异源表达酸土脂环酸芽孢杆菌DnaJ基因提高大肠杆菌胁迫抗性

构建重组质粒p ET28a-Dna J,转化大肠杆菌BL21(DE3),利用IPTG诱导重组大肠杆菌表达酸土脂环酸芽孢杆菌Dna J,对重组大肠杆菌和对照大肠杆菌分别进行热及酸、碱胁迫处理,研究酸土脂环酸芽孢杆菌Dna J基因异源表达对重组大肠杆菌胁迫抗性的影响。试验结果表明,经过热、酸胁迫后,重组大肠杆菌的存活率明显高于对照菌株,而在碱胁迫条件下,两种菌株的存活率相差不大,说明酸土脂环酸芽孢杆菌Dna J基因在大肠杆菌中异源表达能显著提高宿主菌对高温及酸胁迫的抗性,为发酵工业生产中构建高产抗逆工程菌株提供基因素材和理论依据。     

酸土环脂芽孢杆菌检测与控制技术研究进展

酸土环脂芽孢杆菌是造成果汁变质的一种主要腐败菌。在生产实践中快速、准确的检测手段必不可少,同时对该菌进行控制是提高产品质量、减少污染的重要环节,特别是对其耐热芽孢的抑制。通过对酸土环脂芽孢杆菌生长、代谢特性的分析,重点阐述了该菌污染的检测技术和菌种的分离、鉴定方法;同时从果汁生产环节入手探讨了控制该菌的各种方法,并指出今后应该进行的研究方向。     

酸土脂环酸芽孢杆菌Fe-SOD基因克隆及生物信息学分析

酸土脂环酸芽孢杆菌具有嗜酸耐热的独特生理特性,从该属细菌中分离到的多种酶类都具有耐酸热稳定性,是筛选耐酸耐热酶的重要菌种资源。超氧化物歧化酶(SOD)由于具有重要的生理功能而广泛用于食品、药品及化妆品等行业。为了深入探讨酸土脂环酸芽孢杆菌SOD的分子生物学特性,本研究利用同源克隆、交错式热不对称PCR技术和生物信息学方法进行Fe-SOD基因克隆、测序及序列分析。结果表明,Fe-SOD基因(GenBank序列号:JN614998)ORF全长为606bp,编码202个氨基酸,其推测氨基酸序列与来源于酸热脂环酸芽孢杆菌DSM446的Fe-SOD(ACV58017.1)序列相似性最高,为78%。整个蛋白序列含有Fe/MnSODs家族的5个模体,SOD活性中心含有金属离子结合配基His27、His82、Asp164和His168,具有Fe/MnSODs家族典型的蛋白结构特征。酸土脂环酸芽孢杆菌Fe-SOD基因的克隆和生物信息学分析为进一步构建原核表达载体、获得生产耐酸热Fe-SOD的基因工程菌株奠定基础。     

酸土脂环酸芽孢杆菌DnaK基因克隆及生物信息学分析

利用同源克隆和基因组步移技术克隆得到酸土脂环酸芽孢杆菌(DSM 3922T)DnaK基因全序列,其ORF全长1 854 bp,编码617个氨基酸,推测其氨基酸序列与来源于A.acidocaldarius LAA1的分子伴侣蛋白DnaK同源性最高,相似性达86%。利用同源建模对DnaK的二级结构和超二级结构的预测结果显示,该伴侣蛋白主要由α-螺旋和β-折叠组成,基本不含无规则卷曲,具有耐热蛋白质的特征。     

培养基成分对酸土脂环酸芽孢杆菌生长及芽孢形成的影响

本研究通过单因素和双因素交互实验,研究多种营养因子对酸土脂环酸芽孢杆菌生长和芽孢形成的影响。结果表明,葡萄糖、酵母浸粉、Mn²⁺、Mg²⁺对酸土脂环酸芽孢杆菌的菌体生长和芽孢形成影响显著(p<0.05)。其中高浓度的Mn²⁺对酸土脂环酸芽孢杆菌的菌体生长有抑制作用,但能促进其芽孢形成。而Ca²⁺、NH₄⁺和K⁺单一作用时,对其菌体生长和芽孢形成影响不显著(p>0.05),但Ca²⁺与K⁺或者NH₄⁺协同作用时,可以促进酸土脂环酸芽孢杆菌的菌体生长和芽孢形成。研究结果揭示了培养基中的营养成分对该菌菌体生长和芽孢形成的影响,为控制该菌芽孢污染及酸性果蔬产品保藏提供实验和理论依据。     

