壳聚糖对细菌细胞膜及膜蛋白的作用

研究壳聚糖对受试菌株大肠杆菌(E.coli)和金黄色葡萄球菌(St. aureus)的抑制作用;通过测定壳聚糖作用前后菌液的相对电导率变化和以1-N- 苯萘胺为荧光探针荧光强度的变化,分别考察壳聚糖对E.coli 和St. aureus 细胞膜和E.coli 外膜渗透性的影响;运用荧光分光光度计研究壳聚糖对E.coli 细胞膜中色氨酸(Trp)荧光强度的影响。结果表明,壳聚糖具有很好的抑菌性能,能改变细菌细胞膜的渗透性,从而破坏细胞膜。壳聚糖对细胞膜中Trp 的荧光具有明显的猝灭作用,且对细胞膜蛋白的猝灭作用 属于静态猝灭。     

壳聚糖与表面活性剂复合物的抑菌活性及抑菌机理研究

本文对壳聚糖与表面活性剂十二烷基磺酸钠（SDS）、十六烷基三甲溴化铵（CTAB）、曲拉通（TritonX-100）的复合物对大肠杆菌和金黄色葡萄球菌的抑菌活性进行了研究。结果表明：壳聚糖与CTAB、TritonX-100复合物对大肠杆菌抑菌活性强干壳聚糖,合成的三种复合物对金黄色葡萄球菌抑菌活性强于壳聚糖。其中壳聚糖-CTAB复合物抑菌活性最强,且织物经该复合物的整理后,也具有了较强的抗菌作用,故选择壳聚糖-CTAB的复合物考察抑菌机理。通过测定壳聚糖-CTAB作用后菌液中O．D260nm和0．D280nm、细胞膜蛋白荧光强度的变化,分别研究细菌细胞膜的完整性和细胞膜蛋白结构的变化。结果发现,壳聚糖-CTAB影响了细胞膜蛋白结构,使细胞膜破坏,从而达到抑菌作用。     

壳聚糖对革兰氏阴性菌抑菌机理的初步研究

体外抑菌法研究壳聚糖对绿脓杆菌、奇异变形杆菌和大肠杆菌的抑菌性能。同时,对细菌表面的亲水性和负电荷进行研究,以阐述革兰氏阴性菌表面性质与壳聚糖对其抑菌性能之间的关系。以大肠杆菌作为代表性菌株,考察了壳聚糖对革兰氏阴性菌的抑菌机理。通过测定菌液中加入疏水性荧光探剂1-N-苯萘胺(NPN)后荧光强度的改变,考察壳聚糖对外膜渗透性的影响。此外,采用红外吸收光谱(FT-IR),对壳聚糖和大肠杆菌的作用产物进行表征。结果表明：革兰氏阴性菌表面的亲水性越好,所带的负电荷越多,壳聚糖表现出较好的抑菌性能;壳聚糖可增加细胞外膜的渗透性,壳聚糖与细胞膜间发生静电作用而使细胞膜破坏,最终导致菌体的死亡。     

壳聚糖对金黄色葡萄球菌抑菌机理的研究

体外抑菌法研究了壳聚糖分子量、脱乙酰度以及pH值对金黄色葡萄球菌(St.aureus)抑制作用的影响。通过测定壳聚糖作用前后菌液中碱性磷酸酶、β-糖苷酶活性和核酸、蛋白质含量,研究了细胞内容物的泄露以及细胞膜完整性;运用透射电子显微镜和激光共聚焦荧光显微镜从超微结构探讨了壳聚糖对St.aureus的作用位点。结果发现细胞膜是壳聚糖对St.aureus作用的位点,壳聚糖改变了细胞膜的渗透性而使细胞膜破坏,伴随大量细胞内容物泄露。     

新型广谱米酒乳杆菌素对金黄色葡萄球菌的抑菌作用研究

测定了由米酒乳杆菌C2产生的广谱细菌素对金黄色葡萄球菌的最小抑菌浓度和杀菌浓度,以金黄色葡萄球菌ATCC63589为模式菌株,过细菌素对细胞渗透性影响及采用原子力显微镜研究了这种细菌素的作用机理.结果表明,种细菌素对金黄色葡萄球菌的最小抑菌浓度和杀菌浓度分别为20AU/mL和80AU/mL.同时杀菌浓度的细菌素引起了胞内紫外吸收物质的大量泄露,引起细胞表面的严重皱折和破裂.因此,推断杀菌浓度下细菌素导致了金黄色葡萄球菌ATCC63589细胞壁和细胞膜的破坏,起胞内物质的泄露,终导致细胞的死亡.     

