微生物降解玉米赤霉烯酮的研究进展

玉米赤霉烯酮（zearalenone,ZEN）是霉变谷物中常见的霉菌毒素之一,主要出现在霉变的玉米、小麦等谷物中,给畜禽和人类带来一定程度的健康危害,如生殖毒性、免疫毒性、肝毒性和肾毒性等。目前,解决玉米赤霉烯酮污染问题的方法包括物理、化学和生物3个途径。虽然传统的物理和化学脱毒方法已经运用在许多的饲料生产中,但同时也存在着二次污染的风险。生物降解法是一种利用微生物吸附和降解玉米赤霉烯酮的脱毒方法,具有安全环保、高效、特异性强和脱毒率高的特性,且不影响谷物的营养价值,已成为玉米赤霉烯酮降解研究的热点。本文主要介绍了近年来降解玉米赤霉烯酮的微生物种类,并将其归纳分类,从微生物的脱毒能力、脱毒方法和脱毒产物进行了叙述,综述了微生物脱毒的优点及前景,以期为微生物降解玉米赤霉烯酮的理论研究及实际应用提供新的视角。     

下期论文预告

我国科研机构对玉米赤霉烯酮(ZON)等真菌毒素的研究报道和综述论文不少,但是往往只关注它们的检测、毒理、危害和防治措施,对它们的生物合成和降解过程很少报道。     

玉米赤霉烯酮降解菌的鉴定及其降解特性

【背景】玉米赤霉烯酮(Zearalenone,ZEN)是一种具有类雌激素作用的霉菌毒素,常会污染谷物和饲料,严重威胁动物和人类的健康。生物脱毒作为理想的去除ZEN的方法,广受关注,然而相关菌株较少,仍有待进一步筛选。【目的】明确一株玉米赤霉烯酮降解菌的生物学分类地位,并优化其赤霉烯酮降解菌降解条件。【方法】通过菌株的16S rRNA基因序列比对,构建系统发育进化树,并开展了相关培养条件的单因素优化和玉米赤霉烯酮降解动力曲线的绘制。【结果】实验菌株WLB-29经鉴定为斯塔普氏菌属(Stappia),其16S rRNA基因序列在GenBank上登录号为MT196321,该序列与模式菌株Stappia indica B106T相似性最高为97.47%,初步确定为斯塔普氏菌属潜在新种。单因素优化表明,菌株降解玉米赤霉烯酮的最佳条件为LB培养基、37℃培养、pH 8.0、2%接种量和玉米赤霉烯酮初始浓度为10mg/L,在此条件下培养144h后,玉米赤霉烯酮的降解率最高可达92.56%。【结论】菌株WLB-29具有较好的ZEN降解作用,为进一步解析菌株降解ZEN作用机理提供了研究基础,也为进一步开发利用菌株开展ZEN的生物脱毒提供了新的菌株资源。     

玉米赤霉烯酮脱毒菌PA26-7的分离鉴定及其应用效果评价

【背景】玉米赤霉烯酮(zearalenone,ZEN)是广泛污染粮谷类作物的一种雌激素类真菌毒素,不仅给农业经济带来巨大损失,还能通过食物链对人和动物健康造成危害。【目的】从微生态制剂中筛选获得能够高效降解玉米赤霉烯酮的菌株,优化其脱毒条件,测定其在饲料中的实际脱毒效果及对饲料中植酸、维生素含量变化的影响。【方法】从微生态制剂中分离出玉米赤霉烯酮降解菌,通过细胞计数试剂盒-8 (cell counting kit-8, CCK-8)测定菌株降解玉米赤霉烯酮产物的细胞毒性和雌激素活性,通过高效液相色谱法测定分离株在培养基和饲料中的解毒效果,以及分离株在霉变的豆粕、麸皮和成品饲料中固态发酵前后维生素的含量变化,通过三氯化铁比色法测定饲料脱毒前后植酸的含量变化。【结果】从微生态制剂中筛选出一株通过分泌胞外酶高效降解玉米赤霉烯酮的贝莱斯芽孢杆菌(Bacillus velezensis) PA26-7,该菌株在培养基起始pH 4.0-8.0、培养温度25-60℃条件下均可降解玉米赤霉烯酮,产物的细胞毒性和雌激素活性均较ZEN弱。PA26-7经固态发酵72h后,饲料原料(豆粕和麸皮)及霉变的成品鸡...     

