Trends in Food Science & Technology 19 (2008) 124e130
Review
* Corresponding author.
0924-2244/$ - see front matter � 2007 Elsevier Ltd. All rights reserved.doi:10.1016/j.tifs.2007.10.001
Microbial fermented
tea e a potential
source of natural
food preservatives
Haizhen Moa, Yang Zhub,* and
Zongmao ChencaDepartment of Food Science, Henan Institute of
Science and Technology, 453003 Xinxiang,
Henan Province, ChinabFood and Bioprocess Engineering Group, WageningenUniversity and Research Centre, P.O. Box 8129, 6700
EV, Wageningen, Netherlands (Tel.: D31 343 538103;
fax: D31 343 538405; e-mail: [email protected])cChinese Tea Research Institute, Chinese Academy of
Agricultural Sciences, 310008 Hangzhou, Zhejiang
Province, China
Antimicrobial activities of microbial fermented tea are much
less known than its health beneficial properties. These anti-
microbial activities are generated in natural microbial fermen-
tation process with tea leaves as substrates. The antimicrobial
components produced during the fermentation process have
shown inhibitory effects against several food-borne and patho-
genic bacteria. With the trend of increasing use of natural and
biological preservatives in food products, natural antimicrobial
agents from microbial fermented tea may offer an innovative
and interesting measure for such applications. However,
a breakthrough in this field can only be realised after several
critical aspects are clarified and further studied. Only then,
the application of these potential, novel and natural antimicro-
bial substances from microbial fermented tea can be industri-
alized. The present review describes some unique microbial
fermentation of tea and the antimicrobial activities formed
during the fermentation process. Moreover, future needs in
research and development of these antimicrobial compounds
from microbial fermentation of tea are discussed for potential
industrial applications.
IntroductionTea is one of the most consumed beverages worldwide.
In fact, there are mainly three different kinds of tea. In Chinaand many Southeast Asian countries, green tea is preferred.In Western countries and the rest of the world, the consump-tion of black tea is the highest. Another kind of tea is Kom-bucha, which is a sweet-sour tea beverage made actuallyfrom tea extract supplemented with sugar and fermentedwith yeast and acetic-acid bacteria. Fermentation processis necessary to produce black tea and Kombucha. Blacktea can be further subdivided into naturally oxidized tea(although the term of fermentation is often used), and micro-bial fermented tea. Fermentation process for producing mostblack teas is actually an oxidation process catalyzed byenzymes that are originally present in tea leaves (Fowler,Leheup, & Cordier, 1998). Because no microorganismsare involved in this kind of enzyme-oxidized black tea,the use of the term fermentation is obviously not completelycorrect. Microbial fermentation in the production of blacktea, however, is found to be carried out solely in certain Chi-nese tea assortments. Therefore, this kind of tea is littleknown outside China, whereas Kombucha is exceptionallywell known because of its increasing interests in the West.
Recently, several microbial fermented teas got noticed inthe Western world, probably not only because of trade ex-pansions between China and the West, but also because ofseveral health beneficial claims associated with microbialfermented tea. A few studies reveal that extracts from mi-crobial fermented teas contain natural antimicrobial com-ponents that have inhibitive effect on several food-bornepathogen and spoilage bacteria (Greenwalt, Ledford, &Steinkraus, 1998; Mo, Xu, Yan, & Zhu, 2005; Sreeramulu,Zhu, & Knol, 2000; Sreeramulu, Zhu, & Knol, 2001;Steinkraus, Shapiro, Hotchkiss, & Mortlock, 1996).
Application of natural antibacterial agents has been in-creasingly noticed as a novel trend in biological preserva-tion of foods in recent years (Schillinger, Geisen, &Holzapfel, 1996). Although plants can provide a vast sourceof natural preservatives, many potential biological preserva-tives are originated from traditional food fermentations. Themost well known example is probably the production ofnisin by lactic acid bacteria. Some potential and unexploredfood fermentation processes are certainly new possibilitiesfor innovative applications in the modern society.