不同分子量壳聚糖对细胞膜通透性的影响

研究了三种分子量壳聚糖对大肠杆菌和金黄色葡萄球菌的最低抑菌浓度,通过对壳聚糖处理后菌悬液电导率,紫外吸收物质和β-半乳糖苷酶活性的检测,探讨壳聚糖对细菌细胞膜通透性的影响。结果表明,不同分子量壳聚糖对E.coil和S.aureus细胞膜通透性均有不同程度的影响,影响能力依次为CS1>CS2>CS3。     

枯草芽孢杆菌细菌素A32的抑菌机理研究

将酸沉淀法提取的枯草芽孢杆菌细菌素A32作用于大肠杆菌和金黄色葡萄球菌,利用牛津杯法、分光光度法、扫描电镜技术和电导率法、SDS-PAGE电泳法探讨细菌素A32的抑菌活性和抑菌机理。结果显示,细菌素A32对大肠杆菌和金黄色葡萄球菌均具有抑菌性,最小抑菌质量浓度为1.25 mg/mL;对指示菌的生长曲线、细胞形态结构、细胞膜渗透性、细胞膜通透性、细胞膜和细胞壁完整性、细胞中蛋白质的合成均有影响;且随着细菌素A32质量浓度的增大,对指示菌的影响也逐渐增大。以上现象表明,细菌素A32的抑菌性主要是通过破坏革兰氏阴、阳性菌壁膜的通透性和完整性,使内容物外漏,进而影响菌体代谢和生长。孔道形成理论是细菌素A32对革兰氏阴、阳性菌的作用机理。研究结果为细菌素A32作为绿色生物防腐剂的开发应用奠定了理论基础。     

二氢杨梅素对金黄色葡萄球菌细胞渗透性的影响研究

以金黄色葡萄球菌为供试菌,通过测量培养液电导率以及谷丙转氨酶、谷草转氨酶、碱性磷酸酶的酶活,研究二氢杨梅素对细胞渗透性的影响。试验结果表明:二氢杨梅素对电导率、谷草转氨酶和谷丙转氨酶均有一定的影响,但对碱性磷酸酶影响很小,说明二氢杨梅素的加入,能引起金黄色葡萄球菌细胞膜渗透性的变化,导致菌体内离子等电解质外泄,但影响有限,不能使细胞膜完全丧失选择渗透性,推测细胞膜可能为其作用靶点之一,具体作用方式还需要进一步研究。     

山茶油抑菌性能和机理的研究

以大肠杆菌和金黄色葡萄球菌为实验菌,研究了山茶油的抑菌性能和抑菌机理。通过测定山茶油的最低抑菌浓度、菌生长曲线的影响来评定山茶油的抑菌性能;通过测定菌液中碱性磷酸酶、电导率、还原糖和蛋白质含量的变化,从菌体细胞膜和细胞壁的损伤等方面来阐述山茶油的抑菌机理。结果表明:山茶油对大肠杆菌和金黄色葡萄球菌的最低抑菌浓度(MIC)为0.5%和1%,山茶油对大肠杆菌的抑制效果强于金黄色葡萄球菌;山茶油可使大肠杆菌和金黄色葡萄球菌细胞壁和细胞膜受到损伤,并引起菌体细胞溶出物、蛋白质以及离子的泄露,从而导致菌体的死亡。     

