臭氧对耐酸耐热菌作用的研究

以耐酸耐热菌为研究对象,研究了臭氧对它的致死作用,并观察了臭氧作用时间、温度、pH等对臭氧杀菌效果的影响,为果汁行业实际应用臭氧杀菌提供实验依据。      

臭氧对耐酸耐热菌作用的研究

以耐酸耐热菌为研究对象,研究了臭氧对它的致死作用,并观察了臭氧作用时间、温度、pH等对臭氧杀菌效果的影响,为果汁行业实际应用臭氧杀菌提供实验依据。     

PCR技术检测沙门氏菌反应条件的优化

沙门氏菌是引起细菌性食物中毒的重要病源之一,因此建立一种快速、简便、灵敏的检测方法十分重要。该研究以沙门氏菌的侵袭蛋白基因invA为靶细胞设计引物,进行PCR扩增并对PCR反应体系中引物量、模板量、dNTP、退火温度和PCR循环数等进行优化,以确定适宜的PCR反应体系和PCR扩增反应程序。     

PCR法快速检测耐热霉菌雪白丝衣霉

以耐热性霉菌——雪白丝衣霉为研究对象,根据Gen-bank公布的beta-tubulin基因序列,设计两对引物,经过PCR筛选,确定引物Bn-1用于建立雪白丝衣霉的PCR检测方法。对PCR法的扩增条件退火温度和引物浓度进行优化,59℃的退火温度和0.2μM/PCR反应体系的引物浓度为最佳条件;考察该PCR方法的灵敏度和特异性,基因组DNA的灵敏度为1ng/PCR反应体系,检测特异性好。该方法为雪白丝衣霉的快速检测提供了一种重要的技术手段。     

耐热过氧化氢酶基因工程菌的构建及其发酵条件

利用PCR扩增技术以嗜热脂肪芽孢杆菌IAM11001染色体DNA为模板，扩增得到嗜热脂肪芽孢杆菌过氧化氢酶编码基因perA。再与经EcoRⅠ酶切的温控表达载体pBV220连接，构建重组质粒，并转化宿主大肠杆菌JM109，得到耐热过氧化氢酶基因工程菌，然后对该工程菌在LB培养基和半合成培养基中的发酵条件进行了优化。结果表明：在LB培养基中，诱导时期和酶合成最佳诱导时间均为5h，最大产酶量达到72．9U／mL；在半合成培养基中，于30℃培养4h，再于42℃诱导培养6h，产酶量最高达到131U／mL。     

苹果汁中耐热菌培养基的优化及生长曲线

通过对苹果汁中分离耐热菌和德国标准耐热菌菌株基础培养基的正交优化,确定最佳的培养基。得出最佳培养基配比是(质量分数):德国标准菌:蛋白胨0.5%,果糖0.2%,酵母粉0.2%,MgSO4·2H2O0.1%,CaCl20.5%,KH2PO40.12%,MnSO4·4H2O0.05%;分离菌:蛋白胨0.5%,葡萄糖0.2%,酵母粉0.2%,MgSO4·2H2O0.1%,CaCl20.5%,KH2PO40.12%,MnSO4·4H2O0.05%。并计算得出吸光度与细菌浓度之间的关系公式及用优化培养基培养绘制耐热菌生长曲线。     

苹果汁中耐热菌培养基的优化及生长曲线

通过对苹果汁中分离耐热菌和德国标准耐热菌菌株基础培养基的正交优化,确定最佳的培养基。得出最佳培养基配比是(质量分数):德国标准菌:蛋白胨0.5%,果糖0.2%,酵母粉0.2%,MgSO4·2H2O0.1%,CaCl20.5%,KH2PO40.12%,MnSO4·4H2O0.05%;分离菌:蛋白胨0.5%,葡萄糖0.2%,酵母粉0.2%,MgSO4·2H2O0.1%,CaCl20.5%,KH2PO40.12%,MnSO4·4H2O0.05%。并计算得出吸光度与细菌浓度之间的关系公式及用优化培养基培养绘制耐热菌生长曲线。      

双重PCR检测黄秋葵储藏期间青霉和链格孢菌条件的优化

以黄秋葵为材料,分离纯化在贮藏过程中引起腐烂的主要病原真菌青霉和链格孢菌。根据基因库上的序列,针对青霉和链格孢菌在18SRNA的序列设计2对引物,建立了用于检测黄秋葵青霉和链格孢菌双重PCR检测方法,并对PCR扩增条件进行了优化,优化条件如下:引物Alt4、Alt5和Pen F'、Pen R的比例为2∶1、退火温度为52℃、Mg~(2+)浓度为2.5 mmol/L、d NTP浓度为0.08 mmol/L、循环数为35、延伸时间为50 s、退火时间为30 s。结果表明,该方法灵敏度较高、特异性强。该试验可以为黄秋葵在储藏期间的病害防治提供重要指导依据。     

耐热纤维素酶产酶条件的优化

在原始发酵培养条件下,枯草芽孢杆菌的酶活力达到1.0U/ml。对影响其产酶水平的培养基中的碳源、氮源、无机盐等发酵培养条件进行了单因子试验研究,并对碳、氮、磷三因素进行正交试验,对原始培养基进行了优化,结果在CMC-Na1.5%,酵母粉1.0%,K2HPO 40.15%,NaCl0.5%,MgSO 40.05%,蛋白胨1.5%,初始pH为7.0的优化培养基中,于37℃,200转/分的转速下,培养48小时,产生的纤维素酶活力达到1.5U/ml。     

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.     

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。     

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。     

Inhibition of Alicyclobacillus acidoterrestris spores by natural compounds

The aim of this study was to evaluate the antimicrobial effectiveness of some natural compounds (cinnamaldehyde, eugenol, limonene) and sodium benzoate against two strains of Alicyclobacillus acidoterrestris (c8 and γ4). The antimicrobial compounds (10–500 ppm) were solved in malt extract broth, inoculated separately with 10³ spores mL⁻¹ of each strain; the samples were incubated at 44 °C and the outgrowth of spores was evaluated every day by measuring the absorbance of the medium at 420 nm; inoculated samples without active compounds were used as controls. The results pointed out that limonene was not effective in inhibiting the outgrowth of A. acidoterrestris spores; 100 ppm of cinnamaldehyde or sodium benzoate slowed the spore germination, whereas 500 ppm of eugenol inhibited the growth of microbial targets for 13 days. Strain c8 was more resistant than isolate γ4 and cinnamaldehyde was the most effective compound in inhibiting the germination of A. acidoterrestris spores.     

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"。     

Alicyclobacillus acidoterrestris: an increasing threat to the fruit juice industry?

The quality of food can be defined in various ways with processors and consumers considering flavour, odour and appearance as well as extended shelf life among the most important of its attributes. Spoilage of food by bacterial contamination may occur at any point during the processing, altering any one, or all, of these characteristics, rendering the product unusable. Alicyclobacillus spp. and Alicyclobacillus acidoterrestris, in particular, are emerging food spoilage organisms in the fruit juice and fruit juice products industry. Spores of the latter are able to survive heat treatments presently used in the industry and their elimination from products may be used as a measure of the effectiveness of any processing protocol to remove potential spoilage. This paper reviews the history, methods of detection, both traditional and rapid and the protocols that may be effective in controlling the growth of the organism and hence spoilage in the finished product.     

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。     

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.     

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。