100kV/cm高压脉冲电场杀菌系统设计与试验

设计了100kV／cm高场强脉冲电场（PEF）杀菌系统，以荧光假单胞菌为试验靶细胞，经PEF作用180s，在透射电子显微镜（TEM）下观察处理前后的对比情况，发现经PEF处理过的菌体细胞壁发生了破坏，细胞膜上形成不可修复的穿孔，细胞内含物和细胞碎片泄漏，导致细胞质和细胞核物质的流失，致使细胞死亡。结果表明，所设计的PEF杀菌系统可用于液态食品的杀菌处理实验。     

热压结合处理对绿色魏斯氏菌生理特性的影响

为了探究热压结合处理在绿色魏斯氏菌致死过程中发挥的作用。本实验采用超高压(350 MPa、25℃、10 min)和热压结合(350 MPa、50℃、10 min)对绿色魏斯氏菌进行压力处理,研究了热压结合处理对绿色魏斯氏菌生理特性、细胞形态特征、细胞膜的通透性等的影响。结果显示,热压结合处理使菌落总数从超高压处理时的7.67(lg(CFU/mL))显著下降到5.71(lg(CFU/mL))(P0.05),协同的热处理使菌体表面出现严重褶皱、菌体内含物变性凝固加剧,使细胞膜损伤,从而改变膜的通透性,导致细胞外紫外吸收物质显著增加(P0.05)。因此,热压结合处理在超高压致死细菌中会加剧细胞膜的形变及损坏,增加细胞膜的通透性,从而导致细胞内紫外吸收物质的流出,实验结果为热压结合灭活绿色魏斯氏菌提供了理论依据。     

DMDC对假单胞菌浮游菌和被膜菌的杀菌效果及其应用

为探究二甲基二碳酸盐(DMDC)对食源性有害微生物的控制,本研究分析DMDC对生鲜食品特定腐败菌荧光假单胞菌和隆德假单胞菌的浮游菌和被膜菌的杀菌效果.通过API ZYM试剂盒、XTT检测试剂盒、共聚焦显微镜观察分析DMDC处理对假单胞菌存活性、胞内酶活、成熟生物被膜的影响,并评价该处理对生鲜蔬菜品质的影响.结果表明:与...     

超声结合热处理对金黄色葡萄球菌及其生物膜的抗菌和抗生物膜机制

将超声（ultrasound,US）与热处理（heat,HT）相结合（UH）,研究其对金黄色葡萄球菌游离细胞和生物膜细胞的杀菌效果,并分析其抗菌和抗生物膜机制。结果表明：US联合70 ℃ HT（UH70）可显著降低金黄色葡萄球菌游离细胞和生物膜细胞的数量（P＜0.05）;扫描电子显微镜结果表明,UH70对菌体细胞的细胞膜完整性具有明显的破坏作用,激光共聚焦扫描显微镜显示,UH70导致细胞的通透性急剧增加,从而导致胞外ATP、核酸和蛋白质含量显著增加（P＜0.05）;此外,UH70能引起细胞内呼吸链脱氢酶活性显著降低,引起生物膜胞外多糖含量显著降低（P＜0.05）。综上所述,US结合HT与单独的US或HT相比具有较强的杀菌效果,它可以通过破坏金黄色葡萄球菌菌体细胞的细胞膜完整性,引起细胞膜通透性的改变,造成ATP、核酸、蛋白质外泄,降低细菌呼吸链脱氢酶的活性,达到协同杀菌目的。     

脉冲强光对黑曲霉细胞膜损伤及胞内酶活性的影响

通过脉冲强光处理黑曲霉菌悬液,研究菌体细胞膜通透性的变化以及胞内酶活性的变化。结果表明：经脉冲强光处理后菌悬液的蛋白质溢出量、丙二醛的漏出量与对照相比均有显著增加,透射电子显微镜观察到细胞膜和细胞壁表面凹凸不平,出现褶皱。此外,脱氢酶活力与另外胞内3种主要抗氧化酶(超氧化物歧化酶、过氧化氢酶、谷胱甘肽过氧化物酶)活力也都有不同程度的下降。因此,霉菌屏障结构破坏所造成的细胞膜渗透压和通透性的变化可使霉菌胞内物质发生改变,甚至导致菌体死亡。     

