【作者】 张敏；

【导师】 冯镇； 易华西；

【作者基本信息】 东北农业大学 ， 食品工程（专业学位）， 2016， 硕士

【摘要】 植物乳杆菌(Lactobacillus plantarum)作为发酵剂和潜在的益生菌被广泛的用于发酵食品的生产。这引起了人们对Lactobacillus plantarum的益生特性、高密度培养以及代谢调控等方面的广泛研究兴趣。目前,关于Lactobacillus plantarum代谢调控特性的研究还存在显著不足,尤其是氨基酸代谢方面。虽有一些专为Lactobacillus plantarum设计的合成培养基,但这些培养基的氨基酸含量极不均衡,个别氨基酸的浓度偏高,这使得细胞氨基酸消耗量和利用率与总体氨基酸含量相比时非常小,导致结果不准确。对Lactobacillus plantarum氨基酸消耗模式和氨基酸代谢规律研究的缺乏,也阻碍了更加廉价的复合均衡培养基设计和生物过程的优化。本论文的前期工作研究表明,与控制pH(6.0)发酵相比,在不控制pH条件下培养Lactobacillus plantarum KDFR27,其具有较强的发酵活力。为了阐明此现象,本研究以Lactobacillus plantarum KDFR27为目标菌株,在化学合成培养基中对其进行增殖培养,分析了控制pH(6.0)和不控制pH的培养方式对Lactobacillus plantarum KDFR27的生长动力学、主要胞内代谢特征(胞内pH、ATP、H~+-ATP酶活性、NADH和NADPH含量)及氨基酸消耗模式与需求模式的影响,以期为后续的高密度培养奠定基础。就控制pH和不控pH培养对Lactobacillus plantarum KDFR27生长动力学和胞内代谢特征的影响而言,控制pH和不控制pH的最终生物量分别为7.65×107CFU/ml和9.16×108CFU/ml。不控制pH培养Lactobacillus plantarum KDFR27时,其整个生长期的胞内pH均显著高于控制pH的胞内pH,其对数期的pH梯度比控制pH条件时高1.35,稳定期的ATP浓度比控制pH时高36.66μmol/gprot,延迟期和稳定期的H~+-ATP酶活性分别比控制pH时高2.71和2.05μmolPi/mgprot/hour,稳定期的NADH含量比控制pH时高1.77×10-3nmol/104cells,延迟期和对数期的NADPH含量分别比控制pH时高0.57和1.56×10-3nmol/104cells。以上试验结果说明不控制发酵pH有助于增加Lactobacillus plantarum KDFR27的最终生物量,提高胞内pH、pH梯度、稳定期胞内ATP浓度及延迟期和稳定期的细胞内H~+-ATP酶活性、稳定期胞内NADH含量及延迟期和对数期的细胞内NADPH含量。控制pH培养Lactobacillus plantarum KDFR27其大部分氨基酸的总消耗量高于不控制pH时的氨基酸总消耗量。控制pH培养Lactobacillus plantarum KDFR27时,整个生长过程中消耗最多的氨基酸分别是Gly、Glu、Ala和Asp,均高于950μmol/gdw,消耗最少的氨基酸分别是Tyr、Met、His和Arg,均在350~500μmol/gdw之间;不控制pH时,整个生长过程中消耗最多的氨基酸分别为Ser、Gly、Glu和Ala,均高于1300μmol/gdw,消耗最少的氨基酸分别是His、Tyr、Ile和Lys,均在300~400μmol/gdw之间。说明控制pH和不控制pH培养可显著影响Lactobacillu plantarum KDFR27对氨基酸的选择性消耗。控制pH和不控制pH培养Lactobacillus plantarum KDFR27,除Pro外其余氨基酸在不控制pH时的总需求量均高于控制pH时的总需求量,尤其是Ala,不控制发酵pH时的总需求量是控制发酵pH时的8.82倍,说明不控制pH培养Lactobacillus plantarum KDFR27时,其对氨基酸的总需求量较高,且对Ala影响显著。除Ala外,两种发酵条件下Lactobacillus plantarum KDFR27对Glu、Asp和Gly的总需求量都较大,均高于150μmol/gdw;对Pro、Tyr和His的总需求量都最小,在25~65μmol/gdw之间。控制pH和不控制pH培养Lactobacillus plantarum KDFR27的氨基酸总消耗量分别超过氨基酸总需求量的约8和4倍,说明在不控制pH培养Lactobacillus plantarum KDFR27时氨基酸的有效利用率更高。更多还原

