Journal of the Science of Food and Agriculture J Sci Food Agric 87:1502–1504 (2007)

Analysis of characteristic aromaof fungal fermented Fuzhuan brick-tea bygas chromatography/mass spectrophotometryXiangqun Xu,1 Haizhen Mo,2 Mingchao Yan3 and Yang Zhu4∗1Department of Applied Chemistry, Zhejiang Sci-Tech University, Hangzhou 310018, Zhejiang Province, China2Department of Food Science, Henan Institute of Science and Technology, Xinxiang 453003, Henan Province, China3The Management Committee of Hangzhou High-Tech Industry Development Zone, Hangzhou 310012, Zhejiang Province, China4Applied Plant Research, Wageningen University and Research Centre, PO Box 16, NL-6700 AA Wageningen, The Netherlands

Abstract: Fuzhuan brick-tea is a popular fermented Chinese dark tea because of its typical fungal aroma.Fungal growth during the production process is the key step in achieving the unique colour, aroma and tasteof Fuzhuan brick-tea. To further understand the generation of the characteristic aroma, changes in the mainvolatile compounds of Fuzhuan brick-tea during the fungal growth stage were studied by gas chromatography/massspectrophotometry. The results showed that the content of volatile compounds, especially aldehyde compoundswith stale aroma such as (E)-2-pentenal, (E)-2-hexenal, 1-penten-3-ol, (E, E)-2,4-heptadienal and (E, Z)-2,4-heptadienal, increased significantly in fermented tea samples. The concentration of terpene alcohols with floweraroma also increased notably during the fermentation process. The compounds with stale and flower aromas incombination with some volatile components of the raw material contributed to the characteristic ‘fungal/flower’aroma of Fuzhuan brick-tea. Microbial metabolism during the fermentation process probably played the key rolein the generation of characteristic aromatic compounds of Fuzhuan brick-tea. 2007 Society of Chemical Industry

Keywords: volatile compounds; Fuzhuan brick-tea; GC/MS, fermented tea

INTRODUCTIONFuzhuan brick-tea is a pressed dark tea very pop-ular within ethnic groups in the border regions ofsouthern China. It is one of a small number ofmicrobial fermented teas manufactured via a fun-gal fermentation process and has a strong taste withspecific ‘fungal/flower’ aroma1,2 The manufacturingprocess of classic Fuzhuan brick-tea involves steam-ing, piling, pressing, fermentation (fungal growth)and drying. Pretreated tea leaves are pressed into‘brick’ form before fermentation. It is fermented withmicro-organisms that enter the fermentation processas natural flora or so-called ‘golden flora’ based on thecolour of fungal growth within the tea under controlledtemperature and moisture.3 Fungal growth during theprocess is considered to be the key step in achiev-ing the unique colour, aroma and taste of Fuzhuanbrick-tea.4,5 In the fungal growth stage, complex bio-chemical changes take place to form the characteristicaroma. Although Fuzhuan brick-tea is very popularwithin certain ethnic groups in China and southeast-ern Asia, there have been few studies on its microbialfermentation process and analysis/characterisation ofthe fermentation metabolites, i.e. volatile compounds.A better understanding of the fermentation processand the metabolites produced should help in improv-ing and standardising the process for the productionof traditional fermented Fuzhuan brick-tea. In order

to understand the generation of the compounds thatimpart desirable flavour characteristics, we studied thearoma pattern and development of key volatile com-pounds of Fuzhuan brick-tea during the fermentationprocess by gas chromatography/mass spectrophotom-etry (GC/MS).

MATERIALS AND METHODSRaw material and samplingSamples of Fuzhuan brick-tea during fermentationwere obtained in situ from Yiyang Tea Manufactory,Hunan Province, China. The raw material was rawdark tea made from tea leaves by steaming, piling andpressing. The fermented product was harvested after16 days of fermentation of the pressed raw dark tea.

Preparation of aroma concentrateEach 50 g tea sample was extracted with 1 L of distilledwater and 50 mL of diethyl ether by the continuoussteam distillation/solvent extraction (SDE) method for20 min.6 The extract was dehydrated over anhydroussodium sulfate and concentrated to yield about 100 mgof aroma concentrate.

