fungal community associated with fermentation and storage of fuzhuan brick-tea

9

Click here to load reader

Upload: aiqing-xu

Post on 05-Sep-2016

221 views

Category:

Documents


0 download

TRANSCRIPT

Page 1: Fungal community associated with fermentation and storage of Fuzhuan brick-tea

International Journal of Food Microbiology 146 (2011) 14–22

Contents lists available at ScienceDirect

International Journal of Food Microbiology

j ourna l homepage: www.e lsev ie r.com/ locate / i j foodmicro

Fungal community associated with fermentation and storage of Fuzhuan brick-tea

Aiqing Xu a,b, Yuanliang Wang a, Jieyu Wen a, Ping Liu a, Ziyin Liu a, Zongjun Li a,c,⁎a Institute of Food Science and Technology, Hunan Agricultural University, Changsha, Hunan 410128, Chinab School of Life Science, Hunan University of Science and Technology, Xiangtan, Hunan 411201, Chinac National Research Center of Engineering & Technology for Utilization of Functional Ingredients from Botanicals, Hunan Agricultural University, Changsha, Hunan 410128, China

⁎ Corresponding author at: Institute of Food ScieAgricultural University, Furong District, Changsha, Huna731 84673870.

E-mail address: [email protected] (Z. Li).

0168-1605/$ – see front matter © 2011 Elsevier B.V. Adoi:10.1016/j.ijfoodmicro.2011.01.024

a b s t r a c t

a r t i c l e i n f o

Article history:Received 16 October 2010Received in revised form 29 December 2010Accepted 18 January 2011

Keywords:Fuzhuan brick-teaFungal communityDenaturing gradient gel electrophoresisEurotium cristatumDebaryomyces hanseniiAnthraquinone

Chinese Fuzhuan brick-tea is a unique microbial fermented tea characterized by a period of fungal growthduring its manufacturing process. The aim of the present study was to characterize, both physicochemicallyand microbiologically, traditional industrial production processes of Fuzhuan brick-tea. Fermenting teasamples were collected from the largest manufacturer. Physicochemical analyses showed that the low watercontent in the tea substrates provided optimal growth conditions for xerophilic fungi. The fungal communitiesexisting in teamaterials, fermenting tea, and stored teas weremonitored by polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) targeting the D1 region of the 26S rRNA genes, followed bysequencing of the amplicons. Results revealed that the microorganisms were from, or closely related to, thegenera Eurotium, Debaryomyces, Aspergillus, Verticillium, Pichia, Pestalotiopsis, Rhizomucor and Beauveria. Thisis the first report of Debaryomyces participating in the processing of Fuzhuan brick-tea. We concluded that thedominant genera Eurotium, Debaryomyces and Aspergillus are beneficial fungi associated with thefermentation of Fuzhuan brick-tea. The genus Beauveria was present in the stored Fuzhuan brick-tea,which may help protect tea products from insect spoilage. The remaining four genera were of minorimportance in the manufacturing of Fuzhuan brick-tea. The predominant Eurotium species, a strain namedEurotium sp. FZ, was phenotypically and genotypically identified as Eurotium cristatum. High performance thinlayer chromatography analysis of anthraquinones showed that emodin existed in all the dark tea samples, butphyscion was only detectable in the tea fermented by E. cristatum. The PCR-DGGE approach was an effectiveand convenient means for profiling the fungal communities in Fuzhuan brick-tea. These results may helppromote the use of microbial consortia as starter cultures to stabilize and improve the quality of Fuzhuanbrick-tea products.

nce and Technology, Hunann 410128, China. Tel./fax: +86

ll rights reserved.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Other than water, tea is the most popular beverage in the worldtoday, and China is one of the major tea producing and exportingcountries. Conventional teas are classified into four categories basedon the degree of fermentation: non-fermented green tea and yellowtea, partially fermented Oolong-type tea, fully fermented black tea,and post-fermented dark green tea. Except for the last one, the so-called fermentation in tea processing is a misnomer but still adoptedconventionally, since the fermentation involved in those teas has beenshown to be an enzymatic oxidation process induced by the tea leavesand not by microorganisms. Microbial fermentation is only involvedin the processing of post-fermented tea in which a pile fermentationtreatment allows microbiologically induced auto-oxidation and non-enzymatic auto-oxidation to complete the process (Hui et al. 2004).

Dark green teas are microbial fermented teas native to China. Themajor kinds of commercial dark green teas include Pu'er, raw darkgreen, and Fuzhuan brick-tea. Raw dark green tea is made from theleaves of Camellia sinensis var. sinensis, and is mainly used as amaterial reprocessed into Fuzhuan brick-tea. Fuzhuan brick-tea is anindigenous microbial fermented tea traditionally produced mainly inthe Hunan province of China, and throughout history has been anecessary beverage for the ethnic groups living in the border regionsof southern and western China (Chen, 2000). Fuzhuan brick-tea is notjust a beverage but also a folk medicine because of its remarkableantidysentery effects (Mo et al., 2008a,b; Ling et al., 2010).Antibacterial tests on extracts of Fuzhuan brick-tea have showninhibitory effects on certain food-borne spoilage and pathogenicmicroorganisms (Mo et al., 2008a,b). The discovery of the specialbeneficial physiological effects of Fuzhuan brick-tea has attracted tealovers throughout China, Korea and Japan.

Fuzhuan brick-tea is characterized by a unique “fungal fermenta-tion” stage during themanufacturing process. A schematic descriptionof Fuzhuan brick-tea production was given by Mo et al. (2008a,b) andour investigations into the key industrial processes of Fuzhuan brick-

Page 2: Fungal community associated with fermentation and storage of Fuzhuan brick-tea

15A. Xu et al. / International Journal of Food Microbiology 146 (2011) 14–22

tea production are illustrated in Fig. 1. Raw dark green tea and teabranchlets were mixed together (Fig. 1A), moistened by steaming andpiled up to 2 m in height and subjected to temperatures as high as80 °C overnight (Fig. 1B). The pretreated tea materials were thenpartitioned and pressed into desired sizes of brick-tea before beingplaced in the fermentation room for 15–17 days (Fig. 1C). Conse-quently, some microorganisms existing in the environment grewwithin the tea matrix. At the end of the fermentation stage manyyellow fungi, called “golden flora” (Fig. 1C1), appeared within the teaindicating that fermentation was successful. Finally, the tea productswere packaged and stored in a dry warehouse for ripening (ageing)for at least half a year. Fungal growth is considered to be the key stagein the manufacture of Fuzhuan brick-tea, as complex biochemicalchanges take place producing its characteristic aroma and flavour(Wan, 2003).

The fungi that grew during spontaneous fermentation have beenidentified as a mixture of several microorganisms, with Eurotium spp.dominant (Mo et al., 2008a,b; Ling et al. 2010). To date, the reportedresults of microbiota investigation in Fuzhuan brick-tea production werebased on culture-dependent methods (Wen and Liu, 1991), and fungalidentification relied upon morphological approaches (Qi and Sun, 1990).

