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...     

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。     

春化冬小麦的玉米赤霉烯酮结合蛋白(简报)(英文)

玉米赤霉烯酮(Zearalenone,简称ZEN)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,以及动物雌性激素雌二醇可与ZEN竞争结合ZBP。其它植物激素不能与ZBP发生竞争结合。蔗糖密度梯度离心分析表明ZBP沉降于4-5S区。     

真菌毒素玉米赤霉烯酮生物降解的研究进展

玉米赤霉烯酮(Zearalenone,ZEN)及衍生物是一类主要由镰刀菌属的真菌产生的非甾体雌激素类真菌毒素,广泛存在于玉米、大麦、小麦和高粱等谷物饲料及其副产品中,严重危害牲畜及人类健康,迫切需要相关的技术对ZEN进行降解脱毒。传统的物理化学方法不能有效去除谷物中的毒素,并会破坏谷物的营养成分,影响食物口感,甚至造成二次污染,因此利用生物工程技术对ZEN及其衍生物进行脱毒是未来解决这一问题的主要方法。文中简要介绍了ZEN及衍生物和降解ZEN的微生物种类、降解特性,然后详细介绍了目前研究的ZEN降解酶种类、解析唯一的蛋白结构及其异源表达和应用情况,以期为通过分子酶工程和发酵工程等生物工程技术降低ZEN降解酶的成本提供指导,从而提高食品安全。     

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。     

玉米赤霉烯酮生物合成和降解的研究进展

介绍了玉米赤霉烯酮及其6个衍生物的分子结构,以及玉米赤霉烯酮的生物合成途径。阐述了玉米赤霉烯酮生物合成的基因序列群以及基因簇的表达方式。论述了玉米赤霉烯酮钝化和降解过程, 探讨了玉米赤霉烯酮水解酶在基因工程中的应用研究, 并为今后玉米赤霉烯酮生物工程降解研究提出了建议。     

来源于麦氏喙枝孢霉的玉米赤霉烯酮水解酶在毕赤酵母中的高效表达及活性分析

【背景】玉米赤霉烯酮(zearalenone,ZEN)及其衍生物是一群具有雌激素活性的霉菌毒素,广泛存在于被霉菌污染的谷物中,造成食品业和畜牧业的巨大损失。利用专一性高的水解酶进行生物转化可有效去除玉米赤霉烯酮。【目的】构建高效表达玉米赤霉烯酮水解酶的酵母系统,以促进玉米赤霉烯酮水解酶的研究和工业应用。【方法】将来源于麦氏喙枝孢霉(RhinocladiellamackenzieiCBS650.93)的Rmzhd基因转入毕赤酵母中,筛选获得高效表达菌株,通过高效液相色谱分析发酵液中重组酶的性质。【结果】发酵液中Rm ZHD对ZEN的酶活力为16.67 U/m L,对α-ZOL的酶活力为9.85 U/m L。SDS-PAGE检测表达产物的分子量,与理论值30.7k D符合,且发酵上清液蛋白纯度高。Rm ZHD的最适p H值为9.6,最适温度为45°C,并具有较好的耐热性。【结论】研究结果为玉米赤霉烯酮水解酶的异源表达及其潜在的工业应用提供了一定的指导。     

酪丁酸梭菌清除饲料中玉米赤霉烯酮的能力

研究酪丁酸梭菌吸附清除饲料中玉米赤霉烯酮的能力,为其在农作物饲料中的脱毒作用奠定基础。首先通过单因素实验确定初始菌质量浓度、pH、温度和时间的影响,并探究热、酸处理后的菌株清除玉米赤霉烯酮的能力,再采用微生物适应性进化方法驯化酪丁酸梭菌,以提高其脱毒效果。结果表明:当初始质量浓度为1.0×10~(11)个/mL的原始菌悬于pH 4.0的培养基,37℃孵育24 h时,对玉米赤霉烯酮毒素的清除效果分别为活菌组31.9%、热处理组44.2%、酸处理组50.2%,说明原始菌对玉米赤霉烯酮具有一定的清除能力。而驯化后的菌株经过热、酸处理,经验证当菌体密度为1.0×10~(11)个/mL时,酸处理后的驯化菌株对毒素的吸附效果最好,吸附率可达98.5%,有望被运用于实际生产中,从而减少饲料中的毒素含量,有利于家禽体内毒素的清除,降低毒素积累。     

