This study will be conducted at the Department of Endocrinology and Metabolism, Kyoto Prefectural University of Medicine (Kyoto, Japan) and HP (htyearstps://kpum-1nai.jp/metabo/). Trial design is a double-blind crossover interventional study. The principal investigator or the person in charge will explain the study in a written form and obtain written consent from the participants themselves. Healthy adults (age 20 years or older on the date of consent, regardless of gender) who have been fully informed of the contents of this study and have given their consent are eligible. Healthy participants will be recruited through public recruitment. After that, the participants will be randomized by a simple randomization method to the high-quality matcha: Shitimeinomukashi, sold by Horiishichimeien (Uji, Japan, https://uji-shichimeien.co.jp/) group and the processing matcha: no name sold by Kyoeiseicha Co., Ltd. (Osaka, Japan, https://www.kyoeiseicha.co.jp/company/index.html) group by a person in charge who is not involved in any other procedure of this study (Figure 1).
The following inclusion and exclusion criteria will be followed.
Participants who fulfil the following will be included:
(1) Participants who are able to drink at least one cup of matcha green tea daily
(2) Participants who have not received any antibiotics, undergone dietary interventions, and changed their oral medications in the last 3 months, and
(3) Participants between 20 and 80 years of age at the time of obtaining consent.
Participants who fall into any of the following categories will be excluded:
(1) Pregnant women, women who may be or intend to become pregnant, and women who are breastfeeding.
(2) Participants with a history of anemia or those undergoing treatment for it
(3) Participants who have a confirmed diagnosis of or are under treatment for chronic diseases, such as diabetes, hyperlipidemia, and hypertension
(4) Participants with a history of cardiovascular disease, renal disease, liver disease, abnormal thyroid function, or malignancy, and
(5) Participants with diagnoses of or participants are who are undergoing treatment for allergic diseases, such as asthma or immune disorders
The following is a description of the intervention (Table 1 and Figure 1).
Visit 0: After obtaining consent from 20 healthy participants sing an explanatory document and filling out a registration form, 10 participants will be randomly assigned to the high-quality–matcha-first and processing-matcha-first groups.
Visit 1: After the registration, fasting blood and urine samples will be collected and the vascular endothelial function and dietary habits will be evaluated. At the same time, stool samples will be collected, and stool characteristics at the time of collection will be evaluated. The first day is set as the day after stool sample collection.
From the first day, 2 g of matcha dissolved in water or hot water is to be consumed every morning.
Visit 2: Visit 2 will be performed 28–35 days after Visit 1. Stool samples will be collected, and stool characteristics at the time of stool collection will be evaluated. Fasting blood and urine samples will be collected, and vascular endothelial function and dietary habits will be evaluated.
Visit 3: Visit 3 will be performed 28 days (21–35 days) after Visit 2. Fasting blood and urine samples will be collected, and vascular endothelial function and dietary habits will be evaluated. At the same time, stool samples will be collected, and stool characteristics at the time of stool collection will be evaluated. The first day is set as the day after stool sample collection. From the first day, 2 g of matcha, which is of the other type, dissolved in water or hot water is to be consumed every morning.
Visit 4: Visit 4 will be performed 28–35 days after Visit 3. Fasting blood and urine samples will be collected, and vascular endothelial function and dietary habits will be evaluated. During the study period, the participants will be advised to maintain the same dietary, drinking, smoking, and exercise habits as before;
Data collection and management
Samples of gut microbiota, blood, urine, and feces, will be obtained at each visit. In addition, vascular endothelial function will also be evaluated at each visit.
Fecal sample collection and the analyses of gut bacterial composition will be performed as previously described (10). Fecal samples will be collected in a guanidine thiocyanate solution (feces collection kit; Techno Suruga Lab, Shizuoka, Japan). Isolation of genomic DNA and analysis of DNA by 16S rRNA metagenomic sequencing will be performed by Bioengineering Lab. Co. Ltd. (Kanagawa, Japan). The amplicon sequence variant (ASV) table will be generated using Quantitative Insights Into Microbial Ecology 2 (QIIME2) (11). QIIME2 was developed on the basis of a plugin architecture that allows third parties to contribute functionality (https://library.qiime2.org). The prediction of the functional profiles from the 16S rRNA dataset will be performed using the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) software (http://picrust.github.com) (12). The 16S rRNA gene amplicons will be functionally annotated and mapped onto networks in the KEGG database (release 70.0), and functional profiles of the gut microbiota will be predicted.
Serum and urinary samples
The blood samples will be obtained after overnight fasting, and the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), total cholesterol, triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), high-sensitivity C-reactive protein (hsCRP), fasting plasma glucose (FPG), and hemoglobin A1c (HbA1c) will be assessed.
Urinary samples from the second morning’s urine will also be obtained and urine creatinine, sodium, chloride, and potassium levels will be evaluated. Proteinuria, hematuria, glucosuria, urinary ketone, urinary power of hydrogen (pH), and urine specific gravity will be defined using a urine dipstick test.
Dietary assessment (survey of ingested foods and nutrients)
In this study, food and nutrient intake will be evaluated using a brief-type self-administered diet history questionnaire (BDHQ) (13) (14). The BDHQ has been sufficiently validated, enabling relatively simple surveying of the long-term dietary state; additionally, it is suitable for large-scale epidemiological studies.
Metabolomic and lipidemic analyses
Metabolomic and lipidemic analyses will be performed on serum, urine, and fecal samples. The samples will be derivatized using the online solid-phase derivatization system SPE-GC/MS (AiSTI SCIENCE Co., Ltd. Wakayama, Japan) or the fatty acid methylation kit (Nacalai Tesque, Inc. Kyoto, Japan) and analyzed using the GC/MS system (Agilent 7890 B +7000D, Agilent Technologies, Inc. Santa Clara, United States). Pathway analyses will be performed to identify changes in metabolites due to the consumption of high-quality matcha and processed matcha.
Vascular endothelial function
Peripheral arterial tonometry signals will be obtained using the EndoPAT 2000 device (Itamar Medical Inc., Caesarea, Israel) (15) (16). Endothelial function will be measured via the reactive hyperemia-peripheral arterial tonometry (RH–PAT) index (17).
Primary outcome is that to investigate the difference in the changes in the gut microbiota between the high-quality and processing-matcha participant groups, the weighted average differences (WAD) method will be used (18). In this method, genera are ranked by comprehensively assessing higher expression, higher weights, and fold changes. The WAD method was found to be effective in transcriptome analysis. Secondary outcomes are to investigate the difference in the functional profiles of the gut microbiota between the high-quality and processing matcha participant groups, the WAD method will be used. In addition, changes in the metabolites of serum, urine, and fecal samples, dietary intake, fecal condition, and peripheral vascular endothelial function will also be evaluated by paired t-test or Wilcoxon signed-rank test. Furthermore, multiple regression analyses will be carried out to check the correlation of gut microbiota with diet, stool properties, serum and urinary data, and vascular endothelial function.