【Abstract】 Lactobacillus plantarum were widely used in the production of fermented food as a leavening agent and potential probiotics. This aroused people’s extensive interests in researching probiotic properties, high-density culture, metabolic regulation and other aspects of Lactobacillus plantarum. Now, there still existed some prominent inadequacies in metabolic regulation of Lactobacillus plantarum, especially in amino acid metabolism. Although there were some synthetic mediums designed for Lactobacillus plantarum, the amino acid content of these media was very unbalanced, the concentrations of individual amino acids were higher than others, which made the consumption and utilization of the intracellular amino acids was very small when compared with the overall amino acid content, leading to the inaccurate results. Lacking of researches on amino acids consumption patterns and amino acid metabolism obstructed the more inexpensive media design and optimization of complex biological processes.Preliminary researches for this paper showed that Lactobacillus plantarum KDFR27 had strong fermentation activity under the uncontrolled pH fermentation conditions compared to the controlled pH(6.0) fermentation conditions. In order to clarify this phenomenon, we used Lactobacillus plantarum KDFR27 as the target strains which proliferated in the CDM medium. We analyzed the effect of the controlled(pH6.0) and uncontrolled fermentation pH conditions to growth kinetics, the main intracellular metabolic characteristics(intracellular pH, ATP, H~+-ATPase activity, NADH and NADPH content), acid consumption patterns and demand patterns of Lactobacillus plantarum KDFR27.As to the growth kinetics and intracellular metabolic characteristics, the final biomass under the controlled and uncontrolled pH fermentation conditions were respectively 7.65×107CFU/ml and 9.16×108CFU/ml. During the whole growth period, the intracellular pH under the uncontrolled pH condition was significantly higher than controlled pH condition, and the pH gradient was 1.35 higher than controlled pH condition. During the stationary phase the ATP concentration under the uncontrolled pH condition was higher than controlled pH condition at 36.66μmol/gdwprot, and during the lag phase and stationary phase the activity of the H~+-ATPase under the uncontrolled pH condition was respectively higher than controlled pH condition at 2.71 and 2.05μmol Pi/mgprot/hour. During stationary phase, the concentration of NADH under uncontrolled pH condition was higher than controlled pH condition at 1.77×10-3nmol/104 cells, and during the lag phase and logarithmic phase, the concentration of NADPH under uncontrolled pH condition was respectively higher at 0.57 and 1.56 ×10-3nmol/104 cells. The results above stated that uncontrolled pH condition contributed to increase the final biomass of Lactobacillus plantarum KDFR27, intracellular pH, pH gradient, the concentration of intracellular ATP during stationary phase and the activity of the H~+-ATPase during lag phase and stationary phase, as well as the concentration of intracellular NADH during stationary phase and the concentration of intracellular NADPH during lag phase and logarithmic phase.The total consumption of most amino acid under the controlled pH condition was higher than uncontrolled condition. During the four growth phases, the most consumed amino acids, under controlled pH condition, were Gly, Asp, Ala and Thr, whose consumption were more than 950μmol/gdw; while Tyr, Met, Met and Tyr were less consumed, whose consumption were ranging from 350 to 500μmol/gdw. Under uncontrolled pH condition, Glu, Gly, Glu and Ser whose consumption were all more than 1300μmol/gdw were most consumed; while Leu, Met, Lys and His were less consumed respectively whose consumption were ranging from 300 to 400μmol/gdw. This result indicated that different pH condition may significantly affect the selective consumption of Lactobacillus plantarum KDFR27 to most amino acid.When Lactobacillus plantarum KDFR27 grew under controlled and uncontrolled incubation conditions, except for Pro, the aggregate demand of amino acids under uncontrolled pH condition was higher than controlled pH condition. Especially Ala, whose aggregate de mand under uncontrolled pH condition was 8.82 times higher than controlled pH condition. This result demonstrated that Lactobacillus plantarum KDFR27 had greater aggregate demand for amino acids under uncontrolled pH incubation condition and Ala was signif icantly influenced by this condition. Except for Ala, Lactobacillus plantarum KDFR27 had greater aggregate demand for Glu, Asp and Gly which were all higher than 150μmol/gdw, under these two different incubation condition, and had less aggregate demand for Pro, Tyr and His which were ranging from 25 to 65μmol/gdw. Under controlled and uncontrolled pH conditions, the aggregate consumption of amino acids was over the aggregate demand 8 times and 4 times respectively, which showed that Lactobacillus plantarum KDFR27 had higher effective utilization ratio of amino acids under uncontrolled pH condition.更多还原