This article reveals the background information of micro-bial fermented Puer tea and Fuzhuan brick-tea, as well as
125H. Mo et al. / Trends in Food Science & Technology 19 (2008) 124e130
fermented tea beverage Kombucha. The importance of theirantibacterial effects on several food-borne bacteria are givenin detail. Potential applications and research needs of thesemicrobial fermented teas are also critically discussed. Theantimicrobial effects of tea catechins or polyphenols havebeen intensively described previously elsewhere (An et al.,2004; Bandyopadhyay, Chatterjee, Dasgupta, Lourduraja,& Dastidar, 2005; Cushnie & Lamb, 2005; Friedman,Henika, Levin, Mandrell, & Kozukue, 2006; Hamilton-Miller,1995; Hirasawa & Takada, 2004; Kim, Ruengwilysup, &Fung, 2004; Si et al., 2006; Taguri, Tanaka, & Kouno, 2004;Yamamoto, Matsunaga, & Friedman, 2004; Yilmaz, 2006)and so this topic is not included here.
Microbial fermentation and antimicrobial effect ofPuer tea, Fuzhuan brick-tea and Kombucha
Both Puer tea and Fuzhuan brick-tea are microbial fer-mented black teas, whereas Kombucha is a fermented drinkof tea extract supplemented with sucrose and fermentedwith yeasts and acetic-acid bacteria. All three teas haveshown obvious antibacterial effects.
Fermentation process and antimicrobialcharacteristics of Puer tea
Puer tea is a unique Chinese microbial fermented tea ob-tained through indigenous tea fermentation where micro-organisms are involved in the manufacturing process. Aschematic description of Puer tea production is shown inFig. 1. This tea is different from common black tea that un-dergoes a natural oxidation process through enzymes orig-inally existing in tea leaves (Fowler et al., 1998). Theprocess of Puer tea manufacture is characterized by solid-state fermentation with natural flora as inoculums of whichthe fungus Aspergillus niger plays a key role (Mo et al.,2005; Xu, Yan, & Zhu, 2005). The processing of Puer teais quite different from that of black tea, although they areboth called fermented teas. During black tea processing,fresh tea leaves are rolled and cut before drying so that
Wetting and cooling Ripening Evaluatio
Loose Puer tea product
Press
Pasteurization
DryingSteaming and pressing
Collecting of fresh tea leaves Inactivation of
GradingDrying Steaming treatment
Fig. 1. Schematic description o
tea polyphenols in tea leaves come into contact with thetea polyphenol oxidases and then oxidized in the conse-quent fermentation process. During the Puer tea fermenta-tion process, fresh leaves are fixed by heat in a drum toinactivate polyphenol oxidases. The fixed leaves are thenrolled and partially dried. The partially dried leaves arepiled up in humid conditions (moisture content w40% at25e60 �C) for a few weeks, during which the tea polyphe-nols are more intensively oxidized by the action of micro-organisms and environmental oxygen than in black teafermentation process, resulting in low concentrations oftea polyphenols and tea catechins (Liang, Zhang, & Lu,2005).
Puer tea has not only a unique flavour but also severalhealth beneficial properties, such as suppressing fatty acidsynthase expression (Chiang et al., 2006), acting as an in-hibitor of lipid and nonlipid oxidative damage and alsoexhibiting metal-binding ability, reducing power, and scav-enging effect for free radicals (Duh, Yen, Yen, Wang, &Chang, 2004; Jie et al., 2006). Kuo et al. (2005) reportedthat Puer tea and oolong tea can lower the levels of triglyc-eride more significantly than that of green tea and black tea,whereas Puer tea and green tea are more efficient thanoolong tea and black tea in lowering the level of totalcholesterol. Liang et al. (2005) reported that Puer tea sup-presses the genotoxicity induced by nitroarenes, lowers theatherogenic index and increases HDL (high density lipo-protein) e total cholesterol ratio. Rather recently, antimuta-genic and antimicrobial activities of Puer tea were alsoreported (Wu, et al., 2007). Although Puer tea has a historyof production and consumption for hundreds of years, fewstudies can be found on its microbiological properties,especially during the fermentation process.
