吡咯喹啉醌研究新进展

吡咯喹啉醌（pyrroloquinoline quinone,PQQ）是细菌脱氢酶中的一种氧化还原辅助因子,又是一种抗氧化剂,能避免细胞内氧化反应以及体外生物活性物质产生活性氧导致的细胞损伤,为细胞的生长发育提供营养与维生素,同时使细胞具有抗氧化的耐受性。它对植物病原真菌起到生物控制剂的作用,能诱导蛋白激酶参与哺乳动物细胞分化发育过程。PQQ能通过增加不溶性磷酸盐的利用率来提高作物产量,它与氧化还原循环功能有很强相关性,具有抗神经退行性、抗癌、信号传导等功能。     

HPLC法测定吡咯喹啉醌(PQQ)中咪唑并吡咯喹啉(IPQ)的含量

目的:建立吡咯喹啉醌(PQQ)中IPQ的含量测定方法。方法:采用TCI Kaseisorb LC ODS 2000色谱柱(4.6mm×150mm,5μm);流动相为乙腈-缓冲溶液(配制10mmol的磷酸氢二钾和15mmol的四丁基溴化铵水溶液,用磷酸调节p H至7.4)[28∶72];紫外检测器,检测波长为259nm;流速为1.0m L/min;进样量为20μL。结果:IPQ在0.05μg/m L～0.2μg/m L范围内线性关系良好,相关系数大于0.999。IPQ的回收率在104%～113%之间,RSD最大为3%。结论:该方法具有良好的专属性、检测灵敏度、精密度、线性和准确度,适用于PQQ中IPQ的质量控制。     

吡咯喹啉醌功能特性及其产品研究进展

吡咯喹啉醌作为一种氧化还原酶的辅酶,广泛存在于各种生物体组织中,其具有独特的理化性质,在各类生命体生理功能方面发挥着重要的作用。目前吡咯喹啉醌的安全性已经得到了大量证据支持,所以多个国家已经将其批准应用于食品饮料中。大量的动物实验和临床研究表明,吡咯喹啉醌可以改善大脑认知功能、提升运动恢复能力、修复肝损伤、改善骨质疏松症以及其他功能。本文对吡咯喹啉醌安全性和法规现状、生物学功能和在食品中应用情况等方面进行详细阐述,以期为吡咯喹啉醌产品的功能研究及开发应用提供参考。     

吡咯喹啉醌连续发酵工艺初探

吡咯喹啉醌(pyrroloquinoline quinine, PQQ)的微生物发酵生产中,常用补料分批发酵法,但是其发酵前期菌体生长较慢,发酵后期产量下降较快,整个发酵周期的生产效率较低。为了提高PQQ的发酵生产效率,初步研究了使用生丝微菌(Hyphomicrobium sp.)进行单级连续发酵生产PQQ的工艺。以PQQ补料分批发酵时产率最高的时刻作为连续发酵的起点,分别进行了PQQ的恒浊法连续发酵和恒化法连续发酵的研究。实验结果表明,以0.008 7 h^-1的稀释率进行PQQ恒浊法连续发酵更好,发酵液中的菌体浓度能够保持稳定且发酵产量较高。与补料分批发酵相比,恒浊法连续发酵可以提高PQQ的生产效率(80.7%),同时还提高了底物甲醇的转化率(36.5%),具有良好的工业应用前景。     

pCEC法检测运动营养品中吡咯并喹啉醌的含量

建立一种快速、高效的加压毛细管电色谱法检测运动营养品中吡咯并喹啉醌含量的分析方法。样品经20%的乙腈溶液提取后,以乙腈-15mmol/L pH4.7磷酸钾缓冲液（15∶85,v/v）作为流动相,260nm检测波长下检测,在电压强度+2kV条件下,外标法峰面积定量。结果表明,吡咯并喹啉醌标准溶液在0.05μg/mL～2.00μg/mL浓度范围内线性良好,相关系数R2为0.9995,检出限为0.5μg/kg,定量限为1.5μg/kg,加标回收率达到86.4%～98.2%,相对标准偏差（RSD）为2.17%～4.35%。该方法具有前处理简单、检测速度快的优点,适用于运动营养品中吡咯并喹啉醌含量的测定。     

