【作者】 程波财；

【导师】 柳建设；

【作者基本信息】 中南大学 ， 微生物学， 2010， 博士

【摘要】 禾谷镰刀霉(Fusarium graminearum)是谷物中最为常见的镰刀菌,它们产生的脱氧雪腐镰刀菌烯醇(呕吐毒素)(Deoxynivalenol,DON)、玉米赤霉烯酮(Zearalenone, ZEN)和T-2毒素等单端孢霉烯族毒素是粮食中最常见的一类污染性霉菌毒素,进入人和动物体的DON和ZEN分别产生呕吐、雌激素效应等毒性,还引起免疫毒性、细胞毒性、胚胎毒性和致崎致癌性。霉菌引起的粮食和饲料污染腐烂和真菌毒素的污染给全世界造成了巨大的经济损失。常用的物理和化学方法不仅收效甚微,还增加了不安全性。本论文首先利用禾谷镰刀菌作为指示菌株,通过在益生菌中大面积的筛选并鉴定,获得具有抑制镰刀菌能力的唾液乳杆菌(Lactobacillus. salivarius ssp. salivarius)、发酵乳杆菌(L. fermentum)和卷曲乳杆菌(L. crispatus)三株乳杆菌。针对未曾报道的发酵乳杆菌(L. fermentum)和卷曲乳杆菌(L. crispatus),我们进行了禾谷镰刀菌和尖孢镰刀菌的孢子萌芽、菌丝体生长抑制试验以及相关的机理研究。实验发现：发酵乳杆菌和卷曲乳杆菌上清液能不同程度地抑制禾谷和尖孢镰刀菌孢子萌芽及菌丝体生长。但这种抑制作用随时间的延长而逐渐丧失；上清液中的酸对抑制禾谷和尖孢镰刀菌生长有显著性作用,发酵乳杆菌培养上清中的细菌素或蛋白类物质也对抑制起了部分的作用。其次,深入我国粮食主产地(安徽和河南省)进行DON和ZEN的污染情况的调查采样。经检测、比较分析发现：真菌毒素ZEN和DON污染比较严重,DON和ZEN的检出率分别为72.3%和73.4%,小麦中DON检出率甚至高达97.6%；DON平均污染水平为379.2μg/kg,低于国家限量标准,而ZEN的平均污染水平为178.1μg/kg,高于国家限量标准两倍。再次,本文利用快速灵敏的间接竞争ELISA方法在59株乳酸菌和芽孢杆菌中进行筛选具有降解DON毒素潜能的益生菌株。通过选择合适的稀释倍数减弱培养基干扰后,筛选获得枯草芽孢杆菌ZZ(Bacillus subtilis ZZ)(降解率98%)和地衣芽孢杆菌DY(B.licheniformis DY)(降解率30.7%)有效降解真菌毒素DON的两株益生菌,机理研究发现：芽孢杆菌上清中某些对热敏感的蛋白类成分起着主要的作用。对降解时间、温度和振荡转速进行的优化,确定了枯草芽孢杆菌ZZ和地衣芽孢杆菌DY清除DON的最佳条件：37℃,180rpm振荡共培养12h。本文还利用间接竞争ELISA方法和HPLC方法,在分离获得的64株乳酸菌,104株瘤胃微生物,13株双歧杆菌和5株芽孢杆菌中筛选得到了一株能有效降解真菌毒素ZEN的腊样芽孢杆菌(B.cereus);同时利用选择性培养基的方法从采自安徽、河南以及其它部分省市粮食产地的土壤和玉米样品中筛选获得6株具有不同降解能力的菌株分别为：鼠李糖短杆菌(Brachybacterium rhamnosum),罗尔斯通氏贪铜菌(Ralstonia metallidurans),粪肠球菌(Enterococcus faecalis),蜡样芽胞杆菌(B. cereus),藤黄微球菌(Micrococcus luteus),洋葱伯克霍尔德菌(Burkholdeia cepacia)。其中以蜡样芽孢杆菌(降解率97.6%)和藤黄微球菌(降解率43.2%)效果比较好。因此,后续试验对这两株菌进行温度、孵育时间、培养基、起始pH值、碳源和金属离子添加量等条件的优化。蜡样芽胞杆菌在pH值为6.5的营养肉汤培养基,37℃培养96h,可以完全降解ZEN,而且试验表明蜡样芽孢杆菌的上清液中存在某种物质能有效降解液体中的ZEN,而不是菌体细胞壁的吸附；产物分析表明：ZEN没有形成有毒的α-zearalenol、β-zearalenol、α-Zearalanol和β-Zearalanol中间代谢产物。藤黄微球菌在pH值为7的LB培养基中(添加0.05M MnCl2),37℃培养120h,能降解99%的ZEN。最后,本文从粉红螺旋聚孢霉(G roseum)中扩增文献报道的目的片段zhd101基因。获得的序列与NCBI上公布的基因序列有9个碱基差异。通过原核表达获得了ZEN-JJM (ZEN降解酶),该酶与文献报道的ZHD101蛋白存在着二级结构上的差异,但ZEN降解实验证实ZEN-JJM具有完全降解ZEN的能力。更多还原

