【作者】 黄桂东；

【导师】 曹郁生；

【作者基本信息】 南昌大学 ， 食品科学与工程， 2011， 博士

【摘要】 乳酸菌是一类非常重要的食品微生物,在食品中的应用可以追溯到数千年以前。它具有提高食品营养价值,改善食品风味,延长保存时间的功能,还具有多种有益于人类健康的活性。短乳杆菌是其中之一。近年来,短乳杆菌做为发酵剂、益生菌等被广泛应用在食品工业生产中。但是由于自身产酸,在保存、食用过程中也会遇到外界酸性环境的影响,使得短乳杆菌不可避免的面临酸胁迫。酸胁迫对短乳杆菌的存活、生长及生理活性有很大的影响。因此,为了生存、增殖并发挥其有益宿主健康的功能,短乳杆菌必须具有应对酸胁迫的能力,并对酸胁迫产生应激反应来保护其生存、增殖。如果能够找到提高乳酸菌耐酸能力的方法或途径,将会极大地推动乳酸菌在食品工业生产中的应用。从理论意义上,对酸胁迫下乳酸菌的蛋白质组学的深入研究,不仅可以了解乳酸菌在酸应激反应中蛋白质的表达变化,而且可以了解生物在逆境中的某些相关基因和蛋白质的功能,进而分析揭示其酸应激反应的机制。Lactobacillus brevis NCL912是本实验分离得到的一株乳酸菌,具有高效转化L-谷氨酸钠成为γ-氨基丁酸的能力。本论文主要研究了以下内容。1.观察了L. brevis NCL912在酸性环境下的生长及在pH 2.0极端酸性环境中的耐酸性能。结果表明,L. brevis NCL912在pH 3.5～6.4的酸性环境中可以生长,其最适生长pH是5.0。在pH 2.0的极端酸性培养基中,该菌能够存活4-6 h。2.比较了添加与未添加L-谷氨酸钠培养基培养的L. brevis NCL912的生长、存活、γ-氨基丁酸产量及谷氨酸脱羧酶活力,并对该菌的酸应激反应机制进行了分析。结果表明,添加L-谷氨酸钠培养基培养的L. brevis NCL912生长较好。在pH 3.5～5.0条件下,L. brevis NCL912可以完全利用培养基中添加的L-谷氨酸钠生成γ-氨基丁酸,γ-氨基丁酸的产量分别达10.77,11.44,10.94 g/L。在两种培养基中细菌的存活率和谷氨酸脱羧酶活力存在显著性差异（p<0.05）,添加L-谷氨酸钠培养基培养的细菌耐酸性强,酶活力高。说明L-谷氨酸钠对该菌在酸性环境中的生长、存活有重要的影响,其原因可能与γ-氨基丁酸的生成有关,即谷氨酸脱羧酶-γ-氨基丁酸逆向运输系统可能是该菌的酸应激反应机制。3.乳酸菌的酸应激反应是一个涉及到多个基因和蛋白质的复杂的网络调控体系。它不仅与依赖外部氨基酸的酸应激系统有关,而且菌体内部其他蛋白质也可能发生改变以应对酸胁迫。目前对短乳杆菌酸应激反应中菌体蛋白的差异表达及功能分析报道较少。因此,从蛋白质组学水平,对L. brevis NCL912在酸胁迫下菌体蛋白质的变化进行了研究。本研究得到了均匀、背景清晰、分辨率高、重复性好的L. brevis NCL912的双向凝胶电泳图谱。对pH 5.0和pH 4.0条件下培养的该菌总蛋白质电泳图谱进行比较,发现有25个差异表达蛋白,其中8个蛋白点得到了质谱鉴定。其中7个蛋白点表达上调,1个蛋白点表达下调。表达上调的7个蛋白点包括：酸应激蛋白2个,分别是通用应激蛋白UspA蛋白和含CBS结构域蛋白；与蛋白质合成有关的蛋白3个,分别是50S核糖体蛋白L10,核糖体循环因子和SSU核糖体蛋白S30P；与代谢相关的蛋白是依赖NADP的3-磷酸甘油醛脱氢酶；与核苷酸合成有关的蛋白是次黄嘌呤核苷酸脱氢酶。表达下调的蛋白是一个假设蛋白LVIS₀520,其功能未知。在酸胁迫条件下,L.brevis NCL912 CBS结构域蛋白基因的mRNA表达水平呈显著上升（p<0.05）,与蛋白质图谱上蛋白表达水平的变化一致,说明该菌蛋白质和mRNA表达水平之间有较好的相关性。4.应用蛋白质组学技术,对添加L-MSG培养基培养的L. brevis NCL912在酸胁迫条件下的差异蛋白表达也进行了探讨。结果表明,在酸胁迫条件下,添加L-MSG培养基培养的L.brevis NCL912有26个蛋白发生了差异表达,其中11个蛋白得到了鉴定。这11个蛋白包括：通用应激蛋白UspA蛋白、热应激蛋白GrpE、3-磷酸甘油醛脱氢酶、核苷-二磷酸-糖差向异构酶、3-磷酸甘油酰基转移酶PlsX.磷脂结合蛋白、S-核糖同型半胱氨酸酶、延伸因子Tu、RNA结合蛋白以及假设蛋白BRAFLDAFT₆4486。转录学水平的验证表明该菌蛋白质和mRNA表达水平之间有较好的相关性。5.对添加与未添加L-谷氨酸钠培养基培养的L. brevis NCL912已鉴定蛋白质的功能进行了分析比较,发现在酸胁迫条件下,无论是否添加底物氨基酸,L.brevis NCL912都会启动一些共同的应激过程来保护自身,如蛋白质的合成、糖代谢及应激蛋白表达量的变化等。而外源氨基酸的添加可能会诱导某些与细胞膜及信号转导有关的蛋白表达发生改变,从而有利于细胞能够在酸性环境中生存和增殖。这表明酸胁迫应激过程是一个复杂的网络调控体系,不仅与外部氨基酸的添加有关,还涉及到菌体内部其他蛋白质的变化。为进一步了解乳酸菌酸应激反应机制提供了一定的参考和依据。更多还原

