nutrients
ArticleDrinking Refined Deep-Sea Water Improves the Gut
Ecosystem with Beneficial Effects on Intestinal Healthin Humans: A Randomized Double-Blind
Controlled Trial
Hiroaki Takeuchi 1,2, *, Keiro Higuchi 3 , Yu Yoshikane 4 , Ryo Takagi 2 , Shinji Tokuhiro 2 ,Koichi Takenaka 5 , Wataru Oboshi 1 , Asako Kimura 1 , Jahirul Md. Islam 1 , Ayami Kaneko 1 ,Shouichi Sato 1 and Satoshi Ishizuka 3
1

2
3
4
5

*

Department of Medical Laboratory Sciences, Health and Sciences, International University of Health andWelfare Graduate School, 4-3 Kouzunomori, Narita-City 286-8686, Chiba, Japan;
oboshi@iuhw.ac.jp (W.O.); a-kimura@iuhw.ac.jp (A.K.); jahir3683@gmail.com (J.M.I.);
1657021@g.iuhw.ac.jp (A.K.); s-shouichi@iuhw.ac.jp (S.S.)
Department of Kochi Medical School, Kochi University, Kohasu, Oko-cho, Nankoku-City 783-8505, Kochi,
Japan; rtakagi@ako-kasei.co.jp (R.T.); jm-tokuhirosh@kochi-u.ac.jp (S.T.)
Center for Regional Collaboration, Kochi University, 2-17-47 Asakurahonmachi, Kochi-City 780-8073, Kochi,
Japan; khiguchi@river.ocn.ne.jp (K.H.); zuka@kochi-u.ac.jp (S.I.)
Department 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
DyDo-T Beverage Co. Ltd., 1310-1 Hanechou-ko, Muroto-City 781-6741, Kochi, Japan;
takenaka@dt-beverage.com
Correspondence: htake@iuhw.ac.jp; Tel.: +81-476-20-7762

Received: 10 July 2020; Accepted: 27 August 2020; Published: 31 August 2020


Abstract: World health trends are focusing on a balanced food and beverage intake for healthy
life. Refined deep-sea water (RDSW), obtained from deep-sea water collected offshore in Muroto(Japan), is mineral-rich drinking water. We previously reported that drinking RDSW improves humangut health. Here, we analyzed the effect of drinking RDSW on the gut ecosystem to understandthis effect. This was a randomized double-blind controlled trial. Ninety-eight healthy adults weredivided into two groups: RDSW or mineral water (control). The participants consumed 1 L of either
water type daily for 12 weeks. A self-administered questionnaire and stool and urine samples werecollected through the intervention. The following were determined: fecal biomarkers of secretory
immunoglobulin A (sIgA), five putrefactive products, and nine short-chain-fatty-acids (SCFAs) as
the primary outcomes; and three urinary isoflavones and the questionnaire as secondary outcomes.
In post-intervention in the RDSW group, we found increased concentrations of five SCFAs anddecreased concentrations of phenol and sIgA (p < 0.05). The multiple logistic analysis demonstrated
that RDSW significantly affected two biomarkers (acetic and 3-methylbutanoic acids) of the five
SCFAs mentioned above (p < 0.05). Similarly, the concentrations of urinary isoflavones tended toincrease in post-intervention in the RDSW group. Constipation was significantly alleviated in the
RDSW group (94%) compared with the control group (60%). Drinking RDSW improves the intestinal
environment, increasing fecal SCFAs and urinary isoflavones, which leads to broad beneficial effects
in human.
Keywords: healthy life; refined deep-sea water; abdominal discomfort; short-chain-fatty-acid;isoflavone; gut ecosystem