苍术挥发油杀菌活性评价及抑菌机制

研究苍术挥发油体外杀菌活性及抑菌机制。以大肠埃希氏菌、金黄色葡萄球菌、白假丝酵母为供试菌,采用悬液定量杀菌实验测定苍术挥发油的杀菌活性,并测定对3种供试菌胞外碱性磷酸酶（AKP）、可溶性蛋白质含量和生长曲线的影响,从而阐述苍术挥发油的杀菌机制。结果表明,苍术挥发油对3种常见致病菌均有显著的抑制或杀灭作用。3种致病菌悬液中AKP、可溶性蛋白质等大分子物质含量比对照组显著增加,经苍术挥发油处理过的3种菌生长受到明显抑制。苍术挥发油能够通过改变菌体细胞膜通透性,破坏菌体完整结构发挥体外杀菌活性。     

热压结合处理对低温火腿中耐压腐败菌的抑制作用

本文主要探究热压结合处理(50 ℃,350 MPa保压10 min)对低温火腿中绿色魏斯菌和肠膜明串珠菌的抑制作用。通过薄层平板计数法对热压结合处理的杀菌效果进行判断,使用流式细胞技术判断细胞的生理状态,通过扫描电镜(scanning electron microscope,SEM)和透射电镜(transmission electron microscope,TEM)观察细胞形态结构的变化,利用紫外分光光度法测定热压结合处理对细菌细胞膜通透性的影响。结果表明:经热压结合处理后,绿色魏斯菌和肠膜明串珠菌的存活率分别为-4.74和-6.61lg(CFU/mL),其生长变得迟缓,对数期生长速率降低。扫描电镜结果显示热压结合处理对两种菌的形态造成明显的变形,透射电镜结果显示细胞内部出现团状聚集和透电子区。热压结合处理对细胞膜产生损伤,细胞膜通透性变大,导致细胞外紫外吸收物质也极显著增加(p<0.001),细胞处于亚致死状态。热压结合处理会对细胞膜产生一定的形变及损坏,使得细胞内核酸和蛋白质流出,同时细胞内部也会发生蛋白变性和细胞质皱缩,达到抑制耐压菌生长繁殖的目的。     

乳酸链球菌素(Nisin)与超高压结合对E.coli的协同杀菌效应

本文探究了乳酸链球菌素(Nisin)与超高压(High pressure processing,HPP)结合处理对E.coli O157:H7产生的协同杀菌效果,并分析其协同作用机制。以革兰氏阴性菌E.coli为目标菌,用0.005%或0.01% Nisin结合350 MPa/2 min超高压处理,通过扫描电镜和透射电镜分别观察E.coli细胞外部形态与内部结构、激光共聚焦显微镜与流式细胞仪检测细胞膜通透性。结果表明,与单独超高压处理相比,0.005%或0.01% Nisin结合350 MPa/2 min超高压对E.coli具有明显协同杀菌效果,E.coli菌落数下降6~8个数量级,细胞外形变化不明显,但细胞膜和内部结构受到了更严重的破坏,细胞膜完整的细菌仅占总数的2.13%~5.68%,细胞发生更多内溶物外泄和出现更多的空腔。因此,Nisin和HPP存在协同杀菌机制,推测HPP首先破坏E.coli细胞外膜,Nisin作用于外膜受损的细胞进而破坏细胞膜,细胞内溶物外泄形成空腔,从而导致细胞死亡。     

抗菌肽Jelleine-I对采后柑橘果实绿霉病的控制作用

研究抗菌肽Jelleine-I对指状青霉（Penicillium digitatum）生长的影响及可能的作用机理,并采用损伤接种的方法评价Jelleine-I对柑橘果实采后绿霉病的控制效果。结果表明,Jelleine-I能明显抑制P. digitatum的生长,其最小抑菌浓度和最小杀菌浓度分别为6.25 mmol/L和12.5 mmol/L;同时,其能有效控制柑橘果实采后绿霉病的发生。经Jelleine-I处理后的P. digitatum菌丝体细胞膜通透性增加,细胞间隔膜损坏,细胞内绿色SYTOX Green荧光强度增加,细胞间蓝色Calcofluor White荧光减少。此外,经Jelleine-I处理后的P. digitatum菌丝体胞外电导率以及胞内核酸物质泄漏量均显著增加（P＜0.05）。综上,Jelleine-I通过破坏P. digitatum细胞膜结构完整性,引起胞内物质泄漏,加速P. digitatum死亡,从而有效控制柑橘果实采后绿霉病的发生。     

酚酸对水产品腐败希瓦氏菌的抑菌作用

比较了没食子酸、原儿茶酸和绿原酸3种酚酸对腐败希瓦氏菌的抑制活性,并通过扫描电镜、流式细胞仪、细胞膜完整性和通透性测定等探讨了没食子酸对腐败希瓦氏菌的抑菌作用。结果表明:3种酚酸中没食子酸对腐败希瓦氏菌的抑制活性最强,其最小抑菌浓度和最小杀菌浓度分别为1.25,2.50mg/mL。经最小抑菌浓度没食子酸处理后,腐败希瓦氏菌细胞形态改变,表面有明显凹陷;细胞凋亡率明显增高;核酸和蛋白泄漏增加,细胞膜完整性遭到破坏;细胞膜通透性增加,胞内电解质大量泄漏至培养液中。     

