A randomized, double-blinded study evaluating effect of matcha green tea on human fecal microbiota

Abstract

Matcha green tea is made from powdered green tea leaves. Unlike regular green tea, Matcha green tea is believed to exert beneficial effects on the gut microbiota, as it is richer in nutrients such as tea catechins and insoluble dietary fiber. In the present study, we aimed to investigate the effects of consumption of Matcha green tea on the gut microbiota. Human participants were randomly assigned to a placebo (n = 16) or a Matcha green tea (n = 17) drink group and asked to drink the treatments for two weeks. Feces were collected from the participants pre- and post-treatment and fecal microbiota composition was analyzed by 16S rRNA metagenomic sequencing. The beta-diversity of microbial composition significantly (p<0.05) changed in MGT group but not in placebo group. In addition, the number of unique bacterial genera significantly (p<0.05) changed in the Matcha green tea group was 30, while it was only 3 in the Placebo group. Increase and decrease in abundances of Coprococcus and Fusobacterium, respectively, in the gut microbiota of Matcha green tea group, conferred potential health benefits to the host. The present study was registered in the UMIN Clinical Trial Registry (UMIN000043857).

Introduction

The gut microbiota is a complex microbial community that exists in the gastrointestinal tracts of humans and animals and regulates their health. Dysbiotic gut microbiotas are usually associated with gastrointestinal diseases such as the irritable bowel syndrome and the inflammatory bowel disease (IBD),⁽¹⁾ as well as with various systemic disorders such as diabetes and autism.^(2,3) Therefore, recent research has focused on finding strategies to prevent diseases by maintaining the homeostasis of the gut microbiota and ultimately to achieve a higher quality of life for humans. Studies have found that, while factors such as genetics, age, levels of exercise and stress and antibiotic administration can affect it in various degrees, nutrition in particular can have a high impact on the wellness of the gut microbiota.^(4–6)

Unlike regular green tea, which is a leaf infusion product, matcha green tea (MGT) is made from ground-up Japanese green tea (Camellia sinensis) leaves. MGT contains higher amounts of dietary fibers, carotenoids and fat-soluble vitamins such as vitamin K than does regular green tea.^(7,8) MGT leaves are cultivated in a sun-shaded condition for several weeks before harvest, which contributes to increase amino acids, including theanine, which enables MGT to consume as a thicker suspension, providing twice higher amount of catechins and caffeine than green tea.⁽⁷⁾ MGT is thought to be a potential functional food for the gut microbiota.^(9–11) In this context, it is believed that catechins found in green tea possess anti-pathogenic properties, which result in their well-known improving effect on the gut microbiota.⁽¹²⁾ In addition, in contrast with regular green tea, MGT contains dietary fiber, which is used as dietary substrate by many enteric bacteria.⁽¹³⁾ The recommended dietary fiber intake of Japanese adult males is at least 21 g/day and that of females is around 18 g/day.⁽¹⁴⁾ By contrast, the average dietary fiber intake of those in their twenties is only 16 g/day, which is considered deficient.⁽¹⁵⁾ Therefore, it is theorized that MGT could have a greater, positive modulative effect on the gut microbiota than do regular green tea and catechin supplements alone.

Caffeine- and theanine-rich composition of MGT has attracted attention for its effects on brain function.⁽¹⁶⁾ Clinical trials have shown that MGT improves cognitive function not only in the elderly but also in young adults.^(16,17) Vitamin K contained in MGT has also been reported to improve cognitive function and prevent aging of the cortical capillaries, suggesting that MGT may be useful in improving brain function.^(7,18,19) In addition, theanine in MGT has been suggested stress reducing function in mice and humans.^(20,21) Recent growing evidences are suggesting the existence of bidirectional gut-brain axis and stress is considered as a factor of gut microbiota modulation.^(22,23) These findings suggest that the effect of MGT on brain function like relaxant effect may contribute to changes in gut microbiota through brain-gut axis.

