Brick dark tea a review of the manufacture, chemical constituents and bioconversion

Brick dark tea:a review of the manufacture,chemical constituents and bioconversion of the major chemical components during fermentationWen-Jun Zheng ·Xiao-Chun Wan ·Guan-Hu BaoReceived:24December 2014/Accepted:16March 2015/Published online:28March 2015©Springer Science+Business Media Dordrecht 2015Abstract Brick dark tea is a unique brick-formed tea compressed from the older,coarse and rough leaves and branches of Camellia sinensis var.sinensis and C.sinensis var.assamica mainly in Hunan,Sichuan and Yunnan provinces in China.Researches on brick dark tea have become increasingly popular owing to its special health benefits.A series of biochemical reactions occur during the special pro-duction—microbial fermentation stage and multitudinous components have been detected.Many of the functional components have been isolated and identified from brick dark tea.In this paper,modern manufacturing techniques about different kinds of brick dark tea and their effects on transformation of the chemical constituents,as well as the chemical components were reviewed and discussed.Keywords Brick dark tea (BDT)·Fuzhuan brick tea (FBT)·Processing ·Chemical components ·Biochemical conversionIntroductionTea is one of the most popular beverages consumed worldwide.Researches on its chemical components and biological activities have been widely carried out (Wan 2003;Bao et al.2013).Traditionally,tea is divided into six types:green tea,white tea,yellow tea,oolong tea,black tea,and dark tea.All types of tea are normally produced from Camellia sinensis ,the major varieties being C.sinensis var.sinensis and C.sinensis var.assamica (Harbowy and Balentine 1997;Wang et al.2000).According to their degree of fermentation,tea can be classified into non-fermented tea (green tea),partially-fermented tea (white tea,yellow tea,and oolong tea),fermented tea (black tea),and post-fermented tea (dark tea)(Yue et al.2014;Zhu et al.2015).Particularly,dark tea is a unique microbial fermented tea,and its production is limited only to the areas of China and Japan (Jiang et al.2011;Zhu et al.2015;Yue et al.2014).In China,dark tea is divided into Brick dark tea (BDT),Bowl tea,and Tuo tea according to their shapes.Among these tea,BDT has been more attractive not only for its typical fungal aroma,but also for its health-promoting function,such as anti-obesity (Li et al.2013),antibacteria (Chosa et al.1992),antihyperlipidemia (Lu and Hwang 2008;Wang and Ho 2009),antihy-perglycemia (Du et al.2012;Yamashita et al.2012),antioxidation (Duh et al.2004;Cheng et al.2013;Zhang et al.2011a ,b ;Bao et al.2013),anti-mutagenW.-J.Zheng ·X.-C.Wan ·G.-H.Bao (&)State Key Laboratory of Tea Plant Biology and Utilization,Anhui Agricultural University,130Changjiang West Road,Hefei 230036,Anhui Province,Chinae-mail:baoguanhu@Phytochem Rev (2015)14:499–523DOI 10.1007/s11101-015-9402-8(Wang et al.2005;Jie et al.2006;Zhang et al.2011a,b) and so on.Studies on BDT processing technology as well as its biological components have progressed recently. With the rapid growing popularity of this unique tea, it is necessary to review the recent advances, comprehensively.Therefore,the present paper at-tempted to access to the developments in BDT production and the bioconversion of major chemical constituents from BDT.Meanwhile,the chemical components of brick dark tea(BDT)were also listed in this paper.The definition of BDT and its typesThe definition of BDTBrick dark tea(BDT,also called border-sale tea),a kind of brick-formed tea,is a typical compressed tea. This tea is made by older,coarse and rough leaves and branches of C.sinensis var.sinensis or C.sinensis