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Bioactivities and Sensory Evaluation of Pu-erh Teas Made from Three Tea Leaves in an 1

Improved Pile-Fermentation Process 2

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Running title: Bioactivities of pile-fermentation-improved Pu-erh tea 4

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Yuh-Shuen Chen1*, Bing-Lan Liu2, Yaw-Nan Chang3 6

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1Department of Food Nutrition, HungKuang University, Taichung, Taiwan 433, R.O.C. 8

2Department of Applied Chemistry, Chaoyang University of Technology, Taichung, Taiwan 9

413, R.O.C. 10

3Department of Biotechnology, National Formosa University, Yunlin, Taiwan 632, R.O.C. 11

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* Corresponding author. e-mail: chenys@sunrise.hk.edu.tw 15

phone: +886-4-2631-8652 ext. 1600 fax: +886-4-2471-8398 16

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Two commercial Pu-erh teas, 15-year-old Ta-Huang-In and 25-year-old Ta-Hon-In, were 2

used for screening some species of fungi, yeasts, and bacteria, and six of them were isolated 3

and identified as Actinoplanes aurantiacus, Actinoplanes pallidoaurantiacus, Actinoplanes 4

purpeobrunneus, Streptomyces bacillaris, Streptomyces cavourensis subsp. cavourensis, and 5

Streptomyces cinereus. They were selected for inoculated into thee tea leaves (Yunnan from 6

China, TTES-12 and C. S. Oolong from Taiwan) and fermented for 180 days. The total 7

polyphenols and GABA content, and DPPH-radical scavenging effects were determined to 8

examine the tea-infusion quality. The samples inoculated with S. cinereus had the highest 9

total polyphenols content and maximum capacity to scavenge DPPH radicals; the highest 10

GABA content was obtained while the sample inoculated with S. bacillaris. Further 11

comparison of these samples with two commercial Pu-erh teas of different ages (Ta-Huang-In, 12

15-year storage and Ta-Hon-In, 25-year storage) showed that DPPH-radical scavenging 13

activity and GABA content of S. bacillaris and S. cinereus fermented tea leaf were higher 14

than these two commercial teas. Sensory evaluation also demonstrated that the taste, flavor, 15

and overall acceptance did not had significant differenced between the experimental tea 16

leaves and commercial samples. The present studies revealed that the fresh tea leaves 17

inoculated with the suitable microbes in short period of time will possess a high quality 18

tea-infusion as long-term storage Pu-erh tea. 19

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[Keywords: Pu-erh tea, pile fermentation, bioactivities, DPPH-radical scavenging 21

activity, γ-aminobutyric acid (GABA) content, sensory evaluation 22

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Tea is the most consumed drink in the world only next to water. Green tea is a 2

non-fermented tea and contains more catechins than black tea or oolong tea. Catechins are 3

strong antioxidants in vitro and in vivo. Recent studies suggested that green tea not only 4

contributes to reducing the risk of cardiovascular diseases and some forms of cancer but also 5

offers other benefits such as better oral hygiene, lower hypertension, reduction in body 6

weight, antibacterial and antiviral activity, protection from UV (ultraviolet) ray, bone density 7

increase, anti-fibrotic effect, and neuroprotection (1). 8

Pu-erh tea is reddish to brownish red, full-bodied, and markedly fragrant. These 9

properties become more prominent as fermentation continues and the leaves age. Among 10

several hundred kinds of tea, Pu-erh tea best represents the history and culture of China with 11

its cradle in Yunnan, China. Zhao Xueming, in the Qing Dynasty of China, mentioned in 12

complementary for Ben Cao Gang Mu (a Chinese herb pharmacopoeia), “Pu-erh tea is mild 13

and fragrant … it has incomparable perfume, which is very good for drinkers, helping in 14

digestion and eliminating expectoration”. Pu-erh tea does be good to stomach and incretion, 15

with notable effects, and it also is able to cure many disorders such as stomach upsets and 16

colds. 17

Pu-erh tea is one kind of post-fermentation tea, which emanates a moldy odor. In recent 18

