生物技术对黄曲霉毒素B1的脱除及机制研究进展

黄曲霉毒素是危害最大的真菌毒素之一,而黄曲霉毒素B₁(AFB₁)是黄曲霉毒素中毒性最大的一种,具有高毒性、致癌、致畸和致突变的作用,对动物和人类健康造成危害。为了有效脱除AFB₁,综述了近年来研究的具有解毒作用的微生物,介绍了采用生物技术脱除AFB₁的方法,重点探讨了生物脱除AFB₁机制。用于脱除AFB₁的菌株有芽孢杆菌、假单胞菌、乳酸菌、非产毒曲霉等,可采用单菌种发酵、多菌种协同发酵和菌-酶协同作用脱除AFB₁。生物脱除AFB₁机制主要为降解脱除和吸附脱除。可进一步筛选具有高优良能力的菌株以及更深层次地研究微生物脱毒的机制,探索微生物制剂对AFB₁的降解作用。     

西藏高原粮油作物曲霉菌污染及菌株产毒力研究

为探明西藏高原粮油作物曲霉菌污染状况及黄曲霉菌产毒能力,连续5年对西藏青稞、小麦、花生3种作物中曲霉菌污染情况进行分析,并对其分离到的黄曲霉菌株开展产毒力研究,结果表明,204份样品中,共分离出15种曲霉菌,曲霉菌污染率呈花生>青稞>小麦。青稞、小麦中曲霉属优势种均为黑曲霉(Aspergillus niger),真菌毒素主要为杂色曲霉毒素和赭曲霉毒素;花生优势种为黄曲霉(A.flavus);仅受黄曲霉毒素污染。来源于不同作物的黄曲霉菌,其产毒类型也有差异,麦类作物产毒菌株以产黄曲霉毒素B₁(AFB₁)、黄曲霉毒素B₂(AFB₂)为主;花生产毒菌株以产AFB₁、AFB₂、AFG₁、AFG₂为主。     

黄曲霉毒素B1降解菌的筛选鉴定及有效降解组分初探

黄曲霉毒素B₁(AFB₁)是具有强毒性、强致癌性的一种真菌毒素。为了筛选出AFB₁降解菌,将实验室保藏的真菌菌株与AFB₁共培养,选取降解率最高的菌株,经形态学和ITS rDNA分析确定其种属,分离菌株不同组分(菌悬液、菌体、孢子液、发酵液)后探究其降解AFB₁的有效组分,并将有效组分菌体破碎后作进一步探究。结果表明:培养72 h后的郝克氏青霉MD1菌悬液和未破碎菌体对AFB₁降解率分别为98.15%和28.20%,菌体破碎后对AFB₁的降解效果更好,培养24 h后对AFB₁的降解率为55.40%。因此,郝克氏青霉MD1的菌悬液和菌体内的活性组分能有效降解AFB₁。本研究筛选出的郝克氏青霉MD1扩大了AFB₁降解菌的菌种库,为AFB₁的生物降解提供了一定的参考价值。     

湘派卤制品卤汁中黄曲霉毒素产毒菌的筛选鉴定及其产毒能力测定

以湘派卤制品卤汁为研究对象，对其贮存过程中产黄曲霉毒素的微生物(产毒菌)进行筛选鉴定并对产毒能力进行测定。采用平板稀释涂布法和紫外荧光法对湘派卤汁进行产毒菌的初筛；对初筛菌株的ITS序列进行PCR扩增、测序，测序结果经NCBI同源性对比、MEGA6.0软件构建系统发育树进行分子生物学鉴定；应用HPLC-MS/MS检测各产毒菌培养液中黄曲霉毒素(aflatoxin, AFs)含量，确定各产毒菌的产毒能力。经平板稀释涂布法、紫外荧光法和分子生物学共筛选鉴定出5株产毒菌株，其中有4株是黄曲霉(Aspergillus flavus),1株为寄生曲霉(Aspergillus parasiticus);经HPLC-MS/MS法确定4株黄曲霉均不产生AFs,寄生曲霉产生黄曲霉毒素B₁(AFB₁)和黄曲霉毒素G₁(AFG₁)。AFB₁在第3天和第7天的含量分别为6.92、8.08μg/L,AFG₁在第3天和第7天的含量分别为5.80、7.24μg/L;湘派卤汁由于贮存条件...     

