过氧化物酶A4-Prx在毕赤酵母中的优化表达

以来源于不动杆菌Acinetobacter sp.SM04的过氧化物酶A4-Prx的毕赤酵母工程表达菌株GS115/pPIC9K-A4-Prx为研究对象,优化其表达培养条件以提高该菌株对于目的蛋白A4-Prx的表达量。本论文首先研究了培养基成分与诱导条件对表达量的影响,结果表明培养基的pH、甘氨酸浓度和诱导温度对外源蛋白的产量均有显著影响。采用Box-Behnken设计,利用Design Expert软件进行二次回归分析得到了目的蛋白的最优表达条件为:诱导培养基pH 7.0、甘氨酸浓度为0.11%及诱导温度30℃。在此优化条件下重组蛋白的理论表达量达129.87 mg/L,约为未优化下的2倍,实验验证实际表达量达128.94 mg/L,且重组表达的过氧化物酶A4-Prx对酒糟蛋白饲料(DDGS)和食品中的玉米赤霉烯酮毒素(Zearalenone,ZEA)具有高效降解能力。本研究为过氧化物酶的工业化高密度发酵奠定了基础,推动生物降解ZEA研究的进展。     

原料乳中玉米赤霉烯酮胶体金免疫试纸条检测方法的建立

采用柠檬酸三钠法合成胶体金,与抗玉米赤霉烯酮单抗(ZEA-m Ab)制成抗体-胶体金标记物,包被于胶体金结合垫上。玉米赤霉烯酮半抗原和蛋白的偶联物(ZEA-BSA)和羊抗鼠Ig G分别喷涂于硝酸纤维素膜作为检测线(T线)和质控线(C线),建立原料乳中玉米赤霉烯酮(ZEA)的胶体金免疫试纸条检测法。结果显示,试纸条的灵敏度为30n g/m L,与其他毒素无交叉反应,10 min内即可肉眼观察结果。检测添加有玉米赤霉烯酮的原料乳样品,该试纸条的检测限为50 ng/m L。方法可用于原料乳中玉米赤霉烯酮的快速检测。     

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。     

啤酒酵母β-D-葡聚糖吸附毒素玉米赤霉烯酮(ZEA)的研究

本文用碱法,碱酸法和酶碱法从啤酒废酵母中提取碱不溶性葡聚糖,对产品进行了定性定量分析,得到的产品主要为β-D-葡聚糖,且不含甘露糖.研究了三种产品对真菌毒素玉米赤霉烯酮（ZEA）的吸附作用,实验结果表明,酶碱法提取的葡聚糖具有较好的吸附效果,在反应条件：β-D-葡聚糖100μg/ml,玉米赤霉烯酮40μg/ml,37℃,200r/min振荡2h,吸附量最大可达到2.296μg/mg葡聚糖.     

昌黎产区酿酒葡萄果表的酵母分离及发酵性能

以河北省昌黎县葡萄酒产区的酿酒葡萄"赤霞珠"果实为材料,筛选具有产区特色的酵母菌株。对葡萄果表的酵母富集后采用WL培养基进行初筛,通过TTC显色和杜氏小管产气测定,获得性能优良的酵母菌株CLY03和CLY04,经形态观察和理化试验初步确定为伊萨酵母属和酵母属;设置耐受条件分别为酒精浓度10%~18%、p H值3.0~5.0、葡萄糖浓度20%~30%、SO2浓度150~300 mg/m L、培养温度在28~49℃时测定其发酵性能;以LAFFORT菌株为对照,对发酵后葡萄酒样进行理化分析和感官评定。结果表明,各耐受条件下两菌株都能正常启动发酵,其中菌株CLY04对各条件的耐受能力优于菌株CLY03,而菌株CLY03的致死温度较菌株CLY04高;两菌株都有较高的酒精转化能力,发酵后溶液残糖量和挥发酸含量都在允许范围内,菌株CLY04发酵的葡萄酒感官评定优于对照菌株,但两菌株的香气和典型性得分都高于对照菌株。由此菌株CLY03和CLY04发酵的葡萄酒体现了产区的品质和特色,具有潜在的应用价值。     