几种壳聚糖的抑菌性能

通过壳聚糖的抑菌实验和几种壳聚糖的最低抑菌浓度的测定 ,比较了相同脱乙酰度不同分子量 ,以及分子量相近脱乙酰度不同的壳聚糖对金黄色葡萄球菌、枯草杆菌、大肠杆菌和假单胞菌的抑菌作用。结果表明 ,实验中用到的壳聚糖都对金黄色葡萄球菌有很强的抑菌作用 (抑菌率接近 10 0 % ,最低抑菌浓度为 0 0 3% ) ;对于其他 3种细菌 ,脱乙酰度相同 (为 75 3%或 93 7% ) ,粘均分子量不同 (在 4 0～ 80万之间 )的壳聚糖 ,抑菌作用随分子量的升高而增强 ;而分子量相近脱乙酰度不同的壳聚糖对上述 4种细菌的抑菌作用差别不大 ;在 pH 5 5左右至 pH 6 0左右壳聚糖能够发挥最强的抑菌作用 ;总体看来 ,壳聚糖对金黄色葡萄球菌的抑菌作用最强 ,其次是对假单胞菌和枯草杆菌 ,壳聚糖对大肠杆菌的抑菌作用相对弱一些 ;实验条件下的壳聚糖对上述 4种细菌的抑菌作用普遍比苯甲酸钠强。     

Chitosan kills bacteria through cell membrane damage

The bactericidal activity of chitosan (CS) acetate solution against Escherichia coli and Staphylococcus aureus was evaluated by the enumeration of viable organisms at different incubation times. Morphologies of bacteria treated with CS were observed by transmission electron microscopy (TEM). The integrity of the cell membranes of both species and the permeabilities of the outer membrane (OM) and inner membrane (IM) of E. coli were investigated by determining the release from cells of materials that absorb at 260 nm, changes in the fluorescence of cells treated with the fluorescent probe 1-N-phenylnaphthylamine (NPN) and release of cytoplasmic beta-galactosidase activity. In addition, the interaction of CS with synthetic phospholipid membranes was studied using gel permeation chromatography (GPC), UV-VIS spectrophotometery, Fourier-transform infrared spectroscopy (FT-IR) and thermal analysis. Results showed that CS increased the permeability of the OM and IM and ultimately disrupted bacterial cell membranes, with the release of cellular contents. This damage was likely caused by the electrostatic interaction between NH(3)(+) groups of CS acetate and phosphoryl groups of phospholipid components of cell membranes.     

Chitosan disrupts the barrier properties of the outer membrane of gram-negative bacteria.

The mode of antimicrobial action of chitosan (polymeric beta-1,4-N-acetylglucosamine) on gram-negative bacteria was studied with special emphasis on its ability to bind to and weaken the barrier function of the outer membrane (OM). Chitosan (250 ppm) at pH 5.3 induced significant uptake of the hydrophobic probe 1-N-phenylnaphthylamine (NPN) in Escherichia coli, Pseudomonas aeruginosa and Salmonella typhimurium. The effect was reduced (E. coli, salmonellae) or abolished (P. aeruginosa) by MgCl2. No NPN uptake was observed during exposure of the salmonellae to chitosan at pH 7.2. Chitosan also sensitized P. aeruginosa and the salmonellae to the lytic effect of sodium dodecyl sulfate (SDS); such sensitization was not blocked by MgCl2 and was reversible by washing chitosan-treated cells prior to SDS exposure. Chemical and electrophoretic analyses of cell-free supernatants of chitosan-treated cell suspensions showed that interaction of chitosan with E. coli and the salmonellae involved no release of lipopolysaccharide (LPS) or other membrane lipids. However, chitosan rendered E. coli more sensitive to the inhibitory action of dyes and bile acids used in selective media. Highly cationic mutants of S. typhimurium were more resistant to chitosan than the parent strains. Electron microscopy showed that chitosan caused extensive cell surface alterations and covered the OM with vesicular structures. Chitosan thus appeared to bind to the outer membrane, explaining the loss of the barrier function. This property makes chitosan a potentially useful indirect antimicrobial for food protection.     