The denaturing gradient gel electrophoresis (DGGE) technique isan effective and convenient means for determining populationstructures. A combination of DNA-based molecular techniquesallowed us to monitor changes in overall microbial compositionduring fermentation and showed the relationship between samples.Ercolini (2004) described the technique and reported on state-of-the-art applications of this technique to food and food-related ecosystems.A very recent study using DGGE analysis as a culture-independentmethod to investigate the fungi which play a role in Pu'er teafermentation revealed that Aspergillus niger and Blastobotrys adenini-vorans are the major fungi involved (Abe et al., 2008).

The goal of our study was to investigate the fungal diversity ofindustrially fermented Fuzhuan brick-tea, applying culture-dependentand -independent methods. We also aimed to gain informationregarding microbial populations in complicated ecosystems sincesome fastidious and slow-growing microorganisms are difficult todetect with defined media. This knowledge could help elucidate the

Fig. 1. Photographs and flow diagram of the production process of Fuzhuan brick-tea. (Topshelves in the fermentation room and (C1) a piece of fresh Fuzhuan brick-tea with golden

metabolism of dominant fungal populations, and the relationshipbetween the fungi and tea matrices, which could improve the qualityof Fuzhuan brick-tea.

2. Materials and methods

2.1. Tea leaf samples and process characterization

Sampling of fermenting Fuzhuan brick-tea, produced from late June toearly July 2009, was carried out in a major Fuzhuan brick-tea productionfactory, the Yiyang Fu Cha Industry Development Co. Ltd. (Yiyang, HunanProvince, China). Sampling depended on the stage of fermentation: pilingtea materials (S1), fermenting brick-tea in the fermentation room for0 days (S2), 3 days (S3), 6 days (S4), 9 days (S5),12 days (S6), and 15 days(S7), and newly made tea products (S8) at day 17. The samples werepackaged in sterile polyethylene bags, transported to the laboratory andstored at −20 °C until required. Additional samples included raw darkgreen tea (S9), ageing or aged Fuzhuan brick-tea I–V (S10–S14) producedby other manufacturers in China.

Tea leaf samples undergoing fermentation (S1–S8) were subjectedto physicochemical and microbial analysis. The water content of theleaf samples was calculated after the weight of the samples no longerchangedwhilst drying at 105 °C. The tea branchlets to leaves ratio wasmeasured as this greatly influences the degree of brick tightness, animportant parameter associated with air exchange, which providesfor functional aerobic fungi growth.

For fungal enumeration, a dilution plating technique was applied. A25 g tea sample was suspended in a 225 mL sterile 0.1% peptone waterwith several glass beads, homogenized for approximately 20 min at roomtemperature, then diluted 10-fold in 0.1% peptone water. An aliquot(0.1 mL)of thedesireddilution for each samplewasplatedonRoseBengalChloramphenicol Agar (Atlas, 2004), incubated for 7 days at 25 °C, fungalcounts carried out and the Eurotium highlighted.

2.2. DNA extraction from tea samples

Five grams of each tea sample was suspended in a 245 mL sterilesaline (0.85% NaCl) with glass beads (3 mmdiameter) and vortexed at

) (A) The raw dark green tea; (B) the piling tea materials; (C) fermenting brick-tea onflora (yellow dots). (Bottom) A flow diagram of Fuzhuan brick-tea making process.

Page 3: Fungal community associated with fermentation and storage of Fuzhuan brick-tea

16 A. Xu et al. / International Journal of Food Microbiology 146 (2011) 14–22

150 rpm for 30 min at room temperature. The suspension was thenfiltered through two layers of gauze. The filtrate was collected in a30 mL centrifuge tube and centrifuged (10,000×g, 10 min, 4 °C). Theresulting pellet was resuspended in a 1.0 mL sterile saline andtransferred into a sterile mortar and pestle with 0.5 g quartz grains,then ground repeatedly to fine powder in liquid nitrogen. The samplepowder was washed with 4 mL pre-warmed hexadecyltrimethylammonium bromide (CTAB) extraction buffer [1% (w/v) CTAB, 0.7 MNaCl, 1% (v/v) 2-mercaptoethanol, 2% (w/v) polyvinylpyrrolidone,0.03% proteinase K, and 0.1 M Tris–Cl, 10 mM EDTA pH 8.0] andtransferred into a 10 mL centrifuge tube. The suspension wasincubated at 65 °C for 30 min then cooled to room temperature andmixed with an equal volume of chloroform–isoamyl alcohol (24:1, v/v). The mixture was centrifuged at 10,000×g for 10 min at roomtemperature. The supernatant was transferred to a fresh tube and anequal volume of ice-cold isopropanol was added. Samples wereincubated at −20 °C for at least for 30 min to allow the total nucleicacids to precipitate, followed by centrifugation (10,000×g, 10 min,4 °C). Pelleted nucleic acids were then washed in ice-cold 70% (v/v)ethanol and air-dried until all the ethanol had evaporated prior toresuspension in 70 μL TE buffer (pH 8.0). Contaminating RNA wasremoved using RNase A for 30 min at 37 °C and the DNA solutionstored at −20 °C until required.

2.3. Polymerase chain reaction (PCR) amplification of the 26S rRNA genefragment for DGGE

For DGGE analysis, approximately 250 nucleotides of the D1 regionfrom the 26S rRNA gene was amplified by PCR with the universalprimers NL1F-GC and LS2R, synthesized by Dingguo BioTechnologiesCo, Ltd. (Beijing, China) according to the sequence informationreported by Cocolin et al. (2000) and Abe et al. (2008). The PCRswere performed using a MyCycler thermal cycler (Bio-Rad, Hercules,CA, USA), and amplifications were carried out in a final volume of50 μL containing 5.0 μL 10× PCR buffer (Mg2+ free), 5.0 μL 25 mMMgCl2, 1 μL dNTP Mixture (10 mM each dNTP), 0.5 μL Taq polymerase(5 units/μL; Promega, Shanghai, China), 1.0 μM each primer, and 1 μLof extracted DNA and sufficient sterilized distilled water. Reactionswere run for 30 cycles of denaturation at 95 °C for 60 s, annealing at52 °C for 45 s, and extension at 72 °C for 60 s. An initial denaturationat 95 °C for 5 min and a final extension step at 72 °C for 10 min werealso carried out. A 6 μL volume of each PCR mixture was analysed byagarose gel electrophoresis in 0.5× Tris–borate–EDTA (TBE) buffer.