var.assamica mainly in China and Japan(Zhu and Liu1989).In modern manufacture of BDT,high quality dark raw tea or sun-dried leaves involve a solid-state fermentation stage,which is considered to be the most important procedure to the unique characteristics of the mellow taste,the stableflavor, and the special fungal aroma in tea infusion.BDT,mainly produced in Shanxi,Hubei,Hunan, Sichuan,Yunnan provinces in China,is a kind of highly-praised goods in the ancient Tea-Horse road. Together with milk and meat,BDT has been the necessities in Northwest Chinese life and been popular among Central Asia and Russian regions (Zhu and Liu1989).Types of BDT productsAccording to its origin and processing methods,BDT products can be categorized into several types: Heizhuan brick tea,Huazhuan brick tea,Fuzhuan brick tea(FBT),Qingzhuan brick tea,Kangzhuan brick tea,and Pu-erh brick tea(shown in Fig.1)(Wan2003).Heizhuan brick tea(same as“Bazi”)and Huaz-huan brick tea(same as“Qianliang”),pressed from crude dark tea,are mainly produced in Anhua, Hunan.The differences between these two kinds of dark tea are that there are patterns on the surface of Huazhuan brick tea while there is none on Heizhuan brick tea.Fuzhuan brick tea(FBT)is produced by Yiyang Tea Manufactory in Hunan Province and Xianyang Jingwei Fu Tea Manufactory in Shanxi Province(Zhu and Liu1989).FBT is pressed using crude dark tea in Hunan or sun-dried leaves in south of Shanxi. Distinct from other teas,“goldenflora”would appear on FBT after the fungal fermentation process(Ling et al.2010;Luo et al.2013;Zhu et al.2015).Produced by Zhaoliqiao Tea Manufactory in Hubei,Qingzhuan brick tea is also named as “Chuanzi tea”as there is a Chinese character“川”on its surface.This BDT is pressed with high quality Hubei old tea(Zhu and Liu1989).Mainly produced in Yingjing,Ya’an and other counties in Sichuan province,Kangzhuang brick tea has round corners(Zhu and Liu1989).Pu-erh brick tea is produced by the sun-dried leaves of broad-leaf tea species(C.sinensis var. assamica)in Dali,Yunnan province(Zhu and Liu 1989).Manufacture of BDTThe manufacture of tea contributes greatly to its characterization(Xu et al.2013).The fresh tea leaves of BDT are plucked from C.sinensis var.sinensis and C.sinensis var.assamica.According to the varieties of BDT,the manufacture of tea is not all the same in detail.However,the major process of producing BDT can be divided into two parts:processing of fresh tea leaves and processing of raw dark tea.Processing of fresh tea leavesDark tea in Hunan commonly use leaves from C. sinensis var.sinensis with one bud and three or four leaves.The old tea in Hubei and dark tea in Sichuan have low standard on its fresh leaves as their only requirement is that there is no deadwood or diseased leaves in fresh leaves(Zhu and Liu1989).After plucking,the fresh leaves are spread indoors to inhibit respiratory heat.The following manufac-turing process of fresh tea leaves is summarized as steaming,rolling,microbial fermentation and drying, after which the raw dark tea isfinally made(Fig.2a). In the stage of steaming,about10kg of fresh leavesare heated at temperature of300°C until the color turns from fresh green into dark green,and the leaves become supple and the peduncle cannot easily be broken off.During this period,the activity of endogenous enzymes such as Polyphenoloxidase (PPO)and Peroxidase(POD)declined,correspond-ingly,owing to high temperature(Takeo and Uritani 1966).Microbial fermentation is considered to be the key step for manufacture of dark tea.During the microbial fermentation process,the rolled leaves should be stacked up in a room with the temperature above25°C for24–40h and its relative humidity (RH)is