Effects of Exogenous Enzymatic Treatment during Processing on the Sensory Quality of Summer Tieguanyin Oolong Tea from the Chinese Anxi County

Introd uction Tea, a popular healthy beverage consumed around the world, is classifi ed into six types: green tea, yellow tea, dark tea (including brick tea and pu-erh tea), white tea, oolong tea and black tea based on the degree of fermentation (oxidation) during processing (1). These types of tea have diff erent quality characteristics including colour, aroma, taste and appearance. Many studies have shown the positive eff ects of green and black tea on oxidative and infl ammatory stress, insulin resistance and lipid metabolism (2). Oolong tea has prov en its eff ects on antioxi-dation, lipid reduction and anti-obesity (3). The fresh tea leaves are rich in catechins (fl avan-3-ols), caff eine and theanine (N-ethyl-γ-l-glutamine), which contribute to the main health-promoting functions and sensory quality of tea. Moreover, there are abundant gly-ISSN 1330-9862 original scientifi c paper Summary I n order to att enuate the bitt er taste and improve the aroma of the summer tieguanyin oolong tea from the Chinese Anxi county, the eff ects of processing treatment with exoge-nous laccase and α-galactosidase on tea sensory quality and related compounds were investigated. The solutio ns of laccase and/or α-galactosidase were sprayed on the tea leaves before the fi rst drying process. The sensory evaluation results showed that the sensory quality of the tea was signifi cantly enhanced with the enzymatic treatment. The combined application of laccase a t 8.25 and α-galactosidase at 22 U per kg of fresh tea shoots achieved the most satisfying sensory quality. Further analysis of fl avour-related constituents was carried out by HPLC and GC-MS. The HPLC analysis showed that the contents of cate-chins and total polyphen ols were reduced, compared to the untreated group, by 11.9 and 13.3 % respectively, and the total soluble sugars and water extract content were increased by 19.4 and 6.6 % respectively, aft er the treatment with both enzymes. The decrease of cat-echins and total polyphenols reduced the bitt erness and astringency of the summer tea, while the increase of total soluble sugars and water extract content improved the sweetness and mellow taste. The aromatic compound data from GC-MS showed that the total essential oil content in these tea samples co-treated with laccase and α-galactos idase increased signifi cantly, in which aldehydes, alcohols, esters and alkenes increased by 23.28, 37 .05, 20.10 and 38.99 %, respectively. Our data suggest that the exogenous enzymatic treatment can enhance the summer …


