壳聚糖金属配合物的抑菌特性及机理研究（英文）

制备了壳聚糖(CS)与金属离子Zn(II)、Ni(II)和Co(II)的配合物,通过红外光谱和紫外-可见吸收光谱进行了结构性能的表征。体外抑菌法研究了壳聚糖金属配合物对细菌S.aureu和E.coli的抑菌活性。结果表明:壳聚糖金属配合物的抑菌活性较壳聚糖增强,且与所含的金属离子种类有关,其中CS-Zn体现出更强的抑菌活性。因此,选CS-Zn为代表通过测定细胞内溶物的OD260nm判断细胞膜的完整性、荧光探针1-N-苯萘胺(NPN)的荧光变化来判断细胞外膜的渗透性,以研究其对大肠杆菌(E.coli.)的抑菌机理。透射电镜(TEM)结果表明CS-Zn能够破坏细菌细胞膜,使细胞内溶物溢出。     

来自穿膜肽的新肽的抗菌活性及抑菌机制

[目的]研究基于穿膜肽和抗菌肽构效关系改造获得的新肽P7的抗菌活性及其对大肠杆菌(E.coli)的抑菌机制.[方法]微量稀释法和溶血实验分析P7的抑菌活性及其对正常细胞的细胞毒性;采用膜荧光探针、流式细胞术和扫描电镜分析P7对E.coli膜通透性、膜完整性的影响和细胞超微结构变化;通过激光共聚焦分析P7在E.coli细胞中的定位;凝胶阻滞实验测定P7与E.coli基因组DNA结合能力.[结果]P7比母肽显示更强的抑菌活性,最低抑菌浓度范围为4-32 μmol/L,且在作用浓度范围内具有较弱的溶血活性.P7可以增加E.coli外膜和内膜的通透性,使E.coli细胞膜的完整性和细胞表面结构受损.同时P7可以穿过E.coli细胞膜在细胞质聚集并与基因组DNA结合.[结论]P7通过增加E.coli内外膜通透性,穿过细胞膜与胞内DNA结合发挥抑菌活性.     

肉桂不同植物部位精油成分分析及抑菌活性研究

本研究选用肉桂(Cinnamomum cassia)的桂皮、桂叶、桂枝、果实、花萼5种植物部位为研究对象,通过气质联用对各部位精油的主要成分组成进行了分析比较。在此基础上,从抑菌圈、最低抑菌浓度(MIC)、最低杀菌浓度(MBC)等方面比较各部位精油对金黄色葡萄球菌(Staphylococcus aureus)、大肠杆菌(Escherichia coli)抑制活性。结果表明,各部位精油中的主要成分均为反式肉桂醛、邻甲氧基肉桂醛等,但各成分的含量存在明显差异,以花萼中的反式肉桂醛含量最高(87.68%)。各部位精油对两种供试微生物均具有显著抑菌效果,以桂枝中所含精油的抑菌效果最佳,对E.coli和S.aureus的MIC分别为0.05%和0.025%,MBC同样也分别为0.05%和0.025%。     

茶多酚对大肠杆菌抑菌机理的研究

茶多酚(tea polyphenols,TP)是一种具有广谱抗菌作用的活性质,以大肠杆菌(Escherichia coli,E.coli)为研究对象,采用牛津杯实验研究茶多酚的最小抑菌浓度,并采用结晶紫实验、电导率测定、离子泄漏测定来分析TP对E.coli细胞膜通透性的影响。通过琼脂糖凝胶电泳分析TP对E.coli DNA的影响。结果显示,TP对E.coli的最小抑菌浓度为40μg/m L;结晶紫实验中,随着浓度的增大,OD590也随之增大;随着处理时间的增长,电导率随之增大,离子泄漏随之增多,说明TP可以作用于E.coli的细胞膜,能够改变其通透性。通过琼脂糖凝胶电泳分析可知TP处理后和空白对照组相比较,DNA条带出现变暗、拖尾现象,说明TP能够作用于E.coli的遗传物质DNA。     

太行菊提取物的抑菌作用研究

本文对太行菊叶片不同溶剂提取物进行抑菌作用研究。结果表明,太行菊乙醇提取液抑菌效果好于水提液。其中,80%乙醇提取液抑菌效果最显著,对S.aureus和E.coli的MIC分别为25 mg/m L和50 mg/m L,S.aureus对提取物更加敏感。     