2.4. Perpendicular DGGE analysis

The DCode Universal Mutation Detection system (Bio-Rad) wasused for DGGE analysis. Each PCR-amplified sample obtained withprimers NL1F-GC and LS2R (20 μL) was mixed with an equal volume of2× loading buffer and loaded into wells. Electrophoresis was performedin polyacrylamide gels [8% (w/v) acrylamide-bisacrylamide at 37.5:1],using a denaturing gradient from 30 to 70% [100% corresponding to 7 Murea and 40% (w/v) deionized formamide], increasing in the directionof the electrophoretic run. The electrophoretic run was carried out at aconstant temperature of 60 °C in 1× Tris–acetate–EDTA (TAE) buffer for5 min at 200 V, then 16 h at 80 V. Following electrophoresis, the gelwasstained for 10 min in 250 mL of 1× TAE containing 0.5 μg/mL ethidiumbromide solution, and visualized under ultraviolet light. Images weredigitally captured with a Kodak Gel Logic 212 Imaging and AnalysisSystem (Carestream Health Inc., Rochester, NY, USA).

2.5. Sequencing of DGGE bands and analysis

Representative bands were excised from DGGE gels with a sterilescalpel and placed into 1.5 mL microcentrifuge tubes and stored at−20 °C. The band was immersed into an appropriate amount of

diffusion buffer (0.5 M ammonium acetate, 10 mM magnesiumacetate, and 1 mM EDTA pH 8.0, 0.1% SDS) and incubated at 50 °Cfor 30 min to allow the DNA to diffuse from the gel followed byrecovery of the DNA from the solution using a QIAEX II Gel ExtractionKit (Qiagen, Shanghai, China). For sequencing, the eluted DNA wasamplified using the same primer pairs but without the GC clamp usingthe aforementioned conditions. The re-amplified PCR products wereligated into the pGEM-T Easy plasmid (Promega, USA) and trans-formed into Escherichia coli DH5α competent cells according to themanufacturer's instructions.White transformants were selected on LBagar supplemented with X-gal, IPTG and ampicillin, and cultured in LBbroth with aminobenzylpenicillin. Positive clones were detected byplasmid PCR and liquid cultures were used to determine the plasmidsequence. DNA sequencing was accomplished by a commercial facility(Shanghai Sangon Biological Engineering Technology and Service Co.Ltd., China) and the sequence trace files were analysed using Chromas2.31 (Technelysium Pty Ltd., Tewantin, QLD, Australia). Theirsimilarity with eukaryotic rRNA gene sequences stored in theGenBank databases was determined with the BLAST program(www.ncbi.nlm.nih.gov/BLAST).

2.6. Isolation and characterization of the dominant fungi in Fuzhuanbrick-tea

A dilution plating technique was used to isolate the yellow fungifrom Fuzhuan brick-tea to enable identification. A 100 μL samplesuspension was inoculated onto a Petri dish containing dichloranglycerol agar (DG18) (Hocking and Pitt, 1980), followed by cultivationin an incubator for 7 days at 25 °C. The fungal colonies were initiallyclassified and counted. The Eurotium spp. strains were isolated andsubcultured on M40Y agar (Atlas, 2004). For initial identification ofthe fungi to a genus or species level, characteristics of colonies wereobserved with the naked eye, and reproductive structures (ascocarps,asci and ascospores) were examined under a light microscope or ascanning electron microscope (Raper and Fennell, 1965; Qi, 1997; Pittand Hocking, 2009).

2.7. Identification of isolates to a species level by the multilocus sequencetyping method

The ascospores of Eurotium strains maintained on an M40Y agarslant were subcultured in CZA170 broth (Atlas, 2004) at 150 rpm and28 °C for 7 days. The mycelium pellet was harvested by vacuumfiltration through sterile filters and rinsed twice by suspending insterile deionized water to remove residual medium, lyophilized andstored at−20 °C. For subsequent DNA extraction, approximately 0.5 gof lyophilized mycelium was placed in a sterile mortar and pestle andfurther ground into powder in liquid nitrogen. DNA extractions usedfor the template were made from the powdered sample according toStirling (2003). For multilocus sequence identification, the four loci ofβ-tubulin (BT2), calmodulin (CF), ITS and lsu rDNA (ID), and RNApolymerase II (RPB2) described in Peterson (2008) were amplifiedusing the primers and conditions specified by Serra et al. (2008). Theamplicons were cloned into the pGEM-T Easy plasmid and sequenced.Each of the DNA sequences from the four loci was aligned with thecorresponding sequence of the fifteen type strains (Table 1) obtainedfrom GenBank using ClustalW 1.83. For the β-tubulin sequences, a24 bp leader sequence (5′-gat cga tcg atc gat cga tcg atc-3′) wasinserted before the first base of the sequences, forcing ClustalW toalign the first two bases of the sequence even though a variable intronsequence follows immediately after the first two bases. Afteralignment the leader elements were trimmed off (Peterson, 2008).A coelongate tandem DNA sequence, BT2-CF-ID-RPB2, was concate-nated from aligned sequence data from all four loci for each strain.PAUP (Phylogenetic Analysis Using Parsimony) version 4.0b10 wasused to generate phylograms for the combined datasets and construct

Page 4: Fungal community associated with fermentation and storage of Fuzhuan brick-tea

Table 1GenBank accession numbers of the four loci sequences from type strains of Eurotiumspp. retrieved to identify isolates in Fuzhuan brick-tea.

# Type strains β-Tubulin Calmodulin ID region RNA Polymerase II

1 E. amstelodamiNRRL 90

EF651897 EF652017 EF652076 EF651963

2 E. carnoyi NRRL 126 EF651903 EF651985 EF652057 EF6519423 E. chevalieri

NRRL 78EF651911 EF652002 EF652068 EF651954

4 E. cristatumNRRL 4222

EF651914 EF652001 EF652078 EF651957

5 E. echinulatumNRRL 131

EF651907 EF651998 EF652060 EF651939

6 E. halophilicumNRRL 2739

EF651926 EF652034 EF652088 EF651982

7 E. herbariorumNRRL 116

EF651887 EF651989 EF652052 EF651934

8 E. intermediumNRRL 82

EF651892 EF652012 EF652074 EF651958

9 E. leucocarpumNRRL 3497

EF651925 EF652023 EF652087 EF651972

10 E. niveoglaucumNRRL 127

EF651905 EF651993 EF652058 EF651943

11 E. repens NRRL 13 EF651915 EF652005 EF652048 EF65195012 E. rubrum NRRL 52 EF651920 EF652009 EF652066 EF65194713 E. tonophilum NRRL

5124EF651919 EF652000 EF652081 EF651969

14 E. xerophilum NRRL6131

EF651923 EF651983 EF652085 EF651970

15 A. flaschentraegeriNRRL 5042

EF652113 EF652130 EF652150 EF652102

Fig. 2. Changes in water content and amount of Eurotium colonies during Fuzhuanbrick-tea processing.