about85%,which would lead to complex oxidation,condensation,and degradation of chemical components in tea.Drying is thefinal step of making raw dark tea.In this step,moisture content of raw dark is12%in Hunan dark tea,13%in Hubei old tea,14%in both Sichuan and Shanxi dark tea(Zhu and Liu1989;Ji and Xu1993).Processing of raw dark teaProcessing of raw dark tea is divided into softening with steam,piling,brick-pressing,and drying.Specially,FBT has a unique procedure before drying: fungal fermentation(Fig.2b).In this stage,many fungi,such as Eurotium spp.,Saccharomyces spp., and Aspergillus spp.,are cultivated in raw brick tea. When fungal fermentationfinished,FBT was covered by“goldenflora”(in Fig.1)—Eurotium spp.inside. According to Xu et al.(2011),the dominant genera—Eurotium,Debaryomyces and Aspergillus—were beneficial fungi associated with the fermentation of FBT.The manufacture of BDT in Yunnan has been reported by Lv et al.(2013),so details on this kind of tea before2013will not be shown in this paper. Microorganism in BDTAs post-fermented tea,microorganism plays a significant role in tea quality.Recently,more atten-tion has been taken to study this issue.Chen found E.cristatum in both Kangzhuan brick tea(KBT)produced in Sichuan and Qingzhuan brick tea(QBT)produced in Hubei(Chen et al.2006).In 2008,Fu and Qi separated and identified Bacillus, Fig.1Types of BDT productsFig.2a Processing of fresh tea leaves.b Fungal fermentation processingCandida,Actinomyces,Staphylococcus,and Mucor as the dominant microorganisms in KBT produced in Sichuan.Chen et al.(2013)studied the fungi in FBT produced in Shanxi province.In their report,they identified the purified strain as E.cristatum and its anamorph was Aspergillus cristatellus,synonym as A. cristatus or A.spiculosus.Hu et al.(2011)studied the dominant microorganisms in18kinds of FBT produced by different factories in Hunan province, and found that there werefive major superior strains in FBT:E.cristatum,E.costiforme,E.amstelodami, E.chevalieri and E.herbariorumIu,which proved that Eurotium spp.was the dominant fungi in FBT.According to the investigation on microbes during the pile-fermentation process of Yunnan Pu-erh tea, the main microbes were Rhizopus,Saccharomyces, Aspergillus,Penicllium,Bacterium,etc.(Sun et al. 2008;Zhou et al.2004;Zhu et al.2008).Later in 2005,Zhao and Zhou reported that A.niger was the most dominant microorganism among these mi-crobes,the second was Saccharomyces and the last was Bacterium.Chemical constituents of BDTAlthough BDT is very popular among certain ethnic region in China and southeastern Asia,there have been few studies on its microbial fermentation process and the changes of chemical components during microbial fermentation until the upsurge about “Pu-erh Hot”(Zhang et al.2013).To better understand microbial fermentation pro-cess and the metabolites during the process of the fermented BDT,many scientists have studied chemi-cal constituents from these tea by high-speed counter-current chromatography(HSCCC)(Yanagida et al. 2006),high performance liquid chromatography (HPLC)(Wang et al.2009a,b),near infrared spectroscopy(NIRS)(Zhou et al.2009),fourier transform infrared spectroscopy(FTIR)(Ning et al. 2011),metabolomic analysis(Lee et al.2011;Xu et al.2015),high performance liquid chromatography coupled to mass spectrometry(HPLC–MS)(Luo et al.2013;Xu et al.2015)and chromatography coupled to mass spectrometry(GC–MS)(Xu et al. 2007;Dong et al.2008).Now,many constituents have been separated and identified.The major compounds isolated and identified from BDT were shown pounds in Pu-erh brick tea before 2013were listed by Lv et al.