微波对花生黄曲霉毒素B1的消减作用及压榨花生油品质的影响

对花生进行微波处理，研究微波对花生中黄曲霉毒素B₁(AFB₁)的降解效果及压榨花生油品质的影响。结果表明：微波温度、微波时间与花生AFB₁降解率均呈正相关关系；Na₂CO₃喷涂协同微波处理，可以明显增强微波对花生AFB₁的降解效果，降解率达85.67%,但降解率会随着AFB₁初始含量先上升再趋于恒定。Na₂CO₃协同微波处理后所得花生油的酸价、过氧化值均符合相关标准要求，且营养品质高、氧化稳定性好。     

微生物法针对毒性位点降解黄曲霉毒素B1的研究进展

文章主要探究了黄曲霉毒素B₁(AFB₁)的毒性位点及毒性机理,并对比了微生物法破坏AFB₁毒性位点从而降解AFB₁的优势。重点综述了微生物法中的脱毒酶法对AFB₁的降解机制。同时,对脱毒酶法联合其他方法的复合应用进行展望。     

贵州秋季栽培不同荞麦品种成熟期果实中黄曲霉及其毒素的分离与鉴定

本研究以贵阳秋季栽培的2个米苦荞(F.tataricum,贵米苦荞18-1号和贵黑米苦荞12号)、2个多苦荞(F.tatari-cymosum,贵多苦荞003C和贵多苦荞60)、2个甜荞(F.esculentum,贵红花甜荞2号和1412-1)、2个常规苦荞(F.tataricum,定苦荞1号和六苦2017)为材料。对其成熟期种子果壳和籽粒进行了黄曲霉分离鉴定,并采用高效液相色谱法对所有品种果壳和籽粒中分离出的黄曲霉菌株进行AFB₁、AFB₂、AFG₁和AFG₂毒素的检测。结果表明,所有品种果壳中均没有分离出黄曲霉菌落;4类荞麦籽粒中仅米苦荞分离出了黄曲霉菌落,共分离出4株黄曲霉菌株。其中贵米苦荞18-1号黄曲霉带菌率为1.56%,贵黑米苦荞12号黄曲霉带菌率为0.78%。分离菌株形态学和ITS序列扩增产物测序结果与已知黄曲霉菌序列完全一致。毒素检测结果表明不同品种之间产毒素差异显著,所有品种籽粒中只有米苦荞中检出4种毒素,贵米苦荞18-1号产AFB₁最高为(5.861±0.055) μg/kg、AFB₂最少为(1.605±0.052) μg/kg,贵黑米苦荞12号产AFB₁最高为(14.475±0.533) μg/kg、AFG₂最少为(3.393±0.151) μg/kg;籽粒产毒量远大于分离菌株产毒量;各分离出菌株之间产毒素能力差异显著,最大产AFT为(11.102±0.095) μg/kg、最小产AFT为(1.794±0.024) μg/kg。上述结果显示供试米苦荞籽粒带菌来源可能是由于果壳开裂籽粒外露后部分籽粒被直接侵染所致。所得结果可为米苦荞中黄曲霉抗性育种研究及荞麦种子的保存、运输、储藏等研究奠定基础。     