玉米赤霉烯酮降解酶毕赤酵母表达载体的构建及其表达

目的构建毕赤酵母表达载体pPIC9-ZEN-jjm,筛选高效分泌表达活性目的蛋白的菌株。方法克隆ZEN-jjm基因,经EcoRⅠ和NotⅠ双酶切连接至pPIC9中,电转化至毕赤酵母GS115。利用RDB培养基和甲醇诱导表达进行筛选。HPLC检测表达蛋白降解玉米赤霉烯酮的活性。结果测序表明ZEN-jjm成功插入pPIC9中,SDS-PAGE表明获得1株高效表达目的蛋白的重组酵母,其分子量约29 kDa。HPLC表明其能有效地降解玉米赤霉烯酮。结论玉米赤霉烯酮降解酶在毕赤酵母中获得了高效分泌表达。     

玉米赤霉烯酮的生理作用（简报）

玉米赤霉烯酮抑制种子萌发和出苗;经玉米赤霉烯酮浸种后播于田间的春小麦三叶期至四叶期间,叶长和叶绿素含量以及最后的产量性状均高于对照;低浓度的玉米赤霉烯酮有促进萝卜黄化了叶增重和变绿的作用。     

春化冬小麦的玉米赤霉烯酮结合蛋白（简报）

玉米赤霉烯酮是八十年代初从高等植物中鉴定出的一种微量生理活性物质,已证明它在植物成花过程中起重要作用。为了阐明玉米赤霉烯酮的作用机制,我们用放射配体竞争结合分析法研究了春化冬小麦的ＺＥＮ特异结合蛋白。结果表明在春化冬小麦胚芽中存在着可溶性的ＺＥＮ特异结合蛋白（ＺＢＰ）。结合反应的ＰＨ范围在６－８,加热、蛋白酶和尿素处理破坏结合活性。玉米赤霉烯酮的同系物α－玉米赤霉醇和β－玉米赤霉醇,以及动物雌性激     

来源于Rhinocladiella mackenziei的玉米赤霉烯酮水解酶的表达纯化与酶学性质

【背景】玉米赤霉烯酮(Zearalenone,ZEN)是污染最广泛的霉菌毒素之一,对饲料行业和畜牧业造成了巨大的经济损失。目前研究最为广泛的玉米赤霉烯酮降解酶ZHD101因其热稳定性较差,无法满足工业应用上的要求。【目的】为实现玉米赤霉烯酮降解酶在工业上的应用,寻找酶学性质更突出的ZEN降解酶。【方法】基于对Gen Bank数据库的挖掘,发现一个来源于麦氏喙枝孢霉(Rhinocladiella mackenziei CBS 650.93)的Rmzhd基因,构建p ET-46-Rmzhd质粒。利用大肠杆菌表达体系和亲和层析、离子交换纯化体系对蛋白进行表达和纯化,通过高效液相凝胶色谱分析酶学性质。【结果】发现一个新的ZEN水解酶Rm ZHD,RmZHD在pH 8.6和45°C条件下的活性最高,而且具有较高的耐热性。结构分析表明,较高的盐桥数目和溶剂暴露脯氨酸含量可能是造成其高耐热性的原因。【结论】本研究为促进玉米赤霉烯酮降解酶在工业上的应用打下基础。     

冬小麦种子萌发过程中的结合态玉米赤霉烯酮

冬小麦种子萌发过程中的结合态玉米赤霉烯酮陈新建（河南农业大学农学系,郑州１５０００２）孟繁静（北京农业大学生物学院,北京１０００９４）关键词结合态玉米赤霉烯酮,冬小麦玉米赤霉烯酮（ｚｅａｒａｌｅｎｏｎｅ）是玉米赤霉菌（Ｇ～ｔｈｅ－）的一种次生代谢产物...     