基于核酸适体-纳米金银染放大的玉米赤霉烯酮快速检测方法研究

玉米赤霉烯酮(Zearalenone,ZEA)具有较强的生殖毒性、致突变和致畸作用。以ZEA适体为识别元件,构建了基于纳米金诱导聚集和银染放大的ZEA适体比色可视化检测方法。结果表明,在优化条件下,ZEA浓度在5～200 ng/m L范围内与体系的吸光度值呈良好的线性关系,其线性回归方程为y=0.248 6+0.000 461 56x (R2=0.990 2),最低检测限为5 ng/m L,且方法特异性良好。进一步通过银染作用将该方法的灵敏度提高了50倍。经对比,该方法对实际样品的检测结果与酶联免疫法基本一致,为食品中ZEA的快速检测提供了简便有效的新策略。     

玉米赤霉烯酮降解菌的筛选与鉴定

采用富集驯化的方法从猪粪便中分离出一株能够高效降解玉米赤霉烯酮(zearalenone, ZEN)的菌株,命名为ZJ-2019-1。通过16S rDNA序列分析方法对其进行了鉴定,研究了反应时间、培养基初始pH、温度、毒素浓度等因素对ZJ-2019-1降解ZEN的影响,同时对该菌株清除ZEN的机理进行了初步探索。结果表明:ZJ-2019-1为枯草芽孢杆菌(Bacillus subtilis);该菌株在48 h内能将LB培养基中初始浓度为10 mg/L的ZEN去除99.7%;该菌株降解ZEN的最适温度为37℃,当反应温度低于27℃时,该菌株对ZEN的清除率明显降低;当培养基初始pH 5.0～9.0时,ZJ-2019-1对ZEN的清除率能达到88%以上,其中初始pH 7.0为降解反应的最适pH;当初始ZEN浓度在20 mg/L以内时,ZJ-2019-1对ZEN的清除率都在95%以上;ZJ-2019-1对ZEN的清除作用主要源于胞外酶的活性。     

高效液相色谱法对玉米中玉米赤霉烯酮的测定

研究了高效液相色谱法测定玉米中玉米赤霉烯酮的方法.样品借鉴了GB/T 19540-2004中提取玉米赤霉烯酮的方法,通过Oasis HLB净化柱对提取液净化,以agilent extent C18色谱柱为分离柱,乙腈-水(V水:V乙腈=55:45)为流动相进行荧光检测(λex=235 nm,λem=460 nm).玉米赤霉烯酮的质量浓度在12～2 400μ/kg范围内呈良好线性,相关系数为0.9994,对添加高、中、低3个浓度玉米赤霉烯酮的玉米样品进行加标回收试验,平均回收率分别为96.736%、93.839%、86.240%,变异系数在1%～10%之间,最低检测限为10μ/kg.此方法对玉米中玉米赤霉烯酮的检测是可行的,且可给谷物中玉米赤霉烯酮检测方法优化提供参考.     

竞争性酶联免疫法快速检测高产奶牛精料补充料中玉米赤霉烯酮

目的 建立竞争性酶联免疫方法检测饲料中玉米赤霉烯酮的分析方法。方法 样品经过60%的甲醇溶液提取, 提取液经过离心后, 取25 ?L提取液, 加入475 ?L稀释缓冲液混合后即为试样液, 取50 ?L试样液采用酶联免疫吸附方法进行检测。在450 nm波长处检测吸光度值。结果 本方法在3 h内完成了饲料中玉米赤霉烯酮含量的测定。在加标浓度为100、200和500 ?g/kg的加标水平下, 玉米赤霉烯酮酶在饲料中的加标回收率为99.5%~122.1%, 变异系数为1.4%~3.0%。结论 该方法快速、准确、灵敏, 适用于饲料中玉米赤霉烯酮的快速筛查检测。     

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.     