氧化葡萄糖酸杆菌生物制造吡咯喹啉醌进展

吡咯喹啉醌（PQQ）是继黄素核苷酸和吡啶核苷酸之后,在膜束缚的细菌脱氢酶中发现的第三种辅基,具有抗氧化、抗病毒和提高免疫力等多种生理功能,在食品、医药及农业等行业具有广泛的应用前景。该文综述了氧化葡萄糖酸杆菌（Gluconobacter oxydans）中PQQ的生理作用及生物合成的分子机制,表明了氧化葡萄糖酸杆菌作为出发菌株生产PQQ具有潜在优势,对今后工业化生产PQQ具有积极的指导意义。     

变形假单胞菌吡咯喹啉醌合成基因簇的克隆与分析

从变形假单胞菌JUIM01中克隆到吡咯喹啉醌(PQQ)合成基因簇,阐明了其基因组成和生物学信息。根据已报道的假单胞菌的基因组进行简并引物设计,采用LA-PCR技术克隆变形假单胞菌的PQQ合成基因簇,对克隆的基因片段进行测序并使用生物信息学方法进行综合分析。结果表明:克隆到的基因片段全长为11 659 bp,其中包括pqqF、pqqA、pqqB、pqqC、pqqD、pqqE、pqqM、pqqH和pqqI共9个基因,编码PQQ生物合成的前体短肽PqqA和合成途径的相关酶;这些基因与荧光假单胞菌Pf0-1的PQQ合成基因簇的基因组成类似,相应基因的序列一致性达41%～94%。本研究中首次从变形假单胞菌中克隆到PQQ合成基因簇,并对其进行生物信息学分析,为变形假单胞菌的PQQ生物合成途径和胞内再生机制的研究奠定了基础,进而为提高2KGA的生产强度提供了理论支撑。     

Methylopila sp. YHT-1鉴定及其发酵产吡咯喹啉醌

以甲醇为唯一碳源,从土壤中筛选分离出一株分泌吡咯喹啉醌(PQQ)菌株YHT-1。通过形态学特征、生理生化特征和16S r RNA基因序列分析,鉴定为Methylopila sp.菌。经摇瓶发酵初步优化后,YHT-1在3 L发酵罐中发酵4 d,PQQ产量达113.6 mg/L。发酵产物经DEAE阴离子交换初步纯化、低温结晶,得到PQQ粗品,并经HPLC和紫外光谱分析,证实为PQQ。     

还原态吡咯喹啉醌清除氮自由基能力研究

探讨了不同质量浓度及不同反应时间条件下还原态吡咯喹啉醌（Reduced pyrroloquinoline quinone,PQQH₂）对ABTS自由基清除率的变化规律,建立一级和二级反应动力学模型,并以常规抗氧化剂维生素C作为对照,研究了PQQH₂对ABTS自由基的清除性能。结果表明：PQQH₂浓度越高,时间越长,对ABTS自由基清除率越高,反应25 min,70 mg/L PQQH₂对自由基清除率可达到90%以上。准二级动力学模型拟合的线性相关系数为0.959~0.996,更符合PQQH₂对ABTS自由基的清除特性。PQQH₂清除ABTS自由基的IC₅₀值为90 μmol/L,是维生素C的51.14%,当保温时间一定时,相同质量浓度的维生素C对ABTS自由基清除能力的损失是PQQH₂的2~3倍;当加热温度一定时,相同质量浓度的维生素C对ABTS自由基的清除能力损失约为PQQH₂的3~7倍。综上,PQQH₂的抗氧化能力优于维生素C。     

吡咯喹啉醌高通量检测方法的建立与优化

吡咯喹啉醌(Pyrroloquinoline quinone,PQQ)是一种新型辅酶,具有广阔的应用前景。传统PQQ检测方法在检测发酵液中PQQ浓度时都存在一定的缺陷,且检测效率偏低。本研究在大肠杆菌(Escherichia coli BL21(DE3))中表达E.coli K-12来源的葡萄糖脱氢酶(PQQGDH),并通过亲和层析分离纯化PQQGDH,得到了高浓度、高纯度的PQQGDH。通过酶液和PQQ样品用量的双因素正交实验对传统重组酶法的反应体系加以改进,得到适用于96孔板高通量检测的反应体系。结果表明30μL纯化后酶液、2μL PQQ样品配合1.2 m L的显色剂组成的显色体系检测效果最佳。该方法线性范围大且具有较高的精确度和重现性。本研究中利用高浓度、高纯度的PQQGDH配合96孔板和酶标仪,所建立的PQQ高通量检测方法,不仅降低了实验误差,而且大幅提高了PQQ发酵液样品的检测效率。     