Mo et al. (2005) have done a microbiological analysis onsamples from the indigenously fermented Puer tea. Micro-bial counting and identification revealed that A. niger wasthe dominating microorganism during the fermentation.Antimicrobial activity of fermentation samples shows
Inspection and packaging
n and grading
ed tea product Storage and ripening
Inspection and packaging
enzymes Dispersion of tea blocks
Natural inoculation and fermentation
f Puer tea manufacture.
126 H. Mo et al. / Trends in Food Science & Technology 19 (2008) 124e130
inhibitory effect on several food-borne bacteria, includingspore-forming bacteria Bacillus cereus, Bacillus subtilis,Clostridium perfringens and Clostridium sporogenes. Theantimicrobial activity increases with the course of the fer-mentation. This implies that certain metabolites of A. nigergrowing on tea leaves have the feature of inhibiting certainfood-borne bacteria. As the fermentation process willdecrease the content of tea polyphenols and catechins, theincrease in antimicrobial activity of Puer tea results obvi-ously in the formation of antimicrobial components otherthan tea polyphenols and catechins, or in their conversionto other new compounds.
Fermentation process and antimicrobial characteristicsof Fuzhuan brick-tea
Fuzhuan brick-tea is another kind of microbial fer-mented tea uniquely found in China (Xu, Mo, Yan, &Zhu, 2007). A schematic description of Fuzhuan brick-teaproduction is given in Fig. 2.
In a similar manner to studying Puer tea, we analyzedthe microbiological composition and tested the antimicro-bial activity of extracts from the indigenously fermentedFuzhuan brick-tea (unpublished data). Microbial countingand identification revealed that Aspergillus spp., Penicil-lium spp. and Eurotium spp. were the main microorganismsisolated from the samples during fermentation and Euro-tium spp. was the dominating fungus during the fermenta-tion. Antibacterial tests of extracts of fermented teashowed inhibitory effect on several food-borne bacteria,including spore-forming bacteria B. cereus, B. subtilis,C. perfringens and C. sporogenes. The antibacterial activityincreased with the course of the fermentation. This impliesthat certain metabolites of the fungi growing on tea leaveshad the feature of inhibiting certain food-borne bacteria.
Yao, Tan, Zhang, Su, & Wei (1998) developed a bulkacoustic wave bacterial growth sensor to study inhibitoryeffects of tea by continuous monitoring of disturbances inProteus spp. growth in the aqueous extracts of variousteas, including Fuzhuan brick-tea. The kinetic parameters,such as asymptote, maximum specific growth rate, lagtime, and generation time were accurately estimated by us-ing the growth response model. This model characterizesantimicrobial properties of the tested teas. All the parame-ters were changed via the inhibitory effects by tea. Theseinhibitory effects have also been examined by using thepour plate count technique for validation of the model.
Collecting of fresh tea leaves Enzyme inactivatio
Fungal fermentation Drying P
Natural oxidationMixing Steaming
Fig. 2. Schematic description of Fu
Both results show that, in addition to the antimicrobialproperties of tea polyphenols and catechins, the inhibitoryeffects are probably due to the metabolites produced duringthe fermentation processing.
