexanal > nonanal. ( E )-2-hexenal and ( E )-2-heptenal were tested further in an A. flavus -inoculated corn kernel assay. Both compounds prevented colonization by A. flavus and eliminated AFB1 production when exposed to compound volumes < 10 μL as also shown in the solid media assay. The results suggest that soybeans react to lipase by production of potent antifungal volatiles.     

Glufosinate-ammonium reduces growth and aflatoxin B1 production by Aspergillus flavus

The herbicide glufosinate-ammonium (GA) [butanoic acid, 2-amino-4-(hydroxymethylphosphinyl)-ammonium salt] was tested at concentrations from 2 to 2,000 g GA per ml for activity against growth and aflatoxin B1 (AFB) production by the mycotoxigenic fungus Aspergillus flavus Link:Fr. The highest concentration (2,000 microg GA per ml) reduced colony diameter of A. flavus strain AF13 by 80%. AFB1 production was inhibited by 90% at this concentration. Reduction in mycelial dry weight and AFB1 production in response to GA application ranged from 17.2 to 97.1% and from 39.1 to 90.1%, respectively. Of four concentrations tested, 2 microg GA per ml was weakly inhibitory. In the kernel screening assay, AFB1 production was inhibited 60 to 91% when kernels were preimmersed or immersed 5 days after incubation in 200 microg GA per ml. Both concentrations (2 and 200 microg GA per ml) reduced seed germination by 25 to 50%. Results indicate that GA has an inhibitory effect on growth and AFB1 production by A. flavus.     

Occurrence of Aspergillus spp. and aflatoxin B1 in Malaysian foods used for human consumption

Malaysian population widely consumes the cereal-based foods, oilseeds, nuts, and spices in their daily diet. Mycotoxigenic fungi are well known to invade food products under storage conditions and produce mycotoxins that have threat to human and animal health. Therefore, determining toxigenic fungi and aflatoxin B(1) (AFB1) in foods used for human consumption is of prime importance to develop suitable management strategies and to minimize risk. Ninety-five food products marketed in Penang, Malaysia were randomly collected from different supermarkets and were analyzed for presence of Aspergillus spp. by agar plate assay and AFB1 by enzyme-linked immunosorbent assay (ELISA). A. flavus was the dominant fungi in all foods followed by A. niger. Fifty-five A. flavus strains were tested for their ability to produce aflatoxins on rice grain substrate. Thirty-six (65.4%) strains out of 55 produced AFB1 ranging from 1700 to 4400 μg/kg and 17 strains (31%) produced AFB2 ranging from 620 to 1670 μg/kg. Natural occurrence of AFB1 could be detected in 72.6% food products ranging from 0.54 to 15.33 μg/kg with a mean of 1.95 μg/kg. Maximum AFB1 levels were detected in peanut products ranging from 1.47 to 15.33 μg/kg. AFB1 levels detected in all food products were below the Malaysian permissible limits (<35 μg/kg). Aspergillus spp. and AFB1 was not detected in any cookies tested. Although this survey was not comprehensive, it provides valuable information on aflatoxin levels in foods marketed in Malaysia.     

基于形态特征和多基因序列鉴定储藏稻谷的优势真菌

稻谷储藏期间,真菌生长会影响稻谷质量,严重时会产生真菌毒素,引起食品安全问题。确定储藏期间稻谷的真菌群落组成并准确鉴定优势真菌,是实现有效防控危害真菌的前提,对于保障稻谷的品质和食品安全具有重要意义。基于此,利用传统的微生物分离培养方法,确定了不同地区粮仓内储存稻谷的真菌群落组成,并结合模式菌株和国际权威菌株序列,采用形态学观察及多基因联合系统发育分析方法准确鉴定我国储藏稻谷的优势种。结果表明,南方地区优势种为黄曲霉Aspergillus flavus、黑曲霉A. niger和蒙地曲霉A. montevidensis（阿姆斯特丹曲霉A. amstelodami）,北方地区优势种是多育曲霉Aspergillus proliferans和蒙地曲霉A. montevidensis。并阐述了南北方储藏稻谷主要优势种的详细鉴定过程及相关资料,为粮食中主要危害真菌物种的准确鉴定提供参考。     

Evaluation of Chenopodium ambrosioides oil as a potential source of antifungal, antiaflatoxigenic and antioxidant activity

Essential oil extracted from the leaves of Chenopodium ambrosioides Linn. (Chenopodiaceae) was tested against the aflatoxigenic strain of test fungus Aspergillus flavus Link. The oil completely inhibited the mycelial growth at 100 microg/ml. The oil exhibited broad fungitoxic spectrum against Aspergillus niger, Aspergillus fumigatus, Botryodiplodia theobromae, Fusarium oxysporum, Sclerotium rolfsii, Macrophomina phaseolina, Cladosporium cladosporioides, Helminthosporium oryzae and Pythium debaryanum at 100 microg/ml. The oil showed significant efficacy in inhibiting the aflatoxin B1 production by the aflatoxigenic strain of A. flavus. During in vivo investigation it protected stored wheat from different storage fungi for one year. Chenopodium oil also exhibited potent antioxidant activity when tested by ABTS method. All these observations suggest the possible exploitation of the Chenopodium oil as potential botanical fungitoxicant in ecofriendly control of post harvest biodeterioration of food commodities from storage fungi.     