years, many investigations have been carried out to examine the beneficial effects of Pu-erh 19

tea on health: it can act as an antioxidant; it has antimutation (2, 3), antimicrobial (4), and 20

anti-cancer properties (5); it can lower cholesterol (6, 7), blood pressure, and blood sugar (8); 21

it is an anticoagulant as well as an anti-bacterial and anti-viral agent; it can prevent tooth 22

decay and ulcers; it is anti-allergic; it can improve bacterial flora in the intestine; and it can 23

control halitosis (9). 24

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During the fermentation process, the tea catechins are oxidized to orthoquinones which 1

condense to form theaflavins (TFs). These TFs act as oxidizing agents for the substances like 2

gallic acid. Epitheaflavic acids, formed by the oxidation of gallic acid, condense with TFs to 3

produce the polymeric thearubigins (TRs). These TRs are responsible for color, body and 4

taste while TFs content in tea determines the briskness, brightness and quality of the liquor. 5

An ideal fermentation process results in a proper balance of TFs and TRs (2). Among the 6

various components (water-soluble ingredients account for roughly 30-40% of the total dry 7

weight of tea leaves) being estimated and evaluated for the quality of black tea, only TFs 8

content was reported to play a major role in tea quality. Different teas have different chemical 9

ingredients and hence different properties. Also, teas harvested in different seasons differ in 10

their chemical composition. Generally, green tea has the highest proportion of water-soluble 11

ingredients and black tea has the least since it is completely fermented. Water-soluble 12

ingredients in tea include the following nine groups: polyphenols (7), alkaloids (10), proteins 13

and free amino acids (11), carbohydrates (12), pigments (13), lipids (14), minerals (15), 14

vitamins (16), and aromatic compounds (17). Among these, GABA has received more 15

attention because it acts as a relaxant and can boost immunity in humans (18). 16

Traditional Pu-erh tea is made by fermenting tea leaves at room temperature for a long 17

time, i.e. the so-called dry-stored tea. In order to hasten aging, tea is covered with a wet cloth 18

during fermentation, which is called wet-stored tea. Although wet-stored tea made by this 19

method has a shorter fermentation period, it can harbor undesirable microorganisms either 20

harmful to the body or inhibitive to the growth of beneficial microorganisms that enhance the 21

taste of tea. 22

The microorganisms that predominate in piles of Yunnan Pu-erh tea undergoing 23

fermentation are Aspergillius niger, Aspergillius gloucus, and species of Penicllium, Rhizopus, 24

Saccharomyces, and Bacterium. Among them, Aspergillius niger is the most predominant, 25

5

followed by Saccharomyces spp. There are very few Bacillus spp. (19, 20). Because the 1

quality of Pu-erh tea is closely related to the post-fermentation process, the present study 2

isolated some microbes from Ta-Huang-In and Ta-Hon-In, which are known for high-quality 3

Pu-erh tea. Batches of tea leaves were inoculated with individual strains of the isolated 4

microbes. At regular intervals during fermentation, and after it was complete, the tea leaves 5

were assessed in terms of the extent of free-radical scavenging activity and the content of 6

polyphenols and GΑΒΑ to find out the most effective strain for an improved 7

pile-fermentation process that not only enhances the quality of tea but also shortens 8

fermentation time and lowers production cost. The experimental design was depicted in Fig. 9

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MATERIALS AND METHODS 1

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Source of Pu-erh teas Two commercially available Pu-erh teas namely: Ta-Huang-In (of 3

15-year storage) and Ta-Hon-In (of 25-year storage) were used in this study. The teas were 4

supplied by Canghai Tea Shop, Taichung, Taiwan. 5

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Screening and isolation of microbes from Pu-erh teas Five grams of Ta-Huang-In and 7

