Volatile components of essential oils extracted from Pu-erh ripe tea by different extraction methods

International Journal of Food Properties ISSN: 1094-2912 (Print) 1532-2386 (Online) Journal homepage: http://www.tandfonline.com/loi/ljfp20 Volatile components of essential oils extracted from Pu-erh ripe tea by different extraction methods Xuemei Gao, Shidong Lv, Yuanshuang Wu, Jiangbing Li, Wenrui Zhang, Wen Meng, Chen Wang & Qingxiong Meng To cite this article: Xuemei Gao, Shidong Lv, Yuanshuang Wu, Jiangbing Li, Wenrui Zhang, Wen Meng, Chen Wang & Qingxiong Meng (2017) Volatile components of essential oils extracted from Pu-erh ripe tea by different extraction methods, International Journal of Food Properties, 20:sup1, S240-S253, DOI: 10.1080/10942912.2017.1295256 To link to this article: https://doi.org/10.1080/10942912.2017.1295256 2017 Taylor & Francis Group, LLC Accepted author version posted online: 23 Feb 2017. Published online: 24 Jul 2017. Submit your article to this journal Article views: 84 View related articles View Crossmark data Citing articles: 1 View citing articles Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalinformation?journalcode=ljfp20

2017, VOL. 20, NO. S1, S240 S253 https://doi.org/10.1080/10942912.2017.1295256 Volatile components of essential oils extracted from Pu-erh ripe tea by different extraction methods Xuemei Gao a, Shidong Lv b, Yuanshuang Wu a, Jiangbing Li a, Wenrui Zhang a, Wen Meng a, Chen Wang a, and Qingxiong Meng a a Faculty of Life Science and Technology, Kunming University of Science and Technology, Yunnan, People s Republic of China; b Kunming Crain & Oil and Feed Product Quality Inspection Center, Yunnan, People s Republic of China ABSTRACT Pu-erh ripe tea is one of the main tea products in China, which has a unique woody, chestnut fragrance flavour, distinct from other teas. Because of the largely excessive production capacity, it is now possible and necessary to utilise it to produce tea essential oil. In this study, the extraction efficiency of Soxhlet extraction (Soxhlet), ultrasonic-assisted extraction (UAE), simultaneous distillation extraction (SDE), and steam distillation extraction (SD) for extraction oil of Pu-erh ripe tea was investigated. GC-MS and HS-SPME/GC- MS were then applied to identify the volatile compounds in the essential oils and tea samples. The results showed that the extraction ratios of the essential oil were 0.81±0.01 g/kg (Soxhlet), 0.36±0.01 g/kg (UAE), 0.11±0.01 g/kg (SDE), and 0.59±0.01 g/kg (SD). GC-MS identified 40, 38, 35, and 47 volatile compounds in the essential oils extracted by the different methods, whereas 55 compounds were identified by HS-SPME/GC-MS. Therefore, when both the essential oil extraction rate and oil flavour are considered, the best method is SD. ARTICLE HISTORY Received 10 October 2016 Accepted 11 February 2017 KEYWORDS Essential oils; Extraction process; Extraction rate; Gas chromatography-mass spectrometry (GC-MS); Volatile compounds Introduction Tea can be divided into four major categories according to the degree of fermentation: unfermented green teas, semi-fermented oolong and Paochong teas, fully fermented black tea, and microbially fermented dark tea. Yunnan Pu-erh tea is the most representative and popular Chinese dark tea. [1] The tea aroma is one of the most important factors that define tea quality, and it has a great influence on its appreciation by consumers. [2] Pu-erh ripe tea has a unique woody, chestnut fragrance, and/or camphor flavour and amazing tea fusion taste, distinct from green tea, black tea, and other teas. Previous studies showed that Pu-erh tea has many health benefits, and it has been widely accepted and loved by growing numbers of consumers throughout China, Korea, and Japan. [3 5] As this tea has natural flavours and fragrances, although the essential oil contents are generally very low, it has great potential value in food and cosmetics. [6] The extraction of the essential oil is important for the comprehensive utilisation of the excessive amount of tea that is currently produced, for example, before the extraction of tea polyphenols. With the fast and constant expansion of the tea plantation area, tea production has rapidly expanded in recent years. An excess of tea has appeared in China, including Pu-erh ripe tea, and will become an increasingly serious problem in the near future. Furthermore, according to the report of the International Tea Commission (ITC), approximately 25% of raw tea is lost or abandoned during processing. An average of approximately 300,000 tons of tea is wasted every year in China. This mainly includes CONTACT Qingxiong Meng qxmeng@scbg.ac.cn Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, Yunnan, People s Republic of China. Colour versions of one or more of the figures in the article can be found online at www.tandfonline.com/ljfp 2017 Taylor & Francis Group, LLC

