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Citation: Takeuchi, H.; Yoshikane, Y.;
Takenaka, H.; Kimura, A.; Islam, J.M.;
Matsuda, R.; Okamoto, A.;
Hashimoto, Y.; Yano, R.; Yamaguchi,
K.; et al. Health Effects of Drinking
Water Produced from Deep Sea
Water: A Randomized Double-Blind
Controlled Trial. Nutrients 2022,14,
581. https://doi.org/10.3390/
nu14030581
Received: 10 December 2021
Accepted: 20 January 2022
Published: 28 January 2022
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nutrients
Article
Health Effects of Drinking Water Produced from Deep Sea
Water: A Randomized Double-Blind Controlled Trial
Hiroaki Takeuchi 1,* , Yu Yoshikane 2, Hirotsugu Takenaka 3, Asako Kimura 1, Jahirul Md. Islam 1,
Reimi Matsuda 1, Aoi Okamoto 1, Yusuke Hashimoto 1, Rie Yano 1, Koichi Yamaguchi 1, Shouichi Sato 1
and Satoshi Ishizuka 4
1Department of Medical Laboratory Sciences, Health and Sciences, International University of Health and
Welfare Graduate School, 4-3 Kouzunomori, Narita-City 286-8686, Chiba, Japan; a-kimura@iuhw.ac.jp (A.K.);
21s3057@g.iuhw.ac.jp (J.M.I.); 1857070@g.iuhw.ac.jp (R.M.); 1857025@g.iuhw.ac.jp (A.O.);
y.hashimoto@iuhw.ac.jp (Y.H.); 21s1119@g.iuhw.ac.jp (R.Y.); yamaguchi51@iuhw.ac.jp (K.Y.);
s-shouichi@iuhw.ac.jp (S.S.)
2Department of Human Living Sciences, Notre Dame Seishin University, 2-16-9 Ifuku-cho, Kita-ku,
Okayama-city 700-8516, Okayama, Japan; yyoshikane@m.ndsu.ac.jp
3DyDo-T Beverage Co. Ltd., 1310-1 Hanechou-ko, Muroto-City 781-6741, Kochi, Japan;
takenaka@dt-beverage.com
4Center for Regional Sustainability and Innovation, Kochi University, 2-17-47 Asakurahonmachi,
Kochi-City 780-8073, Kochi, Japan; zuka@kochi-u.ac.jp
*Correspondence: htake@iuhw.ac.jp; Tel.: +81-476-20-7762
Abstract:
Global trends focus on a balanced intake of foods and beverages to maintain health.
Drinking water (MIU; hardness = 88) produced from deep sea water (DSW) collected offshore of
Muroto, Japan, is considered healthy. We previously reported that the DSW-based drinking water
(RDSW; hardness = 1000) improved human gut health. The aim of this randomized double-blind
controlled trial was to assess the effects of MIU on human health. Volunteers were assigned to MIU
(n= 41) or mineral water (control) groups (n= 41). Participants consumed 1 L of either water type
daily for 12 weeks. A self-administered questionnaire was administered, and stool and urine samples
were collected throughout the intervention. We measured the fecal biomarkers of nine short-chain
fatty acids (SCFAs) and secretory immunoglobulin A (sIgA), as well as urinary isoflavones. In
the MIU group, concentrations of three major SCFAs and sIgA increased postintervention. MIU
intake significantly affected one SCFA (butyric acid). The metabolic efficiency of daidzein-to-equol
conversion was significantly higher in the MIU group than in the control group throughout the
intervention. MIU intake reflected the intestinal environment through increased production of three
major SCFAs and sIgA, and accelerated daidzein-to-equol metabolic conversion, suggesting the
beneficial health effects of MIU.
Keywords:
health effect; deep sea water (DSW)-based drinking water; body maintenance; short-
chain-fatty-acid; sIgA; daidzein-to-equol conversion; intestinal microbiota
1. Introduction
The utilization of deep sea water (DSW) has expanded to the energy, agriculture,
food, cosmetics, and public health fields [
1
]. DSW obtained from depths of >200 m is
characterized by high purity, stability at low temperatures, high mineral concentrations,
and the presence of bioactive nutritional species [
2
]. Bottled commercial DSW-based
drinking water produced by different methods such as desalinization, is currently available
on the market; this commercial product is gaining popularity due to its potential benefits
to human health, as confirmed by various animal studies [
3
–
6
]. However, clinical trials are
required to clarify the safety and validity of the effects of DSW-based drinking water on
human health.
Nutrients 2022,14, 581. https://doi.org/10.3390/nu14030581 https://www.mdpi.com/journal/nutrients
Nutrients 2022,14, 581 2 of 14
Previous clinical trials using DSW-based drinking water have confirmed that DSW-
based drinking water (RDSW; hardness, 1000 mg/L of Ca/Mg) has various beneficial effects
on human health, for example with regard to hemorheology, allergies, immunology, infectious
diseases (e.g., anti-Helicobacter pylori activity), and the intestinal environment [
7
–
12
]. For
example, a recent clinical trial reported that drinking RDSW improved human health due
to the increased production of short-chain fatty acids (SCFAs) in the intestinal environment
and urinary isoflavones [12].