二甲基二碳酸盐联合动态超高压对模拟果汁的杀菌效果研究

本文研究了二甲基二碳酸盐（DMDC）联合动态超高压对模拟果汁中三种挑战污染菌（大肠杆菌、酿酒酵母、肠膜状明串珠菌）的杀菌效果。结果表明：单独动态超高压对三种挑战污染菌的杀菌效果随着压力增大而显著增强（p〈0.05）,其中在相同处理压力下,对大肠杆菌的杀菌最好,但即使在100 MPa的压力处理下,大肠杆菌也仅下降1.2个对数;向含挑战污染菌的模拟果汁（25 ℃）中添加250 mg/L的DMDC 2 h后,大肠杆菌和酿酒酵母均被完全杀灭,而肠膜状明串珠菌对DMDC具有很强的耐受性,仅下降了1.7个对数,且继续延长DMDC的处理时间,其数量下降也不再明显;当DMDC（250 mg/L）与动态超高压联合处理时,它们对模拟果汁中的肠膜状明串珠菌的杀菌作用具有明显的协同增效效果（p〈0.05）。另外,DMDC与动态超高压先后处理次序对肠膜状明串珠菌的杀菌效果也有影响。     

Combined Effect of Dimethyl Dicarbonate (DMDC) and Nisin on Indigenous Microorganisms of Litchi Juice and its Microbial shelf life

The individual and combined influences of dimethyl dicarbonate (DMDC) and nisin (200 IU/mL) at mild heat on the inactivation of indigenous microorganisms in litchi juice, including bacteria, molds and yeasts (M&Y), were investigated. The fresh litchi juice with or without nisin were exposed to 250 mg/L DMDC at 30, 40, or 45 °C for 0.5, 1, 2, 3, 4, or 6 h. A complete inactivation of M&Y in the litchi juice with or without nisin was achieved as exposed to 250 mg/L DMDC at 30, 40, or 45 °C for 0.5 h. The bacteria, especially Bacillus sp. and Leuconstoc mesenteroides showed higher resistance than M&Y in the litchi juice. Bacillus sp. and Leuconstoc mesenteroides in the litchi juice was not completely inactivated by 250 mg/L DMDC at 30, 40, or 45 °C. However, nisin addition can enhanced the inactivation of these bacteria by DMDC, and nisin and DMDC also showed a synergistic effect on the inactivation of bacteria. M&Y and bacteria were not detected in the litchi juice added with 200 IU/mL nisin as exposed to 250 mg/L DMDC at 45 °C for 3 h. In addition, microbial shelf life of the litchi juice during storage at 4 °C also was evaluated as treated by 250 mg/L DMDC or combination with nisin at 45 °C for 3 h.     

Synergetic effects of high-pressure carbon dioxide and nisin on the inactivation of Escherichia coli and Staphylococcus aureus

Synergetic effects of high-pressure carbon dioxide (HPCD) and nisin on Escherichia coli and Staphylococcus aureus were evaluated. Changes in morphology, interior structure, and membrane permeability were analyzed by scanning and transmission electron microscopy, and flow cytometry. Synergetic effects were found, especially in S. aureus. HPCD alone or with nisin led to morphological and intracellular alterations in both bacteria, but nisin alone led to these damages only in S. aureus. A positive correlation between membrane damage and inactivation was found, but ratios of inactivation were higher, probably because of viable but non-culturable state. Mechanisms were proposed for synergism: for E. coli, outer membrane was damaged first by HPCD, and then HPCD and nisin jointly acted on and destroyed the cytoplasmic membrane, leading to further intracellular damage by HPCD; for S. aureus, HPCD and nisin acted on the cytoplasmic membrane together leading to cell death.Industrial RelevanceEscherichia coli and Staphylococcus aureus are two common microorganisms, which exist widely in the environment and easily contaminate food such as vegetables and dairy products, respectively. Considering heat treatment may destroy some heat-sensitive quality of the products, this study evaluated synergetic effects of high-pressure carbon dioxide (HPCD) combined with the bacteriocin nisin. The investigations provided evidence for potentially combined application of HPCD and nisin to help keep food safe in the industry.     