Effect of the bread-making process on zearalenone levels

The effects of the bread-making process including fermentation with Saccharomyces cerevisiae and lactic acid bacteria (Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus acidophilus and Lactobacillus fermentum) and baking at 200°C on zearalenone (ZEA) levels were investigated. Standard solutions of ZEA were added to flour and then loaves of bread were prepared. Sourdough and three types of yeast including active dry yeast, instant dry yeast and compressed yeast were used for the fermentation of dough. ZEA levels in flour, dough and bread were determined by HPLC with fluorescence detection after extraction and clean-up on an immunoaffinity column. The highest reduction in levels of ZEA was found in the first fermentation (first proof), while the lowest reduction was observed in the baking stage. In addition, the results showed that compressed yeast had the maximum reduction potential on ZEA levels even at the baking stage.     

玉米赤霉烯酮饲粮添加蒙脱石对断奶仔猪脾脏和外周血免疫指标的影响

选择(28±1)日龄、平均体重为(8.84 ±0.21)kg的健康三元(斯格×长×大)杂交断奶仔猪(公母各半)18头,研究1 mg/kg玉米赤霉烯酮(zearalenone,ZEA)污染饲粮对断奶仔猪脾脏和外周血淋巴细胞增殖率和IL-2产量的影响,同时评价改性蒙脱石的脱毒效应.将试验仔猪按照体重随机分为3个处理(公母各半),仔猪采用试验笼单独饲养.试验处理为:Contr.=基础饲粮;ZEA+CZ0=基础饲粮+1 mg/kg ZEA;ZEA+ CZ4=基础饲粮+1 mg/kg ZEA+4g/kg改性蒙脱石.预饲期7d,正式期22 d.结果表明:1)与对照组相比,饲粮添加1 mg/kg ZEA显著降低断奶仔猪脾脏相对重量、外周血和脾脏淋巴细胞增殖率以及IL-2产量(P＜0.05).2)1 mg/kg的ZEA处理饲粮添加4 g/kg改性蒙脱石能够显著改善ZEA诱导的脾脏和外周血淋巴细胞增值率以及IL-2的改变(P＜0.05).结果揭示,1 mg/kg的ZEA足以影响断奶仔猪脾脏的生长发育及其细胞免疫功能,添加4 g/kg改性蒙脱石具有显著的脱毒效应,以上结果对指导动物生产和人类健康具有重要的借鉴意义.     

Binding of zearalenone, aflatoxin B1, and ochratoxin A by yeast-based products: a method for quantification of adsorption performance

A methodology was developed to quantify the efficiency of yeast-based products for adsorption of three mycotoxins: zearalenone (ZEA), aflatoxin B(1) (AFB(1)), and ochratoxin A (OTA). Eight products were tested (yeast cell wall or inactivated yeast). The described experimental protocol based on in vitro tests provided reliable isotherms for each mycotoxin. The most suitable models were the Hill model for ZEA, the Langmuir model for AFB(1), and the Freundlich model for OTA. From these models, original mathematical affinity criteria were defined to quantify the product adsorption performances for each mycotoxin. The best yeast product, a yeast cell wall from baker's yeast, can adsorb up to 68% of ZEA, 29% of AFB(1), and 62% of OTA, depending on the mycotoxin concentrations. The adsorption capacity largely depended both on yeast composition and mycotoxin, but no direct correlation between yeast composition and adsorption capacity was found, confirming that adsorption of mycotoxin on yeast-based products involves complex phenomena. The results of this study are useful for comparing the adsorption efficiency of various yeast products and understanding the mechanisms involved in adsorption.     