GC/MS analysisGC/MS analysis was done using HP 5890–5972GC-MS. The instrument consists of a 5890 Series

∗ Correspondence to: Yang Zhu, Applied Plant Research, Wageningen University and Research Centre, PO Box 16, NL-6700 AA Wageningen, The NetherlandsE-mail: yang.zhu@wur.nl(Received 20 February 2006; revised version received 17 September 2006; accepted 8 October 2006)Published online 24 April 2007; DOI: 10.1002/jsfa.2874

2007 Society of Chemical Industry. J Sci Food Agric 0022–5142/2007/$30.00

Fermented Fuzhuan brick-tea

II Gas Chromatograph, with a model 5972 mass-selective detector (Hewlett Packard, Palo Alto, CA,USA). The column was a 0.25 mm × 50 m capillarycolumn coated with PEG 20M. The column oventemperature was programmed to increase from 50 to190 ◦C at a rate of 2 ◦C min−1. The injection portand the detector were maintained at 200 and 280 ◦Crespectively. The aroma constituents were identifiedby matching GC and MS data with those of authenticcompounds or published data. The relative amountof each compound was calculated as the ratio of thecompound peak area to the internal standard (ethyldecanoate) peak area.1

RESULTS AND DISCUSSIONThe contents of volatile compounds in tea samplesbefore and after fermentation are given in Table 1.Twenty-eight main components were identified,accounting for 91.5% of the essential oil. Among them,aldehydes were dominant, representing 51% of theidentified compounds. The major volatile compoundsin fermented Fuzhuan brick-tea were hexanal, 1-penten-3-ol, (E)-2-pentenal, (E)-2-hexenal, (E, Z)-2,4-heptadienal, (E, E)-2,4-heptadienal and linalooloxides. The two heptadienal isomers, 1-penten-3-ol and (E, E)-2,4-nonadienal with stale aroma andsun-baked flavour were the main contributors tothe characteristic aroma of Fuzhuan brick-tea, inagreement with previous results.7

Figure 1 shows that the main aromatic componentsin fermented Fuzhuan brick-tea and raw dark teasamples were aldehydes, ketones, aliphatic alcohols,terpene alcohols and esters. However, the aromapatterns of tea samples before and after fermentationdiffered, with the proportion of aldehydes to othercompounds being much higher in the fermented tea. Inaddition, the total content of aromatic compounds inthe fermented tea increased significantly. Specifically,aldehydes increased 4.4-fold, aliphatic alcohols 4.7-fold and terpene alcohols 1.9-fold compared withsamples before fermentation (Fig. 1). In normalblack tea, C5 and C6 alcohols and aldehydes areproduced by peroxidation and decomposition ofunsaturated fatty acids during withering and non-microbial fermentation.8,9 In Fuzhuan brick-tea thecontents of (E)-2-pentenal, hexanal, (E)-2-hexenaland 1-penten-3-ol were respectively 9.0, 3.3, 3.3 and7.9 times higher than those in the unfermented tea.Therefore it is hypothesised that severe autoxidationand hydrolysis of unsaturated fatty acids in the rawdark tea took place during fermentation under certainmoisture and temperature conditions. Whether thesefatty acids were oxidised by fungal oxidase generatedvia fermentation requires further investigation. Therewere no enzymes surviving in the raw dark tea, unlikein the case of black tea. During black tea processing,enzymes from fresh tea leaves remain active andplay an important role in biochemical reactions andformation of new compounds, e.g. in peroxidation and

Table 1. Changes in volatile compounds in Fuzhuan brick-tea during

processing

Contenta

No. CompoundRaw

dark teaFermented

teaSimilarity

(%)

1 Ethylpropanoate

0.52 2.28 94

2 2-Pentanone 0.92 3.74 723 Pentanal 0.53 0.39 914 3-Methyl-2-

pentanone0.25 1.37 72

5 1-Penten-3-one 0.93 5.57 906 Hexanal 3.29 10.84 917 Ethylbenzene 0.30 1.11 808 (E)-2-Pentenal 0.58 5.20 819 1-Butanol 0.78 4.04 9010 1-Penten-3-ol 0.77 6.12 7211 Heptanal 0.34 1.41 9112 (E)-2-Hexenal 0.75 2.46 9013 2-Pentyl-furan 0.21 – 8014 1-Pentanol 0.23 0.72 9715 4-Pentenal – 1.34 8016 2-Penten-1-ol 0.23 1.33 7617 6-Methyl-5-

hepten-2-one0.79 0.79 94

18 Nonanal 0.25 – 9419 Linalool oxide I 1.08 2.17 9020 (E, Z)-2,4-

Heptadienal1.54 9.04 91

21 Linalool oxide II 1.15 2.63 9022 (E, E)-2,4-

Heptadienal1.05 6.58 91

23 Benzaldehyde 0.44 0.67 9024 Linaloolb 0.67 0.77 9025 SD: standard

(ethyldecanoate)

