1 Bioactivities and Sensory Evaluation of Pu-erh Teas Made from Three Tea Leaves in an
2 Improved Pile-Fermentation Process
4 Running title: Bioactivities of pile-fermentation-improved Pu-erh tea
6 Yuh-Shuen Chen1*, Bing-Lan Liu2, Yaw-Nan Chang3
8 Department of Food Nutrition, HungKuang University, Taichung, Taiwan 433, R.O.C.
9 Department of Applied Chemistry, Chaoyang University of Technology, Taichung, Taiwan
10 413, R.O.C.
11 Department of Biotechnology, National Formosa University, Yunlin, Taiwan 632, R.O.C.
15 Corresponding author. e-mail: firstname.lastname@example.org
16 phone: +886-4-2631-8652 ext. 1600 fax: +886-4-2471-8398
2 Two commercial Pu-erh teas, 15-year-old Ta-Huang-In and 25-year-old Ta-Hon-In, were
3 used for screening some species of fungi, yeasts, and bacteria, and six of them were isolated
4 and identified as Actinoplanes aurantiacus, Actinoplanes pallidoaurantiacus, Actinoplanes
5 purpeobrunneus, Streptomyces bacillaris, Streptomyces cavourensis subsp. cavourensis, and
6 Streptomyces cinereus. They were selected for inoculated into thee tea leaves (Yunnan from
7 China, TTES-12 and C. S. Oolong from Taiwan) and fermented for 180 days. The total
8 polyphenols and GABA content, and DPPH-radical scavenging effects were determined to
9 examine the tea-infusion quality. The samples inoculated with S. cinereus had the highest
10 total polyphenols content and maximum capacity to scavenge DPPH radicals; the highest
11 GABA content was obtained while the sample inoculated with S. bacillaris. Further
12 comparison of these samples with two commercial Pu-erh teas of different ages (Ta-Huang-In,
13 15-year storage and Ta-Hon-In, 25-year storage) showed that DPPH-radical scavenging
14 activity and GABA content of S. bacillaris and S. cinereus fermented tea leaf were higher
15 than these two commercial teas. Sensory evaluation also demonstrated that the taste, flavor,
16 and overall acceptance did not had significant differenced between the experimental tea
17 leaves and commercial samples. The present studies revealed that the fresh tea leaves
18 inoculated with the suitable microbes in short period of time will possess a high quality
19 tea-infusion as long-term storage Pu-erh tea.
21 [Keywords: Pu-erh tea, pile fermentation, bioactivities, DPPH-radical scavenging
22 activity, γ-aminobutyric acid (GABA) content, sensory evaluation
2 Tea is the most consumed drink in the world only next to water. Green tea is a
3 non-fermented tea and contains more catechins than black tea or oolong tea. Catechins are
4 strong antioxidants in vitro and in vivo. Recent studies suggested that green tea not only
5 contributes to reducing the risk of cardiovascular diseases and some forms of cancer but also
6 offers other benefits such as better oral hygiene, lower hypertension, reduction in body
7 weight, antibacterial and antiviral activity, protection from UV (ultraviolet) ray, bone density
8 increase, anti-fibrotic effect, and neuroprotection (1).
9 Pu-erh tea is reddish to brownish red, full-bodied, and markedly fragrant. These
10 properties become more prominent as fermentation continues and the leaves age. Among
11 several hundred kinds of tea, Pu-erh tea best represents the history and culture of China with
12 its cradle in Yunnan, China. Zhao Xueming, in the Qing Dynasty of China, mentioned in
13 complementary for Ben Cao Gang Mu (a Chinese herb pharmacopoeia), “Pu-erh tea is mild
14 and fragrant … it has incomparable perfume, which is very good for drinkers, helping in
15 digestion and eliminating expectoration”. Pu-erh tea does be good to stomach and incretion,
16 with notable effects, and it also is able to cure many disorders such as stomach upsets and
18 Pu-erh tea is one kind of post-fermentation tea, which emanates a moldy odor. In recent
19 years, many investigations have been carried out to examine the beneficial effects of Pu-erh
20 tea on health: it can act as an antioxidant; it has antimutation (2, 3), antimicrobial (4), and
21 anti-cancer properties (5); it can lower cholesterol (6, 7), blood pressure, and blood sugar (8);
22 it is an anticoagulant as well as an anti-bacterial and anti-viral agent; it can prevent tooth
23 decay and ulcers; it is anti-allergic; it can improve bacterial flora in the intestine; and it can
24 control halitosis (9).
1 During the fermentation process, the tea catechins are oxidized to orthoquinones which
2 condense to form theaflavins (TFs). These TFs act as oxidizing agents for the substances like
3 gallic acid. Epitheaflavic acids, formed by the oxidation of gallic acid, condense with TFs to
4 produce the polymeric thearubigins (TRs). These TRs are responsible for color, body and
5 taste while TFs content in tea determines the briskness, brightness and quality of the liquor.