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     

Streptomyces alboflavus TD-1产挥发性抑菌物质对黄曲霉菌生长及其毒素的抑制作用

利用双皿对扣和气相色谱-质谱法,研究白黄链霉菌TD-1（Streptomyces alboflavus TD-1）在不同葡萄糖浓度培养基中的生长和挥发性有机化合物（volatile organic compounds,VOCs）代谢规律,通过荧光显微镜、扫描电子显微镜及高效液相色谱等方法,研究1-辛烯-3-醇对黄曲霉菌的抑菌机制。结果表明,高浓度葡萄糖（500 mmol/L）培养条件下,白黄链霉菌TD-1的初始生长速率与VOCs产生能力受到明显抑制,当葡萄糖浓度降至50 mmol/L左右,抑制得到解除。利用气相色谱-质谱分析发现,白黄链霉菌TD-1所产生的有效抑菌VOCs包括己醛、1-辛烯-3-醇、2-正戊基呋喃、2-甲基异冰片和苯乙酮等。其中,食品级VOCs 1-辛烯-3-醇在用量15 μL/L时可完全抑制黄曲霉的生长,对黄曲霉菌丝体和孢子形态具有明显的致畸作用,可破坏线粒体膜,抑制黄曲霉毒素B1合成速率至31.98%。说明葡萄糖浓度会影响白黄链霉菌TD-1生长和VOCs产量;1-辛烯-3-醇具有抑制黄曲霉菌生长和黄曲霉毒素B1合成的作用。     

Chemical composition and antifungal activity of essential oil from Cicuta virosa L. var. latisecta Celak

The essential oil extracted from the fruits of Cicuta virosa L. var. latisecta Celak was tested in vitro and in vivo against four foodborne fungi, Aspergillus flavus, Aspergillus oryzae, Aspergillus niger, and Alternaria alternata. Forty-five different components accounting for 98.4% of the total oil composition were identified by gas chromatography-mass spectrometry. The major components were γ-terpinene (40.92%), p-cymene (27.93%), and cumin aldehyde (21.20%). Antifungal activity was tested by the poisoned food technique against the four fungi. Minimum inhibitory concentration against the fungi was 5 μL/mL and percentage inhibition of mycelial growth was determined at day 9. The essential oil had a strong inhibitory effect on spore production and germination in all tested fungi proportional to concentration. The oil exhibited noticeable inhibition on dry mycelium weight and synthesis of aflatoxin B? (AFB?) by A. flavus, completely inhibiting AFB(1) production at 4 μL/mL. The effect of the essential oil on inhibition of decay development in cherry tomatoes was tested in vivo by exposing inoculated and control fruit to essential oil vapor at a concentration of 200 μL/mL. Results indicated that the essential oil from C. virosa var. latisecta (CVEO) has potential as a preservative to control food spoilage.     

Assessment of Cymbopogon citratus (DC.) Stapf Essential Oil as Herbal Preservatives Based on Antifungal,Antiaflatoxin, and Antiochratoxin Activities and In Vivo Efficacy during Storage

Thirty‐five randomly collected samples of stored table grapes (Vitis vinifera L.) from different markets of Gorakhpur city, Uttar Pradesh, India, revealed occurrence of 11 types of fungi. Of which, Aspergillus flavus, Aspergillus niger, and Aspergillus ochraceus were dominant causing severe decay of grapes with 58%, 52%, and 67% incidence, respectively. On screening of 15 essential oils at 0.33 μL/mL, Cymbopogon citratus oil caused 100% mycelial inhibition against aforesaid dominant fungi. Oil was fungistatic at 0.29 μL/mL and exhibited broad fungitoxicity against other fruit rotting fungi associated with collected samples. C. citratus oil completely inhibited the growth and mycotoxin (AFB1 and OTA) secretion of the aflatoxigenic and ochratoxigenic strains of A. flavus, A. niger, and A. ochraceus at 0.8 μL/mL. E‐Citral (52.9%) and Z‐Citral (39.38%) were the major components of C. citratus oil during gas chromatography and gas chromatography‐mass spectrometry analysis. Application of 200 and 300 μL of C. citratus oil on 1 kg of stored grapes showed enhancement of shelf life up to 10 d. The oil did not exhibit any phytotoxic effect on fruits. These results confirm that C. citratus oil could be a natural alternative to commercial fungicide for control of fruit rotting fungi of stored grapes.     

Some probiotic properties of yeast isolates from infant faeces and Feta cheese

The use of modified atmospheres to prevent fungal growth and mycotoxin production in cheese was evaluated. Eight fungal species: Mucor plumbeus, Fusarium oxysporum, Byssochlamys fulva, B. nivea, Penicillium commune, P. roqueforti, Aspergillus flatus and Eurotium chevalieri were inoculated onto cheese and incubated under conditions of decreasing concentrations of O2 (5% to < 0.5%) and increasing concentrations of CO2 (20-40%). Fungal growth was measured by colony diameter and ergosterol content. All fungi examined grew in atmospheres containing 20% and 40% CO2 with 1% or 5% O2, but growth was reduced by 20-80%, depending on species, compared with growth in air. The formation of aflatoxins B1 and B2, roquerfortine C and cyclopiazonic acid was greatly decreased but not totally inhibited in these atmospheres. At 20% or 40% CO2 with < 0.5% O2, only B. nivea exhibited growth, which was very slow. Growth of F. oxysporum, B. fulca, P. commune and A. flavus showed good correlations between colony diameter and ergosterol content. However, for the other species correlations were inconsistent.