Relationship between the Physiological Activity of Japanese Post-Fermented Teas and Lactic Acid Bacteria

: Post-fermented tea is a beverage or food made by fermenting tea leaves with microorganisms. Four types of post-fermented tea are traditionally produced in Japan. Three of these post-fermented teas are produced by lactic acid fermentation in the Shikoku region. Post-fermented tea has physiological activities such as antioxidant, antiallergic, and fat accumulation inhibitory effects. The composition of catechins in post-fermented tea differs from that in green tea. Compared to green tea, epigallocatechin, epigallocatechin gallate, epicatechin


Introduction
Post-fermented tea is produced by fermenting tea leaves with microorganisms.The regions wherein post-fermented tea is traditionally manufactured are limited to Southeast Asia and Japan.In Southeast Asia, they are produced in northern Thailand (e.g., Nan Province, Lampang Province, Chiang Mai Province) and northern Laos, Myanmar, and Yunnan in China.In Japan, they are manufactured in the Shikoku and Toyama prefectures.The type of post-fermented tea depends on the region in which it is manufactured.Famous examples of post-fermented teas in Southeast Asia include Miang tea from Thailand, Lahpet-so tea from Myanmar, and Pu'er tea from China.Examples of post-fermented teas in Japan are Ishizuchi-kurocha (Ehime), Goishi-cha (Kochi), Awa-bancha (Tokushima) in Shikoku, and Batabata-cha (Toyama).
These post-fermented teas are produced in different ways.Batabata-cha from Toyama is produced by fermentation with fungi belonging to the genus Aspergillus [1].The production of the other three Japanese post-fermented teas from Shikoku involve lactate fermentation by lactic acid bacteria [2].The production method common to several post-fermented teas is heating the tea leaves by steaming or boiling them to sterilize and inactivate the enzymes and then growing microorganisms in the tea leaves to change the tea leaf components.For lactic acid fermentation, tea leaves are packed in plastic bags or wooden buckets, and the fermentation process is performed under anaerobic conditions while shutting off Fermentation 2023, 9, 876 2 of 13 the air.In aerobic fermentation, tea leaves are placed in wooden boxes or chambers to allow for molds to grow.Two types of Japanese post-fermented teas, Ishizuchi-kurocha and Goishi-cha, are produced by a two-step fermentation process, in which mold is grown and lactic acid fermentation is performed.One of the major differences between post-fermented tea made in Southeast Asia, Thailand, Laos, and Myanmar and Japanese post-fermented tea lies in sun-drying as the final process.In Japanese post-fermented tea, the fermented tea leaves are dried in the sun.The dried tea leaves are extracted using hot or cold water and drinks, such as green tea or black tea.By contrast, Southeast Asian post-fermented tea is produced without drying the leaves.In Japanese post-fermented tea, changes in the components of tea leaves caused by microorganisms cease during the drying process [2].Conversely, in the case of the post-fermented tea from Southeast Asia, the composition of tea leaves continues to change during distribution.
Traditional production methods do not usually include microorganisms as starters.In any post-fermented tea, raw tea leaves are heated once.During this process, the enzymes in the tea leaves are deactivated, and enzymatic oxidation does not occur in the subsequent steps.In this respect, the process of component change in post-fermented tea differs from that in black and oolong teas.Microorganisms are involved in changes in the components of the post-fermented tea.The components of the tea leaves change owing to the metabolism of the microorganisms.Therefore, the chemical components of post-fermented tea differ from those of nonfermented tea, such as green tea, and fermented tea, such as black tea, by enzymatic oxidation.Post-fermented tea also contains substances that exhibit physiological activities that benefit human health, although the causative agent is unknown.
Microorganisms that contribute to the fermentation of post-fermented tea are derived from the environment; however, because they are sterilized by heating, the contribution of microorganisms adhering to tea leaves is small.However, how microorganisms, particularly lactic acid bacteria, invade and grow during fermentation is still unclear.According to the results of the bacterial flora analysis of Japanese Ishizuchi-kurocha, Lactiplantibacillus plantarum is the dominant species in the bacterial flora of tea leaves after lactic acid fermentation [3].
This paper summarizes the physiological activities reported to benefit human health in post-fermented teas worldwide.In addition, we summarized the characteristic chemical components that exhibit physiological activity and considered the contribution of lactic acid bacteria when producing these components.Pu'er tea and batata tea, the production of which do not involve lactic acid fermentation, were not covered in this study.