6 An ideal fermentation process results in a proper balance of TFs and TRs (2). Among the
7 various components (water-soluble ingredients account for roughly 30-40% of the total dry
8 weight of tea leaves) being estimated and evaluated for the quality of black tea, only TFs
9 content was reported to play a major role in tea quality. Different teas have different chemical
10 ingredients and hence different properties. Also, teas harvested in different seasons differ in
11 their chemical composition. Generally, green tea has the highest proportion of water-soluble
12 ingredients and black tea has the least since it is completely fermented. Water-soluble
13 ingredients in tea include the following nine groups: polyphenols (7), alkaloids (10), proteins
14 and free amino acids (11), carbohydrates (12), pigments (13), lipids (14), minerals (15),
15 vitamins (16), and aromatic compounds (17). Among these, GABA has received more
16 attention because it acts as a relaxant and can boost immunity in humans (18).
17 Traditional Pu-erh tea is made by fermenting tea leaves at room temperature for a long
18 time, i.e. the so-called dry-stored tea. In order to hasten aging, tea is covered with a wet cloth
19 during fermentation, which is called wet-stored tea. Although wet-stored tea made by this
20 method has a shorter fermentation period, it can harbor undesirable microorganisms either
21 harmful to the body or inhibitive to the growth of beneficial microorganisms that enhance the
22 taste of tea.
23 The microorganisms that predominate in piles of Yunnan Pu-erh tea undergoing
24 fermentation are Aspergillius niger, Aspergillius gloucus, and species of Penicllium, Rhizopus,
25 Saccharomyces, and Bacterium. Among them, Aspergillius niger is the most predominant,
1 followed by Saccharomyces spp. There are very few Bacillus spp. (19, 20). Because the
2 quality of Pu-erh tea is closely related to the post-fermentation process, the present study
3 isolated some microbes from Ta-Huang-In and Ta-Hon-In, which are known for high-quality
4 Pu-erh tea. Batches of tea leaves were inoculated with individual strains of the isolated
5 microbes. At regular intervals during fermentation, and after it was complete, the tea leaves
6 were assessed in terms of the extent of free-radical scavenging activity and the content of
7 polyphenols and GΑΒΑ to find out the most effective strain for an improved
8 pile-fermentation process that not only enhances the quality of tea but also shortens
9 fermentation time and lowers production cost. The experimental design was depicted in Fig.
1 MATERIALS AND METHODS
3 Source of Pu-erh teas Two commercially available Pu-erh teas namely: Ta-Huang-In (of
4 15-year storage) and Ta-Hon-In (of 25-year storage) were used in this study. The teas were
5 supplied by Canghai Tea Shop, Taichung, Taiwan.
7 Screening and isolation of microbes from Pu-erh teas Five grams of Ta-Huang-In and
8 Ta-Hon-In were washed with 45 mL of sterilized water. The washing liquid (1 mL) was then
9 plated onto PCA agar (Plate Count Agar, Difco, France). The plates were incubated at 37°C
10 for a week (each sample was performed in triplicate). In another parallel experiment, two
11 grams of Ta-Huang-In and Ta-Hon-In were washed with sterilized water and then
12 homogenized in a blender (CF-15BS, XinGuang Mechanic, Taiwan) after adding 225 mL of
13 sterilized water. A 1 mL of each homogenized sample was incubated as before (three
14 replications). The selection of different colonies of microorganisms from both sources were
15 subcultured using inoculation loops and then incubated for a week at 37°C on NB (Nutrient
16 Broth, Difco, France). Pure cultures were obtained by subculturing one more time and
17 incubated for a week before sending them to the Food Industry Research and Development
18 Institute (FIRDI, Hsinchu, Taiwan) for identification.
20 Pile-fermentation process Three fresh tea leaves (300 g each): Yunnan (China), TTES-12
21 (Taiwan tea), C. S. Oolong (Chein Shin Oolong; Taiwan golden tea) were sterilized by hot air.
22 Subsequently, the tea leaves were inoculated with the selected isolate (104 cfu microbe per
23 gram) obtained from Pu-erh teas. The lots were fermented under controlled conditions (at
24 37°C and 70% relative humidity with partly anaerobic culture) for 180 days. The samples
25 were evaluated for scavenging activity and GABA/total polyphenols content during the
1 fermentation period. For the total plate count, one gram of fermented samples were washed
2 with 99 mL of sterilized water, and diluted with sterilized water to 103x, 104x, and 105x and
3 then 1 mL suspension was plated onto PCA agar (Plate count agar; Difco, France). The plates
4 were incubated in an incubator (Firstek Scientific, Taipei, Taiwan) at 37°C for 48 hrs (each
5 sample was replicated three times), and then was counted by using Microbial counter, the
6 selection of the microbial range was from 25-250 C.F.U. per plate.
