冷胁迫对冷冻干燥罗伊氏乳酸杆菌细胞膜流动性的影响

以DPH(1,6-二苯基-1,3,5-己三烯)为荧光探剂,优化了测定罗伊氏乳酸杆菌细胞膜流动性的条件,其最佳测定条件为:激发波长360nm,发射波长430nm,菌浓度OD0.8,30℃孵育时间为20min。同时研究冷胁迫处理对冷冻干燥存活率。细胞活力及细胞膜流动的影响,研究表明冷胁迫能改善乳酸菌的冷冻干燥存活率,同时在一定的条件下,冻干后细胞的流动性也有所改变,说明经过一定的冷胁迫处理后,增加了细胞的抗性,从而提高细胞的冷冻存活率。     

冷冻干燥影响乳酸菌发酵活力机制的研究进展

乳酸菌是食品中常用的发酵菌株或益生菌,常以冻干粉的形式应用于工业生产。冻干过程中的脱水及低温等条件对乳酸菌形成胁迫,造成菌株活力下降甚至死亡。冻干粉中菌株活菌数或发酵活力是乳酸菌冻干粉的重要指标。目前大部分研究集中在解析菌株抗冻干机制以及提高菌体冻干存活率等技术,本文围绕乳酸菌发酵活力指标,综述了冷冻干燥过程中影响发酵活力的因素,阐述了细胞膜流动性、通透性和细胞膜损伤异质性等对乳酸菌发酵活力的作用及机制,为进一步阐明冷冻干燥影响乳酸菌发酵活力的途径,开发提高乳酸菌冻干粉发酵活力的技术提供依据。     

低聚糖对乳酸菌抗氧化胁迫能力的影响

目的:研究GOS(低聚半乳糖)和xos(低聚木糖)对乳酸菌(L.bulgaricus Fn009)耐H202胁迫能力的影响.方法:利用H2O2建立氧化应激模型,以MTT法(四甲基偶氮唑盐比色法)检测GOS和XOS对乳酸菌存活率影响,以流式细胞技术检测GOS和XOS对乳菌细胞膜完整性的影响,并测定GOS和XOS对乳酸菌抗氧化酶活力和应激蛋白基因表达水平的影响.结果:H202胁迫条件下,GOS和XOS干预组乳酸菌存活率提高了1.83～1.96倍,细胞膜完整率分别提高了17％和13％,T-SOD和GSH-Px酶活力均显著上升(p＜0.05),Dnak、GroEL和Gsp65应激蛋白基因的表达水平显著下调(p＜0.05).结论:GOS和XOS能够提高乳酸菌自身的抗氧化活力,降低H2O2胁迫对菌体细胞膜的损伤程度,增强乳酸菌对氧化胁迫的抗性.     

真空冷冻干燥对乳酸菌损伤机制的研究进展

通过真空冷冻干燥法制备乳酸菌发酵剂,具有活菌数高、发酵活力强、遗传稳定性好等优点,但也会对菌体造成一定的损伤。本文通过分析冷冻干燥过程中细胞膜通透性、细胞膜流动性、酶及蛋白质类物质以及遗传物质等的变化阐述了真空冷冻干燥对乳酸菌造成损伤的相关机制。得到结论如下：在冷冻干燥过程中,冰晶的形成以及磷脂分子的相转变会导致膜的通透性增加;细胞膜脂肪酸成分的改变会造成膜流动性的变化;酶活力的降低及相关蛋白的变性会造成菌体的代谢和生长速率受到影响;DNA结合蛋白结构的改变以及碱基对间的氢键断裂导致DNA超螺旋结构的破坏,这些都会导致乳酸菌发酵活力的下降,甚至死亡。     

乳酸菌的热胁迫研究进展

乳酸菌和其他自由生活的微生物一样,在发酵及食品加工、贮藏过程中经常暴露于各种环境胁迫条件下,包括饥饿胁迫、渗透压胁迫以及热胁迫等,增加胁迫抗性是提高生物量及代谢产物积累的有效策略。热胁迫可能是自然界及工业应用中微生物所面临的最常见压力。目前研究表明来自不同生境的乳酸菌通过基因表达的快速变化对温度的突然升高做出反应,从而导致热休克蛋白的蛋白质水平升高。热休克蛋白在进化过程中具有高度保守性,在正常条件下,其有助于受体调节,细胞骨架稳定及蛋白质的折叠、组装、运输、降解。在热胁迫条件下,其功能变得尤为重要。综述了乳酸菌的热胁迫耐受机制及不同乳酸菌的热胁迫反应,为研究乳酸菌热胁迫应答机制提供一定的理论借鉴,有利于乳酸菌的工业化应用。     