Introd uction
Tea, a popular healthy beverage consumed around the world, is classifi ed into six types: green tea, yellow tea, dark tea (including brick tea and pu-erh tea), white tea, oolong tea and black tea based on the degree of fermentation (oxidation) during processing (1).These types of tea have diff erent quality characteristics including colour, aroma, taste and appearance.Many studies have shown the positive eff ects of green and black tea on oxidative and infl ammatory stress, insulin resistance and lipid metabolism (2).Oolong tea has prov en its eff ects on antioxidation, lipid reduction and anti-obesity (3).
The fresh tea leaves are rich in catechins (fl avan-3--ols), caff eine and theanine (N-ethyl-γ-l-glutamine), which contribute to the main health-promoting functions and sensory quality of tea.Moreover, there are abundant gly-cosides with monoterpene alcohols and aryl alcohols like aglycone, which having been hydrolysed by endogenous glucosidase release the aglycone during processing.These compounds have fl oral or fruity smell and dominate the formation of tea aroma (4).During the processing period of oolong tea, fresh tea leaves are harvested and undergo the basic steps of being withered, bruised (shaken), partially fermented, fi xed, rolled and dried.Oxidation is limited only to the leaf edges through shaking, known a s ′semi-fermentation′.Tieguanyin tea, a kind of oolong tea that originates from Anxi county (Fujian province, PR China), is one of the top ten famous teas in China and is processed from Tieguanyin cultivar.Besides natural conditions and good agricultural practices of tea plantations, the season of tea production plays an important role in quality.According to the harvest time, tieguanyin tea is classifi ed into spring, summer and autumn tea.It has been f ound that summer tea has higher output than spring and autumn tea, but generally has lower quality (5), the diff eren ce being that summer tea has fewer soluble sugars and more polyphenols and caff eine, undesirable bitt er and astringent taste with lower quality aroma.Given the situation, it is necessary and justifi able to fi nd ways to improve the sensory quality of summer tieguanyin tea.
To optimize the unique quality of tea, endogenous enzymes like polyphenol oxidase and glucosidase in tea shoots should be well controlled.However, this is restricted by the quality of fresh leaves and the production process.Many att empts such as changing the processing conditions have been made to improve t he qua lity of summer tea (6,7).In addition, it has been reported that the quality of te a and related tea products has been improved by introducing some exogenous enzymes such as polyphenol oxidase , peroxidase, tannase, cellulase, pectinase, protease and β-glucosidase into tea processing si nce 1990s.For example, Murugesan et al. ( 8) did screening experiments with a series of exo genous enzymes and found that the purifi ed cellulase combi ned with laccase from Trametes versicolor in the ratio of 3:2 (by volume) was the most effective in enhancing the quality of black tea.In 2013, Kumar et al. (9) reviewe d various major enzy matic methods applied in the ready-to-drink black tea production and discussed the advantages and limitations of these methods.As far as green tea is concerned, β-glucosidase could signifi cantly enhance the aromatic constituents in green tea liquor (10), papain and cellulase could reduce its bitt erness (11), and the proteolytic enzyme derived fr om Aspergillus oryzae could hydrolyse proteins in green tea residues (12).Besides, peroxidase, cellulase and Flavourzyme ® were employed by Li and Liu in 2008 (13) in the processing of pu-erh tea to improve its quality.Above all, exogenous enzyme treatment can achieve the purpose of improving tea quality by ch anging the contents of some constituents in the tea.
Laccase is o ne of the copper-containing polyphenol oxidases (p-diphenol oxidase, EC 1.10.3.2), which was fi rst found in sumac juice in 1883 (14) and is widely present in fungi and bacteria (15,16).Its substrates include, among others, polyphenols, methoxy-substituted phenols and diamines.Laccase is able to transform phenols by self-coupling or cross-coupling with polymeric products.Canfora et al. (17) proved that laccase was eff ective in t he transformation of simple and complex phenolic mixtures.Luo et al. (18) studied the eff ect of the kinetics of biocatalytic activity of laccase during oxidation of phenolic compounds and demonstrated that laccase-catalysed oxidation of phenolic compounds was more eff ec tive than that of epicatechin and catechol.Yue (19) performed the study on synthe sising theafl avins by using laccase.Therefore, laccase has been widely used in food industry (20)(21)(22), such as for juice clarifi cation, beer processing stability and food sensory quality improvement.It can deoxygenate oil, remove bitt erness and other unpleasant tastes of cacao, reduce the bitt erness of olives, improve the colour and enhance the quality of black tea (8).
To the best of our knowledge, so far there have been no reports on the eff ect s of processing treatment of exogenous laccase and α-galactosidase on the sensory quality of summer tieguanyin tea from the Anxi county in China.In order t o improve the fragrance quality of the tea by reducing bitt erness and astringency, we investigated the effects of laccase and α-galactosidase on the fi nal tea quality.The main constituents related to its taste and aroma were also analysed.

Materials
Summer fresh tea shoots (Camellia sinensis cv.Tieguanyin), consisting of dormant bud and three or four adjacent leaves, were hand-plucked in July 2011 from the fi elds of the Xianghua Tea Factory, Anxi Tie Guan Yin Group, Anxi, Fujian province, PR China.

