铜绿假单胞菌M19降解黄曲霉毒素B1的产物研究（网络首发、推荐阅读）

以实验室筛选保藏的黄曲霉毒素B1（Aflatoxin B1,AFB1）降解菌M19产生的降解酶PADE为对象,对其AFB1降解液进行薄层色谱及荧光光谱分析（LC-MS）,分析降解产物可能的结构变化,并利用液相质谱检测AFB1降解产物。薄层色谱检测结果：有机相和水相降解液均未检测到新的荧光吸收物质,表明降解产物没有荧光吸收;荧光光谱检测结果：AFB1降解产物的荧光明显减弱;液相质谱检测结果：发现质荷比为227.18的物质P。结果推断：AFB1降解过程中内酯键断裂,产生一个分子量为226的降解产物P,利用Xcalibur软件分析其分子式为C14H10O3,并根据AFB1降解后的LC-MS图谱分析以及AFB1降解产物的相关文献对降解途径进行假设。     

铜绿假单胞菌M19降解黄曲霉毒素B1的产物研究

以实验室筛选保藏的黄曲霉毒素B1(Aflatoxin B1,AFB1)降解菌M19产生的降解酶PADE为对象,对其AFB1降解液进行薄层色谱及荧光光谱分析(LC-MS),分析降解产物可能的结构变化,并利用液相质谱检测AFB1降解产物。薄层色谱检测结果:有机相和水相降解液均未检测到新的荧光吸收物质,表明降解产物没有荧光吸收;荧光光谱检测结果:AFB1降解产物的荧光明显减弱;液相质谱检测结果:发现质荷比为227.18的物质P。结果推断:AFB1降解过程中内酯键断裂,产生一个分子量为226的降解产物P,利用Xcalibur软件分析其分子式为C14H10O3,并根据AFB1降解后的LC-MS图谱分析以及AFB1降解产物的相关文献对降解途径进行假设。     

黄曲霉毒素B1降解菌的筛选鉴定及降解酶挖掘

目的 筛选、鉴定一株高效降解黄曲霉毒素B1 (aflatoxin B1, AFB1)菌株,并对其降解特性和降解酶基因进行分析。方法 通过形态学观察、生理生化鉴定及同源性分析,对具有高效降解AFB1能力的菌株进行鉴定。通过菌株发酵液、上清液、细胞悬液、胞内提取物对AFB1降解能力的不同确定降解活性部位,使用冷冻干燥方法探索其粗酶制剂对AFB1降解效果。结合全基因组分析进行降解酶基因的挖掘。结果 通过鉴定,菌株HNGD-B3为贝莱斯芽孢杆菌(Bacillus velezensis),菌株上清液对AFB1降解效果最好,72 h降解率为88.24%±2.02%。上清液蛋白酶K处理后,对AFB1降解效果大幅下降,热处理后上清液降解效果基本无变化,判断其降解活性成分为胞外酶。粗酶制剂可显著提高AFB1降解效率,结合全基因组分析与Blastp比对筛选得到3条具有AFB1降解潜力的候选基因序列。结论 菌株HNGD-B3对AFB1具有良好降解效果,在生物降解真菌毒素具有较大潜力。     

施氏假单胞菌F4对黄曲霉毒素B_1的酶解作用及其降解产物的初步分析

施氏假单胞菌F4能高效降解黄曲霉毒素B1(aflatoxin B1,AFB1)。研究了F4的AFB1降解活性、降解动力学以及蛋白酶K和SDS对其降解性能的影响。F4细胞悬液与毒素共培养72 h后降解率达80.03%;蛋白酶K处理对降解率没有影响,SDS处理的细胞悬液基本丧失了降解活性。不同时间点的降解液上清液仍能有效降解残留AFB1,其中以60 h的降解液上清液活性较好,与残留AFB1继续作用48 h后降解率达84.30%;而经蛋白酶K处理后降解率仅为45.42%。低浓度AFB1诱导对菌体的降解活性没有影响。上述结果提示,F4通过胞内酶作用降解AFB1。高效液相色谱对产物分析表明,F4可将AFB1酶解为至少2种产物。     