重组抗冻肽在乳酸乳球菌中表达及其抗冻活性研究

为了优化抗冻蛋白SF-P的重组表达,利用乳酸链球菌素Nisin对已构建的重组乳酸乳球菌进行表达。通过对诱导时间、诱导pH、诱导温度和诱导剂Nisin浓度等诱导表达条件进行优化,利用SDS-PAGE和Western blot确定最佳的表达条件;通过比较冷冻胁迫前后菌体的生长状况、发酵活力和细胞内钠钾离子含量的变化,研究重组菌在冷冻胁迫作用下的生理功能特性。结果表明:优化后的最佳表达条件为pH 7.0,诱导剂Nisin浓度15ng/mL,温度25℃,诱导时间6h;SF-P2诱导重组菌株可以显著改善乳酸菌由于经受冷冻胁迫导致的对数生长延滞期和稳定生长延滞期的增加,表现出较强的酸化活力,并且可以有效降低冷冻胁迫过程对乳酸菌细胞膜通透性的影响,起到保护细胞生理功能的作用,表明SF-P2诱导重组菌具有显著的抗冷冻胁迫保护作用。     

蛋白组学技术在乳酸菌环境胁迫应激研究中的应用

文中介绍了蛋白质组学新技术原理及常用技术路线,着重论述了该技术对乳酸菌在各种不同环境胁迫应激研究中的应用,从酸胁迫、冷胁迫、胆盐胁迫、渗透压胁迫及氧化胁迫等方面分别做了应用分析,并对该技术在分子生物学领域尤其是乳酸菌分子生物学研究方面未来的发展做了展望。     

铜离子对酿酒酵母能量代谢及细胞膜功能的影响

以优良葡萄酒酿酒酵母为研究对象,采用葡萄汁模拟发酵体系,较为系统地研究了在不同浓度铜离子的作用下酵母的代谢特征。同时,从能量代谢(ATP、NADH/NAD+)、细胞膜的通透性和完整性的角度分析了发酵过程中铜离子对酵母的作用机制。结果表明,Cu~(2+)胁迫处理后葡萄糖和果糖的利用率以及乙醇的生成率降低,胞内ATP、NADH/NAD~+、核酸、蛋白质的含量以及荧光强度都比对照高。说明经Cu~(2+)胁迫处理后酵母的能量代谢受到抑制、细胞膜的完整性受损,从而导致了糖和乙醇代谢的减缓以及部分酵母菌的死亡。     

GDL诱导大豆分离蛋白冷凝胶对益生菌的保护

主要研究了葡萄糖酸内酯(GDL)诱导大豆分离蛋白冷凝胶在胃酸pH环境下对乳酸菌的保护情况.利用GDL诱导形成的大豆分离蛋白冷凝胶在胃酸pH环境下作用30min后乳酸菌的存活率为评价指标,用以评价大豆分离蛋白冷凝胶对乳酸菌的保护情况通过正交实验得出,在pH1.2的胃酸环境下,大豆分离蛋白冷凝胶的成胶条件为变性温度90℃、SPI浓度9％、GDL浓度1.2％,对乳酸菌的保护效果最佳.活菌落菌落计数的数量级由初始的107变为105 cfu/mL,仅下降了2个数量级,乳酸菌的存活率达到26.61％,得到显著提高.实验结果表明,在胃酸pH环境下GDL诱导的大豆分离蛋白冷凝胶能很好的保护乳酸菌,为益生菌和大豆分离蛋白在食品中的应用提供了参考.     

哈密瓜脱氢抗坏血酸还原酶基因的鉴定及冷胁迫表达分析

为阐明哈密瓜脱氢抗坏血酸还原酶(dehydroascorbate reductase,DHAR)基因的基本性质及在响应冷胁迫过程中的表达水平,基于生物信息学对2个哈密瓜DHAR基因(CmDHAR)的基本信息、多序列比对、系统进化关系、共线性关系、基因结构域特征、蛋白质三级结构、蛋白质-蛋白质相互作用及冷胁迫下的抗坏血酸...     