S241 poor quality or poor taste tea, tea leaves pulverised by the processing, and coarse and old tea leaves that are pruned in winter. It is urgent to find new, diverse applications for the excess tea. Essential oils are complex mixtures of low molecular weight compounds extracted from plants by steam distillation or various solvents. [7] Essential oils are important products of the agricultural industry. They are commonly used as flavouring agents in food products, drinks, perfumeries, pharmaceuticals, and cosmetics. [8 10] Approximately 3,000 essential oils have been produced from at least 2,000 plant species, of which 300 are important from a commercial point of view. A production of 40,000 60,000 tonnes per annum with an estimated market value of US$700 million indicates that the production and consumption of essential oils are increasing around the world. [11] The essential oil extracted from tea has a very wide range of applications in the fields of food, cosmetics, flavours and fragrances, and the cigarette industry. [2,12,13] The extraction of essential oil from abandoned tea can reduce the amount of wasted tea and can bring stupendous economic benefits to tea farmers and the essential oil industry. To date, no study has been reported on the rate and chemical difference of tea essential oils in different extract processes. Therefore, it is necessary to study the volatile composition and the rate of tea extraction using difference extract methods and to analyse the aroma differences. Many extraction methods have been developed to extract essential oils, including microwave hydrodiffusion and gravity (MHG), solvent-free microwave extraction (SFME), microwave-assisted extraction (MAE), subcritical water extraction (SWE), ultrasonic-assisted extraction (UAE), supercritical CO 2 fluid extraction (SFE-CO 2 ), and Soxhlet extraction. Simultaneous distillation extraction (SDE) is the most comprehensively studied method for the extraction of essential oils from tea, although these studies have mostly been conducted in laboratories. [14 17] However, this method is a lengthy process and consumes large amounts of organic solvents. [18] In recent years, a simple, rapid, solvent-free, and low-cost technique, named solid-phase micro extraction (SPME), was developed. This technique is based on the distribution coefficient of the analytes between the sample matrix, the gas phase, and a fibre coated with an adsorbent polar or apolar high polymer. [19] Since its introduction, this technique has been widely used to analyse the volatile compounds of food, such as fruit, [20] sauce, [21] and wine. [22] In previous studies, a fully automatic HS-SPME method for the determination of aroma compounds from Pu-erh ripe tea and black tea was investigated and showed good repeatability, sensitivity, and selectivity. [18,23,24] Based on these works, we attempted to identify the optimal technique for the extraction of essential oils from Pu-erh ripe tea by systematically comparing the different processes. The volatile compounds were identified and compared to explore the differences in the aroma compounds of the essential oils extracted by the different methods and to determine whether and which types of volatile compounds are lost in the corresponding extraction processes. In this study, essential oils were extracted from Pu-erh ripe tea by four different processes respectively, and the aroma composition in the essential oils was analysed by gas chromatography-mass spectrometry (GC- MS). In addition, fully automatic HS-SPME was used to identify the original aroma composition from Pu-erh ripe tea as a reference [14 16] Furthermore, the colour and odour of the extractions were compared. Materials and methods Materials, instruments, and equipment Pu-erh ripe tea powder was obtained by using a high-speed multifunctional disintegrator of 40 mesh (Yunnan Green Tea Industry Co. Ltd.), ethanol (AR), dichloromethane (AR), sodium chloride, sodium sulphate anhydrous (Kunming Bei erji Science and Technology Co. Ltd.), and deionised water (laboratory made). An SDE apparatus was purchased from Beijing Glass Instrument Factory (Beijing, China) and was similar to the design of the Lickens-Nickerson apparatus. An essential oil extractor (Beijing Tian

S242 X. GAO ET AL. Lian Harmonious Instrument Co., Ltd.), a simultaneous distillation and extraction device (Beijing Heng Aode Instrument Limited Company), probe-type ultrasonic generator (Nanjing First European Instrument Manufacturing Co., Ltd.) at a frequency 20 khz and a rotating speed of 300 rpm, Soxhlet extractor (Shanghai Dafeng Glassware Co. Ltd.), Rotary evaporator (Shanghai Ilover Instrument Co. Ltd.), electronic balance (DENVER company), high-speed multifunction grinder (Shanghai Arrow Machinery Co., Ltd.), and Agilent 7890A-5975 (GC-MS) were also used. Experimental methods Simultaneous distillation extraction A total of 30 g tea powder was immersed in a 1000 ml flask with 250 ml of distilled water, and 50 ml of dichloromethane, used as the extraction solvent, was placed in another flask. Both flasks were placed in a Lickens-Nickerson apparatus and heated to their boiling points. Each extraction was carried out for 3 h after the two arms started to reflux. After cooling to ambient temperature, the extract was collected and the flask was washed with dichloromethane (3 1 ml), which was then added to the extract. The combined extract was dried over anhydrous sodium sulphate overnight and filtrated. The filtrate was then concentrated to approximately 0.5 ml under a gentle stream of highpurity nitrogen, and the volume was brought to 1 ml with dichloromethane. [24 27] The concentrated extraction was stored at 80 C prior to analysis. The essential oil was analysed by GC-MS after filtering through a 0.45 µm membrane. Ultrasonic-assisted extraction Thirty grams of powdered Pu-erh ripe tea was dissolved in 500 ml dichloromethane, and the mixture was soaked for 3 h before ultrasonic extraction was performed three times. The ultrasonic frequency was 40 khz at 50 