(2013),so in this paper, new compounds found in Pu-erh brick tea reported only after2013were listed.These BDT compounds can be divided into different categories according to their chemical structures(the compounds in the following paragraph were listed in“Appendix”).Flavan-3-ols and their derivatives(1–39)Commonly,the term“catechins”refers to(+) -catechin(C,1),(−)-epicatechin(EC,2),gallocatech-in(GC,3),epigallocatechin(EGC,4),(+)-catechin gallate(CG,5),epicatechin-3-O-gallate(ECG,6), (−)-epigallocatechin-3-O-gallate(EGCG,7),(−)-epi-catechin8-C-glucoside(EC-8-Glc,8),epiafzelechin (EA,9),epiafzelechin(EAG,10),(−)-epigallocatech-in-3-O-(4″-O-Me)gallate(EGCG4″Me,11), gallocatechin3-O-gallate(GCG,12)(Davis et al. 1996;Kumar and Rajapaksha2005).EC(2),ECG (6),EGC(4)and EGCG(7)were the main catechins responsible for the antioxidant actions of green tea and their levels were reduced by oxidation and polymerization during enzymatic reactions in black or oolong tea and microbial post-fermentation in Pu-erh tea and Fuzhuan brick tea(Cabrera et al.2003; Lin et al.2008;Xie et al.2009;Zuo et al.2002;Luo et al.2013;Zhu et al.2015;Xu et al.2015).The content of catechin monomers of BDT was far less than those in green tea,oolong tea,white tea and black tea(Peterson et al.2005;Wu et al.2010;Zhu et al.2015;Xu et al.2015).The residual main polyphenols were gallic acid(GA,60)and epigallo-catechin(EGC,4)in FBT compared to those of green tea and black tea(Zhu et al.2015;Wu et al.2010;Fu et al.2011;Liang et al.2005).In the report of Wu et al.(2010),there were about20%decrease of catechins and catechin monomers parallel with total catechins,except for(−)-catechin gallate(CG,5) which was too low to be detected during the stage when raw dark tea turned into dark tea.Other derivatives of catechins were(R)-6-oxo-4-((2R,3R)-3,5,7-trihydroxychroman-2-yl)-3,6-dihydro-2H-pyran-2-carboxylic acid(13)(Jiang et al.2011), teasperin(14)(Kanegae et al.2013),teadenol B(15) (Kanegae et al.2013),which were identified in fermented tea.According to Jiang et al.(2011), catechin degradation in microbial fermented teaincluded polymerisation and oxidative cleavage of aromatic rings and the degradation of tea catechins was related to the production of humic acids.Recently,six new derivatives of catechins:fuz-huanin A(16),fuzhuanin B(17),fuzhuanin C(20), fuzhuanin D(21),fuzhuanin E(22)and fuzhuanin F (23),together with teasperol(18),xanthocerin(19) have been isolated and identified in FBT(Luo et al. 2013;Zhu et al.2015).As Luo et al.reported in2013, fuzhuanin B(17)showed antiproliferative activity and teasperol(18)showed bactericidal activity.Among these components,13–23are B-ring fission derivatives of catechin which were only found in post-fermented tea and were considered to be the characteristic constituents of post-fermented tea(Zhu et al.2015).Later in2014,Tao et al.isolated four new phenylpropanoid-substitutedflavan-3-ols,puerins C-F(24–27),which were formed in Pu-er tea post-fermentation process,and were isolated for thefirst time from tea and Theaceae plant.Tian et al.(2014) identified newflavan-3-ol derivatives,8-car-boxymethyl-(+)-catechin(36),8-carboxymethyl-(+)-catechin methyl ester(37),6-carboxymethyl-(+)-catechin(38),and6-carboxyl-(−)-gallocatechin (39),from the n-BuOH-soluble fraction of ripe Pu-erh tea.Wang et al.