**4. The characteristics of Chungtaejeon**

The post-fermented tea has softer taste, an increased anti-bacterial effect, antioxidant activity, and lower cholesterol production effects by fermentation of microorganisms due to higher amounts of gallic acid, methoxy phenolic compound, and polyflavonoids [2–7]. The Chungtaejeon changes to blue color during the fermentation process (**Figure 1**).

The raw tea leaves of wild *Camellia sinensis* are dried overnight in a well-ventilated room. The steaming process is carried out for about 15 minutes after removing impurities in the leaves. The steamed tea leaves are pulverized with a motor. After pulverization, the coin-shaped tea balls dry for 2–3 days in a well-ventilated room. The center of coin-shaped tea ball is punched by bamboo stem and connected by a thread to dry it in the sun, ondol room, or shade. Dried Chungtaejeon is kept in a pot to prevent loss of aroma and to avoid moisture. We analyzed bacterial community from raw leaves to products of a 1-year fermented tea (**Figure 2**).

#### **Figure 2.**

*Flow diagram of PCR-DG-DGGE (double gradient-denaturing gradient gel electrophoresis) analysis for microbial communities.*



*Bioactive Compounds in Nutraceutical and Functional Food for Good Human Health*

#### *The Characteristics of Korean Traditional Post-Fermented Tea (Chungtaejeon) DOI: http://dx.doi.org/10.5772/intechopen.91855*

DNA extractions were carried out in accordance with eukaryotic microalgal nucleic acids extration (EMNE) method. The bacterial universal primers 27F (5′-GAG TTT GAT CMT GGC TCA G-3′) and 518R (5′-WTT ACC GCG GCT GCT GG-3′) were used to amplify the 16S rRNA genes for NGS analysis. The primer sequences and PCR conditions for Roche 454 are described in Pitta et al. [8]. After the PCR reaction, purification was carried out using PCR purification kit (Biofact, Daejeon, Korea). NGS was performed with a Roche 454 GS-FLX plus (454 Life Sciences) according to the procedure described by Galan et al. [9]. To compare modified methods with traditional ones for the overall bacterial communities of the Chungtaejeon, operational taxon unit (OTU)-based and phylogeny-based analyses were performed.

Catechins, gallic acid and caffeine contents were analyzed by HPLC (Agilent 1216 Infinity LC) using column (ZORBAX Eclipse plus C18, 4.6 × 250 mm).

We studied the structure of the fermentation bacterial community of Korean traditional post-fermented tea that was investigated by metagenome analysis using 16S rRNA gene during manufacturing (**Table 1**). The Acidobacteria was changed

#### **Figure 3.**

*Phylogenetic tree on 16S rRNA gene sequence using the neighbor-joining methods showing the Korean traditional post-fermented tea (Chungtaejeon). \*: 16S rRNA gene clone library, \*\*:\*\*: DG-DGGE (double gradient-denaturing gradient gel electrophoresis).*

#### **Figure 4.**

*Dominant bacterial OTUs detected in the Korean traditional post-fermented tea (Chungtaejeon) 16S rRNA gene clone libraries.*

from 42.86% before steaming raw leaves to 7.55% after steaming and then ranges from 2.44% after crushing to 66.6% (9 day). The alpha-probacteria range from 1.89 to 32.1%. The gamma-probacteria increased from 25.0% before steaming raw leaves to 97.6% after crushing and then from 33.3 to 66.6% during forming and fermentation by manufacturing process. Therefore, dominant bacteria during manufacturing were gamma-probacteria for Chungtaejeon. The microbial community size was the largest for Chungtaejeon compared with other teas [10, 11]. Also, phylogenetic analysis suggested that the taxonomic affiliation of the dominant species in the post-fermented tea was gamma-proteobacteria (**Figure 3**) for fermentation [10]. Our results were similar to the report of Kim et al. [10, 11] during manufacturing of Korean traditional post-fermented tea (**Table 1**).

However, the structure of the bacterial community involved in the production of oriental traditional post-fermented tea (Chungtaejeon) was investigated using 16S rRNA gene analysis (**Figure 2**). The main microorganisms in fermentation process of Chungtaejeon (**Figure 1**) are identified as *Pantoea sp*. and *Klebsiella oxytoca*, respectively (**Figures 3** and **4**). Phylogenetic analysis suggested that the taxonomic affiliation of the dominant species in the Chungtaejeon was γ-proteobacteria (**Figure 3**). Comparison of bacterial (A), fungal (B), and archaeal (C) community

#### **Figure 5.**

*Comparison of bacterial (a), fungal (B), and archaeal (C) community size using the real-time PCR from the various oriental post-fermented tea (Chungtaejeon). A, D and Puer1 ~ 4 were post-fermented tea for Korea and China, respectively.*


*The Characteristics of Korean Traditional Post-Fermented Tea (Chungtaejeon) DOI: http://dx.doi.org/10.5772/intechopen.91855*

#### **Table 2.**

*Dominant microorganism different of oriental post-fermented tea by metagenome analysis (%).*

size using the real-time PCR from the various oriental post-fermented tea (Chungtaejeon), other Korean post-fermented tea and puer-tea from China is shown in **Figure 5**. The bacterial community size was the highest for Chungtaejeon. It is higher about 100 times in Chungtaejeon (2.65 × 108 ± 1.35 × 108 copy/ul) compared with other Korean tea and puer-tea. Also, the fungal community size was higher about seven times in the Chungtaejeon than in the other post-fermented teas. However, the archaeal community size was highest about six times in the Chungtaejeon. Therefore, the bacteria, fungal and archaeal community size were highest for Chungtaejeon in the seven post-fermented teas. As a result, the microbial communities of Chungtaejeon were the largest compared with other teas.

Also, cluster analysis confirmed that microbial population present in both Korean and Chinese post-fermented teas groups into the same class [11]. The dominant microorganism present in Korean post-fermented tea was bacterium, while for the Chinese post-fermented tea, it was fungus [5–7, 11]. However, dominant microorganism different from oriental post-fermented tea by metagenome analysis was a acidobacteria and alpha-proteobacteria for Puer 1 and Puer 2, firmicutes for Awabancha and gamma-proteobacteria for Chungtaejeon (**Table 2**).

Tea is classified as green tea, semi-oxidation tea, oxidation tea and post-fermented tea, depending on its manufacturing methods. Post-fermented tea has softer taste, an increased anti-bacterial effect, antioxidant activity, and lower cholesterol production effects by fermentation of microorganisms due to higher amounts of gallic acid. The gallic acid content was 1.67 mg/g for green tea and 21.98 mg/g for puer-tea [4, 12–15].