C, and the extraction time was 20 min. The filtrate was then placed in a rotary evapourator to evapourate the dichloromethane and was then combined with the extract. [19,28 30] The concentrated extract was stored at 80 C prior to analysis. The essential oil was analysed by GC-MS after filtering through a 0.45 µm membrane. Soxhlet extraction method (Soxhlet) A total of 30 g tea powder dissolved in 450 ml dichloromethane was placed into a Soxhlet extractor. Soxhlet extraction was conducted three times for 8 h. The Soxhlet extracts were placed in a rotary evapourator to evapourate the dichloromethane, and then the extracts were combined. [31,32] The concentrated extract was stored at 80 C until analysis. The essential oil was analysed by GC-MS after filtering through a 0.45 µm membrane. Steam distillation A total of 30 g tea powder was dissolved in 1000 ml water and placed in an essential oil extractor, and the essential oil was extracted according to the method in appendix XD in the Chinese pharmacopoeia (edition 2005). [33] After extraction for 5 h, the combined extract was dried over anhydrous sodium sulphate overnight and filtrated. Finally, the essential oil of the Pu-erh ripe tea was collected. [33 35] The concentrated extract was stored at 80 C prior to analysis. The essential oil was analysed by GC-MS after filtering through a 0.45 µm membrane. Fully automatic headspace-solid phase microextraction (HS-SPME) In a previous study, [18] the HS-SPME parameters for Pu-erh ripe tea were validated and optimised; therefore, the same method and parameters were adopted in this work to extract the volatile compounds of Pu-erh ripe tea. A detailed explanation of the HS-SPME parameters is as follows. A total of 2.0 g tea sample was placed in a 20 ml sealed headspace vial with 5 ml boiling water, and the temperature of the headspace vial was kept at 80ºC using an electric hot plate. Then, a 65 μm polydimethylsiloxane/divinylbenzene (PDMS/DVB)-coated fibre (Supelco, Inc., Bellefonte, PA) was

S243 exposed to the sample headspace for 60 min. The volatile compounds that were absorbed on the SPME fibre were desorbed in the GC-MS injector at 250ºC for 3.5 min and were then immediately analysed by GC-MS. After adsorption, the SPME fibre was transferred to the GC injection port at 250ºC for 30 min. GC-MS analysis conditions An HP 7890A GC instrument integrated with an Agilent 5975C MSD mass spectrometer (Agilent, Santa Clara, CA, USA) was used to identify the volatile compounds in the tea samples. The capillary column was an Agilent HP-5MS (30 m 0.25 mm i.d. 0.25 μm film thickness), helium (purity > 99.999%) was used as the carrier gas, and the flow rate was 1 ml/min. The injector temperature was 250 C, and the injection mode was splitless. The GC oven temperature was held at 50 C for 5 min, increased to 210 C at a rate of 3 C/min, maintained at 210 C for 3 min, and finally increased to 230 C at 15 C/min. The mass spectrometer conditions were as follows: [18,36,37] ionisation energy, 70 ev; ion source temperature, 230 C; quadrupole temperature, 150 C; quadrupole mass spectrometer scan range, 30 500 atomic mass units (amu); solvent delay time, 2.8 min. Data analysis Identification of the peaks was performed by searching the National Institute of Standards and Technology (NIST) 08.L MS library (a minimum match quality of 95% was used as the criterion). The retention indices (RI) were compared with published reports. [38 42] The relative percentages of the detected peaks were obtained by peak-area normalisation, and all relative response factors were taken as one. The Kovats retention index (RI) for each aroma compound was calculated with a homologous series of n-alkanes (C8-C40; Sigma-Aldrich, USA) under the same GC-MS conditions. Results and discussions Comparison of the extraction rates of tea essential oil by the different processes As shown in Table 1, the time consumed for essential oil extraction by the Soxhlet extraction method was 8 h, twice as long as that of the UAE method. The SDE method required 6 h, and the SD method required 5 h. The highest yield of essential oil was obtained by the Soxhlet extraction method, that is, 0.81 ± 0.01 g/kg, more than 2 times yield of that of the UAE method. The SD method yielded 0.59 ± 0.01 g/kg, and the lowest yield was 0.11 ± 0.01 g/kg by SDE. However, according to the previously developed methods to extract volatile compounds of tea, the extraction yield accounted for only 0.01% 0.05% of the dry matter quantity. [2,43,44] We believe the higher efficiency in our study is due to the repeated extraction procedures. Although the repetition requires more energy and time, it improves the extraction yield of the essential oil. In this experiment, the basic principles of the SDE device were derived from the design of Likens and Nickerson (1964), with reference to H S Thomas report. [45] Although this experiment obtained essential oil with a high concentration of aroma, it has some scorched and stuffy flavours (Table 3) that are different from the original flavour of the tea, and the extraction rate is relatively low. This Table 1. Time consumption and extraction rate for extracting the essential oil by different methods. Extraction method Raw material quantity (g) Time consumption (h) Extraction rate (g/kg) Soxhlet 30 8 0.81 ± 0.01 UAE 30 4 0.36 ± 0.01 SDE 30 6 0.11 ± 0.01 SD 30 5 0.59 ± 0.01 Extraction rate (g/kg) = Oil quantity/tea powder (mean value ± standard deviation, n = 3).