产漆酶食用菌的筛选及食用菌漆酶降解黄曲霉毒素B1的研究进展

每年有大量的谷物被黄曲霉毒素B1(aflatoxin B₁,AFB₁)污染,对人类和牲畜健康造成极大的危害。生物法降解AFB₁具有底物特异性强、反应条件温和等优点,受到社会的广泛认可。在降解谷物中AFB₁时,宜采用食用安全的微生物,以避免引入新的污染物,因此,产漆酶的食用菌是一种较好的选择。基于此,本文从美国国家生物技术信息中心数据库和Uniprot数据库(Unified Protein Database)中筛选出具有漆酶基因序列或氨基酸序列的食用菌,对已报道的能够产漆酶的食用菌进行总结,并概述产漆酶食用菌的实验筛选方法,探讨食用菌漆酶的结构和理化性质,综述食用菌漆酶的作用和食用菌漆酶降解AFB₁的研究进展,以期为食用菌漆酶安全、高效、绿色降解AFB₁提供理论支持。     

低温等离子体对黄曲霉毒素B1的降解效能

目的:确定低温等离子体降解黄曲霉毒素B₁（Aflatoxin B₁，AFB₁）最佳工艺条件，并探究其在农产品中应用的可行性。方法:选取低温等离子体不同激发条件（峰值电压、工作频率、作用时间），研究其对溶液中AFB₁的降解效果。通过Center Composite Design（CCD）法进行响应面试验，获取最优降解组合及各因素交互作用机制，并考察此条件下玉米中AFB₁降解效果。结果:当AFB₁浓度为1000 μg/L时，其降解率随峰值电压及作用时间（除90~120 s）的增加，工作频率的下降而极显著升高（P<0.01）。响应面优化后最佳工艺条件为峰值电压160 kV、工作频率50 Hz、作用时间165 s，此时AFB₁降解率为99.62%。此外，将优化后的降解条件在受AFB₁污染的玉米（23.18±0.06 μg/kg）中进行应用，发现180 s处理时间下，其降解率可达39.29%。结论:通过CCD法确定了低温等离子体技术降解AFB₁最优工艺，证实了其在玉米中的降解效果。表明低温等离子体技术在降低谷物黄曲霉毒素污染方面具有巨大潜力。     

射频联合热风加热对玉米籽粒中黄曲霉毒素B1降解及玉米品质的影响

为研究射频-热风联合处理玉米中的黄曲霉毒素B₁(aflatoxin B₁,AFB₁)降解效果,分析不同初始水分质量分数(19.05%、22.25%、25.55%)、射频加热温度(55、65、75、85℃)和加热持续时间(10、15、20、25 min)对玉米籽粒中AFB₁降解效果及玉米品质的影响。结果表明：射频-热风联合处理可有效降解AFB₁,在高水分含量下,加热温度和时间一定,初始水分含量越高,AFB₁残留量越高;初始水分含量一定,随温度增加和时间延长,AFB₁残留量随之减少,且加热持续时间对降解效果的影响大于加热温度;射频-热风联合处理玉米籽粒过程中,对其品质有一定影响,随温度升高和时间延长,与对照组相比,蛋白质、脂肪含量无显著差异(P>0.05),黏度系数显著降低(P<0.05);低场核磁共振分析表明,射频加热过程中水分迁移效果明显,且水分迁移特性与玉米的糊化特性、蛋白质、脂肪含量显著相关(P<0.05)。     

Overexpression of Aspergillus aculeatus cellobiohydrolase I in Aspergillus oryzae

To express the cbhI gene, encoding Aspergillus aculeatus cellobiohydrolase I (CBHI), in Aspergillus oryzae, a plasmid was constructed. The strain that displayed the strongest CBHI activity among the transformants produced about 941 mg/l in liquid culture. It was confirmed by a PCR method that the plasmid was integrated at the niaD locus.     