Nutrients 2020, 12, 2646; doi:10.3390/nu12092646

www.mdpi.com/journal/nutrients

Nutrients 2020, 12, 2646

2 of 15

1. Introduction
Current world trends in healthy life in human focus on a balanced intake of safe foods andbeverages. We purchase many foods and beverages on the market. However, some such products,
promoted without suitable verification, may not exert any beneficial effect or may even be harmful.
Deep-sea water (DSW) is obtained from depths of over 200 m. It is highly pure, with low
temperature stability, high mineral concentration, and active nutritional species [1]. Bottled commercial
DSW-based drinking water obtained by different methods, e.g., desalinization, is available on themarket. According to studies involving animal models, the DSW-based drinking water exerts
hypocholesterolemic and antiatherogenic effects [2], probably by modulating lipid metabolism [3,4].
It exerts additional beneficial effects including inhibition of hyperlipemia [2], hypertension [5],obesity [6], diabetic mellitus [7], duodenal ulcer [8], and cataract formation [9,10].
We previously performed clinical trials to investigate the effects of refined deep-sea water (RDSW),
a commercial bottled DSW-based drinking water, made from DSW collected offshore in Muroto(Kochi Prefecture, Japan) in humans. RDSW is widely consumed in Japan for reasons beyond simple
hydration. Clinical trials confirmed diverse beneficial effects of RDSW in humans in broad areas
such as hemorheology, allergy, immunology, and infectious diseases (e.g., anti-Helicobacter pylori
activity) [11–17]. No adverse effects of even long-term consumption (one year) of 1 L/day were reported
in any clinical trials. RDSW alleviates constipation and abdominal discomfort, and is also safe for
concurrent administration with pharmaceutical agents [18]. Hence, drinking RDSW is beneficial
for humans and its use is expanding to include the cosmetic industry and public health. However,
the mechanism through which RDSW exerts such various beneficial effects in human is unclear.
The gut ecosystem is composed of microbiota, microbiota-derived metabolites, and the ingesta,
and incorporates the microbe–microbe and host–microbe interactions. It plays a fundamental role in thewell-being of human and influences the development of various diseases, including neuropsychiatricdisorders [19–24]. Many factors of human life, such as the working and living conditions, and diet, affect
the gut ecosystem. Probiotics, prebiotics, and functional and/or healthy foods, including fabaceous
meals, are widely consumed to maintain and support the intestinal environment. Similar, there isa growing research interest worldwide into fecal microbiota transplantation, as a natural clinicaltreatment for gastrointestinal diseases that modulates the gut microbiota [25–27]. The application of
fecal microbiota transplantation has been expanded to various clinical fields, such as autoimmune
disease, hepatitis, metabolic syndrome, and mental disorders [28–31].
Gut microbes produce metabolites, such as isoflavones. Equol is a metabolite of the soy isoflavone
daidzein. It exerts the highest physiological effect of all isoflavones on the host health. The productionof equol is limited by the existence of specific bacteria and therefore, equol levels depend on thegut microenvironment [32]. One of the current research topics is how the gut environment could be
supported to enhance equol production.
Collectively, the above indicate that the gut ecosystem plays an important role in human, like anyorgans in the body, critically influencing the human health. The aim of the current clinical trial was toinvestigate whether RDSW influences the biomarkers associated with the human gut ecosystem, toprovide new insights into the understanding of the broad beneficial effects of drinking RDSW.
2. Materials and Methods
2.1. Clinical Study Design
The study was designed as a randomized double-blind controlled trial to compare the human gut
ecosystem in individuals drinking RDSW or mineral water using a self-administered questionnaireand stool and urine analyses. The study was performed in 2016–2018 at Muroto, Kochi, Japan.
The study, having severe restrictions on term and budget, was approved as feasible by the ethics
committee of Kochi University (approval number 28-93), and was therefore conducted in accordancewith the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.

Nutrients2020,
2020,12,
12,2646
x FOR PEER REVIEW
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of15
16
33of

accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later
amendments.
The questionnaire
stool
and urine
sampleswerecollected
the experimentThe
questionnaireand stool andandurine
sampleswerecollected
throughthethroughexperiment(pre- and(pre- and post-intervention).
Thetrial
clinicalwas registered
Japan Primary
Registries
post-intervention).
The clinical
was trial
registeredin Japan inPrimary
Registries
NetworkNetwork(JPRN)(JPRN) (UMIN000022002
2016/04/20).
We on
focusedon the inchangesin fecal biomarker
concentrations(UMIN000022002
2016/04/20).
We focused
the changes
fecal biomarker
concentrations
throughthroughthe intervention
in asthisas outcome,the primary
outcome,and thequestionnairetheintervention
in this study
the study
primary
and thequestionnaire(theimprovement(theofimprovementandof constipation)
andconcentrations
urine biomarker
as the secondary outcomes.
constipation)
urine biomarker
as concentrations
the secondary outcomes.
2.2.
2.2. Subjects
Subjects
A
A totaltotal ofof115
115participants
participantswere
wereenrolled
enrolledin
inthisthisclinicalclinicalstudy
study(industry–academia–government(industry–academia–government
collaboration
Muroto,Kochi,
Japan(Figure1). We1).
gathered
the healthy
excludedcollaborationproject)
project)in in
Muroto,Kochi,
Japan(FigureWe gathered
the adults;
healthyweadults;
weparticipants
with any illnesses
and illnesses
any prescription
andcommercialanddrugsuse. The pregnant
persons
excluded participants
with any
and anyprescriptioncommercial
drugs use.
The
andsupplement
users
were
also excluded.
obtaining
written informed
consent,written
98 healthy
adultspregnant
personsandsupplement
usersAfter
werealso excluded.
After obtaining
informedwererandomly
dividedintowere
two randomly
groups: a divided
group drinking(n a= group
49; meanage: 48RDSW
years;(nrange:
consent,
98 healthy
adultsinto twoRDSW
groups:drinking
= 49;
27–72
years;
19
men
and
30
women)
andagroup
drinking
mineral
water
(n
=
49;
mean
age:
44
years;
mean age: 48 years; range: 27–72 years; 19 men and 30 women) and a group drinking mineral water
range
22–71
years;
andrange
28 women).
lattergroupandwas28thecontrol group.
Thegroupcharacteristics(n = 49;mean
age:2144men
years;
22–71 The
years;
21 menwomen).
The latter
was theofparticipants
this characteristics
clinical study are
in Table
Theclinicaldifferencesin pre-intervention
items1.such
controlgroup.inTheof shown
participants
in1.thisstudy
are shown in Table
The
as
age,
sex,
body
mass
index
(BMI),
andbiomarker
concentrations
betweenthetwogroupsallocateddifferences in pre-intervention items such as age, sex, body mass index (BMI), and biomarker
wereanalyzed using
the Mann–Whitneytest. These
showedno statisticaldifferences
and these
concentrations
betweenthe two groupsUallocatedwereanalyzedusing theMann–Whitney
U datatest.
werein thenofollowing
Theseusedshowedstatisticalstudy.
differences and these data were used in the following study.

FigureFigure 1.1. A
A totaltotal 98
98 healthy
healthy adults
adults were
were enrolled
enrolled fromfrom 115
115 adults
adults who
who participatedparticipated inin thisthis clinicalclinicalstudy(industry–academia–government
collaboration
project)
heldin
Muroto-city,
Kochi,
Japan.
study (industry–academia–government collaboration project) held in Muroto-city, Kochi, Japan.
The
The
subjects
werehealthy
adultswithoutanyillnesses,and
andnonoprescription
prescriptionand
andcommercial
commercial drugs
drugs use.
subjects
werehealthy
adultswithoutanyillnesses,
use.
Pregnant
supplement
users
were
excluded
fromfrom
this study.
RDSW,
refinedrefined
deep-seawater.
Pregnantpersons
personsand
andsupplement
users
were
excludedthis study.
RDSW,
deep-seawater.

Nutrients 2020, 12, 2646

4 of 15

Table 1. Characteristics of participants in pre-intervention of two groups (average).
RDSW

Age (year)
BMI (kg/m2 )(Body Mass Index)

sIgA (µg/g) (secretory immunoglobulin A)

Mineral Water (Control)

Total(n = 49)Men(n = 19)

Women(n = 30)

Total(n = 49)Men(n = 21)

Women(n = 28)

48.1

45.9

49.4

44.2

44.2

44.2

24.8

26.8

23.6

24.1

25.6

23.1

Total
(n = 49)Men(n = 19)

Women(n = 30)

Total(n = 46)Men(n = 19)

Women(n = 27)

656

810

558

536

571

512

6.01
51.59
0.60
25.93
2.69

10.02
43.78
0.36
26.31
2.31

3.47
56.53
0.75
25.69
2.94

5.72
62.78
1.03
27.08
3.69

9.63
27.26
0.70
20.57
2.72

3.68
87.78
1.26
31.67
4.37

0.39
0.02
0.00
2.73
1.22
0.10
0.95
0.19
0.20

0.65
0.04
0.00
3.09
1.48
0.10
0.98
0.18
0.21

0.22
0.01
0.01
2.50
1.05
0.10
0.93
0.20
0.19

0.30
0.06
0.00
2.88
1.29
0.11
0.87
0.22
0.18

0.33
0.12
0.01
3.43
1.40
0.08
0.86
0.14
0.16

0.29
0.02
0.00
2.49
1.21
0.13
0.87
0.28
0.20

Total
(n = 48Men(n = 18)