8 DPPH-radical scavenging activity Radical scavenging activity (RSA) of tea infusions
9 against stable DPPH was determined by a slightly modified DPPH-radical scavenging assay
10 (21, 22). Samples representing the teas (pile-fermented tea samples and those sold in market,
11 i.e. Ta-Huang-In and Ta-Hon-In) were ground to powder and then passed through a 60-mesh
12 sieve. The oven-dried powder, in lots of 10, 50, 100, 150, 200, and 250 mg, was extracted
13 with 10 mL of hot water (70°C). After 5 min, the infusion was filtered through a filter paper
14 to obtain the sample liquor. A 25 µL of sample liquor diluted to 4 mL with methanol and then
15 mixed with a 0.5 mL of freshly prepared 1 mM DPPH solution. After 30 mins incubation, the
16 absorbance at 517 nm was measured. The DPPH radical scavenging activity was calculated
17 by a formula I = [(AB - AA)/AB] × 100, where I is the DPPH radical scavenging activity (in %);
18 AB is the absorbance of blank sample (t = 0 min); AA is the absorbance of sample solution (t =
19 30 mins). For the comparison, DPPH scavenging activity was also determined according to
20 previously reported (23, 24). The experiments were monitored by analytic HPLC on a model
21 PU-L2130 instrument (Hitachi, Tokyo, Japan) with a 250 × 4.6 mm i.d., 5 µm Mightsil RP-18
22 GP column (Kanto Chemical. CO., Inc.). The mobile phase was solvent A (100% MeOH) and
23 solvent B (Ultrapure water). Elution conditions were 0-55 min of 10-70% A to B (linear
24 gradient) and 55-60 min of 70-100% A to B (linear gradient) at a flow rate of 1.0 mL/min
25 using a photo diode array detector (Hitachi L-2455) at 254 nm. As for online DPPH
1 radical-scavenging analysis, the flow of DPPH reagent (50 mg/L in methanol) was set to be
2 0.5 mL/min, and the induced bleaching was detected photometrically as a negative peak at
3 517 nm.
6 Superoxide scavenging activity Superoxide radical generated by the xanthine/xanthine
7 oxidase system was determined spectrophotometrically by monitoring the product of
8 nitroblue tetrazolium (NBT). The tea infusions were mixed with 0.15 mM NADH and 0.74
9 mM NBT in sequence and then added promptly to 0.05 mM PMS to obtain a reduced form of
10 NBT. The amount of NBT in the reduction state was determined by absorbance at a
11 wavelength of 560 nm. A reduction in absorbance is therefore proportional to a sample’s
12 ability to eliminate superoxide anion oxygen free radicals (25). The scavenging activities of
13 tea infusion were measured by the following equation:
light absorption value of sample
14 light absorption value of control unit without sample × 100
Scavenging activity (%) = 1 -
16 Quantitative analysis of GABA content Tea liquor was prepared as described above with
17 200 mg of dry tea powder. The sample was derivated by the method reported elsewhere (26,
18 27) period to HPLC analysis. The tea liquor (1 mL each) was mixed with 0.6 mL of 0.1 M
19 buffer and 1 mL of 0.3% 2-hydroxy naphthaldehyde (derivatization reagent). The mixture
20 was incubated for 10 mins in a water bath maintained at 80°C and then cooled to room
21 temperature thereafter. Methanol was then added to make the final volume to 5 mL. Twenty
22 microlitres of each sample were analyzed using HPLC and a LiChrosper®100 RP18 column
23 (5 µm, 250 × 4 mm). The mobile phase comprised MeOH and H2O (62:38) with a flow rate
24 of 1.0 mL/min. The detection wavelength was 330 nm. Each sample was determined in
1 triplicate and the average value of the peak area at different values of chroma was calculated.
2 Recovery and precision were also tested to validate the method.
4 Determination of total polyphenols content Total polyphenols content was quantified
5 using the Folin-Ciocalteau colorimetric assay. Three grams of fermented tea samples were
6 extracted with 150 mL hot water (70°C). The tea infusion (50 µL) was mixed with 2 mL of
7 2% sodium carbonate solution for 2 mins. Subsequently, a 100 µL of 50% Folin-Ciocalteu
8 reagent (Sigma) was added into the mixture. After incubation at room temperature for 30
9 mins, the absorbance was measured at 750 nm. Total polyphenols were determined using a
10 gallic acid (Sigma) calibration curve and the results were expressed as gram per 100 g of
11 gallic acid equivalents.
13 Sensory evaluation Sensory evaluation was performed in the Department of Food
14 Nutrition at the HungKuang University (Taichung County, Taiwan). Tea sample was
15 extracted with 500 mL of boiling water for 5 min. In preparation for the sensory evaluation,
16 tea infusions were individually presented in covered small porcelain cups to each panelist.
17 The judges were not informed of the experimental approach and the samples were
18 blind-coded with random numbers. Panelists were instructed to smell and drink the tea
19 infusions, and pause for 30 s between samples. A 10-point hedonic scale, from 9 (excellent,
20 extremely acceptable) to 0 (poor, extremely unacceptable), was employed to evaluate each
21 tea infusion sample on taste, and flavor. The sensory attributes of tea infusion were compared
22 with those of the commercial Pu-erh products (Ta-Huang-In and Ta-Hon-In). Tea infusion
23 samples receiving overall scores of more than 4 were considered acceptable, while a score
24 between 3 and 4 was considered the borderline of acceptability. The overall acceptance of the
25 tea infusions were also evaluated by those consumers using a 10-point hedonic scale. At least
1 sixty panelists participated in each test, with about total 86-102 people participating in this
1 RESULTS AND DISCUSSION
3 Screening and isolation of microbes that enhance the taste of Pu-erh tea The
4 predominant microbes that had been isolated in traditional Pu-erh tea were: Aspergillius
5 niger, A. gloucus, and species of Penicllium, Rhizopus, Saccharomyces, and Bacterium.