Fermentation process and antimicrobial characteristicsof Kombucha
Kombucha is a slightly sweet-sour flavoured tea bever-age, obtained by fermentation of sweetened boiled teawith a mixed culture of yeasts and acetic-acid bacteria(Aidoo, Nout, & Sarkar, 2006). A flowchart of Kombuchapreparation is given in Fig. 3. Kombucha originated innortheast China (former Manchuria region) and later spreadto Russia and the rest of the world. Kombucha is also fre-quently called ‘‘tea fungus’’ in the literature, although thereis actually no fungus involved in the fermentation (Benk,1988; Liu, Hsu, Lee, & Liao, 1996; Mayser, Fromme,Leitzmann, & Grunder, 1995; Sievers, Lanini, Weber,SchulerSchmid, & Teuber, 1996; Steinkraus et al., 1996).This beverage reportedly has a number of health benefits,for example, against metabolic disease, arthritis, psoriasis,constipation, indigestion, and hypertension, but there arefew solid scientific evidences available yet for its efficacy(Dufresne & Farnworth, 2000; Ernst, 2003; Greenwalt,Steinkraus, & Ledford, 2000; Hartmann, Burleson, Holmes,& Geist, 2000; Pauline et al., 2001; Ram et al., 2000). Byvirtue of the numerous health-promoting aspects reportedand the easy and safe preparation of this beverage athome, it has gained popularity as other traditional beverages.Kombucha is a symbiotic growth of bacteria (Acetobacterxylinum, Acetobacter xylinoides, Bacterium gluconicum)and yeast strains (Schizosaccharomyces pombe, Saccharo-mycodes ludwigii, Saccharomyces cerevisiae, etc.) culturedin a sugared tea (Chen & Liu, 2000; Chu & Chen, 2006;Greenwalt et al., 1998; Teoh, Heard, & Cox, 2004). The ex-act microbiological composition also depends on the sourceof inoculums of the tea fermentation. Growth patterns ofthese microorganisms during the fermentation process ofKombucha are not well documented. Cellulose producedduring the fermentation by A. xylinum appears as a thinfilm on top of the sugared tea broth where the cell mass ofbacteria and yeasts is attached. This fungus-like mixture ofmicroorganisms and cellulose is likely why Kombucha isalso called ‘‘tea fungus’’. Glucose liberated from sucrose ismetabolized for the synthesis of cellulose and gluconicacid by Acetobacter strains. Fructose is metabolized into
n and grinding Grading and bagging
ackaging & storage
Softening with steam Cooling andbrick-making
zhuan brick-tea manufacture.
Black tea
Water Boiling
Sucrose and glucose
CoolingTake out tea
Inoculation with tea fungus Fermentation Product
Fig. 3. Schematic description of Kombucha manufacture.
127H. Mo et al. / Trends in Food Science & Technology 19 (2008) 124e130
ethanol and carbon dioxide by yeasts. Ethanol is oxidized toacetic acid by Acetobacter strains.
It is also reported that the fermentation process inducesthe synthesis of B complex of vitamins and folic acid(Bauer-Petrovska & Petrushevska-Tozi, 2000). The pHvalue of Kombucha decreases during the fermentation pro-cess following the increase in organic acid content (Blanc,1996; Sievers et al., 1996). The resultant low pH and pres-ence of antimicrobial metabolites reduce the competition ofother bacteria, yeasts and filamentous fungi.
The antimicrobial activity of Kombucha was testedagainst a number of pathogenic microorganisms (Sreeramuluet al., 2000). Staphylococcus aureus, Shigella sonnei, Escher-ichia coli, Aeromonas hydrophila, Yersinia enterolitica,Pseudomonas aeruginosa, Enterobacter cloacae, Staphylo-coccus epidermis, Campylobacter jejuni, Salmonella enteriti-dis, Salmonella typhimurium, B. cereus, Helicobacter pylori,and Listeria monocytogenes were found to be sensitive toKombucha. According to the literature on Kombucha, aceticacid is considered to be responsible for the inhibitory effecttowards a number of microbes tested (Greenwalt et al.,1998; Sreeramulu et al., 2000; Steinkraus et al., 1996). How-ever, Sreeramulu et al. (2000, 2001) found that Kombuchaexerts antimicrobial activities against E. coli, S. sonnei,S. typhimurium, S. enteritidis, and C. jejuni, even at neutralpH and after thermal denaturation of Kombucha. This findingsuggests the presence of antimicrobial compounds other thanacetic acid or large proteins in Kombucha.
Sreeramulu et al. (2001) further characterized the anti-microbial compounds in Kombucha. During the fermenta-tion process of 14 days, several metabolites were analyzedevery two days in Kombucha. Levels of acetic acid andgluconic acid were found to increase with fermentationtime. No lactic acid or ethanol was detected. Systematicinvestigation of the antimicrobial activity in Kombuchaconfirmed the presence of antimicrobial compounds otherthan organic acids or proteins (enzymes) produced duringfermentation, or the tannins originally present in the teabroth. Synergistic activity of antimicrobial componentsand low pH due to the increase of organic acids probablystrengthen the inhibitory effect of Kombucha.