Quantification of Fusarium graminearum and Fusarium culmorum by real-time PCR system and zearalenone assessment in maize

Zearalenone (ZEA) is a mycotoxin produced by some species of Fusarium, especially by Fusarium graminearum and F. culmorum. ZEA induces hyperoestrogenic responses in mammals and can result in reproductive disorders in farm animals. In the present study, a real-time PCR (qPCR) assay has been successfully developed for the detection and quantification of Fusarium graminearum based on primers targeting the gene PKS13 involved in ZEA biosynthesis. A standard curve was developed by plotting the logarithm of known concentrations of F. graminearum DNA against the cycle threshold (Ct) value. The developed real time PCR system was also used to analyze the occurrence of zearalenone producing F. graminearum strains on maize. In this context, DNA extractions were performed from thirty-two maize samples, and subjected to real time PCR. Maize samples also were analyzed for zearalenone content by HPLC. F. graminearum DNA content (pg DNA/ mg of maize) was then plotted against ZEA content (ppb) in maize samples. The regression curve showed a positive and good correlation (R2=0.760) allowing for the estimation of the potential risk from ZEA contamination. Consequently, this work offers a quick alternative to conventional methods of ZEA quantification and mycological detection and quantification of F. graminearum in maize.     

Comparison of the sequestering properties of yeast cell wall extract and hydrated sodium calcium aluminosilicate in three in vitro models accounting for the animal physiological bioavailability of zearalenone

The sequestration/inactivation of the oestrogenic mycotoxin zearalenone (ZEA) by two adsorbents – yeast cell wall extract (YCW) and hydrated sodium calcium aluminosilicate (HSCAS) – was studied in three laboratory models: (1) an in vitro model was adapted from referenced methods to test for the sequestrant sorption capabilities under buffer conditions at two pH values using liquid chromatography coupled to a fluorescence detector for toxin quantification; (2) a second in vitro model was used to evaluate the sequestrant sorption stability according to pH variations and using ³H-labelled ZEA at low toxin concentration; and (3) an original, ex vivo Ussing chamber model was developed to further understand the transfer of ZEA through intestinal tissue and the impact of each sequestrant on the mycotoxin bioavailability of ³H-labelled ZEA. YCW was a more efficient ZEA adsorbent than HSCAS in all three models, except under very acidic conditions (pH 2.5 or 3.0). The Ussing chamber model offered a novel, ex vivo, alternative method for understanding the effect of sequestrant on the bioavailability of ZEA. The results showed that compared with HSCAS, YCW was more efficient in sequestering ZEA and that it reduced the accumulation of ZEA in the intestinal tissue by 40% (p < 0.001).     

基于核酸适体-纳米金银染放大的玉米赤霉烯酮快速检测方法研究（网络首发、推荐阅读）

玉米赤霉烯酮（Zearalenone,ZEA）具有较强的生殖毒性、致突变和致畸作用。以ZEA适体为识别元件,构建了基于纳米金诱导聚集和银染放大的ZEA适体比色可视化检测方法。结果表明,在优化条件下,ZEA浓度在5~200 ng/mL范围内与体系的吸光度值呈良好的线性关系,其线性回归方程为y= 0.248 6+0.000 461 56x (R2=0.990 2),最低检测限为5 ng/mL,且方法特异性良好。进一步通过银染作用将该方法的灵敏度提高了50倍。经对比,该方法对实际样品的检测结果与酶联免疫法基本一致,为食品中ZEA的快速检测提供了简便有效的新策略。     

Monoclonal-based enzyme-linked immunosorbent assay for the detection of zearalenone in cereals

A monoclonal antibody against zearalenone (ZEA) was produced and used successfully to develop a direct competitive enzyme-linked immunosorbent assay (DC-ELISA) for the analysis of ZEA in cereals. This DC-ELISA had a limit of detection of 0.15 +/- 0.02 microg l(-1) and an IC50 value of 1.13 +/- 0.16 microg l(-1). Matrix interference was minimized by dilution of the sample extract before ELISA assays. Aqueous methanol (80%) gave good extraction efficiencies, and the recovery from spiked rice, barley, and corn samples averaged between 87 and 112%. Although ZEA was detected in seven (9%) of 80 rice samples and in eight (16%) of 50 barley samples, the concentration of ZEA in samples was around or below the limit of detection of DC-ELISA. Among 38 corn samples, ZEA was detected in nine (24%) samples in the range 41.0-909.8 microg kg(-1). Re-analysis of the ELISA-positive corn samples by high-performance liquid chromatography (HPLC) confirmed that seven (18%) corn samples were positive. The ZEA results for corn showed very good agreement between DC-ELISA and a commercial AgraQant zearalenone kit (r2 = 0.98). Thus, the monoclonal antibody-based DC-ELISA could be applied to the preliminary screening of ZEA contamination when analysis of a large sample number is needed.     