藤黄微球菌降解真菌毒素玉米赤霉烯酮的研究

目的研究并优化藤黄微球菌降解真菌毒素玉米赤霉烯酮(ZEN)的因素条件。方法采用HPLC的检测方法对藤黄微球菌降解真菌毒素玉米赤霉烯酮的影响因素(培养基、温度、pH、摇床转速、培养时间和金属离子等)进行优化研究。结果藤黄微球菌在0.05 mol/LMnCl2、初始pH为7.0的LB培养基中,37℃,180 r/min,连续培养120 h,能降解99%的ZEN毒素(初始浓度为2μg/ml)。结论藤黄微球菌降解真菌毒素ZEN的能力与培养基成分、pH和添加的金属离子种类密切相关。     

Induction of a zearalenone degrading enzyme caused by the substrate and its derivatives

The Fusarium toxin zearalenone (ZON) is very harmful to animal and man due to its estrogenic effect, immunotoxicity and genotoxicity. Therefore, it is of high importance to establish a system for the detoxification of ZON. In large screening programmes, only the mycoparasiteGliocladium roseum (DSM 62726) was found to be capable of detoxifying ZON, by not yet characterized enzyme(s). It is the only known microorganism hydrolyzing the lactonic bond within the macrocyclic ring system of ZON. The resulting products are less toxic because they loose their estrogenic capacity. The extent of toxin degradation is enhanced when enzyme production inG roseum is induced by the substrate ZON itself and its derivatives. ZON and its derivatives differ in the ability to induce enzyme production. This was investigated underin vitro conditions. Differences were found in the required amount of the inducing substances and time optimum of induction in order to get maximal degradation of ZON. The regulation and biochemical properties of the enzyme are to be characterized as a prerequisite to develop applications aimed at the detoxification of ZON in food and feeding-stuff. Our aim is to isolate the unknown enzyme which is capable of the detoxification of this mycotoxin.     

Excretion kinetics and metabolism of zearalenone in broilers in dependence on a detoxifying agent

Two experiments were carried out with male broilers to examine excretion kinetics of zearalenone (ZON) and its metabolites and their occurrence in blood plasma and bile fluid after a single oral dose of ZON (approximately 6 μg/kg BW) from naturally contaminated wheat (406 μg ZON per kg). In addition, this ZON bolus was administered either in the absence or presence of a detoxifying agent (Mycofix®‐Plus, Biomin GmbH, Herzogenburg, Austria). Specimens were sampled after administration of the zearalenone bolus at different times of up to 48 h.

Excretion of zearalenone and α‐zearalenol as the only detectable metabolite of ZON peaked at approximately 6.5 h after administration of the bolus. Cumulative excretion of both substances amounted to approximately 58% of ZON intake after 48 h, when a plateau was achieved. The incomplete recovery could have been due to a partial total degradation of ZON in the digestive tract, undetected sulfate conjugates of ZON or its metabolites, to other unknown and undetected metabolites or to incomplete analytical recovery from the matrix, and needs to be examined further.

Peak concentrations of zearalenone and a‐zearalenol in bile were detected in the time period of approximately 2 to 6 h after bolus, whereas ZON and metabolite concentrations in blood plasma were around or lower than the detection limits. Mycofix®‐Plus supplementation seemed to have only minor or no effects on the parameters examined.     

Effects of long-term storage on Fusarium toxin concentrations in wheat--sources of error of the analytical results

The concentrations of the Fusarium toxins deoxynivalenol (DON) and zearalenone (ZON) of a heavily contaminated wheat grain batch were followed over a period of 1 year by taking samples 15 times every 28 days. The air temperature and relative humidity at the top of the wheat batch ranged between 7 and 22 degrees C and 44 and 55%, respectively, and corresponded to a variation in the moisture content of the wheat grain between 11.5 and 12.3%. None of these fluctuations were related to ZON and DON concentrations, which varied between 0.46 and 0.66 and 15.0 and 19.5 mg/kg DM. Therefore, the data were used to analyse the error sources for the analytical results. It was found that the variance proportions due to sampling and sample preparation plus analysis were not similar for DON and ZON. The variance proportion due to sampling was found to be 0.62 for ZON, which corresponded to a variance proportion of 0.38 due to sample preparation plus analysis. In contrast, the latter variance proportion for DON was estimated to be 1.0 and consequently completely superimposed the sampling error. It is concluded that long-term storage of contaminated wheat grain does not affect the concentrations of DON and ZON considering the measured fluctuations in ambient temperature, relative humidity and moisture content of the grain. Therefore, no degradation of DON and ZON occurred during the storage of wheat for a period of one year under ambient conditions. The effects of sampling and sample preparation plus analysis on the final analytical results are different for DON and ZON and require further consideration.     