Production Method of Japanese Post-Fermented Teas
There are four types of post-fermented tea that have been traditionally produced in Japan since at least the 19th century.The production methods for these Japanese postfermented teas are different from each other.An outline of the production method is shown in Figure 1.The names of these four types of Japanese post-fermented tea are based on the production region and production method; they are not defined by their components.Each post-fermented tea is produced by multiple producers.Therefore, the microbial flora, as well as the components, vary during fermentation depending on the producer.As a result, like wine, the flavor varies depending on the producer, year of production, or production lot.The production methods of Awa-bancha and Ishizuchi-kurocha were designated as Intangible Cultural Heritage of Japan in 2021 and 2023, respectively, and research on the production method of these post-fermented teas has since been conducted.
Awa-bancha is a post-fermented tea produced in Tokushima, Shikoku Island of Japan.It is mostly produced in Naka and Kamikatsu in the southwestern Tokushima.In addition, in recent years it has also been produced in Yamashiro, Miyoshi, in the western Tokushima.The tea leaves are harvested in early summer, boiled, and then kneaded with a tea roller.After that, the tea leaves are placed in a plastic or wooden barrel, weighted with stones or blocks, and lactic acid fermentation is carried out anaerobically.After fermentation, the product is dried by solar drying.mented teas in Southeast Asia, and it is the only method employed for the production of Ishizuchi-kurocha and Goishi-cha worldwide.
Batabata-cha, produced in Toyama in the Hokuriku District, Japan, is made by steaming the tea leaves and then stacking them to create a chamber for aerobic fermentation.Every 2 to 3 days, the tea leaves are mixed 10 to 15 times.This process is called "Kirikaeshi".And after 20 to 30 days of aerobic fermentation, they are dried by solar drying to then become the final product.In the production process of these four types of Japanese post-fermented tea, fungi of the genus Aspergillus and lactic acid bacteria of the genus Lactiplantibacillus play a major role in aerobic and anaerobic fermentation, respectively, as a dominant species.

Chemical Composition of Post-Fermented Tea
The chemical composition of post-fermented tea differs from that of green tea.Postfermented tea also contains caffeine and catechins, which are the major chemicals found Ishizuchi-kurocha is a post-fermented tea produced in Saijo, Ehime, on the Shikoku Island.In early summer, the tea leaves are harvested with the branches and steamed in a steamer for about an hour.The leaves are then placed in a wooden box and subjected to primary fermentation (aerobic fermentation) for several days.After primary fermentation, the tea leaves undergo kneading, are packed into plastic buckets or wooden buckets, weighted with stones, and subjected to lactic acid fermentation under anaerobic conditions for one to several weeks.After fermentation, the product is dried by solar drying.
Goishi-cha, produced in Kochi on Shikoku Island, is made using a method similar to Ishizuchi-kurocha, but without the kneading process.Tea leaves are harvested in early summer, steamed, and then the tea leaves are fermented by aerobic conditions.After that, the tea leaves are stacked and lactic acid fermentation is performed under anaerobic conditions.After fermentation, the product is dried by solar drying.Two-step fermentation with aerobic fermentation and subsequent anaerobic fermentation is not seen in postfermented teas in Southeast Asia, and it is the only method employed for the production of Ishizuchi-kurocha and Goishi-cha worldwide.
Batabata-cha, produced in Toyama in the Hokuriku District, Japan, is made by steaming the tea leaves and then stacking them to create a chamber for aerobic fermentation.Every 2 to 3 days, the tea leaves are mixed 10 to 15 times.This process is called "Kirikaeshi".And after 20 to 30 days of aerobic fermentation, they are dried by solar drying to then become the final product.
In the production process of these four types of Japanese post-fermented tea, fungi of the genus Aspergillus and lactic acid bacteria of the genus Lactiplantibacillus play a major role in aerobic and anaerobic fermentation, respectively, as a dominant species.