6 Amongst, A. niger was present in the greatest number, followed by species of Saccharomyces
7 whereas the least population was Bacterium (19, 20). In this studied, 30 strains were obtained
8 from Ta-Huang-In and 24 strains from Ta-Hon-In in the preliminary screening. These
9 included: Penicllium, Rhizopus, Saccharomyces, Aspergillius, and Bacterium (mainly
10 actinomycete and photosynthetic bacteria). At least 15 strains of fungi, bacteria, and yeasts
11 that enhanced the quality of tea leaves were observed. The beneficial microbes isolated from
12 traditional Pu-erh tea and deployed for fermentation can enhance the flavor of Pu-erh tea
13 were subjects for the identification. Strains that led to a bitter taste, astringency, and smell of
14 grass, mold, or soil were discarded.
15 The strains were further identified by Bioresource Collection and Research Center, FIRDI,
16 as belonging to Actinoplanes and Streptomyces. These two species were used for further study.
17 Six strains namely: Streptomyces bacillaris, Actinoplanes pallidoaurantiacus, Actinoplanes
18 purpeobrunneus, Actinoplanes aurantiacus, Streptomyces cavourensis subsp. cavourensis,
19 and Streptomyces cinereus were selected for inoculated into thee tea leaves (i.e. Yun Nan,
20 TTES-12, C.S.Oolong) for 180 days. The results were compared with the commercial Pu-erh
21 products (i.e. Ta-Huang-In and Ta-Hon-In) in terms of DPPH-radical scavenging activity,
22 GABA and total polyphenol contents. Following fermentation period for 180 days, the results
23 suggested that these 6 strains could not only enhance the three tea leave’s characteristic taste
24 and flavor (see below), also exhibited a potential in the improvement of scavenging activity
25 as well as GABA contents (see below).
2 DPPH-radical scavenging activity Human cells suffer oxidation damage following
3 prolonged and constant exposure to free radicals and reactive oxygen. The damage to cell or
4 death of cells causes cancer, aging, arteriosclerosis, and diabetes (28). Tea leaf contains many
5 ingredients, such as vitamins C and E, β-carotene, catechin, and other polyphenols that can
6 eliminate the free radicals; among these ingredients, catechin shows very high antioxidation
7 capacity, which is held to be closely related to its anti-cancer and anti-tumour properties.
8 The antioxidation ability, expressed as DPPH-radical scavenging capacities, of tea
9 leaves following fermentation with different strains and for varying duration is shown in
10 Table 1. The DPPH• test is a non-enzymatic method currently used to provide basic
11 information on the ability of tea infusions to scavenge free radicals. This could be obtained
12 from the reduction of DPPH• by an antioxidant results in a loss of absorbance at 517 nm.
13 Generally, the capacity to scavenge DPPH radicals increased with the duration of
14 fermentation. The results indicated that when Yun Nan, TTES-12, and C.S.Oolong inoculated
15 with S. cinereus were observed the highest antioxidant potential, as measured by the DPPH
16 assay after 180 days cultivation, followed by tea leave inoculated with S. bacillaris, and A.
17 aurantiacus. When the A. pallidoaurantiacus, A. purpeobrunneus, or S. cavourensis subsp.
18 cavourensis was inoculated, the tea infusions exhibited the lowest scavenging capacity. Yet,
19 the scavenging activity contributed from six selected microbes was increased with the
20 fermentation time (up to 180 days) in three tea leaves studied (Table 1). These were
21 coincidence with the early studied (29).
22 Furthermore, the commercial Pu-erh teas (i.e. Ta-Huang-In and Ta-Hon-In) and
23 microbes fermented tea leaves exhibited the DPPH radical-scavenging activity in a
24 concentration-dependent manner (Fig. 2). Again, when the tea leave of Yun Nan, TTES-12,
25 and C.S.Oolong inoculated with S. cinereus were observed the highest DPPH-radical
1 scavenging activity, whereas the Ta-Huang-In shown the lowest scavenging activity at all
2 cases studied. As can be seem from Fig. 2, the Yun Nan, TTES-12, and C.S.Oolong fermented
3 with S. cinereus, S. bacillaris, or A. aurantiacus were demonstrated a better antioxidation
4 ability as compared with Ta-Huang-In and Ta-Hon-In after 180 days fermentation.