Women(n = 30)

Total(n = 48)Men(n = 21)

Women(n = 27)

1.74
2.16

1.98
2.34

1.56
2.04

3.28
3.99

2.50
3.27

3.89
4.55

Total
(n = 19)Men(n = 6)

Women(n = 13)Total(n = 17)Men(n = 9)Women(n = 8)

2.57

2.53

2.58

2.30

1.98

2.66

Putrefaction (µg/g)
Phenolp-Cresol
4-Ethylphenol
Indol
SkatolSCFA (short-chain-fatty-acids, mg/g)
Succinic acid
Lactic acid
Formic acidAcetic acid
Propionic acid
Isobutyric acid
Butyric acid
3-Methylbutanoic acid
Valeric acid

Urine Isoflavones
Daidzein (mg/g-Cre)
Genistein (mg/g-Cre)

Equol (mg/g-Cre)

2.3. Ingestion Schedule
Bottled RDSW, Amami no Mizu, Hardness 1000 (Ako Kasei Co., Ltd., Hyogo, Japan) [15],and mineral water, the most popular water consumed in Japan, were used. Both waters include no
calory, protein, fat, carbohydrate and vitamin. RDSW and mineral water were hard- and soft-typewater, respectively. We purchased both bottled waters on the market, changed the labels in the project
office, and prepared the masked water for this study. The participants consumed 1 L of either watertype, daily, for 12 weeks.
2.4. Evaluation
A self-administered questionnaire was used to assess the general gut health status of theparticipants. The following biomarkers were analyzed in the collected fecal samples: secretory
immunoglobulin A (sIgA), five putrefactive products (phenol, p-cresol, 4-ethylphenol, indole,
and skatole), and nine short-chain-fatty-acids (SCFAs, succinic, lactic, formic, acetic, propionic,
isobutyric, butyric, 3-methylbutanoic, and valeric acids). The analyses were performed at TechnoSuruga
Laboratory Co., Ltd. (Shizuoka, Japan) [33,34]. Three isoflavones (genistein, daidzein, and equol) wereanalyzed in urine. We analyzed the data in these biomarkers with non-parametric analysis to assessthe differences in the change between pre- and post-interventions. Based on the differences in theNutrients 2020, 12, 2646

