玉米赤霉烯酮内酯水解酶的鉴定、改造及应用

玉米赤霉烯酮(zearalenone,ZEN)及其衍生物是世界范围内污染最严重的一种真菌毒素,对人类生命和财产安全构成了极其严重的威胁。传统的物理和化学脱毒方式因存在脱毒不彻底、破坏食品质构以及导致营养成分流失等弊端而无法满足实际生产应用。近年来,生物酶法降解真菌毒素因微生物来源广泛、酶反应条件温和以及脱毒效率高等诸多优势而逐渐被研究和报道。特别地,玉米赤酶烯酮内酯水解酶(lactonase)因具有较高的ZEN降解活力、明确的脱毒机理、不可逆的脱毒过程和阐明的晶体结构等优点而被广泛研究。文章对微生物来源的内酯水解酶的基因挖掘、性质鉴定、分子改造以及应用探索方面进行了综述。旨在阐明目前内酯水解酶在玉米赤霉烯酮脱毒工业应用中存在的优势与挑战,并为其未来在玉米赤霉烯酮生物降解方面的研究提供参考。     

玉米赤霉烯酮生物脱毒与降解的研究进展

玉米赤霉烯酮（ZEN）是由镰刀菌属（Fusarium. sp）产生的污染范围比较广的一种毒素,它在自然界中广泛存在。物理、化学脱毒法成本高且对人类、动植物有一定的损害,生物法因其具有高效性、无污染且降解率高而被科学界关注。该文概述了玉米赤霉烯酮的物理、化学和生物脱毒方法,并对脱毒后的产物进行了简要介绍,对不同降解菌株的降解机制以及降解酶基因的表达和产物毒性进行了综述。     

降解玉米赤霉烯酮芽孢杆菌的筛选和鉴定

从400株芽孢菌中筛选到对玉米赤霉烯酮(ZEN)有较好清除作用的两株芽孢杆菌373-2和411-1,分别与玉米赤霉烯酮(15μg/m L)共培养8 h和6 h后,将其完全清除。通过16S rDNA序列分析鉴定,373-2和411-1分别为一株新种芽孢杆菌和枯草芽孢杆菌(Bacillus subtilis)。对两株菌的玉米赤霉烯酮清除机理进行初步分析,结果表明,清除作用主要源自菌株胞外酶的降解活性。     

小豆内生真菌的筛选鉴定及其产玉米赤霉烯酮的可能性探究

玉米赤霉烯酮(zearalenone,ZEN)是目前全球范围污染谷物最广泛的霉菌毒素之一,对人体和动物健康都会产生极大威胁,目前从市售小豆检测到ZEN。【目的】筛选暴露ZEN的小豆内生真菌,结合经典方法和分子生物学方法鉴定所筛选内生真菌,并探索菌株潜在的ZEN产生风险。【方法】以暴露ZEN的小豆为样本,将分离纯化后的内生真菌进行经典形态观察;同时扩增真菌核糖体保守序列ITS1、ITS4,镰刀菌特异基因序列FU1、FU2确定小豆中镰刀菌种属,同时通过ZEN玉米赤霉烯酮产毒控制基因pks13进行PCR扩增,分析菌株ZEN合成的潜在风险。【结果】鉴别出3株镰刀菌,其中2株初步鉴定为砖红镰刀菌(Fusarium lateritium)、1株为花腐镰刀菌(Fusarium napiforme),未检测出ZEN合成关键基因;1株玛利节菱孢霉(Arthrinium marii),分子检测具有产ZEN玉米赤霉烯酮毒素的可能性。将以上4株菌分别回接至ZEN的产毒培养基,LC-MS/MS法未检测出ZEN毒素。【结论】暴露ZEN的小豆具有内生镰刀菌,存在可能产ZEN的内生节菱孢霉;回接培养基未检测到ZEN,但不排除小豆产生ZEN的风险。     