酸胁迫对鼠伤寒沙门氏菌抗酸性、细胞膜及膜蛋白的影响

目的：以鼠伤寒沙门氏菌CGMCC 1.1190为对象，研究柠檬酸反复胁迫处理对其抗酸性、细胞膜及其膜蛋白的影响。方法：将CGMCC 1.1190分别在经柠檬酸调节到pH值为3.0、2.7、2.5的胰蛋白胨大豆肉汤培养基中胁迫处理并转接12 次培养后，获得3 株CGMCC 1.1190的抗酸性菌株，测定了这3 株抗酸性菌株的D值、菌落形态、个体形态、膜通透性、膜流动性和膜蛋白的变化。结果：这3 株抗酸性菌株的D值随着酸处理次数的增加不断增大，酸胁迫的pH值越低，D值越大，菌落形态与个体形态变化越明显；与原始对照菌株相比，当这3 株抗酸性菌株置于pH 2.0的强酸环境下时，其碘化丙啶染色区域的死菌比例显著降低（P＜0.05），表明酸胁迫pH值越低，这3 株抗酸性菌株的细胞膜对H＋通透性越低；随着酸胁迫pH值的降低，CGMCC 1.1190抗酸性菌株细胞膜磷脂的熔点升高，表明细胞膜磷脂的相变温度升高，细胞膜流动性降低；十二烷基硫酸钠-聚丙烯酰胺凝胶电泳结果表明，这3 株抗酸性菌株分子质量35～180 kDa范围内的细胞膜蛋白表达量增加，在135 kDa和180 kDa处均增加了特异条带。结论：随着酸胁迫pH值的降低，CGMCC 1.1190抗酸性增加，菌落变小，个体形态变长，细胞膜通透性和流动性均降低，部分膜蛋白表达量和表达种类增加，这些变化有利于其适应在酸胁迫环境下生长，提高生存能力。     

Altered hydrophobicity and membrane composition in stress-adapted Listeria innocua

Exposure of Listeria innocua to acid and starvation stress decreases sensitivity to the quaternary ammonium compound cetrimide, whereas exposure to cold and heat stress increases sensitivity to this compound. Changes in membrane lipids occur in response to certain types of stress, and these changes likely impact cell sensitivity to chemical sanitizers. The present study included an assessment of the effects of acid, starvation, cold, and heat stress on net cell hydrophobicity and fatty acid composition in L. innocua. Net cell hydrophobicity was determined by measuring absorbance of stress-adapted cell suspensions after partitioning with the nonpolar solvent n-hexadecane. Free fatty acids extracted from stress-adapted suspensions were analyzed by gas chromatography. Adaptation to acid and starvation increased net cell hydrophobicity and decreased membrane fluidity, which was correlated with reductions in anteiso fatty acids and in ratios of anteiso to iso fatty acids. Conversely, cold-stressed populations exhibited decreased net cell hydrophobicity and increased membrane fluidity with a corresponding increase in C15:C17 and anteiso:iso ratios and in C18 unsaturated fatty acids. No significant changes in net cell hydrophobicity or membrane fluidity were observed in heat-stressed cells, which exhibited increased sensitivity to cetrimide, suggesting another mechanism for altered cell sensitivity. These findings indicate that the efficacy of cetrimide against Listeria is partially dependent on the physiological state of the organism following exposure to various environmental stresses.     

Modifications in membrane fatty acid composition of Salmonella typhimurium in response to growth conditions and their effect on heat resistance

The effects of growth temperature (in the range 10-45 degrees C) and acidification up to pH 4.5 of the culture medium (Brain Heart Infusion, BHI) with different organic acids (acetic, citric and lactic) and hydrochloric acid on membrane fatty acid composition and heat resistance of Salmonella typhimurium CECT 443 were studied. The heat resistance was maximal in cells grown at 45 degrees C (cells grown in non-acidified BHI showed a D58-value of 0.90 min) and decreased with decreasing growth temperature up to 10 degrees C (D58-value of 0.09 min). The growth of cells in acidified media caused an increase in their heat resistance. In general, acid adapted cells showed D-values of between 1.5 and 2 times higher than the corresponding for non-acid adapted control cells. This cross-protection response, which has important implications in food processing, was not dependent on the pH value and the acid used to acidify the growth medium. A membrane adaptation corresponding to an increase in the unsaturated to saturated fatty acids ratio (UFA/SFA) and membrane fluidity was observed at low growth temperature. Moreover, the acidification of the growth medium caused a decrease in UFA/SFA ratio and in the C18:1 relative concentration, and an increase in cyclopropane fatty acids (CFA) content mainly due to the increase in cyc19 relative concentration. Thus, acid adapted cells showed CFA levels 1.5 times higher than non-acid adapted control cells. A significant proportion of unsaturated fatty acids were converted to their cyclopropane derivatives during acid adaptation. These changes in membrane fatty acid composition result in cells with decreased membrane fluidity. A clear relation between membrane fatty acid composition and heat resistance was observed. In general, D-values were maximum for cells with low UFA/SFA ratio, and, consequently, with low membrane fluidity. Moreover, CFA formation played a major role in protecting acid adapted cells from heat inactivation. However, changes observed in membrane fatty acid composition are not enough to explain the great thermotolerance of cells grown at 45 degrees C. Thus, other mechanisms, such as the synthesis of Heat Shock Proteins, could be responsible for this increase in the bacterial heat resistance.     