S244 X. GAO ET AL. result is similar to the conclusions of Shimoda Mitsuya. [46] Incubating the tea with solvents at the high temperature in a closed system for a long time may have altered some compounds, especially the heat-sensitive compounds. For instance, glycoside compounds hydrolyse and release linalool and geraniol and β-carotene degradation generates β-ionone, [41,47] which results in obviously different flavours in the extracted oil, compared to the original tea. Chemical composition of Pu-erh tea essential oil from the different extraction processes The aroma compounds of the essential oils prepared under four conditions and HS-SPME were analysed by GC-MS. The TICs are presented in Fig. 1. The identified compounds and their relative percentage contents (%) are summarised in Table 2. A total of 96 volatile compounds were detected, including alcohols, aldehydes, ketones, hydrocarbons, phenolic compounds, esters, and acids. The essential oil extracted by the SD method contained 47 aroma compounds; the essential oils extracted by Soxhlet extraction method, the UAE method, and the SDE method can detect 40, 38, and 35 aroma compounds, respectively. The contents of caffeine (15.02%), nerolidol (13.13%), benzyl alcohol (3.84%), 1,2,4-trimethoxybenzene (3.14%), isophytol (2.59%), and 1,2,3-trimethoxybenzene (2.35%) were relatively high in the Soxhlet extract. The contents of caffeine (19.74%), 2,6-dimethoxyphenol (12.12%), 6-methyl-5-hepten-2-one (2.65%), 1-octen-3-ol (2.39%), and linalool (1.34%) were relatively high in the UAE extract. The caffeine (23.85%), 2-pentylfuran (10.85%), 2-methoxynaphthalene (9.09%), 1-octen-3-ol (7.34%), phenethyl alcohol (3.43%), nerol (1.66%), and 1,2,3,4- tetramethoxybenzene (1.18%) contents were relatively high in the SDE extract. The caffeine (33.27%), nerolidol (17.10%), phytol (12.97%), fluorene (6.63%), 1-octen-3-ol (4.12%), and isophytol (2.23%) contents were relatively high in the SD extract. The comparison results of the aroma compounds prepared by four processes are shown in Fig. 2. Further analysis showed that the main aroma constituents of the essential oils were alcohols, esters, aldehydes, ketones, hydrocarbons, and acids. In terms of the quantity of identified aroma compounds, the highest was obtained by SD, in which 14 alcohols, 7 esters, 4 aldehydes, 3 ketones, 1 hydrocarbon, and 3 acids were found. Ester compounds were the most abundant type in the oil extracted by the Soxhlet method, followed by hydrocarbons and alcohols, and the content of aldehydes was the lowest. The hydrocarbon content was the highest in the oil extracted by UAE, followed by alcohols, and the contents of acids and aldehydes were the lowest. The hydrocarbon content was the highest in the oil extracted by SDE, followed by alcohols and esters, and the content of acids was the lowest. The ketones content was the highest in the oil extracted by SD, followed by SDE, and the Soxhlet method ketone content was the lowest. This shows that the compositions of the essential oils extracted by different extraction processes were different with respect to type and contents. A comparison of aroma-active compounds extracted from the Pu-erh tea samples by the four applied techniques revealed that HS-SPME was more efficient in extracting alcohols, methoxyphenolic compounds, ketones, hydrocarbons, and phenolic compounds, whereas the SDE extracts contained more acids. This finding was in agreement with the results obtained in previous studies of Pu-erh tea. [24] The aroma-active compounds from Table 2 with similar sensory descriptors were grouped into seven general aroma categories based on their primary aroma character: floral, stale/ musty, woody, fruity, green, rancid/pungent, and miscellaneous. To compare the relative aroma intensity of each aroma category, the higher average intensity value of the same compound was selected from the results of HS-SPME and SDE during calculation. Alcohols (floral aroma notes), methoxyphenolic compounds (stale/musty aroma notes), and ketones (woody or floral aroma notes) play a vital role in the special flavour of Pu-erh ripe tea. [27] The results of the compounds detected in the essential oils extracted by four different processes were compared. Although SD shares a same extraction principle with SDE, the limitation of the technique is clear in Table 2. Thirty-nine of aroma compounds, including methyl linoleate, phytone, α-calacorene, cis-jasmone, γ-terpinene, cis-2-(2-pentenyl) furan, and 1-pentanol, were the only volatile compounds that could be detected in the essential oil extracted by SD method, whereas

S245 Figure 1. TICs of Pu-erh tea essential oil by different extraction processes. (A) headspace-solid phase microextraction (HS-SPME); (B) ultrasonic-assisted extraction (UAE); (C) simultaneous distillation extraction (SDE); (D) Soxhlet extraction; (E) steam distillation extraction (SD).