蜜环菌对玉米加工副产物中玉米赤霉烯酮降解效果

以蜜环菌Am-07-22 为发酵菌株,以玉米皮和玉米黄粉为主要研究对象,以玉米赤霉烯酮的降解率为指标,考察不同发酵时间、发酵温度、料液比、接种量对玉米皮和玉米黄粉中玉米赤霉烯酮降解率的影响,并研究蜜环菌Am-07-22 对玉米皮和玉米黄粉不同比例混合物的毒素降解效果及产物中蛋白质和多糖的含量变化。结果表明：蜜环菌降解玉米皮中玉米赤霉烯酮的最佳条件为发酵温度27 ℃、料液比1∶1.5（g/mL）、接种量10%,玉米赤霉烯酮的降解率为93.63%,蜜环菌降解玉米黄粉中玉米赤霉烯酮的最佳条件为发酵温度27 ℃、料液比1∶2（g/mL）、接种量12.5%,玉米赤霉烯酮的降解率为96.60%。蜜环菌Am-07-22 固态发酵不同质量比的玉米皮、玉米黄粉（1∶1、1∶2、2∶1）中玉米赤霉烯酮的降解率分别为95.93%、96.62% 和96.97%,质量比为2∶1 的玉米皮、玉米黄粉中蛋白质和多糖含量提高,分别提高91% 和52%。蜜环菌Am-07-22 不仅对玉米加工副产物中玉米赤霉烯酮有良好的降解效果,同时提高产物中蛋白质和多糖含量。     

玉米赤霉烯酮生物降解研究进展

玉米赤霉烯酮是一种由镰刀菌产生的具有雌激素作用的真菌毒素,是世界上污染范围最广的一种镰刀菌素.由于物理、化学脱毒法存在较大弊端,生物去毒方法因其特异性、高效性及环境友好而日益被科学界所关注.目前玉米赤霉烯酮生物降解主要是微生物降解作用,本文紧跟国内外玉米赤霉烯酮的生物降解情况,文章介绍了降解菌株的种类,降解能力和最佳降解条件,包括降解产物毒性,降解酶基因的发掘,以及降解菌株和酶的应用方向及前景.     

粮食中玉米赤霉烯酮检测方法的研究进展和展望

<正>玉米赤霉烯酮(ZEN)起初发现于玉米赤霉病之中,由禾谷镰刀菌等菌种产生的,后经突变衍生出多种毒素种类。玉米赤霉病烯酮作用危害主要是自身的雌激素导致,其会对动植物造成生殖毒害与致畸突变,必须严格控制粮食中ZEN的含量。玉米赤霉烯酮检测是控制粮食中ZEN含量有效方法,现可行的检测方法有多种,总体上可分为三类,即化学检测、免疫胶体检测以及其他方法。     

玉米赤霉烯酮降解酶ZLHY6活性影响因素研究

以玉米赤霉烯酮降解酶ZLHY6为研究对象,采用高效液相色谱(HPLC)法,检测玉米赤霉烯酮(ZEN)的降解率,考察温度、pH值、金属离子对酶活性的影响.结果表明,该酶最适反应温度为37℃;酶降解反应的最适pH值为8.5,并对酸性环境有一定耐受能力;Mg2+对该酶有激活作用,Cu2+、Mn2+、Co2+、Ca2+对该酶有抑制作用.     

合理运用各种检测方法 准确测定饲料及食品中玉米赤霉烯酮

正玉米赤霉烯酮(ZEN)又称F-2毒素,是一种由禾谷镰刀菌以及雪腐镰刀菌等菌种产生的一种有毒代谢物,会从赤霉病玉米中分离并且影响到食品的质量。玉米赤霉烯酮具有雌激素作用,主要作用于生殖系统,可使家畜、家禽和实验小鼠产生雌性激素亢进症。     