The intestinal environment comprises the microbiota, microbiota-derived metabolites,
and ingesta, and includes the microbe–microbe and host–microbe interactions, which play a
fundamental role in human health [
13
]. Healthy foods and beverages, including probiotics
and supplements, are widely consumed to maintain and support the intestinal environment
and microbiota [
14
–
17
]. Recently, fecal microbiota transplantation has been included in
the treatment of autoimmune diseases, hepatitis, metabolic syndromes, and mental disor-
ders [
18
–
21
] via modulation of the gut microbiota [
22
,
23
]. Gut microbes produce various
metabolites, such as isoflavones, that are beneficial to human health [
24
–
26
]. Isoflavone
and other metabolite contents vary among individuals due to differences in the intestinal
environment, including microbial identity and activity, stability, and variations in the con-
centrations of endogenous compounds that modulate biotransformation pathways [
27
,
28
].
Equol, which is produced from daidzein by gut microbes, is one of the most physiologically
active isoflavones. However, only 30–50% of the human population produces equol; a
regional difference exists due to the frequency of soybean consumption. Current research
has focused on manipulating the gut environment to enhance equol production.
The purpose of the present study was to assess whether DSW-based drinking water
(MIU; hardness, 88) could modulate intestinal microbe biomarkers in healthy adults.
2. Materials and Methods
2.1. Clinical Study Design
This randomized double-blind controlled trial was designed to compare the intestinal
environment of individuals in response to drinking MIU vs. mineral water using a self-
administered questionnaire and stool and urine sample analysis. The study was conducted
in Muroto, Kochi, Japan, from 2018 to 2020. The study protocol, although severely restricted
in terms of time and budget, was approved by the Ethics Committees of Kochi University
(approval no. 28–93) and the International University of Health and Welfare (approval no.
18-lo-100) and was conducted in accordance with the ethical standards described in the
1964 Declaration of Helsinki and its later amendments. The questionnaire and stool and
urine samples were collected before and after the intervention.
2.2. Participants
The study cohort included 114 healthy adults residing in Muroto, Kochi, Japan, who
agreed to participate and submitted a signed consent form (Figure 1). Potential participants
with any current illness, those using any prescription or commercial drugs or dietary
supplements, and pregnant women were excluded from the study. Of the 107 healthy
adults who met the inclusion criteria, 82 who correctly completed the questionnaire were
randomly divided into 2 groups: the MIU group (n= 41) and the mineral water (control)
group (n= 41). The characteristics of the study participants are presented in Table 1. There
were no significant differences in terms of age, sex, body mass index, and biomarker
concentrations between the 2 groups (Mann–Whitney Utest).
Nutrients 2022,14, 581 3 of 14
Table 1. Preintervention characteristics of the participants from the 2 groups.
MIU Mineral Water (Control)
Total (n= 41) male (n= 17) female (n= 24) Total (n= 41) male (n= 19) female (n= 22)
Age (year) 43 (33–53) 47 (33–52) 42.5 (32–53) 42 (33–57) 37 (37–58) 47 (37–58)
BMI(Kg/m2)22.6 (20.7–26.4) 23.5 (22.5–27.2) 21.7 (20.3–23.2) 42 (33–57) 37 (37–58) 47 (37–58)
22.9 (21.5–25.5) 22.4 (21.3–25.1) 23.2 (21.9–26.4)
sIgA (µg/g) 408 (209–651) 394 (202–538) 492 (207–678) 555 (169–1042) 449 (159–1016) 614 (21.9–26.4)
Putrefaction (µg/g)
Phenol 1.3 (0.50–6.70) 4.6 (1.07–13.45) 0.8 (0.45–5.20) 1.4 (0.60–7.20) 2 (0.72–13.55) 1.3 (0.45–5.90)
p-Cresol 28.2 (9.15–69.45) 19.7 (6.00–65.50) 40.7 (10.07–81.30) 59.2 (21.20–90.98) 57.4 (23.80–
111.42) 60.65 (19.70–78.90)
4-Ethylphenol 2.3 (1.63–4.15) 2.7 N/A 2.3 N/A 1.7 (0.70–2.90) 1.7 (1.50–7.80) 0.7 (0.70–2.47)