副干酪乳杆菌Z17-壳聚糖复配对大肠杆菌O157:H7的抑菌效果及作用机制

目的：研究副干酪乳杆菌Z17-壳聚糖复配对草莓中大肠杆菌O157:H7抑菌活性及作用机制。方法：采用流式细胞术、傅里叶变换红外光谱、拉曼光谱及扫描电子显微镜技术分析副干酪乳杆菌Z17-壳聚糖对大肠杆菌O157:H7细胞膜的影响。结果：质量分数1.0%壳聚糖溶液与副干酪乳杆菌Z17复配处理能有效去除草莓上的大肠杆菌O157:H7,减菌率达99%;壳聚糖溶液与副干酪乳杆菌Z17共同作用3 h使大肠杆菌O157:H7 DNA胞外释放量达（381.00±3.53）ng/μL,细胞膜破损率为58.3%;细胞壁膜中脂肪酸、蛋白、肽聚糖、糖苷环、多糖结构成分被破坏;细胞膜局部位移变薄,大分子物质黏附于菌体细胞表面,细胞表面出现孔洞,胞内物质泄漏,最终导致菌体死亡。结论：副干酪乳杆菌Z17-壳聚糖能够有效地抑制草莓中大肠杆菌O157:H7,其抑菌作用靶点为大肠杆菌O157:H7的细胞膜,研究可为大肠杆菌O157:H7的生物防治提供参考。     

Mechanism of action of Spanish oregano, Chinese cinnamon, and savory essential oils against cell membranes and walls of Escherichia coli O157:H7 and Listeria monocytogenes

The mechanism of the antimicrobial action of Spanish oregano (Corydothymus capitatus), Chinese cinnamon (Cinnamomum cassia), and savory (Satureja montana) essential oils against cell membranes and walls of bacteria was studied by the measurement of the intracellular pH and ATP concentration, the release of cell constituents, and the electronic microscopy observations of the cells when these essential oils at their MICs were in contact with Escherichia coli O157:H7 and Listeria monocytogenes. E. coli O157:H7 and L. monocytogenes, two pathogenic foodborne bacteria, were used as gram-negative and gram-positive bacterial models, respectively. Treatment with these essential oils at their MICs affected the membrane integrity of bacteria and induced depletion of the intracellular ATP concentration. Spanish oregano and savory essential oils, however, induced more depletion than Chinese cinnamon oil. An increase of the extracellular ATP concentration was observed only when Spanish oregano and savory oils were in contact with E. coli O157:H7 and L. monocytogenes. Also, a significantly higher (P < or = 0.05) cell constituent release was observed in the supernatant when E. coli O157:H7 and L. monocytogenes cells were treated with Chinese cinnamon and Spanish oregano oils. Chinese cinnamon oil was more effective to reduce significantly the intracellular pH of E. coli O157:H7, whereas Chinese cinnamon and Spanish oregano decreased more significantly the intracellular pH of L. monocytogenes. Electronic microscopy observations revealed that the cell membrane of both treated bacteria was significantly damaged. These results suggest that the cytoplasmic membrane is involved in the toxic action of essential oils.     

Antibacterial activity of N-alkylated disaccharide chitosan derivatives

Antibacterial activity of the water-soluble N-alkylated disaccharide chitosan derivatives against Escherichia coli and Staphylococcus aureus was investigated. It was found that the antibacterial activity of chitosan derivatives was affected by the degree of substitution (DS) with disaccharide and the kind of disaccharide present in the molecule. Regardless the kind of disaccharide linked to the chitosan molecule, a DS of 30-40%, in general, exhibited the most pronounced antibacterial activity against both test organisms. E. coli and S. aureus were most susceptible to cellobiose chitosan derivative DS 30-40% and maltose chitosan derivative DS 30-40%, respectively, among the various chitosan derivatives examined. Although the disaccharide chitosan derivatives showed less antibacterial activity than the native chitosan at pH 6.0, the derivatives exhibited a higher activity than native chitosan at pH 7.0. Antibacterial activity of the chitosan derivatives (DS 30-40%) against E. coli increased as the pH increased from 5.0 and reached a maximum around the pH of 7.0-7.5. The effect of pH on the antibacterial activity of chitosan derivatives against S. aureus was not as pronounced as that observed with E. coli. Population reduction of E. coli or S. aureus in nutrient broth increased markedly upon increasing the concentration of chitosan derivatives from 0 to 500 ppm. No marked increase in population reduction was noted with further increase in the concentration of chitosan derivatives even up to 2000 ppm.     

