Study On Extracting Theanine By Surfactant Aqueous Two-phase System

Theanine is one of the most promising functional ingredients in home to natural medicine, natural health products, functional beverage segment, but the body can not synthesize. Currently obtain the theanine only chemical synthesis and extraction separation from the tea, chemical synthesis is not easy to get raw materials, there is pollution,toxic and difficult to purify ( D and L-type ), and other shortcomings; Theanine from tea extract, theanine content of only 1% to 2%, extraction complex, high production costs, and also easy is polluted by Pb2+, Cu2+, S2-. Taking into account the production of tea polyphenols waste contains some theanine, if it can be separated out by a lower cost, not only won the high value theanine, and can be very good to solve polyphenols manufacturer of industrialwaste outlet. Therefore, obtained theanine from the polyphenols waste safely and effectively has important practical significance.In recent years, the aqueous two-phase has been widely used in the biochemical field. Because aqueous two-phase system not only can meet the required of biological material water environment, but also it is easy to enlarge the process. This advantage has been initially unfolded in bioseparation technology by the polymer aqueous two-phase systems. Due to surfactant aqueous two-phase system superior than the polymer two-phase system in some respects, such as low surfactant concentration, high selectivity, with the scalability, etc. However, there is no the studying currently that surfactant aqueous two-phase systems extract theanine, which is a area to be studied and developed.This paper to the production of polyphenols obtained waste as raw material, first, it studied the analysis of detection of theanine in tea polyphenols waste, and analyzed the main component of the polyphenols waste raw materials, then elected suitable method for testing theanine in raw materials. Second, the paper used cationic surfactant (SDS) and anionic surfactant (CTAB) preparate the aqueous two-phase, and explored the physical and chemical properties of the aqueous two-phase, then extracted theanine in the raw materials with the aqueous two-phase system. Finally, producted the phase diagram of non-ionic surfactant (PEG) and (NH4)2SO4, explored the aqueous two-phase system at different molecular weight and concentration of PEG, different concentrations of (NH4)2SO4, different temperatures, and extracted the theanine of raw materials with the aqueous two-phase system. The main results of this work can be summarized as follows:1. The research with analytical detection method of theanine: The applicability that the HPLC method, TLC and the ninhydrin color test detect theanine in polyphenols raw materials was studied, the results show that: HPLC method is more suitable for testing theanine in the raw material, testing conditions: C18 column 210nm, 0.08% trifluoroacetic acid solution as mobile phase. 2. The analysis of Polyphenols waste raw materials composition: The polyphenols waste ingredients: solid insoluble about 8.4%, theanine about 2.3%, approximately 42.1% of the carbohydrate, protein 4.3%, polyphenols of about 1.8%, remaining inorganic salts, pectin and a small amount of other impurities. Therefore, the theanine in the raw material was extracted, taking into account the distribution of the main component carbohydrate and protein, at the same time.3. The study with SDS/CTAB aqueous two-phase system extraction theanine:1) SDS/CTAB/H2O ternary phase diagram has been divided into three categories:①the homogeneous district, SDS / CTAB of mixed system molar ratio from 100/0 to 75/25 and 25/75 to 0/100;②the precipitation district, SDS / CTAB of mixed system molar ratio from 67/33 to 33/67;③the aqueous two-phase district include two parts: SDS / CTAB of mixed system molar ratio from 74/26 to 68/32 and 32/68 to 26/7. ATPSa district, when the total SAA concentration less than 0.046mol / L, it can not form aqueous two-phase system; ATPSc district, when the total SAA concentration less than 0.037mol / L, it can not form aqueous two-phase system. The phase volume ratio R increases with increasing of CTAB molar content, the phase volume ratio R decreases with the decreasing of the total concentration of SAA in ATPS.2) Adding NaBr and Na2SO4 in mixed system of SDS /CTAB, can make the phase separation time shortened to1/5 of no salt, and the phase interface is more stable. NaBr is more outstanding than Na2SO4 in forming ATPS time, ATPS interfacial stability and width of ATPS district. Therefore, NaBr is added to the SDS / CTAB ATPS when the theanine is extracted from polyphenols raw materials.3) With decreasing of CTAB molar content, partition coefficient of theanine decreases gradually to 0.056, the lowest partition coefficient of proteins in the upper and lower phase was 21.9, the highest to 111.5, partition coefficient of carbohydrate gradually decreases until 0, so molar content of CTAB as low as possible on the basis of the forming ATPS.4) In the conditions of SDS / CTAB = 2.8/7.2, increasing the concentration of polyphenols waste, the distribution coefficient of theanine and sugar decreasing gradually, the partition coefficient of the protein gradually increasing, when polyphenols waste concentration is 36mg/mL, the extraction rate of theanine and sugars in the next phase is 100%, protein and other colored substances were in the upper phase, it is better to separat theanine and sugars with protein and other color substances in the raw materials waste of polyphenols.4. The study with PEG / (NH4)2SO4 aqueous two-phase extraction theanine 1) The greater PEG mass fraction, the smaller Na2SO4 mass fraction of the formation of aqueous two-phase which is required; PEG mass fraction under certain conditions, the greater the molecular weight of PEG, the lower the mass fraction of the (NH4)2SO4; (NH4)2SO4 mass fraction under certain conditions, the order with mass fraction of forming aqueous two-phase required for the different molecular weight PEG as follows: PEG1000>PEG4000> PEG6000> PEG10000; Increasing the molecular weight of PEG, can shorten the time of forming aqueous two-phase. PEG4000 was selected after comprehensive evaluation.2) Within forming ATPS, increasing the PEG4000 concentration did not affect the partition coefficient of theanine and protein, but apparently increased the distribution coefficient of the sugars, the mass fraction of (NH4)2SO4 was selected as 15% after comprehensive Considering.3) Elevated temperature, time of forming ATPS shortening, while the partition coefficient of theanine, protein and carbohydrate decreases. Therefore, elevated temperature had no effect to extract theanine, but can make the time of forming ATPS shorten, selected extraction temperature 30℃after comprehensive Considered.4) The appropriate conditions that PEG / (NH4)2SO4 aqueous two-phase extraction theanine in polyphenols raw materials are, the average molecular weight of PEG was 4000, the quality percentage of PEG is 10%, the quality percentage of (NH4)2SO4 was 15%, 30℃. Under these conditions, the distribution coefficient of theanine K1 = 0.16, the distribution coefficient of protein K2 = 0.28, the distribution coefficient of sugar K3 = 9.8, the extraction rate of theanine in the low phase was 89.5%. Therefore, the system can separate the theanine and protein with sugars and other color impurities in the polyphenols raw materials.5. The researched results were consolidated as follows: Surfactant aqueous two-phase system extracted the theanine in polyphenols raw materials, it is required to use the two ATPS in this paper (SDS / CTAB and PEG / (NH4)2SO4) in order to separate theanine with protein, carbohydrates, that is the two systems using at the same time can extract the theanine from the waste of polyphenols raw materials entirely.