17A. Xu et al. / International Journal of Food Microbiology 146 (2011) 14–22

genealogical trees as detailed described by Peterson (2008) andDettman et al. (2003). MrBayes 3.1.2 was used to calculate theposterior probabilities of branches on the consensus tree. Thephylogenetic relationship between the Eurotium sp. isolate and theknown type strains was ascertained from the tree.

2.8. Determination of the major anthraquinone derivatives in dark teasby high performance thin layer chromatography (HPTLC)

2.8.1. Samples and extraction preparationSamples subjected to anthraquinone analysis included represen-

tative ready-made dark teas on the market such as Pu'er tea, rawdark green tea, Fuzhuan brick-tea, newly-made tea fermented byE. cristatum exclusively, and the fresh biomass of E. cristatum. To maketea fermented by E. cristatum exclusively, the ripe ascospores ofE. cristatum grown on M40Y agar for 2 weeks were inoculated on 20 gcrushed raw dark green tea in 500 mL Erlenmeyer flasks, andincubated at 28 °C for 9 days. The fresh biomass of E. cristatumcomprised hyphae and spores of E. cristatum grown on M40Y agar for14 days. To prepare extractions, the dried and powdered samples(1.0 g) were extracted with 10 mL methanol–chloroform (80:20, v/v)at 25 °C for 15 min and this was repeated three times. The combinedextract was centrifuged at 10,000×g for 10 min and the supernatantwas filtered through a 0.45 μm filter. The filtrate was incubated in ahot water bath (70 °C) to vaporize the organic solvent. The obtaineddry extraction was re-dissolved in methanol–chloroform (80:20, v/v)to prepare a 10 mg/mL stock solution, and diluted to a 1.0 mg/mLworking solution prior to sample loading on HPTLC plates.

2.8.2. HPTLC procedureA Camag HPTLC system (Muttenz, Switzerland) equipped with a

semi-automatic TLC applicator (Linomat 5), TLC scanner 3 (winCATSversion 1.1.2), ultraviolet cabinet and twin-trough glass chamber(24.5 cm×8 cm×22.5 cm) was used for the analysis. The glass-backed silica Gel-G60 HPTLC plates (20 cm×10 cm) (QingdaoHaiyang Chemical, China) were activated at 110 °C for 30 min andstored in a desiccator before use. The samples were applied using a

Linomat 5 fittedwith a 100 μL syringe to 6 mmbands, 10 mm from thebottom with a 6 mm space between bands. A 20 μL volume of eachsample working solution (1.0 mg/mL) was taken and applied to theHPTLC plate in duplicate. Three standard samples (HPLC purity) wereemodin, physcion and chrysophanol, purchased from ChengduMansite Pharmaceutical Co. Ltd. (Chengdu, China). Each standardwas dissolved in methanol to make a stock solution (0.2 mg/mL),diluted to a 20 μg/mL working solution, and 4 μL and 8 μL loaded ontothe HPTLC plate in two lanes. The HPTLC plates were developed in theCamag twin-trough glass chamber pre-saturated with the developingsolvents, petroleum ether–ethyl acetate–glacial acetic acid(87:6.4:3.6, v/v/v). The plate was developed to a height of about9 cm from the base in the developing solvents. After development, theplate was removed, dried and spots were visualized under ultravioletlight (300 nm). Quantitative evaluation of the plate was performed inthe reflectance/absorbance mode at 430 nm with a slit width of6 mm×0.4 mm, a scanning speed of 20 mm/s and data resolution at100 μm/step. Peak heights and peak areas were recorded for all thelanes.

3. Results

3.1. Characterization of Fuzhuan brick-tea production

Typically the entire fermentation process of Fuzhuan brick-tealasts about 17 days. In practice, the fermentation process is dividedinto two stages, the fungal growth period (days 0–12) and the dryingperiod (days 13–17). Key technological parameters included prepa-ration of the tea leaves and branchlets at the appropriate ratio prior topiling overnight, adjustment of the water content before pressing intotea bricks, as well as temperature and moisture control in thefermentation room for optimal fungal growth conditions. To inves-tigate the standard production of Fuzhuan brick-tea, a batch ofsamples (S1–S8) was taken from the largest manufacturer. Experi-mental results indicated that the ratio of tea branchlets to tea leavesranged from 20 to 25%. The changes in water content and amount ofEurotium colonies during the fermentation process are shown in Fig. 2.To commence fermentation, the water content was adjusted up toabout 20% (S2, day 0) from 16% (S1, piled materials) with littlefluctuation until day 12 (S6) and maintained at 25–28 °C. Thexerotolerant Eurotium spp. grew rapidly under favourable conditionsand achieved a maximal 6 log CFU/g dry weight at days 9–12. Thedrying period followed fermentation was characterized by an increasein temperature of 2–3 °C a day. As a result, water content andEurotium counts were reduced gradually (S7, day 15), and eventuallythe final tea product (S8, day 17) with very low water content (below7%) resulted.

Page 5: Fungal community associated with fermentation and storage of Fuzhuan brick-tea

Table 2Identities of bands obtained from the fungal community of the Fuzhuan brick-tea.

Bands #a Size (bp) Accession number Closest relative % Identity b

a 197 HQ154060 Verticillium sp. 97b 202 HQ154059 Debaryomyces spp. 99c, d, e 203 HQ154058 Eurotium spp. 100f 203 HQ154057 Pichia spp. 94g 199 HQ154056 Pestalotiopsis spp. 95h 204 HQ154055 Aspergillus niger 100i 205 HQ154054 Rhizomucor pusillus 100j 197 HQ154053 Beauveria spp. 99

a Bands were extracted from the DGGE gel shown in Fig. 3.b Percentage of identical nucleotides between the sequences retrieved from the

DGGE gel and the closest relative found in GenBank.

18 A. Xu et al. / International Journal of Food Microbiology 146 (2011) 14–22

3.2. PCR-DGGE

Specific separation of the amplified D1 region of the 26S (28S)rRNA gene by DGGE was used to determine the fungal diversity inindustrial Fuzhuan brick-tea and related dark teas. Fig. 3 shows theDGGE profiles obtained for the DNA extracted from 14 tea samples.Lane 9 showed more than ten bands, suggesting that the stored Rawdark green tea (S9) harbours the most diverse microbiota. Lanes 1–8 corresponding to samples S1–S8 were closely related to Fuzhuanbrick-tea. The band patterns in lanes 1–8 reflected the fungal diversityin tea processing. A few faint bands were visualized in lane 1 (S1).There were fewmicroorganisms detectable at this stage in the presentstudy using the diluted plate culture method. Lanes 2–8 representedfermenting tea samples taken from the fermentation room at 2-dayintervals. At the initial fermentation stage (day 0, Lane 2), eight bandswere clearly evident. Four distinct bands (bands b, c, d and e in Fig. 3)appeared consistently at the bottom of the DGGE image from lane 3(day 3, S3) to lane 8 (day 17, S8). Strictly speaking, bands c and d wereexcised from the top and the bottom of the same wider band. Thesebands were also observed in lane 10 (S10) and lane 12 (S12), the twotea samples were produced by the same company as S8, but weredifferent in weight at 800 g and 1500 g per piece, respectively. Theother brands of Fuzhuan brick-tea, as indicated in lanes 11 (S11), 13(S13) and 14 (S14), had a comparative shadowy fingerprint at theposition of bands c–e on the gel. Obviously, the band b existed in allfourteen lanes but with a slight discrepancy in band brightness.According to the distribution of the bands, we speculated that themicrobes related to bands b–f and h play important roles in thefermentation process of Fuzhuan brick-tea.