Indol 19.4 (11.80–31.75) 19.4 (9.32–32.37) 19.45 (12.60–31.25) 22.8 (11.85–35.90) 30.4 (15.20–41.70) 17.3 (10.00–27.60)
Skatol 2.75 (1.20–7.80) 2.8 (1.27–7.02) 2.7 (0.57–12.12) 4.8 (1.35–10.00) 2.7 (0.09–0.24) 5.8 (1.40–10.00)
SCFA (mg/g)
Succinic acid 0.19 (0.09–0.47) 0.21 (0.11–0.36) 0.16 (0.08–0.50) 0.12 (0.08–0.24) 0.14 N/A 0.11 (0.07–0.24)
Lactic acid 0.19 (0.08–0.68) 0.23 (0.13–0.82) 0.12 (0.07–0.44) 0.11 (0.08–0.17) 0.08 (0.07–0.15) N/A
Formic acid N/A N/A N/A N/A N/A N/A
Acetic acid 3.19 (1.85–4.03) 2.72 (1.65–3.77) 3.27 (2.16–4.04) 2.63 (1.97–3.37) 2.63 (1.90–3.65) 2.59 (1.99–3.69)
Propionic acid 1.02 (0.76–1.28) 1.01 (0.72–1.28) 1.04 (0.79–1.35) 1.12 (0.87–1.57) 1.38 (0.88–1.66) 1.07 (0.85–1.45)
Isobutyric acid 0.16 (0.12–0.20) 0.19 N/A 0.13 (0.11–0.19) 0.15 (0.13–0.19) 0.17 (0.130–0.21) 0.14 (0.130–0.155)
Butyric acid 0.77 (0.54–1.27) 0.73 (0.54–1.05) 0.78 (0.53–1.31) 0.84 (0.54–1.48) 1.03 (0.56–1.80) 0.81 (0.52–1.23)
3-Methylbutanoic acid 0.2 (0.14–0.26) 0.18 (0.14–0.30) 0.21 (0.14–0.25) 0.2 (0.15–0.27) 0.21 (0.16–0.33) 0.2 (0.130–0.25)
Valeric acid 0.21 (0.13–0.31) 0.25 (0.18–0.30) 0.18 (0.12–0.32) 0.17 (0.14–0.29) 0.22 (0.18–0.38) 0.15 (0.13–0.20)
Urine Isoflavones
Daidzein (mg/g-Cre) 0.9 (0.37–2.15) 0.88 (0.57–1.36) 0.92 (0.36–2.70) 0.79 (0.33–1.60) 0.9 (0.48–1.34) 0.76 (0.19–1.71)
Genistein (mg/g-Cre) 1.11 (0.41–2.10) 1.11 (0.41–1.59) 1.12 (0.54–2.41) 1.03 (0.49–1.76) 1.15 (0.75–2.04) 0.87 (0.46–1.43)
Total (n= 20) male (n= 7) female (n= 13) Total (n= 12) male (n= 8) female (n= 4)
Equol (mg/g-Cre) 0.5 (0.00–1.73) 0.21 (0.00–5.52) 0.57 (0.26–1.52) 0.98 (0.35–2.14) 1.9 (1.08–2.72) 0.32 (0.19–0.48)
Equol (g/g-Da) 0.72 (0.00–1.72) 0.73 (0.00–3.59) 0.72 (0.15–0.88) 1.82 (0.37–3.01) 2.4 (1.06–4.76) 0.31 (0.15–0.93)
Equol (g/g-E + D) 0.42 (0.00–0.63) 0.42 (0.00–0.75) 0.42 (0.13–0.47) 0.63 (0.27–0.74) 0.71 (0.50–0.83) 0.23 (0.13–0.40)
N/A, less than n= 6; The data was shown as median and IQR in parentheses; non-parametric analysis (Mann-Whitney U test).
Nutrients 2022,14, 581 4 of 14
Figure 1.
Flow diagram of this clinical study. A total 107 healthy adults were enrolled from Muroto,
Kochi, Japan. Potential participants with any current illness, those using any prescription or com-
mercial drugs or dietary supplements, and pregnant women were excluded. Participants in the
experimental group consumed MIU (hardness, 88) and those in the control group consumed mineral
water (hardness, 0–20).
2.3. Ingestion Schedule
The study participants in the MIU group consumed bottled MIU water (Dydo-miu;
hardness, 88; Dydo-Takenaka Beverage Co., Ltd., Kochi, Japan) (Supplementary Table S1),
whereas those in the control group consumed mineral water (hardness, 0–20). The most
popular mineral water consumed in Japan was used. Neither of the types of water had
calories, proteins, fats, carbohydrates, or vitamins. Both bottled waters were commercially
available in Japan and the labels were changed to mask the type of water. The participants
were instructed to consume 1 L of the assigned water type daily for 12 weeks.
2.4. Evaluation
A self-administered questionnaire was implemented to assess the general health status
of the participants. We analyzed the following fecal biomarkers: secretory immunoglobulin
A (sIgA), 5 putrefactive products (phenol, p-cresol, 4-ethylphenol, indole, and skatole), and
9 SCFAs (succinic, lactic, formic, acetic, propionic, isobutyric, butyric, 3-methylbutanoic,
and valeric acids) at TechnoSuruga Laboratory Co., Ltd. (Shizuoka, Japan) [
29
,
30
]. Three
urinary isoflavones (genistein, daidzein, and equol) were measured in the urine samples.
Nonparametric analysis was conducted to assess the differences in these biomarkers before
Nutrients 2022,14, 581 5 of 14
and after the intervention. Based on the changes in the biomarker concentrations through-
out the intervention period, multiple logistic regression analysis was performed to evaluate
the relationship between the water type and biomarkers.
