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- MATERIALS AND METHODS
Green tea is one of the most popular beverages in the world. Its beneficial health effects and components have been extensively reviewed. However, little is known about the influence of green tea consumption on the human intestinal microbiota (HIM), which plays a crucial role in human health. Ten volunteers who did not usually consume green tea, drank it for 10 days and then stopped drinking it for 7 days. Their fecal samples were collected at three time points: before beginning the 10-day green-tea regime, at the conclusion of that 10 days, and 7 days after stopping the regime. Their fecal samples were analyzed by terminal restriction fragment length polymorphism with specific primer-restriction enzyme systems for HIM and by using a real-time PCR method for the Bifidobacterium species. Although the HIM of each subject was relatively stable, the proportion of Bifidobacterium species played an important role in the classification of their fecal microbiota. Although there were inter-individual differences in the Bifidobacterium species, an overall tendency for the proportion of bifidobacteria to increase because of green tea consumption was noted. However, little change was observed in the composition of Bifidobacterium species in each sample. This suggests that the change in proportion was induced, not by an inter-species transition, but by an intra-species increase and/or decrease. In conclusion, green tea consumption might act as a prebiotic and improve the colon environment by increasing the proportion of the Bifidobacterium species.
The HIM, a community of microbial cells that outnumber the eukaryotic cell population of a host by a factor of 10, is closely related to our health status (1, 2). The HIM plays a crucial role in nutrient absorption, development of our immune systems, and resistance to exogenous pathogens (1, 3–7).
Despite intensive studies, it has been difficult to determine the characteristics and composition of the HIM because of inter-individual diversity and continuous alterations in the community, which is influenced by environmental exposure (8), diet (9, 10), drugs (e.g., antibiotics) (11), and health status (12, 13). In fact, these factors can control the composition of the HIM. In particular, dietary ingredients directly affect the composition of the HIM by supplying nutrients.
After water, tea is the most commonly consumed beverage in the world. Based on the process by which it is manufactured, there are four types of tea: green, black, oolong, and white tea. Green tea, they type most often consumed in Asia, is made by precluding oxidation of tea polyphenols (14, 15). The polyphenols are composed mainly of several kinds of catechins, including epigallocatechin gallate, epigallocatechin, epicatechin gallate, epicatechin, gallocatechin, gallocatechin gallate, and catechin. Interest in the health effects of green tea and its components has been increasing, and many beneficial effects, both direct and indirect, have been reported (16–19).
Modification of HIM can have an indirect effect on human health. Green tea may improve the balance of HIM, resulting in beneficial influences on the health of the host. Because of green tea's important effects on the host, several studies have examined its influence on the human intestinal bacteria, and the effects of its constituents (14, 20, 21). However, there have been only a few studies of green tea's in vitro effects, and little is known about how drinking green tea for a prescribed period modifies the HIM.
In the present study, we assessed changes in the HIM of 10 volunteers who drank green tea instead of water for 10 days. We selected Bifidobacterium species based on their contribution to the data classification and analyzed them quantitatively and qualitatively. We analyzed the fecal samples by T-RFLP with specific primer-restriction enzyme systems and used an adapted real-time PCR method.
- Top of page
- MATERIALS AND METHODS
Researchers have studied the effects of green tea and tea phenols on health extensively. However, very few studies have investigated the relationship between drinking green tea and HIM under in vivo conditions and by molecular analyses. In this study, we used T-RFLP and real-time PCR to demonstrate how 10 days of green tea ingestion modifies the HIM. To minimize the effects of green tea consumed before the experiment, we selected 10 volunteers who did not usually drink tea.
Bacterial composition analyzed by T-RFLP showed inter-individual variation among subjects and intra-individual variation between the three time points. These variations might be due partly to differences in dietary intake, which we did not control during this experiment. However, we noted little change in the fecal microbiota of some subjects across all three time points despite their variable dietary intake, suggesting that dietary differences have little effect.
Data mining was used to calculate contribution scores, which were classified into two groups based on the average proportion of each OTU in each subject. This analysis suggested that changes in the fecal microbiota population resulting from green tea consumption is mainly attributable to OTU124, which we presumed corresponded to bifidobacteria; these organisms have been studied extensively for a long time because of their probiotic health benefits (27, 28). Additional real-time PCR analysis showed that drinking green tea increased the proportion of bifidobacteria in eight of ten subjects; the proportion of bifidobacteria in five of these eight subjects decreased after green tea ingestion had stopped. This is in agreement with other reports describing bifidobacterial growth in humans consuming green tea (29–31).
Operational taxonomic unit 853 had the highest contribution score. Though the average proportion of OTU853 was low, several subjects, such as C, D, H and I, had decreased proportions of OTU853 after green tea consumption and increased proportions after tea drinking had stopped. Moreover, OTU853 was not detected in samples collected after green tea consumption in these four subjects. These patterns might have affected the statistical analysis and calculation of high contribution scores. In two subjects, F and G, we did not detect OTU853 throughout this experiment. Non-detection of OTU853 in many fecal samples lowered the average proportion. On the other hand, we did not observe a correlation between OTU469 and green tea consumption in spite of the high proportion of OTU469 in all subjects.