中国被毛孢降解黄曲霉毒素B1的特性研究

目的 分析中国被毛孢（Hirsutella sinensis）8#菌株降解黄曲霉毒素B1 (AFB1)的能力。方法 考察该菌株培养液、菌丝体悬液和上清液去除AFB1的能力;利用洗脱和萃取法区分生物降解和可逆吸附AFB1;研究不同初始AFB1浓度、温度、pH和金属离子对其降解能力的影响;对AFB1降解液进行高效液相色谱和薄层色谱分析。结果 H.sinensis培养液和菌体降解效果显著（P<0.05）,96 h后对初始浓度100 ng/mL的AFB1降解率分别是96.90%±4.39% 和 97.93%±2.92%。磷酸盐缓冲液洗脱液和甲醇萃取液均未检测到AFB1,证实了该菌生物降解AFB1。菌株AFB1降解效果与初始浓度密切相关。当反应温度25 ℃,pH 7.0时,培养液作用72 h后降解率为99.90%±0.18%。同时,反应液中加入Fe2+有利于降解,而Mg2+却起到了抑制作用。高效液相色谱和薄层层析对产物分析表明,该菌株可将AFB1降解为至少为1种产物。结论 中国被毛孢8#菌株对AFB1有良好的降解作用,可用于生物降解真菌毒素的潜力菌株。     

重组漆酶降解黄曲霉毒素B1分子对接分析及产物结构解析

以重组漆酶lac3基因同源性最高的3KW7作为模板进行同源模建,采用分子对接预测漆酶与黄曲霉毒素B1（aflatoxin B1,AFB1）的结合模式,结果显示漆酶与AFB1可以相互作用,氢键是其关键作用力,漆酶可用于黄曲霉毒素的降解。随后,通过实际的降解实验进行验证,响应面优化获得AFB1降解率最优的条件为底物AFB1 1 μg、孵育时间15 h、孵育温度34 ℃、酶活力2 U,降解率可达91.08%。在此条件下利用超高效液相色谱-飞行时间串联质谱分析AFB1降解产物结构,发现4 个主要降解产物,根据其二级质谱信息和精确分子质量,推测出降解产物的分子式分别为C16H22O4、C14H16N2O2、C7H12N6O和C24H30O6。     

等离子体降解花生中黄曲霉毒素的影响因素

采用低温射频等离子体技术处理黄曲霉毒素B1(AFB1),通过HPLC分析其中AFB1的降解率,探究不同处理时间、处理功率等离子体和不同水分含量、颗粒大小、种类的花生对AFB1降解的影响。结果表明,在相同时间下等离子体处理功率越大,花生中的AFB1降解得越多,400 W时降解率为73.45%;在相同处理功率下,处理时间越长,花生中的AFB1降解越多,8 min后降解率为79.26%;在相同条件下,花生中水分含量越高,AFB1的降解率越高,含水量达到40%时降解率达到50%;花生的颗粒越小,AFB1的降解率越高,花生粉的降解率达到近60%;含油量越高的花生,其AFB1降解率越低,花生的含油量为45%时降解率只有45%。得出等离子体技术降解AFB1时,外界条件对降解有较大影响,该结果对实际应用有指导作用。     

抗黄曲霉毒素B_1纳米抗体的原核表达、纯化及活性分析

表达和纯化抗黄曲霉毒素B1(Aflatoxin B1,AFB1)纳米抗体(G8),并分析纳米抗体的热稳定性及生物学活性,建立基于纳米抗体检测AFB1的ELISA方法。将编码抗AFB1纳米抗体的基因亚克隆至原核表达载体,转化至大肠杆菌BL21(Rosetta,DE3),IPTG诱导表达后,SDS-PAGE分析蛋白表达情况;采用酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)分析纳米抗体的热稳定性、有机溶剂耐受性、盐离子耐受性及耐酸碱性。结果显示,在大肠杆菌中成功表达可溶性的抗AFB1纳米抗体,表达量为80 mg/L,在25~65℃之间,抗体具有良好的热稳定性。在5%甲醇、p H7.4、10 mmol/L PBS条件下,建立了G8-ELISA检测AFB1的方法,该方法的半抑制浓度(IC50)为4.61 ng/m L,线性范围为0.95~42.45 ng/m L。     

降解黄曲霉毒素B1菌株的发酵条件优化及降解机制

目的：鉴定一株降解黄曲霉毒素B1（aflatoxin B1,AFB1）的霉菌HSK8,通过优化其发酵条件提高AFB1降解率,并初步探索其降解机制。方法：通过形态学和内部转录间隔区（internal transcribed spacer,ITS）、18S rRNA和26S rRNA序列分析对该菌株进行鉴定,并通过单因素试验对发酵条件进行优化,采用薄层层析对AFB1降解产物进行研究。结果：经鉴定该株霉菌为夏孢生枝孢（Cladosporium uredinicola）。其发酵条件经优化确定为：发酵温度28 ℃、装液量75 mL/250 mL、接种量15%、发酵时间36 h、初始pH 8.0。薄层层析观察到一个不同于AFB1的荧光斑点。结论：发酵条件优化后,AFB1降解率从40.68%提升到68.96%,提高了69.52%;AFB1降解产物能在365 nm波长处发出蓝色荧光。     