5 The typical UV and DPPH radical quenching chromatograms of the fermented samples
6 after 180 days were shown in Fig. 3. From the comparison of the chromatograms of authentic
7 compounds, the peaks with retention times at 2.54, 17.76, 20.86, 22.31, 29.03, and 37.89 min
8 in the UV chromatogram, and at 2.54, 17.77, 20.97, 22.41, 29.07, and 37.93 min in the DPPH
9 chromatogram were identified as gallic acid, chlorogenic acid, epicatechin, epigallocatechin,
10 caffeic acid, and epicatechin gallate, respectively (Fig. 3B). The chromatograms show that
11 antioxidants from tea samples can be identified and most of them did not observed in
12 non-inoculated sample (Fig. 3A). Yet, the more rapidly the absorbance decreases at 517 nm,
13 the more potent the antioxidant activity of the compound in terms of hydrogen donating
14 ability (23). Obviously, sample without S. cinereus fermented exhibited a lower decrease in
15 absorbance at 517 nm (Fig. 3A). This implied that an improved pile-fermentation process
16 employed a selected microbe into fresh tea leaves will bearing a splendid DPPH• scavenging
17 activity as compared with non-inoculated or the commercial long-term storage Pu-erh teas.
19 Superoxide scavenging activity In a non-enzymatic system, a superoxide negative radical
20 is produced when phenazine methosulfate (PMS) reacts with dihydronicotinamide-adenine
21 dinucleotide (NADH). The superoxide negative radical reacts with nitro bluetetrazolium
22 (NBT) to form a diformazan compound, which has its maximum absorption peak at 560 nm.
23 Therefore, a change in absorbance at 560 nm can be used for calculating the production of
24 superoxide negative radical. If a sample decreased in the absorbance, it indicates that the
25 sample has the capacity to capture superoxide negative radical. The values obtained by
1 inoculating the samples of rough tea with different strains and following different
2 fermentation periods are shown in Table 2. In general, the scavenging activity did not differ
3 greatly from strains or fermentation periods. Almost over 90% superoxide radical (O2·-)
4 scavenging activity was observed from 60 days fermentation thereafter, revealed that the O2·-
5 was readily scavenged by tea infusions (Table 2).
6 In fact, the scavenging of O2·- by tea infusions may be attributed to their various
7 phenolic constituents. Cotelle et al. (30) reported that flavones, a class of flavonoids, bearing
8 hydroxyl groups at positions 3′, 4′ or 3′, 4′, 5′ scavenged O2·-. These authors also reported
9 that flavonoids containing one hydroxyl group in the C-7 position of ring A could inhibit
10 generation of O2·- in the xanthine/xanthine oxidase system by inhibiting the enzyme. Phenolic
11 acids and their derivatives are also reported to be an excellent free radical-scavenger. The
12 radical-scavenging ability of phenolic acids is mediated by the number of hydroxyl groups in
13 the molecule. The electron-withdrawing property of the carboxylic acid group in benzoic acid
14 has a negative influence on hydrogen-donating ability of hydroxybenzoates (31). Therefore,
15 the high O2·- scavenging properties of tea infusion, as presented in Table 2, may also be
16 attributed to both neutralization of superoxide radicals via hydrogen donation and inhibition
17 inoculate of xanthine oxidase by various phenolic components present in the tea infusions.
19 GABA content in Pu-erh teas and fermented tea leaves GABA is one of the major
20 inhibitory neurotransmitters in the central nervous system and known to mediate pre-synaptic
21 inhibition of primary afferent fibers in the motor system and may also be involved in
22 post-synaptic forms of motor neuron inhibition (32). Amino acid neurotransmitters are critical
23 for the function of the central nervous system; they act rapidly, responding within a few
24 milliseconds, and play an important role in brain function and neurological diseases. In recent
25 studies, GABA was fed to rats through enriched soy products, was reported to exert an
1 antihypertensive effect (33). GABA occurs naturally in many kinds of foods at low levels,
2 whereas in fermented food products GABA levels were higher. GABA is used as a functional
3 food ingredient in different kinds of foods in Japan because of its health benefits (18).
4 In recent years, processed tea such as Jiayelong tea has been attracting increasing
5 attention from researchers and the industry because it was believed to be rich in GABA.
6 These products are also favored by consumers. The GABA content of Pu-erh teas and tea
7 leaves inoculated with different strains was estimated by HPLC, the results were presented in
8 Fig. 4. In the case of Yun Nan, the GABA content of samples inoculated with A.
9 pallidoaurantiacus and A. purpeobrunneus turned out to be lower than that Ta-Hon-In and
10 Ta-Huang-In. However, the GABA contents of tea leaves inoculated with S. cinereus and S.
11 bacillaris was markedly higher than that of non-fermented teas. When the TTES-12 was
12 inoculated with S. cinereus, S. bacillaris, or A. aurantiacus, the GABA content displayed
13 excellent levels (Fig. 4 middle panel). This is coincidence with the DPPH-radical scavenging
14 activity profiles (Table 1). The similar observation was found in the case of C.S.Oolong (Fig.
15 4 bottom panel). Taken account all the samples, when Yun Nan, TTES-12, or C.S.Oolong
16 inoculated with S. bacillaris the GABA contents were 712, 813, and 8632 mg/100g,
17 respectively, which were extraordinary higher than that Ta-Hon-In (291 mg/100g) and
18 Ta-Huang-In (467 mg/100g). Jeng et al. also had similar observation during 42 days
19 fermentation (29).