大肠杆菌四氢嘧啶合成途径的构建与优化

为了构建重组工程菌株发酵合成四氢嘧啶,解决野生型菌株对高盐环境的依赖,作者克隆了来自伸长盐单胞菌(Halomonas elongata ATCC 33173)的四氢嘧啶合成相关基因簇ectABC并在大肠杆菌(Escherichia coli,E.coli)中重构了四氢嘧啶的合成途径.通过宿主比较发现,与E.coli W...     

肉制品中致泻大肠埃希菌的分子特征分析

目的 了解国内肉制品中致泻大肠埃希菌（DEC）的流行特征。方法 运用全基因组测序技术对86株DEC进行分子特征分析,明确优势病理类型、序列型和血清型。通过全基因组单核苷酸多态性分析,确定我国肉制品来源DEC菌株之间的系统发育关系。结果 肉制品中致泻大肠埃希菌的病理类型以EAEC为主,86株DEC可分为8种毒力基因型,包括48个ST型（包括6个新ST型）和55个O:H血清型;ST11和CC10为优势序列型和克隆复合体,O157、O15和O6为优势血清群。DEC菌株间表现为高度的遗传异质性,相同病理类型的同源性较低,处在不同的进化分支上。结论 DEC的病理类型与ST型及血清型间未见对应关系,通过对ST型及血清型的监测不能直接对病理类型进行判断,但数量最多的ST型及血清型揭示了DEC的主要危害特征与防控点。可根据全基因组测序结果补充和优化DEC的判定方法,为今后DEC的溯源及流行病学调查提供数据支撑。     

核酸-免疫层析法快速检测肠道侵袭性大肠埃希氏菌

目的 建立快速检测肠道侵袭性大肠埃希氏菌(EIEC)的核酸-免疫层析方法。方法 利用不对称聚合酶链式反应技术制备目标单链DNA,结合免疫层析技术分型检测EIEC的毒力基因invE和大肠埃希氏菌标志基因uidA。结果在不对称PCR体系中,uidA和invE最佳上下游引物比例为1∶3,最佳引物浓度(下游)分别为0.2μmol/L和0.25μmol/L,最佳扩增循环次数为40。本方法最低检测限为3.97×10^-3ng/μL的基因组DNA,特异性与PCR-凝胶电泳法相当。结论 本方法具有操作简便、快速、成本低、检测结果准确以及特异性良好等优点,可分型检测EIEC、非EIEC大肠埃希氏菌和非大肠埃希氏菌,适用于基层实验室使用。     

鸡源大肠埃希氏菌mcr-1基因携带及分析

目的 了解在农业农村部禁止使用多黏菌素作为动物促生长使用后四川部分地区鸡源大肠埃希氏菌（E.coli）mcr-1基因的携带情况,为制定进一步防控措施提供依据。方法 采集四川部分地区市场售卖点肉鸡直肠拭子,用含有多黏菌素（终浓度4 μg/mL）的EC肉汤增菌接种含多黏菌素（终浓度4 μg/mL）的麦康凯平板,挑取可疑菌落,采用PCR方法鉴定菌株并检测mcr-1基因;微量肉汤稀释法测定mcr-1基因阳性菌株对临床常见抗菌药物耐药情况。脉冲场凝胶电泳（PFGE）对mcr-1基因阳性菌株进行同源分析。耐药基因质粒结合实验验证mcr-1基因传播途径。结果 从70份肉鸡样本中的13份检出mcr-1基因阳性大肠埃希氏菌,检出率18.57%（13/70）,对实验的13种抗生素,除13株mcr-1阳性菌株对头孢西丁有12株敏感以外,对其他抗生素都表现出不同程度的耐药,其中四环素和甲氧苄啶/磺胺甲恶唑耐药率最高,达到了100%（13/13）;其次是氨苄西林和氯霉素,耐药率为84.62%（11/13）。PFGE显示13株mcr-1阳性大肠埃希氏菌分属13个不同的型别;质粒结合实验显示mcr-1基因能够通过质粒传播。结论 mcr-1基因在鸡大肠内大肠杆菌中检测率比较高,且鸡大肠中mcr-1阳性大肠埃希氏菌的耐药情况比较严重。     