5 of 15change in fecal biomarker concentrations through the intervention, we performed multiple logistic
regression analysis to evaluate the relationship between these waters and biomarkers.
Self-Administered Questionnaire
All participants answered questions on their general gut health and eating habits, such as
constipation, abdominal discomfort, medicine intake, and consumption of unusual foods and drinks.
Constipation was defined according to World Gastroenterology Organization [35].
2.5. Measurement of Samples
2.5.1. Measurement of Fecal sIgA
The feces (0.1 g) were placed in a 2.0 mL-tube with zirconia beads and suspended in a mixtureof 0.1 mM perchloric acid with 3% phenol. Samples were heated at 80 ◦ C for 15 min, vortex-mixed
at 5 m/s for 45 s using FastPrep 24 (MP Biomedicals, Santa Ana, CA, USA), and then centrifuged at
15,350× g for 10 min. The supernatant was passed through a 0.45 µm filter. The supernatant was
employed for the measurements of sIgA and SCFAs. The sIgA levels were measured using Human IgA
ELISA Quantitation kit (E80-102, Bethyl Laboratories Inc., Montgomery, TX, USA), according to the
manufacturer’s instructions, and a microreader (Varioskan Flash, Thermo Fisher Scientific, Waltham,
MA, USA).
2.5.2. Measurement of Fecal Putrefactive Products
The feces (0.1 g) was suspended in 2.5 mL of phosphate buffer containing 0.4 mg/L
4-isopropylphenol as an internal standard. Samples were heated at 85 ◦ C for 15 min, mixed with
2.5 mL of acetonitrile and 1 g NaCl, shaken for 30 min, and then centrifuged at 1300× g for 10 min.
Then, 1 mL of the supernatant was dehydrated and purified by passing through a sodium sulfate
drying cartridge (Bond Elut LRC, Agilent Technologies, Tokyo, Japan), a C18 cartridge (Smart SPE
C18-30, AiSTI Science, Wakayama, Japan), and a PSA cartridge (Smart SPE PSA-30, AiSTI Science,
Wakayama, Japan), and placed in a vial.
The indole and phenol levels were determined by gas chromatography/mass spectrometry(QP-2010, Shimadzu, Kyoto, Japan) and a capillary column (Inert cap WAX, 30 m × 0.25 mm × 0.25 µm,
GL Science, Tokyo, Japan). Helium was used as the carrier gas at a flow rate of 1.11 mL/min. The injector
temperature and the interface temperature were maintained at 240 and 230 ◦ C, respectively. The oventemperature program was as follows: 70 ◦ C for 2 min; then 20 ◦ C/min to 200 ◦ C, held for 3 min;10 ◦ C/min to 240 ◦ C; and finally, 240 ◦ C, held for 16 min. For the analysis, 1 µL of the extract was
injected in the splitless mode. The mass spectrometer was operated in electron impact ionization mode
at 70 eV. The measurements were recorded using an absolute calibration curve (range: from 0.02 to
5.0 mg/L). Data were acquired in the selected ion-monitoring mode for quantification.
2.5.3. Measurement of Urinary Isoflavones
Genistein, daidzein, equol, and propyl 4-hydroxybenzoate were purchased from Cayman Chemical,
Combi-Blocks, Wako Pure Chemical, and Daicel, respectively. β-glucuronidase/sulphatase type H-2
from Helix pomatia was purchased from Sigma-Aldrich Japan. All other chemicals were analyticalgrade or HPLC grade. Samples of urinary genistein, daidzein, and equol levels were prepared andmeasured as described elsewhere [36], with modifications. Briefly, urine (800 µL) was mixed with
80 µL of 1 M sodium acetate (pH 4.5) and 8 µL of β-glucuronidase/sulfatase solution (Sigma-Aldrich
Japan), and incubated at 37 ◦ C for 4 h to hydrolyze the conjugated forms of isoflavones. As an internal
standard, 80 µL of propyl 4-hydroxybenzoate (100 µg/mL) was added the mixture, and the analytes
were extracted with 1.5 mL of dichloromethane and evaporated at 50 ◦ C. The residue was dissolvedin 400 µL of methanol; 20 µL were injected into the HPLC system (Shimadzu Co., Ltd., Koto, Japan).
The HPLC conditions were as follows: column, TSK-gel ODS-100V (4.6 mm i.d. × 250 mm, Tosoh Co.,

