超高压处理对枯草芽孢杆菌超微结构的影响

 研究了超高压处理对枯草芽孢杆菌（Bacillus subtilis AS 1.140）超微结构的影响,探讨其营养体以及芽孢的灭活机制。在经过500 MPa、60 ℃下保温保压20 min超高压处理后,采用透射电子显微镜进行观察,比较处理前后超微结构的变化。观察结果表明：超高压处理后,枯草芽孢杆菌的营养体细胞壁皱缩、出现缺口,胞浆泄漏、结构层次感消失、出现大片透电子区;其芽孢外壳被破坏、出现缺口,芽孢内含物结构紊乱、泄漏、出现部分透电子区;甚至内含物质完全泄漏,出现细胞壁或孢子外壳残留。     

He-Ne激光在异种间原生质体融合中的应用

用632.8nm 12mW的氦氖激光及聚乙二醇（PEG）复合诱导融合枯草芽孢杆菌与黑曲霉原生质体.为了提高枯草芽孢杆菌的糖化酶产酶能力,对两亲本在融合中采用不同的激光照射时间,选育出的融合子与枯草芽孢杆菌亲本相比,糖化酶酶活提高了2倍,并通过酯酶同工酶谱分析对融合子进行了鉴定,结果表明:融合子遗传性状与亲本相比发生了显著变化,通过传代培养,融合子具有良好的遗传稳定性.     

傅里叶变换红外光谱对枯草芽孢杆菌的光学特性研究

使用傅里叶变换红外光谱(FTIR)技术,测量了两种不同浓度的枯草芽孢杆菌的红外透过率谱,根据朗伯-比尔定律计算出它们的质量消光截面,通过算出复折射率的虚部,再使用KK(Kramers-Kronig)关系,导出复折射率的实部,并对实验结果作了分析和讨论。通过研究枯草芽孢杆菌复折射率的测量和分析方法,对于进一步研究生物气溶胶的吸收和散射特性、拓宽生物气溶胶的测量和遥测技术方法,具有重要的意义。     

阵列式等离子体射流处理芽孢的实验研究北大核心CSCD

研制了一套单极性微秒脉冲阵列式等离子体射流系统,该系统可在大气压下激发产生等离子体射流,实现大面积的灭菌处理。该系统可产生峰值电压20 kV、频率15 kHz的高压脉冲,激发产生的射流均匀稳定,覆盖面积达37.7 cm2,射流长度达6 cm,射流功率为40.05 W,处理5 min可使射流覆盖范围内的枯草芽孢杆菌的芽孢基本全部失去活性。考察了不同参数对灭菌效率的影响,结果表明,灭菌率与工作电压、脉冲频率、处理时间呈正相关,在氦气氛围下有较好的灭菌效果。SEM显示等离子体射流能够对枯草芽孢杆菌的芽孢外壳结构造成损坏,导致芽孢无法正常代谢,最终死亡。     

快中子辐照对枯草芽孢杆菌DNA损伤研究

采用脉冲场凝胶电泳技术检测并定量分析了CFBR-Ⅱ快中子脉冲堆产生的快中子在不同剂量和剂量率条件下, 对枯草芽孢杆菌黑色变种(ATCC 9372)DNA双链断裂的诱导. 通过DNA释放百分比PR值、DNA断裂水平L值、断裂DNA平均分子量和DNA片段分布等指标的分析, 结果表明：在不同的辐射条件下, DNA片段均明显分布于两个区域, 表明枯草芽孢杆菌黑色变种DNA分子上可能存在对中子辐射较为敏感的位点; 并且随着中子辐射剂量和剂量率的变化, DNA~释放百分比PR值、DNA断裂水平L值和各片段区双链断裂的含量也会发生一定规律性的变化.     

基于傅里叶变换近红外光谱的脂环酸芽孢杆菌种间分类鉴定

傅里叶变换近红外光谱(Fourier transform near infrared spectroscopy,FT-NIR)可以反映微生物细胞的分子振动信息,特异性鉴别不同类别的微生物。为了建立准确、有效的脂环酸芽孢杆菌种间分类鉴定的方法,文章基于FT-NIR技术进行了如下探究:(1)收集了7株不同种的标准菌近红外漫反射光谱数据并进行预处理,运用化学计量学中的主成分分析(principal component analysis,PCA)和线性判别分析(linear discriminant analysis,LDA)对其种间水平区分与判别的可行性进行探索。结果表明:PCA模型能对7株标准菌进行正确区分,LDA模型Ⅰ判别准确率为100%,初步证明该方法可以对脂环酸芽孢杆菌种间水平进行分类鉴定。(2)为了提高模型的稳健性和实用性,在上述标准菌建模的样品中加入分离菌,用41株菌的光谱信息依照上述方法进行数据分析后建立LDA种间判别模型Ⅱ。结果表明:选取其中15个样本进行评估,模型Ⅱ准确率为86.67%,菌种信息更全面、可信性更高。因此,FT-NIR技术结合化学计量学方法可以准确、有效地进行脂环酸芽孢杆菌的种间分类鉴定。     