Bands a–j were excised from the DGGE gel and were successfullysequenced. Their GenBank accession numbers are shown in Table 2. ABlastN search of these sequences indicated that these bands werederived from the closest relatives listed in Table 2. Eight bandsshowed high similarities (at least 97%) with the genera Eurotium,Debaryomyces, Aspergillus, Verticillium, Rhizomucor and Beauveria. Two

Fig. 3.DGGE profiles of amplified 26S rRNAD1 regions obtained from tea samples. Lanes1–14 correspond to samples S1–S14. Lanes 1–8 represent processed Fuzhuan brick-tea;9, tea materials; and 10–14, ageing or aged Fuzhuan brick-tea. DNA fragments of thelarge subunit rDNA were amplified by PCR and subjected to DGGE analysis.Representative bands (indicated a–j) were excised for cloning and sequencing.

bands showed a moderate similarity of 95% and 94% to Pichia andPestalotiopsis, respectively.

Band b is derived from a fungus closely related to the genusDebaryomyces. These yeasts had not been observed by previousmicrobiota investigations of Fuzhuan brick-tea. Band c was derivedfrom Eurotium spp., and identical nucleotide sequences were found inbands d and e, but with different melting behaviours in the gel. Theseclearest bands testified that the Eurotium spp. were the dominantfungi in the production of Fuzhuan brick-tea. However, it was notpossible to identify which species of Eurotium was actually presentdue to the limited sequence information provided by the D1 region of26S rRNA gene. These results corresponded with the dilution platemethod from our study and previous reports that Eurotium spp. weredominant in high-quality Fuzhuan brick-teas.

Bandhappeared to relate toAspergillus nigeron thebasis of sequenceanalysis. A. niger strains were commonly isolated from the raw darkgreen and Fuzhuan brick-teas. They were considered to be the mainfunctional fungi in the piling fermentation of Raw dark green tea,whereas they are regarded as contaminant fungi in the production ofFuzhuan brick-tea all through the ages, since overgrowth of the blackmoulds would deteriorate the appearance and decrease the tea quality.The fingerprinting results show that A. niger participated in the teaprocessing and acted as a subordinate partner under normal conditions.

The other faint bands appearing in lanes 2–8 (S2–S8) revealed themicrobial diversity and complexity to some extent in the fermentationprocess Fuzhuan brick-tea. Among these bands, f and g were derivedfrom Pichia spp. and Pestalotiopsis spp., respectively, according tosequencing results of the excised band. Pestalotiopsis spp. are plantpathogenic fungi, with P. guepinii the cause of grey leaf spot, die back,stem canker and petal rot of tea (Camellia sp.) (Mehrotra and Aneja,1990; Lu, 2001).

The bright band i located in the middle of lane 9 was derived fromRhizomucor pusillus, indicating that it was one of the importantfunctional fungi for raw dark green tea production. Another clear bandappeared in lane 9 and was parallel to band a in lane 2 correspondingto Verticillium spp., which had been found before in mycobiotainvestigations of the raw dark green tea production process.

The band j was found in the stored readymade Fuzhuan brick-teas,samples S10–S14 (lanes 10–14), and was a fingerprint of Beauveriaspp.. These saprophytic and entomogenous fungi were believed toconfer a great benefit in the protection of aged tea from insectspoilage.

In this study, eight genera of fungi, Eurotium, Debaryomyces,Aspergillus, Verticillium, Pichia, Pestalotiopsis, Rhizomucor and Beau-veria in the Fuzhuan tea samples were identified by DGGE analysis.Excluding Rhizomucor and Beauveria, fingerprints of the remaining sixgenera were detected in the fermenting tea samples (S3–S8).Eurotium, Debaryomyces and Aspergillus were consistently visible,with the genus Debaryomyces not previously reported in Fuzhuanbrick-tea. The genus Rhizomucor which was one of the important

Page 6: Fungal community associated with fermentation and storage of Fuzhuan brick-tea

19A. Xu et al. / International Journal of Food Microbiology 146 (2011) 14–22

functional fungi in raw dark green tea had never been detectedpreviously. This is the first report of the genus Beauveria in storedready made Fuzhuan brick-tea.

3.3. Isolation and characterization of the dominant fungi in Fuzhuanbrick-tea

A large number of macroscopic yellow dots appearing within thetea at the end of fermentation mark successful fermentation withsuperior product quality. It is known that these dots are the typicalascomata of the genus Eurotium. Enumeration of the yellow colonieson DG18 agar reached 105CFU/g dry weight in ready made Fuzhuanbrick-tea samples. Some strains were subcultured randomly from theDG18 plates and further purified with single spore inoculation onM40Y agar. A representative strain was designated Eurotium sp. FZ.The isolates grew rapidly and preferentially on M40Y of three testedmedia: M40Y, CZA200 and CZA30. The colonies were greater than 8.5,4–5, and less than 3 cm in diameter, respectively, 2 weeks afterinoculation and grew well at 37 °C. According to the morphologicalfeatures (Fig. 4), the strains described here have cleistothecia thatwere morphologically typical of the genus Eurotium and have thesame ascospore type corresponding most closely with E. cristatum asdescribed by Raper and Fennell (1965), Qi (1997) and Pitt andHocking (2009).

3.4. Identification of the dominant fungus using multilocus sequencetyping (MLST)

Previous identification of the Eurotium spp. isolated from Fuzhuanbrick-tea has mainly relied on morphology, without support ofmolecular techniques. We used molecular methods of identification

Fig. 4. Morphological features of Eurotium sp. FZ isolated from Fuzhuan brick-tea. (A) A colo(B1) Ascocarp wall one layer thick, composed of large polygonal cells (×1000); (C) eight asc(×5000).

in our study. For identification of the strain Eurotium sp. FZ to thespecies level, DNA sequences from the four loci, ID, RPB2, BT2 and CFwere deposited in GenBank under the accession numbers HQ148160,HQ148161, HQ148162 and HQ148163, respectively. It was impossibleto distinguish the fifteen type strains from each other simply relyingon the alignment of individual sequences due to low divergence. Forinferring the fungal phylogeny, all data should be considered together,whether the loci are fully congruent or not. DNA comparisons shouldbe based on longer nucleotide sequences, thereby making it easier toprecisely define a species. A phylogenetic tree produced from amaximum parsimony heuristic search using the combined alignmentof 3502 characters from all four loci is shown in Fig. 5. Seventeenspecies were distinguished and separated into four main clades in thetree diagram on the basis of congruence analysis of the four loci. Thearrangement of branches in the consensus tree closely resembles thetree diagram in Peterson (2008). The sibling species of Eurotium sp.isolated from Fuzhuan brick-tea was E. cristatum, as seen in clade 2.