2.5. Self-Administered Questionnaire
A total of 86 participants, including 4 who did not submit urine and fecal samples,
answered the questions regarding general gut health and eating habits (i.e., constipa-
tion (evacuation frequency, incomplete evacuation, straining at stool, dyschezia, etc.),
abdominal discomfort, medication use, and consumption of unusual foods and beverages).
Constipation was defined in accordance with the guidelines of the World Gastroenterology
Organization [31].
2.6. Measurement of Fecal and Urine Samples
Measurements of the samples were taken as previously described [
12
]. Fecal sample
analyses were performed at TechnoSuruga Laboratory Co., Ltd. [29,30].
2.6.1. Fecal sIgA
Here, 0.1 g of each fecal sample suspended in a mixture containing 0.1 mM perchloric
acid and 3% phenol was heated and vortexed, followed by centrifugation (15,350
×
g,
10 min) according to previous protocol [
12
]. The supernatant was collected and filtered
(pore size, 0.45
µ
m) to measure sIgA and SCFA contents. sIgA levels were measured using
the Human IgA ELISA Quantitation kit (E80–102; Bethyl Laboratories Inc., Montgomery,
TX, USA) and a microplate reader (Varioskan Flash; Thermo Fisher Scientific, Waltham,
MA, USA).
2.6.2. Fecal Putrefactive Products
For this, 0.1 g of each fecal sample suspended in 2.5 mL of phosphate buffer including
0.4 mg/L of 4-isopropylphenol as an internal standard was employed to the previous
procedures [
12
]. Briefly, 1 mL of the supernatant was dehydrated and purified with
3 cartridges such as sodium sulfate drying cartridge (Bond Elut LRC; Agilent Technologies,
Tokyo, Japan), C18 cartridge (Smart SPE C18-30; AiSTI Science, Wakayama, Japan), and
PSA cartridge (Smart SPE PSA-30; AiSTI Science).
The levels of indole and phenol were determined by a single quadrupole gas
chromatograph–mass spectrometer (QP-2010; Shimadzu, Kyoto, Japan) equipped with
a capillary column (Inert cap WAX; GL Science, Tokyo, Japan). Helium was used as the
carrier gas. The injector and interface temperatures were maintained at 240
◦
C and 230
◦
C,
respectively. For the analysis, 1
µ
L of the extract was subjected to the splitless mode. The
mass spectrometer was operated in the electron impact ionization mode at 70 eV. The
measurements were recorded, and data were obtained from the selected ion-monitoring
mode for quantification.
2.6.3. Analysis of Intestinal Microbiota
The fecal samples suspended in a buffer containing 4 M guanidium thiocyanate,
100 mM Tris-HCl, and 40 mM ethylenediaminetetraacetic acid were pulverized as previ-
ously described [
12
]. Following this, DNA was extracted from the suspension using the
Magtration System 12GC and GC series MagDEA DNA 200 (Precision System Science
Co., Ltd., Matsudo, Japan). The final DNA concentration (10 ng/
µ
L) was subjected to
the analysis of the microbial community structure by terminal restriction fragment length
polymorphism and next-generation sequencing using the MiSeq system (Illumina, San
Diego, CA, USA) at TechnoSuruga Laboratory Co., Ltd. [
29
,
30
,
32
]. Bioinformatic analy-
sis was performed using the Ribosomal Database Project (RDP) Multiclassifier tool and
Metagenome@KIN software (World Fusion Co., Ltd., Tokyo, Japan) based on data from
bacterial species as determined by RDP taxonomic analysis.
Nutrients 2022,14, 581 6 of 14
2.6.4. Urinary Isoflavones
Measurement of urinary isoflavones (genistein, daidzein, and equol) was performed
according to the previous procedures [
12
]. Briefly, the mixture containing 800
µ
L urine,
80
µ
L 1 M sodium acetate and 8
µ
L
β
-glucuronidase/sulfatase solution was hydrolyzed,
followed by addition of 80
µ
L of propyl 4-hydroxybenzoate as an internal standard, and
then the analytes were extracted. A 20
µ
L of the residue dissolved in 400
µ
L of methanol
was subjected to a high-performance liquid chromatography system (Shimadzu Co., Ltd.,
Koto, Japan) and evaluated under the previous conditions [
12
]. The detection limit of
urinary isoflavones was as follows; 100 ng/mL for genistein and daidzein, and 200 ng/mL
for equol. The urinary isoflavones were corrected with urinary creatinine (expressed as
mg/g-Cre
). The equol was corrected for the presence of daidzein (equol/daidzein ex-
pressed as
g/g-D
). The metabolic efficiency of daidzein-to-equol conversion was calculated
as equol/ equol + daidzein (expressed as g/g-E + D) [33].
2.7. Statistical Analysis
Throughout the study, we basically performed statistical analysis with nonparametric
analyses unless otherwise indicated whenever necessary. The normality test was performed
using the Kolmogorov–Smirnov method to assess the normal distribution. Differences
in the preintervention biomarker concentrations between the MIU and mineral water
(control) groups were identified by the Mann–Whitney U test. There were no significant
differences in the baseline characteristics of the participants among the two groups (Table 1).