新收获小麦中玉米赤霉烯酮监测与分析

玉米赤霉烯酮(ZEN)是真菌毒素中的一种,国标要求其在小麦和小麦粉中的限量为60μg/kg。监测近3年泰州市小麦中玉米赤霉烯酮发现,泰州市小麦中玉米赤霉烯酮含量均值远低于国家标准限值,卫生状况良好。对玉米赤霉烯酮和呕吐毒素相关性进行统计分析后发现,两种真菌毒素存在显著的正相关,方程为y=0.019x-9.007,相关系数R~2为0.9365。     

清除玉米赤霉烯酮菌株的筛选、鉴定及机理初探

通过对我国赤霉病高发区田间取样,筛选具有清除玉米赤霉烯酮(Zearalenone,ZEN)活性的菌株。获得2株清除效果较好的细菌A5、D6,其中D6活性较高。通过生理特征分析、16S r DNA序列比对、GC含量及BIOLOG分析等方法,2株菌都被鉴定为红球菌,DNA-DNA同源性分析表明,这2株菌均为Rhodococcus qingshengii。灭活细胞与ZEN共培养,发现细胞无清除ZEN能力,证明A5、D6不具有吸附ZEN的活性;菌液上清液及细胞内容物与ZEN共培养,证明D6菌株中具有清除ZEN的活性成分为胞内蛋白类物质。同时,通过HPLC荧光检测,发现疑似ZEN代谢产物峰。通过筛选获得了2株可通过胞内酶活降解ZEN的红球菌,为生物法清除真菌毒素提供参考。     

两种强启动子在枯草芽孢杆菌中调控表达研究

通过玉米赤霉烯酮(ZEN)降解酶基因ZLHY6的表达水平及降解酶的活性评价,比较了两种组成型强启动子P43与Plap S调控异源基因表达的效果。结果表明,在枯草芽孢杆菌(Bacillus subtilis)Bs 168中,Plap S调控的降解酶基因得到了高效表达,在发酵12 h时降解酶活性达到最高值,酶活为219.02 U/m L。由启动子Plap S介导的ZEN降解酶基因表达载体p WBZ7可以在Bs 168中稳定遗传,为降解酶的高效分泌表达奠定了基础。     

Aerobic and anaerobic in vitro testing of feed additives claiming to detoxify deoxynivalenol and zearalenone

Deoxynivalenol (DON) and zearalenone (ZEN) are mycotoxins produced by fungi of the genus Fusarium which frequently contaminate maize and grain cereals. Mycotoxin-contaminated feed endangers animal health and leads to economic losses in animal production. Several mycotoxin elimination strategies, including the use of commercially available DON and ZEN detoxifying agents, have been developed. However, frequently there is no scientific proof of the efficacy of such adsorbents and degrading products. We therefore tested 20 commercially available products claiming to detoxify DON and/or ZEN either by biodegradation (4 products) or a combination of degradation and adsorption (16 products) under aerobic and anaerobic conditions at approx. pH 7. Under the applied conditions, a complete reduction of DON and consequent formation of the known non-toxic metabolite DOM-1 was exclusively observed in samples taken from the anaerobic degradation experiment of one product. For all other products, incubated under aerobic and anaerobic conditions, a maximum DON reduction of 17% after 72 h of incubation was detected. Aerobic and anaerobic incubation of only one tested product resulted in complete ZEN reduction as well as in the formation of the less-toxic metabolites DHZEN and HZEN. With this product, 68–97% of the toxin was metabolised within 3 h. After 24 h, a ZEN reduction ≥ 60% was obtained with four additional products during aerobic incubation only. Six of the 20 investigated products produced α- and/or β-ZEL, which are metabolites showing similar oestrogenic activity compared to ZEN. Aerobic and anaerobic degradation to unknown metabolites with unidentified toxicity was obtained with 10 and 3 products, respectively. The results of our study demonstrate the importance of in vitro experiments to critically screen agents claiming mycotoxin detoxification.     

