【作者】 钱静亚；

【导师】 李树君；

【作者基本信息】 江苏大学 ， 农产品加工及贮藏工程， 2013， 博士

【摘要】 随着生活水平的提高,人们对食品质量的要求越来越高,传统的热加工已逐渐不能满足人们的需求。脉冲磁场作为非热加工技术之一,能够在低温下杀灭食品中的微生物,保持食品的风味及营养成分,已成为食品杀菌领域的研究热点之一。论文以枯草芽孢杆菌为例,就脉冲磁场对微生物灭活作用及其机理进行了研究,主要包括脉冲磁场对枯草芽孢杆菌的灭活效果、脉冲磁场对枯草芽孢杆菌细胞结构和功能的影响,初步探讨了灭活机理。主要研究结果如下：(1)微生物的不同生长期、磁场强度、脉冲数对枯草芽孢杆菌灭活效果均有影响。枯草芽孢杆菌在对数生长期对脉冲磁场最为敏感；随着磁场强度(>1.5T)和脉冲数(>15)的增加,枯草芽孢杆菌残留率呈下降趋势；但在磁场强度(≤1.5T)和脉冲数(≤15)相对较低时,脉冲磁场对枯草芽孢杆菌没有灭活作用,反而促进了其生长；对培养10h的枯草芽孢杆菌,采用磁场强度3.3T、脉冲数30个灭活时残留率最低为33.87%。对灭活曲线分别用Bigelow模型、Weibull模型和Hulsheger模型进行拟合,发现Weibull模型最好地拟合了脉冲磁场作用下枯草芽孢杆菌残留率的动力学曲线,R2最高为0.967。(2)介质的温度(5～45℃)、pH(4.5～8.0)和水分活度(0.88～1.00),介质中NaCl、糖类(葡萄糖、乳糖)、蛋白胨、乙醇等成分对脉冲磁场灭活枯草芽孢杆菌均会造成一定的影响,低温(5～20℃)或较高温介质(45℃)对脉冲磁场的灭活作用具有促进作用,酸性(4.5～6.5)或碱性介质(7.5～8.0)也促进了脉冲磁场的灭活作用,介质水分活度越低,灭活作用越强。枯草芽孢杆菌的残留率随着NaCl浓度(1.2%～1.8%)、乙醇浓度(15%～45%)的升高而降低,随着葡萄糖(6%～10%)、乳糖(3%～6%)和蛋白胨含量(2%～6%)的增大而上升。(3)加热(50～100℃)、超声(200W,5-30min)、nisin (100～350IU/mL)处理联合3.0T,30个脉冲数的脉冲磁场灭活枯草芽孢杆菌比单一处理方法灭活效果要好。且先脉冲磁场处理再加热、超声、nisin处理的灭活效果比先加热、超声、nisin处理后再脉冲磁场处理的灭活效果要好。联合灭活后,枯草芽孢杆菌的形态发生改变,细胞产生萎缩现象。(4)以DPH作为探针,在35℃标记45min后采用荧光偏振法测定枯草芽孢杆菌细胞膜的荧光偏振度和荧光各异向性的变化,结果发现脉冲磁场提高了细胞膜的荧光偏振度和荧光各异向性,枯草芽孢杆菌的细胞膜流动性下降,而细胞膜流动性的下降会引起细胞膜功能的丧失,这可能是导致细胞死亡的原因之一。而以碘化丙啶为染料,用流式细胞仪测定细胞膜的渗透性时发现脉冲磁场提高了细胞膜的渗透性,膜渗透性的增加也可能导致细胞失活。(5) Fura-2/AM探针可成功的负载于枯草芽孢杆菌细胞中,经脉冲磁场处理后,荧光强度增加；荧光比例法测定细胞[Ca2+]i后发现[Ca2+]i随着磁场强度和脉冲数的增加而增加；随着细胞[Ca2+]i的不断增加,细胞膜的渗透性也不断增加,细胞膜的破坏和细胞[Ca2+]i的超载可能是细胞最终死亡的原因之一。(6)扫描电镜和透射的观察结果显示脉冲磁场使枯草芽孢杆菌发生变形,破坏了细胞的细胞壁和细胞膜,同时使细胞胞内物质出现缩合现象；260nm和280nm处的紫外吸光值随着强度和脉冲数的增加不断增大,说明核酸、蛋白等胞内物质在脉冲磁场作用下出现了外泄；经脉冲磁场作用后,ERIC-PCR指纹图谱上不同程度地出现了新的条带,说明脉冲磁场造成了DNA的断裂；枯草芽孢杆菌胞内的游离氨基酸总量经3.0T,30个脉冲数的脉冲磁场处理后增加,这可能是因为脉冲磁场使胞内蛋白发生了降解。胞内物质的外泄、DNA的断裂、蛋白质的降解等都可能导致细胞死亡。(7)采用蛋白质组学方法,比较未经脉冲磁场处理和经3.3T,30个脉冲处理的枯草芽孢杆菌的蛋白质组,发现共有34个差异蛋白点,其中质的差异有9个(在处理组中消失或特有),量的差异有25个；膜蛋白的破坏再次证明脉冲磁场破坏了细胞膜结构；对成功鉴定的19个蛋白进行GO分析和KEGG分析后得知蛋白信息分为细胞定位、分子功能、生物过程三大类,有8个蛋白有pathway信息,共参与16个pathway;脉冲磁场对枯草芽孢杆菌的有机物代谢和能量代谢产生影响；脉冲磁场导致枯草芽孢杆菌某些生理学功能的丧失或改变,这可能是细胞死亡的又一大原因。更多还原