The measured values of all biomarkers of the 2 groups before and after the intervention
were compared using the Wilcoxon signed-rank test (p< 0.05) as appropriate (Table 2).
Fecal formic acid was excluded from the statistical analysis due to the limited number
of samples. Based on the differences in the changes to fecal biomarker concentrations
throughout the intervention period, multiple logistic regression analysis was performed
to evaluate the relationship between the water types and fecal biomarkers (p< 0.05).
Multiple logistic regression analysis was performed using adequate data excluding extreme
values. Participants with detectable (
≥
200 ng/mL) and undetectable equol levels were
classified as equol producers and equol nonproducers, respectively. Equol nonproducers
were excluded from the statistical analysis for equol level assessment. Daidzein-to-equol
conversion efficiency and relative abundance of equol-producing bacteria detected in the
equol producers were assessed using the Wilcoxon signed-rank test. All analyses were
performed with BellCurve for Excel ver. 3.20 (Social Survey Research Information Co., Ltd.,
Tokyo, Japan).
Table 2. The values of fecal biomarkers in the 2 intervention groups.
MIU (n= 41) Mineral Water (Control) (n= 41)
Preintervention Postintervention Preintervention Postintervention
sIgA (µg/g) 408 (209–651) 515 (319–1039) 555 (169–1042) 479 (215–893)
Putrefaction (µg/g)
Phenol 1.30 (0.50–6.67) 1.75 (0.85–6.20) 1.40 (0.60–7.20) 1.63 (0.75–4.70) ↑*
p-Cresol 28.19 (9.15–69.45) 42.56 (12.60–95.72) 59.18 (21.20–90.97) 44.80 (18.22–105.77)
4-Ethylphenol 2.32 (1.62–4.15) 1.92 (0.70–2.20) 1.68 (0.70–2.90) 2.24 (0.75–8.45)
Indol 19.36 (11.80–31.75) 21.44 (14.57–41.10) ↑* 22.79 (11.85–35.90) 18.78 (8.50–28.45)
Skatol 2.76 (1.20–7.80) 1.98 (1.30–6.85) 4.81 (1.35–10.00) 5.50 (2.30–14.75)
SCFA (mg/g)
Succinic acid 0.19 (0.09–0.46) 0.11 (0.080–0.220) ↓** 0.12 (0.08–0.23) 0.14 (0.085–0.345) ↑*
Lactic acid 0.19 (0.08–0.68) 0.12 (0.10–0.24) ↓* 0.11 (0.08–0.17) 0.17 (0.08–0.53)
Formic acid 0.24 N/A 0.18 N/A 0.25 N/A 0.25 N/A
Acetic acid 3.19 (1.85–4.02) 3.00 (1.94–4.21) 2.63 (1.97–3.66) 1.99 (1.72–3.20) ↓**
Propionic acid 1.02 (0.75–1.28) 1.19 (0.81–1.53) 1.12 (0.87–1.57) 0.99 (0.67–1.26) ↓**
Isobutyric acid 0.15 (0.12–0.20) 0.15 (0.12–0.20) 0.15 (0.13–0.19) 0.15 (0.12–0.21)
Butyric acid 0.77 (0.54–1.26) 0.89 (0.51–1.11) 0.84 (0.54–1.48) 0.61 (0.33–1.04) ↓**
3-Methylbutanoic acid 0.19 (0.14–0.26) 0.20 (0.14–0.33) 0.20 (0.15–0.27) 0.25 (0.17–0.34)
Valeric acid 0.21 (0.13–0.31) 0.20 (0.14–0.27) 0.17 (0.14–0.29) 0.21 (0.15–0.32)
Nutrients 2022,14, 581 7 of 14
Table 2. Cont.
MIU (n= 41) Mineral Water (Control) (n= 41)
Preintervention Postintervention Preintervention Postintervention
Urine Isoflavones (n= 20) (n= 12)
Equol (mg/g-Cre) 0.50 (0.00–1.73) 1.90 (0.50–5.53) 0.98 (0.35–2.14) 1.30 (0.14–2.00)
Equol (g/g-Da) 0.72 (0.00–1.72) 2.74 (0.53–4.06) 1.82 (0.37–3.01) 0.31 (0.00–2.60)
Equol (g/g-E + D) 0.42 (0.00–0.63) 0.73 (0.34–0.80) ↑# 0.63 (0.27–0.74) 0.25 (0.17–0.80)
*, p< 0.05; **, p< 0.01; #, p< 0.1; N/A, less than n= 6; The data was shown as median and IQR in parentheses.;
non-parametric analysis (Wilcoxon rank sum test); ↑: increase; ↓; decrease.
3. Results
3.1. Self-Administered Questionnaire
The questionnaire (n = 86) revealed that three and two participants in the MIU and
control groups, respectively, suffered from constipation as per the guidelines of the World
Gastroenterology Organization prior to the study [
31
]. Drinking water ameliorated the
symptoms of constipation in all (100%) and none (0%) of the individuals in the MIU and
control groups, respectively. Improvement in constipation was observed only in the MIU
group, although the small number of samples limited the suitability of the definition
of constipation.