S246 X. GAO ET AL. Table 2. GC-MS analysis results of the tea essential oils from different extraction methods. c Relative percentage content [%(range)] No RI a Compound b Soxhlet UAE SDE SD HS-SPME Odour note 1 803 2,3-Butanediol - 15.92 16.73-0.11 2 805 1-Pentanol - - - 0.05-3 843 (Z)-3-Hexen-1-ol - 0.05 0.09-0.12 Mushroom d 4 861 Hexyl alcohol - 0.12-0.09-5 884 2-Heptanone 0.08 - - 0.04 1.20 6 957 Benzaldehyde 5.36 0.07 - - - 7 979 1-Octen-3-ol 1.24 2.39 7.34 4.12 0.18 Mushroom d 8 985 6-Methyl-5-hepten-2-one - 2.65-0.09 1.77 Fat d, orange d 9 989 2-Pentylfuran - - 10.85 0.04 0.29 Fruit, earthy d 10 997 α-phellandrene 0.08 0.08 11 998 Cis-2-(2-pentenyl)furan - - - 0.04-12 1018 1,2,3-Trimethylbenzene - - 2.02 0.03 0.16 13 1026 Limonene - - - 0.07 0.13 14 1034 Benzyl alcohol 3.84 - - 0.10 - Fruit d, smoke d 15 1037 (E)-3,7-Dimethyl-1,3,6-octatriene 0.20 0.12 1.49 0.60 0.50 16 1042 Phenyl acetaldehyde 0.36 - - - 0.30 17 1048 Ocimene - 0.06 0.04 - - Sweet d 18 1056 γ-terpinene - - - 0.04-19 1064 Acetophenone 0.08 - - - - 20 1068 (E)-2-Octen-1-ol - - 1.72 0.05 0.13 21 1072 Linalool oxide I - - - - 0.34 Flower d, wood e 22 1087 Linalool oxide II - - - - 2.85 Flower d, wood e 23 1092 (E,E)-3,5-octadien-2-one 0.12 0.04-0.04 1.04 24 1098 Linalool - 1.34-0.76 0.15 Flower, lavender d, wood d 25 1100 Hotrienol 1.43 - - 0.97 26 1104 Nonanal - - 0.05 - - 27 1110 Phenylethyl alcohol 0.45-3.43 0.37 0.31 Rose e 28 1135 Benzene acetonitrile 0.09 0.02-0.07 3.61 29 1137 2,5-Pyrrolidinedione, 1-ethyl- - 0.06 0.06 - - 30 1149 1,2-Dimethoxybenzene - 0.06-0.68 0.39 31 1169 Linalool oxide III - - - - 0.35 Flower d, wood e 32 1175 Linalool oxide IV - - - - 2.89 Flower d, wood e 33 1178 Naphthalene 0.28 0.06 2.93-0.20 34 1188 α-terpineol - 0.04 0.35 0.06 1.24 Flower, clove e 35 1190 Methyl salicylate 0.15 0.03 0.20 0.22 1.38 Green, fat, herb e 36 1200 Dodecane - 0.15 - - - 37 1205 Decanal - - 1.02 - - 38 1218 β-cyclocitral - - 0.30-0.88 39 1228 Nerol - - 1.66 0.15 0.69 Sweet, rose d 40 1241 3,4-Dimethoxytoluene 0.34 0.09 - - 1.96 Mouldy d 41 1256 Geraniol - 0.02 0.36-1.06 Rose d 42 1260 2-Methoxybenzylalcohol - - 0.31-0.98 43 1265 3,5-Dimethoxytoluene - 0.09-0.04 2.02 44 1287 2-Methylnaphthalene 1.41 0.15 - - - 45 1289 Indole - - 0.08 0.05 0.57 Mothball, burnt d 46 1302 1-Methylnaphthalene - 0.06 - - - 47 1304 Isopropyl salicylate - 0.03 - - - 48 1316 1,2,3-Trimethoxybenzene 4.29 2.35 - - 27.43 Wood, herb e 49 1325 4-Ethyl-1,2-dimethoxybenzene 1.33 - - 0.08 0.24 50 1334 2,6,6-Trimethyl-1-cyclohexene-1-ethanol - - 0.05 - - 51 1351 2,6-Dimethoxyphenol 2.66 12.12 - - 0.87 Medicine, phenol, smoke d 52 1362 γ-nonanolactone 0.09 53 1369 Butanoic acid butyl ester 0.18 0.17 0.07 54 1375 1,2,4-Trimethoxybenzene 3.14 1.19-0.15 0.87 Wood, herb e 55 1387 β-guaiene - 0.11 0.76-1.27 Wood, spice d 56 1397 Cis-jasmone - - - 0.07-57 1400 Tetradecane - 0.17-0.10 0.54 58 1406 1,2,3-Trimethoxy-5-methylbenzene 0.74 0.18 - - 2.98 59 1409 α-cedrene - - 0.99 - - Wood e 60 1417 β-caryophyllene 0.69 0.28 - - 8.25 Mint e, spice d, wood d 61 1428 α-ionone 0.29 - - - Wood d,violet d 62 1433 1,2-Benzopyrone 0.64 0.33 (Continued )

S247 Table 2. (Continued). No RI a Compound b Soxhlet UAE SDE SD HS-SPME Odour note Relative percentage content [%(range)] 63 1438 Dihydro-β-ionone - - 0.09-0.69 64 1442 1-Methoxynaphthalene - - 0.07 - - 65 1447 2-Methoxynaphthalene - 0.72 9.09-1.88 Orange, sweet d 66 1449 1,2,3,4-Tetramethoxybenzene - - 1.18-1.06 Herb e 67 1455 Geranyl acetone - - - - 0.88 Magnolia, green d, fruit e 68 1468 5-Methoxy-6,7-dimethylbenzofuran 0.07 0.17 0.98 69 1487 β-ionone - - 1.02-0.65 Seaweed, violet, flower d 70 1492 2-Tridecanone - - - 0.05-71 1497 Methyl isoeugenol - - - 0.09-72 1500 Pentadecane 0.36 - - 0.05-73 1506 Dibenzofuran - - - 0.13-74 1508 α-farnesene - - 0.09 - - Flower, herb e, green d 75 1528 Dihydroactinidiolide 0.11 - - 0.27 - Musk d, coumarin d 76 1543 α-calacorene 0.50 77 1554 Nerolidol 13.13 6.61-17.10 - Apple, rose, green d 78 1572 Fluorene - 2.70-6.63-79 1598 Cedrol 1.29 - - - 0.27 Wood e, sweet d 80 1600 Hexadecane 11.18 - - - 1.57 Alkane d 81 1649 Methyl jasmonate 0.14 1.24 8.01 82 1653 α-cadinol - - - - 1.09 83 1659 2,2,5,5 -Tetramethyl-1,1 -biphenyl 6.26-1.70 0.88-84 1664 2-Methylhexadecane 0.50 - - - 0.36 85 1700 Heptadecane 1.43 0.87 Alkane d 86 1828 Isopropyl myristate - 0.06 0.10 - - 87 1840 Caffeine 15.02 19.74 23.85 33.27 2.98 88 1846 Phytone - - - 0.51-89 1918 Farnesyl acetone - - - 0.15 0.64 Sweet d, rose d 90 1927 Hexadecanoic acid methyl ester - - 1.34 - - 91 1949 Isophytol 2.59 - - 2.23 1.36 Flower d, herbs e 92 1975 Hexadecanoic acid 0.16 18.47 - - 0.19 93 2000 Eicosane 0.01 - - - - 94 2093 Methyl linoleate - - - 0.05-95 2095 Linolenic acid 0.12 - - - 0.38 96 2122 Phytol - - - 12.97 3.22 Flower d, balsam e a RI, retention indices as determined on an HP-5MS column using a homologous series of n-alkanes. b Compounds are listed in order of retention time. c Relative content, percent normalised peak areas; - not detected. d http://www.flavornet.org/flavornet.html. e Liu Shuwen. Synthetic spices manual [M]. China light industry press. 