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。     

牛樟芝发酵液提取物抗菌活性研究

牛樟芝作为一种珍稀食用和药用菌,具有极大的开发潜力。该研究以麦芽浸粉肉汤液体培养基(BD,美国BD公司)对牛樟芝菌丝体进行摇床培养60 d后,收获发酵液并用乙酸乙酯对其进行萃取,浓缩至干获得提取物;同时,采用抑菌圈法评价培养物对13种致病细菌抗菌活性(蜡样芽孢杆菌、缓慢芽孢杆菌、无乳链球菌、短小芽孢杆菌、福氏志贺氏菌、枯草芽孢杆菌、金黄色葡萄球菌、藤黄微球菌、副溶血性弧菌、溶血性葡萄球菌、铜尿假单胞菌、乙型副伤寒沙门氏菌、大肠埃希菌),并检测相应致病细菌的最低抑制浓度(MIC)。结果表明:牛樟芝麦芽浸粉肉汤发酵液提取物对供试的13种致病菌均有抑菌活性;在供试的13种致病菌中,提取物对缓慢芽孢杆菌、短小芽孢杆菌、枯草芽孢杆菌、副溶血性弧菌、藤黄微球菌5种致病菌的最低抑制浓度值均小于80μg·m L~(-1),其中对藤黄微球菌的最低抑制浓度最低为66.5μg·m~(-1);随着培养时间的增加,提取物的抗菌活性也增加。这说明牛樟芝菌丝体在液体培养条件下,能够产生广谱高效抑菌活性的次生代谢产物。该研究结果为牛樟芝进一步的有效利用开发奠定了理论基础。     

Feather degradation by Bacillus sp. FK 46 in submerged cultivation

Cultivation conditions affecting feather degradation by Bacillus sp. FK 46 were investigated. The results showed that feather was almost completely degraded under the following conditions: 1% whole chicken feather as a substrate at the initial medium pH of 9 with 5% bacterial inoculum, at a temperature of 37 degrees C and a shaking speed of 250 rev/min. Glucose, methanol, Tween 80 and Triton X-100, however, had no effect on feather degradation. After feather was degraded, its residue and fermented broth would become a protein feed for animals.     

黄曲霉毒素B1降解菌的分离鉴定及其降解特性

【目的】黄曲霉毒素是一类强毒、致癌的真菌次级代谢产物。本文旨在筛选出能高效降解黄曲霉毒素B1(AFB1)的细菌。【方法】以AFB1结构类似物香豆素为惟一碳源进行AFB1降解菌株初筛,得到的活性菌株的培养液分别与AFB1标准品(2.5μg/mL)共同作用,以AFB1降解率为指标进行复筛。对降解活性最好的菌株通过形态、生理生化特性以及16S rRNA序列分析进行初步鉴定;并对细胞浓度、pH、温度、金属离子等对菌株降解活性的影响进行考察。【结果】初筛获得了10株在香豆素培养基上生长良好的细菌,复筛发现这些菌均具有良好的AFB1降解活性,其中从金毛羚牛粪便中筛选出的菌株F4降解活性最好,去除AFB1能力达到90.03%。根据F4菌株16S rRNA序列同源性分析,结合形态、生理生化特性,初步确定菌株F4为施氏假单胞菌(Pseudomonas stutzeri)。F4的降解活性与细胞浓度呈正相关。当pH 7.0,35℃,菌细胞作用72 h后毒素降解率达到82.91%。Mg2+可增强F4的降解活性,降解率提高7.68%,而Cu2+可抑制其降解活性,降解率降低51.1%。【结论】筛选到能高效降解AFB1的施氏假单胞菌(Pseudomonas stutzeri)F4,F4降解毒素的活性物质主要存在于菌体细胞,其作用受到温度、pH等的影响,可能是一种胞内酶。     