臭氧降解玉米中黄曲霉毒素B1效果及降解动力学研究

玉米是我国重要的食品和饲料原料,当收获、加工和储藏等措施不当时,可能会造成黄曲霉毒素B1（aflatoxin B1,AFB1）污染玉米这一突出问题,AFB1已被国际癌症机构定为1级致癌物。尽管目前已建立了一些物理、化学和生物降解AFB1的方法,但高效、安全、经济的绿色降解方法仍很少。本研究以AFB1污染的玉米为试样,研究臭氧对玉米中AFB1的降解效果。结果表明：AFB1降解率随着臭氧质量浓度的增加和处理时间的延长而显著提高;当水分质量分数为20.37%的玉米经90 mg/L的臭氧处理40 min后,AFB1含量由77.6 μg/kg降低到21.42 μg/kg,降解率达72.4%。臭氧降解AFB1的动力学模拟结果表明,臭氧降解AFB1符合一级动力学模型。玉米中AFB1降解速率常数按以下次序递减：k90 mg/L＞k65 mg/L＞k40 mg/L。实验得到臭氧降解AFB1的动力学方程、反应速率常数、决定系数和半衰期,为最优地控制臭氧降解AFB1的反应条件奠定了理论和实践基础,也为臭氧降解AFB1污染玉米的应用提供了技术保障。     

Distribution of aflatoxins and fumonisins in dry-milled maize fractions

The aim was to evaluate the distribution of aflatoxins and fumonisins in fractions derived from the dry-milling of contaminated maize. Two maize lots with different contamination levels were processed and sampled: the first (maize 1) had aflatoxin B₁ (AFB₁) and fumonisin B₁ (FB₁) levels of 3.6 and 5379 µg kg^?1, respectively; the second (maize 2) had corresponding levels of 91.1 and 8841 µg kg^?1, respectively. The cleaning step reduced AFB₁ and FB₁ levels by 8 and 11% in maize 1 and by 57 and 34% in maize 2. The subsequent removal of bran and germ led to a further decrease in contamination levels in the products destined for human consumption. In the latter, AFB₁ was uniformly distributed, while FB₁ was concentrated in the finer size fractions. Contamination of raw maize 1 (3.6 µg kg^?1) was below the European Union AFB₁ limit of 5 µg kg^?1 for unprocessed maize, but among the final products only coarse flour (1.7 µg kg^?1) was within the European Union limit of 2 µg kg^?1, while grits and fine flour showed higher levels (2.7 and 2.5 µg kg^?1, respectively). As regards cleaned maize, a different distribution of the two toxins was observed in the kernels: AFB₁ contamination was more superficial and concentrated in germ, while FB₁ contamination affected the inner layers of the kernels.     

基于监测过氧化氢酶活性对储藏玉米AFB1污染的早期预警

早期预警是防控储藏玉米污染黄曲霉毒素B1(AFB_1)的重要手段。将不同含水量的玉米置于各种温度环境下进行储藏试验,研究过氧化氢酶活性变化与玉米污染黄曲霉毒素B1(AFB_1)的关系,结果表明,两者数据变化的趋势一致,AFB_1是过氧化氢酶活性的二次曲线函数(相关性系数大于0.95)。储藏温度和玉米含水量等条件可改变所含霉菌产生AFB_1和升高过氧化氢酶活性的速度,但不影响数值变化的趋势。在各种储藏条件下,过氧化氢酶活性变化可比玉米中AFB_1含量显著变化的起始点提前4~21d。因此,借助检测过氧化氢酶活性的方法可以早期预警储藏玉米污染AFB_1。     