Chemical Composition of Post-Fermented Tea
The chemical composition of post-fermented tea differs from that of green tea.Postfermented tea also contains caffeine and catechins, which are the major chemicals found in tea leaves.Table 1 summarizes the caffeine and catechin contents of Japanese postfermented teas reported to date.The caffeine content is lower than that of green tea.While green tea uses young leaves in early summer from May to June, post-fermented tea uses hardened tea leaves for fermentation from July to August.The caffeine content of post-fermented tea is lower than that of green tea.The caffeine content in Ishizuchi-kurocha decreases after steaming.In one manufacturing lot of Ishizuchi-kurocha, the amount of caffeine in raw tea leaves was 6.5 g/100 g, while it was 2.7 g/100 g after steaming and 3.5 g/100 g after primary fermentation and 3.3 g/100 g after secondary fermentation [2].Caffeine is derived from raw tea leaves, as no decrease in caffeine is observed even after fermentation compared to tea leaves before fermentation.In addition, the tea leaves used as raw materials for Awa-bancha are harvested ~2 m later than green tea; therefore, the amount of caffeine in the leaves may change.It may also depend on cultivation methods such as soil type and fertilization.Nevertheless, the contribution of microorganisms to caffeine levels is likely to be low.
The content of catechin, a typical polyphenol in tea, is generally lower in postfermented tea than in green tea.In particular, the epicatechin (EC) and epicatechin gallate (ECg) contents, which are abundant in green tea, are low, and Ishizuchi-kurocha contains nearly no ECg.The catechin content was reduced by fermentation.In one lot of Ishizuchi-kurocha, the total catechin content of fresh tea leaves before fermentation was 9.6 g/100 g, whereas the contents after steaming, primary fermentation, and secondary fermentation were 6.2 (64.6% of raw tea leaves), 4.6 (47.9%), and 3.8 g/100 g (39.6%), respectively.In particular, the EC content was 0.98 g/100 g in raw tea leaves but 0.41 g/100 g after steaming and was not detected after primary fermentation.The ECg content was 2.4, 0.56, and 0.19 g/100 g after steaming, primary fermentation, and secondary fermentation, respectively, compared to 4.7 g/100 g in raw tea leaves [4].In one lot of Awa-bancha, the total amount of catechins in raw tea leaves was 2969.2 mg/100 g whereas, after lactic acid fermentation, it was 1838.6 mg/100 g [5].In Awa-bancha, the total amount of catechins in tea leaves after fermentation decreased to ~55-84% compared to that before fermentation [5].By contrast, the EGC and EGCg levels increased after fermentation in some Awa-bancha producers.In addition, pyrogallol is present in Ishizuchi-kurocha, Goishi-cha, and Awa-bancha [6].
Post-fermented tea also contains amino acids.Ishizuchi-kurocha contains ~4 g/100 g amino acids.It has high Thr and Ser contents, accounting for ~42% and ~31% of the total amino acid mass, respectively [4].In addition, the Glu (3.8%), Ala (2.3%), Val (2.1%), and Leu (3.9%) contents are relatively high.By contrast, Aps, Ala, Glu, and Ser are predominant in other reports [7,8].Some studies reported the theanine content to be high [7].In Awabancha, the total amino acid content varied depending on the producer, ranging from 75.6 to 980.6 mg/100 g [5].It decreased after fermentation compared to that in raw tea leaves.The total amino acid content of Awa-bancha was as high as 80% of that before fermentation and as low as 4%.Ser, Ala, Glu, and Leu were relatively abundant in Awa Bancha.In contrast to Ishizuchi-kurocha, Thr is not present.
The amino acids in Ishizuchi-kurocha include not only the L form but also the D form.D-Ala was the most abundant amino acid, followed by D-Glu.D-Asp was not present in some production lots [8].D-Ser acts in the brain and is involved in memory and learning [9].D-Ser also improves kidney function [10].D-Ala, D-Ser, and D-Arg inhibit lipid accumulation in hepatocytes [11].The bioactivity of D-amino acids may also be present in post-fermented teas, including lactic acid fermentation, such as Ishizuchi-kurocha.
Regarding organic acids, Ishizuchi-kurocha contains the most lactic acid at 2135 mg/ 100 g, oxalic acid at 1094 mg/mL, succinic acid, and tartaric acid [5].In Awa-bancha, the amount of lactic acid was the highest, at ~600-5200 mg/100 g.Acetic acid, oxalic acid, and sometimes succinic acid are included [5].The content was converted to mg/100 g DW of leaves from the values in each report.n.d.: not detected or traced.n.t.: not tested.All data were obtained for post-fermented teas from different production years, production lots, or producers.