黑曲霉、米曲霉和解脂假丝酵母协同处理竹片

在相同发酵工艺条件下,探索黑曲霉、米曲霉、解脂假丝酵母、黑曲霉 米曲霉、黑曲霉 解脂假丝酵母、米曲霉 解脂假丝酵母、黑曲霉 米曲霉 解脂假丝酵母七组中的最优处理竹片茵种,然后确定其最佳发酵工艺,最后进一步研究接种量和接种比例的影响。研究结果表明,黑曲霉 米曲霉 解脂假丝酵母是最优处理竹片菌种,其最佳处理工艺为温度35℃,pH7.0,定时通氧,添加营养液,处理时间8d,接种量影响不大,接种比例以2:1:1(黑曲霉:米曲霉:解脂假丝酵母)为佳。     

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.     

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。     

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。     

Cladal relatedness among Aspergillus oryzae isolates and Aspergillus flavus S and L morphotype isolates

Aspergillus flavus is the main etiological agent for aflatoxin contamination of crops. Its close relative, A. oryzae, does not produce aflatoxins and has been widely used to produce fermented foods. We compared the phylogeny of A. oryzae isolates and L- and S-type sclerotial isolates of A. flavus using single nucleotide polymorphisms in the omtA gene in the aflatoxin biosynthesis gene cluster and deletions in and distal to the norB-cypA intergenic region as phylogenetic signals. Aflatoxin-producing ability and sclerotial size also were weighted in the analysis. Like A. flavus, the A. oryzae isolates form a polyphyletic assemblage. A. oryzae isolates in one clade strikingly resemble an A. flavus subgroup of atoxigenic L-type isolates. All toxigenic S-type isolates closely resemble another subgroup of atoxigenic L-type isolates. Because atoxigenic S-type isolates are extremely rare, we hypothesize that loss of aflatoxin production in S-type isolates may occur concomitantly with a change to L-type sclerotia. All toxigenic L-type isolates, unlike A. oryzae, have a 1.0 kb deletion in the norB-cypA region. Although A. oryzae isolates, like S-type, have a 1.5 kb deletion in the norB-cypA region, none were cladally related to S-type A. flavus isolates. Our results show that A. flavus populations are genetically diverse. A. oryzae isolates may descend from certain atoxigenic L-type A. flavus isolates.     

Nannocystis exedens: a potential biocompetitive agent against Aspergillus flavus and Aspergillus parasiticus.

This study examined the potential for controlling toxigenic Aspergillus flavus and Aspergillus parasiticus by biological means using a myxobacterium commonly found in soil. The ability of Nannocystis exedens to antagonize A. flavus ATCC 16875, A. flavus ATCC 26946, and A. parasiticus NRRL 3145 was discovered. Cultures of aflatoxigenic fungi were grown on 0.3% Trypticase peptone yeast extract agar for 14 days at 28 degrees C. When N. exedens was grown in close proximity with an aflatoxigenic mold, zones of inhibition (10 to 20 mm) developed between the bacterium and mold colony. A flattening of the mold colony on the sides nearest N. exedens and general stunting of growth of the mold colony were also observed. When N. exedens was added to the center of the cross-streak of a mold colony, lysis of the colony by the bacterium was observed after 24 h. Microscopic observations revealed that N. exedens grew on spores, germinating spores, hyphae, and sclerotia of the molds. These results indicate that N. exedens may be a potential biocontrol agent against A. flavus and A. parasiticus.     

High-yields heterologous production of the novel Aspergillus fumigatus elastase inhibitor AFUEI in Aspergillus oryzae

AFUEI, an elastase inhibitor produced by Aspergillus fumigatus strongly inhibits the elastolytic activity of A. fumigatus etc. To purify AFUEI, we constructed a strain that overproduces AFUEI by introducing the gene encoding AFUEI (Genbank accession no. AB546725) under control of the amyB promoter into the heterologous host Aspergillus oryzae. A. oryzae TF-4 displayed strong elastase inhibitory activity and produced considerably more AFUEI than that of A. fumigatus. Furthermore, AFUEI could be purified using culture broth and single ultrafiltration (UF) treatment, allowing for the effective production of AFUEI for use in clinical trials.