Stress resistance of biofilm and planktonic Lactobacillus plantarum subsp. plantarum JCM 1149

The purpose of this study was to investigate the change in resistance of biofilm and planktonic food spoilage lactic acid bacteria (LAB) to environmental stresses, which strongly inhibit bacterial growth and are important in food preservation or in disinfection. The stress responses of biofilm and planktonic cells of Lactobacillus plantarum subsp. plantarum JCM 1149, which was used as a model spoilage bacterium, in various organic acids (namely, acetic acid, citric acid, lactic acid, and malic acid), ethanol, and sodium hypochlorite, were investigated using survival tests. The bacterial cells in biofilms showed greater resistance to all treatments than the planktonic bacterial cells in either the stationary or logarithmic phase. The planktonic bacterial cells showed reduced resistance to acetic acid after the cell suspension was diluted; however, intriguingly, the bacterial cells in biofilms maintained their resistance to acetic acid even after they were suspended or the cell suspension was diluted. These findings suggested the risk for food spoilage due to LAB derived from biofilms and suspended or diluted in foods, and demonstrated the importance of controlling biofilms of LAB in the food industry.     

培养温度对腐败希瓦氏菌DSM6067生长动力学及细胞膜理化特性的影响

低温是保障水产品质量安全的重要方法,然而仍有部分耐冷菌能适应低温环境,继续生长繁殖并最终导致水产品腐败变质。为了解不同培养温度对腐败希瓦氏菌生长动力学参数及细胞膜理化特性变化的影响,以水产品中典型的耐冷腐败微生物腐败希瓦氏菌模式菌株DSM6067为研究对象,对细菌的生长情况、细胞膜脂肪酸组成、膜蛋白含量、细胞微观结构和细胞膜流动性进行分析。结果表明,腐败希瓦氏菌模式菌株DSM6067分别在30,10℃和4℃培养条件下经3.35,25.94 h和122.03 h的延滞期后进入对数生长期;腐败希瓦氏菌虽在低温环境下培养增速较为缓慢,但能够适应低温环境继续生长。腐败希瓦氏菌在相应培养温度下的对数生长中期,30℃条件下细菌细胞膜不饱和脂肪酸总量仅43.36%,其中C16:1(棕榈油酸)为26.62%,而4℃培养下细菌细胞膜不饱和脂肪酸总量为51.04%,其中单不饱和脂肪酸C16:1含量高达46.66%,低温培养下细菌细胞膜中C16:1含量显著增加;细菌细胞膜蛋白表达量随培养温度的降低而有所上升;腐败希瓦氏菌微观结构观察显示,低温培养下的细菌细胞膜较为平滑,胞内空泡较少,细胞体积略大;在低温适应过程中,细菌细胞膜流动性增强。腐败希瓦氏菌细胞膜理化特性所产生的变化为细菌更好地适应低温环境生长提供了一定的物质基础。     

Generation of aroma compounds in sourdough: effects of stress exposure and lactobacilli-yeasts interactions

The effects of the interaction between Saccharomyces cerevisiae LBS and Lactobacillus sanfranciscensis LSCE1 and of their responses to acid, oxidative or osmotic stress on alcohol and aroma production were assessed. The exposure of S. cerevisiae LBS and L. sanfranciscensis LSCE1 cells to oxidative, acid or osmotic sub-lethal stress gave rise to a common or specific responses. Gamma-decalactone, 2(5H)-furanones and aldehydes were overproduced by LAB following oxidative stress. The acid stress induced both in yeasts and LAB, as well as in their co-cultures, a relevant accumulation of isovaleric and acetic acids and higher alcohols. A cross-exposure of yeasts and LAB to their preconditioned media, generated in S. cerevisiae a release of esters including esters of long-chain unsaturated fatty acids coming from membrane phospholipids. These esters were excreted also by yeasts following a pressure stress.