Ta-Hon-In were washed with 45 mL of sterilized water. The washing liquid (1 mL) was then 8

plated onto PCA agar (Plate Count Agar, Difco, France). The plates were incubated at 37°C 9

for a week (each sample was performed in triplicate). In another parallel experiment, two 10

grams of Ta-Huang-In and Ta-Hon-In were washed with sterilized water and then 11

homogenized in a blender (CF-15BS, XinGuang Mechanic, Taiwan) after adding 225 mL of 12

sterilized water. A 1 mL of each homogenized sample was incubated as before (three 13

replications). The selection of different colonies of microorganisms from both sources were 14

subcultured using inoculation loops and then incubated for a week at 37°C on NB (Nutrient 15

Broth, Difco, France). Pure cultures were obtained by subculturing one more time and 16

incubated for a week before sending them to the Food Industry Research and Development 17

Institute (FIRDI, Hsinchu, Taiwan) for identification. 18

19

Pile-fermentation process Three fresh tea leaves (300 g each): Yunnan (China), TTES-12 20

(Taiwan tea), C. S. Oolong (Chein Shin Oolong; Taiwan golden tea) were sterilized by hot air. 21

Subsequently, the tea leaves were inoculated with the selected isolate (104 cfu microbe per 22

gram) obtained from Pu-erh teas. The lots were fermented under controlled conditions (at 23

37°C and 70% relative humidity with partly anaerobic culture) for 180 days. The samples 24

were evaluated for scavenging activity and GABA/total polyphenols content during the 25

7

fermentation period. For the total plate count, one gram of fermented samples were washed 1

with 99 mL of sterilized water, and diluted with sterilized water to 103x, 104x, and 105x and 2

then 1 mL suspension was plated onto PCA agar (Plate count agar; Difco, France). The plates 3

were incubated in an incubator (Firstek Scientific, Taipei, Taiwan) at 37°C for 48 hrs (each 4

sample was replicated three times), and then was counted by using Microbial counter, the 5

selection of the microbial range was from 25-250 C.F.U. per plate. 6

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DPPH-radical scavenging activity Radical scavenging activity (RSA) of tea infusions 8

against stable DPPH was determined by a slightly modified DPPH-radical scavenging assay 9

(21, 22). Samples representing the teas (pile-fermented tea samples and those sold in market, 10

i.e. Ta-Huang-In and Ta-Hon-In) were ground to powder and then passed through a 60-mesh 11

sieve. The oven-dried powder, in lots of 10, 50, 100, 150, 200, and 250 mg, was extracted 12

with 10 mL of hot water (70°C). After 5 min, the infusion was filtered through a filter paper 13

to obtain the sample liquor. A 25 µL of sample liquor diluted to 4 mL with methanol and then 14

mixed with a 0.5 mL of freshly prepared 1 mM DPPH solution. After 30 mins incubation, the 15

absorbance at 517 nm was measured. The DPPH radical scavenging activity was calculated 16

by a formula I = [(AB - AA)/AB] × 100, where I is the DPPH radical scavenging activity (in %); 17

AB is the absorbance of blank sample (t = 0 min); AA is the absorbance of sample solution (t = 18

30 mins). For the comparison, DPPH scavenging activity was also determined according to 19

previously reported (23, 24). The experiments were monitored by analytic HPLC on a model 20

PU-L2130 instrument (Hitachi, Tokyo, Japan) with a 250 × 4.6 mm i.d., 5 µm Mightsil RP-18 21

GP column (Kanto Chemical. CO., Inc.). The mobile phase was solvent A (100% MeOH) and 22

solvent B (Ultrapure water). Elution conditions were 0-55 min of 10-70% A to B (linear 23

gradient) and 55-60 min of 70-100% A to B (linear gradient) at a flow rate of 1.0 mL/min 24

using a photo diode array detector (Hitachi L-2455) at 254 nm. As for online DPPH 25

8

radical-scavenging analysis, the flow of DPPH reagent (50 mg/L in methanol) was set to be 1

0.5 mL/min, and the induced bleaching was detected photometrically as a negative peak at 2

517 nm. 3

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Superoxide scavenging activity Superoxide radical generated by the xanthine/xanthine 6

oxidase system was determined spectrophotometrically by monitoring the product of 7

nitroblue tetrazolium (NBT). The tea infusions were mixed with 0.15 mM NADH and 0.74 8

mM NBT in sequence and then added promptly to 0.05 mM PMS to obtain a reduced form of 9