ChallengesAlthough several antimicrobial activities were found in
the extracts of Puer tea and Fuzhuan brick-tea samples
during the fermentation process, as well as Kombucha,critical arguments still remain.
Antimicrobial components generated by microbialfermentation in tea
The first question is whether tea catechins or polyphe-nols have played the role of inhibition of microbialgrowth. This doubt can be excluded by the fact that theantibacterial activity increases with the fermentationtime; namely, the longer the fermentation is, the more an-tibacterial activity the sample has. This implies that theactivity comes from fungal metabolism and not from theoriginally present catechins or polyphenols of tea leaves.Nevertheless, Chou, Lin, and Chung (1999) reported thatconventional fermentative processed teas have decreasedantimicrobial activity with the fermentation time andthis implies that the originally present tea catechins orpolyphenols lose their antimicrobial activity during theenzymatic oxidation. In their study, B. subtilis, E. coli,Proteus vulgaris, Pseudomonas fluorescens, Salmonellaspp. and S. aureus were used to test the antimicrobial ac-tivity of various tea extracts. Among the six test micro-organisms, P. fluorescens was the most sensitive to theextracts, whereas B. subtilis was the least sensitive. Ingeneral, antimicrobial activity decreased when the extentof tea fermentation increased. The antimicrobial activitiesof tea extracts with different extents of fermentation var-ied with testing microorganisms. Extract of green tea, theunfermented or non-oxidized tea, showed the strongestantimicrobial activity followed by the partially fermentedtea products such as Longjing, Tieh-Kuan-Ying, Pao-chung, and oolong teas. On the other hand, black tea,the completely fermented (oxidized) tea, showed the leastantimicrobial activity. It was also noted that extracts ofoolong tea prepared in summer exhibited the strongest an-timicrobial activity, followed by those prepared in spring,winter and fall (Chou et al., 1999). It must be pointed outthat none of the teas used by these authors is microbialfermented tea although the term of fermentation is oftenused for black teas (Fowler et al., 1998). This indicatesthe possibility that fungus involved in microbial tea fer-mentation contributes to the formation of antimicrobialmetabolites other than tea catechins or polyphenols thatare originally present in unfermented or green tea leaves.Moreover, the results of Yao et al. (1998) also confirmthat the antimicrobial effect of microbial fermentedFuzhuan brick-tea is not catechins or polyphenols thatare actually fully oxidized during the process.
Thermo-stable characteristics of antimicrobialcomponents
The second question is whether the antimicrobial com-pounds are large proteins or enzymes that may have the in-hibitory activity. This question is answered by the fact thatsample extraction with hot water has wider antibacterialspectrum and stronger activity, and this confirms that the
128 H. Mo et al. / Trends in Food Science & Technology 19 (2008) 124e130
antimicrobial compounds are unlikely large proteins or en-zymes, because they are otherwise inactivated througha thermal treatment. Sakanaka, Juneja, and Taniguchi(2000) studied the inhibitory action of tea polyphenols to-wards the development and growth of bacterial spores.Among the tested Bacillus bacteria, tea polyphenols showantibacterial effects towards Bacillus stearothermophilus,which is a thermophilic spore-forming bacterium. Theheat resistance of B. stearothermophilus spores is reducedby the addition of tea polyphenols. Clostridium thermoace-ticum, an anaerobic spore-forming bacterium, also exhibitsreduced heat resistance of its spores in the presence of teapolyphenols. Epigallocatechin gallate, a main componentof tea polyphenols, shows strong activity against bothB. stearothermophilus and C. thermoaceticum. The heat re-sistance of these bacterial spores is more rapidly decreasedby the addition of tea polyphenols at high temperatures.This phenomenon also indicates that the potential antimi-crobial agents produced by microbial fermentation in teaare thermo-stable compounds, at least stable at 80 �C dur-ing the extraction time needed. With this thermo-stable fea-ture, this kind of natural preservatives can be used in foodproducts that will go through pasteurisation and short-timeheat treatment without losing the inhibitory activity. Thiscan be a very interesting measure to control thermophilicspore-forming bacteria.