降解玉米赤霉烯酮菌株的鉴定及其发酵条件优化

从土壤样品中得到一株能降解玉米赤霉烯酮(zearalenone,ZEN)的菌株ZENL09,对该菌株的形态特征、生化理化特性和16r sRNA进行分析鉴定,并通过单因素实验得出ZENL09的较优的发酵条件。结果表明:ZENL09为枯草芽孢杆菌(Bacillus subtilis),其较优的发酵条件为:发酵时间24 h,发酵温度33℃,摇床转速200 r/min,装液量25 mL/250 mL,接种量3%,在此条件下,ZENL09发酵上清液对ZEN的降解率达到了96%。     

玉米赤霉烯酮降解菌的分离及降解特性研究

采用富集培养的方法从沼气污泥中筛选到一株可以高效降解玉米赤霉烯酮(zearalenone,ZEN)的菌株,命名为ASAG16。通过生理特征和16S rDNA序列分析的方法对其进行鉴定,研究了培养基、时间、pH、金属离子不同因素对ASAG16降解玉米赤霉烯酮的影响。结果表明:ASAG16为香茅醇假单胞菌;ASAG16在LB培养基中降解ZEN的效果最好,培养6 d后降解率达到91.59%;在pH 4的LB培养基中,ZEN的降解率为20.83%,在pH 5~9时,ZEN的降解率都在75%以上,降解率随pH的升高而逐渐增加;为检测不同金属离子对ASAG16降解效能的影响,在培养基中分别添加10 mmol/L的Mg~(2+)、Ca~(2+)、Mn~(2+)、Cu~(2+)、Zn~(2+)、Co~(2+)、Ba~(2+)和EDTA,ZEN的降解率分别为84.48%、86.92%、78.94%、16.22%、18.93%、18.23%、45.61%、0.72%,其中Mg~(2+)、Ca~(2+)、Mn~(2+)对ASAG16降解ZEN效果影响不明显,而Cu~(2+)、Zn~(2+)、Co~(2+)、Ba~(2+)和EDTA严重抑制ASAG16对ZEN的降解。     

西藏酵母对玉米赤霉烯酮的清除效果及作用机理研究（网络首发、推荐阅读）

为研究清除粮食真菌毒素的绿色方法并克服生物法稳定性差的难题,以西藏酵母菌种为试验对象,筛选对玉米赤霉烯酮（Zearalenone,ZEN）具有较好清除效果的菌种,并研究其作用机理。结果表明：西藏胶红酵母对ZEN具有较好的降解效果,且随菌液浓度的升高其降解效果逐步提高;对酵母不同处理液的毒素清除效果评价,发现该酵母对ZEN的清除机理主要为吸附作用,同时伴有一定的胞内生物降解作用;采用液质联用对ZEN的降解产物进行测定,发现其降解产物可能为玉米赤霉烯醇和玉米赤霉酮,降解产物的安全性需进一步验证;经西藏胶红酵母的胁迫耐受试验,表明具有一定的低温和氧化胁迫耐受能力。     

Detoxification of zearalenone and ochratoxin A by ozone and quality evaluation of ozonised corn

Zearalenone (ZEN) and ochratoxin A (OTA) are secondary toxic metabolites of fungi that can contaminate a wide range of food and feedstuff. In this study, the effects of ozone treatment on ZEN and OTA and the quality of ozonised corn are investigated. Ozone significantly affects ZEN and OTA solutions. ZEN was undetectable 5 s after being treated with 10 mg l^–1 ozone. However, OTA was resistant to ozonation with a degradation rate of 65.4% after 120 s of treatment. Moreover, ZEN and OTA solutions were difficult to degrade after being dried by a nitrogen stream. Results showed that ozone effectively degraded ZEN and OTA in corn. The degradation rates of ZEN and OTA in corn increased with ozone concentration and treatment time. The degradation of ZEN and OTA at different ozone concentrations appropriately conformed to first-order kinetics with an R² value > 0.8749. Furthermore, under the same conditions, corn with increased moisture content (MC) (19.6%) was more sensitive to ozone than corn with a low MC (14.1%). When treated with 100 mg l^–1 ozone for 180 min, ZEN and OTA in corn with 19.6% MC decreased by 90.7% and 70.7%, respectively. To evaluate the quality of ozonised corn, subsequent quality experiments were conducted using corn samples treated at different times with 100 mg l^–1 ozone. The MC of corn decreased after ozone treatment. The whiteness and yellowness of the corn increased and decreased with increasing time, respectively. The fatty acid value of the corn increased significantly (p ≤ 0.05) after 180 min of treatment. This study verified that ozone can effectively degrade ZEN and OTA in corn, but slightly affected corn quality.     