环介导等温扩增技术检测食品中酸土环脂芽孢杆菌

目的：利用环介导等温扩增技术建立食品中酸土环脂芽孢杆菌快速检测方法。方法：针对酸土环脂芽孢杆菌16S序列设计特异引物,再优选反应体系,用显色法检测实验结果。结果：该方法能够在63 ℃条件下1 h内检出食品中酸土环脂芽孢杆菌,所设计的引物有良好的特异性;灵敏度达6.7 CFU/mL（弱阳性）。结论：该方法具有高效、特异性强和敏感性高等特点,可满足酸土环脂芽孢杆菌快速检测筛选的要求。     

脂环酸芽孢杆菌对苹果汁品质的影响

为了考察分离自浓缩苹果汁生产线的脂环酸芽孢杆菌对苹果汁品质的影响,将已经分纯并作初步鉴定的18株脂环酸芽孢杆菌和DSM3922接入100%苹果汁中,37℃培养,定期取样观察其感官品质的变化,并测定其OD360nm和浊度。试验结果表明:所有试验菌株均能使苹果汁产生浑浊、沉淀和异味,并且使吸光值和浊度增大;随着培养时间的延长,苹果汁变质程度加剧;不同菌株使苹果汁变质的程度不同,其中编号为7-1、2-1、DSM3922、3-5的4株菌比其它菌株更易使苹果汁腐败变质。     

肌苷对酸土脂环酸芽孢杆菌生长及生物膜形成的影响

由嗜酸耐热酸土脂环酸芽孢杆菌污染酸性果汁引起的营养损失、风味劣变、沉淀等质量问题是制约果蔬加工及出口贸易的瓶颈。本文通过光密度测定、生物被膜测定、扫描电子显微镜检测、平板菌落计数、果汁理化特性及感官品质鉴定等方法,研究肌苷对酸土脂环酸芽孢杆菌生长的抑制效果及对橙汁品质的影响。结果表明,不同浓度的肌苷在酸性条件下均能高效抑制该菌营养菌体的生长繁殖,且对鲜榨橙汁的酸度、糖度、色度及口感等理化特性和感官品质无显著影响。低至5 mmol/L的肌苷添加量即可降低该菌的生物被膜形成能力,损伤菌体的细胞壁和细胞膜,导致菌体裂解死亡。在初始污染菌含量高达105 CFU/mL时,仍能将菌体的数量降低1个数量级,控制营养菌体的数量,使之不能产生达到嗅觉阈值的愈创木酚量,肌苷有望成为酸性果汁生产中抑制酸土脂环酸芽孢杆菌污染的新型高效防腐剂。     

脂环酸芽孢杆菌的研究进展

概述了脂环酸芽孢杆菌的特性和分类,介绍和分析了脂环酸芽孢杆菌分离鉴定的方法及其对果汁工业的危害情况及控制方法的研究进展。     

基于近红外光谱的橙汁中酸土脂环酸芽孢杆菌的生长预测

酸土脂环酸芽孢杆菌（Alicyclobacillus acidoterrestris）是引起橙汁劣变的主要微生物,为研究酸土脂环酸芽孢杆菌在橙汁中的生长规律,利用近红外光谱获取橙汁中酸土脂环酸芽孢杆菌含量的信息,采用标准化（autoscale）、多元散射校正（multiplicative scatter correction,MSC）、标准正态变换（standard normal variate,SNV）、去趋势化（detrend）对光谱进行预处理,结合化学计量学,构建近红外光谱与酸土脂环酸芽孢杆菌含量预测模型。在此基础上,将近红外光谱转换为酸土脂环酸芽孢杆菌预测菌落数据,并采用“一步法”直接基于预测菌落数构建橙汁中酸土脂环酸芽孢杆菌的生长模型。结果表明,利用标准化进行光谱预处理建立的偏最小二乘（partial least squares,PLS）模型对橙汁中酸土脂环酸芽孢杆菌含量的预测效果相对较好,其预测决定系数（prediction determination coefficient,Rp2）与预测均方根误差（root mean square error of prediction...     