Fate of aflatoxin B(1) in contaminated corn gluten during acid hydrolysis

BACKGROUND: Aflatoxins are a group of mycotoxins that cause serious chronic disease outbreaks and contaminate several food products such as corn and its by‐product, corn gluten. The aim of the current study was to evaluate the effect of hydrochloric acid (HCl) on aflatoxin B₁ (AFB₁) degradation in contaminated corn gluten under different HCl concentrations, hydrolysis temperatures and hydrolysis times. RESULTS: During the wet milling process the highest AFB₁ level (45.68 µg kg^?1) (37.86%) was found in corn gluten fraction. Treatment with 1 mol L^?1 HCL at 110 °C resulted in degradation of AFB₁ by 27.6% (33.07 µg kg^?1) after 4 h and reached 42.5% (26.26 µg kg^?1) after 8 h. Increasing HCl concentration from 1 to 3 mol L^?1 HCl resulted in increased degradation of AFB₁, while complete degradation occurred in the presence of 5 mol L^?1 HCl after 4 h at 110 °C. Meanwhile, half‐life time of AFB₁ was recorded after 2 h at 100 °C and was < 2 h at 110 °C in the presence of 3 mol L^?1 HCl. CONCLUSION: It could be demonstrated that the manufacture of hydrolyzed vegetable protein is a suitable method for decontamination of aflatoxin in highly contaminated grains, especially gluten fractions. The hydrolysis reaction could be considered in terms of first‐order reaction kinetics of AFB₁ degradation. Copyright © 2010 Society of Chemical Industry     

Feasibility of Detecting Aflatoxin B1 on Inoculated Maize Kernels Surface using Vis/NIR Hyperspectral Imaging

The feasibility of using a visible/near‐infrared hyperspectral imaging system with a wavelength range between 400 and 1000 nm to detect and differentiate different levels of aflatoxin B₁ (AFB₁) artificially titrated on maize kernel surface was examined. To reduce the color effects of maize kernels, image analysis was limited to a subset of original spectra (600 to 1000 nm). Residual staining from the AFB₁ on the kernels surface was selected as regions of interest for analysis. Principal components analysis (PCA) was applied to reduce the dimensionality of hyperspectral image data, and then a stepwise factorial discriminant analysis (FDA) was performed on latent PCA variables. The results indicated that discriminant factors F₂ can be used to separate control samples from all of the other groups of kernels with AFB₁ inoculated, whereas the discriminant factors F₁ can be used to identify maize kernels with levels of AFB₁ as low as 10 ppb. An overall classification accuracy of 98% was achieved. Finally, the peaks of β coefficients of the discrimination factors F₁ and F₂ were analyzed and several key wavelengths identified for differentiating maize kernels with and without AFB₁, as well as those with differing levels of AFB₁ inoculation. Results indicated that Vis/NIR hyperspectral imaging technology combined with the PCA–FDA was a practical method to detect and differentiate different levels of AFB₁ artificially inoculated on the maize kernels surface. However, indicated the potential to detect and differentiate naturally occurring toxins in maize kernel.     

Effect of dietary acids on the formation of aflatoxin B2a as a means to detoxify aflatoxin B1

Aflatoxin B₁ (AFB₁) is a class 1 carcinogen and a common food contaminant worldwide with widely uncontrolled human exposure. The ability of organic acids to transform AFB₁ into a known detoxified form, aflatoxin B_2a (AFB_2a), was investigated using high performance liquid chromatography-electrospray ionisation-time of flight mass spectrometry (HPLC/ESI/TOF/MS). The identity of the transformation product was confirmed by accurate mass measurement, chromatographic separation from other aflatoxins, H¹-nuclear magnetic resonance (NMR) and infrared (IR) spectroscopy. Of the weak acids tested, citric acid was found to be the most effective for AFB_2a formation. At room temperature, 1 M citric acid was able to convert > 97% of AFB₁ to AFB_2a over 96 h of treatment. Up to 98% transformation was achieved by boiling AFB₁ in the presence of citric acid for 20 min. AFB₁ hydration after ingestion was explored by spiking AFB₁ into simulated gastric fluid containing citric acid. Under these conditions, > 71% of AFB₁ was hydrated to AFB_2a and did not show any reversion to the parent compound after being transferred to a neutral solution. These results provide a basis for a practical and effective method for detoxification of AFB₁ in contaminated foods.     

Degradation of aflatoxin B1 in peanut meal by electron beam irradiation

The effects and safety of electron beam irradiation (EBI) treatment on the detoxification of aflatoxin B₁ (AFB₁) in the peanut meal were evaluated in this article. The AFB₁ degradation was predominantly affected by both initial AFB₁ and water concentrations. The degradation of AFB₁ in the selected concentrations (0.5–5 ppm) was proven to follow pseudo first-order reaction kinetics (R² > 0.95). The AFB₁ degradation was faster when the initial concentration was 5 ppm and the moisture content was 21.47%, in comparison with the initial concentration of 1 ppm and 0.5 ppm and the moisture content of 14.32% and 8.74%, respectively. The Ames and cytotoxicity tests were employed to evaluate the residual toxicity of EBI-treated peanut meal. The mutagenic activity of EB-treated samples was completely lost compared with that of untreated samples and the degradation products in peanut meal has almost no cell toxicity.     