不同实验方法检测常用有机溶剂对细菌活性的影响及其安全使用限量

【目的】采用不同实验方法测定常用有机溶剂二甲基亚砜(DMSO)、丙酮和乙醇对细菌活性的影响,以指导抗菌类药物体外抑菌实验所用溶剂的选择和添加限量。【方法】采用常规体外抑菌实验方法(纸片扩散法、肉汤稀释法),并参照生长曲线法检测有机溶剂DMSO、丙酮和乙醇对大肠杆菌(Escherichia coli)及金黄色葡萄球菌(Staphylococcus aureus)的抑菌作用,采用扫描电子显微镜(SEM)观察溶剂作用后的细菌形态变化。【结果】3种溶剂对E.coli和S.aureus抑菌率达到20%时,在肉汤稀释法下,DMSO、丙酮、乙醇的浓度(体积比)分别为1.00%、0.25%、2.00%和1.00%、1.00%、0.50%;在生长曲线法下,溶剂浓度(体积比)分别为0.50%、1.00%、0.50%和1.00%、0.50%、0.50%;而在纸片扩散法下,32%(体积比)DMSO和32%(体积比)乙醇对E.coli产生明显抑菌圈,但3种溶剂对S.aureus均无抑菌圈出现。3种方法比较后得出:当3种溶剂的抑菌率达到20%时,溶剂浓度(体积比)均低于0.5%,对细菌整体生长活性影响较小。SEM结果表明控制溶剂使用限量可有效减少其对E.coli生长过程的影响。【结论】相对于DMSO和丙酮,乙醇对微生物生长繁殖能力的影响更加明显;采用相同浓度有机溶剂时,液态条件下(肉汤稀释法和生长曲线法)微生物受到有机溶剂的影响较大。     

嗜酸乳杆菌S-层蛋白联合抗生素对Escherichia coli和Staphylococcus aureus的抑制作用研究

旨在检测嗜酸乳杆菌S-层蛋白以及S-层蛋白与抗生素联用对大肠杆菌(Escherichia coli)和金黄色葡萄球菌(Staphylococcus aureus)的抑制作用。采用液体发酵培养法获得嗜酸乳杆菌菌体,LiCl法提取S-层蛋白粗提物,凝胶过滤层析法纯化S-层蛋白,分别用E.coli和S.aureus处理Caco-2细胞2 h后,考察S-层蛋白在不同浓度和不同作用时间条件下对E.coli和S.aureus的抑制作用,并考察S-层蛋白联合抗生素对E.coli和S.aureus的抑制作用,实验分组:(1)空白对照;(2)嗜酸乳杆菌组;(3)S-层蛋白组;(4)抗生素组;(5)嗜酸乳杆菌+抗生素组;(6)S-层蛋白+抗生素组。结果显示,液体发酵得到嗜酸乳杆菌菌体,提取并纯化得到S-层蛋白;S-层蛋白对E.coli和S.aureus有很好的抑制效果,具有浓度依赖性,高浓度下抑制率达到42.2%和31.7%,差异极显著(P<0.01),且在E.coli和S.aureus作用的短时间内干预效果明显,0 h时的抑制率分别达到59.3%和48.4%;S-层蛋白联合抗生素的抑菌率分别达到81.7%和79.3%,差异极显著(P<0.01),效果优于单独使用抗生素。嗜酸乳杆菌S-层蛋白具有较强的抑菌作用,可以与抗生素联用,有望开发称为一种新型的抗菌药物。     

唾液乳杆菌ZDY159a抑菌机制的研究

目的对唾液乳杆菌(Lactobacillus salivarius)ZDY159a的产酸能力和体外抑菌影响因素进行初步研究。方法采用共培养法和牛津杯法,以大肠埃希菌(Escherichia coli O157:H7)NCTC 12900和金黄色葡萄球菌(Staphylococcus aureus)CMCC 26003作为指示菌。结果唾液乳杆菌ZDY159a发酵产生的乳酸和乙酸均具有较强抑菌活性;发酵全液的抑菌活性强于发酵上清液;低p H与发酵上清液的抑菌活性呈正相关;蛋白酶和过氧化氢酶处理可降低发酵上清液的抑菌活性。结论唾液乳杆菌ZDY159a的抑菌活性与发酵过程中产生的有机酸、细菌素(蛋白或多肽类物质)和过氧化氢有关,且菌体可提高发酵上清液的抑菌活性。     