3.2. Analysis of Fecal sIgA, Putrefactive Products, and SCFAs
The values of fecal biomarkers throughout the intervention period are summarized in
Table 2. Overall, in the postintervention period, the sIgA concentration increased in the MIU
group and decreased in the control group compared with the concentrations in preinterven-
tion period. Thus, the preintervention data of the two subgroups were analyzed in detail
to evaluate the sustainable effect on intestinal immune status between the low and high
sIgA subgroups with the median (<500 and
≥
500
µ
g/g, respectively). The postintervention
sIgA concentration significantly increased in both low-value subgroups. Conversely, the
sIgA concentration significantly decreased in the high-value subgroup of the control group
but remained unchanged in the high-value subgroup of the MIU group(Figure 2). Among
putrefactive products in the postintervention period, indol significantly increased in the
MIU group and phenol significantly increased in the control group.
Differences in the changes in SCFAs concentrations throughout the intervention pe-
riod were analyzed using the Mann–Whitney U test (Table 2). In few minor SCFAs, an
increase/decrease of amounts was observed. In particular, the levels of succinic acid and
lactic acid decreased in the postintervention period in the MIU group but not in the control
group. However, overall, the differences in the changes in the nine SCFAs among the two
groups were similar. On the other hand, the total amounts of the three major SCFAs (acetic,
propionic, and butyric acids) slightly increased during the postintervention period in the
MIU group. A decrease was only observed in the control group (p< 0.1, Wilcoxon signed-
rank test), with a 23% difference between the two groups (Figure 3a). The populations of the
responders whose concentrations of three major SCFAs increased in the postintervention
period were significantly higher in the MIU group than in the control group, as determined
by the Chi-squared test (Figure 3b). Notably, there were no significant differences between
males and females.
Based on the differences in the changes to the fecal biomarker concentrations through-
out the intervention period, multiple logistic regression analysis was performed to evaluate
the relationship between the two types of water and fecal biomarkers. The results revealed
that MIU significantly affected only one biomarker (butyric acid). In addition, MIU more
noticeably impacted the intestinal concentrations of the three major SCFAs. Among the
82 participants, formic acid was detected in the range of 0.01–0.02 mg/mL (limit of de-
tection, 0.01 mg/mL) in relatively few samples, suggesting that only minute amounts of
formic acid are produced in the human intestine.
Nutrients 2022,14, 581 8 of 14
Figure 2.
Differences in the changes to sIgA concentrations throughout the intervention period in the
MIU (
a
) and control (
b
) groups. Participants in the MIU and control groups was further classified
into 2 subgroups: low and high sIgA preintervention levels (<500 vs.
≥
500
µ
g/g). The concentration
of sIgA throughout the intervention period significantly increased in both low-value subgroups
irrespective of the water type. However, sIgA significantly decreased in the high-value subgroup of
the control group but remained unchanged in the MIU group. Open bar, preintervention; hatched
bar, postintervention. Bar depicts standard deviation. * p< 0.05; ** p< 0.01.
Figure 3.
Effect of MIU or control water on fecal biomarker concentrations of 3 SCFAs (acetic acid,
propionic acid, and butyric acid) throughout the intervention period. (
a
) The concentrations of the
SCFAs decreased in the control group (* p< 0.1). There was a 23% difference between the 2 groups.
The top and bottom of each box indicate the 25th and 75th percentiles, and the solid line within the
box is a median. Whiskers depict the minimum and maximum values. pre, preintervention; post,
postintervention, (
b
) The proportions of responders were significantly higher in the MIU group than
in the control group. * p< 0.05; ** p< 0.01.
Nutrients 2022,14, 581 9 of 14
3.3. Analysis of Urinary Isoflavones
The results of urinary isoflavone analysis of the 41 and 42 participants in the MIU
and control groups, respectively, are presented in Figure 1and Table 1. The focus of this
analysis was the differences in the changes to equol concentrations, which is among the
most physiologically active isoflavones [
34
]. Throughout the intervention period, urinary
equol was detected in 20 and 12 participants (who were identified as equol producers) in
the MIU and control groups, respectively. Overall, equol was identified in 38.6% (32/83)
of participants. Interestingly, among the 32 equol producers, 8 participants (6 in the
MIU group and 2 in the control group, respectively) became equol producers during the
intervention period.
If urinary isoflavones were not detected in a sample, the value was considered 0.
The equol value was corrected with creatinine (mg/g-Cre) and daidzein (g/g-D). In ad-
dition, the metabolic efficiency of daidzein-to-equol conversion was calculated as equol/
equol + daidzein (g/g-E + D), as mentioned above. The changes in equol concentrations
throughout the intervention period between the two groups are presented in Table 2.
All three evaluations revealed increased equol concentrations in the MIU group. The
metabolic efficiency of daidzein-to-equol conversion significantly increased in the MIU
group compared with the control group (p< 0.1, Wilcoxon) (Figure 4).
Figure 4.