Enzymatic treatment
The basic manufacturing process for summer fresh tea shoots according to Chinese National Standard GB/T 19598-2006 (29) includes the following steps: plucking→ withering and cooling→bruising and rolling→fi xation→ twisting and wrapping→fi rst drying→full dryin g.For enzymatic tr eatment, 10 kg of half-dried tea were sprayed with 220 mL o f aqueous solution containing enzymes prior to full drying, rolled for 2 min and left for 10 min in tea baking box, and then dried fully at 70-80 °C.
As shown in Table 1, the sample not treated with the enzymes was labelled CK, the groups treated with laccase (0.05, 0.1 and 0.2 g/L) A1, A2 and A3 respectively, the groups treated with α-galactosidase (0.1, 0.2 and 0.4 g/L) B1, B2 a nd B3, and t he groups treated wi th both laccase and α-galactosidase (in g/L: laccase 0.075+α-galactosidase 0.1 and la ccase 0.075+α-galactosidase 0.2) C and D respectively.Three parallel experiments were done at each concentration.

Sensory evaluation
The tea samples were examined and scored independently according to Chinese National Sta ndard GB/T 23776-2009 (30 ) by a tea tasting panel consisting of six experts from the Department of Tea Scienc e, Anhui Agricultural University, Hefei, PR China.The tea tasters fi rstly evaluated the appearance of the dry leaves, including colour, size and twist.A mass of 5.0 g of tea samples was infused three times (for 2, 3 and 5 min) in a 110-mL tea tasting porcelain cup with 110 mL of freshly boiled water, and then the infusions were poured three more times into 110-mL tea tasting porcelain bowls for quality assessment.Finally, the tea tasters assessed the aroma, colour and taste of the infusion based predominantly on the infusion time of 3 min.The grading system was based on the maximum score of 100 for each quality att ribute (aroma and taste).

Determination of total polyphenoli c content
The total polyphenols, caff eine, catec hins, free amino acids, tea polysaccharides and water extracts of the tea samples were examined by National Tea Quality Supervision and Inspection Center, Fujian, PR China.The total polyphenolic content (TPC) was determined by spectrophotometry (722S, Shanghai Analytical Instrument Company, Shanghai, PR China), using gallic acid as the standard, according to the method described in the International Standard ISO 1 4502-1:2005 (31).Briefl y, 1.0 mL of the diluted samp les was transferred in duplicate to separate tubes contai ning 5.0 mL of a 1:10 dilution of Folin-Ciocalteu reagent in water.Then, 4.0 mL of a sodium carbonate solution (7.5 %, by mass per volume) were added.The tubes were then allowed to stand at room temperature for 60 min before absorbance was measured at 765 nm against water.The TPC was expressed as gallic acid equi valents (GAE) in g per 100 g of material.The concentr ation of polyphenols in the samples was derived from a standard curve of gallic acid ranging from 10 to 60 μg/mL (Pearson's correlation coeffi cient: R 2 =0.998).

Determination of caff eine and catec hins
Caff eine and catechins were quantifi ed by Chinese National Standard GB/T 8312-201 3 (32) and International Standard ISO 14502-2:2005, respectively (33).Caff eine and catechins were analysed by a HPLC system (2695 / 29 96, PDA detector, Waters, Milford, MA, USA) equipped with a reversed phase Waters C 18 column (5 μm, 250 mm× 4.6 mm i.d.) at 35 °C and 278 nm.Concentrations of caffeine and catechins were quantifi ed by their peak areas against those of standards prepared from authentic compounds.

Determination of water extract, tea polysaccharides and free amino acids
Water extract was determined according to Chinese National Standard GB/T 8305-2013 (34).Briefl y, a volume of 50 mL of tea extra ct prepared as described above was evaporated in a dish on boiling water bat h to partially dry, and further dried in an oven at 103 °C to complete dryness and then weighed aft er cooling down to room temperature in a silica gel desiccator.
Tea polysaccharides were determined by an anthrone-sulphuric acid method described by Wang et al. (35).A volume of 4 mL of 0.033 % anthrone in concentrated sulphuric acid was added to 1 mL of tea polysaccharide solution and heated for 7 min at 100 °C.The absorbance was measured at 620 nm aft er cooling for 30 min, and glucose was used as the standard.The result was expressed in g of glucose per 100 g of material and derived from a standard curve of glucose ranging from 0.025 to 0.2 mg/mL (Pearson's correlation coeffi cient: R 2 =0.999).
The content of free amino acids was de termined spectrophotometrically at 570 nm with ninhydrin colourimetry, using theanine as standard, according to Chinese National Standard GB/T 8314-2013 (36).It was expressed in g of theanine per 100 g of material and derived from a standard curve of theanine ranging from 0.004 to 0.012 mg/ mL (Pearson's correlation coeffi cient: R 2 =0.985).