Genomic and phenotypic characterization of Escherichia coli isolates recovered from the uterus of puerperal dairy cows

The role of Escherichia coli in the pathogenesis of the puerperal uterine infection of the cow is largely unknown. It is proposed that E. coli favors the persistence of Arcanobacterium pyogenes and gram-negative bacteria that are pivotal to the establishment of the infection. Here, we report the genomic and phenotypic characteristics of 72 E. coli isolates recovered from the uterus of dairy cows with normal puerperium (n = 12; 35 isolates) or clinical metritis (n = 18; 37 isolates), in an attempt to identify characteristics that are related to the establishment of uterine infection. We evaluated DNA fingerprints generated by repetitive element sequence-based PCR, phylogenetic grouping, the presence of 15 virulence factor genes, in vitro biofilm formation and its relationship to curli fimbriae expression, and cellulose production. We found a wide genetic diversity (40 clonal types), including types common to normal puerperium and clinical metritis cows (n = 6), as well as types specific to normal puerperium (n = 14) or clinical metritis (n = 20) cows. Isolates were assigned to phylogenetic groups B1 (58%), A (31%), and D (11%). Only 4 virulence factor genes were detected (hlyE, hlyA, iuc, and eaeA). In vitro biofilm formation was significantly affected by culture medium and incubation temperature. Curli fimbriae expression and cellulose production, although related to biofilm formation, were not required for it. None of the evaluated E. coli characteristics were significantly related to the establishment of the uterine infection. In conclusion, data presented in this paper indicate that E. coli isolates recovered from the uterus of puerperal cows present a wide genetic diversity, do not belong to a known pathogenic group, and have a low potential of virulence and persistence. This corroborates the putative role of the bacterium in the pathogenesis of the puerperal uterine infection of the cow.     

Inactivation of Escherichia coli in orange juice using ozone

This research investigated the efficacy of gaseous ozone for the inactivation of Escherichia coli ATCC 25922 and NCTC 12900 strains in orange juice. Orange juice inoculated with E. coli (10⁶ CFU mL^− 1) as a challenge microorganism was treated with ozone at 75–78 µg mL^− 1 for different time periods (0–18 min). The efficacy of ozone for inactivation of both strains of E. coli was evaluated as a function of different juice types: model orange juice, fresh unfiltered juice, juice without pulp, and juice filtered through 500 µm or 1 mm sieves. Fast inactivation rates for total reduction of E. coli were achieved in model orange juice (60 s) and in juice with low pulp content (6 min). However, in unfiltered juice inactivation was achieved after 15–18 min. This indicated that juice organic matter interferes with antibacterial activity of gaseous ozone. The effect of prior acid (pH 5.0) exposure of E. coli strains on the inactivation efficacy of ozone treatment was also investigated. There was a strain effect observed, where prior acid exposure resulted in higher inactivation times in some cases by comparison with the control cells. However, the overarching influence on inactivation efficacy of ozone was related to the pulp content. Generally, the applied gaseous ozone treatment of orange juice resulted in a population reduction of 5 log cycles.

Industrial relevance

To facilitate the preservation of unstable nutrients many juice processors have investigated alternatives to thermal pasteurisation, including un-pasteurised short shelf life juices with high retail value. This trend has continued within the European Union. However within the US recent regulations by the FDA have required processors to achieve a 5-log reduction in the numbers of the most resistant pathogens in their finished products. Pathogenic E. coli may survive in acid environments such as fruit juices for long periods. This study demonstrates that the use of ozone as a non-thermal technology is effective for inactivation of E. coli and acid exposed E. coli in orange juice. Information on the design of the ozone treatment for inactivation of E. coli which results into safe juice products is also among the main outputs of this work. Ozone auto-decomposition makes this technology safe for fruit juice processing.     

Biofilm formation by Escherichia coli in hypertonic sucrose media

High osmotic environments produced by NaCl or sucrose have been used as reliable and traditional methods of food preservation. We tested, Escherichia coli as an indicator of food-contaminating bacterium, to determine if it can form biofilm in a hyperosmotic environment. E. coli K-12 IAM1264 did not form biofilm in LB broth that contained 1 M NaCl. However, the bacterium formed biofilm in LB broth that contained 1 M sucrose, although the planktonic growth was greatly suppressed. The biofilm, formed on solid surfaces, such as titer-plate well walls and glass slides, solely around the air–liquid interface. Both biofilm forming cells and planktonic cells in the hypertonic medium adopted a characteristic, fat and filamentous morphology with no FtsZ rings, which are a prerequisite for septum formation. Biofilm forming cells were found to be alive based on propidium iodide staining. The presence of 1 M sucrose in the food environment is not sufficient to prevent biofilm formation by E. coli.     