While the effects of green tea and its biologically-active compounds (e.g., catechins) on the gut microbiota have previously been investigated, that of MGT intake has not been thoroughly studied.^(11,24–26) Moreover, to the best of our knowledge, the association of MGT intake and the gut microbiota alteration is yet to be reported. Therefore, in the present study, we aimed to examine the effect of continuous MGT consumption on the gut microbiota and elucidate the possible use of MGT as a functional food.

Materials and Methods

Ethics statements and research subjects

The present study was registered in the UMIN Clinical Trial Registry (UMIN000043857). The experimental protocol was approved by the Ethical Committee of the Kyoto Prefectural University and conducted as per their guidelines (approval number: 149 and 2018/Mar/30) and conducted according to the guidelines of the Declaration of Helsinki. Healthy male and female at university-age and postgraduate-age (18–25 years old) were enrolled in this study. Written informed consents were obtained from all participants. Thirty-three people living in Kyoto, Japan (10 males, 23 females, age 18–25) were initially recruited for the experiment and randomly allocated to MGT (n = 17) and placebo (n = 16) groups.

Experimental design

Prior to the start of the experiment, we supplied either 28 MGT (containing 1.5 g of MGT) or 28 placebo (containing fragrance and Gardenia pigment) capsules to each participant in the MGT and the Placebo groups, respectively. The participants prepared drinks with the supplied capsules and 80 ml of hot water, using a Dolce gust machine (Nestlé Japan Ltd., Tokyo, Japan). The experiment participants drank the treatments in the morning and the evening for 2 weeks, following their daily routine. The capsules were collected after the use, and participants who had not used all capsules were excluded from further analysis. Feces were collected from the participants pre- (Week 0) and post-consumption (Week 2) of the experimental drinks. On the samples collection days, the participants filled up forms with questions about the defecation frequency per week and fecal formation, as per the Bristol Stool Form Scale (BSFS).⁽²⁷⁾ A flow-chart describing this experimental procedure is provided in Fig. 1.

Fig. 1.

Flow-chart of the experimental design.

Sample collection and DNA extraction

Fecal samples of the size of grains of rice were collected using stool collection brush and storage tube sets (Wako Pure Chemicals, Osaka, Japan). The storage tubes contained DMSO/EDTA/saturated sodium chloride (DESS).⁽²⁸⁾ After mixing well, the samples were stored at room temperature for up to 14 days until DNA extraction.⁽²⁸⁾

To extract the DNA from the samples, they were first centrifuged (10,000 × g for 3 min at room temperature) and the supernatants removed. Next, the fecal pellets were washed with phosphate buffered saline. Then, whole bacterial DNA was extracted using a Quick Gene DNA Tissue Kit S (KURABO, Okayama, Japan). DNA extraction was conducted exactly as instructed in the manufacturer’s manual (KURABO).

16S rRNA metagenomic analysis

Library preparation, DNA sequencing and sequence data analysis were carried out as previously described.⁽³⁾ For the heat-denaturing process, 10 pM of the library combined with phiX Control (ver. 3, Illumina; expected 20%) was used.

During the sequence data analysis step, the obtained Fastq data was quality filtered, and USEARCH,⁽²⁹⁾ VSEARCH,⁽³⁰⁾ and Quantitative Insights Into Microbial Ecology (QIIME)⁽³¹⁾ were used to remove dereplication, singletons, to define OTU (operational taxonomic units), and to assign taxonomy to each out, as previously reported.⁽³²⁾ Calculation and visualization of the alpha and beta diversity indices were conducted using the R package “phyloseq”.⁽³³⁾

Statistical analysis

Physical characteristics (age, BMI) between the Placebo and the MGT groups were compared with the Wilcoxon rank sum test. The BSFS data, the defecation frequency data, the alpha diversity of the fecal microbiotas and the relative abundances (%) of bacterial genera obtained pre- and post-consumption of the experimental drinks were compared with the Wilcoxon signed rank sum test. Comparison of the beta diversity in the feces was carried out using a permutational multivariate analysis of variance (PERMANOVA) in QIIME ver. 1.9.1. Values are expressed as the means ± SD. Differences between the means were considered to be significant if p<0.05. All statistical analyses were carried out using R (ver. 4.0.2), unless otherwise specified.