Determination of aromatic compounds
The aromat ic compounds were analysed and determined by brewed extraction method reported by Kawakami et al. (37).In brief, a simultaneous steam distillation and extraction apparatus was used to extract volatile compounds with ethyl ether as the extraction solvent, and then the extracts were concentrated.Finally, the concentrates were determined by gas chromatography-mass spectrometry (GC-MS, QP2010S, Shimadzu, Kyoto, Japan).Aromatic compounds were extracted as follows: 30 g of tea sample were mixed with 500 mL of deionized water in a 1-litre fl ask, and 1 mL of ethyl decanoate solution (50 μg/mL in ethyl ether) was added as an inte rnal standard for GC-MS quantitative analysis.Then, the volatile constituents were extracted applying simultaneous distillation and extraction method using a modifi ed Likens- -Nickerson apparatus.A volume of 30 mL of redistilled ethyl ether was used as the extraction solvent, and the extraction was continued for 1 h aft er the sample began boiling in the fl ask.The ethyl ether phase was then transferred into a 50-mL glass tube and dehydrated with 5 g of anhydrous sodium sulphate overnight.The dehydrated ethyl ether phase was then concentrated to 1 mL.The concentrate was used for GC-MS analysis.
The fl avour substances were analysed by GC-MS, with DB-5 chromatographic column (30 m×0.25 mm×0.25 μm i.d.).The oven temperature was kept at 50 °C for 2 min, then raised to 60 °C at a rate of 1 °C per min, maintained for 2 min, then raised to 150 °C at a rate of 3 °C per min, and maintained for 5 min, then raised to 250 °C at a rate of 10 °C per min and maintained for 7 min.The injector temperature was 250 ° C. The temperature of the detector was 280 °C.Highly purifi ed helium was used as carrier gas with a constant fl ow rate at 1.0 mL/min.The split ratio was 1:10 and the injection volume was 1 μL.Mass spectrometry was performed in electron ionization mode by scanning the mass spectra at 70 eV in an m/z range from 35 to 400.The aroma constituents were identifi ed by matching GC and MS data with those of authentic compounds and the literature.The relative amount of each compound was calculated by the ratio of the compound peak area to the internal standard (ethyl decanoate) peak area.

Data analysis
The experimental data about taste were shown as the average value±standard deviation (N=3).Data were analysed using data processing system (DPS) for analysis of variance (ANOVA), with statistically signifi cant diff erence at p<0.05.
The aromatic constituents were identifi ed by NIST l47 and NIST 27 libraries (38).

Results and Discussion
Tea taste and aroma are the most important indices to eval uate the sensory quality of oolong tea.Taste refl ects the coordination eff ects of various water-soluble substances in tea infusion, which include soluble phenols, caff eine, amino acids, soluble sugars, etc. Sensory aroma character-istics of tea depend on the total amount of essential oil and the abundance of various aromatic compounds.
Summer tieguanyin tea has plain and thin but bitt er and astringent taste and plain aroma.In order to improve the quality of summer tea, the exogenous enzymes laccase and α-galactosidase were added to adjust the content of one or several taste and aromatic factors.