基于光谱学分析的生物磷参与暹罗芽孢杆菌对铀的去除行为及机制研究

以暹罗芽孢杆菌为研究对象,它具有较高的表面积/体积比,吸附性能良好。前人关于暹罗芽孢杆菌的研究多集中在它降解纤维素淀粉或抗菌方面,关于暹罗芽孢杆菌与放射性核素的作用及机制基本未涉及。利用等离子发射光谱和等离子发射光谱-质谱研究溶液初始pH值、铀初始浓度、菌体用量等因素对暹罗芽孢杆菌去除铀的影响及作用过程中菌体释放的生物磷与铀去除的关系;利用红外光谱和扫描电镜对与铀作用前后的暹罗芽孢杆菌形貌及基团变化进行表征;利用X射线光电子能谱和扫描电镜能谱分析菌体表面元素分布情况和元素价态,进而探讨暹罗芽孢杆菌对铀的去除机制。结果表明,由于不同pH条件下暹罗芽孢杆菌生长活性、铀存在形态和磷元素释放量的不同,其对铀的去除差异很大。在pH 5.0时,暹罗芽孢杆菌对铀的去除效果最好。菌体用量增加有利于暹罗芽孢杆菌对铀的去除。对实验结果进行Langmuir和Freundlich等温吸附拟合后发现:暹罗芽孢杆菌对铀的去除行为符合Langmuir等温吸附模型;铀浓度实验获得的最大吸附量高于理论计算的最大吸附量,说明暹罗芽孢杆菌对铀的去除可能是物理和化学行为的共同作用。暹罗芽孢杆菌能够有效去除水体中的铀,实验获得的最大去除率为96.5%,最高吸附量为450.3 mg·g~(-1),高于大部分已报道的用于吸附铀的芽孢杆菌。对反应前后菌体的扫描电镜测试发现,与铀作用后暹罗芽孢杆菌表面出现鳞片状沉淀, X射线光电子能谱和扫描电镜能谱分析表明该沉淀为含磷铀物质。结合红外光谱分析,推测暹罗芽孢杆菌去除铀的机制为:首先,通过静电作用铀被快速吸引到暹罗芽孢杆菌表面,随后以配位的形式被菌体上的磷酸基团、氨基、羟基、羧基等活性基团吸附,同时与菌体释放的含磷酸盐类物质相互作用,形成含磷铀沉淀而被固定至细菌表面。在此过程中,少部分六价铀被菌体释放的胞内物质还原成四价铀而发生沉降。推测菌体表面沉淀可能为铀的磷酸盐沉淀和含磷化合物与铀的络合物形成的混合物。     

拉曼光谱测定单个细菌芽孢吡啶二羧酸浓度及机理研究

应用激光镊子拉曼光谱技术(LTRS)测定19株芽孢杆菌的单个细菌芽孢吡啶二羧酸(DPA)浓度,并验证了系统的重现性。一束30 mW,785 nm的近红外激光导入倒置显微镜,形成光镊,随机俘获水溶液中的单个芽孢,同时收集其拉曼光谱信号。细菌芽孢的DPA是以与钙离子形成的鳌合物(Ca-DPA)形式存在,收集到的芽孢拉曼光谱信号反映的是Ca-DPA信息,选用信号最强、与Ca-DPA浓度成线性关系的1 017 cm-1作为其定量的谱峰。通过1 017 cm-1的峰强,推算出Ca-DPA的浓度。结果显示,单个芽孢的拉曼光谱能够很好地反映出单个芽孢的特征信息,不同种、同种不同菌株之间的芽孢的DPA浓度有所不同,同一菌株的不同芽孢个体之间DPA浓度也存在差异。该方法不需要复杂的分离、纯化过程,可在水溶液中直接俘获单个芽孢并收集其拉曼光谱信号,便可得到单个芽孢的信息,读出DPA的浓度水平。     

基于单细胞光学技术探究微波和高温协同作用对微生物影响机制的研究

微波(300 MHz～30 GHz)对生物组织具有明显的热效应和生物效应,能极大程度上抑制微生物芽孢的生长,但从单分子层面研究微波和温度对干燥芽孢的影响机制鲜有报道。采用激光拉曼光镊技术(激光波长为532 nm,功率为2.5 mW),测量并对比不同温度下(50～125℃)微波处理(2 450 MHz, 150 W)芽孢的平均拉曼光谱,发现温度处于125℃时,1 017 cm^-1峰强明显下降,表明高温和微波对芽孢内膜造成了损伤。该研究还使用原子力显微镜观察不同条件下芽孢的表面形貌特征,发现处理后的芽孢表面均有附着物,极有可能为CaDPA,温度越高现象越明显,而且随着温度的上升,芽孢的尺寸也随之下降,说明微波和高温的协同作用,导致芽孢核芯分子的泄露,同时也加剧了水分子蒸发使体积减小。该研究的结论为研究微波和温度协同作用对芽孢的影响提供了特征参数,也为应用单细胞光学技术开展理化因子对微生物影响机制的研究开拓思路。     