3.5. Determination of the major anthraquinones in dark teas by HPTLC

A number of anthraquinone pigments were isolated from culturesof Aspergillus cristatus (anamorphic E. cristatum). These fungalanthraquinones had multiple biological activities, with emodin andphyscion among them (Anke et al., 1980a,b). Similarly, the four majoranthraquinone derivatives namely physcion, chrysophanol, emodinand chrysophanol glycoside are the pharmacologically active sub-stances in Rheum emodi (Singh et al., 2005). Both Rheum emodi andaged dark teas are regarded as traditional Chinese medicines.Considering the ingredients of Fuzhuan brick-tea consisted of thetransformed tea substrate, biomass, and the metabolites of thetransforming fungi, which were predominantly E. cristatum, it is

ny grown on M40Y agar in a 9 cm Petri dish at 28 °C for 14 days; (B) Ascocarps (×40);ospores released from one ascus (×400); (D) Cleistothecia (×200); and (E) Ascospores

Page 7: Fungal community associated with fermentation and storage of Fuzhuan brick-tea

Fig. 5. Bayesian consensus tree depicting the relationship between Eurotium sp. isolated from Fuzhuan brick-tea and the type strains of Eurotium. The tree was based on combinedBT2, CF, ID and RPB2 data. Numbers at the nodes are themaximum parsimony bootstrap values (1000 replicates) and Bayesian posterior probabilities with Aspergillus flaschentraegeriused as an outgroup.

20 A. Xu et al. / International Journal of Food Microbiology 146 (2011) 14–22

doubtful whether anthraquinones were bioactive substances in tea.We determined the occurrence of the major anthraquinones inFuzhuan brick-tea and related samples using HPTLC.

The resulting HPTLC diagrams (Fig. 6) showed a clear separation ofthe three compounds, emodin (band a), physcion (band b) andchrysophanol (band c) with Rf values of 0.28, 0.43, and 0.50,respectively, in an optimized developing solvent consisting ofpetroleum ether–ethyl acetate–glacial acetic acid (87:6.4:3.6, v/v/v).All the compounds were used as references for the identification andquantification of anthraquinones present in themethanol–chloroform(80:20, v/v) extract of four dark teas and the biomass of E. cristatum.The occurrence of the three anthraquinones was detected at 430 nm

Fig. 6. HPTLC diagrams. (Left) HPTLC photograph of the anthraquinones detected at 300 nm.and 2, Pu'er tea; 3 and 4, raw dark green tea; 5 and 6, Fuzhuan brick-tea; 7 and 8, tea fermenand 12, 160 ng of each standard sample; (Right) HPTLC chromatogram obtained from anthraqvalues 0.28, 0.43 and 0.51, respectively.

and quantity comparison showed in Table 3. Emodin was present inall five samples in descending order of peak areas: raw dark green tea,Pu'er tea, tea fermented by E. cristatum, Fuzhuan brick-tea andbiomass of E. cristatum. The physcion contained in the biomass of E.cristatum far exceeded that of tea fermented by E. cristatum, with nophyscion found in the remaining three samples. Chrysophanol wasnot detectable in any of the samples.

An unknown intense fluorescent band (band d in Fig. 6) waspresent in the extraction of biomass of Eurotium sp. FZ (lanes 9 and 10,Fig. 6). We believed that it could be regarded as an indicativesubstance from Eurotium. This band appeared in only two tea samples,the tea fermented by Eurotium (lanes 7 and 8, Fig. 6) and Fuzhuan

Spots a, b and c represent the emodin, physcion and chrysophanol, respectively. Lanes 1ted by E. cristatum; 9 and 10, biomass of E. cristatum; 11, 80 ng of each standard sample;uinone standards scanned at 430 nm, peaks 1, 2 and 3 correspond to spots a, b, c with Rf

Page 8: Fungal community associated with fermentation and storage of Fuzhuan brick-tea

Table 3Occurrence of anthraquinones in different dark teas and E. cristatum detected at 430 nm.

Pu'er tea Raw dark green tea Fuzhuanbrick-tea

Tea fermented byE. cristatum

Biomass ofE. cristatum

Standard 80 ngeach

Standard 160 ngeach

Emodin ++ a (1255.8±10.9) +++ (2354.6±224.0) + (675.9±23.1) ++ (1227.7±30.9) + (573.7±72.5) (1895.9) (3333.9)Physcion – – – + (791.4±1.4) +++ (7434.2±235.7) (717.2) (1436.6)Chrysophanol – – – – – (1481.5) (2889.1)

a –, +, ++ and +++ indicated increasing amounts of anthraquinones. Numbers in parentheses were average peak areas detected at 430 nm, (n=2).

21A. Xu et al. / International Journal of Food Microbiology 146 (2011) 14–22

brick-tea (lanes 5 and 6, Fig. 6). This phenomenon further recon-firmed that the Eurotium spp. were important fungi in the productionof Fuzhuan brick-tea.

4. Discussion

The industrial fermentation process of Fuzhuan brick-tea wasexamined to elucidate the fungal community structure in tea leaves.The present investigations have shown that several eukaryoticmicroorganisms are present during the traditional fermentationprocess in the production of Fuzhuan brick-tea. The main factorsthat determine fungal growth on (or within) a food matrix are wateractivity (aw), pH, temperature, oxygen and other microorganisms.Physicochemical analysis results showed that the best environmentalconditions were provided during processing that were suitable forgrowth of xerotolerant microorganisms.

Our PCR-DGGE approach indicated that the fermenting Fuzhuanbrick-tea was dominated by the genera Eurotium, Debaryomyces andAspergillus. Beauveria occurred in the stored Fuzhuan brick-tea. Forthe DGGE profile, the disperse migration of a DNA fragment occurred,as indicated by bands c–e. One application of DGGE is for the isolationof DNA fragments associated with CpG islands, owing to the reducedrate of strand dissociation of partially melted DNA fragmentscontaining many CpG sites (Shiraishi et al., 1995). Tost (2008)presumed that CG-rich regions such as CpG islands are morerefractory to denaturation, and single-stranded CG rich DNA isprone to form complex secondary structures even after denaturation.The 203 nt fragment of HQ154058 showed 58.6% G-C content and aTm of 99.2 °C (%GC method) as analysed by Oligo software (version6.71). It had a total of 16 CpGs along the sequence, whilst the CpGsfrom the other bands sequenced varied from 7 to 12 and lacked thesequence CGCG. These CpG islands that exist in the double-strandedDNA fragment induced discrete melting behaviour. As a result ofstrand dissociation in the denaturant, at least two types of partiallymelted DNA fragments with steady structure were aggregated intotwo bands on the gel as bands c–e.