【Abstract】 Lactic acid bacterial (LAB) are important food microorganisms that have been used in food production for thousands years. LAB can improve food nutritions, flavors, textures and preservation, and are also of value in maintaining and promoting human health. Lactobacilli are Gram-positive rods belonging to the group of lactic acid bacteria. In recent years, the industrial applications of Lactobacilli as starters, producers, and probiotics have greatly increased. However, the growth of Lactobacilli is characterized by the production of organic acids, which accumulate and lead to a reduction of pH in its growth environment. As the probiotics, these bacteria also encounter a transient acidic environment in the stomach after consumption. Acid stress greatly affects the growth and bioactivities of Lactobacilli. Therefore, they must possess the acid tolerance ability and be able to induce a series of acid stress responses to survive and grow in acidic environments prior to performing their health benefits. It would greatly drive the appilication of lactic acid bacterial in food industry when the acid tolerance ability of Lactobacilli could be enhanced. The proteomics studies on lactic acid bacterial under acid stress may not only provide a better understanding the changes of the protein expressed in acid stress response, but comprehending the correlative genes and proteins under stress which will obtain the further insight into acid stress response mechanism. The results are as follows:1. The growth and survival of Lactobacillus brevis NCL912, a high y-aminobutyric acid (GABA)-yielding bacterium isolated from fermented vegetables, were investigated at different acidic pH levels. The results show that L. brevis NCL912 grows at pH 3.0-6.4, and the optimal growth pH is 5.0. L. brevis NCL912 could be survive for 4-6 h at pH 2.0.2. The growth, survival, GAB A yield and glutamate decarboxylase activity of L. brevis NCL912 in the media with or without the addition of sodium L-glutamate were compared. The strain attained higher cell concentrations in the media with L-MSG than that without L-MSG in the same growth period. The addition of sodium L-glutamate significantly enhances acid resistance and glutamate decarboxylase activity (p<0.05). Sodium L-glutamate is an important factor for the growth and survival of L. brevis NCL912 at different acidic pHs. It was implied that the acid stress response of L. brevis NCL912 was related to glutamate decarboxylase-GABA antiporter system. 3. Acid stress responses involve a very comprehensive network system of genes and proteins. Therefore, the differentially expressed proteins of L. brevis NCL912 under acid stress were investigated at the global proteomic level by using the two-dimensional gel electrophoresis and mass spectrum. The results showed that 25 proteins were differentially expressed under acid stress. Among them, eight protein spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, of which 7 were up-regulated and 1 was down-regulated. The function of down-regulated protein was unknown and the putative functions of up-regulated proteins were categorized as stress response proteins, DNA repair, protein synthesis and glycolysis. Real-time flurescent quantitative PCR was used to further validate these differentially expressed proteins at the mRNA level and a positive correlation between the content of the proteins and their mRNA levels was found.4. The differential protein expression of L. brevis NCL912 in the media with sodiun L-glutamate under acid condition were investegated by 2-dimensional gel electrophoresis and mass spectrometry. Real-time flurescent quantitative PCR was used to estimate the expression of selected proteins at the mRNA level. The results showed that 26 proteins were differentially expressed under acid stress. Among them, eleven protein spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, of which 10 were up-regulated and 1 was under-shifted. According to the putative functions, the up-regulated proteins were categorized as stress response proteins and the other related to protein synthesis and glycolysis which were related to acid stress induction. Another cluster of cell membrane formation-related proteins and cell communication-related proteins might be induced by sodium L-glutamate. Real-time flurescent quantitative PCR data revealed a positive correlation between the content of the proteins and their mRNA levels.5. The proteins and their functions of L. brevis NCL912 in the media with or without sodium L-glutamate were compared and analyzed. The analysis of bioinformatics showed that L. brevis NCL912 might launch the common acid resistance systems such as the protein synthesis, glycometabolism and stress proteins to protect the cell against acid stress which is irrelevant to the sodium L-glutamate. However, the exogenous amino acid (sodium L-glutamate) might also induce the proteins which were involved in the cell membrane formation and signal transporter. It was implied that the acid resistance-responsive system is a very complex network system which is not only related to the exogenous amino acid, but also the expression of the cell proteins.更多还原