大孔吸附树脂对家蝇抗菌肽的吸附与分离纯化

通过比较6种不同型号的大孔吸附树脂对家蝇蛋白的吸附解吸特性,发现D101大孔吸附树脂的性能优于其他5种,吸附流速、浓度影响大孔吸附树脂的动态吸附性能。D101大孔吸附树脂对未经诱导的家蝇蛋白的吸附量可达217．18mg／g（以干树脂总量为基准）,洗脱率为76．48％。吸附后的大孔吸附树脂用15％、35％、55％的乙醇溶液阶段洗脱,各洗脱组分的疏水性逐渐增大,蛋白质含量也明显增加。用E.coli、S．aureus和B．subtilis对各洗脱组分进行抑菌活性测定,抑菌活性随洗脱组分的疏水性增加而增大。测得55％乙醇洗脱组分的抑菌活性最大,其中对E.coli的抑菌圈直径达5．8mm。     

生物化学教材中E. coli DNA聚合酶Ⅲ的组成和结构变化

所有生物化学教材均把大肠杆菌(Escherichia coli,E. coli) DNA聚合酶Ⅲ当做DNA复制的模式分子介绍，但新的研究结果发现，最新探索的E. coli DNA聚合酶Ⅲ的结构与原来描述的结构已有很大的不同。我国目前的生物化学教材对E. coli DNA聚合酶Ⅲ的结果描述仍旧采用旧的模型，已经落后于新的研究发现，需要及时更新。为促进我国生物化学教材建设，本文介绍了新版国际生物化学教材《Lehninger Principles of Biochemistry》描述的E. coli DNA聚合酶Ⅲ结构，以供教学交流。     

壳聚糖对大肠杆菌的抑制作用规律及抗菌机理初探

考察了不同分子量壳聚糖对大肠杆菌的抑菌性能,利用壳聚糖的席夫碱反应对其氨基加以保护,探讨了壳聚糖对大肠杆菌的抗菌机理。研究结果表明:壳聚糖分子量越小,对大肠杆菌的抗菌作用越明显;壳聚糖对大肠杆菌的抑菌作用与其氨基的质子化有关。     

Synthesis, characterization, and antibacterial activity of N,O-quaternary ammonium chitosan

N,N,N-Trimethyl O-(2-hydroxy-3-trimethylammonium propyl) chitosans (TMHTMAPC) with different degrees of O-substitution were synthesized by reacting O-methyl-free N,N,N-trimethyl chitosan (TMC) with 3-chloro-2-hydroxy-propyl trimethyl ammonium chloride (CHPTMAC). The products were characterized by ¹H NMR, FTIR and TGA, and investigated for antibacterial activity against Staphylococcus aureus and Escherichia coli under weakly acidic (pH 5.5) and weakly basic (pH 7.2) conditions. TMHTMAPC exhibited enhanced antibacterial activity compared with TMC, and the activity of TMHTMAPC increased with an increase in the degree of substitution. Divalent cations (Ba²⁺ and Ca²⁺) strongly reduced the antibacterial activity of chitosan, O-carboxymethyl chitosan and N,N,N-trimethyl-O-carboxymethyl chitosan, but the repression on the antibacterial activity of TMC and TMHTMAPC was weaker. This indicates that the free amino group on chitosan backbone is the main functional group interacting with divalent cations. The existence of 100 mM Na⁺ slightly reduced the antibacterial activity of both chitosan and its derivatives.     

Synthesis of methylated chitosan containing aromatic moieties: Chemoselectivity and effect on molecular weight