20 Generally, commercial tea products claiming to have higher GABA content are produced
21 using anaerobic fermentation to improve GABA content. However, in this studied, the
22 fermentation was only partly anaerobic or oxygen-limited (because it was carried out in a
23 container with the opening sealed). As a results, the GABA contents even two folds higher
24 than that Ta-Hon-In. By making the process completely anaerobic and inoculated with S.
25 bacillaris, the GABA content can be increased further so that the teas will fetch a better price.
2 Total polyphenols content Table 3 shows the total polyphenols content of three tea leaves
3 inoculated with different strains. As can be seem from this table, the total polyphenol content
4 was increased with the duration of fermentation. Although samples inoculated with S.
5 bacillaris and S. cinereus did not have the highest total polyphenols content in the initial
6 stages of fermentation, strain S. bacillaris surpassed all the rest on the 60th day and at the end
7 of fermentation (180 days). Meaning while, 104 CFU/g of S. cinereus was counted at
8 beginning of fermentation (day 0) and reached to 1.2×106 CFU/g at the end of fermentation
9 (day 180). The best strain contributed to the total polyphenol content in fermented tea
10 infusions still goes to S. cinereus. The highest total polyphenol content (8620 mg/100g) was
11 found in C.S.Oolong tea infusion fermented with S. cinereus. When TTES-12 fermented with
12 S. cinereus also shows a high polyphenol level (8125 mg/100g) follow by C.S.Oolong
13 fermented with S. bacillaris (8120 mg/100g). Thus, S. bacillaris and S. cinereus deserve
14 further research and discussion. Significantly difference in polyphenol content was also found
15 in GABA levels, consequently indicating a possible correlation between S. bacillaris and S.
16 cinereus topped the rest on DPPH radical-scavenging activity (Fig. 2) as well.
18 Sensory evaluation The effects of fermentation strains on sensory evaluation were
19 summarized in Table 4. No significant difference between 6 strains of inoculated samples and
20 commercial samples on taste, flavor and overall acceptance of tea infusions were observed.
21 Yet, the Yun Nan, TTES-12, or C.S.Oolong fermented with S. cinereus remain exhibited the
22 better results on sensations as compared to other microbes. The sensory results were even
23 better than the commercial samples. These results highlight consistent changes of DPPH
24 radical scavenging activity (Table 1) and total polyphenols content (Table 3) in response to
25 the inoculation conditions: both the sensations depend upon the microbe used and
1 fermentation duration. This results leading to the conclusion that the microbe effect on these
2 sensations were strongly dependent on the original qualitative of species incorporated.
4 Summary At least fifteen strains of microbes that could enhance the quality of tea leaves
5 as found in preliminary screening test. Six of them were isolated and identified as S.
6 bacillaris, A. pallidoaurantiacus, A. purpeobrunneus, A. aurantiacus, S. cavourensis subsp.
7 cavourensis, and S. cinereus. They were selected for inoculated into thee tea leaves (Yun Nan,
8 TTES-12, and C.S.Oolong). The tea leaves inoculated with isolated microbes were possess a
9 strong DPPH• scavenging properties which were either comparable or superior to those of
10 commercial Pu-erh teas at the same concentration. These effects may attributable to varying
11 hydrogen-donating and metal-chelating capacities of the phenolic compounds present in the
12 tea infusions.
13 Inoculation with these microbes, especially strains S. bacillaris, A. aurantiacus, and S.
14 cinereus and subsequent fermentation up to 180 days increased total polyphenols content of
15 tea leaves and their capacity to scavenge DPPH radical. In particularly, the samples
16 inoculated with S. cinereus had the highest total polyphenols content and maximum capacity
17 to scavenge DPPH radical (Tables 1 & 3); the highest GABA content was obtained while the
18 sample inoculated with S. bacillaris (Fig. 3). Further comparison of these samples with two
19 commercial Pu-erh teas of different ages (Ta-Huang-In, 15-year storage and Ta-Hon-In,
20 25-year storage) showed that DPPH-radical scavenging activity and GABA content of S.
21 bacillaris and S. cinereus fermented tea leaf were higher than that Ta-Huang-In and
22 Ta-Hon-In teas. The GABA content in inoculated samples was higher than that in tea leaves
23 produced without fermentation. The effects observed were concentration-dependent. These
24 microbes, originally isolated from traditional Pu-erh teas and utilized for unraveling the
25 microorganisms involved in tea infusions taste and antioxidant activity, have multiple effects
1 that are only now being fully appreciated. In addition to their ability to exhibit the high
2 DPPH-radical scavenging activity, these microbes also appear to have additional activities,
3 including GABA and total polyphenol contents, which continue to expand their range of
4 potential uses. The sensory evaluation results suggests that the tea leaves inoculated the
5 appropriate microbes were maintain the taste, flavor, and overall acceptance of tea infusion.