基于贝叶斯方法的设备级电磁脉冲效应评估

在电磁脉冲效应试验中,由于所获得的效应数据量一般较少,利用经典的数理统计方法对效应阈值场强进行概率拟合非常困难。基于贝叶斯数理统计方法,对油气管道数据采集与监视控制系统的中心控制系统客户端的电磁脉冲效应数据拟合分析。选择了Weibull分布模型和正态分布模型作为假设模型,并依次求得这两种模型参数的先验分布、似然函数和后验分布。通过拟合优度检验,最后基于贝叶斯信息准则选择形状参数、尺度参数分别为8.87,21.11的Weibull模型作为更合理的阈值场强概率分布模型。     

Moderate hydrostatic pressure–temperature combinations for inactivation of Bacillus subtilis spores

We report the effect of using moderate hydrostatic pressure, 40–140?MPa, at moderate temperature (38–58°C) to inactivate Bacillus subtilis spores in McIlvaine's citric phosphate buffer at pH 6. We have investigated several parameters: pressure applied, holding time, pressure cycling, and temperature. The kinetics of spore inactivation is reported. The results show that spore inactivation is exponentially proportional to the time the sample is exposed to pressure. Spore germination and inactivation occur at the hydrostatic pressures/temperature combinations we explored. Cycling the pressure while keeping the total time at high pressure constant does not significantly increase spore inactivation. We show that temperature increases spore inactivation at two different rates; a slow rate below 33°C, and at a more rapid rate at higher temperatures. Increasing pressure leads to an increase in spore inactivation below 95?MPa; however, further increases in pressure give a similar rate kill. The time dependence of the effect of pressure is consistent with the first-order model (R²?>?0.9). The thermal resistance values (Z_T) of B. subtilis spores are 30°C, 37°C, and 40°C at 60, 80, 100?MPa. The increase in Z_T value at higher pressures indicates lower temperature sensitivity. The pressure resistance values (Z_P) are 125, 125 and 143?MPa at 38°C, 48°C, and 58°C. These Z_P values are lower than those reported for B. subtilis spores in the literature, which indicates higher sensitivity at pressures less than about 140?MPa. We show that at temperatures <60°C, B. subtilis spores are inactivated at pressures below 100?MPa. This finding could have implications for the design of the sterilization equipment.     

Pressure inactivation of microorganisms

Abstract

The kinetics of inactivation by hydrostatic pressure is measured for Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, bacteriophage T4, and the spores of Bacillus subtilis. Temperature and pressure effects are discussed as well as the interrelation between germination and inactivation in the case of bacterial spores.     

Fluorescence-based methods for the detection of pressure-induced spore germination and inactivation

The application of high pressure (HP) provides an opportunity for the non-thermal preservation of high-quality foods, whereas highly resistant bacterial endospores play an important role. It is known that the germination of spores can be initiated by the application of HP. Moreover, the resistance properties of spores are highly dependent on their physiological states, which are passed through during the germination. To distinguish between different physiological states and to detect the amount of germinated spores after HP treatments, two fluorescence-based methods were applied. A flow cytometric method using a double staining with SYTO 16 as an indicator for germination and propidium iodide as an indicator for membrane damage was used to detect different physiological states of the spores. During the first step of germination, the spore-specific dipicolinic acid (DPA) is released [P. Setlow, Spore germination, Curr. Opin. Microbiol. 6 (2003), pp. 550–556]. DPA reacts with added terbium to form a distinctive fluorescent complex. After measuring the fluorescence intensity at 270 nm excitation wavelength in a fluorescence spectrophotometer, the amount of germinated spores can be determined. Spores of Bacillus subtilis were treated at pressures from 150 to 600 MPa and temperatures from 37 °C to 60 °C in 0.05 M ACES buffer solution (pH 7) for dwell times of up to 2 h. During the HP treatments, inactivation up to 2log ₁₀ cycles and thermal sensitive populations up to 4log ₁₀ cycles could be detected by plate counts. With an increasing number of thermal sensitive spores, an increased proportion of spores in germinated states was detected by flow cytometry. Also the released amount of DPA increased during the dwell times. Moreover, a clear pressure-temperature-time-dependency was shown by screening different conditions. The fluorescence-based measurement of the released DPA can provide the opportunity of an online monitoring of the germination of spores under HP inside the HP vessel. Implementation can be done using diamond anvil cells, units with inspection glasses or by inserting an optical fiber into the HP vessel. The analytical methods used can help to understand the complex mechanism of germination and inactivation of bacterial spores. Due to its universal, process-independent character, the application of these methods is feasible for established and emerging technologies.     

