Alicyclobacillus属细菌及其在生物冶金中的研究进展

脂环酸芽孢杆菌(Alicyclobacillus)属细菌广泛存在于酸性果汁饮料、矿山、热泉和火山附近土壤等酸热环境中,具有嗜酸耐热的典型特征。多项研究表明该属细菌具有广泛的底物利用特性,可以利用有机物进行异养生长,也可以氧化Fe2+、元素硫以及硫化矿等进行自养生长,在厌氧或微缺氧的条件下还可以还原Fe3+以及针铁矿等铁的沉淀物中的Fe?,因此该属细菌在生物冶金中具有巨大应用潜力,而目前关于该属细菌在生物冶金中的研究报道很少。对Alicyclobacillus属细菌的分离、生理生化特征以及在生物冶金领域中的国内外研究现状进行概述和分析,并展望了未来该属细菌在生物冶金领域中的研究方向。     

一株针对高温烟气的嗜热嗜酸同步脱硫脱氮菌的分离鉴定

为了解决高温烟气在洗尘降温后温度较高(50~65 ℃)而致常温微生物(30 ℃)净化效果大幅度下降的难题,将取自云南腾冲酸性热泉的泥水菌样通过质量浓度为2 000~3 000 mg/m 3的SO2和500~2 000 mg/m 3的NOx诱导驯化90 d后,分离得到一株嗜热嗜酸的同时脱硫脱氮菌NS。该菌革兰氏染色为阳性,呈短杆状,大小为1.5~2.0 μm,其最适生长温度为65 ℃,最适生长pH值为4.0。经16S rDNA 序列分析表明,NS菌株与Alicyclobacillus sendaiensis的相似度达99%。该菌株在最适生长条件下培养60 h 后,能同步利用硫代硫酸盐和硝酸盐,对硫代硫酸盐和硝酸盐的代谢率分别超过80%和85%,代谢产物为硫酸盐、单质硫和亚硝酸盐。     

微波提取苹果汁中脂环酸芽孢杆菌DNA的PCR检测方法研究

根据Genbank公布的酸土脂环酸芽孢杆菌(Alicyclobacillus acidoterrestris DSM 3922T)鲨烯环化酶序列自行设计一对引物,利用微波技术直接从果汁样品中提取目标菌DNA,对引物特异性、微波法提取DNA的扩增效果及检测灵敏度进行探讨。结果表明：微波功率1000W、处理时间30s、经5000r/min离心2min即可获得酸土脂环酸芽孢杆菌基因组DNA,所得模板质量符合聚合酶链式反应检测要求,目的条带清晰,检测时间仅为2h,检测限为200CFU/mL,有望真正应用于苹果汁生产的在线检测。     

利用电子鼻检测6种商业果汁饮料中脂环酸芽孢杆菌的污染

脂环酸芽孢杆菌具有嗜酸耐热的特性和产芽孢的能力,在果汁中萌发繁殖会使产品出现异味,导致腐败。本文将筛选得到的分离菌株XC-6和购自德国菌种保藏中心的标准菌株DSM 3922分别等量接种到6种果汁饮料,采用FOX 4000电子鼻结合主成分分析对接种样品与正常样品进行对比分析,发现电子鼻可快速有效鉴别出经不同处理的样品,其中第一主成分累积方差贡献率均大于95%。GC-MS分析表明接种样品相较于正常样品,挥发性物质图谱存在着差异,特征表向物质含量的差异可以被电子鼻用来鉴别正常样品与污染样品。在水蜜桃乳饮料和水果牛奶混合饮料中均检出了脂环酸芽孢杆菌的特征代谢产物邻乙氧基苯酚和愈创木酚,它们的含量显著高于正常样品。本实验表明电子鼻可以根据样品气味图谱的不同而对污染样品做出准确的鉴别,是可以将电子鼻作为早期快速检测脂环酸芽孢杆菌污染的检测工具。     

Effects of pH, cinnamaldehyde and heat-treatment time on spore viability of Alicyclobacillus acidoterrestris

A three variables–five levels central composite design was used to study the effects of pH (3.5, 4.0, 4.5, 5.0 and 5.5), cinnamaldehyde (0, 40, 80, 120 and 160 ppm) and heating temperature (80, 84, 88, 92 and 96 °C) on the spores of Alicyclobacillus acidoterrestris in malt extract broth (MEB). The heat shock resulted in a slight decrease of spore number from 3.2 to 2.5 log CFU mL⁻¹ and occurred at 80–85 °C depending on the pH of the medium. Otherwise, cinnamaldehyde acted as an additional hurdle within the storage time; the critical amounts to be used were included in the range 41.35–44.42 ppm of cinnamaldehyde and were related positively to the pH, i.e. the critical amount of active compound decreased with the decreasing of the pH.     