Chemical and toxicological alterations of zearalenone under ozone treatment

Our work mainly studied the elimination of zearalenone (ZEN) from corn flour by ozone treatment. We found four degradation products, after separation and identification by UPLC-ESI-QTOF-MS, named as Compound 1 (m/z 335.18), Compound 2 (m/z 351.19), Compound 3 (m/z 321.19) and Compound 4 (m/z 367.18). Then, one main transformation pathway was predicted on the basis of molecular weight and the reaction mechanism of ozone. We had also detected two of the degradation products by our modified UPLC-MS/MS method in real samples—contaminated corn flour. We screened the toxicity of the parent ZEN compound as well as the ozone induced decomposition products with BALB/c mice to evaluate the safety of our method in vivo. Therefore, aqueous ozone was demonstrated to effectively degrade ZEN and chemical structure alternations of ZEN after ozone treatment were predicted in both pure ZEN and real contaminated-corn flour samples. Considering the presence of the by-products formed, differences in the toxic effects on mice exposed to ZEN before and after the ozone treatment emphasise the significance of further study on the quality and toxicity of real food samples after ozone treatment.     

响应面法优化Paenibacillus sp.JX426产黄原胶降解酶发酵培养基

从土壤中分离筛选出1株具有较强黄原胶降解能力的类芽孢杆菌(Paenibacillus sp.)JX426.为了获得高活力的黄原胶降解酶,研究借助Minimb15软件,采用Plackett-Burman试验设计方法、最陡爬坡试验设计方法和响应面分析方法对菌株JX426进行了液体发酵条件的优化.首先通过Plackett-Burman方法对7个相关影响因素的效应进行了评价,并筛选出有显著正效应的黄原胶、CaCl2添加量和有显著负效应的酵母粉添加量等3个因素,然后利用最陡爬坡试验设计方法和响应面分析方法确定了上述3个因素的最佳工艺参数,即黄原胶、CaCl2和酵母粉的添加量分别为0.39%、0.02%和0.042%.试验结果表明,在最佳浓度和组成条件下,黄原胶降解酶的酶活能达到4.20U/mL,较优化前的3.05U/mL提高了37.7%,为黄原胶降解菌的实际应用奠定了基础.     

降解柠檬苦素菌株的筛选鉴定及其发酵条件的优化

为解决柠檬苦素脱苦问题,筛选了可降解柠檬苦素的菌株并优化该菌株的产酶条件。从多年生橘园的土壤和发酵时期的醋醅中筛选并分离出8株可降解柠檬苦素的菌株,对较好降解柠檬苦素的C-1-5、C-1-2-2、JJ-3菌株进行形态学观察和分子生物学鉴定,在单因素实验的基础上,选JJ-3菌株进行响应面法优化的发酵工艺。筛选得到的8株菌中C-1-5、C-1-2-2、JJ-3菌株的柠檬苦素降解率分别为27%、36%、38%。鉴定C-1-5菌株为纤维菌属(Cellulomonas sp.),JJ-3菌株和C-1-2-2菌株为苍白杆菌属(Ochrobactrum sp.)。单因素优化后C-1-5、JJ-3、C-1-2-2菌株柠檬苦素降解率分别提高至65.90%、72.79%、74.66%。通过响应面试验,JJ-3菌株最佳发酵工艺条件是:pH3.5,温度30℃,氮源为硝酸铵,菌接种量3×108 CFU/mL。在此工艺条件下,柠檬苦素降解率为78.90%。优化后的菌株具有较高的降解率,可为柠檬苦素酶的研究提供帮助。