An Improved Bacteriological Method for the Detection of Sulfonamide Residues in Food

The addition of trimethoprim to Mueller-Hinton medium was found to improve the sensitivity of test bacteria towards sulfonamides by 20—50 times. Depending on the test bacteria used, the optimal concentrations of trimethoprim added to the medium are 1 μg per ml (Micrococcus luteus), 0.25 μg per ml (Bacillus stearothermophilus var. calidolactis) and 0.1 μg per ml (Bacillus megaterium). Using Micrococcus luteus or Bacillus megaterium a concentration of 0.25 μg sulfanilamide per ml may be detected, and 0.1 μg per ml may be detected when Bacillus stearothermophilus var. calidolactis is used.     

Detection of lysozyme-like enzymatic activity secreted by an immune-responsive mosquito cell line

Using molecular approaches, we have recently shown that the C7-10 mosquito cell line from Aedes albopictus, and the Aag-2 line from Aedes aegypti, secrete a variety of immune peptides into the culture medium, including cecropins, defensins, transferrin, and lysozyme. The diversity of these peptides makes it difficult to quantify the relative activities of each molecule, because possible synergistic interactions may occur. Using a microtiter plate assay with live bacteria, we now show that C7-10 cells secrete an activity that is more potent against the Gram-positive bacterium, Micrococcus luteus, than against Gram-negative Escherichia coli. This lysozyme-like activity is accompanied by production of a lytic zone in an agarose plate assay containing commercially available, lyophilized M. luteus. Properties of the lysozyme-like activity from C7-10 cells included a broad pH optimum from 5.5 to 6.5, and heat-sensitivity above 42 degrees C. Amounts of secreted activity increased during the initial 24h of incubation with heat-killed bacteria. During this induction, lysozyme-like activity was found primarily in the cell culture supernatant.     

The antimicrobial activity of Aspergillus fumigatus is enhanced by a pool of bacteria

In a screening program for new antibiotic producers, a strain of Aspergillus fumigatus was isolated from Brazilian soil samples. A pool of autoclaved bacteria was added to part of the fungus culture on the second day of fermentation to increase antibiotic production. The chloroform extract from the culture broth to which the pool of autoclaved bacteria was added showed an increase of 55%, 63% and more than 100% in activity against Staphylococcus aureus, Candida albicans and Micrococcus luteus, respectively. Also, the HPLC chromatographic profiles of the chloroform extracts from both culture conditions were different. Two active compounds were isolated from the broth of the culture grown in the presence of pooled bacteria and were identified as 3,4-dimethoxyphenol and 1,3,5-trimethoxybenzene.     

Antimicrobial activity of a nisin-activated plastic film for food packaging

AIMS: To determine the effectiveness of a packaging film coated with nisin to inhibit Micrococcus luteus ATCC 10240 in tryptone soya broth (TSB) and the microbiota of raw milk during storage. A further aim was to examine the release of nisin from the activated film. METHODS AND RESULTS: An active package, obtained from nisin-treated film, was filled with 1 l of M. luteus ATCC 10240 (ML) suspension in TSB and stored at 4 and 25 degrees C for 2 days. After 24 h at 25 degrees C there was a remarkable reduction of M. luteus ATCC 10240 compared with the control, while at 4 degrees C a slight reduction was observed. Moreover, microbial growth was controlled when 1 l of three different kinds of milk was poured into the active package and stored at 4 degrees C for 7 days. The most significant results were observed in raw milk and pasteurized milk with a reduction of 0.9 log and 1.3 log, respectively. The release experiments showed that nisin release from the film was unpredictable, but it was favoured by low pH and high temperature. CONCLUSIONS: It appears that nisin-coated films were effective in inhibiting M. luteus ATCC 10240 in TSB and the bacterial flora in milk, and the release of nisin was pH and temperature dependent. SIGNIFICANCE AND IMPACT OF THE STUDY: Nisin-activated film may control bacterial growth, maintaining food quality, safety and extending the shelf-life of food products.     