【Abstract】 The increasing demand for higher quality products where a thermal process is hardly adoptable has significantly intensified with the improvement of living standard. The pulsed magnetic field (PMF) is a non-thermal method that is capable of inactivating microorganisms at lower temperatures and preserves the sensory and nutritional quality of the fresh-like food products, and is being one of research focuses in the field of food sterilization. This paper aims to study the effects on inactivation and on the cell structure and function of Bacillus subtilis by PMF. The mechanisms about PMF inactivation were also discussed preliminarily. The main conclusions were as follow:(1) Bacillus subtilis was most sensitive to the PMF at logarithmic phase. The survival rate of B. subtilis generally decreased with increasing intensity (>1.5T) and pulse numbers (>15). However, lower intensity (≤.1.5T) and pulse numbers (≤15) of PMF enhanced bacterial growth. A minimum survival rate of33.87%was obtained at an intensity of3.3T and30pulses with10h culture time of B. subtilis. Compared with the Bigelow and Hiilsheger models, the Weibull distribution model provided the best fit for B. subtilis inactivation data in relation to PMF treatments with the highest R2being observed at0.967.(2) Lower (5～20℃) and higher (45℃) temperature, and acidic (pH4.5～6.5) or alkaline (pH7.5～8.0) medium all increased the inactivation influence of PMF. The lower water activity, the stronger inactivation effect on B. subtilis. The survival rate decreased with increasing of NaCl (1.2%～1.8%) and ethanol concentration (15%～45%), the higher of concentration, the better effect of inactivation. The survival rate increased with increasing of glucose (6%～10%), lactose (3%～6%) and peptonein (2%～6%) content, the higher of concentration, the worse of inactivation.(3) The effect of PMF with the intensity of3.0T and30pulses combined with heat(50～100℃), ultrasonic(200W,5～30min), and nisin(100～350IU/mL) on the inactivation of B. subtilis was better than the effect of PMF used alone. The inactivation effect was better when B. subtilis were treated by PMF followed by heat, ultrasonic, and nisin than treated by heat, ultrasonic, and nisin followed by PMF. The morphology of B. subtilis changed and the cells shrunk after the combined treatments.(4)1,6-diphenyl-1,3,5-hexatriene (DPH) was used as fluorescence probe to label the membrane of B. Subtilis at35℃for45min to determine the changes of fluorescence polarization and fluorescence anisotropy of B. Subtilis membrane by fluorescence polarization technique. The results indicated that fluorescence polarization and fluorescence anisotropy increased after PMF treatment, meaning that membrane fluidity decreased which would result in the lost of the membrane function. This was one of the perhaps mechanisms for the cell death. The membrane permeability evaluated by propidium iodide staining and flow cytometry measurement enhanced after PMF treatment, which also suggested one of the reasons for cell inactivation.(5) Fura-2acetoxymethyl ester (Fura-2/AM), a ratiometric indicator was loaded in the cell of B. subtilis successfully. Fluorescence intensity improved when cell was treated by PMF. Intracellular free Ca2+ions assessed by ratiometric methods increased with increasing of the intenstiy and pulse numbers. The membrane permeability of cell also increased with increasing of [Ca2+]i The destruction of the cell membrane and [Ca2+]i overload in cell were perhaps the reasons for the cell death.(6) The SEM and TEM observation indicated that PMF caused a deformed morphology and damaged cell wall and membrane with the condensation of cell intracellular material. OD260and OD280values increased continuously with increasing of the intensity and pulsed numbers, suggesting the leakage of intracellular contents such as DNA and proteins. ERIC-PCR fingerprint showed DNA had been fragmented in different degree after PMF treatment. The total contents of free acids in cell increased after PMF treatment at the intensity of3.3T with30pulses, which implied that PMF treatment degradated the cell proteins. The leakage of intracellular conten, the fracture of DNA and the degradation of protein might result in the cell death.(7) Proteomic method was used for compare the protein profile of B. subtilis PMF-treated (3.3T with30pulses) and PMF-untreated. It was found that34proteins expressed differentially, including9proteins with qualitative difference(disappeared or appreared in PMF-treated samples) and25proteins with quantity difference. The destruction of the membrane protein proved that PMF destroyed the structure of the cell membrane again. GO and KEGG pathway analysis were employed to analyzed the different proteins.19proteins identified by mass spectrometry successfully were classed as cellular component, molecular function and biological process.8proteins had pathway information and participated in18pathways. Organic and energy metabolism of B. subtilis were effected by PMF and the loss or change of some physiology function might be one of the causes leading to the cell death.更多还原