(2014)separated eight8-C N-ethyl-2-pyrrolidinone substitutedflavan-3-ols,puerins I–VIII (28–35)in the aqueous extract of Pu-erh tea.In their report,they quantitatively analyzed the changes of C, EC,GC,EGC and theanine during the post-fermen-tation process.According to the results,they hypothesized that the N-ethyl-2-pyrrolidinone moiety of puerins was derived from the structure of theanine, and the biosynthetic pathway of puerins I–VIII was correlated with theanine and catechins.The changes on the above16new catechin derivatives from Pu-erh tea all happened on the A ring,which was different from those B ringfission derivatives of catechin from FBT whose changes were on the B ring,which maybe caused by different microbial species based on the different processing procedures(Structures shown in Fig.3a).Flavones and their glycosides(40–59)Tea chemical constituents comprise2–5%flavones and their glycosides(Wan2003).They are derivatives offlavone.(2R,3R)-taxifolin distylin(40)was separated from endophytic fungus in teaand ampelopsin(41)was identified in post-fermentedtea(Freudenberg and Weinges1958;Agusta et al.2005).In2013,kaempferol(42)was isolated from“Yamabukinadeshiko”tea extract by Kanegae et al.Quercetin(43)and myricetin(44)wasfirst separatedin Pu-erh tea by Zhao et al.(2011).Flavonoids in BDT are predominately present astheir glycosides,and the majorflavonoid glycosidesin BDT appear to be O-linked glycosides,one C-linked glycosides together with two compoundscharacterized by both C-and O-glycosylation.The onlyflavone C-glycosides was vitexin-2″-α-L-rhamnopyranoside(45)together with its anions wereidentified as vitexin-2″-α-L-rhamnopyranoside-7-oxy-gen anion(46).These two compounds werefirstseparated from Crataegus(Sun et al.2002),and werelater discovered in black tea and Fuzhuan brick tea(Scharbert et al.2004;Luo et al.2013).Chafurosides A(47)and chafurosides B(48)werefirst separated from oolong leaves,and later thesecomponents were also identified in dark tea(Hitoshiet al.2009).The quercetin glycosides are the majorflavone glycoside in BDT.In addition,BDT is alsoenriched in kaempferol glycosides.Theflavoneglycosides were listed as follows:kaempferol3-O-α-L-rhamnopyranoside(49),biorobin(50),quercetin-3-O-robinobioside(51),kaempferol-3-O-[β-D-glucopy-ranosyl-(1→3)-O-α-L-rhamnopyranosyl-(1→6)-O-β-D-galactopyranoside](52),nicotiflorin(53),rutin (54),myricetin-3-O-rutinoside(55),kaempferol-3-O-[β-D-glucopyranosyl-(1→3)-O-α-L-rhamnopyra-nosyl-(1→6)-O-β-D-glucopyranoside](56), quercetin-3-O-[β-D-glucopyranosyl-(1→3)-O-α-L-rhamnopyranosyl-(1→6)-O-β-D-glucopyranoside] (57),quercetin-3-O-[β-D-glucopyranosyl-(1→3)-O-α-L-rhamnopyranosyl-(1→6)-O-β-D-glucopyra-noside]-7-oxygen anion(58)and kaempferol-3-O-glucoside(59)(Kanegae et al.2013;Zhao et al.2011; Scharbert et al.2004;Farkas et al.1976;Brasseur and Angenot1986;Tomczyk et al.2002;Zhou and Yang 2000;Tanikawa et al.2011;Finger et al.1991). Specially,compound53wasfirst detected inflowers and leaves of Ficaria verna(Tomczyk et al.2002). Compounds55and56werefirst isolated from petals of Clitoria ternatea and Wabisuke camellia cv.Tarokaja I,respectively(Tanikawa et al.2011; Kazuma et al.2003)(Structures shown in Fig.3b).Fig.3a Flavan-3-ols and their derivatives(1–39).b Flavones and their glycosides(40–59).c Phenolic acids and derivatives (60–72).d Phenylpropanoids and hydrolysable tannins(73–78).e Terpenoids(79–119).f Caffeine,pyrimidines,and pyrrole (120–125).g Alkans,aryl compounds,and fatty acids(126–135).h Anthraquinones,phenylpropanoids and esters(136–139).i Aldehydes,alcohols,and ketones(140–162).j Minor compounds(163–164)Fig.3continuedFig.3continuedFig.3continuedPhenolic acids and derivatives(60–72)Compared the content of chemical components in dark tea with other tea,such as black tea,the content of small molecules in dark tea is much higher than that of black tea(Zhang et al.2011a,b;Drynan et al. 2010;Zhang et al.1995;Zou et al.2009).Gallic acid(GA,60)is a characteristic phenolic compound as well as one of the most important phenolic acids in BDT with notable bioactivity(Sak-agami and Satoh1997).2,3-dihydroxyl-1-(4-hydroxy-3-methoxyphenyl)-propan-1-one(61)showed