Influences of extract concentrationThe third question to answer is whether the conclusion
is solid enough that the extract has no inhibitory effect onyeasts and fungi. In fact, Mo et al. (2005) used only oneextract concentration of the antimicrobial compounds forthe test. Therefore, it is critical whether a higher concen-tration of the extracts will have inhibitory effect on yeastsand fungi. Other challenges might arise when a higherconcentration of the fungal antimicrobial metaboliteswill be applied as natural preservatives in food products.First, the flavour of such natural preservatives mighthave impact on the food products concerned. These natu-ral preservatives should be desirably colourless and taste-less so that they will not bring about any off flavourtroubles. Second, whether there are interactions betweenthese natural preservatives and nutritional components inthe concerned food products. Ideally, these natural preser-vatives should not bring about any anti-nutritional effects.Davis (1990) described the anti-nutritional effect of caf-feine and Eggum, Pedersen, and Jacobsen (1983) reportedthe anti-nutritional effect of tannins. Lule and Xia (2005)indicated in a review on phenolic compounds that severalanti-nutritional effects can happen despite the positiveeffect of these compounds. However, little is known ifmicrobial fermented tea still has the anti-nutritional effecton other food components after weeks of fungal fermen-tation and oxidation. This, however, can be easily testedin future studies.
Perspectives and future research needs of microbialfermentation in tea
It is clear that microbial fermentation of tea results incertain metabolites that have inhibitory effects againsta number of bacteria. However, before these antimicrobialmetabolites can be used as natural preservatives in foodproducts, several critical issues must be clarified.
The responsible microorganisms for microbialfermentation in tea
First, it is necessary to confirm the responsible fungi,yeast and bacteria for the production of inhibitive compo-nents. Although certain microorganisms are claimed to beresponsible for the antimicrobial activity in Puer tea, Fuz-huan brick-tea and Kombucha, little is known about the fer-mentation of these teas by a pure culture of the claimedmicroorganisms. When a pure culture of the claimed micro-organism is applied in the fermentation on the same sub-strate, and can generate the same inhibitory effect againstcertain microorganisms, we can be sure that these claimedmicroorganisms are really responsible for the activity. Fur-thermore, the confirmation of the responsible role of theclaimed microorganisms will stimulate and promote thestandardization of the tea fermentation process and improvethe industrial efficiency.
Characterization of antimicrobial componentsSecond, we need to further characterize the antimicro-
bial agents produced by microbial fermentation of tea. Al-though several characterization attempts have been made toexclude the possibilities that the antimicrobial agents aretea catechins, large proteins and organic acids, the accurateformula of the compounds remains unknown. The identifi-cation and characterization of the antimicrobial compoundsare further of importance for understanding the metabolicpathways how compounds in tea leaves are transformedinto antimicrobial agents. When this metabolism is clari-fied, to overproduce these agents will be feasible.
Standardization and optimization of the processThird, as for Puer tea and Fuzhuan brick-tea, both are
produced through a solid-state fermentation process; stan-dardization and optimization are necessary. Solid-state fer-mentation is described as a process where no free water ispresent (Smits et al., 1999). During a solid-state fermenta-tion process, water will be evaporated to release heat pro-duced by microbial metabolism and this brings about thelimitation of growth and production, as a result of reducedwater activity. In a typical solid-state fermentation process,moisture, heat transfer (affecting substrate temperature) andmicrobial growth are closely interactive with each other(Smits, Rinzema, Tramper, Van Sonsbeek, & Knol, 1996;Smits et al., 1999; Von Meien & Mitchell, 2002). In an un-controlled traditional indigenous fermentation process suchas Puer tea and Fuzhuan brick-tea manufacture, not onlythe natural floras for inoculation, but also the season of
129H. Mo et al. / Trends in Food Science & Technology 19 (2008) 124e130
production strongly affect the product quality and consis-tency. Therefore, a standardized fermentation process willnot only ensure food safety but also the product quality.Furthermore, during the standardization and optimizationof the process, more insight will be obtained for the meta-bolic mechanism of the fungi involved, how they produceantimicrobial metabolites and eventually an overproductionof these useful natural preservatives can be expected.
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