Results

Experiment participants and evaluation of feces

During the experimental period, one participant from the Placebo group was removed after one week, because they accidentally failed to take the treatment. Therefore, the number of participants in the Placebo group totaled to 15 (Fig. 1). The number of subjects analyzed was 5 males and 10 females in the Placebo group and 5 males and 12 females in the MGT group, with no significant gender differences between the two groups by chi-square test.

Prior to the start of the experiment, it was confirmed that there were no significant differences in ages (20.9 ± 2.0, vs 21.5 ± 1.8 years old; p = 0.36) and the body mass indices (BMI) (20.9 ± 3.3 vs 21.3 ± 3.0; p = 0.53) between the participants in the Placebo and MGT groups. The BSFS and Defecation frequency are described below (Table 1). There were no significant pre- and post-experiment changes in these two fecal parameters.

Table 1.

Defecation frequency and Bristol Stool Form Scale pre- and post-consumption of experimental drinks

	Placebo group				MGT group
	Pre-consumption (Week 0)	Post-consumption (Week 2)	p value		Pre-consumption (Week 0)	Post-consumption (Week 2)	p value
Defecation frequency (times per week)	9.73 ± 13.32	11.20 ± 16.75	0.44		6.65 ± 4.14	6.65 ± 4.05	>0.99
Bristol Stool Form Scale	3.29 ± 1.68	3.14 ± 1.56	>0.99		3.71 ± 1.10	3.53 ± 1.23	>0.99

Analysis of the fecal microbiota

The alpha diversity of the fecal microbiotas is shown below (Table 2). Both experimental groups showed significantly lower Chao1 (bacterial genus richness) and Shannon (bacterial genus abundant equality) indices post-consumption of the experimental drinks.

Table 2.

Alpha diversity index of fecal microbiotas pre- and post-consumption of experimental drinks

	Placebo group				MGT group
	Pre-consumption (Week 0)	Post-consumption (Week 2)	p value		Pre-consumption (Week 0)	Post-consumption (Week 2)	p value
Chao1	508.39 ± 82.69	428.58 ± 42.68	<0.01**		560.60 ± 122.75	447.55 ± 68.48	<0.01**
Shannon	5.48 ± 0.66	4.85 ± 0.33	<0.01**		5.66 ± 0.87	4.87 ± 0.65	<0.01**

**p<0.01.

As for the beta diversity, a principal coordinate analysis (PCoA) showed that the fecal microbiota composition two weeks post-MGT consumption significantly changed when compared with that pre-MGT consumption (Fig. 2). In contrast, the microbiota composition of the Placebo group showed no changes between the pre- and the post-placebo drink consumption periods (Fig. 2). A comparison of the bacterial composition in the feces during the pre- and post-experimental drink consumption periods showed that the abundances of 52 bacterial genera changed in the MGT group. In Placebo group, the number of bacterial genera of which abundance significantly changed during pre- and post-experimental period was 25, of which 22 bacterial genera were common with MGT group (Supplemental Table 1*). When excluding these commonly changed bacterial genera, only three bacterial genera, including two unclassified Erysipelotrichaceae and Streptcoccus, significantly decreased in the Placebo group (Table 3) during two weeks experimental drink consumption period. In contrast, the abundances of 30 bacterial genera, including Fusobacterium and Coprococcus, changed in the MGT group (Table 4).

Fig. 2.

Beta-diversity index of fecal microbiotas in Placebo and MGT groups pre- and post-consumption of experimental drinks. A principal coordinate analysis (PCoA) was conducted based on weighted UniFrac distances. There was no significant change in beta-diversity in the placebo group (p = 0.42), while there was a significant change in the MGT group over the study period by PERMANOVA (p<0.001). The ellipses enclosing the clusters indicate a 95% confidence interval.

Table 3.