The role of alkylhydroxybenzenes in the adaptation of Micrococcus luteus to heat shock

The response of the gram-positive bacterium Micrococcus luteus to heat shock (45 degrees C, 15 min) and the adaptogenic activity of alkylhydroxybenzenes (AHB), which are extracellular growth-regulating substances of these bacteria, were studied. The perception of stress and the postshock behavior of M. luteus cells proved to depend on the growth phase and medium. The magnitude of stress response was more pronounced in cultures grown on synthetic medium than in cultures grown on rich medium (nutrient broth). During exponential or linear growth, the cells were more sensitive to the temperature effect than during decelerated growth. In linearly growing M. luteus cultures, the amount of total intra- and extracellular alkylhydroxybenzenes, the anabiosis inducers, increased in response to heat shock. AHB redistribution between cells and culture liquid occurred in the course of stress and after stress. In micrococci exposed to heat shock, an increase in the AHB concentration both in cells and culture liquid is likely a defense reaction of stress resistance. This conclusion was confirmed in the experiments with the addition 30 min before the heat shock of a chemical analogue of the anabiosis inducer, C7-AHB (12 mM), which protected M. luteus cells so that their intense growth was observed after shock without any lag. The protective effect of AHB is a result of their ability to form complexes with enzyme macromolecules and stabilize them. The data obtained extend the knowledge of the stress-protective functions of low-molecular-weight autoregulators and of the role of intercellular communications in the stress response of bacterial cultures.     

Influence of pH,temperature and culture media on the growth and bacteriocin production by vaginal Lactobacillus salivarius CRL 1328

AIMS: To study the influence of pH, temperature and culture medium on the growth and bacteriocin production by vaginal Lactobacillus salivarius subsp. salivarius CRL 1328. METHODS AND RESULTS: The study was performed using a complete factorial experimental design. Lactobacillus salivarius was cultivated in LAPTg and MRS broths, adjusted to specific initial pH, and at different temperatures of incubation. The growth, which was evaluated by the Gompertz model, was higher in MRS broth than in LAPTg broth. The initial pH of the culture medium and the temperature had a dramatic effect on the production of bacteriocin. The optimal conditions for bacteriocin production were different to those for optimal growth. The decrease in the pH of the culture medium was parallel to the growth; pH had similar final values in both the MRS and the LAPTg broths. CONCLUSIONS: The optimal growth conditions were recorded in MRS broth, with an initial pH of 6.5 and a temperature of 37 degrees C. The maximum bacteriocin activity was obtained in LAPTg after 6 h at 37 degrees C, and at an initial pH of 6.5 or 8.0. SIGNIFICANCE AND IMPACT OF THE STUDY: The application of a complete factorial design, and the evaluation of the growth parameters through the Gompertz model, enabled a rapid and simultaneous exploration of the influence of pH, temperature and growth medium on both growth and bacteriocin production by vaginal Lact. salivarius CRL 1328.     

Interaction of bacteria with mercuric compounds

We investigated the interaction of mercuric compounds with the bacteria Corynebacterium ammoniagenes, Micrococcus luteus, and Mycobacterium smegmatus capable of producing hydroxylamines (R-NOH) and 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate (MECP), which are prone to form free radicals. The interaction of these substances with Hg2+ ions and their dynamics during the mercuric poisoning of bacteria was studied by EPR and NMR. Under stress conditions induced by lowering pH or generation of active oxygen species, the bacteria and, especially, their mutants with enhanced sensitivity to oxidative stress, were found to respond to exposure to 1-3 micrograms/ml HgCl2 and p-chloromercuribenzoate by a several-fold increase in their viability. The data obtained were interpreted in terms of the involvement of the sulfhydryl groups of bacterial surface proteins in this phenomenon. The interaction of bacteria with mercuric compounds may affect the pathogenesis of tuberculosis and other diseases.