Sensory evaluation results of tea s amples
The sensory evaluation results of the samples CK, A1, A2, A3, B1, B2, B3, C and D are presented in Table 2 (the sensory tests were conducted according to Chinese National St andard GB/T 14487-2008 (39)) and they indicate that enzymatic treatments had signifi cant eff ects on the sensory quality.
The taste of sample CK was described as bitt er and astringent with signifi cant yin fl avour, and its score was 83.In contrast, astringe nc y decreased with the increase of laccase concentration, while bitt erness decreased with the increase of α-galactosidase concentration.The taste scores of A1, A2, A3, B1, B2 and B3 were 84, 86, 87, 87, 89 and 82, respective ly; sample C was mellow and slightly bitt er, with score of 91; sample D was mellow, sweet and with signifi cant yin fl avour, with a taste score of 93.
Th e aroma of sample CK was described as clean and pure and its sc ore was 85; the aroma became stronger with the increase of α-galactosidase concentration, but sample B3 was described as light; the aroma scores of A1, A2, A3, B1, B2 and B3 were 86, 88, 87, 89, 92 and 89, respectively; both samples C and D had intense and lasting aroma, and their scores were 91 and 93, respectively.In summary, these results indicate that the sensory quality of sample D was the best.Both laccase and α-galactosidase showed signifi cant eff ects on improving the sensory quality of Anxi summer tieguanyin tea.Laccase had a positive eff ect on the taste only, while α-galactosidase had a positive eff ect mainly on the aroma and only partly aff ected the taste.Moreover, the e xperiments sh owed that the concentration of enzymatic solution was an important factor to infl ue nce the eff ects of exogenous enzymatic treatment on the sensory quality.When the mixed enzyme solution with 0.075 g/L of laccase and 0.2 g/L of α-galactosidase was used, the tea received the highest score.

Eff ects of enzymatic treatment on the taste of tieguanyin tea
As shown in Fig. 1, the contents of tea polyphenols in samples A1, A2 and A3, which were treated with diff erent conc entrations of laccase, were 12.3, 11.9 and 11.5 %, l ower than the sample CK (13.4 %), their catechin content was 7.93, 7.29 and 6.73 % respectively, wh ich was lower than that of the sample CK (8.32 %), while the contents of tea polyphenols and catechin signifi c antly decreased along with the increase of laccase concentration.Ther efore, laccase treatment can reduce the content of tea polyphenols and this eff ect is concentration-dependent.
Similar results can also be observed in the samples treated with α-galactosidase, marked B1, B2 and B3.The content of tea polyphenols in sample CK was 13.4 % and it slightly decreased to 12.8, 12.8 and 12.7 % aft er a treatme nt with diff erent concentrations of α-galactosidase (Fig. 1).However, the catech in content in samples B1, B2 and B3 ( 8.24, 8.23 and 8.16 %, respectively) was slightly lower com pared with sample CK (8.32 %), but this was not stati stically signifi cant.These results demonstrated that the eff ects of α-gala ctosidase treatment on the content of tea polyphenols and catechins were relatively weak.
When the combination of these two enzymes (samples C and D) was used, the content of both tea polyphenols and catechins decreased signifi cantly compared with sample CK.When the concentration of α-galactosidase (sample C) was 0.1 g/L, the reduction rate was similar to that of the samples treated with laccase only.However, after 0.2 g/L of α-galactosidase (sample D) was sprayed together with laccase, lower content of tea polyphenols and catechins was determined, which indicated that although the eff ect of α-galactosidase alone on the summer tea was weak, it was able to enhance the eff ects of laccase on the content of tea polyphenols and catechins when these two enzymes were applied together.
As shown in Fig. 1, the total soluble sugars of samples treated with laccase alone (samples A1, A2 and A3), α-galactosidase alone (samples B1, B2 and B3) or the combination of the two enzymes (samples C and D) were signifi cantly higher than in the CK sample (6.5, 6.9 and 7.The increased level of water extract in samples treated with laccase was higher than in the samples treated with α-galactosidase, but the increased level of total soluble sugars in samples treated with laccase was lower than in samples treated with α-galactosidase.Unlike these two results above, the content of caff eine and free amino acids was basically the same whether they were treate d with laccase or α-galactosidase.
It can be seen from the above results that the content of s oluble polyphenols decreased aft er laccase treatment.Aft er enzyma tic treatment, the total soluble sugars and the content of water extract increased signifi cantly, while the change of caff eine and free amino acids was not obvious.Decrease of catechins and polyphenols in tea could eff ectively reduce the bitt erness and astringency of the s ummer tea; increase of the total soluble sugars and the cont ent of t he water extract could balance the fl avour of tea infusion.The reason why α-galactosidase treatment could result in an increase of total soluble sugars may be that α-galactosidase can hydrolyse α-glycosidic bond specifi cally, as has been previously reported in the literature.Both laccase and α-galactosidase can destruct the cell wall structure and promote the release of intracellular nutrients, so the content of the water extract was significantly increased aft er enzymatic treatment.
Low quality of summer tea is diffi cult to improve only by adjusting the processing parameters; however, it has been reported that a treatment with exogenous enzymes can achieve the purpose by changing the contents of some constituents in the tea.The study found that the reason for the bitt erness and astringency of Anxi summer tieguanyin tea is that the summer tea has lower soluble sugars and higher polyphenol content (40).Research has also shown that there are positive eff ects of the higher total soluble sugar ratio of tea polyphenols.T he above results show that appropriate exogenous laccase and α-galactosidase treatment could achieve the expected result of adjusting the content of polyphenols and total soluble sugars, which was proven by the sensory evaluation results.

