International Journal of Food Microbiology 48 (1999) 125–130

Antimicrobial activity of tea as affected by the degree offermentation and manufacturing season

a , a b*Cheng-Chun Chou , Lon-Leu Lin , King-Thom ChungaGraduate Institute of Food Sciences and Technology, National Taiwan University, Taipei, Taiwan

bDepartment of Microbiology and Molecular Cell Sciences, The University of Memphis, Memphis, TN, USA

Accepted 1 March 1999

Abstract

Bacillus subtilis, Escherichia coli, Proteus vulgaris, Pseudomonas fluorescens, Salmonella sp. and Staphylococcus aureuswere used to test the antimicrobial activity of tea flush extract and extracts of various tea products. Among the six testorganisms, P. fluorescens was the most sensitive to the extracts, while B. subtilis was the least sensitive. In general,antimicrobial activity decreased when the extents of tea fermentation increased. The antimicrobial activities of tea flushextract and extracts of tea products with different extents of fermentation varied with test organisms. Tea flush and Greentea, the unfermented tea, exerted the strongest antimicrobial activity followed by the partially fermented tea products such asLongjing, Tieh-Kuan-Ying, Paochung, and Oolong teas. On the other hand, Black tea, the completely fermented tea, showedthe least antimicrobial activity. It was also noted that extracts of Oolong tea prepared in summer exhibited the strongestantimicrobial activity, followed by those prepared in spring, winter and fall. 1999 Elsevier Science B.V. All rightsreserved.

Keywords: Tea; Degree of fermentation; Antimicrobial activity

1. Introduction reported that black tea infusion inhibited the growthof Shigella dysenteriae and Salmonella typhosa but

There have been many reports on the beneficial not Staphylococcus aureus. Ryu (1982) showed theeffects of tea on human health in the past decade inhibitory effects of tea powder on the growth of(Blot et al., 1996; Katiyar and Mukhtar, 1996; Segal, some pathogenic bacteria. Nisiyama and Kozaki1996; Balentine et al., 1997; Dreosti et al., 1997). (1974) demonstrated the growth inhibition of variousThe inhibitory effect of tea infusion on the growth of lactic acid bacteria by the infusion of green tea.microorganisms has also been reported. Das (1962) Sakanaka et al. (1997) presented the data regarding

the antimicrobial activity of Green tea extract onvarious microorganisms. Chou and Lin (1987) re-*Corresponding author. Tel.: 1886-2-2363-0231 Ex. 2717; fax:ported that tea flush extract inhibited the growth of1886-2-2362-0849.

E-mail address: fstcchou@ccms.ntu.edu.tw (C.-C. Chou) various food spoilage and pathogenic bacteria. In

0168-1605/99/$ – see front matter 1999 Elsevier Science B.V. All rights reserved.PI I : S0168-1605( 99 )00034-3

126 C.-C. Chou et al. / International Journal of Food Microbiology 48 (1999) 125 –130

addition, Vanos et al. (1987) also noted that somepathogens were unable to multiply in instant teasolution, although spoilage organisms could grow.

There are different kinds of tea products availablein the markets, and each is prepared differently, i.e.the tea leaves are not fermented or are fermented tovarious extents. Furthermore, tea products are manu-factured with tea leaves harvested at differentseasons. These variations affect the flavor and aromaof tea products (Kan, 1980), and, therefore, may alsoaffect their antimicrobial activities. In this paper, wereport on the antimicrobial activities of different teaproducts which were made with various degrees offermentation. The antimicrobial activity of Oolongtea, a popular partially fermented tea, prepared indifferent seasons was also compared.

Fig. 1. Processes for the preparation of dry tea flush, Green,Paochung, and Black teas.

2. Materials and methodstea leaves continue and are completed during the

2.1. Test organisms next stage of black tea processing, traditionallyreferred to as fermentation. Black tea is regarded as a

Bacillus subtilis, Escherichia coli, Proteus vul- completely fermented tea, while Green and Paoch-garis, Pseudomonas fluorescens, Salmonella sp. and ung teas are considered as unfermented and partiallyStaphylococcus aureus were from culture collections fermented teas, respectively. On the other hand,of the Graduate Institute of Food Science and Oolong, Teih-Kuan-Ying, and Longjing teas obtainedTechnology, National Taiwan University. These from the retail market are all regarded as partiallyorganisms were grown at 378C except for Pse. fermented teas with various degrees of fermentation.fluorescens, which was grown at 288C. Each organ-ism was first transferred twice at an interval of 24 h 2.3. Preparation of tea flush and tea extractson nutrient agar (Difco, MI) slants. Cultures weregrown in nutrient broth for 12 h and served as the Dry tea flush and various tea products were firstsource of inoculum for each experiment. ground into powder using a Krups Garantie-Karte

KM75 grinder (Germany). The extracts were then2.2. Tea flush and tea products prepared by extracting one part of the above powder

with ten times the volume (w/v) of distilled water atFresh tea flush and various tea products, including 508C for 1 h and filtered through Whatman No. 1

Paochung, Black, Green, Oolong, Teih-kuan-Ying, filter paper. The filtrates were lyophilized (tempera-and Longjing teas, were tested in this study. Each tea ture , 2 408C, vacuum 0.1 bar) into powder.product possessing a different characteristic flavor isconsumed by the consumer according to individual 2.4. Growth and survival testspreference. They were either obtained directly from atea farmer who prepared these tea products with tea Growth and survival studies were performed inleaves of Camellia sinensis var. Chin-Hsin-Oolong 250-ml screw cap Erlenmeyer flasks containing 50upon our special request or from the retail market. ml of nutrient broth. Various amounts of tea extract