N-Arylated chitosans were synthesized via Schiff bases formed by the reaction between the primary amino group of chitosan with aromatic aldehydes followed by reduction of the Schiff base intermediates with sodium cyanoborohydride. Treatment of chitosan containing N,N-dimethylaminobenzyl and N-pyridylmethyl substituents with iodomethane under basic conditions led to quaternized N-(4-N,N-dimethylaminobenzyl) chitosan and quaternized N-(4-pyridylmethyl) chitosan. Methylation occurred at either N,N-dimethylaminobenzyl and N-pyridylmethyl groups before the residual primary amino groups of chitosan GlcN units were substituted. The total degree of quaternization of each chitosan varied depending on the extent of N-substitution (ES) and the sodium hydroxide concentration used in methylation. Increasing ES increased the total degree of quaternization but reduced attack at the GlcN units. N,N-dimethylation and N-methylation at the primary amino group of chitosan decreased at higher ES’s. Higher total degrees of quaternization and degrees of O-methylation resulted when higher concentrations of sodium hydroxide were used. The molecular weight of chitosan before and after methylation was determined by gel permeation chromatography under mild acidic condition. The methylation of the N,N-dimethylaminobenzyl derivative with iodomethane was accompanied by numerous backbone cleavages and a concomitant reduction in the molecular weight of the methylated product was observed. The antibacterial activity of water-soluble methylated chitosan derivatives was determined using Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) bacteria; minimum inhibitory concentrations (MIC) of these derivatives ranged from 32 to 128 μg/mL. The presence of the N,N-dimethylaminobenzyl and N-pyridylmethyl substituents on chitosan backbone after methylation did not enhance the antibacterial activity against S. aureus. However, N-(4-N,N-dimethylaminobenzyl) chitosan with degree of quaternization at the aromatic substituent and the primary amino group of chitosan of 17% and 16–30%, respectively, exhibited a slightly increased antibacterial activity against E. coli.     

Antibacterial characteristics and activity of acid-soluble chitosan

The antibacterial activity of chitosan was investigated by assessing the mortality rates of Escherichia coli and Staphylococcus aureus based on the extent of damaged or missing cell walls and the degree of leakage of enzymes and nucleotides from different cellular locations. Chitosan was found to react with both the cell wall and the cell membrane, but not simultaneously, indicating that the inactivation of E. coli by chitosan occurs via a two-step sequential mechanism: an initial separation of the cell wall from its cell membrane, followed by destruction of the cell membrane. The similarity between the antibacterial profiles and patterns of chitosan and those of two control substances, polymyxin and EDTA, verified this mechanism. The antibacterial activity of chitosan could be altered by blocking the amino functionality through coupling of the chitosan to active agarose derivatives. These results verify the status of chitosan as a natural bactericide.     

Effect of abiotic factors on the antibacterial activity of chitosan against waterborne pathogens

To assess the adaptability of chitosan (from agricultural waste) as a natural disinfectant, its antibacterial activity against bacteria associated with waterborne diseases was investigated by varying such abiotic conditions, as pH and ionic strength and by adding different amounts of acid solvent, metal ions, and EDTA. Two major waterborne pathogens, Escherichia coli and Staphylococcus aureus, were examined. Results showed that organic acids with low carbon number were better solvents for chitosan than were inorganic acids. The effect of pH below 6 on the antibacterial activity of chitosan was significant. The antibacterial activity of chitosan increased with ionic strength but decreased with the addition of metal ions. The addition of Zn(2+) ions inhibited the antibacterial activity of chitosan the most, while the addition of Mg(2+) ions inhibited the antibacterial activity of chitosan the least. This was due to the chelating capacity of chitosan toward metal ions. The antibacterial activity of chitosan against E. coli was enhanced by EDTA. However, the antibacterial activity of chitosan against S. aureus was partially suppressed by EDTA. The antibacterial activity of chitosan was also dependent on its charges and solubility. The antibacterial mechanism of chitosan has currently been hypothesized as being related to surface interference. The results show that the chitosan is a potential bactericide under various environmental conditions.     

Surface structure of chitosan and hybrid chitosan-amylose films-restoration of the antibacterial properties of chitosan in the amylose film

The surface structure of films prepared by casting aqueous solutions of mixtures of water soluble chitosan (WSC) and amylose as well as a fully deacetylated chitosan was studied. Zeta potential measurements indicated that the surface of WSC and fully deacetylated chitosan films is positively charged but very weakly, whereas, a film of amylose blended with WSC exhibited an obvious positive charge. X-ray photoelectron spectra of these films suggest that less amino groups are exposed on the surface of WSC and fully deacetylated chitosan films, whereas, more amino groups are exposed on the surface of a WSC film blended with amylose. A sheet structure in which free amino groups are less exposed on the surface of the film of WSC or fully deacetylated chitosan is proposed. This accounts for the loss of antibacterial activity of chitosan on the WSC film surface. When blended with amylose, the morphology of the film may be disrupted, resulting in strong antibacterial properties.     