Monoclonal-based enzyme-linked immunosorbent assay for the detection of zearalenone in cereals.

A monoclonal antibody against zearalenone (ZEA) was produced and used successfully to develop a direct competitive enzyme-linked immunosorbent assay (DC-ELISA) for the analysis of ZEA in cereals. This DC-ELISA had a limit of detection of 0.15 +/- 0.02 microg l(-1) and an IC50 value of 1.13 +/- 0.16 microg l(-1). Matrix interference was minimized by dilution of the sample extract before ELISA assays. Aqueous methanol (80%) gave good extraction efficiencies, and the recovery from spiked rice, barley, and corn samples averaged between 87 and 112%. Although ZEA was detected in seven (9%) of 80 rice samples and in eight (16%) of 50 barley samples, the concentration of ZEA in samples was around or below the limit of detection of DC-ELISA. Among 38 corn samples, ZEA was detected in nine (24%) samples in the range 41.0-909.8 microg kg(-1). Re-analysis of the ELISA-positive corn samples by high-performance liquid chromatography (HPLC) confirmed that seven (18%) corn samples were positive. The ZEA results for corn showed very good agreement between DC-ELISA and a commercial AgraQant zearalenone kit (r2 = 0.98). Thus, the monoclonal antibody-based DC-ELISA could be applied to the preliminary screening of ZEA contamination when analysis of a large sample number is needed.     

Monoclonal-based enzyme-linked immunosorbent assay for the detection of zearalenone in cereals

A monoclonal antibody against zearalenone (ZEA) was produced and used successfully to develop a direct competitive enzyme-linked immunosorbent assay (DC-ELISA) for the analysis of ZEA in cereals. This DC-ELISA had a limit of detection of 0.15?±?0.02 µg l^?1 and an IC₅₀ value of 1.13?±?0.16 µg l^?1. Matrix interference was minimized by dilution of the sample extract before ELISA assays. Aqueous methanol (80%) gave good extraction efficiencies, and the recovery from spiked rice, barley, and corn samples averaged between 87 and 112%. Although ZEA was detected in seven (9%) of 80 rice samples and in eight (16%) of 50 barley samples, the concentration of ZEA in samples was around or below the limit of detection of DC-ELISA. Among 38 corn samples, ZEA was detected in nine (24%) samples in the range 41.0–909.8 µg kg^?1. Re-analysis of the ELISA-positive corn samples by high-performance liquid chromatography (HPLC) confirmed that seven (18%) corn samples were positive. The ZEA results for corn showed very good agreement between DC-ELISA and a commercial AgraQant® zearalenone kit (r ²?=?0.98). Thus, the monoclonal antibody-based DC-ELISA could be applied to the preliminary screening of ZEA contamination when analysis of a large sample number is needed.     

A survey of zearalenone in corn using Romer Mycosep™ 224 column and high performance liquid chromatography

A survey of zearalenone (ZEA) in corn from various regions of Brazil was carried out by the analysis of 380 corn samples, of which 30 samples (7.8%) were found to be contaminated in the range of 46.7-719 μg/kg. ZEA was extracted with acetonitrile-water (84:16, v/v), cleaned-up on a Romer Mycosep™ 224 column, separated, detected and quanti® ed by high performance liquid chromatography (HPLC). The in-house method characteristics of linearity, accuracy, precision, and detection limit were defined by means of recovery tests with spiked corn samples in the range of 35.8-716 μg/ kg and the analysis of a naturally-contaminated sample (n = 7). The mean recovery for ZEA was 99.4% and the relative standard deviation (RSD) varied from 0.7 to 26.6% in the range studied. The method has been shown to be accurate, quick and reliable for determination of zearalenone in corn.