Physiological Activity of Post-Fermented Tea
Post-fermented tea exhibits various physiological activities.Like green tea, postfermented tea also shows antioxidant activity.Catechins are important components responsible for the antioxidant activity of green tea [17].In post-fermented tea, these catechins decrease in tea leaves after fermentation.In post-fermented tea, which includes lactic acid fermentation, the strength of antioxidant activity is inferior to that of green tea, but antioxidant activity is maintained compared to the reduction in catechins [12].In black tea, theaflavin is produced by the polymerization of catechins and exhibits antioxidant activity [18].Polymers of catechins, such as Tea Brown, may be involved in the antioxidant activity of post-fermented tea.It exhibits O 2− scavenging and radical-scavenging activities [15].The antioxidant activity of Goishi-cha is comparable to that of green tea and significantly higher than that of black tea and oolong tea [19].However, the catechin content of Goishi-cha is lower than that of green tea.The antioxidant activity of post-fermented tea is mainly related to substances different from those in green tea and black tea.One of the main components of Goishi-cha is pyrogallol [6].The contribution of catechins to the antioxidant activity of Goishi-cha was ~5%, whereas that of pyrogallol was 28.2%, as suggested in [20].By contrast, Batabata-cha showed hardly any antioxidant activity [12].Compared to other post-fermented teas, Batabata-cha exhibits a lower catechin content [4], but whether it is converted to brown tea is unknown.In addition, batata tea has no antioxidant activity; the reason for this is also unclear.
Ishizuchi-kurocha exerts antiallergic effects.Ishizuchi-kurocha extract inhibits the IgE-mediated degradation of RBL-2H3 cells.Ishizuchi-kurocha also suppresses allergic reactions in mice experiments [21].Theabrownins contained in Ishizuchi-kurocha are involved in this effect.Theabrownins are produced by the polymerization of catechins, although their chemical structure is unclear.The polymerization of catechins generally involves the oxidases present in tea leaves.This reaction is well-known in black and oolong teas.Theaflavins are responsible for the antioxidant activity of black tea.
Goishi-cha effectively prevents hyperlipidemia and arteriosclerosis [22].In an experiment in which cholesterol-loaded rabbits orally ingested Goishi-cha, the serum LDL cholesterol and lipid peroxide levels in the Goishi-cha group were significantly lower than those in the green tea and tap water groups at 9-10 w.In contrast, no significant differences were observed in HDL cholesterol and triglyceride levels [19].At this time, the fat-stained area ratio in the aorta was lower than that in the tap water group, and foam cell accumulation was mild.We speculate that this effect is related to the antioxidant activity of Goishi-cha.Ishizuchi-kurocha also inhibits fat accumulation in 3T3-L1 cells [4].
In addition, in an experiment in which C57BL/6J mice were provided a high-fat diet and orally ingested Goishi-cha, the Goishi-cha group showed an inhibitory effect on increases in cholesterol, TNF-α, and IL-6 compared to the water-administered group.In addition, the decrease in adiponectin levels was suppressed.Although no significant difference was observed in visceral fat mass compared to the water-administered group, the inflammatory adipocytokines may be suppressed by reducing oxidative stress [22].