no in-hibition activity of antibacterial assay at the concentration of800μg mL−1by hole plate diffusion method(Luo et al.2013;Baderschneider and Winter-halter2001).Pyrogallol(62)(Zhu et al.2015),recently isolated from FBT,previously isolated from green tea,was examined for toxicity towards egg-bearing adults of Caenorhabditis elegans,and was tested toxic(Mukai et al.2008).There were plenty of methyl ethers of phenolic compounds such as2-methylphenol(63), 3-ethylphenol(64),2,5-dimethylphenol(65),4-ethyl-guaiacol(66),gentisic acid(67),2-methoxy-4-propylphenol(68),1-(2-hydroxy-5-methoxyphenyl) ethanone(69),3,4,5-trimethoxybenzaldehyde(70)in BDT(Lin et al.2006;Xu et al.2005;Zhu et al. 2015).These compounds were considered to be the products of degradation and methylization of tea catechins by the microorganisms and chemically transformed into new compounds during the fermen-tation(Xu et al.2005;Guerin and Olerich1975). Benzyl2-neohesperidosyloxy-6-hydroxybenzoate (71)and(7R,8S)-dihydrodehydrodiconiferyl alcohol 9-O-β-D-glucopyranoside(72)were two simple phe-nolic compounds with an aglycon found in FBT(Luo et al.2013)(Structures shown in Fig.3c). Phenylpropanoids(73–77)and hydrolysable tannins(78)5,7-Dihydroxycoumarin(73)showed significant an-tioxidation and antibacterial activity(Luo et al. 2013).Liu et al.(2013)isolated salicifoliol(74), (+)-matairesinol(75),symplicosigenol(76)and p-hydroxy ethyl cinnamate(77)from Qianliang tea,a kind of dark tea fully fermented from the leaves of C. sinensis var.sinensis for thefirst time.Strictin(78),which was separated from FBT by Luo et al.(2012),was defined as hydrolysable tannins (Structures shown in Fig.3d).Terpenoids(79–120)Terpenoids are considered to contribute to the characteristic aroma of BDT including monoterpenes, sesquiterpenes,diterpenes,and triterpenes.Wan(2003)listed some of the monoterpenes in BDT.They were linalool oxide B(79),linalool oxide C(80),linalool oxide D(81),limonene(82),β-terpinene(83),p-mentha-2,4(8)-diene(84),cis-β-ocimene(85),β-myrcene(86)and trans-β-ocimene (87).Yue et al.had listed them in the TMDB in2014.From identified sesquiterpenes,Luo et al.(2013) tested dihydrophaseic acid(88)and epidihydropha-seic acid(89)about their antibacterial activities. Results showed that there was no antibacterial activity at the concentration of800μg mL−1.Other compounds listed by Wan(2003)wereα-gurjunene (90),longifolene(91),α-cedrene(92),α-muurolene (93),α-humulene(94)andα-curcumene(95).These compounds,together with monoterpenes,were con-sidered to be the major active ingredients for tea aroma.They were listed in TMDB by Yue et al. (2014).Neophytadiene(96)and6,10,14-trimethylpentade-can-2-one(97)was the only diterpenes found in FDT (Wan2003).Reports about compound96demon-strated that it could be as a measure contributed to experimental tobacco smoke carcinogenesis(Guerin and Olerich1975).Oleanane-type triterpenes were the major triterpe-nes separated and identified in BDT.3β,6α,13β-trihydroxy olean-7-one(98)and3β-acetoxy-6α,13β-dihydroxyolean-7-one(99)were thefirst two exam-ples of triterpenoids possessing a6-hydroxy-7-one function found in FBT(Ling et al.2010).In their report,compound98performed no cytotoxic activity against A-549,Bel-7402,and HCT-8cells.In addi-tion,in the paper,β-amyrone(102)and3β-O-(8-hydroxyoctanoyl)-12-oleanene(103)were also separated from FTB.Friedelin(100)and canophyllol(101)isolated from FBT(Zhu et al.2015),previously isolated from Endodesmia calophylloides(Ngouamegne et al. 2008),were tested for antiplasmodial activity against the W2strain of Plasmodium falciparum,and weredemonstrated a direct link to antiplasmodial activity in vitro with IC50values