| Germinated brown rice is the rice products that got0.5-1mm long germs and endosperms with the bran layers. The brown rice was put in a certain temperature and humidity environment and trained up to germinated to a certain extent, then the enzymes of the rice were inactivated. At this time, A large number of nutrients that were not existed in brown rice are found in germinated brown rice, active substances with special physiological functions are significantly increased, meanwhile, eating qualities are improved which were obviously lacking in the brown rice, y-aminobutyric acid (GABA) is a water soluble non-protein amino acids and it wildly present in plants and animals. GABA has a high physiological activity. GABA is the important inhibitory transmitter substance existing in the higher animals’nervous systems, it can directly take part in neural activities as neurotransmitters or neurotransmitter precursors and it also participates in a variety of body metabolic activities. Amount of GABA is produced in brown rice germination process, GABA content increased by10times compared with white rice. Germinated brown rice is an important source of human body obtained GABA. Therefore, a series of studies like to increase the GABA content in germinated brown rice by optimizing the process conditions, to introduce a more accurate method of GABA assay, to choose the best extraction method extracted GABA from the germinated brown rice and to purificate GABA with cation exchange resin have a very important significance.The paper decided to adopt the HPLC method to precise determine the GABA content because of the electrochemical and UV-visible light insensitivity of GABA. GABA is an amino acid, it can rapidly derivatization react with o-phthalaldehyde (OPA) and come out to1-thio-2-alkyl isoindole adduct under the conditions of alkaline conditions and with the exist of the thiol reagent3-mercaptopropionic acid, the derivatives can be determined by HPLC with fluorescence or ultraviolet. In this paper, the OPA pre-column derivatization and HPLC-UV338nm detection were used to detect the GABA content in germinated brown rice. The abilities of extracting GABA were accurate analyzed in different methods such as vortex oscillation extraction and reflux extraction. Finally we found the detect conditions are as follows: the column was a C18reverse phase column, retention time was22min. Linear equations is y=4.72×10-7x+3.95×10-3, regression coefficient R2=0.9991. A good linear relationship was obtained between the concentration of GABA from0.02to0.2mg/mL, average regression coefficient were100.00%. The GABA contents came from different extraction methods with the same germinated brown rice powder were tested in this method, the result was that the maximum detection value comes from drawing the rice powder with60%ethanol in37℃oscillating water bath for4hours.Brown rice can finally be germinated brown rice after screening, cleaning, disinfecting, soaking, germination, enzyme inactivation under certain conditions. So germination conditions, as well as exogenous additive type and concentration, would make obviously great differences at the content of varieties of substances in germinated brown rice. In this article, we respectively studied the effects that how6factors affect the GABA contents in germinated rice grains during germination, the6factors are as follows: soaking temperature, soaking time, the pH value of soaking liquid, the addition CaCb concentration in the soaking liquid, germination temperature and germination time. We use Plackett-Burman Design to select3key factors from these6factors, which took more important roles related to GABA contents in germinated brow rice. The3key factors are soaking time, the addition CaCh concentration in the soaking liquid and germination time. Then a Central Composite Design was held to optimize brown rice germination conditions for high levels of GABA with these three key factors. After the data processing and analysis, the optimum conditions we got was soaking temperature of35℃, soaking time30h, the pH value of soaking liquid is5, the addition CaCl2concentration is2.12mmol/L, germination temperature of25℃and germination time is12.15h. The forecast for the content of GABA in germinated brown rice is76.294mg/100g. After validation of the verification tests, the prediction accuracy is up to98.76%.GABA is an acidic substance, but also with basic groups-amino. In the acidic environment, amino combined a proton to be the cationic state which easy to be adsorbed by the resin; under alkaline conditions, carboxyl ionizated a proton to be the anion state with negatively charge, so that the cation exchange resin adsorption decreased. In this paper, so we use strong acidic732cation exchange resin to separate and sublime the GABA in germinated brown rice can reach a wonderful effect. The sample flow velocity greater, the adsorption effect would be worse, so we choose3mL/min as sample flow velocity so as to taking the adsorption effect and processing time into account at the same time. The adsorption effect is significantly when the pH value of crude extract indicated acidic, while the pH value indicated alkaline,732strong acid cation exchange resin barely adopt GABA, so we adjust pH value of crude extract to3in our experiment. The pH value of eluent plays a very important role in the process of elution. The elution efficiency is obviously better in alkaline conditions than that under acidic environment. In this article we adjust pH value of eluent to8-9can perfectly elute GABA which adsorbed on the cation exchange resin. |
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