2010. c Table 3. Sensory evaluation on the different essential oils samples from the four extraction methods. Extraction method Colour Persistence Transparency Degree of flavour Taste Soxhlet Yellow Short General transparency Can sniff out Miscellaneous taste UAE Yellow Long General transparency Can identify Commonly SDE Light yellow Medium More transparent Slightly stronger Fragrant SD Brown Longer Turbid Very strong Very fragrant not in that oil extracted by SDE and the other methods. This is due to its open steam distillation system and following step-liquid-liquid extraction. During this procedure, not only high-volatile components such as benzyl alcohol, linalool oxides, and linalool had a chance to escape from the distillation system, but the analytes hardly transferred to organic phase. [25] Decanal, α-farnesene, 2,6,6-trimethyl-1-cyclohexene-1-ethanol, hexadecanoic acid methyl ester, α-cedrene, and nonanal were only detected in the essential oil extracted by SDE. This may attribute to the fact that SDE was a closed and continuous extraction system, in which the target components can be thoroughly transferred to organic phase. Eicosane, γ-nonanolactone, and acetophenone were only detected in the essential oil extracted by the Soxhlet method. As for extraction of the low-volatile components in

S248 X. GAO ET AL. Figure 2. The number of the types of aroma compounds in different methods. Pu-erh tea, such as 1,2,4-trimethoxylbenzene, SDE is less poor compared with Soxhlet extraction, probably due to the characteristics of high-boiling point and incompatibility with water steam. Soxhlet extraction was a classical technique to extract essential oil from natural product. [48] Table 2 shows that it can almost exhaustively extract all the 1,2,3- trimethoxylbenzene and 1,2,4-trimethoxylbenzene from Pu-erh tea compared with SDE; nevertheless, it is very poor for benzyl alcohol, linalool oxides, linalool, phenethyl alcohol, and geraniol. These findings indicate Soxhlet was suitable for high-boiling point (low-volatile) compounds such as essential oil. [49,50] As for the trace highvolatile component, SDE was appreciated. Therefore, in addition to the extraction ratio, the compositions of the essential oils were different by each extraction technique. Aroma compound loss in different extraction methods As shown in Table 2, there were obvious differences between HS-SPME method and other four extraction methods with respect to the contents and types of aroma compounds. A total of 55 aroma compounds were detected in the Pu-erh ripe tea by HS-SPME. Moreover, compared with the four other extraction methods, HS-SPME had the highest number of aroma compounds, which covered 96.49% of the total compounds detected in all five extraction methods. Similar to our results, previous studies have shown a total of 77 volatile compounds were determined in 18 green teas, [51] mainly including alcohols, ketones, hydrocarbons, methoxy-phenolic compounds, and so forth. Among these, the methoxy-phenolic compounds were the most abundant components in the Puerh ripe tea. [42] The essential oils extracted by Soxhlet extraction, UAE, SDE, and steam distillation contained 40, 38, 35, and 47 aroma compounds, respectively. The aroma compounds lost from the tea essential oil do not reflect the true tea aroma compounds because secondary reactions may occur during the incubation at high temperature. Linalool oxide I (0.34%), linalool oxide III (0.35%), geranyl acetone (0.88%), α-cadinol (1.09%), linalool oxide II (2.85%), and linalool oxide IV (2.89%) were the only aroma compounds that were extracted and then detected by the HS-SPME method. Theses linalool oxides are present as primeverosides in teas and are liberated by specific enzymes (primeverosidases), [35] which along with α-terpineol are very important odourants that provide a floral and sweet scent in Pu-erh tea. Among these volatile compounds, α-cadinol has a woody

S249 fragrance and contributes to the unique flavour of Pu-erh tea. Moreover, geranyl acetone provides a special floral and woody odour and is a unique volatile compound in Pu-erh tea. [52] In contrast, isophytol and phytol, with high molecular weights, were only detected in the Pu-erh tea essential oil extracted by SD. Although they were extracted by SD method in high proportions, these two compounds were thought to be less important for the aroma flavour of Pu-erh tea because of their low volatility. Compared with HS-SPME, most volatile compounds are lost in the evapouration procedure of the SDE process, that is, 20 aroma compounds were missing. Seventeen aroma compounds were lost in UAE extraction, and eight aroma compounds were lost in the SD method, which was the lowest loss. When using SPME, a large number of hydrocarbons (Fig. 3) were detected and constituted a significant proportion of the total volatile compounds in Pu-erh tea. As shown in Table 1, alkanes were present as the richest hydrocarbons, but they had a minor effect on the aroma characteristics