摘要
本论文主要研究了发酵液中γ-氨基丁酸(γ-aminobutyric acid, GABA)的测定方法、GABA产生菌的选育及产酸条件优化,主要内容及结果如下:
1)论文首先研究了GABA的测定方法,即用纸层析法定性分析GABA,纸层析-比色法定量分析GABA。确定了纸层析分析方法中展开剂和显色剂的组成,展开剂组分正丁醇∶冰醋酸∶水(V/V)=2∶1∶1为最优比例,显色剂茚三酮浓度为8g/L时较为合适,此时GABA和谷氨酸的Rf值有明显差异,分离效果最好。并确定了最佳分析条件:当波长在513nm处,显色温度为80℃、显色时间和洗脱时间均为30min时,实验效果最明显。并制作了GABA含量与吸光度值的关系曲线,即GABA标准曲线,为常规检验样品中的GABA含量提供了可靠的实验依据。进一步对样品重复性实验、加标回收率等进行了分析,试验结果表明,纸层析-比色法定量GABA的试验方案重复性较好,相对标准偏差仅为0.272%,加标回收试验的平均回收率为98.1%,说明此法测定具有较高的可靠性,为后续研究工作奠定了基础。
2)实验从酸菜中分离、筛选到一株野生菌(乳酸菌35#),它能够将L-谷氨酸钠转化为GABA,产量为1.85g/L,是一株很有潜力高产GABA的菌株。随后对35#菌的形态学特征及生理生化特征进行了研究,在MRS培养基上30℃培养2d后,发现菌落较小,呈圆形乳白色,不透明,中间隆起,边缘整齐,周边有小皱纹,表面湿润光滑。其分类学研究发现:菌株为G+,兼性厌氧,石蕊牛乳试验使石蕊变红,不液化明胶,不产硫化氢,V-P试验阳性,不产过氧化氢酶,吲哚试验阴性,甲基红试验阳性,葡萄糖发酵产酸不产气,蔗糖、果糖、乳糖、甘露糖等发酵阳性,最适生长温度28~34℃,最适生长pH6.5~7.5。根据《伯杰细菌鉴定手册》,该菌株初步鉴定为乳酸链球菌。
3)实验还对乳酸菌35#的发酵培养基和发酵条件进行了优化,得出培养基的组成(g/L):蔗糖10,丁二酸钠10;胰蛋白胨5,酵母膏5;L-谷氨酸钠10;优化后的最佳发酵条件:培养基初始pH6.5、培养温度32℃、接种量8%、培养时间为静置培养48h,比国内其他研究GABA高产乳酸菌种发酵周期缩短24h,在最佳培养基和发酵条件下,发酵液中GABA的含量达4.57g/L,比优化前有了显著提高。
The study mainly investigated the method for detection ofγ-aminobutyric acid (GABA), the breeding of GABA-producer 35# and the optimization of medium contents and fermentation conditions for 35#. Its contents and results are as follows:
The method for detection of GABA was investigated in the beginning of this study. The paper chromatography accompanying colorimetry was used to measure the concentration of GABA. Then the composition of exhibition layer system and color system included in paper chromatography was determined. The best ratio of the exhibition layer agent was butanol: acetate:water (V/V) =2:1:1, and the proper concentration of coloring agent ninhydrin was 8g/L. It was found that the there was evident difference between Glu and GABA on Rf, which signified the condition for isolation was the best. And then we determined the best analytic conditions: the wavelength at 513nm, chromogenic temperature at 80℃, chromogenic time 30min, preserving time of eluent 30min. The relation curve of GABA content and OD513 was made out, which was also called standard curve of GABA and provided reliable experimental data for detecting the GABA content in samples. Furthermore, the repeated test and value add recovery rate of the sample was carried out. It turned on that using paper chromatography and colorimetry to quantitatively analyse GABA had a better repeatability, and the relative standard deviation was only 0.27%, meanwhile, the average value add recovery rate was 98.1%. These results signified that this method had higher reliability, which provided favorable base for the further study.
In this study, a wilding bacterium (lactobacillus 35#) was isolated from pickled vegetable, which could transform L-MSG to GABA, and the GABA content of the culture reached 1.85g/L. Then the morphological characteristic and physiological characteristic of lactobacillus 35# was studied. After 2 days cultivation on MRS medium at 30℃, the germ was small, round, ivory-white and opaque, it was swelled up in the center, the edge was tidy, the periphery contained small crease, the surface was moist and smooth. It was found by its classification that the strain was a G+, facultative anaerobes, making the litmus red, glutin test negative, H2S test negative, V-P test positive, catalase test negative, indole test negative, M-R test positive, it produced acids but no gas by glucose zymolysis; sucrose, fructose, lactose, mannose test positive; the optimum growth temperature at 28~34℃, the optimum growth pH at 6.5~7.5. Basing on the Bergey’s Manual of Systematic Bacteriology, this strain was preliminarily identified as Streptococcus lactis.
The experiment optimized the culture medium and conditions for lactobacillus 35#. Based on the results, a piece of better medium constitution as follows(g/L): sucrose 10, sodium succinate 10,tryptone 5, yeast extract 5; L-MSG 10; and the better fermentation conditions as follows: initial pH6.5, temperature 32℃, seed volume 8%, fermentation time 48h, which is less than other internal study for 24h. The content of GABA was over 4.57g/L in the optimum culture conditions, there was prominent improvement on the yield.
引文
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