Effects of long-term storage on Fusarium toxin concentrations in wheat - sources of error of the analytical results

The concentrations of the Fusarium toxins deoxynivalenol (DON) and zearalenone (ZON) of a heavily contaminated wheat grain batch were followed over a period of 1 year by taking samples 15 times every 28 days. The air temperature and relative humidity at the top of the wheat batch ranged between 7 and 22°C and 44 and 55%, respectively, and corresponded to a variation in the moisture content of the wheat grain between 11.5 and 12.3%. None of these fluctuations were related to ZON and DON concentrations, which varied between 0.46 and 0.66 and 15.0 and 19.5 mg/kg DM. Therefore, the data were used to analyse the error sources for the analytical results. It was found that the variance proportions due to sampling and sample preparation plus analysis were not similar for DON and ZON. The variance proportion due to sampling was found to be 0.62 for ZON, which corresponded to a variance proportion of 0.38 due to sample preparation plus analysis. In contrast, the latter variance proportion for DON was estimated to be 1.0 and consequently completely superimposed the sampling error. It is concluded that long-term storage of contaminated wheat grain does not affect the concentrations of DON and ZON considering the measured fluctuations in ambient temperature, relative humidity and moisture content of the grain. Therefore, no degradation of DON and ZON occurred during the storage of wheat for a period of one year under ambient conditions. The effects of sampling and sample preparation plus analysis on the final analytical results are different for DON and ZON and require further consideration.     

A sensitive and inexpensive yeast bioassay for the mycotoxin zearalenone and other compounds with estrogenic activity

Zearalenone (ZON) is a nonsteroidal estrogenic mycotoxin produced by plant-pathogenic species of Fusarium. As a consequence of infection with Fusarium culmorum and Fusarium graminearum, ZON can be found in cereals and derived food products. Since ZON is suspected to be a cause of human disease, including premature puberty syndrome, as well as hyperestrogenism in farm animals, several countries have established monitoring programs and guidelines for ZON levels in grain intended for human consumption and animal feed. We developed a low-cost method for monitoring ZON contamination in grain based on a sensitive yeast bioassay. The indicator Saccharomyces cerevisiae strain YZRM7 is unable to grow unless an engineered pyrimidine biosynthetic gene is activated by the expressed human estrogen receptor in the presence of exogenous estrogenic substances. Deletion of the genes encoding ATP-binding cassette (ABC) transporters Pdr5p and Snq2p increases net ZON uptake synergistically. Less than 1 microg of ZON per liter of medium is sufficient to allow growth of the indicator strain. To prevent interference with pyrimidines potentially present in biological samples, we also disrupted the genes FUR1 and URK1, blocking the pyrimidine salvage pathway. The bioassay strain YZRM7 allows qualitative detection and quantification of total estrogenic activity in cereal extracts without requiring further cleanup steps. Its high sensitivity makes this assay suitable for low-cost monitoring of contamination of maize and small grain cereals with estrogenic Fusarium mycotxins.     

A Sensitive and Inexpensive Yeast Bioassay for the Mycotoxin Zearalenone and Other Compounds with Estrogenic Activity

Zearalenone (ZON) is a nonsteroidal estrogenic mycotoxin produced by plant-pathogenic species of Fusarium. As a consequence of infection with Fusarium culmorum and Fusarium graminearum, ZON can be found in cereals and derived food products. Since ZON is suspected to be a cause of human disease, including premature puberty syndrome, as well as hyperestrogenism in farm animals, several countries have established monitoring programs and guidelines for ZON levels in grain intended for human consumption and animal feed. We developed a low-cost method for monitoring ZON contamination in grain based on a sensitive yeast bioassay. The indicator Saccharomyces cerevisiae strain YZRM7 is unable to grow unless an engineered pyrimidine biosynthetic gene is activated by the expressed human estrogen receptor in the presence of exogenous estrogenic substances. Deletion of the genes encoding ATP-binding cassette (ABC) transporters Pdr5p and Snq2p increases net ZON uptake synergistically. Less than 1 μg of ZON per liter of medium is sufficient to allow growth of the indicator strain. To prevent interference with pyrimidines potentially present in biological samples, we also disrupted the genes FUR1 and URK1, blocking the pyrimidine salvage pathway. The bioassay strain YZRM7 allows qualitative detection and quantification of total estrogenic activity in cereal extracts without requiring further cleanup steps. Its high sensitivity makes this assay suitable for low-cost monitoring of contamination of maize and small grain cereals with estrogenic Fusarium mycotxins.     