The processes for the preparation of dry tea flush powders were also added. A five-tenth ml aliquot ofand Green, Paochung, and Black teas by the tea a 12-h old culture was added and incubated at thefarmer are summarized in Fig. 1. The chemical and temperatures indicated above. At selected time inter-biochemical reactions initiated during the rolling of vals, samples from culture flasks were removed,

C.-C. Chou et al. / International Journal of Food Microbiology 48 (1999) 125 –130 127

serially diluted with 0.1% (w/v) peptone water (pH to agree with most literature reports (Das, 1962;7.0), and pour plated with nutrient agar. Colonies Ryu, 1982; Chou and Lin, 1987). Results obtainedthat appeared on plates were counted after 24 h of from this study also demonstrated that tea productsincubation. Each experiment was repeated twice. The with various degrees of fermentation exhibit differentpercentage of inhibition was calculated according to extents of antimicrobial activities (Figs. 2 and 4).Rico-Munoz and Davidson (1983) and expressed as Tea beverage is an infusion of the leaves of C.follows sinensis. Generally speaking, tea is made by crushing

and drying fresh tea leaves to affect ‘fermentation’,% inhibition 5 (Log CFU/mlcontrol prior to final processing. At present, there is a great

2 Log CFU/ml ) /Log CFU/ml variety of tea products in the retail market. Classifi-treated control

cation of processed tea has been fairly well estab-lished, based on quality and processing methods

3. Results which cause various degrees of fermentation andoxidation of the polyphenols (Hara et al., 1995).

Fig. 2 indicates the inhibitory effects of tea flush Black tea is the product of complete fermentation,extract and extracts of various kinds of products on whereas Green tea, an unfermented tea, is preparedthe growth of the tested organisms. In general, Green when fresh leaves are processed rapidly to preventtea, the unfermented tea, and tea flush extracts fermentation. Longjing (a variety of Green tea),exerted the highest antimicrobial activities, while Tieh-Kuan-Ying (a variety of Pouchong tea), Pouch-extract of Black tea, the completely fermented tea, ong, and Oolong teas are partially fermented withseemed to have the least inhibitory activity. Among increasing degrees of fermentation. Fermentation ofthe organisms tested, Pse. fluorescens was most tea leaves causes a reduction in the concentration ofsusceptible to the inhibitory effect of these extracts at catechins. During fermentation, some catechins com-the added concentration (0.1%), whereas B. subtilis bine to form complex theaflavins and other undefinedwas least sensitive. E. coli, Sta. aureus, Pro. vul- flavonoids, which provide distinctive flavor and colorgaris, and Salmonella sp. were all inhibited to a to the tea beverage. Green tea contains approximate-similar extent. ly 30% catechins (w/w), the completely fermented

Fig. 3 shows the inhibitory effects of the extracts Black tea beverage typically contains approximatelyof various tea products obtained from a retail market 9% catechin, 4% theaflavin, 3% flavonols, and 15%against Sta. aureus. Longjing tea extract was found undefined catechin condensate products (Wiseman etto exert the strongest antimicrobial activity against al., 1997). The survey made by Taiwan Tea Ex-the test organism, followed by Tieh-Kuan-Ying, perimental Station showed that the average catechinPaochung, and Oolong teas. Black tea showed the contents in tea products in the Taiwan area were asleast antimicrobial activity. follows: Green tea 17.1%, Paochung tea 16.3%,

The inhibitory effect of Oolong, a popular tea Oolong tea 13.3%, and Black tea 10.5% (Kan,product in Taiwan, prepared at different seasons was 1980). Hwang and Hwang (1995) also noted that thetested against Sta. aureus and results are presented in content of catechins, including epicatechin (EC),Fig. 4. Although, the preparation procedure was the epigallocatechin (EGC), epicatechin–gallate (ECG),same, it was found that extract of Oolong tea and epigallocatechin-3-o-gallate (EGCG) in teaprepared from tea leaves harvested in summer ex- products obtained from the market decreased as theerted the strongest inhibitory effect, followed by degree of fermentation increased. Green tea has thethose harvested in spring, winter, and fall. highest content of catechins followed in descending

order by Ti-Kuang-Ying, Oolong, and Black teas.Chou and Lin (1987) fractionated tea flush and

4. Discussion found that the fraction exhibiting the strongestantimicrobial activity contained mainly EGC and

The present findings, indicating that tea extracts ECG. Furthermore, Hara et al. (1995) demonstratedare inhibitory to the growth of various microorga- the antimicrobial activity of tea catechins includingnisms including both Gram (1) and Gram (2), seem EGC, EC, EGCG, and ECG against the growth of

128 C.-C. Chou et al. / International Journal of Food Microbiology 48 (1999) 125 –130

Fig. 2. Inhibition of bacterial growth by extracts of tea flush and various tea products. Medium was supplemented with 0.1% (w/v) ofextract powder.

C.-C. Chou et al. / International Journal of Food Microbiology 48 (1999) 125 –130 129

Fig. 4. Behavior of Staphylococcus aureus in presence of extractsFig. 3. Behavior of Staphylococcus aureus in presence of variousof Oolong tea prepared at different seasons. Medium was sup-tea extracts. Medium was supplemented with 0.2% (w/v) ofplemented with 0.2% (w/v) of extract powder. n, control; j,extract powder. s, control; d, Black tea; h, Oolong tea; j,spring tea; h, summer tea; d, fall tea; s, winter tea.Paochung tea; n, Teih-Kuan-Ying tea; m, Longjing tea.

Clostridium botulinum. Therefore, the decreased products may be closely related to catechin content,antimicrobial activity of the extracts from increasing- which is affected by the degree of fermentation andly fermented tea as shown in Figs. 2 and 3, may be the manufacturing season.attributed to the least amount of catechins in thesetea products.

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