Impact of sodium chloride on Escherichia coli O157:H7 and Staphylococcus aureus analysed using transmission electron microscopy

Abundant literature information is available on sodium chloride, NaCl, as an antimicrobial and a preservative, however, information on NaCl effects on bacterial cell morphology is lacking. The effect of NaCl, on Escherichia coli O157:H7 and Staphylococcus aureus cells individually grown in a laboratory medium was examined using transmission electron microscopy (TEM). Cultures were grown in brain heart infusion (BHI) broth containing dissolved 0%, 5%, or 10% (w/v) commercially obtained fine (FN) and extra coarse (EC) grade granular NaCl. The pathogens were incubated at 35 degrees C for 12 and 24 h. Then, a mixture of five strains of each pathogen per treatment was prepared. Samples were centrifuged, pellets collected, fixed immediately with glutaraldehyde, and prepared for TEM examination. Cells morphology on TEM micrographs verified that the magnitude of morphological damage to E. coli O157:H7 cells was significantly greater than that of S. aureus cells. More cell injury occurred as NaCl concentration increased from 5% to 10%. Generally, S. aureus maintained its cellular structure and no severe cell wall or plasma membrane damage and/or shrinkage was observed. At 10% NaCl, the damage to E. coli O157:H7 cells was extensive, and the pathogen seemed to have lost its cellular integrity. Although NaCl affected the morphology of E. coli O157:H7 and S. aureus, the coarse grade of NaCl seemed to have a milder effect with respect to cell damage, especially on S. aureus. The 24 h-old cultures were more susceptible to NaCl treatment compared to the 12 h-old cells. Thus, the age of the cells has an impact on their resistance to salt--the environmental stressor.     

Effect of slightly acidic electrolyzed water for inactivating Escherichia coli O157:H7 and Staphylococcus aureus analyzed by transmission electron microscopy

The use of different available chlorine concentrations (ACCs) of slightly acidic electrolyzed water (SAEW; 0.5 to 30 mg/liter), different treatment times, and different temperatures for inactivating Escherichia coli O157:H7 and Staphylococcus aureus was evaluated. The morphology of both pathogens also was analyzed with transmission electron microscopy. A 3-min treatment with SAEW (pH 6.0 to 6.5) at ACCs of 2 mg/liter for E. coli O157:H7 and 8 mg/liter for S. aureus resulted in 100% inactivation of two cultures (7.92- to 8.75-log reduction) at 25°C. The bactericidal activity of SAEW was independent of the treatment time and temperature at a higher ACC (P > 0.05). E. coli O157:H7 was much more sensitive than S. aureus to SAEW. The morphological damage to E. coli O157:H7 cells by SAEW was significantly greater than that to S. aureus cells. At an ACC as high as 30 mg/liter, E. coli O157:H7 cells were damaged, but S. aureus cells retained their structure and no cell wall damage or shrinkage was observed. SAEW with a near neutral pH may be a promising disinfectant for inactivation of foodborne pathogens.     

克氏螯虾壳聚糖对细菌表面作用机理研究

目的：研究克氏螯虾壳聚糖对细菌表面的作用机理。方法：将不同浓度克氏螯虾壳聚糖与大肠埃希菌及金黄色葡萄球菌作用3.5h,检测细菌上清液中草酰乙酸氨基转移酶活性、总蛋白及核酸含量;用透射电镜观察细菌与壳聚糖作用1h后的形态变化;将62.5mg/L壳聚糖与含十二烷基磺酸钠的菌液作用,观察溶菌效果。结果：克氏螯虾壳聚糖与细菌作用后上清液中草酰乙酸氨基转移酶活性、总蛋白和核酸含量显著高于对照组(p<0.01);透射电镜下发现两种细菌都有菌内物质泄漏及形态变化发生;62.5mg/L壳聚糖能增加十二烷基磺酸钠的溶菌作用 (p<0.01)。结论：克氏螯虾壳聚糖能破坏细菌的表面结构,影响屏障功能,使菌内物质泄漏,并能协同SDS对细菌的溶菌作用。