EFFECT OF PEDIOCIN DT10 ON LEUCONOSTOC MESENTEROIDES OZ-N3 CELLS

Pediococcus pentosaceous DT10, isolated from ready‐to‐eat fish products, produced a bacteriocin active against Gram‐positive bacteria, many of which are associated with food spoilage and food‐related health hazards. The bacteriocin, named pediocin DT10, was partially purified and was observed to kill sensitive Leuconostoc mesenteroides cells by acting on their cytoplasmic membrane. Exposure of cell suspensions of L. mesenteroides to pediocin DT10 produced cell‐viability loss, as shown by the reduction in colony‐forming units after treatment. The activity of pediocin DT10 against L. mesenteroides cells was bactericidal in nature, and also induced an important efflux of intracellular material. The transmission electron microscopy of ultrathin sections of L. mesenteroides cells confirmed the way pediocin DT10 causes lysis of the sensitive L. mesenteroides cells.     

Transmission electron microscopy of Listeria innocua treated by pulsed electric fields and nisin in skimmed milk

Pulsed electric fields (PEF) is a nonthermal food preservation process where organoleptic and nutritional properties of the food are maintained. PEF is known to inactivate microorganisms by causing dielectric breakdown of the cell membrane, thus altering the functionality of the membrane as a semipermeable barrier. The extent of damage of the cell membrane, whether visible in the form of a pore or as loss of membrane functionality leads to the inactivation of the microorganism. The objective of this study was to investigate under transmission electron microscopy (TEM) the morphological changes on Listerit innocua as a result of PEF treatment in skimmed milk containing nisin. L. innocua was subjected to PEF at selected electric field intensities of 30, 40, and 50 kV/cm. L. innocua was treated by PEF in both skimmed milk with and without 37 IU nisin/ml. L. innocua treated by PEF in skimmed milk exhibited an increase in the cell wall roughness. cytoplasmic clumping, leakage of cellular material, and rupture of the cell walls and cell membranes. L. innocua subjected to PEF in skimmed milk containing 37 IU nisin/ml exhibited an increased cell wall width. At the highest electric field intensity, 50 kV/cm, elongation of the cell length was observed. There were no morphological differences between cells treated by PEF in skimmed milk with or without nisin. The combination of PEF and nisin exhibit an additive effect in the morphological damage observed on L. innocua. Pore formation was observed on L. innocua for an electric field intensity of 40 kV/cm. The inactivation of L. innocua was a consequence of rupture of the cell membrane and loss of cell membrane functionality.     

Membrane damage and enzyme inactivation of Lactobacillus plantarum by high pressure CO2 treatment

Physiological changes of Lactobacillus plantarum (KFRI 815) by high pressure CO2 treatment were investigated to examine the relevance to microbial inactivation. Characteristic properties of the cells measured in this study included salt tolerance, release of UV-absorbing substances, Mg and K ions, proton permeability, glycolysis, H+-ATPase and constitutive enzymes, and dye uptake. The cells treated with high pressure CO2 of 7 MPa at 30 degrees C for 10 min showed the irreversible cellular membrane damages including loss of salt tolerance, leakage of UV-absorbing substances, release of intracellular ions, collapse of proton permeability and uptake of Phloxine B dye. L. plantarum cells after CO2 treatment also exhibited reduced glycolytic activity and inactivation of some constituent enzymes. However, H+-ATPase of the cell membrane maintained its initial specific activity of about 2.50 U/mg protein even though viability of the cells was reduced by several log cycles after high pressure CO2 treatment.     

Comparative Study on Antibacterial Effect of Pediocin ACCEL and Nisin Against Fluorescencestained Listeria monocytogenes BCRC 14845

ABSTRACT: Effects of pediocin ACCEL (Class IIa, pediocin-like bacteriocins) and nisin on carboxyfluorescein diacetate (cFDA) -stained Listeria monocytogenes BCRC 14845 was carried out. Decrease of fluorescence intensity in stained cells and increase in efflux were observed with the increase in bacteriocin concentrations up to 12.5 μg/mL. No further decreases in fluorescence intensity and cell numbers were obtained when the concentration of bacteriocins was over 12.5μg/mL. The leakage of efflux was further confirmed by the release of intracel-lular ultraviolet-absorbing substances. Comparing the leaking time of fluorescence, the intensity of cFDA in nisin-treated cells within 1 min incubation was almost comparable to that in pediocin-treated samples after 40 min incubation. Considering the leakage of cFDA, UV-absorbing substances, changes in cell numbers during incubation of cFDA-stained cells, and restoration ability, nisin seemed to reveal mainly bacteriostatic effect and partially bactericidal effect on L. monocytogenes and to cause easier-to-repair membrane damage. However, pediocin ACCEL appeared to have both bacteriostatic and bactericidal effects on L. monocytogenes and to cause more difficult-to-repair membrane damage.