Eff ects of enzymatic treatment on the aroma of tieguanyin tea
Fresh tea leaves are virtually odourless and the aroma components of tea are generated mainly because of biochemical and chemical reactions during tea processing.In our experiments, aft er the addition of exogenous enzymes, the aroma precursors were transformed into aroma components.Sixty-seven compounds were identifi ed which can be divide d into eight groups according to their structure: 6 aldehydes, 16 alcohols, 20 esters, 7 ketones, 1 acid, 5 alkenes, 3 alkanes and 9 other compounds.Almost all the compounds have been identifi ed as the aroma compounds of tea by Hara et al. (41).The main volatile constituents of the samples are shown in Table 3 and Fig. 2. The results illustrate that the relative content (values given as the ratio of the component peak area to the internal standard (ethyl decanoate) peak area) of the total essential oils in samples A1 (108.19), A2 (113.41),A3 (117.65 ), B1 (111.00),B2 (129.83),B3 (135.84),C (117.15) and D (123.26) was higher than that in sample CK (97.95).Compared to those in the cont rol grou p, the total essential oils of tea sample D increased signifi cantly, amo ng which aldehydes increased by 23.28 %, alcohols by 3 7.05 %, esters by 20.10 % and alkenes by 38.99 %.
The main constituents of the essential oils were aldehydes, alcohols, esters and alkenes, which accounted for 70-80 %.Among them, the contents of aldehydes, alcohols, esters and alkenes in the samples trea ted with laccase and α-galactosidase were obviously higher than in the CK sample.The samples treated with α-galactosidase had a higher content of aldehydes and alcohols, but lower content of este rs than th ose treated with laccase.
Nerolidol, indole and α-fa rnesene were found to be the most important odour-active compounds in oolong tea (37).The smell of nerolidol is like the light fragrance of roses and apples, which is very sweet, refreshing and lasti ng.Indole can enhance the fl avour of tea.The organoleptic character of farnesene is soft and mellow.Compar ed with sample CK, all these three compounds were increased in sample D (53.34 vs. 38.09% of nerolidol, 11.52 vs. 10.33 % of indole, and 12.90 vs. 9.21 % of farnesene).Benzonitrile is considered as the unique constituent of tieguanyin (42), which was also detected in this study.Therefore, these odourants were the most important components of tieguanyin tea.The contents of these components in the samples treated with α-galactosidase were much higher than in those treated with laccase and the sample CK.It is obvious that α-galactosidase plays an important role in tea aroma formation.
Alcohols accounted for about half of the total essential oils.Linalool, phenylethyl alcohol, nerolidol and phytol were the main constituents of oolong tea.It i s known that terpene alcohols, such as linal ool can be generated by the hydrolysis of their glycosides, which are precursors of tea aroma, through the enzymatic action (43).Fig. 2 shows that the linalool, phenylethyl alcohol and nerolidol content greatly increased in the samples B2 and B3.Phenylethyl alcohol was considered as a tea aroma precursor (44), which increased in samples treated with enzymes.Nerolidol is the major component of volatile oil contributing to the odour of tieguanyin and is the most important indicator of the aroma of Fujian oolong tea (45).The content of phytol, the hydrolysed product of chlorophyll, was 8.51 % in sample A3, which was much higher than that in sample CK (6.01 %).In the above sensory evaluation, the colour of sample D was golden yellow, brighter than that of sam ple CK, and this change resulted from the eff ect of laccase.
Aldehydes accounted for above 10 % of the total essential oils.(E)-2-hexenal, nonanal, palmitaldehyde and diallyl acetal belong to aliphatic aldehydes.(E)-2-hexenal is known to possess a strong green note.The relative level of (E)-2-hexenal is much higher in sample D (0.39) than in sample CK (0.25).Nonanal gives a pleasant rose note.Benzeneacetaldehyde has a characteristic hyacinth-like odour.The contents of nonanal, benzaldehyde and benzeneacetaldehyde in sample D were much higher than in sample CK.
Esters, the main constituents of the essential oils, always have a characteristic pleasant fl owery aroma.There were 20 kinds of esters detected in the tea samples.In green tea, oolong tea and black tea, (Z)-3-hexenyl hexanoate has been repor ted as one of the most important aroma compounds exhibiting a green note, although its concentration was not fairly high (46)(47)(48).The relative level of (Z)-3-hexenyl hexanoate was much hig her in the samples treated with laccase than in sample CK.The contents of m ethyl palmitate, methyl linoleate and methyl linolenate in samples treated with α-galactosidase and laccase were much higher than in sample CK.These components showed lasting aroma (49).Farnesene is the most abundant alkene in tieguanyin o olong tea.It largely increased in samples treated with α-galactosidase or laccase.However, the exception was obser ved in sample D, in which the content of alkanes decreased.
Research shows that the bett er quality tea has a highe r content of total essential oils and higher boiling point of aroma components (50).Therefore, these parameters can be used as a quality standard for tieguanyin tea from Anxi county.In this experiment, the total essential oil content of samples treated with laccase and α-galactosidase was signifi cantly higher than in sam ple CK.The content of the characteristic aroma components with h igh boiling point, such as nerolidol, α-farnesene, phenylethyl alcohol, benzeneacetaldehyde, (Z)-3-hexenyl hexanoate, and methyl palmitate showed the same trend.This in dicates that the appropriate exogenous enzyme treatment can improve the aroma of summer tieguanyin tea from Anxi county, which was confi rmed by the results of sensory evaluation.