NBT. The amount of NBT in the reduction state was determined by absorbance at a 10

wavelength of 560 nm. A reduction in absorbance is therefore proportional to a sample’s 11

ability to eliminate superoxide anion oxygen free radicals (25). The scavenging activities of 12

tea infusion were measured by the following equation: 13

100 sampleut unit witho control of valueabsorptionlight

sample of valueabsorptionlight - 1 (%)activity Scavenging ×

= 14

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Quantitative analysis of GABA content Tea liquor was prepared as described above with 16

200 mg of dry tea powder. The sample was derivated by the method reported elsewhere (26, 17

27) period to HPLC analysis. The tea liquor (1 mL each) was mixed with 0.6 mL of 0.1 M 18

buffer and 1 mL of 0.3% 2-hydroxy naphthaldehyde (derivatization reagent). The mixture 19

was incubated for 10 mins in a water bath maintained at 80°C and then cooled to room 20

temperature thereafter. Methanol was then added to make the final volume to 5 mL. Twenty 21

microlitres of each sample were analyzed using HPLC and a LiChrosper100 RP18 column 22

(5 µm, 250 × 4 mm). The mobile phase comprised MeOH and H2O (62:38) with a flow rate 23

of 1.0 mL/min. The detection wavelength was 330 nm. Each sample was determined in 24

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triplicate and the average value of the peak area at different values of chroma was calculated. 1

Recovery and precision were also tested to validate the method. 2

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Determination of total polyphenols content Total polyphenols content was quantified 4

using the Folin-Ciocalteau colorimetric assay. Three grams of fermented tea samples were 5

extracted with 150 mL hot water (70°C). The tea infusion (50 µL) was mixed with 2 mL of 6

2% sodium carbonate solution for 2 mins. Subsequently, a 100 µL of 50% Folin-Ciocalteu 7

reagent (Sigma) was added into the mixture. After incubation at room temperature for 30 8

mins, the absorbance was measured at 750 nm. Total polyphenols were determined using a 9

gallic acid (Sigma) calibration curve and the results were expressed as gram per 100 g of 10

gallic acid equivalents. 11

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Sensory evaluation Sensory evaluation was performed in the Department of Food 13

Nutrition at the HungKuang University (Taichung County, Taiwan). Tea sample was 14

extracted with 500 mL of boiling water for 5 min. In preparation for the sensory evaluation, 15

tea infusions were individually presented in covered small porcelain cups to each panelist. 16

The judges were not informed of the experimental approach and the samples were 17

blind-coded with random numbers. Panelists were instructed to smell and drink the tea 18

infusions, and pause for 30 s between samples. A 10-point hedonic scale, from 9 (excellent, 19

extremely acceptable) to 0 (poor, extremely unacceptable), was employed to evaluate each 20

tea infusion sample on taste, and flavor. The sensory attributes of tea infusion were compared 21

with those of the commercial Pu-erh products (Ta-Huang-In and Ta-Hon-In). Tea infusion 22

samples receiving overall scores of more than 4 were considered acceptable, while a score 23

between 3 and 4 was considered the borderline of acceptability. The overall acceptance of the 24

tea infusions were also evaluated by those consumers using a 10-point hedonic scale. At least 25

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sixty panelists participated in each test, with about total 86-102 people participating in this 1

study. 2

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RESULTS AND DISCUSSION 1

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Screening and isolation of microbes that enhance the taste of Pu-erh tea The 3

predominant microbes that had been isolated in traditional Pu-erh tea were: Aspergillius 4

niger, A. gloucus, and species of Penicllium, Rhizopus, Saccharomyces, and Bacterium. 5