Cleavage of Zearalenone by Trichosporon mycotoxinivorans to a Novel Nonestrogenic Metabolite

Zearalenone (ZON) is a potent estrogenic mycotoxin produced by several Fusarium species most frequently on maize and therefore can be found in food and animal feed. Since animal production performance is negatively affected by the presence of ZON, its detoxification in contaminated plant material or by-products of bioethanol production would be advantageous. Microbial biotransformation into nontoxic metabolites is one promising approach. In this study the main transformation product of ZON formed by the yeast Trichosporon mycotoxinivorans was identified and characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and LC-diode array detector (DAD) analysis. The metabolite, named ZOM-1, was purified, and its molecular formula, C₁₈H₂₄O₇, was established by time of flight MS (TOF MS) from the ions observed at m/z 351.1445 [M-H]⁻ and at m/z 375.1416 [M+Na]⁺. Employing nuclear magnetic resonance (NMR) spectroscopy, the novel ZON metabolite was finally identified as (5S)-5-({2,4-dihydroxy-6-[(1E)-5-hydroxypent-1-en-1-yl]benzoyl}oxy)hexanoic acid. The structure of ZOM-1 is characterized by an opening of the macrocyclic ring of ZON at the ketone group at C6′. ZOM-1 did not show estrogenic activity in a sensitive yeast bioassay, even at a concentration 1,000-fold higher than that of ZON and did not interact with the human estrogen receptor in an in vitro competitive binding assay.Zearalenone (ZON) is the main member of a growing family of biologically important “resorcylic acid lactones” (RALs), which have been found in nature. ZON is produced by several Fusarium species, which colonize maize, barley, oat, wheat, and sorghum and tend to develop ZON during prolonged cool, wet growing and harvest seasons (38). Maize is the most frequently contaminated crop plant, and therefore, ZON can be found frequently in animal feeding stuff. Occurrence, toxicity, and metabolism data of ZON were summarized by the European Food Safety Authority (EFSA) (5) and in recent reviews (12, 38).The potent xenohormone ZON leads to hyperestrogenism symptoms and in extreme cases to infertility problems, especially in pigs (15). Ovarian changes in pigs have been noted with toxin levels as low as of 50 μg/kg in the diet (1). Ruminants are more tolerant to ZON ingestion; however, hyperestrogenic syndrome, including restlessness, diarrhea, infertility, decreased milk yields, and abortion, have been well documented with cattle and sheep (4, 29).Because widespread ZON contamination in feed can occur in problematic years, efficient ways to detoxify are desirable. The transformation of mycotoxins to nontoxic metabolites by pure cultures of microorganisms or by cell-free enzyme preparations (3) is an attractive possibility. Microbial metabolization of ZON to alpha-ZOL and beta-ZOL cannot be regarded as detoxification, because both ZOL products are still estrogenic (14). Also, formation of ZON-glucosides and -diglucosides (8, 17) and ZON-sulfate (7) cannot be considered true detoxification but rather formation of masked mycotoxins, because the conjugates may be hydrolyzed during digestion (11, 23), releasing ZON again (2).As the estrogenic activity of ZON and its derivates can be explained by its chemical structure, which resembles natural estrogens (20), it can be expected that cleavage of the lactone undecyl ring system of ZON results in permanent detoxification.El-Sharkawy and Abul-Hajj (9) were the first to report inactivation of ZON after opening of the lactone ring by Gliocladium roseum. This filamentous fungus was capable of metabolizing ZON in yields of 80 to 90%. Also Takahashi-Ando et al. (31) described the degradation reaction of ZON with Clonostachys rosea (synonym of G. roseum). A hydrolase (encoded by a gene designated ZHD101) cleaves the lactone ring, and as recently proved (37; unpublished data) by subsequent decarboxylation of the intermediate acid, the compound 1-(3,5-dihydroxyphenyl)-10′-hydroxy-1′E-undecene-6′-one is formed. In contrast to ZON and 17β-estradiol, which showed potent estrogenic activity, this cleavage product did not show any estrogenic activity in the human breast cancer MCF-7 cell proliferation assay (16). Further details, e.g., on the conditions of the maximum activity of ZHD101 and its exploitation in genetically modified grains, can be found in later published work of this research group (32, 33).Only a few authors reported the loss of estrogenicity in microbial metabolites of ZON, which are based on reactions other than cleavage of the lactone undecyl ring system. El-Sharkawy and Abul-Hajj demonstrated (10) that binding to rat uterine estrogen receptors requires a free 4-OH phenolic group (devoid of methylation or glycosylation). Loss of estrogenicity was, for instance, observed with 2,4-dimethoxy-ZON, one of the metabolites produced by Cunninghamella bainieri ATCC 9244B. Nevertheless, this rule cannot be generalized, as 8′-hydroxyzearalenone formed by Streptomyces rimosus NRRL 2234, despite having a free 4-phenolic hydroxyl group, did not bind to the estrogen receptor. Also, other authors reported that 8′-hydroxyzearalenone and 8′-epi-hydroxyzearalenone are nonestrogenic (13). However, so far, no practical application in feed or food detoxification has been found for the microorganisms producing these compounds.It has been shown previously that the yeast Trichosporon mycotoxinivorans has a very high capability to degrade both ochratoxin A (OTA) and ZON (22, 26, 27). When T. mycotoxinivorans is used as a feed additive preparation, microbial degradation of the mycotoxins is assumed to take place in the gastrointestinal tract of the animal after consumption of contaminated feed. The protective effect of T. mycotoxinivorans against OTA toxicity has already been shown with broiler chicken (24).In the present study we report the isolation, analytical characterization, and structure elucidation, as well as the evaluation, of the estrogenic activity of the main degradation product of ZON produced by T. mycotoxinivorans.     