Expression of DnaJ Gene in Alicyclobacillus acidoterrestris under Stress Conditions by Quantitative Real-Time PCR

Abstract: This article describes the cloning, sequence analysis and expression of the DnaJ gene from Alicyclobacillus acidoterrestris. The genome walking technique was used to clone the full‐length sequence of DnaJ and quantitative real‐time PCR was used to analyze DnaJ expression under stress conditions. AadnaJ (GenBank accession nr: HQ893544) containing an open reading frame of 1137 bp encoding 378 amino acid residues was cloned from A. acidoterrestris DSM 3922^T. The nucleotide sequence of AadnaJ shows 77% homology with the DnaJ of A. acidocaldarius LAA1. The DnaJ expression level was upgraded rapidly under heat or acid stress. Its mRNA expression level reached a peak value at 25 min after the onset of heat stress (70 °C) and at 1 h after the onset of acid stress (pH = 1). Acid stress at pH 1 for 25 and 60 min led to the DnaJ expression levels 2.1 times and 35.7 times above that of the control, respectively. In response to cold stress at 0 °C, the DnaJ expression level decreased drastically to 0.04 times that of the control level after 1 h. The expression patterns of DnaJ in response to the stress conditions shown here explained the heat and acidity endurance of A. acidoterrestris. Practical Application: This study directly addresses the role of the DnaJ gene in temperature and acid endurance in A. acidoterrestris. This provides a basis for the development of genetic and molecular techniques that may minimize the adverse effects of A. acidoterrestris in fruit juice production. This study also sheds light on the design of heat‐ and acid‐tolerant recombinases and the understanding of the molecular mechanisms underlying heat and acid resistance in A. acidoterrestris.     

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。     

对羟基苯甲酸丙酯对果蝇超氧化物歧化酶(SOD)基因表达的影响

探讨对羟基苯甲酸丙酯(PP)对果蝇超氧化物歧化酶(SOD)基因表达的影响,用含不同质量浓度(0.00、0.20、0.50、1.00、2.00g/L)的PP培养基饲喂果蝇10d后,提取不同PP组果蝇总RNA,采用逆转录PCR方法在mRNA表达水平上研究SOD基因。结果表明:PP抑制了果蝇体内抗氧化酶Cu,Zn-SOD、Mn-SOD基因的表达,与对照组相比具有显著性差异(P<0.05);随着PP质量浓度的升高,雌果蝇体内Cu,Zn-SOD基因与雄果蝇体内Mn-SOD基因的表达量随之降低,而Mn-SOD基因在雌果蝇体内总体表达量提高。故PP的毒性作用可能与抑制抗氧化酶基因的表达有关,而这种作用因果蝇性别而异。     

欧姆加热对嗜酸耐热菌的杀灭作用研究

本实验探讨了欧姆加热对嗜酸耐热菌的杀灭作用。利用自行设计的批式欧姆加热装置,对苹果汁中嗜酸耐热菌进行处理,分析了欧姆加热的电压、pH值、时间及加热体积等对杀菌效果的影响,并利用扫描电镜观察微生物细胞壁膜结构的变化,利用电导率仪和紫外吸收分光光度计测定菌悬液的成分变化。结果表明欧姆加热可以有效的杀灭苹果汁中的嗜酸耐热菌,杀菌率随电压、加热体积的升高而增大,随pH值的降低而增大。通过环境扫描电镜可观察到嗜酸耐热菌的表面出现凹陷和破损,电导率仪和紫外吸收分光光度计测定细胞内容物的溢出,据此推测欧姆加热造成了嗜酸耐热菌的"电穿孔"。     

Characterization of a wild strain of Alicyclobacillus acidoterrestris: heat resistance and implications for tomato juice

This article reports the characterization of a wild strain of Alicyclobacillus acidoterrestris and describes the implications of the heat resistance of this microorganism in tomato juice. The strain (labeled as A. acidoterrestrisγ4) showed pH and temperature ranges for growth typical of the species (3.0 to 6.0 for the pH and 35 to 60 °C for the temperature); heat resistance in tomato juice was as follows: D(T) values of 40.65, 9.47, and 1.5 min (at 85, 90, and 95 °C, respectively) and z-value of 7 °C. A treatment at 70 °C for 15 min was found to be optimal for spore activation, whereas Malt Extract Agar, acidified to pH 4.5, showed good results for spore recovery. Concerning the implications of heat resistance of A. acidoterrestris on tomato juice, high temperatures required for spore inactivation determined a general decrease of the antioxidant activity (increase of the redox potential and reduction of the chain-breaking activity), but not the formation of brown compounds (namely, hydroxymethylfurfural), thus suggesting an effect on the secondary antioxidants (carotenoids and ascorbic acid) rather than on lycopene. PRACTICAL APPLICATION: Alicyclobacillus acidoterrestris is an emerging spore-forming microorganism, capable of causing spoilage in tomato juice. Due to their high thermal resistance, spores could be used as targets for the optimization of heat processing; this article reports on the assessment of thermal resistance of a wild strain of A. acidoterrestris, then focusing on the effect of the thermal treatment necessary to inactivate spores on the quality of tomato juice.     