Amongst, A. niger was present in the greatest number, followed by species of Saccharomyces 6

whereas the least population was Bacterium (19, 20). In this studied, 30 strains were obtained 7

from Ta-Huang-In and 24 strains from Ta-Hon-In in the preliminary screening. These 8

included: Penicllium, Rhizopus, Saccharomyces, Aspergillius, and Bacterium (mainly 9

actinomycete and photosynthetic bacteria). At least 15 strains of fungi, bacteria, and yeasts 10

that enhanced the quality of tea leaves were observed. The beneficial microbes isolated from 11

traditional Pu-erh tea and deployed for fermentation can enhance the flavor of Pu-erh tea 12

were subjects for the identification. Strains that led to a bitter taste, astringency, and smell of 13

grass, mold, or soil were discarded. 14

The strains were further identified by Bioresource Collection and Research Center, FIRDI, 15

as belonging to Actinoplanes and Streptomyces. These two species were used for further study. 16

Six strains namely: Streptomyces bacillaris, Actinoplanes pallidoaurantiacus, Actinoplanes 17

purpeobrunneus, Actinoplanes aurantiacus, Streptomyces cavourensis subsp. cavourensis, 18

and Streptomyces cinereus were selected for inoculated into thee tea leaves (i.e. Yun Nan, 19

TTES-12, C.S.Oolong) for 180 days. The results were compared with the commercial Pu-erh 20

products (i.e. Ta-Huang-In and Ta-Hon-In) in terms of DPPH-radical scavenging activity, 21

GABA and total polyphenol contents. Following fermentation period for 180 days, the results 22

suggested that these 6 strains could not only enhance the three tea leave’s characteristic taste 23

and flavor (see below), also exhibited a potential in the improvement of scavenging activity 24

as well as GABA contents (see below). 25

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DPPH-radical scavenging activity Human cells suffer oxidation damage following 2

prolonged and constant exposure to free radicals and reactive oxygen. The damage to cell or 3

death of cells causes cancer, aging, arteriosclerosis, and diabetes (28). Tea leaf contains many 4

ingredients, such as vitamins C and E, β-carotene, catechin, and other polyphenols that can 5

eliminate the free radicals; among these ingredients, catechin shows very high antioxidation 6

capacity, which is held to be closely related to its anti-cancer and anti-tumour properties. 7

The antioxidation ability, expressed as DPPH-radical scavenging capacities, of tea 8

leaves following fermentation with different strains and for varying duration is shown in 9

Table 1. The DPPH• test is a non-enzymatic method currently used to provide basic 10

information on the ability of tea infusions to scavenge free radicals. This could be obtained 11

from the reduction of DPPH• by an antioxidant results in a loss of absorbance at 517 nm. 12

Generally, the capacity to scavenge DPPH radicals increased with the duration of 13

fermentation. The results indicated that when Yun Nan, TTES-12, and C.S.Oolong inoculated 14

with S. cinereus were observed the highest antioxidant potential, as measured by the DPPH 15

assay after 180 days cultivation, followed by tea leave inoculated with S. bacillaris, and A. 16

aurantiacus. When the A. pallidoaurantiacus, A. purpeobrunneus, or S. cavourensis subsp. 17

cavourensis was inoculated, the tea infusions exhibited the lowest scavenging capacity. Yet, 18

the scavenging activity contributed from six selected microbes was increased with the 19

fermentation time (up to 180 days) in three tea leaves studied (Table 1). These were 20

coincidence with the early studied (29). 21

Furthermore, the commercial Pu-erh teas (i.e. Ta-Huang-In and Ta-Hon-In) and 22

microbes fermented tea leaves exhibited the DPPH radical-scavenging activity in a 23

concentration-dependent manner (Fig. 2). Again, when the tea leave of Yun Nan, TTES-12, 24

and C.S.Oolong inoculated with S. cinereus were observed the highest DPPH-radical 25

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scavenging activity, whereas the Ta-Huang-In shown the lowest scavenging activity at all 1

cases studied. As can be seem from Fig. 2, the Yun Nan, TTES-12, and C.S.Oolong fermented 2

with S. cinereus, S. bacillaris, or A. aurantiacus were demonstrated a better antioxidation 3

ability as compared with Ta-Huang-In and Ta-Hon-In after 180 days fermentation. 4