Lactic Acid Bacteria in Japanese Post-Fermented Tea
Traditional post-fermented tea production does not include lactic acid bacteria as starters.Lactic acid bacteria, which are responsible for lactic acid fermentation, enter the production site and multiply by making the tea leaves anaerobic.The temperature during lactic acid fermentation is ~15-30 • C throughout the year in Southeast Asia, where rice is grown all year round.In Japan, it is made in the summer, and the temperature is ~25-30 • C. In post-fermented tea, which is manufactured using a mold-based process, the inside of the tea leaves rises to ~40 • C owing to the fermentation heat caused by the mold; therefore, a stable temperature environment can be obtained.The lactic acid bacteria begin to proliferate during fermentation by fungi and then proliferate in an anaerobic environment.Lactic acid bacteria grow under anaerobic conditions at different temperatures.In Japan, postfermented tea is not produced in winter when the temperature is low because lactic acid bacteria cannot proliferate.The lactic acid bacteria involved in the lactic acid fermentation of post-fermented tea originate from the environment of the manufacturing site, but where they usually live and how they enter tea leaves remains unknown.Table 2 shows the strains belonging to the Lactobacillaceae family reported to date from post-fermented tea containing lactic acid.The most commonly isolated lactic acid bacteria are Lactiplantibacillus plantarum, regardless of the production location and method of post-fermented tea.Among these, L. plantarum is the most commonly isolated species.In several post-fermented teas, L. plantarum is the dominant species.In addition, Lactiplantibacillus pentosus has often been isolated.Interestingly, the dominant Awa-bancha species differed depending on the production area [23].L. pentosus is the dominant species in Awa-bancha, produced in the southwestern part of Tokushima Prefecture (Kamikatsu/Naga).By contrast, L. plantarum dominates in the Awa-bancha produced in the eastern part (Miyoshi).Nearly all the Ishizuchi-kurocha isolates were L. plantarum [4,24].These two strains are isolated every year from post-fermented tea in Japan, regardless of the year of manufacture.In addition, in the same year, it was isolated as the dominant species regardless of the production lot.Levilactobacillus brevis was isolated from Ishizuchi-kurocha.In Ishizuchi-kurocha, L. plantarum accounts for ~80-100% of the lactic acid bacteria, and L. brevis accounts for ~0-20% [3].L. brevis was not detected in any of the production lots.L. plantarum is rarely found in Ishizuchi-kurocha or Awa-bancha in Miyoshi.In addition, L. brevis is rarely found in Awa-bancha in Kamikatsu or Naka [23].Interestingly, other major species of L. plantarum, such as L. paraplantarum, are rarely found.The lactic acid bacteria flora is regionally dependent, regardless of the year of production.The reason for which the dominant species of lactic acid bacteria differ depending on the production area remains unknown.Moreover, it had the same bacterial flora without adding lactic acid bacteria every year.L. plantarum promotes the production and growth of lactic acid by promoting the decarboxylation of malic acid in the presence of catechin [25].The promotion of lactic acid production implies that the pH decreases rapidly.Catechins exhibit antibacterial activity against some pathogenic bacteria; however, their antibacterial activities are extremely low against lactic acid bacteria [25,26].Among the lactic acid bacteria, the animal-derived lactic acid bacteria such as Lactobacillus acidophilus and Limosilactobacillus vaginalis did not grow in grape seed extracts rich in catechins and epicatechins, whereas the growth of L. plantarum was affected [27].Thus, L. plantarum grows preferentially during fermentation, and polyphenol metabolism occurs.However, the details of how lactic acid bacteria are selected and what factors are important for selective pressure remain unclear.