Conclusions
In conclusion, laccase and α-galactosidase significantly improved the sensory quality of tieguanyin tea from Anxi county, China, harvested in summer, which has been confi rmed by sensory evaluation by the tea tasting experts and the results of main fl avour substance analysis.Laccase had positive eff ect on the taste and α-galacto sidase on the aroma.Spraying the tea leav es with the solution of the two enzymes could improve the tea quality, but the aroma of the tea remained similar to that treated with α-galactosidase alone.Treatment of tea with laccase and α-galactosidase has been rarely reported.Our study demonstrated that this treatment could improve the sensory quality of summer tieguanyin tea from Anxi county eff ectively.The results also show that the exogenous enzymatic treatment method can be potentially applied in other tea production processes, widening the application range of laccase and α-galactosidase.

Fi g. 2 .
h o l s E s t e r s K e t o n e s A c i d s A l k e n e s A l k a n e s O t h e r s Comparison of aroma profi les of samples of tieguanyin tea from Anxi county treated with diff erent enzyme concentrations (for sample abbreviations see legend of Fig. 1) *Values are give n as the ratio of the component peak area to the internal standard (ethyl decanoate) peak area

Table 1 .
Concentrations of enzymes used for the treatment of diff erent summer tieguanyin tea samples

Table 2 .
Results of sensory evaluation of the summer tieguanyin tea from Anxi county aft er diff erent enzymatic treatments For sample abbreviations see legend of Fig.1

Table 3 .
Aroma compounds and relative contents of tieguanyin tea samples from Anxi county aft er diff erent enzymatic treatments