Nutrients 2020, 12, 2646
Nutrients 2020, 12, x FOR PEER REVIEW

6 of 15
6 of 16

acetate/methanol
mobile phase B, ethyl acetate/methanolv/v); elution,
Ltd.,
Tokyo, Japan);(0.4:800:18:182,
mobile phase v/v);
A, phosphate/water/ethyl
acetate/methanol (20:980,(0.4:800:18:182,v/v); alineargradientof
0–60%
mobile
phase
B
in
30
min;
flow
rate,
1.0
mL/min;columntemperature,mobile phase B, ethyl acetate/methanol (20:980, v/v); elution, a linear gradient of 0–60% mobile phase B40
UV detection,
280mL/min;
nm. Thecolumn
detection
limits wereng/mL
for genistein200◦ C; UVin °C;
30 min;flow rate, 1.0
temperature,
40 100detection,
280 nm.and
Thedaidzein,
detection andlimitsng/mL
forequol.
The
amountsofdaidzeinandgenistein
were
corrected
forthepresenceofcreatinine,
were 100 ng/mL for genistein and daidzein, and 200 ng/mL for equol. The amounts of daidzein andand arewere
expressed
as mg/g-Cre.
The amountof equolwaspresence
ofamount
daidzeingenisteincorrected
for the presenceof creatinine,andarecorrected
expressedfor
as themg/g-Cre.
The
ofas
equol/daidzein,
andexpressed
as
mg/mg
daidzein.
equol was corrected for the presence of daidzein as equol/daidzein, and expressed as mg/mg daidzein.
2.6.
Statistical
Analysis
2.6.
Statistical
Analysis
Participants
wererandomly
assigned
two groups.
Wethe
assessed
the indifferencesin preParticipants
wererandomly
assignedin two ingroups.
We assessed
differences
pre-intervention
intervention
biomarker concentrations
betweenRDSWwaterand mineralgroupsallocated by nonbiomarker
concentrations
between RDSW
and mineralgroups water
allocatedby non-parametricparametricwith Mann–Whitney
U test.
We founddifference
no significant
difference
and thesedata
analysiswith analysis
Mann–Whitney
U test. We foundno significant
and these
data were
utilized
wereutilizedinfollowing
analyses
in
this
study
(Table1).
Forallbiomarkers
inthis
study,
in following analyses in this study (Table 1). For all biomarkers in this study, the measured valuesthemeasuredvalueswere between
staticallyprecompared
between pre- andin thegroupswere
staticallycomparedand post-interventionsin post-interventions
the two groups using
the two
Wilcoxon
using the test
Wilcoxon
signed-rank
testas(significance,< fecal0.05),biomarkers,
as appropriate.
For
fecal
signed-rank(significance,p < 0.05),
appropriate. pFor
formicacid
wasbiomarkers,
excluded
formicwas excluded
fromstatisticalanalysisdueon
to thesmall
size (n =in5).
on inthefrom
the acidstatisticalanalysis dueto thesmall
size (n =5). Baseddifferences
theBasedchangedifferencesin thechange in fecal
biomarker
concentrations
through themultiple
intervention,
weregression
performed
fecalbiomarker
concentrations
throughthe intervention,
we performed
logistic
multiplelogistic
regression
analysistoevaluatetherelationshipbetweenthesewatersfecal
analysis to evaluate the relationship between these waters and fecal biomarkers (significance, pand
< 0.05).
biomarkers(significance,p < 0.05).
Thewasmultiplelogistic
regression
analysis
was performed
using
The
multiple logistic
regression
analysis
performed
using
adequate
data without
the extreme
adequate
withoutof
thetheextremevalues.
Theincreased
proportionof theinresponder
with increased
change invalues.
Thedata
proportion
responderwithchangefecal biomarker
concentrations
fecal biomarker
concentrations
throughusing
the intervention
analyzed using
the χ2-test(significance,
throughthe intervention
was analyzed
the χ2 -test was(significance,p < 0.05).
For theurinaryp < 0.05). For
the urinary
isoflavones,
subjects
withdetectable(≥200equol
ng/mL)
and were
undetectableisoflavones,
subjectswith detectable(≥200
ng/mL)
andundetectablelevelsclassifiedequolaslevelswere classified
as equol producers
and non-producers,
respectively. were
Equolexcluded
non-producers
were
equol
producers
and non-producers,
respectively.
Equol non-producers
from theexcluded
from theanalysisfor equollevel assessment.
All analyses
performed
withstatisticalanalysis
forstatisticalequol levelassessment.
All analyses
were performed
with were
BellCurve
for Excel
BellCurve
for Survey
Excel ver.
3.20 (Social
Survey ResearchCo., Ltd., Tokyo, Japan).
ver.
3.20 (Social
Research
Information
Co., Ltd., Information
Tokyo, Japan).

3. 3.
Results
Results
3.1.
Self-Administered
Questionnaire
3.1.
Self-Administered
Questionnaire

The improvement rate

The
questionnaire(n(n= =98)
The
questionnaire98)revealed
revealedthat
that1717andand1515subjects
subjectsininthe
theRDSW
RDSWandandcontrol
controlgroups,
groups,respectively,
sufferedfromconstipation
prior
to
the
study
per
the
definitionof
constipation.
respectively, suffered from constipation prior to the study per the definition of constipation. Drinking
Drinking
water ameliorated
the symptomsof constipationin 16/17
and (60%)
9/15 (60%)
individuals
water ameliorated
the symptomsof constipationin 16/17(94%)(94%)
and 9/15
individuals
in thein RDSWthe RDSW
andcontrolgroups,respectively.
The
improvement
rate
wassignificantly
higherintheand control groups, respectively. The improvement rate was significantly higher in the RDSW
RDSW
group
than
that
in
the
controlgroup(Figure2).
group than that in the control group (Figure 2).