Contribution of Lactic Acid Bacteria to Bioactivity
The physiological activity observed in Japanese post-fermented tea is owed to the direct effects of the lactic acid bacteria, such as immunostimulation by bacterial cells and indirect effects by metabolites.
The antioxidant activity of post-fermented tea is very likely attributed to polyphenols and metabolites represented by catechins.Pyrogallol is involved in the antioxidant activity of Goishi-cha.Pyrogallol in post-fermented tea is produced from gallic acid by decarboxylase [6].The pyrogallol content in Goishi-cha increases significantly during aerobic fermentation, mainly by Aspergillus sp.No increase was observed during subsequent lactic acid fermentation, but a slight decrease was observed [20].The pyrogallol contents of Ishizuchi-kurocha and Goishi-cha were higher than that of Awa-bancha, suggesting that aerobic fermentation is important for the production of pyrogallol.Aspergillus sp. is the primary microorganism involved in the aerobic fermentation of Ishizuchi-kurocha and Goishi-cha [30,32].However, the antioxidant activity of Batabata-cha produced via aerobic fermentation with Aspergillus sp. is remarkably low [12].In addition, because pyrogallol is produced even in Awa-bancha, which does not undergo aerobic fermentation, pyrogallol is probably produced during lactic acid fermentation.L. plantarum and L. pentosus are the primary fungi involved in lactic acid fermentation in Japanese post-fermented tea.L. pentosus isolated from Awa-bancha grew in tea extract supplemented with glucose [33].Although the metabolism of catechins has not been investigated in lactic acid bacteria derived from post-fermented tea, L. plantarum exhibits galloyl-esterase, decarboxylase, and benzyl alcohol dehydrogenase activities.Upon adding grape seed-extracted polyphenols containing large amounts of catechin and epicatechin to water, it decomposes to produce gallic acid and pyrogallol [27].Lentilactobacillus hilgardii isolated from wine produces pyrogallol in the presence of catechins or galls [34].L. plantarum degrades gallic acid, chlorogenic acid, epicatechin, and catechin [35].L. plantarum also degrades theaflavin-3,3digallate (TFDG) in black tea [36].Circumstantial evidence suggests that the Aspergillus sp. and L. plantarum groups of lactic acid bacteria are involved in catechin metabolism and pyrogallol production in post-fermented tea.
The antiallergic effects of Ishizuchi-kurocha are associated with theabrownin, a catechin polymer [21].By contrast, the L. plantarum FG4-4 strain isolated from Awa-bancha was ingested orally in a mouse model of atopic dermatitis.Th1-type cytokines such as -γ were increased [37].These results indicate that L. plantarum in Awa-bancha may improve the Th1/Th2 balance and reduce allergic reactions.Suppression of allergic reactions by post-fermented tea may involve lactic acid bacteria and bacterial cells.
In post-fermented tea, the concentration of GABA increased during fermentation.When the GABA-producing ability of lactic acid bacteria isolated from Ishizuchi-kurocha was evaluated, high GABA production was observed in L. brevis.L. plantarum produces lower levels of GABA than L. brevis [4].GABA production by these lactic acid bacteria varied at the strain level, suggesting that the variation in the post-fermentation GABA content depends on the lactic acid bacterial strain involved.L. brevis produces antifungal agents and reduces aflatoxin production [38].L. plantarum produces antifungal substances active against molds, including Aspergillus sp.[39,40].Although the antifungal activity of lactic acid bacteria in Japanese post-fermented tea has not yet been investigated, it may contribute to the selection and control of fungi in post-fermented tea.
Lactic acid bacteria also participate in D-amino acid synthesis.Several fermented foods, including lactic acid-fermented foods, contain D-amino acids [41].Ishizuchi-kurocha contains D-amino acids such as D-Ala, D-Asp, and D-Glu.Lactic acid bacteria retain amino acid racemases, and L. plantarum and L. brevis isolated from Ishizuchi-kurocha retain glutamate racemase, alanine racemase, and aspartate racemase genes [8].By contrast, Ishizuchi-kurocha exhibits different D-amino acid contents depending on the production lot, which is likely related to the lactic acid flora during fermentation.
However, the amount of caffeine in Japanese post-fermented tea was lower than that in green tea.Lactic acid bacteria do not contribute to the low caffeine content in postfermented tea compared to green tea and black tea.Green tea and black tea are made from young leaves of tea trees in May-June, whereas Japanese post-fermented tea is made from hardened tea leaves in July-August.Differences in tea leaf harvesting times may affect the caffeine content.