of Pu-erh because of their relative lack of odours. Dodecane and cyclododecane were detected in the lowest proportions of the hydrocarbons, but the result was not consistent with the study described by Lv et al, [41] who reported that the most abundant hydrocarbon in Pu-erh tea was hexatriacontane, followed by 2-methyl-naphthalene and naphthalene. As shown in Fig. 3, aldehydes were also present in high proportions in Pu-erh tea, and they were mainly extracted by SPME. The aldehydes, originating from thermal Strecker oxidative degradation of amino acids and fatty acids, [53] had an important role in the overall odour because of their relatively low odour threshold values. [25] Geranyl acetone, β-ionone, and dihydro-β-ionone were detected as the major ketones in Pu-erh tea when using SPME method. Although they were detected at low relative percentages, ketones played an important role in the odour of Pu-erh tea because most of the ketones delivered unique odours. Among the ketones, β-ionone was identified as a significant active odour that offered a complex fruity and woody scent. [54] Other compounds showed significant differences in their relative percentages when five methods were compared, as shown in Table 2. SPME was more efficient for the extraction of ketones than other four methods. These results indicated that these aroma compounds have certain selectivity to the tea aroma extraction method; HS-SPME/GC-MS is the best method to detect the volatile compounds in tea. Aroma comparison and sensory quality evaluation of essential oil The quality evaluation indicators for essential oils include colour, persistence, transparency, degree of flavour, and taste. [55 57] In our work, the quality of the essential oils was evaluated by 10 professionally reviewers in spice and essence, and the results are shown in Table 3. The evaluation index weight sequence was: taste of flavour > degree of flavour > persistence > colour > transparency. The comprehensive evaluation results indicate that the SD method was the best extraction Figure 3. Composition comparison of Pu-erh ripe tea essential oils extracted by five different methods.

S250 X. GAO ET AL. Figure 4. The appearance and aroma characteristics of the tea essential oils obtained by four extraction methods. process, followed by SDE, and Soxhlet extraction had the lowest final score. Therefore, compared with other three methods, the essential oil extracted by SD showed the best quality. As shown in Fig. 4, the appearance and aroma characteristics of the essential oil extracted by SD were different from the original tea. One possible reason is that during the high-temperature incubation, a series of chemical reactions occurred, such as oxidation, condensation, and group transfer, which altered the distillation product aroma. [41,47] In this experiment, α-terpineol, phenethyl alcohol, R-limonene oxide, phytol, and indole had higher contents in the SD extract. α- Terpineol gives a special floral and woody odour to Pu-erh tea. Regarding the problems mentioned above and to reduce the influence of temperature on the tea aroma compounds, UAE was also used in this experiment. Compared with other three methods, the essential oils extracted by UAE better reflect the natural flavour of the tea. Compared with SDE, the extraction rate of essential oil was 3.13 times higher, although the total number of aroma compounds recovered by SDE was greater than that of the UAE method. Compared with Soxhlet extraction, UAE was simple and easy to scale, and the total number of recovered aroma compounds was higher than that of the Soxhlet extraction method. However, the extraction rate of essential oil was 2.28 times lower than that of the Soxhlet method. In addition, some of the compounds in the essential oils extracted by the Soxhlet method may not be aroma compounds, especially the compounds with low volatility or heat sensitivity; for example, 1,2,3-trimethoxybenzene, 1,2,4-trimethoxybenzene, and 3,4,5-trimethoxy toluene are characteristic aroma compounds of Yunnan Pu-erh ripe tea that can be detected. These results are similar to previous studies. [41,58] Pu-erh ripe tea is rich in compounds with methyl phenyl groups, which have a special contribution to the fragrance of Pu-erh ripe tea. The International Tea Commission data showed that the production of dry tea was 2,278,000 tonnes in 2015, an increase of 186,000 tonnes or 8.9 % compared to the previous year. This trend will continue for at least 5 years. According to our experiment on essential oil extraction yield estimation, even though the extraction rate is relatively low, nearly 34,000 tonnes of essential oil from abandoned tea could be obtained every year, which will open a new pathway for the utilisation of tea and result in great economic value. Because the distinct flavour of Pu-erh ripe tea is thought to be formed by the pile fermentation process, [1] to meet the market demands of the essential oil, more Pu-erh ripe tea could be produced by the appropriate the pile fermentation process, if necessary.