FigureAlleviationofofconstipation
constipation
drinking
RDSW
or mineralIn theFigure2.2. Alleviation
by bydrinking
RDSW
or mineralwater.water.
In the study,
17 study,
subjects
17 (RDSW
subjects group)(RDSW andgroup)
and15
subjects(controlgroup)
reported
constipation
inthequestionnaire.
15 subjects (control group) reported constipation in the questionnaire. The
The
constipationimprovement
improvementrate
ratewaswas94%
94%andand 60%60% in the RDSW
constipation
RDSW group
groupandandcontrol
controlgroup,group,respectively.
respectively.
Hatchedbar,
RDSWgroup;
black
bar,bar,controlgroup;* p *<p0.05.
Hatchedbar,
RDSWgroup;
black
controlgroup;< 0.05.

3.2. Analysis of Fecal sIgA, Putrefactive Products, and SCFAs
3.2. Analysis of Fecal sIgA, Putrefactive Products, and SCFAs
Three out of 49 subjects in the control group were excluded because of an insufficient volume
Three out of 49 subjects in the control group were excluded because of an insufficient volume ofof feces. Consequently, samples for 49 subjects from the RDSW group and 46 subjects from thefeces. Consequently, samples for 49 subjects from the RDSW group and 46 subjects from the control

Nutrients 2020, 12, 2646

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control group (mean age: 44 years; range: 22–71 years; 19 men and 27 women) were included in the
fecal analysis.
The average values of fecal biomarker concentrations through the intervention are summarizedin Table 2. The concentrations of sIgA and phenol significantly decreased in post-intervention in
the RDSW group per the Wilcoxon signed-rank test. However, we found no statistically significant
differences in the change between pre- and post-intervention in the control group.
Table 2. The values of fecal biomarkers of two groups through the intervention (average).
RDSW (n = 49)

sIgA (µg/g)

Mineral Water (Control) (n = 46)

Pre-Intervention

Post-Intervention

Pre-Intervention

Post-Intervention

656

567 ↓ **

536

544

6.01
51.59
0.60
25.93
2.69

3.08 ↓ *
54.22
1.21
25.99
2.65

5.72
62.78
1.03
27.08
3.69

5.77
61.56
0.46
30.50
3.69

0.39
0.02
0.00
2.73
1.22
0.10
0.95
0.19
0.20

0.47 ↑ *0.05 ↑ *0.00
3.04 ↑ *1.33
0.13 ↑ *0.86
0.23 ↑ *
0.19

0.30
0.06
0.00
2.88
1.29
0.11
0.87
0.22
0.18

0.27
0.03
0.02
2.34 ↓ **1.12 ↓ *
0.12
0.78
0.21
0.17

Putrefaction (µg/g)
Phenolp-Cresol
4-Ethylphenol
Indol
SkatolSCFA (mg/g)
Succinic acid
Lactic acid
Formic acid #Acetic acid
Propionic acid
Isobutyric acid
Butyric acid
3-Methylbutanoic acid
Valeric acid* p < 0.05; ** p < 0.01; ↑, increase; ↓, decrease; # formic acid was excluded from analysis.

Regarding to SCFAs, the differences in the change in SCFAs concentrations through the intervention
were analyzed using the Wilcoxon signed-rank test. These demonstrated that five SCFAs (succinic,
lactic, acetic, isobutyric, and 3-methylbutanoic acids) significantly increased post-intervention in the
RDSW group. An increased change in propionic acids was observed in the RDSW group without
any statistically significant difference. By contrast, the concentrations of two biomarkers (acetic acidand propionic acid) significantly decreased post-intervention in the control group. During this study,
the combined amount of nine SCFAs increased from 5.81 to 6.3 mg/g in the RDSW group and decreased
from 5.92 to 5.05 mg/g in the control group (Table 2, Figure 3a). The combined amounts of SCFAs
through the intervention significantly increased in the RDSW group compared with the control group,with a 23% difference between two groups (Figure 3b). The proportion of subjects with increased
SCFAs concentrations post-intervention is shown in Figure 4a. For all SCFAs, except for formic
acid, the proportion of the responders was higher in the RDSW group than in the control group.
The proportion of acetic acid responder was significantly higher in the RDSW group than in the control
group. Next, the proportion of the responders by sex showed the differences between men and women(Figure 4b). The proportion of acetic acid responder was higher in men than in women in the RDSW
group. However, the proportion of isobutyric acid and 3-methylbutanoic acid responders was higher inwomen than in men in the RDSW group. By contrast, we found no significant difference in the control
group. In addition, based on the differences in the change in fecal biomarker concentrations throughthe intervention, multiple logistic regression analysis was performed to evaluate the relationshipbetween these waters and fecal biomarkers. These revealed that RDSW significantly affected only twobiomarkers (acetic acid and 3-methylbutanoic acid). These observations indicated that drinking RDSW
more noticeably impacted SCFA production in the human gut ecosystem than drinking mineral water.