Modification of Bombyx mori silk fabrics by tyrosinase‐catalyzed grafting of chitosan

Tyrosinase could oxidize tyrosyl residues in silk fibroin and result in the production of activated o‐quinone residues, which could facilitate the grafting of the functional amino‐compounds onto silk fibers. In this study, the enzymatic modifications of Bombyx mori silk fibroin with tyrosinase and chitosan were investigated, aiming at improving the properties of silk fabrics, including dyeability, crinkling resistance, and antibacterial activity. The grafting grades of chitosan were evaluated by a color‐development method using bromocresol green. The result indicated that chitosan molecules were not only adsorbed on silk fibers via electrostatic interactions, they also could react with the oxidized silk fibers with tyrosinase. For the silk fabric combinedly treated with tyrosinase and chitosan, tensile strength and crinkling resistance were noticeably increased as compared to that of the chitosan‐treated. The antibacterial activity and its durability measurements revealed the actions of the tyrosinase‐catalyzed grafting of chitosan. The efficacy of the graft reaction might be further enhanced by increasing the accessibility of reactive sites in silk fibers.     

低聚壳聚糖美拉德衍生物抑菌性能研究

本文研究了基于与葡萄糖、麦芽糖和木糖进行美拉德反应的低聚壳聚糖衍生物的抑菌性.测定低聚壳聚糖及其衍生物对大肠杆菌和金黄色葡萄球菌的抑制效果.结果显示:壳聚糖及其衍生物对金黄色葡萄球菌的抑制作用强于对大肠杆菌的抑制作用,且随着浓度增加,对两种菌的抑菌效果增强.大多数壳聚糖衍生物的抑菌效果优于壳聚糖本身,其中CG 1∶1 8 h(低聚壳聚糖的氨基与葡萄糖的羰基的物质量比为1∶1,反应8h)的抑菌效果最好,CM 1∶3 8 h(低聚壳聚糖的氨基与麦芽糖的羰基的物质量比为1∶3,反应8 h)抑菌性最差,这可能与参加反应的还原糖种类、反应物比例以及反应时间相关.     

壳聚糖抑菌机制的初步研究

壳聚糖在医学、食品、环保、日化用品等领域有着广泛而重要的应用.近年来,壳聚糖由于对不同的菌类都具有良好的抑菌效果而被研究者们密切关注.然而,有关壳聚糖抑菌机制的研究却并不多,其抑菌机制也没有被完全阐明.在本研究中,我们发现很多金属离子可以对壳聚糖的抑菌效果产生影响,高浓度金属离子(0.5%)可以使壳聚糖完全丧失抑菌活性.还发现金黄色葡萄球菌和白色念珠菌在壳聚糖的作用下会发生钾离子和ATP的渗漏,而且五万分子量的壳聚糖引起钾离子和ATP的渗漏大约比五千分子量壳聚糖多2到4倍.不同分子量的壳聚糖对金黄色葡萄球菌和白色念珠菌都具有较好的抑菌效果,但是引起钾离子和ATP的渗漏量却存在很大差异,这说明小分子量壳聚糖很可能存在与大分子量壳聚糖不同的抑菌机制.     

Antibacterial activity and mechanism of action of chitosan solutions against apricot fruit rot pathogen Burkholderia seminalis

The in vitro antibacterial activity and mechanism of action of two kinds of acid-soluble chitosan and one water-soluble chitosan against apricot fruit rot pathogen Burkholderia seminalis was examined in this study. Results showed that water-soluble chitosan displayed limited antibacterial activity at four tested concentrations. However, two kinds of acid-soluble chitosan solution at 2.0 mg/mL had strong antibacterial activity against B. seminalis although weak antibacterial activity was observed at a concentration lower than 1 mg/mL. The antibacterial activity of acid-soluble chitosan may be due to membrane disruption, cell lysis, abnormal osmotic pressure, and additional chitosan coating around the bacteria based on integrity of cell membranes test, out membrane permeability assays and transmission electron microscopy observation. In addition, biofilm biomass were markedly reduced after treating with two kinds of acid-soluble chitosan at concentrations of 2.0 and 1.0 mg/mL for 3 and 12 h, indicating the importance of biofilm formation in the antibacterial mechanism of chitosan. Overall, the results clearly indicated that two kinds of acid-soluble chitosan had a potential to control the contamination of apricot fruits caused by B. seminalis.