The typical UV and DPPH radical quenching chromatograms of the fermented samples 5

after 180 days were shown in Fig. 3. From the comparison of the chromatograms of authentic 6

compounds, the peaks with retention times at 2.54, 17.76, 20.86, 22.31, 29.03, and 37.89 min 7

in the UV chromatogram, and at 2.54, 17.77, 20.97, 22.41, 29.07, and 37.93 min in the DPPH 8

chromatogram were identified as gallic acid, chlorogenic acid, epicatechin, epigallocatechin, 9

caffeic acid, and epicatechin gallate, respectively (Fig. 3B). The chromatograms show that 10

antioxidants from tea samples can be identified and most of them did not observed in 11

non-inoculated sample (Fig. 3A). Yet, the more rapidly the absorbance decreases at 517 nm, 12

the more potent the antioxidant activity of the compound in terms of hydrogen donating 13

ability (23). Obviously, sample without S. cinereus fermented exhibited a lower decrease in 14

absorbance at 517 nm (Fig. 3A). This implied that an improved pile-fermentation process 15

employed a selected microbe into fresh tea leaves will bearing a splendid DPPH• scavenging 16

activity as compared with non-inoculated or the commercial long-term storage Pu-erh teas. 17

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Superoxide scavenging activity In a non-enzymatic system, a superoxide negative radical 19

is produced when phenazine methosulfate (PMS) reacts with dihydronicotinamide-adenine 20

dinucleotide (NADH). The superoxide negative radical reacts with nitro bluetetrazolium 21

(NBT) to form a diformazan compound, which has its maximum absorption peak at 560 nm. 22

Therefore, a change in absorbance at 560 nm can be used for calculating the production of 23

superoxide negative radical. If a sample decreased in the absorbance, it indicates that the 24

sample has the capacity to capture superoxide negative radical. The values obtained by 25

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inoculating the samples of rough tea with different strains and following different 1

fermentation periods are shown in Table 2. In general, the scavenging activity did not differ 2

greatly from strains or fermentation periods. Almost over 90% superoxide radical (O2·-) 3

scavenging activity was observed from 60 days fermentation thereafter, revealed that the O2·- 4

was readily scavenged by tea infusions (Table 2). 5

In fact, the scavenging of O2·- by tea infusions may be attributed to their various 6

phenolic constituents. Cotelle et al. (30) reported that flavones, a class of flavonoids, bearing 7

hydroxyl groups at positions 3′, 4′ or 3′, 4′, 5′ scavenged O2·-. These authors also reported 8

that flavonoids containing one hydroxyl group in the C-7 position of ring A could inhibit 9

generation of O2·- in the xanthine/xanthine oxidase system by inhibiting the enzyme. Phenolic 10

acids and their derivatives are also reported to be an excellent free radical-scavenger. The 11

radical-scavenging ability of phenolic acids is mediated by the number of hydroxyl groups in 12

the molecule. The electron-withdrawing property of the carboxylic acid group in benzoic acid 13

has a negative influence on hydrogen-donating ability of hydroxybenzoates (31). Therefore, 14

the high O2·- scavenging properties of tea infusion, as presented in Table 2, may also be 15

attributed to both neutralization of superoxide radicals via hydrogen donation and inhibition 16

inoculate of xanthine oxidase by various phenolic components present in the tea infusions. 17

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GABA content in Pu-erh teas and fermented tea leaves GABA is one of the major 19

inhibitory neurotransmitters in the central nervous system and known to mediate pre-synaptic 20

inhibition of primary afferent fibers in the motor system and may also be involved in 21

post-synaptic forms of motor neuron inhibition (32). Amino acid neurotransmitters are critical 22

for the function of the central nervous system; they act rapidly, responding within a few 23

milliseconds, and play an important role in brain function and neurological diseases. In recent 24

studies, GABA was fed to rats through enriched soy products, was reported to exert an 25

15

antihypertensive effect (33). GABA occurs naturally in many kinds of foods at low levels, 1

whereas in fermented food products GABA levels were higher. GABA is used as a functional 2

food ingredient in different kinds of foods in Japan because of its health benefits (18). 3

In recent years, processed tea such as Jiayelong tea has been attracting increasing 4

attention from researchers and the industry because it was believed to be rich in GABA. 5