Effect of MIU or control water on the metabolic efficiency of daidzein-to-equol conversion
throughout the intervention period. The metabolic efficiency of daidzein-to-equol conversion was
significantly prompted in the MIU group. The top and bottom of each box indicate the 25th and
75th percentiles, and the solid line within the box is the median. Whiskers depict the minimum and
maximum values. pre, preintervention; post, postintervention, * p< 0.1.
3.4. Analysis of Fecal Microbiota in Equol Producers
Fecal microbiota analysis of the 32 equol producers identified 15 equol-producing
bacteria (Table 3) [
34
,
35
]. The metabolic efficiency of daidzein-to-equol conversion was
significantly greater in the equol producers in the MIU group as compared to the control
group throughout the intervention period. Thus, relative abundance of equol-producing
bacteria detected in the equol producers was compared before and after drinking MIU.
Of 15 equol-producing bacteria, the median of relative abundance of Bacteroides ovatus
especially increased from 0.064% (IQR, 0.021–0.262%) to 0.126% (0.034–0.217%) without
statistical significance (Wilcoxon signed-rank test).
Nutrients 2022,14, 581 10 of 14
Table 3.
Detection of equol-producing bacteria in the 32 subjects whose urinary equol levels were
detected in this study.
Adlercreutzia equolifaciens Asaccharobacter celatus Bacteroides ovatus
Bifidobacterium animalis Bifidobacterium breve Bifidobacterium longum
Finegoldia magna Lactobacillus graminis Lactobacillus intestinalis *
Lactobacillus mucosae Lactobacillus sakei Pediococcus pentosaceus *
Slackia equolifaciens Slackia isoflavoniconvertens Streptococcus intermedius
Twenty and 12 from MIU and control groups, respectively. * Not detected in control group.
Furthermore, the differences in intestinal microbes were analyzed, except for 15 known
equol-producing bacteria in the 8 participants who became equol producers during the
intervention period. Intestinal microbiota analysis was able to be performed in 5 of the
8 participants (4 in the MIU group and 1 in the control group, respectively). The results
revealed that the relative abundances of Blautia wexlerae and Streptococcus cristatus increased
throughout the intervention period in the five equol producers (Table 4).
Table 4. List of the increased bacteria detected postintervention in 5 equol producers.
Blautia wexlerae Streptococcus cristatus
Increased bacteria detected in 4 of 5 equol producers
Blautia faecis Butyricicoccus desmolans Clostridium aldenense
Clostridium bolteae Eggerthella lenta Enterococcus avium
Eubacterium hallii Fusobacterium varium Gemella sanguinis
Lactococcus lactis Murimonas intestini Ruminococcus lactaris
Solobacterium moorei
4. Discussion
Current world trends are focused on the balanced intake of foods and beverages to
promote human health. However, the quality of products currently available in the market
is questionable because of a lack of clinical studies. RDSW (hardness, 1000) is reported
to improve the intestinal environment [
12
]. In this study, the average of total amount of
the three SCFAs slightly increased in MIU (hardness, 88); however, MIU mainly increased
sIgA production as a fecal biomarker. It also improved the metabolic efficiency of daidzein-
to-equol conversion and, subsequently, the intestinal environment. Intriguingly, MIU
significantly induced sIgA secretion, and the increased level was continuously maintained
irrespective of the sIgA level in the preintervention period. However, this was not seen in
the control group, indicating that MIU sustainably maintains the intestinal immune status
with inducible sIgA. Furthermore, the metabolic efficiency of daidzein-to-equol conversion
was accelerated in the MIU group. These findings were not observed in a previous clinical
study with RDSW [
12
]. We previously found increased concentrations of five SCFAs in the
RDSW. Thus, the influences differed even with similar DSW-based drinking water, which
was likely due to the hardness and manufacturing process. The reference values of the
constituents (i.e., putrefaction, SCFA, isoflavones) measured in this study are not defined
at present. New investigations could assess these effects on immune and inflammatory
responses, gut microbiota, and microbial products in healthy adults. However, at least in
healthy persons, the increased constituents (IgA and SCFAs) are considered as beneficial to
the body and not a disadvantage. We found no adverse events during this clinical study.
It is generally accepted that isoflavones can ameliorate the symptoms of various syn-
dromes and diseases, including cancers. In particular, the physiological activities of equol
produced by bacterial daidzein conversion in the intestine can reduce the risk of several
diseases [
34
]. Epidemiological evidence suggests that equol production (~30–50% world-
wide) is related to environmental factors, dietary habits, and gut microbes that convert
daidzein to equol (daidzein-to-equol conversion). To date, a limited number of bacte-
ria capable of daidzein-to-equol conversion have been identified [
34
,
35
]. Thus, further
studies are warranted to identify foods, beverages, and as-yet unidentified bacteria capa-
Nutrients 2022,14, 581 11 of 14
ble of daidzein-to-equol conversion to improve the intestinal environment and maintain
human health.