PCR法检测耐热耐酸菌条件的优化

以耐热菌为研究对象 ,对PCR法快速检测耐热菌的扩增条件 :Taq酶浓度、Mg2 +浓度、退火温度、循环温度 /时间、循环次数 5个方面进行了优化。最终得到PCR反应最佳条件 :5 0 μL扩增体系中 ,Taq酶浓度 2U/mL、Mg2 +浓度 2 0mmol/L、退火温度 5 8℃。扩增程序 :94℃预变性 4min后进入PCR循环 ,即 94℃ /3 0S -5 8℃ /3 0S -72℃ /3 0S ,循环 3 5次 ,72℃下延伸 5min。     

Characterisation of the volatile profile of orange juice contaminated with Alicyclobacillus acidoterrestris

A rapid and reliable analytical method, based on the characterisation of the volatile profile by dynamic headspace extraction followed by gas chromatography mass–spectrometry, was developed in order to early detect Alicyclobacillus acidoterrestris spoilage in orange juice. Gas chromatographic peak areas were submitted to multivariate statistical analysis (principal component and linear discriminant analysis) in order to visualise clusters within samples and to detect the volatile compounds able to differentiate contaminated from not-contaminated samples. Significant differences in the volatile profile of the analysed samples were found, assessing the reliability of the proposed method to detect the A. acidoterrestris contamination in orange juice.     

苹果浓缩汁中嗜酸耐热菌多克隆抗体的制备及纯化

从苹果浓缩汁中分离得到嗜酸耐热菌,首次以耐热菌繁殖体及芽胞体分别作为免疫抗原,采用耳静脉注射及肌肉注射2种途径,对8只日本大耳白兔进行免疫,分别获得多克隆抗体。使用试管凝集法分别检测得到的抗体效价及其特异性,并采用饱和硫酸铵沉淀法及DEAE离子交换法纯化多克隆抗体,最终经SDS-PAGE电泳进行鉴定。以耳静脉注射和肌肉注射为免疫途径,制备得到的抗体效价分别达到1∶2560和1∶640。耳静脉注射得到的抗体效价高且免疫周期短,因此,耳静脉注射途径为最佳免疫途径;抗体特异性及纯化效果好。首次建立了苹果浓缩汁中耐热菌的家兔免疫程序,获得了多克隆抗体。     

Formation of guaiacol from vanillin by<Emphasis Type="Italic"> Alicyclobacillus acidoterrestris</Emphasis> in apple juice: a model study

In this study, the formation of guaiacol from vanillin by Alicyclobacillus acidoterrestris in apple juice was investigated. Apple juices spiked with 10 and 100 mg/L of vanillin were inoculated with 1×10³ CFU/mL and 1×10⁵ CFU/mL spores of A. acidoterrestris and incubated at 25 and 46 °C. A. acidoterrestris started to form guaiacol from vanillin when the spore count exceeded the critical level of 1×10⁴ CFU/mL. Increasing the vanillin concentration also increased the concentration of guaiacol formed, especially at 46 °C. The growth of A. acidoterrestris was not favorable at 25 °C. Thus, the formation of guaiacol is strongly limited at room temperature.     

Target selection in designing pasteurization processes for shelf-stable high-acid fruit products

This study is focused on the search for targets and criteria for the design of pasteurization processes for high-acid shelf-stable fruit products, such as juices, nectars, pastes, purees, concentrates, jams, jellies, etc. First, an overview of pasteurization is presented and then, frequently used targets for pasteurization processes are reviewed Enzymes naturally present in fruits, in decreasing order of heat resistance, were pectinesterase, peroxidase, and polyphenoloxidase, and they may be used as pasteurization targets. The heat resistance of each enzyme is strongly dependent on its fruit origin. The most heat resistant micro-organisms capable of spoiling high acid fruit products include ascospores of Neosartorya fischeri, Byssochlamys nivea, Talaromyces flavus, Eupenicillium javanicum, and Byssochlamys fulva moulds, as well as bacterial spores of Clostridium butyricum, Bacillus coagulans, and Bacillus megaterium. These micro-organisms, spores, and enzymes were, in general, less heat resistant than the spores of a particular spoilage micro-organism named Alicyclobacillus acidoterrestris, which has been causing problems in the fruit industry. Therefore, the use of Alicyclobacillus acidoterrestris spores as a reference micro-organism in the design of pasteurization processes for high-acid shelf-stable fruit products is suggested.