These products are also favored by consumers. The GABA content of Pu-erh teas and tea 6

leaves inoculated with different strains was estimated by HPLC, the results were presented in 7

Fig. 4. In the case of Yun Nan, the GABA content of samples inoculated with A. 8

pallidoaurantiacus and A. purpeobrunneus turned out to be lower than that Ta-Hon-In and 9

Ta-Huang-In. However, the GABA contents of tea leaves inoculated with S. cinereus and S. 10

bacillaris was markedly higher than that of non-fermented teas. When the TTES-12 was 11

inoculated with S. cinereus, S. bacillaris, or A. aurantiacus, the GABA content displayed 12

excellent levels (Fig. 4 middle panel). This is coincidence with the DPPH-radical scavenging 13

activity profiles (Table 1). The similar observation was found in the case of C.S.Oolong (Fig. 14

4 bottom panel). Taken account all the samples, when Yun Nan, TTES-12, or C.S.Oolong 15

inoculated with S. bacillaris the GABA contents were 712, 813, and 8632 mg/100g, 16

respectively, which were extraordinary higher than that Ta-Hon-In (291 mg/100g) and 17

Ta-Huang-In (467 mg/100g). Jeng et al. also had similar observation during 42 days 18

fermentation (29). 19

Generally, commercial tea products claiming to have higher GABA content are produced 20

using anaerobic fermentation to improve GABA content. However, in this studied, the 21

fermentation was only partly anaerobic or oxygen-limited (because it was carried out in a 22

container with the opening sealed). As a results, the GABA contents even two folds higher 23

than that Ta-Hon-In. By making the process completely anaerobic and inoculated with S. 24

bacillaris, the GABA content can be increased further so that the teas will fetch a better price. 25

16

1

Total polyphenols content Table 3 shows the total polyphenols content of three tea leaves 2

inoculated with different strains. As can be seem from this table, the total polyphenol content 3

was increased with the duration of fermentation. Although samples inoculated with S. 4

bacillaris and S. cinereus did not have the highest total polyphenols content in the initial 5

stages of fermentation, strain S. bacillaris surpassed all the rest on the 60th day and at the end 6

of fermentation (180 days). Meaning while, 104 CFU/g of S. cinereus was counted at 7

beginning of fermentation (day 0) and reached to 1.2×106 CFU/g at the end of fermentation 8

(day 180). The best strain contributed to the total polyphenol content in fermented tea 9

infusions still goes to S. cinereus. The highest total polyphenol content (8620 mg/100g) was 10

found in C.S.Oolong tea infusion fermented with S. cinereus. When TTES-12 fermented with 11

S. cinereus also shows a high polyphenol level (8125 mg/100g) follow by C.S.Oolong 12

fermented with S. bacillaris (8120 mg/100g). Thus, S. bacillaris and S. cinereus deserve 13

further research and discussion. Significantly difference in polyphenol content was also found 14

in GABA levels, consequently indicating a possible correlation between S. bacillaris and S. 15

cinereus topped the rest on DPPH radical-scavenging activity (Fig. 2) as well. 16

17

Sensory evaluation The effects of fermentation strains on sensory evaluation were 18

summarized in Table 4. No significant difference between 6 strains of inoculated samples and 19

commercial samples on taste, flavor and overall acceptance of tea infusions were observed. 20

Yet, the Yun Nan, TTES-12, or C.S.Oolong fermented with S. cinereus remain exhibited the 21

better results on sensations as compared to other microbes. The sensory results were even 22

better than the commercial samples. These results highlight consistent changes of DPPH 23

radical scavenging activity (Table 1) and total polyphenols content (Table 3) in response to 24

the inoculation conditions: both the sensations depend upon the microbe used and 25

17

fermentation duration. This results leading to the conclusion that the microbe effect on these 1

sensations were strongly dependent on the original qualitative of species incorporated. 2

