| L-theanine is a kind of characteristic non-protein amino acid mainly found in green tea (Camellia sinensis). It has the special physiological and pharmaceutical functions. In this article, the technology of recovering theanine from the raffinate of tea polyphenols or the waste water produced in processing tea polyphenols in industry was studied in order to give some scientific message for the comprehensive utilization of tea and find out a new method to produce theanine.Through ultrafitration, decolorization, ion-exchange and column chromatography, theanine with the purity of was 95.1% was obtained. The main results were as following:(1) The paper chromatography-mottle elution method was compared with high performance liquid chromatography (HPLC) method in order to establish the optimal methods determining the content of theanine. The results obtained from paper chromatography method was higher or lower than HPLC method 5-9 %, thus it was used to the relative quantitative analysis, while HPLC method was used to quantify theanine accurately.(2) The tea polyphenols raffinate was pretreated by sedimentation method and ultrafitration. The loss of impurities by adding saturated neutral Lead acetate is 41.3%, with the recovery of theanine 90.2%;while by ultrafiltration is 50.4% and 95.5% respectively. In experiments of one-factor design, appropriate ultrafiltration conditions were obtained: the polysulfone membrane with the molecular weight cut-off (MWCO) of 6KD;the ultrafiltration pressure 0.09Mpa;the ultrafiltration temperature 35 ℃;volume concentration ratio 4.5;the ultrafiltration time 2h. The solution of 0.5mol/L NaOH and 1% NaClO was applied to refresh the ultrafitration membrane.(3) The resin D301-G was used as a medium on the decolorization of the pre-treatments solution. The optimal decolorization conditions were: the flow rate 4BV/h, at normal temperature, the treatment capacity of D301-G resin 20BV, and the recovery of theanine was 93.5%. The conditions of resin regeneration were also studied. The result was that 1mol/L Hcl (containing 50% acetone) has an effective regeneration performance on the resin.(4) 001 ×7 resin as the adsorption medium on ion-exchange chromatography was adopted to separate L-theanine from the pretreated liquid. The factors effected on exchange capacity of L-theanine was analyzed, and the kinetics of adsorption were studied. By response surface analysis (RSA) the optimal conditions of adsorption in the fixed-bed of 001×7 resin were resulted as following: pH of pretreatment liquid 3.4, concentration of L-theanine 3.0mg/ml, sample flow-rate 1.7BV/h. The Amomomia water with pH11.3 was used as eluant. The content of L-theanine in crude product reached 58%, recovery of theanine was 82%.( 5 ) The crude theanine was further isolated on microcrystalline cellulose column chromatography. The mobile phase was butyl alcohol: glacial acetic acid: water (4:1:5, V/V/V), flow rate 0.3ml/min.The eluate was concentrated and crystallized by absolute ethanol. The crystal was dried at 60℃ and vacuum condition, theanine was purified to 95.1% determined by HPLC.
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