| Theaflavins is a general term for a class of water-soluble phenoxolone compounds with multiple hydroxyl or phenolic hydroxyl groups formed by the oxidation of tea polyphenols.It is the main component of black tea pigments.The currently identified theaflavins include theaflavin(TF1),theaflavin-3-gallate(TF2a),theaflavin-3’-gallate(TF2b)and theaflavin-3,3’-bisgallate(TF3)four monomers,etc.Theaflavins has good effects in regulating blood lipids,scavenging free radicals and anti-oxidation,anti-tumor,anti-viral,bacteriostatic and neuroprotective,its excellent physiological activity makes it an ideal natural raw material for drug development,functional food,and daily chemicals,it is also one of the environmentally friendly plant-based pesticides with independent intellectual property rights developed by China.However,theaflavins has not made breakthroughs in industrialized production technology since its discovery.In this study,on the basis of optimizing the determination of polyphenol oxidase enzyme activity and the establishment of high-performance liquid chromatography for simultaneous analysis and detection of catechins and theaflavins,using exogenous enzyme biological fermentation technology,through screening of efficient,safe and Polyphenol oxidase enzyme source,we further optimized the simulated fermentation synthesis of theaflavins in vitro,and combined column chromatography technology to purify the crude theaflavins,reduced the industrial production costs of theaflavins to form economic benefits,created prerequisites and a good foundation to form social benefits for the subsequent theaflavins products development and utilization.The main results are as follows:1.A method for the simultaneous detection of 11 substances such as GA,EGC,DL-C,EC,EGCG,GCG,ECG,TF1,TF2 a,TF2b and TF3 in black tea was developed using high performance liquid chromatography.Using Wonda Sil-C18 liquid chromatography column(4.6 mm × 250 mm,5μm),through the optimization of mobile phase acid concentration,flow rate and column temperature,mobile phase A was determined to be 0.1%(v/v)formic acid aqueous solution,mobile phase B 0.1%(v/v)formic acid acetonitrile solution with flow rate 1.0 m L/min,injection volume 20 μL,detection wavelength 280 nm,and column temperature 30°C.At the same time,the linear range and correlation coefficient were examined,and the repeatability test,precision test,stability test,and accuracy test were performed.The relative standard deviations were all less than 5%,and the recovery rates were between98.69% and 104.76%.2.The current conditions for measuring the activity of polyphenol oxidase were improved.The results showed that the optimum condition for measuring the activity of polyphenol oxidase was to extract fresh leaves homogenate at 4℃ for 12 h,centrifugate at 8000 rpm for 15 min,and take the supernatant enzyme solution at p H 6.0 and 37°C,add 1.5% catechol as the substrate,terminate the reaction with 3 m L metaphosphoric acid(1 mol/L)after reaction for 10 min,and measure the absorbance value at 460 nm.The standard deviation and relative standard deviation of the results were less than 5%.3.Based on the results of the single factor test,the temperature,p H,and substrate concentration were selected and a three-level Box-Behnken(BBD)test was used to optimize the process conditions for theaflavin synthesis.The optimal synthesis process parameters of theaflavin were as follows: 9.18mg/m L catechin B(EGC 16.50%,EC 4.90%,EGCG 41.61%,ECG 3.04%,total catechin 70.84%)was catalyzed by crown pear polyphenol oxidase with 30 times catechin content at p H 5.45 and 24.9℃ for 30 min,filtered by gauze,extracted by ethyl acetate,dried the ester layer to obtain theaflavin primary product.Three verification experiments were performed,and the average value was 33.01%,which was close to the predicted value 32.49% of the model.The results showed that the model was feasible to optimize theaflavin synthesis conditions.4.It was found that HP-20 macroporous adsorption resin had the best effect of separating and purifying the crude theaflavin,and the optimal process after optimization was: 80 g crude theaflavin was dissolved in 10 times of 80°C hot water.After overnight standing,the supernatant was put on the column at the flow rate of 1 BV/h.After washing with 3 BV water,it was eluted with 1 BV 35% ethanol to remove impurities,and then desorbed with 80% ethanol until the color of eluent was lighter,80% ethanol eluent was collected and dried by rotary evaporation.Purifying theaflavin according to this process,the content of theaflavin increased from 30.49% to 59.64%,and its content increase rate was 95.62%. |
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