6 In summary, the fresh tea leaves inoculated with the suitable microbes in short period of time
7 will possess a high quality tea infusion as long-term storage Pu-erh tea.
4 The work supported by financial contributions of the Ministry of Education, Republic of
5 China (Taiwan) (98-F-29022), and the China Biochemical Co. Ltd. The authors also express
6 their thanks to Canghai tea shop for providing different kinds of commercial Pu-erh tea.
3 1. Cabrera, C., Artacho, R., and Gimenez, R.: Beneficial effects of green tea--a review.
4 J. Am. Coll. Nutr., 25, 79-99 (2006).
5 2. Duh, P. D., Yen, G. C., Yen, W. J., Wang, B. S., and Chang, L. W.: Effects of Pu-erh
6 tea on oxidative damage and nitric oxide scavenging. J. Agric. Food Chem., 52,
7 8169-8176 (2004).
8 3. Jie, G., Lin, Z., Zhang, L., Lv, H., He, P., and Zhao, B.: Free radical scavenging
9 effect of Pu-erh tea extracts and their protective effect on oxidative damage in human
10 fibroblast cells. J. Agric. Food Chem., 54, 8058-8064 (2006).
11 4. Wu, S. C., Yen, G. C., Wang, B. S., Chiu, C. K., Yen, W. J., Chang, L. W., and Duh,
12 P. D.: Antimutagenic and antimicrobial activities of pu-erh tea. LWT, 40, 506-512
14 5. Hayakawa, S., Kimura, T., Saeki, K., Koyama, Y., Aoyagi, Y., Noro, T., Nakamura,
15 Y., and Isemura, M.: Apoptosis-inducing activity of high molecular weight fractions
16 of tea extracts. Biosci. Biotechnol. Biochem., 65, 459-462 (2001).
17 6. Chiang, C. T., Weng, M. S., Lin-Shiau, S. Y., Kuo, K. L., Tsai, Y. J., and Lin, J. K.:
18 Pu-erh tea supplementation suppresses fatty acid synthase expression in the rat liver
19 through downregulating Akt and JNK signalings as demonstrated in human hepatoma
20 HepG2 cells. Oncol. Res., 16, 119-128 (2005).
21 7. Yang, D. J., and Hwang, L. S.: Study on the conversion of three natural statins from
22 lactone forms to their corresponding hydroxyl acid forms and their determination in
23 Pu-erh tea. J. Chromatogr. A, 1119, 277-284 (2006).
24 8. Anderson, R. A., and Polansky, M. M.: Tea enhances insulin activity. J. Agric. Food
25 Chem., 50, 7182-7186 (2002).
1 9. Weisburger, J. H.: Tea and health: the underlying mechanisms. Proc. Natl. Acad. Sci.
2 USA, 220, 271-275 (1999).
3 10. McGaw, L. J., Steenkamp, V., and Eloff, J. N.: Evaluation of Athrixia bush tea for
4 cytotoxicity, antioxidant activity, caffeine content and presence of pyrrolizidine
5 alkaloids. J. Ethnopharm., 110, 66-22 (2007).
6 11. Kakuda, T.: Neuroprotective effects of the green tea components theanine and
7 catechins. Biol. Pharm. Bull., 25, 1513-1518 (2002).
8 12. Cipriani, T. R., Mellinger, C. G., de Souza, L. M., Baggio, C. H., Freitas, C. S.,
9 Marques, M. C., Gorin, P. A., Sassaki, G. L., and Iacomini, M.: A polysaccharide
10 from a tea (infusion) of Maytenus ilicifolia leaves with anti-ulcer protective effects. J.
11 Nat. Prod., 69, 1018-2101 (2006).
12 13. Tanaka, T., Matsuo, Y., and Kouno, I.: A novel black tea pigment and two new
13 oxidation products of epigallocatechin-3-O-gallate. J. Agric. Food Chem., 53,
14 7571-7578 (2005).
15 14. Hartford, O. and Zug, K. A.: Tea tree oil. Cutis, 76, 178-180 (2005).
16 15. Ozcan, M.: Determination of mineral contents of Turkish herbal tea (Salvia aucheri
17 var. canescens) at different infusion periods. J. Med. Food, 8, 110-112 (2005).
18 16. Kittaka-Katsura, H., Watanabe, F., and Nakano, Y.: Occurrence of vitamin B12 in
19 green, blue, red, and black tea leaves. J. Nutr. Sci. Vitaminol., 50, 438-440 (2004).
20 17. Yao, S. S., Guo, W. F., Lu, Y., and Jiang, Y. X.: Flavor characteristics of lapsang
21 souchong and smoked lapsang souchong, a special Chinese black tea with pine smoking
22 process. J. Agric. Food Chem., 53, 8688-8693 (2005).
23 18. Abdou, A. M., Higashiguchi, S., Horie, K., Kim, M., Hatta, H., and Yokogoshi, H.:
24 Relaxation and immunity enhancement effects of gamma-aminobutyric acid (GABA)
25 administration in humans. Biofactors, 26, 201-208 (2006).