1.00 1.00 86

26 1-Octanol 0.27 – 8627 (E)-3,5-

Heptadien-2-one

0.51 – 86

28 Linalool oxide III 0.23 – 8329 Linalool oxide IV 0.27 0.35 7230 (E, E)-2,4-

Nonadienal0.39 0.59 85

31 α-Iononeb 0.43 1.23 8332 6,10-Dimethyl-

5,9-undecadien-2-one

4.21 2.04 95

33 Nerolidolb 0.30 0.67 9234 β-Iononeb 0.51 0.79 9135 Phytol 0.34 – 91

a Values are given as the ratio of the component peak area to theinternal standard (ethyl decanoate) peak area.b Identified with MS and authentic compounds. Other componentswere identified with MS and literature data.

hydrolysis of fatty acids during withering and naturalfermentation (non-microbial but enzymatic reactions).Apart from oxidation and hydrolysis of fatty acids,some aldehydes are derived from decarboxylation

J Sci Food Agric 87:1502–1504 (2007) 1503DOI: 10.1002/jsfa

X Xu et al.

0 10 20 30 40 50 60 70 80

1

2

Content

Aldehydes

Ketones

Aliphatic alcohols

Terpene alcohols

Esters

Figure 1. Comparison of aroma patterns of Fuzhuan brick-tea duringprocessing: 1, before fermentation; 2, after fermentation.

and oxidative deamination of amino acids.10 Thecontents of both (E, Z)-2,4-heptadienal and (E, E)-2,4-heptadienal increased approximately sixfold inFuzhuan brick-tea compared with the raw dark tea.These two compounds are also found in stale teaand in tea with sun-baked flavour (arising fromphotic oxidation). Microbial metabolism during thefermentation process of Fuzhuan brick-tea may causegeneration of the two heptadienal isomers, in view oftheir abundance in Fuzhuan brick-tea samples.

Terpene alcohols, particularly linalool oxides withflower aroma, increased in the fermented tea comparedwith the raw dark tea. It is known that monoterpenealcohols such as linalool can be generated by hydrolysisof their glycosides, which are precursors of tea aroma,through the action of enzymes.11,12 That is the reasonwhy black tea and oolong tea deliver flower and fruitaromas arising from withering during processing. Itis a reasonable deduction that extracellular enzymesfrom fungal growth during fermentation played a keyrole in the formation of more monoterpene alcoholsin Fuzhuan brick-tea, because the raw dark tea itselfhad little surviving enzyme activity. In other words,more terpene alcohols in Fuzhuan brick-tea werederived from precursors in the raw tea by the action ofmicrobial glycosidases developed in the manufacturingprocess.

In conclusion, aldehydes with stale fragrance andterpene alcohols with flower aroma were producedduring the 16 day fungal growth stage of Fuzhuanbrick-tea manufacture. These key compounds con-tributed to the characteristic ‘fungal/flower’ aroma ofFuzhuan brick-tea.

PERSPECTIVESThe exact mechanism of volatile compound formationand change in Fuzhuan brick-tea needs further

clarification, because the fermentation is initiatedwith natural flora comprising a microbial complexwith dozens of different species of fungi, yeasts andbacteria. It is believed that the extracellular enzymesproduced by fungal fermentation, such as oxidase,pectinase, cellulase and protease, contribute to thevolatile changes during fermentation.13 To confirmthis, standardisation of the process using a pureculture of the responsible fungus under well-definedtemperature and moisture control conditions will benecessary. This will form part of our future studies onthe topic.

ACKNOWLEDGEMENTThe authors thank Yi Yang Tea Manufactory, HunanProvince, China for collecting samples.

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