Evaluation of the importance of germinative cycles for destruction of bacillus cereus spores in miniature cheeses

Our objective was to determine the effect of high pressure on inactivation of spores of Bacillus cereus ATCC 9139 inoculated into cheese made of raw cow's milk. Inoculated miniature cheeses were manufactured under controlled bacteriological conditions, vacuum packed and kept at 8 °C for 15 days after pressure treatment. Cheeses were submitted to pressures of 300, 400 or 500 MPa at 30 °C, during 15 min. Some of them were treated with a germination cycle of 60 MPa at 30 °C for 210 min. Lethality was calculated comparing surviving sample counts to control ones. Adding the germinative cycle resulted in higher efficiency, and when applied with 500 MPa, lethality reached 2.0 log cfu/mL. We saw that with both cycles initial counts of spores diminish, but all of them were not inactivated. However, considering that in raw milk mesophilic spore counts are 2.6-2.9 log cfu/mL, this treatment may be useful.     

Increasing Preservation Efficiency and Product Quality through Control of Temperature Distributions in High Pressure Applications

The effectiveness of HP sterilisation is a function of both temperature and pressure. As during pressurisation the product temperature increases, heat transfer to the colder HPP vessel wall occurs and the product fraction near the vessel wall will be colder than the product in the middle of the vessel. The effect of the temperature distribution in the vessel on the inactivation of Bacillus stearothermophilus has been examined. A mathematical model has been built, in which both thermodynamics and inactivation kinetics are integrated. Heat transfer is based on a Finite Element simulation, inactivation kinetics are based on first order kinetics. Based on this model and experiments the effect of an homogeneous temperature distribution on inactivation is demonstrated.     

Pressure inactivation of fungal spores in aqueous model solutions and in real food systems

Abstract

Conidiospores from Penicillium expansum and ascospores from Eurotium repens were exposed to high hydrostatic pressure in isotonic salt solution, apple and broccoli juice. Kinetic measurements were done at 4,25 and 40 or 45°C. The shape of the inactivation curves was strongly dependent on the temperature. Asco- and conidiospores were found to behave in a contrary way. The fastest reduction for conidiospores was found at 4°C, for ascospores best inactivation was achieved at 45°C. High pressure inactivation of spores in apple or broccoli juice was nearly the same as in isotonic salt solution.     

Decrease in optical density as a results of germination of Alicyclobacillus acidoterrestris spores under high hydrostatic pressure

Alicyclobacillus acidoterrestris is a spore-forming bacterium, causing spoilage of juices. The spores of these bacteria have the ability to survive in the typical conditions used for thermal pasteurization. Therefore, the use of other techniques such as high hydrostatic pressure is considered for their inactivation. The effect of hydrostatic pressure of 200–500 MPa, at temperatures 4–50 °C for 15 min, on the dynamics of germination of A. acidoterrestris spores in apple juice and pH 4 buffer was studied. To estimate the share of germinated spores, the method of determining the optical density at a wavelength of 660 nm (OD₆₆₀) was used. Parameters of hydrostatic pressure treatment used in this work affected the dynamics of germination of A. acidoterrestris spores in apple juice, and the temperature had the greatest effect. The results indicate that nutrients present in apple juice can promote the germination of A. acidoterrestris spores.     

Combination of supercritical CO2 and high-power ultrasound for the inactivation of fungal and bacterial spores in lipid emulsions

For the first time, this study addresses the intensification of supercritical carbon dioxide (SC-CO₂) treatments using high-power ultrasound (HPU) for the inactivation of fungal (Aspergillus niger) and bacterial (Clostridium butyricum) spores in oil-in-water emulsions. The inactivation kinetics were analyzed at different pressures (100, 350 and 550 bar) and temperatures (50, 60, 70, 80, 85 °C), depending on the microorganism, and compared to the conventional thermal treatment. The inactivation kinetics were satisfactorily described using the Weibull model.Experimental results showed that SC-CO₂ enhanced the inactivation level of both spores when compared to thermal treatments. Bacterial spores (C. butyricum) were found to be more resistant to SC-CO₂ + HPU, than fungal (A. niger) ones, as also observed in the thermal and SC-CO₂ treatments. The application of HPU intensified the SC-CO₂ inactivation of C. butyricum spores, e.g. shortening the total inactivation time from 10 to 3 min at 85 °C. However, HPU did not affect the SC-CO₂ inactivation of A. niger spores. The study into the effect of a combined SC-CO₂ + HPU treatment has to be necessarily extended to other fungal and bacterial spores, and future studies should elucidate the impact of HPU application on the emulsion’s stability.