1 19. Zhao, L. F., and Zhou, H. J.: Study on the main microbes of Yunnan Puer tea during
2 pile-fermentation process. J. Shangqiu Teach. Coll., 21, 129-133 (2005). (in Chinese)
3 20. Zhao, L. F., Xu, Y. J., and Zhou, H. J.: Research on microbes improve quality and
4 flavor of pu’er tea in solid fermentation. Food Res. Develop., 27, 155-156 (2006). (in
6 21. Cuendet, M., Hostettmann, K., and Potterat, O.: Iridoid glucosides with free radical
7 scavenging properties from Fagraea blumei. Hel. Chim. Acta., 80, 1144-1152 (1997).
8 22. Zhu, Q. Y., Hackman, R. M., Ensunsa, J. L., Holt, R. R., and Keen C. L.:
9 Antioxidative activities of oolong tea. J. Agric. Food Chem., 50, 6929-6934 (2002).
10 23. Bandonieneú, D., and Murkovic, M.: On-Line HPLC-DPPH screening method for
11 evaluation of radical scavenging phenols extracted from apples (Malus domestica L.). J.
12 Agric. Food Chem., 50, 2482-2487 (2002).
13 24. Wu, J. H., Huang, C. Y., Tung, Y. T., and Chang, S. T.: Online RP-HPLC-DPPH
14 Screening method for detection of radical-scavenging phytochemicals from flowers of
15 Acacia confuse. J. Agric. Food Chem., 56, 328-332 (2008).
16 25. Robak, J., and Gryglewski, R. J.: Flavonoids are scavengers of superoxide anions.
17 Biochem. Pharmacol., 37, 837-841 (1988).
18 26. Mengerink, Y., Kutlan, D., Toth, F., Csampai, A., and Molnar-Perl, I.: Advances in
19 the evaluation of the stability and characteristics of the amino acid and amine
20 derivatives obtained with the o-phthaldialdehyde/3-mercaptopropionic acid and
21 o-phthaldialdehyde/N-acetyl-L-cysteine reagents. High-performance liquid
22 chromatography-mass spectrometry study. J. Chromatogr. A, 949, 99-124 (2002).
23 27. Zhang, G., and Bown, A. W.: The rapid determination of γ-aminobutyric acid.
24 Phytochem., 44, 1007-1009 (1997).
25 28. Cadenas, E.: Biochemistry of oxygen toxicity. Annu. Rev. Biochem., 58, 79-110
2 29. Jeng, K-C., Chen, C-S., Fang, Y-P., Hou, R. C-W., and Chen, Y-S.: Effect of
3 microbial fermentation on content of statin, GABA, and polyphenols in pu-erh tea. J.
4 Agric. Food Chem. 55, 8787-8792 (2007).
5 30. Cotelle, N., Bernier, J. L., Catteau, J. P., Pommery, J., Wallet, J. C., and Gaydou,
6 E. M.: Antioxidant properties of hydroxy-flavones. Free Radical Biol. Med., 20, 35-43
8 31. Wettasinghe, M., and Shahidi, F.: Scavenging of reactive-oxygen species and DPPH
9 free radicals by extracts of borage and evening primrose meals. Food Chem., 70, 17-26
11 32. Curtis, D. R., and Lacey, G.: GABA-B receptor-mediated spinal inhibition. Neuro.
12 Report, 5, 540-542 (1994).
13 33. Shizuka, F., Kido, Y., Nakazawa, T., Kitajima, H., Aizawa, C., and Kayamura, H.,
14 and Ichijo, N.: Antihypertensive effect of gamma-amino butyric acid enriched soy
15 products in spontaneously hypertensive rats. Biofactors, 22, 165-167 (2004).
2 Figure Captions
4 FIG. 1. The experimental flow chart of this study.
5 FIG. 2. Concentration dependent of DPPH radical-scavenging activity of tea infusions on
6 commercial Pu-erh teas and tea leaves of Yun Nan (A), TTES-12 (B), C.S.Oolong
7 (C) inoculated with isolated microbes and fermented with an improved process.
8 DPPH radical-scavenging activity for fermented tea leaves was measured after 180
9 days cultivated with isolated microbes. Sample preparation and DPPH analysis see
10 materials and methods section for detail.
11 FIG. 3. The typical UV and DPPH radical quenching chromatograms of tea sample without
12 S. cinereus inoculated (A) and with S. cinereus inoculated (B) after 180 days. The
13 quoted number in each peak represents: 1. Gallic acid (GA), 2. Gallocatechin (GC),
14 3. Chlorogenic acid (CHA), 4. Epicatechin (EC), 5. Epigallocatechin (EGC), 6.
15 Caffeic acid (CA), 7. Epicatechin gallate (ECG).
17 FIG. 4. Comparison of GABA content on commercial Pu-erh teas and tea leaves of Yun Nan
18 (top), TTES-12 (middle), C.S.Oolong (bottom) inoculated with isolated microbes
19 and fermented with an improved process.
20 GABA content for fermented tea leaves was measured after 180 days cultivated
21 with isolated microbes. Each bar represents the mean value of three determinations
22 standard deviation.