| Pu’er tea is a unique historical and tradition kind of tea, originating in Xishuangbanna, Simao, Chuxiong, Honghe, Dali areas. Taking dried raw green tea as materials, which abtained from fresh leaves of Yunnan big-leaf species after fixation, rolling, sun drying processes. Then a variety of shapes of finished tea were made after the fermentation process, presenting its special maroon leaves, thickly red and bright tea infusion, aged fragrant, long-lasting aftertaste sweet. Those all are the unique quality features. Pu’er tea are drawing more and more attention, because of its rich nutritious components, the function of anti-oxidation, anti-cancer, anti-mutagenic, immunity enhancement, lipid lowering, beauty and longevity maintaining, and other significant biological activity. It is also widely used in health food, drug, daily chemical industry. Separation and purification of Pu’er tea and the study of the physiological activities and processing of Pu’er tea are also in full swing worldwide. In this paper, taking Pu’er tea instant powder as the experiment object, comparisons and anlyses of tea segment retentions were made with six kinds of macroporous resin: XAD-16, HZ816, NKA-9, D101, HPD300, HPD600 and two kinds of polyamide resin in 30～60 mesh and 60～100 mesh under different concentrations of ethanol as eluent. Testing the contents of taste substances in each retentate, analyzing the separation effectiveness and separation law of each resin and selecting one with the best extracting performance of Pu’er tea after which the author identified the key compounds separated by resin with UPLC-MS/MS and analyzed their contributions to the taste of Pu’er tea so as to meet different requirements of flavors by components blending based on the property and separation laws of taste substances as well as provide a theoretical basis and a new path for the deep processing products of Pu’er tea. The main findings are as follows:1. Preparation of Pu’er tea instant powderThis paper studied the influences of the different extraction time, extraction temperature and solid-liquid ratio to Pu’er tea water extracts combined with single factor and three-factor-three-level orthogonal experiment to find the optimum conditions for the extraction of Pu’er tea:extraction temperature:90℃ Time 70min; solid-liquid ratio:1:24. Extracting tea substances under the optimum conditions and by freeze-drying to obtain the Pu’er tea powder, the yield was 82.49%.2. Resin screening, separation and parameter optimization:(1) Results of gradient elution:in the aspect of desorption rates, XAD-16, HZ-816, NKA-9, D101 those four kinds of macroporous resins had significant separation effect for polyphenols, caffeine, amino acids, flavonoids, total sugar and theabrownin but poor separation effect for total catechins, theaflavins and thearubigin. Although the overall separation effect of polyamide resin was not very good, the desorption rates for polyphenols and caffeine were much better which allowed it to separate bitter and astringent substances more effectively.(2) Tests of parameters optimizationComparisons of separation effect of Pu’er tea were made between macroporous resin XAD-16, HZ816, NKA-9, D101 and polyamide resin of 30 to 60 mesh,60 to 100 mesh. The result showed that D101 had the best separation effect. Polyphenols, amino acids and total catechins were most enriched in water eluate and 20% and 40% ethanol eluate while caffeine was concentrated in 20% and 40% ethanol eluate; Most of the amino acids were found in the water eluate. Flavonoids, total sugar, total catechins and theabrownin all had their own peaks at each stage. Polyamide resin of 60-100 mesh was used after D101 separation. Caffeine was significantly enriched in the first column volume of water eluate; A large concentration of flavonoids were concentrated in 40% ethanol eluent and polyphenols and catechins in 60% ethanol eluent. Bitter and astringent substances were basically separated. And in this elution order, desorption rates of each substance were up to 85.67%～96.02%. The resulting samples were freeze-dried, and the yield of D101 separated tea instant powder was 91.19% higher than that of polyamide of 60-100 mesh whose yield was 84.26%.3. Conclusions drawn from UPLC-MS/MS test results:(1)104 kinds of substances were identified in Pu’er tea instant powder and 93 kinds with resin separation. Only 8 kinds of gallic glucose and 3 kinds of theaflavin-3’-gallate were not successfully separated by the resin and those who had been separated successfully were basically the same with the earlier identification both in category and quantity. Those substances were 5 kinds of organic acids,16 kinds of non-flavonoid phenolic acids,11 kinds of hydroxy cinnamic quinine esters,16 kinds of flavanols (9 kinds of catechins,3 kinds of catechin glycosides,2 kinds of anthocyanins, 2 kinds of theaflavins),19 kinds of flavonoid glycosides (6 kinds of kaempferols.9 kinds of quercetin glycosides.4 kinds of myricetin glycosides),23 kinds of amino acids and 3 kinds of alkaloids.(2) 93 kinds of substances were separated both by D101 and 60-100 mesh of polyamide resin. The most qualitative components in single samples were gained from the water eluent parts, reseparated by D101 resin and polyamide 60～100 mesh reaching 90 kinds.(3) The UP LC-MS/MS detection results were almost consistent with the results of routine testing. The eluants of 20% ethanol,40% ethanol separated by D101 resin and from water eluant of polyamide resin at 60～100 mesh had strong taste of bitterness, gathering lots of flavanols, flavonoid glycosides, and caffeine. Theanine which shows umami flavor, most gathered in water eluants reseparated by D101 resin and polyamide resin at 60～100 mesh. High content of gallic acid is a symbol of Pu’er tea.4. Contribution of retentates to the taste of Pu’erSensory evaluation found that in samples obtained from D101 resin separation, substances such as amino acids and organic acids were mainly concentrated in water eluant, which showed umami and sour flavor; Bitterness and astringency are mainly concentrated in 20% and 40% ethanol eluant, resulted from the concentration of polyphenols, caffeine, catechins and flavonoids but the consistency of sweet substances is quite poor. They might exist mostly in the form of glycoside and have a higher threshold value which led up to their poor consistency. In the first volume of sample isolated from the polyamide resin 60～100 mesh after D101 separation, a large amount of coffeine were gathered accompanied by strong bitterness; Flavonoids were concentrated in 40% ethanol eluant and polyphenols in 60% ethanol eluant. Bitter substances and astringent substances were basically separated. |
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