Bacterial genera whose relative abundances changed pre- and post-consumption of the placebo drink

Taxonomy			Pre-consumption (Week 0)	Post-consumption (Week 2)	p value
Order	Family	Genus
Erysipelotrichales	Erysipelotrichaceae	Unclassified	1.138 ± 1.251	0.775 ± 1.014	<0.01**
Erysipelotrichales	Erysipelotrichaceae	Other	0.005 ± 0.010	<0.001	0.04*
Lactobacillales	Streptococcaceae	Streptococcus	1.933 ± 1.170	1.145 ± 0.958	0.02*

*p<0.05, **p<0.01.

Table 4.

Bacterial genera whose relative abundances changed pre- and post-consumption of the MGT drink

Taxonomy			Pre-consumption (Week 0)	Post-consumption (Week 2)	p value
Order	Family	Genus
Bacillales	Staphylococcaceae	Staphylococcus	0.002 ± 0.003	0.011 ± 0.018	0.03*
Bacteroidales	[Barnesiellaceae]	Unclassified	1.175 ± 1.022	0.057 ± 0.109	<0.01**
Bacteroidales	[Odoribacteraceae]	Butyricimonas	0.283 ± 0.251	0.025 ± 0.089	<0.01**
Bacteroidales	Rikenellaceae	Unclassified	1.627 ± 1.153	0.464 ± 0.663	<0.01**
Bacteroidales	Rikenellaceae	Alistipes	0.243 ± 0.219	0.003 ± 0.005	<0.01**
Bifidobacteriales	Bifidobacteriaceae	Alloscardovia	0.002 ± 0.003	<0.001	0.03*
Clostridiales	[Mogibacteriaceae]	Unclassified	0.129 ± 0.093	0.045 ± 0.072	<0.01**
Clostridiales	Christensenellaceae	Christensenella	0.007 ± 0.008	<0.001	<0.01**
Clostridiales	Clostridiaceae	Sarcina	0.041 ± 0.039	<0.001	<0.01**
Clostridiales	EtOH8	Unclassified	0.011 ± 0.011	<0.001	<0.01**
Clostridiales	Lachnospiraceae	Coprococcus	1.481 ± 1.320	2.610 ± 1.913	0.01*
Clostridiales	Lachnospiraceae	Epulopiscium	0.003 ± 0.004	<0.001	0.01*
Clostridiales	Peptococcaceae	rc4-4	0.008 ± 0.009	<0.001	<0.01**
Clostridiales	Ruminococcaceae	Butyricicoccus	0.433 ± 0.411	0.902 ± 0.771	0.02*
Clostridiales	Ruminococcaceae	Ruminococcus	5.816 ± 3.775	2.058 ± 2.487	<0.01**
Clostridiales	Ruminococcaceae	Other	0.373 ± 0.283	0.097 ± 0.140	0.02*
Clostridiales	Veillonellaceae	Megasphaera	0.057 ± 0.050	0.017 ± 0.026	<0.01**
Coriobacteriales	Coriobacteriaceae	Eggerthella	0.081 ± 0.061	0.153 ± 0.139	<0.01**
E2	[Methanomassiliicoccaceae]	Methanomassiliicoccus	0.007 ± 0.009	<0.001	0.01*
Erysipelotrichales	Erysipelotrichaceae	Catenibacterium	0.012 ± 0.013	<0.001	<0.01**
Fusobacteriales	Fusobacteriaceae	Fusobacterium	0.163 ± 0.245	0.061 ± 0.095	0.04*
Pasteurellales	Pasteurellaceae	Haemophilus	0.082 ± 0.208	0.115 ± 0.184	0.03*
Pseudomonadales	Moraxellaceae	Acinetobacter	0.041 ± 0.028	0.098 ± 0.095	0.02*
RF32	Unclassified	Unclassified	0.159 ± 0.155	0.014 ± 0.060	<0.01**
SHA-98	Unclassified	Unclassified	0.005 ± 0.006	0.001 ± 0.004	0.02*
Synergistales	Dethiosulfovibrionaceae	Pyramidobacter	0.013 ± 0.015	<0.001	<0.01**
Synergistales	Synergistaceae	Synergistes	0.010 ± 0.013	<0.001	<0.01**
Turicibacterales	Turicibacteraceae	Turicibacter	0.135 ± 0.311	0.038 ± 0.071	0.04*
YS2	Unclassified	Unclassified	0.033 ± 0.033	<0.001	<0.01**
	Unclassified c__Clostridia		0.037 ± 0.035	<0.001	<0.01**

*p<0.05, **p<0.01.