S251 Conclusion In this study, compared with other four different methods, SPME was the most effective method to extract and identify compounds with high volatility, such as low molecular weight alcohols, methoxyphenolic compounds, aldehydes, ketones, and hydrocarbons, which play a major role in the sensory perception of Pu-erh ripe tea. For these scalable extraction methods, most volatile compounds are lost during the evapouration procedure of SDE and UAE methods, and 20 compounds and 17 compounds, respectively, were not detected in the essential oils extracted by these methods. Therefore, SPME is more appropriate for the extraction of volatile compounds from Pu-erh ripe tea than the four other methods, although SPME is currently not scalable and not applicable for the industrial production of essential oil. Fewer compounds were present in the essential oil extracted by SDE and UAE. The extraction rates were also relatively low, but the methods can better maintain the aromatic compounds in the essential oils. In terms of essential oil production, we suggest Soxhlet extraction, which had the highest extraction rate. To preserve the essential oil flavour, we recommend the UAE method, whose extracted aroma is the most similar to the natural composition of Pu-erh ripe tea, has similar flavour profile as the tea itself, and can be easily scaled for industrial essential oil production. However, when both the essential oil extraction rate and oil flavour are considered, the best method is SD. Funding This work was supported by the National Natural Science Foundation of China (No. 31460228) and scientific research funds in Yunnan Province department of education (No. 2014Y089). References 1. Xie, G.; Ye, M.; Wang, Y.; Ni, Y.; Su, M.; Huang, H.; Qiu, M.; Zhao, A.; Zheng, X.; Chen, T.; Jia, W. Characterization of Pu-erh Tea using Chemical and Metabolic Profiling Approaches. Journal of Agricultural and Food Chemistry 2009, 57, 3046 3054. 2. Yang, X.S., Zhao, C.; Zhou, X.; Luo, B.; Yang, F.M.; Hao, X.J. The Constituents of Volatile Oil from Ilex Kudincha. Acta Botanica Yunnanica 2002, 24, 406 408. 3. Jeng, K.C., Chen, C.S.; Fang, Y.P.; Hou, R.C.; Chen, Y.S. Effect of Microbial Fermentation on Content of Statin GABA and Polyphenols in Pu-Erh Tea. Journal of Agricultural and Food Chemistry 2007, 55, 8787 8792. 4. Ling, T.J.; Wan, X.C.; Ling, W.W.; Zhang, Z.Z.; Xia, T.; Li, D.X.; Hou, R.Y. New Triterpenoids and Other Constituents from a Special microbial-fermented Tea-Fuzhuan Brick Tea. Journal of Agricultural and Food Chemistry 2010, 58, 4945 4950. 5. Wu, Y.S.; Lv, S.D.; Lian, M.; Wang, C.; Gao, X.M.; Meng, Q.X. Study of Characteristic Aroma Components of Baked Wujiatai Green Tea by HS-SPME/GC-MS Combined with Principal Component Analysis. CYTA- Journal of Food 2016, 3, 423 432. 6. Cragg, G.M.; Newmann, D.J.; Snader, K.M. Nature Products in Drug Discovery and Development. Natural Product Research 1997, 60, 52 60. 7. Raut, J.S.; Karuppayil, S.M. A Status Review on the Medicinal Properties of Essential Oils. Industrial Crops and Products 2014, 62, 250 264. 8. Liu, N.; Qin, Y.; Song, Y.Y.; Tao, Y.S.; Sun, Y.; Liu, Y.L. Aroma Composition and Sensory Quality of Cabernet Sauvignon Wines Fermented by Indigenous Saccharomyces Cerevisiae Strains in the Eastern Base of the Helan Mountain, China. International Journal of Food Properties 2016, 11, 2417 2431. 9. Huang, Y.Y.; Liu, C.; Xiao, X.D. Quality Characteristics of a Pickled Tea Processed by Submerged Fermentation. International Journal of Food Properties 2015, 19, 1194 1206. 10. Teixeira, B.; Marques, A.; Ramos, C.; Neng, N.R.; Nogueira, J.M.; Saraiva, J.A.; Nunes, M.L. Chemical Composition and Antibacterial and Antioxidant Properties of Commercial Essential Oils. Industrial Crops and Products 2013, 43, 587 595. 11. Patel, S. Plant Essential Oils and Allied Volatile Fractions as Multifunctional Additives in Meat and Fish-Based Food Products: A Review. International Journal of Food Properties 2015, 32, 191 200. 12. Tang, S.Z.; Kerry, J.P.; Sheehan, D.; Buckley, D.J. Antioxidative Mechanisms of Tea Catechins in Chicken Meat System. Food Chemistry 2002, 76, 45 51. 13. Namal Senanayake, S.P.J. Green Tea Extract: Chemistry, Antioxidant Properties and Food Applications-A Review. Journal of Functional Foods 2013, 5, 1529 1541.

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