of7.2and15.0mM, respectively.2-hydroxydiplopterol(104)isolated from FBT (Zhu et al.2015)performed cytotoxicity against K562cell lines with an IC50value of22μM, however,it didn’t exhibit any cytotoxic effects on A-459(IC50260μM)or P388cell lines(IC50 192μM)in SRB assay(Wang et al.2009a,b).13,26-epoxy-3β,11α-dihydroxyolean-12-one(105)and 3β,11α,13β-trihydroxyolean-12-one(106)werefirst found in Japanese post-fermented tea(Huang et al. 2013).In their report,compound105was considered to be thefirst oleanane-type triterpene with an ether linkage between C-13and C-26.The presence of a ketone group at C-12was determined as the charac-teristic feature of compounds105and106.Some other terpenoids,such as taraxastane-3β,20β-diol(107),taraxastane-3β,20α-diol(108),α-spinasterone(109),α-spinasterol(110),22,23-dihy-dro-α-spinasterone(111)and22,23-dihydro-α-spinasterol(112)were identified in fermented dark tea with unknown pharmacological function(Wan 2003;Yue et al.2014).In2012,Luo et al.firstly obtained a new norisoprenoid3R,9R-oxido-5-megastigmene(113) from the CHCl3and n-BuOH fractions of FBT.They suspected that compound113was the C-3/C-9ether derivative of(3R,9R)-3-hydroxy-7,8-dihydro-β-ionyl 6-O-β-D-apiofuranosyl-β-D-glucopyranoside(114),an aroma precursor from the fresh tea leaves.In their report,compound113exhibited no inhibition activity against the tested enteric pathogenic microorganisms, i.e.,enteropathogenic E.coli(EPEC),S.aureus,S. dysenteriae,and S.typhi at a concentration of 800μg mL−1by the hole plate diffusion method (Luo et al.2012).In addition,they identified roseoside(116)and icariside B5(117).These com-pounds,together withα-tocopherol(115)(Wang and Quinn1999),can also be classified as sesquiterpenoid glycosides(116and117)or terpenoid derivative (115)(Luo et al.2012).Linalool oxide A(118),as well asα-phytol(119)which was isolated recently by Luo et al.(2012)from FBT,were both defined as terpenoids(Structures shown in Fig.3e).In Keller’s et al.report(2013),they made a comparison of the antibacterial activity between fermented Fuzhuan brick tea and non-fermented green tea.The results revealed that both FBT and non-fermented green tea did inhibit the growth of S. sonnei,dependently and significantly,compared with negative treated controls(p\0.01),with a minimum inhibitory concentration(MIC)of 5.0and 0.625mg mL−1,respectively.However,there was no growth inhibitory activity observed against E.coli, S.enterica Typhimurium,or P.aeruginosa. Caffeine(120),pyrimidines(121–124)and pyrrole(125)Similar to other teas,the major alkaloids in BDT is 1,3,7-trimethylxanthine(caffeine,120).The content in FBT is2.8%while in other teas is mainly between 1.5and5%,with the concentration in green and black tea being very similar(Zhu et al.2015;Wan2003).The RNA and DNA are metabolized and degraded during manufacture process by tea nucleases natural-ly in tea leaves(Imagawa et al.1982).These catabolic reactions produced derivatives of nucleic acids,such as purines and pyrimidines,which have been detected in very small quantities in tea(Har-bowy and Balentine1997;Hicks et al.1996). Cytosine(121),5-methylcytosine(122),thymine (123)and uracil(124)are the mainly pyrimidines in BDT(Wan2003;Yue et al.2014).Containing a stable cyclic structure,these components are hardly to be degraded(Fu et al.2008).Recently,N-(2-hydroxyphenyl)-2-pyrrolidinone (125)was isolated as a new natural product in Huazhuan brick tea(Liu et al.2013),and was now recognized as the new characteristic component of this tea(Kumar and Rajapaksha2005)(Structures shown in Fig.3f). Alkanes(126–128),aryl compounds(129–132)and fatty acids(133–135)There are a host of small molecules in BDT,such as alkanes,aryl compounds and fatty acid.Wan(2003) listed them as follow:hexadecane(126),heptadecane (127),octadecane(128),benzene(129),toluene (130),ethylbenzene(131),naphthalene(132).Re-cently,some new fatty acids were separated from fermented C.sinensis—FBT.They areα-linolenic acid(133),dodecanamide(134)and linoleamide (135)(Luo et al.2012;Keller et al.2013).Castellano et al.