In this study, MIU increased the metabolic efficiency of daidzein-to-equol conver-
sion, which benefited human health by improving the intestinal environment. Of the
15 equol-producing bacteria detected in 32 subjects, the relative abundance of B. ovatus
especially increased in the MIU group without statistical significance, suggesting that
MIU intake influenced the equol-producing bacteria, including B. ovatus. Furthermore,
fecal microbiota analysis of five participants who became equol producers throughout the
intervention period demonstrated that the proportions of B. wexlerae and S. cristatus had
significantly increased in the postintervention period. Blautia spp., which is dominant in
the human intestine, can ameliorate the symptoms of inflammatory and metabolic diseases
and improve antibacterial activity [
36
,
37
]. These physiological activities of probiotics are
beneficial to human health [
38
]. In particular, B.wexlerae reduces inflammation associated
with obesity-related complications and produces acetic acid as a final product of glucose
fermentation [
38
,
39
]. The relative abundance of B.wexlerae in the postintervention period
was observed in all five equol producers, suggesting that B.wexlerae could be involved
in equol production. However, no study has investigated the relationship between equol
production and Blautia spp. in the intestine. Hence, further studies are needed to inves-
tigate the contribution of B.wexlerae to equol production and/or the metabolic efficiency
of daidzein-to-equol conversion. Previous studies have reported that S. cristatus peptides
repressed expression of the virulence genes of Porphyromonas gingivalis, which had an
inhibitory effect on the oral microbiota [
40
,
41
]. The physiological function of S. cristatus
is mostly unknown; thus,
in vitro
studies are warranted to investigate the activities of
S. cristatus in the intestinal environment to clarify the connection with equol. In addition, to
evaluate the improvement of intestinal microbiota, all 82 fecal samples collected during the
preintervention period were subjected to PCR analysis with specific primers for the amplifi-
cation of methicillin-resistant Staphylococcus aureus (MRSA) [
42
]. The results demonstrated
that three participants in the MIU group were healthy MRSA-carriers and all three were
free of MRSA postintervention, indicating that MIU eventually cleared MRSA from the
intestine, although the results were limited by the small number of MRSA carriers (data not
shown). Furthermore, an increased concentration of indol was observed postintervention
in the MIU group. Indol is a metabolite produced from tryptophan by intestinal microbiota,
and this thus suggests that MIU intake influenced the intestinal microbiota/condition.
The health effects of isoflavones, including equol, are dependent on the quantity and
bioavailability of absorbed nutrients [
43
]. Isoflavones are converted to aglycones (daidzein
and genistein) from glycones (daidzin and genistin) by enzymes (i.e.,
β
-glucosidase) in
intestinal microbes/conditions. The aglycones absorbed in the body physiological func-
tion. Thus, we measured urinary isoflavones (daidzein, genistein, and equol) to evaluate
the health effects. The lifestyle factors of the participants were strictly monitored to as-
sess dietary habits and the consumption of unusual foods, beverages, and supplements
throughout the study period. MIU probably influenced not only the metabolic efficiency of
daidzein-to-equol conversion but also the absorption efficiency via the intestinal environ-
ment. An improvement of constipation was suggested in the MIU group only, although
no significant difference was observed due to the small number of participants fitting the
definition of constipation. Taken together, the findings of this study indicate that MIU
intake induces these biomarkers in the intestinal environment.
5. Conclusions
This clinical study revealed that a long-term intervention with MIU mainly induced
sIgA production and increased the metabolic conversion of daidzein-to-equol, suggesting
an adaptation of the host/microbe which influences human health.
Nutrients 2022,14, 581 12 of 14
Supplementary Materials:
The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/nu14030581/s1, Table S1: Nutrition facts of Dydo-MIU, hardness,
88 (100 mL).
Author Contributions:
Conceptualization, H.T. (Hiroaki Takeuchi); methodology (clinical trial), H.T.
(Hirotsugu Takenaka), S.I., R.M. and A.O.; software and validation, Y.Y. and S.S.; formal analysis,
A.K. and J.M.I.; investigation, Y.H. and R.Y.; resources, S.I.; data curation, K.Y.; writing—original
draft preparation, review and editing, H.T. (Hiroaki Takeuchi); visualization, Y.Y.; supervision, H.T.
(Hiroaki Takeuchi) and Y.Y.; project administration, S.I.; funding acquisition, H.T. (Hiroaki Takeuchi)
All authors have read and agreed to the published version of the manuscript.
Funding:
This research was performed as a practice-based regional employment creation project
and was funded by the Ministry of Health, Labor and Welfare in Japan. This work was partially
supported by JSPS KAKENHI Grant Number 20K07857.
Institutional Review Board Statement:
The study protocol, although severely restricted in terms
of time and budget, was approved by the Ethics Committees of Kochi University (approval no.
28–93) and the International University of Health and Welfare (approval no. 18-lo-100) and was
conducted in accordance with the ethical standards described in the 1964 Declaration of Helsinki and
its later amendments.
Informed Consent Statement:
Informed consent was obtained from all subjects involved in the study.
Acknowledgments:
We thank the Kochi government office and the Muroto city office for supporting
this industry–academia–government collaboration project.
Conflicts of Interest: The authors declare no conflict of interest.
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