Two processes may explain the effect of green tea on the proportion of bifidobacteria in the fecal microbiota. First, green tea can directly affect the composition of the microbiota. Its constituents may act as prebiotics, which cause proliferation of bifidobacteria in the colon. Goto et al. reported the influence of tea catechins on the fecal flora of the elderly (30). Administration of four tea catechins caused significant increases in the amounts of bifidobacteria and lactobacilli, whereas the amounts of Bacteroidaceae, eubacteria, clostridia, and Enterobacteriaceae decreased. In addition, Tzounis et al. reported that (+)-catechin exposure in vitro results in growth of bifidobacteria (20). Moreover, green tea has moderate growth-promoting activity for Bifidobacterium spp., including B. adolescentis, B. longum, B. breve, and B. infantis (31). Based on these effects of green tea and its constituents, green tea may be a promising prebiotic beverage.
Furthermore, selective anti-bacterial properties of green tea and its constituents may act as indirect prebiotics. Researchers have reported both in vitro and in vivo studies of such selective anti-bacterial activity. In one study, growth of Bifidobacterium spp. and Lactobacillus spp. was less affected by several tea polyphenol components, including epicatechin, catechin, and caffeic acid, than was that of other bacterial species such as Clostridium difficile, C. perfringens, and Streptococcus pyogenes (21). In addition, the percentage of Bifidobacterium spp. relative to total bacterial counts increased significantly after intake of tea phenols and volatile fatty acids, including acetic acid and propionic acid, which could reduce the fecal pH and inhibit potential pathogenic species (14, 32). In a study on calves, Ishihara et al. found that although the total bacterial count of Bifidobacterium spp. decreased with the growth time, their relative proportion increased after exposure to green tea extracts because the growth of other bacterial species decreased at a higher rate (29). In summary, although green tea and its components do not directly affect the absolute counts of bifidobacteria, green tea is a prebiotic because it inhibits other bacterial species. It is difficult to judge, through an in vivo study, whether drinking green tea affects the growth of bifidobacteria directly or indirectly. However, as noted in prior reports, it is certain that green tea and its components have a positive effect on the growth of bifidobacteria.
Researchers have used PCR analysis using specific primers to evaluate the effect of green tea consumption on the species composition of bifidobacteria (33–35). Although a previous report had shown that PCR-inhibiting substances prevented amplification with any primers of DNA extracted from some fecal specimens (36), we detected bifidobacteria at least once in every sample in this study. Despite changes in proportions of bifidobacteria to total bacteria caused by drinking green tea, the species compositions were relatively stable. This means that the changes in proportions were not induced by inter-species transition, but rather by intra-species increase and/or decrease. Moreover, we did not observe differences in the relationships among specific Bifidobacterium species and fluctuations in this proportion. For example, although subject A had five species, only a small change in the Bifidobacterium proportion was observed after tea consumption. Subjects C and D, who each had only one species, showed an increase in the proportion of Bifidobacterium. This suggests that competition among species and/or interaction with other species in the colon causes great changes in the compositions and proportions of some bacteria. In addition, other Bifidobacterium species that we did not analyze in this study, such as B. ruminantium and B. gallicum, might have been present.
We detected B. longum subsp. infantis, a species characteristically of infants, in three subjects. This corresponds to the report of Saito et al. (9). Although we did not detect B. longum subsp. infantis after green tea consumption in two subjects, we did detect it in one subject after the tea regimen had been stopped. In subject B, however, we continuously detected B. infantis in all three samples. Moreover, we did not detect B. animalis subsp. lactis in subject E and B. longum subsp. infantis in subject J after green tea ingestion and did not recover these organisms after the 7-day no-tea period. We did not detect B. longum subsp. longum in subject G before the onset of green tea consumption but it was present afterwards. Anti-bacterial or bacterial growth-stimulating factors in the tea may modulate the appearance and disappearance of specific speciesy. Moreover, inter-individual variation in absolute amounts of various species and inter-individual responses to the green tea consumption might influence this. Further investigation of the species-specific influence of green tea is necessary.
The B. catenulatum group and B. longum subsp. longum are reportedly dominant Bifidobacterium species in human adults (36). Our study also showed a similar pattern of bifidobacterial frequencies. However, in this study we did not detect B. bifidum, which others have previously been detected in 38% of adults.
The composition of HIMs change depending on age (37, 38). In this study, we deliberately selected subjects of widely differing ages to elucidate the patterns of influence of different age ranges. However, we did not observe a correlation between age and proportion of Bifidobacterium spp. in this study.
In this study, green tea consumption changed the compositions of fecal microbiotas and demonstrated prebiotic properties. More studies of individuals on controlled diets are necessary to establish the effect of green tea on human fecal microbiota and any concomitant improvements in human health.