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

 2   Improved Pile-Fermentation Process

 3

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

 5

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

 7
     1
 8       Department of Food Nutrition, HungKuang University, Taichung, Taiwan 433, R.O.C.
     2
 9       Department of Applied Chemistry, Chaoyang University of Technology, Taichung, Taiwan

10       413, R.O.C.
     3
11       Department of Biotechnology, National Formosa University, Yunlin, Taiwan 632, R.O.C.

12

13

14
         *
15           Corresponding author. e-mail: chenys@sunrise.hk.edu.tw

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

17

18




                                                    1
 1

 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.

20

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

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

23




                                                     2
 1

 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

17   colds.

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).




                                                    3
 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,


                                                   4
 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.

10   1.




                                                 5
 1                                 MATERIALS AND METHODS

 2

 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.

 6

 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.

19

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


                                                  6
 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.

 7

 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


                                                 7
 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.

 4

 5

 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 -                                                    
                                                                                        

15

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


                                                 8
 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.

 3

 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.

12

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


                                                  9
1   sixty panelists participated in each test, with about total 86-102 people participating in this

2   study.




                                                10
 1                                   RESULTS AND DISCUSSION

 2

 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).


                                                   11
 1

 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


                                                   12
 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.

18

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


                                                13
 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.

18
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


                                                  14
 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.


                                                  15
 1

 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.

17

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


                                                  16
 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.

 3

 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


                                                   17
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.




                                                 18
1

2                                   ACKNOWLEDGEMENTS

3

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.




                                                19
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                                                 23
 1
 2                                          Figure Captions

 3

 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).

16

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.

23

24

25



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