【作者】 王丹；

【导师】 王玉华；

【作者基本信息】 吉林农业大学 ， 食品科学， 2012， 硕士

【摘要】 琥珀酸(Succinic Acid)是一种常见的天然有机酸,又名丁二酸,是三羧酸循环的产物之一,琥珀酸在人体、动物、植物和微生物中广泛存在。琥珀酸用途极为广泛,目前存在几个主要的市场：各种类型的表面活性剂、表面清洁剂、添加剂以及起泡剂；在电镀行业中琥珀酸可用作离子鳌合剂；琥珀酸还可以作为酸化剂、pH改良剂、风味物质和抗菌剂等在食品行业中广为应用；在医药生产行业里可以用于抗生素、氨基酸、维生素以及其他药品的生产；琥珀酸也是聚丁二酸丁二醇酯(PBS)的主要原料,用于生物可降解塑料生产。近几年来,通过微生物发酵法生产琥珀酸,已经发展成为各研究机构的研究热点,其中产琥珀酸放线杆菌因其独有的耐糖耐酸耐盐特性,而被研究者列为国内外琥珀酸发酵研究较多的菌株之一。国内关于琥珀酸放线杆菌的研究内容主要有菌种筛选、菌种诱变、动力学模型以及分批发酵研究。本文对产琥珀酸放线杆菌菌株ATCC55618进行紫外诱变,筛选出耐酸pH3.5的菌株M1,继而对M1采取两轮紫外-亚硝基胍复合诱变,筛选出了高产琥珀酸菌株R1、R2,并对诱变后菌株R2的培养基进行了培养基优化。本研究内容主要包括：(1)采用平板计数法,确定了菌株的最佳紫外诱变时间为50s,将出发菌株ATCC55618厌氧培养至对数生长期,取0.1mL菌液涂布于pH7.0、pH6.7、pH6.4、pH6.1、pH5.8、 pH5.5、pH5.2、pH4.9、pH4.6、pH4.3、pH4.0的培养基平板上,确定产琥珀酸菌株生长的临界pH为4.6,之后对菌株进行紫外诱变,将诱变后的菌液7倍稀释,涂布于pH3.5的固体培养基平板上,筛选出耐pH3.5强酸的菌株M1,其琥珀酸产量为17.01g/L,较原始菌株提高了10.53%。(2)平板计数法确定菌株诱变最佳亚硝基胍浓度500μg/mL,之后用含0.1%的溴甲酚绿变色平板筛选,对M1进行两轮紫外-亚硝基胍复合诱变,分别筛选出琥珀酸高产菌株R1、R2,第一轮复合诱变菌株R1产量较耐酸菌株M1提高率为22.69%,较原始菌株提高率为35.61%,第二轮复合诱变菌株R2产量较R1提高率为16.96%,较M1提高43.50%,较原始菌株提高了58.61%。(3)以菌株R2的菌体生长状况、发酵液pH及琥珀酸产量为指标,分别对碳源、氮源、初始pH、pH调节剂、金属离子等五个因素进行研究,对该菌株的培养基进行优化,检测方式为每3h测一次,共测48h,最终优化结果：最适碳源为葡萄糖,最适氮源为酵母浸粉,最适发酵液初始pH为7.0,最适pH调节剂为NH3·H2O,最适的金属离子为Mn2+。(4)分别对葡萄糖,酵母浸粉,Mn2+三个因素进行单因素试验,确定各组分添加量,葡萄糖添加量分别为20g/L,25g/L,30g/L,35g/L,40g/L,酵母浸粉添加量分别为4g/L,7g/L,10g/L,13g/L,16g/L, Mn2+添加量分别为0.2g/L,0.3g/L,0.4g/L,0.5g/L,0.6g/L,以琥珀酸产量为指标,确定各组分的添加量,优化结果为：葡萄糖30g/L,酵母浸粉10g/L, Mn2+0.3g/L。(5)采用正交试验对单因素试验确定的培养基条件进一步优化,确定三个因素的最适添加量。葡萄糖添加量分别为27g/L,30g/L,33g/L,酵母浸粉添加量分别为8g/L,10g/L,12g/L, Mn2+添加量分别为02g/L,0.3g/L,0.4g/L,以琥珀酸产量为指标,确定最佳添加量分别为葡萄糖27g/L,酵母浸粉10g/L, Mn2+0.2g/L。更多还原

【Abstract】 Succinic acid, a common natural organic acid, is one of the products among the Krebs cycle of organism, it exists widely in the human body, animals, plants and microbes. Succinic acid is used in following fields: surface active agent, chemicals, additives and foaming agent; chelator that is used in electroplating industry; acidification agent in the food industry, pH improver, flavor substances and antiseptics; and it is also used in the production of Medicine, antibiotics, amino acids and vitamins; it is the main raw materials of biodegradable plastic PBS. In recent years, producting succinic acid by microorganism fermentation becomes the focus of research, most research are focused on Actinobacillus succinogenes at home and abroad, Research about Actinobacillus succinogenes in the domestic are mainly includes the screening of strains, Mutagenesis, kinetic model and batch fermentation. In this paper, Actinobacillus succinogenes ATCC55618was irradiated by Mutagenesis Ultraviolet radiation, and through that procedure we obtained a strain M1that can tolerance low pH, then M1was irradiated by both UV and nitrosoguanidine (NTG), strainsRl, R2that are of high producing are obtained, then the strain R2was analyzed among culture media optimization.This study includes:(1) Using plate counting method to determine the proper UV mutagenesis time is50s,0.1mL of culture is at logarithmic phase is coated at medium plates that with pH7.0, pH6.7, pH6.4, pH6.1, pH5.8, pH5.5, pH5.2, pH4.9, pH4.6and of pH4.3, pH4.0, the critical pH4.6of Actinobacillus succinogenes ATCC55618is determined, after UV mutagenesis, a strain M1is obtained, whose yield is17.01g/L, increased by10.53%than the original strain.(2) Using the plate counting method to determine the proper NTG concentration500μg/ml, then M1was irradiated by both UV and nitrosoguanidine (NTG). After two rounds of the compound mutation, high producing strains R1, R2were obtained, the yield of succinic acid of R1is20.87g/L, improved22.69%than M1, improved35.61%than original strain,the yield of succinic acid of R2is24.41g/L, improved16.96%than R1,43.50%than M1,58.61%than the original strain ATCC55618.(3) The influence of the culture media factors on the growth of R2were carbon source, nitrogen source, incipient pH, pH regulator, and metalion pH of fermentation broth. The consequence of the analysis showed that glucose as carbon source, yeast powder as nitrogen source, incipient pH7.0, ammonia water as pH regulator, Mn2+as metalion.(4) Design single factor experiment of the factors:glucose, yeast extract, Mn2+. Addition of glucose was20g/L,25g/L,30g/L,35g/L,40g/L, addition of yeast extract was4g/L,7g/L,10g/L,13g/L,16g/L, addition of Mn2+was0.2g/L,0.3g/L,0.4g/L,0.5g/L,0.6g/L. Succinic acid production among single factor experiments were analyzed. The optimal amount of each component to add, as follows:glucose30g/L, yeast extract10g/L, and Mn2+0.3g/L.(5) To further optimize the conditions of the medium, the amount of the three factors were optimized by orthogonal experiments. Addition of glucose was27g/L,30g/L,33g/L; addition of yeast extract was8g/L,10g/L,12g/L; addition of Mn2+was0.2g/L,0.3g/L,0.4g/L, respectively. Succinic acid production are analyzed. The optimal amount of glucose27g/L, yeast extract10g/L, Mn2+0.2g/L.更多还原