The strain Eurotium sp. FZ was identified as E. cristatum by acombination of morphological attributes and the MLST method. Thesetwo distinct but complementary methods confirmed that the mostcommon fungus associated with Fuzhuan brick-tea was E. cristatum,corresponding with previous results (Qi and Sun, 1990). Some speciesbelonging to the genus Eurotium are ubiquitous food-borne fungi.They are commonly found in preserved foods and cause food spoilage(Pitt and Hocking, 2009). Non-mycotoxigenic strains of E. repens andE. rubrum are used as starter cultures in the manufacture of thetraditional fermented food katsuobushi, from bonito (Katsuwonuspelamis), in Japan (Manabe, 2001; Pitt and Hocking, 2009). Duringkatsuobushi fermentation these xerophilic fungi produce effectiveantioxidants which participate in the suppression of lipid oxidationsuch that the fermented katsuobushi is resistant to lipid oxidation andhas a long shelf-life (Kaminishi et al., 1999). For E. cristatum, majorsubstrates include soil, Fuzhuan brick-tea, Cordyceps (Dong chong xiacao), Chinese drug tablets and logging debris. E. cristatum iswidespread in China but was especially dominant in Fuzhuan brick-tea (Qi, 1997). Except for some anthraquinones (Anke et al., 1980a,b),few reports on its metabolites and production of its mycotoxins are

known. Our HPTLC analysis showed that emodin-like substancesexisted in the Fuzhuan brick-tea, and both emodin and physcion werepresent in the tea fermented by E. cristatum. Moreover, an unknownsubstance (band d, Fig. 6) closely associated to E. cristatum requiresfurther investigation.

In contrast to common filamentous fungi, the yeast component ofFuzhuan brick-tea remains very poorly characterized. The discovery ofPCR-DGGE fingerprinting of Debaryomyces in all experimentalsamples demonstrated that this genus contributes significantly tothe production of Fuzhuan brick-tea.

The genus Debaryomyces is widespread, with present taxonomicclassification showing 19 species within the genus (Kirk et al., 2008).D. hansenii (anamorph Candida famata), is a significant species infoods. It is an osmo-, halo-, xero- and cryotolerant marine yeast. It wasfirst isolated from sea water but also grows in various low aw foodsincluding cheese, processed meat, wine, beer and fruit juices. Thisspecies is very important for the food industry as it is used for surfaceripening of cheese and meat products, over-production of riboflavin(vitamin B2), bioconversion of xylose into the sweetener xylitol, andpotential synthesis of arabinitol and pyruvic acid (Satyanarayana andKunze, 2009). D. hansenii is a prominent yeast species in cheese. Theyeasts species in cheese do not seem to be associated with disease inhealthy humans, and belong to the category of those “generallyregarded as safe” (Jacques and Casaregola, 2008). As a phyllospheremicroorganism, D. hansenii was also found to be involved in thelaboratory-scale tea leave fermentation process, with the ability togrow between 4 and 28 °C, with an optimum of 23 °C (Sansone et al.,2007).

These properties indicate that Debaryomyces could becomedominant in the low water content fermentation process for Fuzhuanbrick-tea and stored products. Their enzyme activities improve teaquality by producing the sweet substance xylitol, vitamins, and otherorganic acids. We speculated that Debaryomyces had a considerableeffect on tea leaf biotransformation during the Fuzhuan tea fermen-tation process. Therefore, we believed thatDebaryomyces sp. would bean indispensable candidate microorganism in a fermentation starterfor artificial fermentation of Fuzhuan brick-tea.

The DGGE fingerprint of Beauveria spp. implicated that theseentomogenous fungi may parasitize insect that invade the tea, andprotected tea against further deterioration. Beauveria is one of themost important genera for biocontrol of insects (Kirk et al., 2008). Forpest control in stored products, strains of B. bassiana outperformedthe other entomopathogens (Wildey et al., 2002). The ‘Mycopest’project demonstrated that B. bassiana is present in UK grain stores,and that under laboratory conditions, good control of a range of insectand mite pests could be achieved (Wakefield et al., 2005). Addition-ally, there is evidence of a consumer demand for treatments that aresustainable, and present lower health risks, such as those employingentomopathogens for the protection of stored products (Hodges,2005). Furthermore, both Beauveria species are considered to be safe(Zimmermann, 2007). Recently, studies on the biological activities ofB. bassiana mycelia showed that hot water extraction contributedsignificantly to the anti-coagulant activity, anti-complementary activity,and stimulation of the intestinal immune system (Park et al., 2008).These properties provide important clues relating to utilization of theentomopathogenic fungi for managing insect pests in the stored tea

Page 9: Fungal community associated with fermentation and storage of Fuzhuan brick-tea

22 A. Xu et al. / International Journal of Food Microbiology 146 (2011) 14–22

product and possibly even to improve the medicinal value of Fuzhuanbrick-tea.

Improvement of fermented product quality with modern fermen-tation technology usually accompanies a change in the traditionalflavour. To maintain a consistent flavour, a critical factor in themodern fermentation process is to prepare suitable fermentationstarters. Practically, a microbial consortium is better than anyindividual culture. Therefore, it is very important to know themicrobial community structure in Fuzhuan brick-tea, from whichwe can select, maintain and grow potential starter cultures. To thisend, more experiments need to be conducted to obtain detailedinformation on the specific function of isolated microorganismsindividually as well as in a mixture, the interaction betweenmicroorganisms and their contribution to the formation of the uniqueflavour of Fuzhuan brick-tea.

Acknowledgements

This study was financially supported by the Key Program ofScience and Technology of Hunan Province of China (Grant No.2008FJ1005).

References

Abe, M., Takaoka, N., Idemoto, Y., Takagi, C., Imai, T., Nakasaki, K., 2008. Characteristicfungi observed in the fermentation process for Puer tea. International Journal ofFood Microbiology 124, 199–203.

Anke, H., Kolthoum, I., Zahner, H., Zahner, H., 1980a. Metabolic products ofmicroorganisms. 185. The anthraquinones of the Aspergillus glaucus group. I.occurrence, isolation, identification and antimicrobial activity. Archives ofMicrobiology 126, 223–230.

Anke, H., Kolthoum, I., Laatsch, H., 1980b. Metabolic products of microorganisms. 192.The anthraquinones of the Aspergillus glaucus group. II. biological activity. Archivesof Microbiology 126, 231–236.

Atlas, R.M., 2004. Handbook of Microbiological Media, 3rd ed. CRC Press LLC., Florida.Chen, Z.M., 2000. A Grand Dictionary of Chinese Tea. China Light Industry Press, Beijing.

