Study On The Determination Of γ-aminobutyric Acid By HPLC And The Pretreatment Of Bioconversion Broth

Bioconversion broth of y-aminobutyric acid (GABA) is a complex system containing GABA that is synthesized by glutamate decarboxylase (GAD) in microbe cells to catalyze irreversible a-decarboxylation of L-glutamate. It is of great importance to establish an economical and applicative post-extraction process for high-purity GABA to improve the quality and market competitiveness of the product. Due to the bioconversion broth of GABA got complex ingredients, improving the preprocessing efficiency was the key that improved the quality of product and reduced the cost. In this study, the pretreatment conditions of GABA bioconversion broth were optimized to lay a foundation for further extraction and purification of GABA, and also, a quantitive analysis method was developed. The main results are as follows:1. Combining the characteristics of bioconversion broth of GABA, a reverse high performance liquid chromatography (HPLC) method for determination of GABA derivatized using O-phthalic aldehyde (OPA) prior to HPLC analysis was developed. Satisfactory separation was achieved at27℃and338nm by using Eclipse XDB-C18column and a gradient system. Under the conditions described above, the linear range was from0.01mmol/l to10.00mmol/1, and GABA was well separated from L-glutamate and other impurities in sample at about19.0min. The assay accuracy of GABA in bioconversion broth was not remarkably changed while adding ethanol in the broth or regulating the pH value of the broth from3.0to8.0. The intra-day and inter-day precisions (%RSD) for retention time were0.78%and0.72%, respectively, while those for peak area (%RSD) were1.64%and1.24%, respectively. Therefore, the method described was of high sensitivity, good repeatability and suitable for the determination of GABA in the bioconversion broth.2. The results of decoloration study showed that atlapulgite was better than active carbon for bioconversion broth of GABA. The content of pigments was decreased by65.32%while directly adding2%of atlapulgite into the bioconversion broth of GABA under the optimal condition that was shaking for20min at40℃. 3. The optimum operation parameters of ultrafiltration were determined using the broth after decoloration. The protein removal ratio and decolorization ratio of the broth with the original pH value after ultrafiltration were88.37%and50.00%, respectively, under the optimal conditions, at0.2Mpa and65rpm. With the increase of concentration ratio, the recovery ratio of GABA improved gradually and reached90.76%, the maximum. Our results indicated that separation and purification of GABA from bioconversion broth by ultrafiltration had achieved the anticipated target.