短波紫外发光二极管处理对脂环酸芽孢杆菌的灭活效果及作用机制

短波紫外发光二极管（ultraviolet-C light-emitting diode,UVC-LED）处理是一种新型的非热杀菌技术。本实验以果汁中常见的致腐菌脂环酸芽孢杆菌（Alicyclobacillus acidoterrestris）为目标菌,研究UVC-LED对脂环酸芽孢杆菌的杀灭作用,通过测定处理后细菌胞内核酸和蛋白质泄漏量、细胞膜通透性、胞内活性氧（reactive oxygen species,ROS）的累积水平以及胞内蛋白质和DNA的损伤情况,进一步探究UVC-LED对脂环酸芽孢杆菌的杀菌机理。结果表明：增加UVC-LED的照射剂量可增强其对脂环酸芽孢杆菌的杀灭效果,当照射剂量增加至50 mJ/cm2时,生理盐水中存活的细菌数量降低4.6（lg（CFU/mL））。通过对存活曲线的模拟,发现UVC-LED对生理盐水中脂环酸芽孢杆菌的杀灭作用既符合log-linear模型,又符合Weibull模型。处于不同生长时期的细菌对UVC-LED的敏感度不同,其中处于对数期的细菌对UVC-LED更敏感。照射处理导致膜通透性的改变以及内容物的泄漏,说明细胞膜结构遭到一定程度的破坏,但是胞内ROS的累积水平没有显著提高（P＞0.05）,拉曼光谱分析表明胞内蛋白结构有所改变,经吖啶橙（acridine orange,AO）染色荧光显微镜观察发现照射处理后菌体DNA的结构变化明显。综上,UVC-LED可通过造成DNA损伤、蛋白结构变化和细胞膜透性改变从而杀灭脂环酸芽孢杆菌,根据破坏程度的不同,推测DNA损伤是细胞死亡的主要原因。     

Sensitive and rapid detection of Alicyclobacillus acidoterrestris using loop-mediated isothermal amplification

BACKGROUND: A loop‐mediated isothermal amplification (LAMP) assay was developed for the rapid detection (within 2 h) of Alicyclobacillus acidoterrestris. The assay detected the species‐specific DNA sequence of the 16S–23S rRNA internal transcribed spacer. RESULTS: The eight strains of A. acidoterrestris were successfully amplified, but six strains of other bacillus Acidocaldarius and 13 bacterial species other than bacillus Acidocaldarius were not. The sensitivity of the LAMP assay was at 4.50 × 10^?2 cfu per tube. This sensitivity is greater than that obtained by polymerase chain reaction (PCR) assay. The LAMP assay was examined further for its ability to detect A. acidoterrestris in juice samples. The results were compared with those of conventional PCR detection. CONCLUSION: Results indicate that the proposed LAMP assay is a rapid, specific and sensitive method for detecting A. acidoterrestris. As the amplification has been conducted under isothermal conditions, only a water bath or heating block is needed to maintain the required temperature. Thus, the method can be generalised and popularised easily in the future. Copyright © 2011 Society of Chemical Industry     

Thermal resistance of Alicyclobacillus acidoterrestris spores in different heating media

The aim of this work was to study the influence of temperature (85, 90, 95 and 100 °C), total soluble solids (SS: 10 and 20°Brix or % by weight of sucrose) and pH (3.5 and 4.0) on decimal reduction time ( D- value) of the Alicyclobacillus acidoterrestris strain DSM2498 spores in apple juice, orange juice and malt extract broth (MEB). The effects of SS and pH on D -values and z- values in each media were insignificant ( P  > 0.05). In apple juice, orange juice and MEB, z- values of A. acidoterrestris for pH 3.5 and pH 4.0 were 12.2 ± 1.3–14.2 ± 3.2 °C, 11.2 ± 0.3–9.4 ± 0.0 °C and 11.9 ± 0.8–10.3 ± 0.4 °C, respectively. z- values of apple juice, orange juice and MEB samples with SS = 10°Brix and SS = 20°Brix were 14.1 ± 3.2–12.2 ± 1.3 °C, 10.2 ± 0.7–10.5 ± 1.1 °C and 11.3 ± 1.5–10.9 ± 0.2 °C, respectively. However, D -values of all samples were affected by temperature significantly ( P  < 0.01). Average D -values of apple juice, orange juice and MEB were 101.2 ± 14.7, 34.4 ± 7.9, 20.3 ± 4.9 and 4.3 ± 1.3 min for 85, 90, 95 and 100 °C. This study demonstrated that A. acidoterrestris spores exhibited high resistance to thermal processing applications. pH and SS of the media did not affect thermal resistance.