【Abstract】 Fusarium graminearum is the most common Fusarium strain for grain contamination, which can produce a group of toxic secondary metabolites, including deoxynivalenol, zearalenone and Toxin-2. Many side effects such as vomiting, estrogenic, immunotoxic, mutagenisity, cytotoxic and genotoxic can be caused by consumption of cereals contaminated with these mycotoxins.Food and feed spoiling moulds cause great economic losses worldwide. A serial of physical and chemical treatments can be applied to prevent or minimize the spoilage of food and contaminations from mycotoxins. However, the efficiency of conversional treatments is low, and a risk of introducing harmful breakdown chemicals is concerned.In the present research, firstly, we used Fusarium graminearum as the contamination indicator, and probiotic strains of Lactobacillus. salivarius ssp. Salivarius, L. fermentum and L. crispatus were screened out for their inhibitory ability on the indicator growth. We also tested the inhibitory effect of L. fermentum and L. crispatus on the spore germination and mycelial growth, which have not been reported so far. Results showed that the spore germination and mycelial growth can be inhibited by L. fermentum and L. crispatus, and the inhibitory capacity was decreased with the prolonging of time. Principal components analysis found that the bacteriocins, proteins and organic acids from the supernatant of culture suspension are the main inhibitor.Secondly, the contamination status of deoxynivalenol and zearalenone in maize and wheat was investigated in Henan and Anhui province. Deoxynivalenol and zearalenone were found in 72.3%and 73.4%of random selected samples respectively in these provinces. Deoxynivalenol was detected 379.2μg per kilogram, which was lower than the national standard, and Zearalenone was 178.1μg per kilogram, which was much higher than the limitation of the national standard.Thirdly, Bacillus subtilis ZZ and B. licheniformis DY were screened out for their degradation ability of deoxynivalenol from 59 Lactobacillus and Bacillus strains by using competitive indirect ELISA. Mechanism analysis showed that the heat sensitive protein substances produced by Bacillus rather than cell itself played the major role. B. subtilis ZZ and B. licheniformis DY can degrade 98% and 30.7% Zearalenone respectively in broth after incubated for 12 h under the optimum condition (37℃,180 rpm).Fourthly, B. cereus was screened out with the ability of degrading zearalenone from 186 bacteria strains including Lactobacillus, Bacillus, Bifidobacterium and rumen microorganisms isolates by competitive indirect ELISA. Meanwhile, three other strains isolated from samples of maize, wheat and soil (collected from Henan and Anhui province) were screened out for their degradation ability in selective medium. They were identified as Brachybacterium rhamnosum, Ralstonia metallidurans, Enterococcus faecalis, B. cereus, Micrococcus luteus and Burkholdeia cepacia. Then the growth condition of B. cereus and M. luteus were optimized since they showed better ability of zearalenone degradation. The B. cereus can completely degrade the zearalenone in liquid BA (pH=6.5) after incubated for 96 h under the optimum condition (37℃, 180 rpm), the protein substances produced by B. cereus were anticipated to be the major degrader. M. luteus can degrade 99% zearalenone in liquid LB (pH=7.0) with 0.05M MnCl2 after incubated for 120 h under the optimum condition (37℃,180 rpm).Finally, a 795 bp DNA fragment (ZEN-jjm) from Gliocladium roseum coding lactonohydrolase was obtained. Sequencing results showed a 9-base difference in the amplified gene compared with reported gene zhd101, resulting in 3 amino acid difference in lactonohydrolase. The degradation capability of expressed protein in the supernatant of disrupted cells was verified by HPLC, and a similar biological function was observed in the expressed protein.更多还原