(in Chinese).Cocolin, L., Bisson, L.F., Mills, D.A., 2000. Direct profiling of the yeast dynamics in wine

fermentations. FEMS Microbiology Letters 189, 81–87.Dettman, J.R., Jacobson, D.J., Taylor, J.W., 2003. A multilocus genealogical approach to

phylogenetic species recognition in the model eukaryote Neurospora. Evolution 57,2703–2720.

Ercolini, D., 2004. PCR-DGGE fingerprinting: novel strategies for detection of microbesin food. Journal of Microbiological Methods 56, 297–314.

Hocking, A.D., Pitt, J.I., 1980. Dichloran-glycerol medium for enumeration of xerophilicfungi from low-moisture foods. Applied and Environmental Microbiology 39,488–492.

Hodges, R., 2005. Consumer acceptance, barriers to application of entomopathogens instored products. Biocontrol of Arthropod Pests in Stored Products, Proceedings ofthe 6th meeting of COST, Action 842, Working Group IV, Locorotondo, Italy,10th–11th June 2005, pp. 36–41.

Hui, Y.H., Meunier-Goddik, L., Hansen, A.S., Josephsen, J., Nip, W.K., Stanfield, P.S.,Toldra, F. (Eds.), 2004. Handbook of Food and Beverage Fermentation Technology.Marcel Dekker, Inc., New York.

Jacques, N., Casaregola, S., 2008. Safety assessment of dairy microorganisms. Thehemiascomycetous yeasts. International Journal of Food Microbiology 126,321–326.

Kaminishi, Y., Egusa, J., Kunimoto, M., 1999. Antioxidant production from severalxerophilous fungi used in Katsuobushi molding. Journal of National FisheriesUniversity 47, 113–120.

Kirk, P.M., Cannon, P.F., Minter, D.W., Stalpers, J.A., 2008. Ainsworth & Bisby's Dictionaryof the Fungi, 10th ed. CAB International, Wallingford.

Ling, T.J., Wan, X.C., Ling, W.W., Zhang, Z.Z., Xia, T., Li, D.X., Hou, R.Y., 2010. Newtriterpenoids and other constituents from a special microbial-fermented tea —

Fuzhuan brick-tea. Journal of Agricultural and Food Chemistry 58, 4945–4950.Lu, J.Y., 2001. Plant Pathogenic Mycology. China Agricultural Press, Beijing, pp. 474–475

(in Chinese).Manabe, M., 2001. Fermented food and mycotoxins. Mycotoxins 51, 25–29.Mehrotra, R.S., Aneja, K.R., 1990. An Introduction to Mycology. New Delhi, New Age

International Publishers. 628–629.Mo, H.Z., Zhang, H., Li, Y.Q., Zhu, Y., 2008a. Antimicrobial activity of the indigenously

microbial fermented Fuzhuan brick-tea. Journal of Biotechnology 136, S722.Mo, H.Z., Zhu, Y., Chen, Z.M., 2008b. Microbial fermented tea — a potential source of

natural food preservatives. Trends in Food Science and Technology 19, 124–130.Park, S.Y., Song, H.H., Lee, Y.G., Yoon, C.S., Lee, C., 2008. Biological activities and partial

characterization of Beauveria bassiana mycelium. Food Science and Technology 17,95–101.

Peterson, S.W., 2008. Phylogenetic analysis of Aspergillus species using DNA sequencesfrom four loci. Mycologia 100, 205–226.

Pitt, J.I., Hocking, A.D., 2009. Fungi and Food Spoilage, 3rd edn. Springer SciencetBusiness Media, LLC., New York.

Qi, Z.T., 1997. Flora Fungorum Sinicorum (Vol. 5) — Aspergillus et Teleomorphi CognatiChinese edition with Latin name index. Science Press of China, Beijing, China. (inChinese).

Qi, Z.T., Sun, Z.M., 1990. Identification of predominant species in brick tea. ActaMycologica Sinica 9, 176–179. (in Chinese).

Raper, K.B., Fennell, D.I., 1965. The Genus Aspergillus. The Williams and Wilkims Co,Baltimore.

Sansone, C., Massardo, D.R., Pontieri, P., Maddaluno, L., Stefano, M.D., Tredici, S.M., Tala,A., Alifano, P., Giudice, L.D., 2007. Isolation of a psychrotolerant Debaryomyceshansenii strain from fermented tea plant (Camellia sinensis) leaves. Journal of PlantInteractions 2, 169–174.

Satyanarayana, T., Kunze, G. (Eds.), 2009. Yeast Biotechnology: Diversity andApplications. Springer Science Business Media B.V, pp. 65–112.

Serra, R., Peterson, S.W., Venancio, A., 2008. Multilocus sequence identification ofPenicillium species in cork bark during plank preparation for the manufacture ofstoppers. Research in Microbiology 159, 178–186.

Shiraishi, M., Lerman, L.S., Sekiya, T., 1995. Preferential isolation of DNA fragmentsassociated with CpG islands. Proceedings of the National Academy of Sciences ofthe United States of America 92, 4229–4233.

Singh, N.P., Gupta, A.P., Sinha, A.K., Ahuja, P.S., 2005. High-performance thin layerchromatography method for quantitative determination of four major anthraqui-none derivatives in Rheum emodi. Journal of Chromatography A 1077, 202–206.

Stirling, D., 2003. DNA extraction from fungi, yeast, and bacteria, In: Bartlett, John M.S.,Stirling, David (Eds.), Methods in Molecular Biology, 2nd edn. : PCR Protocols, 226.Humana Press Inc, Totowa, NJ, pp. 53–54.

Tost, J., 2008. Epigenetics. Caister Academic Press, Norfolk, England.Wakefield, M.E., Cox, P.D., Moore, D., Aquino De Muro, M., Bell, B.A., 2005. Mycopest:

results and perspectives. Biocontrol of Arthropod Pests in Stored Products,Proceedings of the 6th meeting of COST, Action 842, Working Group IV,Locorotondo, Italy,10th–11th June 2005.

Wan, X.C., 2003. Tea biochemistry, 3rd edn. China Agriculture Press, Beijing. (inChinese).

Wen, Q.Y., Liu, S.C., 1991. Evolutionary regulation of dominant fungi in Fuzhuan brick-tea during the fungus growing process. Journal of Tea Science 11, 56–62 (suppl.).

Wildey, K.B., Cox, P.D., Wakefield, M., Price, N.R., Moore, D., Bell, B.A., 2002. The use ofentomopathogenic fungi for stored product pest control — the “MYCOPEST”project. Integrated Protection of Stored Products. IOBC Bulletin 25 (3), 15–19.

Zimmermann, G., 2007. Review on safety of the entomopathogenic fungi Beauveriabassiana and Beauveria brongniartii. Biocontrol Science and Technology 17,553–596.