UV-C inactivation of Escherichia coli at different temperatures

The influence of treatment parameters (dose and temperature), treatment medium characteristics (absorption coefficient, pH and water activity) and microbiological factors (strain, growth phase and UV damage and repair capacity) on Escherichia coli UV-C resistance has been investigated. UV-C doses to inactivate at 25 °C 99.99% of the initial population (4D) of five strains of E. coli in McIlvaine buffer of pH 7.0 with tartrazine added (absorption coefficient of 10.77 cm⁻¹) were 16.60, 14.36, 14.36, 13.22, 11.18 J/mL for strains E. coli STCC 4201, STCC 471, STCC 27325, O157:H7 and ATCC 25922, respectively. The entrance in the stationary growth phase increased the 4D value of the most resistant strain, E. coli STCC 4201, from 13.09 to 17.23 J/mL. Survivors to UV treatments showed neither oxidative damages nor injuries in cell envelopes. On the contrary, the photoreactivation by the incubation of plates for 60 min below visible light (11.15 klx) increased the dose to 18.97 J/mL. The pH and the water activity of the treatment medium did not affect the UV tolerance of E. coli STCC 4201, but the lethal effect of the treatments decreased exponentially (Log₁₀4D = − 0.0628α + 0.624) by increasing the absorption coefficient (α). A treatment of 16.94 J/mL reached 6.35, 4.35, 2.64, 1.93, 1.63, 1.20, 1.02 and 0.74 Log₁₀ cycles of inactivation with absorption coefficients of 8.56, 10.77, 12.88, 14.80, 17.12, 18.51, 20.81 and 22.28 cm⁻¹. The temperature barely changed the UV resistance up to 50.0 °C. Above this threshold, inactivation rates due to the combined process synergistically increased with the temperature. The magnitude of the synergism decreased over 57.5 °C. An UV treatment of 16.94 J/mL in media with an absorption coefficient of 22.28 cm⁻¹ reached 1.23, 1.64, 2.36, 4.01 and 6.22 Log₁₀ cycles of inactivation of E. coli STCC 4201 at 50.0, 52.5, 55.5, 57.5 and 60.0 °C, respectively.

Industrial relevance

Results obtained in this investigation show that UV light applied at mild temperatures (57.5 to 60 °C) could be an alternative to heat treatments for 5-Log₁₀ reductions of E. coli in liquid foods. Since microbial resistance to UV-C light did not depend on the pH and water activity (a_w) of the treatment media, eventual advantages of UV light for pasteurization purposes will be higher in low a_w foods. E. coli STCC 4201 could be considered as a target when UV light processing of foods.     

Improvement of productivity of active horseradish peroxidase in Escherichia coli by coexpression of Dsb proteins

Coexpression of two classes of folding accessory proteins, molecular chaperones and foldases, can be expected to improve the productivity of soluble and active recombinant proteins. In this study, horseradish peroxidase (HRP), which has four disulfide bonds, was selected as a model enzyme and overexpressed in Escherichia coli. The effects of coexpression of a series of folding accessory proteins (DnaK, DnaJ, GrpE, GroEL/ES, trigger factor (TF), DsbA, DsbB, DsbC, DsbD, and thioredoxin (Trx)) on the productivity of active HRP in E. coli were examined. Active HRP was produced by very mild induction with 1 μM isopropyl-β- -thiogalactopyranoside (IPTG) at 37°C, whereas the amount of active HRP produced by the induction with 1 mM IPTG was negligibly small. Active HRP production was increased significantly by coexpression of DsbA-DsbB (DsbAB) or DsbC-DsbD (DsbCD), while coexpression of molecular chaperones did not improve active HRP production. The growth of E. coli cells was inhibited significantly by the induction with 1 mM IPTG in a HRP single expression system. In contrast, when HRP was coexpressed with DsbCD, the growth inhibition of E. coli was not observed. Therefore, coexpression of Dsb proteins improves both the cell growth and the productivity of HRP.