Fusarium toxin residues in physiological samples of piglets

Physiological samples of 100 piglets fed diets containing 0.01, 0.06, 0.15, 0.22 and 0.42 mg ZON and 0.2, 0.8, 1.0, 1.9 and 3.9 mg DON per kg over a period of 35±1.5 days were investigated for concentrations of deoxynivalenol (DON) and zearalenone (ZON) and their metabolites. DON was detected in serum, bile and urine in increasing concentrations corresponding to the diet contamination. The metabolite de-epoxy-DON was detected only in urine. The DON contamination of the diet was closely reflected by the serum concentrations of the piglets. ZON and its metabolite α-zearalenol were detected in bile fluid, liver and urine, while β-zearalenol was only detected in bile fluid. In serum neither ZON nor its metabolites were found. The total concentration of ZON plus its metabolites in the bile fluid corresponded well with the dietary contamination. For all analyses it has to be noted that toxin residues were detectable even in individual samples of piglets fed the control diet containing 0.01 mg ZON/kg and 0.2 mg DON/kg. Presented at the 25^th Mykotoxin Workshop in Giessen, Germany, May 19–21, 2003     

Purification and characterization of a zearalenone degrading enzyme produced by<Emphasis Type="Italic">Gliocladium roseum</Emphasis>

Enzymatic inactivation of fungal toxins is an attractive strategy for the decontamination of food and feeding stuff. A constitutively expressed enzyme opening the lactone linkage within the macrocyclic ring system of zearalenone (ZON) was isolated fromGliocladium roseum. The enzyme has been shown to catalyze the transformation of the mycotoxin ZON and therefore has been named ZON degrading enzyme. The resulting products of the enzymatic reaction are less toxic because they have lost their estrogenic capacity. In this study, we used scanning electron microscopy to evaluate the possible mycoparasitism betweenFusarium graminearum andG. roseum. The ZON-degrading enzyme could be isolated fromG. roseum cultures and biochemically characterized. It has been found to be similar to superoxide-dismutases at its N-teminus.