Discussion

In the present study, we investigated the effect of two weeks MGT consumption on the fecal microbiota. MGT consumption for two weeks did not affect the fecal formation or defecation frequency. However, the beta-diversity resulting from a PCoA indicated that MGT continuously consumed for two weeks possibly affected the gut microbiota composition. Among the bacterial genera whose abundance significantly changed pre- and post-consumption of MGT, Fusobacterium and Coprococcus were noteworthy. For example, Fusobacterium is considered to be a harmful bacterium, as a high proportion of it was reported to be present in the gut microbiota when IBD and colorectal cancer were diagnosed.^(4,34,35) In contrast, Coprococcus has been reported in lower abundance in IBD patients^(36,37) Moreover, Coprococcus produces butyric acid, which is a short-chain fatty acid that is beneficial to the host’s health.⁽³⁸⁾ Therefore, it is believed that the consumption of MGT may have had a beneficial modulatory effect on the human gut microbiota.

Liu et al. (2020)⁽³⁹⁾ reported that epigallocatechin-3-gallate inhibited the growth of Fusobacterium in vitro. Therefore, we theorized that in the present study, catechins contained in MGT and perhaps its bacterial metabolites such as 4-phenylbutyric acid may have played a role in the decrease of Fusobacterium. With respect to Coporococcus, it was reported that the ingestion of insoluble dietary fiber increased its abundance in the gut microbiotas of piglets.⁽⁴⁰⁾ Moreover, using an in vitro digestion and fermentation model, Pérez-Burillo et al. (2020)⁽⁴¹⁾ found increases in the concentration of butyrate and the activity of Coprococcus when they applied a green tea extract to feces suspended in fermentation medium. Therefore, we believed that components of MGT such as insoluble fiber may have helped increase the abundance of Coprococcus. MGT components have been observed to have antioxidant, anti-inflammatory and carbohydrate absorption inhibitory effects,⁽¹¹⁾ which may have also indirectly affected the gut microbiota composition studied in the present work.

In the present study, some changes in the fecal microbiotas pre- and post- consumption of the experimental drinks followed the same trend, regardless of the treatment. For example, the alpha diversity index significantly decreased and the abundance of Bacteroides and Faecalibacterium significantly increased pre- and post-consumption of both experimental drinks, although the reason for these changes remained unclear. Nonetheless, one plausible reason may be the stress caused by an abnormally high temperature observed during the experimental period. To be precise, the present work was conducted in Kyoto, Japan in July 2018, which had an average temperature of 29.8°C, which was about 2.5°C higher than the average temperature previously recorded between 1991 and 2020 (27.3°C).^(42,43)

In conclusion, the present study demonstrated that MGT consumption for two weeks affected the fecal microbiota. An increase of beneficial Coprococcus and a decrease of potential pathogenic Fusobacterium in the gut microbiota due to MGT consumption would likely be beneficial long-term to the host’s health.

Authors Contributions

Conceptualization, RI and YF; methodology, RI and YF; validation, SM and RI; formal analysis, SM; investigation, SM and YK; resources, YF and RI; data curation, RI and SM; writing – original draft preparation, SM; writing – review and editing, RI, TT, and YN; visualization, SM; supervision, RI, TT, and YN; project administration, RI; funding acquisition, RI. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Matcha and Health Research Group.

Conflict of Interest

All authors have no conflict of interest. The funders had no role in the design of the study, the sample collection, analyses or interpretation of data, the writing of the manuscript or in the decision to publish the results.