栽培大豆Rubisco小亚基基因（rbcS）全长cDNA的克隆及原核表达

根据NCBI中发表的大豆Rubisco小亚基基因（rbcS）序列（AF303939-1）设计特异引物,以吉农13大豆为实验材料,采用Trizol法提取叶片总RNA,通过RT-PCR克隆大豆rbcS的cDNA。将该基因与原核表达载体pET-30a（＋）连接,转化大肠杆菌E.coli BL21感受态细胞,双酶切鉴定后筛选阳性克隆,测序结果显示：rbcS全长696bp,其中开放阅读框为537bp,编码178个氨基酸;选择含大豆rbcS cDNA阳性克隆菌液IPTG诱导,经SDSPAGE分析,诱导表达出分子量为29.1kDa的融合蛋白,表达产物大小与预计理论值相符。诱导7h蛋白表达量最高,为64.15%。     

黑曲霉降解黄曲霉毒素B1的研究及转录组分析

为探究黑曲霉FS10对AFB_1的降解机制,使用黑曲霉FS10的不同组分(菌悬液、发酵液、孢子、菌丝体)对AFB_1进行降解,并研究了AFB_1刺激对黑曲霉FS10降解效果的影响;利用扫描电子显微镜观察降解过程中黑曲霉菌丝体的形态变化;用转录组学技术探究AFB_1可能的降解机理。结果表明:黑曲霉FS10能有效降解AFB_1,72 h时菌悬液的对AFB_1脱除率高达98.65%;黑曲霉FS10孢子对AFB_1无明显脱除作用,但菌丝体对AFB_1有一定的吸附能力,发酵液对AFB_1有明显脱除效果;经过AFB_1诱导刺激后黑曲霉FS10降解效果有明显提升,表明AFB_1处理能显著提升黑曲霉FS10对AFB_1的降解能力。微观结构分析表明AFB_1处理在一定程度上影响黑曲霉FS10的形态,但随着时间的延长这种影响逐渐减小。此外,转录组学分析表明AFB_1处理降低了一些能量代谢基因的水平,这可能是黑曲霉FS10的一种自我保护机制,同时蛋氨酸的合成基因上调,推测AFB_1的降解可能与蛋氨酸的合成有关。     

地衣芽孢杆菌耐高温α-淀粉酶基因在大肠杆菌中的克隆及表达

用PCR方法从地衣芽孢杆菌中扩增了耐高温α淀粉酶基因,将扩增的DNA片段插入到大肠杆菌载体pUC19中,构建重组分泌型表达载体pUAM。pUAM中的耐高温α淀粉酶基因在大肠杆菌JM109中得到表达。经SDSPAGE分析显示,蛋白表达产物的分子量为55ku,同核酸序列测定所推导的值相符。     

异淀粉酶产生菌的分离鉴定及异淀粉酶基因的克隆表达

目的:为了进一步提高淀粉的利用率,从微生物中获得高产量异淀粉酶的菌株。方法:本实验从富含淀粉的土壤中筛选出1株高产异淀粉酶的菌株,命名为LZ-5,通过菌落形态观察、生理生化特性实验、16S rDNA序列及gyrB基因分析,构建系统发育进化树并对其进行鉴定;根据NCBI上的迟缓芽孢杆菌(Bacillus lentus)异淀粉酶基因设计引物,扩增出异淀粉酶基因后,以pMD-18T为载体,构建重组质粒,导入到E.coli DH5a感受态细胞,挑选阳性重组质粒进行酶切鉴定及表达产物的检测。结果:LZ-5鉴定为枯草芽孢杆菌,所产异淀粉酶酶活力为10.9 U/mL;测序结果与NCBI上报道的异淀粉酶基因相似度达到96%,表明将枯草芽孢杆菌的异淀粉酶基因成功转化到大肠杆菌中。经IPTG诱导表达,构建的大肠杆菌工程菌,通过细胞破碎仪破碎后,测得异淀粉酶酶活力为28.4 U/mL,是菌株LZ-5的2.6倍。结论:细菌异淀粉酶基因重组表达是解决异淀粉酶活力低的主要策略之一。