(2014)conducted a safe and short-term evaluation of dietary mono-conjugatedα-li-nolenic acid isomers(CLNA;c9-t11-c15-18:3+c9-t13-c15-18:3),and played it in a neonatal pig model. Their results supported that short-term taking in of mono-CLNA was safe in neonatal pigs. However,n-3PUFA reduction in tissues needed further investigating before using long-term nutri-tional supplementation in pigs and other animal models,as well as before it move to clinical trials (Structures shown in Fig.3g).Anthraquinones(136,137)and esters(138,139)In Xu’s et al.(2011)study,emodin(136),firstly investigated by micellar electrokinetic capillary chro-matography in Chinese tea(Zheng et al.2004), inhibited the activity of casein kinase II selectively as a competitive inhibitor.In their report,physcion (137)showed antimicrobial activity.In addition,Liu et al.isolated ethyl4-(sulfooxy) benzoate(138)from Qianliang tea in2013,which was revealed to be thefirstly isolated from Theaceae family.Ethyl propanoate(139)wasfirst found in Pu-erh brick tea(Xu et al.2005)(Structures shown in Fig.3h).Aldehydes(140–154),alcohols(155–157)and ketones(158–162)Some small molecules,which are regarded as the products of degradation,such as aldehydes,alcohols and ketones,are the most important components for the formation of aroma.Aldehydes were listed as follow:phenylacetalde-hyde(140),2-phenylpropanal(141),2-phenyl-2-butenal(142),4′-hydroxyacetophenone(143),2,5-dimethylhexanal(144),trans-2,cis-4-heptadienal (145),(e)-2-octenal(146),hexanal(147),2-hexenal (148),heptanal(149),(e)-hept-2-enal(150),2,4-heptadienal(151),nonanal(152)(Acree and Arn, 2004),trans-2-decenal(153)and trans-2,trans-4-decadienal(154)(Wan2003;Yue et al.2014).Table1Variations of main components content during processing of brick dark tea(%)Chemical constituents FTB KBT PBT ReferencePF AF PF AF PF AFEGC0.880.390.6720.233 2.15–Zuo et al.(2002),Qi et al.(2004)DL-C0.140.050.3330.1110.490.33Zuo et al.(2002),Qi et al.(2004)EC0.340.120.3470.127 1.190.36Zuo et al.(2002),Zhu et al.(2015)EGCG 2.300.710.8870.547 4.04–Zuo et al.(2002),Zhu et al.(2015),Qi et al.(2004) GCG0.120.040.2250.187––Zhu et al.(2015),Qi et al.(2004)ECG0.730.330.2980.28411.31–Zuo et al.(2002),Zhu et al.(2015),Qi et al.(2004) Total catechins 4.51 1.64 2.763 1.36518.31 3.62Liang et al.(2005),Fu et al.(2008),Qi et al.(2004),She et al.(2005)Tea polyphenol8.77 3.988.18 5.9929.739.40Liang et al.(2005),Fu et al.(2008),Qi et al.(2004),Chen et al.(2012),Luo et al.(1998),She et al.(2005)Flavonoids 4.93 2.39––––Wang et al.(1991a,b)Zhang et al.(2006)Crudefiber content24.9820.7418.2214.90––Wang et al.(1991a,b),Zhang et al.(2006) Soluble sugar 4.34 4.0812.20 6.29 5.13 3.86Wang et al.(1991b),Zhang et al.(2006),Fu et al.(2008),She et al.(2005),Qi et al.(2004) Amino acids0.380.24 1.70 1.16 1.800.66She et al.(2005),Qi et al.(2004),Fu et al.(2008) Caffeine 1.29 1.15 1.3 1.27 4.19 5.17Chen et al.(2012),Tian et al.(2011),Wang et al.(2008),Fu et al.(2008)Total content of nitrogen 1.84 2.35––––Wang et al.(1991a)Soluble sugar 4.34 4.0812.20 6.29 5.13 3.86Wang et al.(1991a),Zhang et al.(2006),Fu et al.(2008),She et al.(2005),Qi et al.(2004) Water-extractable substances22.6322.1026.9423.9245.041.0Fu et al.(2008),Chen et al.(2012),Qi et al.(2004)FBT Fuzhuan brick dark tea,KBT Kangzhuan brick dark tea,PBT Pu-erh brick dark tea,PF pre-fermentation,AF after fermentation。