Possibility of the Presence of Bacteriocin in Microbial-Fermented Tea
L. plantarum and L. pentosus were isolated from fermenting Ishizuchi-kurocha and Awa-bancha.Due to the fact that they are always isolated as dominant species, we conclude that these species play a significant role in fermentation.Next-generation sequencing (NGS) results also show that the genus Lactiplantibacillus was dominant during the fermentation of Ishizuchi-kurocha [3] and Awa-bancha [23].Interestingly, L. plantarum and L. pentosus have many similarities, including bacteriocin genes.Bacteriocin is an antimicrobial peptide produced by lactic acid bacteria in addition to lactic acid and various other low-molecularweight organic acids.Bacteriocin is responsible for making Lactiplantibacillus dominant during the fermentation of Ishizuchi-kurocha and Awa-bancha.Bacteriocins are classified into Class I, which contains abnormal amino acids, and Class II, which does not [42].Class I bacteriocins, also known as antibiotics, undergo post-translational modifications of their amino acid chains.Class II bacteriocins are peptides without modified amino acids and are further subdivided into four subclasses.The bacteriocin produced by L. plantarum is known as plantaricin and is usually reported as a class II bacteriocin.Class II bacteriocins are small peptides (<10 kDa); they are heat-stable, hydrophobic molecules with amphiphilic α-helical structures [43] and isoelectric points in the range of 8.3-10.0[44].L. plantarum IYO1511, isolated from Ishizuchi-kurocha, was sequenced at the whole-genome level [45], and the plantaricin A gene was identified (BLLP00000000).Whole genome shotgun sequencing results reveal that L. pentosus AWA 1501, isolated from Awa-bancha, also contains genes encoding bacteriocins by [46] (BOUG01000001.1).Although the role of bacteriocins in tea fermentation has not yet been characterized, an increase in the safety and shelf life of fermented foods and probiotic properties has been reported [47].Studies focusing on the role of bacteriocins during the fermentation of teas can be an interesting topic, enabling a better understanding of the mechanisms of other microbial fermentation foods.

Brewed Tea from Artificial Post-Fermented Tea: Kamigare Lactic Acid Bacteria-Fermented Tea
In Kamigare District, Ibigawa, Gifu, Japan, tea leaves are produced without using agricultural chemicals.First-grade tea leaves are shipped exclusively, and most lowergrade tea leaves are discarded.To utilize these leaves, the original post-fermented tea of Kamigare District was artificially made [48].L. plantarum plays a central role in the fermentation of Ishizuchi-kurocha [3,4,24] and Awa-bancha [5,23].L. plantarum produces antibacterial polypeptides such as plantaricin A and plantaricin EF as bacteriocins, becoming a dominant species in the growing environment.These polypeptides may contribute to the reproducible fermentation of Ishizuchi-kurocha and Awa-bancha.L. plantarum, which encodes bacteriocin, was selected for the artificial fermentation of Kamigare District original post-fermented tea (Figure 2).mentation method following the fermentation method of Awa-bancha.The amino acid contents of Kamigare fermented tea and Awa-bancha were compared.Kamigare fermented tea contains arginine, alanine, proline, cysteine, lysine, tyrosine, and valine.However, in a few cases, cysteine, tyrosine, and lysine were detected among these amino acids [40].A significant difference in amino acid content was observed regarding glutamine, which contributes to good taste.Kamigare-fermented tea exhibits a lower glutamate content, which can be improved.