3

Summary At least fifteen strains of microbes that could enhance the quality of tea leaves 4

as found in preliminary screening test. Six of them were isolated and identified as S. 5

bacillaris, A. pallidoaurantiacus, A. purpeobrunneus, A. aurantiacus, S. cavourensis subsp. 6

cavourensis, and S. cinereus. They were selected for inoculated into thee tea leaves (Yun Nan, 7

TTES-12, and C.S.Oolong). The tea leaves inoculated with isolated microbes were possess a 8

strong DPPH• scavenging properties which were either comparable or superior to those of 9

commercial Pu-erh teas at the same concentration. These effects may attributable to varying 10

hydrogen-donating and metal-chelating capacities of the phenolic compounds present in the 11

tea infusions. 12

Inoculation with these microbes, especially strains S. bacillaris, A. aurantiacus, and S. 13

cinereus and subsequent fermentation up to 180 days increased total polyphenols content of 14

tea leaves and their capacity to scavenge DPPH radical. In particularly, the samples 15

inoculated with S. cinereus had the highest total polyphenols content and maximum capacity 16

to scavenge DPPH radical (Tables 1 & 3); the highest GABA content was obtained while the 17

sample inoculated with S. bacillaris (Fig. 3). Further comparison of these samples with two 18

commercial Pu-erh teas of different ages (Ta-Huang-In, 15-year storage and Ta-Hon-In, 19

25-year storage) showed that DPPH-radical scavenging activity and GABA content of S. 20

bacillaris and S. cinereus fermented tea leaf were higher than that Ta-Huang-In and 21

Ta-Hon-In teas. The GABA content in inoculated samples was higher than that in tea leaves 22

produced without fermentation. The effects observed were concentration-dependent. These 23

microbes, originally isolated from traditional Pu-erh teas and utilized for unraveling the 24

microorganisms involved in tea infusions taste and antioxidant activity, have multiple effects 25

18

that are only now being fully appreciated. In addition to their ability to exhibit the high 1

DPPH-radical scavenging activity, these microbes also appear to have additional activities, 2

including GABA and total polyphenol contents, which continue to expand their range of 3

potential uses. The sensory evaluation results suggests that the tea leaves inoculated the 4

appropriate microbes were maintain the taste, flavor, and overall acceptance of tea infusion. 5

In summary, the fresh tea leaves inoculated with the suitable microbes in short period of time 6

will possess a high quality tea infusion as long-term storage Pu-erh tea.7

19

1

ACKNOWLEDGEMENTS 2

3

The work supported by financial contributions of the Ministry of Education, Republic of 4

China (Taiwan) (98-F-29022), and the China Biochemical Co. Ltd. The authors also express 5

their thanks to Canghai tea shop for providing different kinds of commercial Pu-erh tea. 6

20

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1 Figure Captions 2

3

FIG. 1. The experimental flow chart of this study. 4

FIG. 2. Concentration dependent of DPPH radical-scavenging activity of tea infusions on 5

commercial Pu-erh teas and tea leaves of Yun Nan (A), TTES-12 (B), C.S.Oolong 6

(C) inoculated with isolated microbes and fermented with an improved process. 7

DPPH radical-scavenging activity for fermented tea leaves was measured after 180 8

days cultivated with isolated microbes. Sample preparation and DPPH analysis see 9

materials and methods section for detail. 10

FIG. 3. The typical UV and DPPH radical quenching chromatograms of tea sample without 11

S. cinereus inoculated (A) and with S. cinereus inoculated (B) after 180 days. The 12

quoted number in each peak represents: 1. Gallic acid (GA), 2. Gallocatechin (GC), 13

3. Chlorogenic acid (CHA), 4. Epicatechin (EC), 5. Epigallocatechin (EGC), 6. 14

Caffeic acid (CA), 7. Epicatechin gallate (ECG). 15

16

FIG. 4. Comparison of GABA content on commercial Pu-erh teas and tea leaves of Yun Nan 17

(top), TTES-12 (middle), C.S.Oolong (bottom) inoculated with isolated microbes 18

and fermented with an improved process. 19

GABA content for fermented tea leaves was measured after 180 days cultivated 20

with isolated microbes. Each bar represents the mean value of three determinations 21

standard deviation. 22

23

24

25