Conclusions
Post-fermented tea exhibits characteristic physiological activities caused by its microbial metabolism.The physiological actions of post-fermented tea include antioxidant activity, antiallergic activity, and inhibition of lipid accumulation.Catechins and catechin metabolites contribute to antioxidant and antiallergic activities.Microorganisms, such as lactic acid bacteria, are involved in catechin metabolism.In addition, lactic acid bacteria L. plantarum was isolated from wild grasses growing in Kamigare District.One gram of each grass sample was suspended in 10 mL of sterile distilled water.The suspension was serially diluted with distilled water and smeared onto MRS agar plates.The MRS agars were then incubated under anaerobic conditions at 30 • C for 1-2 d.After single-colony isolation, each bacterial 16SrRNA DNA sequence was analyzed, and L. plantarum was selected.The genes encoding plantaricin A (PlnA) and plantaricin EF (Pln EF) were confirmed using the DNA extracted from the isolated L. plantarum.We selected L. plantarum strain S13-02 isolated from the dandelion harvested in Kamigare District.The DNA extracted from strain S13-02 showed the strongest PCR-amplified DNA band [48].Plantaricin genes are encoded by chromosomes and/or plasmids.Some lower-grade tea leaves are dried and sold as bancha.These dried banchas were selected for fermentation because the product has a long shelf-life and can be obtained throughout the year.For fermentation, two volumes (v/g) of water were added to dried bancha, and L. plantarum S13-02 was inoculated to obtain a final CFU of 106.The fermentation was performed under anaerobic conditions at 37 • C for 4 d, resulting in the highest lactic acid production, and the final concentration of lactic acid was ~140 mM.
Bacterial flora was monitored in the V3-V4 region of the 16SrRNA gene using Illumina NGS to observe the fermentation process.The bacterial community structure in the L. plantarum S13-02-inoculated sample was uniform with Lactobacillus, but the sample without the inoculum showed diverse bacterial genera.The total amount of 16SrRNA DNA in the inoculated samples was much higher than that in the absence of the inoculum.This confirms that the inoculated L. plantarum S13-02 grew well, and the inoculum of bancha was effective in producing Kamigare lactic acid bacteria-fermented tea.This suggests that natural and microbial tea fermentation cannot be achieved by maintaining tea leaves under anaerobic conditions.The traditional microbially fermented teas Ishizuchi-kurocha and Awa-bancha are produced reproducibly, the reasons behind which are still unknown.
Kamigare lactic acid bacteria-fermented tea is produced via a one-step anaerobic fermentation method following the fermentation method of Awa-bancha.The amino acid contents of Kamigare fermented tea and Awa-bancha were compared.Kamigare fermented tea contains arginine, alanine, proline, cysteine, lysine, tyrosine, and valine.However, in a few cases, cysteine, tyrosine, and lysine were detected among these amino acids [40].A significant difference in amino acid content was observed regarding glutamine, which contributes to good taste.Kamigare-fermented tea exhibits a lower glutamate content, which can be improved.

Conclusions
Post-fermented tea exhibits characteristic physiological activities caused by its microbial metabolism.The physiological actions of post-fermented tea include antioxidant activity, antiallergic activity, and inhibition of lipid accumulation.Catechins and catechin metabolites contribute to antioxidant and antiallergic activities.Microorganisms, such as lactic acid bacteria, are involved in catechin metabolism.In addition, lactic acid bacteria isolated from post-fermented tea exhibit antiallergic activity, GABA production,

Figure 1 .
Figure 1. Outline of production methods of Japanese post-fermented teas.

Figure 1 .
Figure 1. Outline of production methods of Japanese post-fermented teas.

Table 1 .
Contents of caffeine and catechins in Japanese post-fermented teas.

Table 2 .
Lactic acid bacteria species isolated from Japanese post-fermented teas.