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16
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The concentration of SCFAs
The concentration of SCFAs

The concentration of SCFAs
The concentration of SCFAs

methylbutanoic acid). These observations indicated that drinking RDSW more noticeably impacted
SCFA productionin thehuman
gut ecosystemthan drinking
mineralwater.
Formicacid wasdetected
methylbutanoicacid).
These
observations
indicatedthat drinking
RDSW
more
noticeably
impactedin samples
fromdetected
only
out
of 95
in athanof95
0.01–0.02
mg/mL
(the
detectable
limit
was(the
0.01
Formicacid wasin samples
from only
5rangeoutdrinkingofsubjects,
inwater.
a rangeof
0.01–0.02
mg/mL
SCFA
production
inthe5human
gutsubjects,
ecosystem
mineralFormicacid wasdetected
mg/mL),
suggesting
that
only
minuteamountsofformicacidareproduced
in
the
human
intestine.
detectable
limit
was
0.01
mg/mL),
suggesting
that
only
minuteamountsofformicacidareproducedinin samples from only 5 out of 95 subjects, in a range of 0.01–0.02 mg/mL (the detectable limit was 0.01
the
human
intestine. that only minute amounts of formic acid are produced in the human intestine.
mg/mL),
suggesting

Figure 3. Effect of drinking RDSW or mineral water on fecal short-chain-fatty-acids (SCFAs)
concentrations
the RDSW
intervention(average).
(a)on
The
combinedamounts of nine
SCFAs
3. Effect
of
water
fecal
short-chain-fatty-acids(SCFAs)Figure3.
Effect throughof drinking
drinking
RDSW oror mineralmineralwateronshort-chain-fatty-acids
increased
in
the
RDSW
group
and
decreasedinthecontrolgroup.
The
following
nine
SCFAs
were
concentrations throughthroughthetheintervention(average).
(a) combined
The combined
amountsof nineSCFAs
concentrations
intervention(average).
(a) Theamountsof nine SCFAs
increased
analyzed,frombottomtotop:
succinic
acid,
lacticacid,
formicacid,
aceticacid,
propionicacid,
inthe RDSW
group
andgroup
decreasedin the control
group.
The
followingnine SCFAs
were
analyzed,
increasedin theRDSW
and decreasedin thecontrolgroup.
The following
nine
SCFAs
were
isobutyric
acid,
butyricacid,
3-methylbutanoic
acid,
and
valeric
acid.
Formicacidisnotlisteddue
frombottomto top:succinic
acid,
lactic acid,acid,
propionic
acid, propionicisobutyric acid,
acid,toanalyzed,frombottomto top:succinic
acid,formic
lactic acid,
acid,acetic
formicacid,
acetic acid,its extremelysmallamounts.
(b)
Thechangeintheandcombinedamounts
SCFAs
observedbutyricacid,
3-methylbutanoic
acid,
and
valeric
acid.
Formicis Formic
notoflisted
to itsextremely
isobutyric
acid,
butyric
acid, 3-methylbutanoic
acid,
valericacid
acid.
aciddueiswas
notlistedduewithtoa23%
difference
betweentwo
groups.
Shadedbar,
allsubjects;
hatchedbar,
RDSWgroup;
black
bar,
small
amounts.
(b) Thechange(b)in Thethe combinedof SCFAs
was observed
with
23% difference
its extremelysmallamounts.
change inamounts
the combined
amountsof SCFAs
wasa observed
withgroup;
pre,pre-intervention;
post,
post-intervention;*hatchedp group;< 0.05.bar,
betweentwo
groups.
Shadedall subjects;
hatchedRDSW
blackbar, control
pre,a control
23% difference
betweentwobar,groups.
Shadedbar, all bar,
subjects;
RDSW
group;group;
black bar,

The proportion of the responder
The proportion of the responder

pre-intervention;
post,
post-intervention;p < 0.05.
control group; pre,pre-intervention;
post,* post-intervention;* p < 0.05.

Figure 4. Cont.

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Figure 4. Participants with increased change in eight SCFAs concentrations post-intervention during
FigureParticipants
withincreasedchangeacid
in eight
concentrations
duringthestudy4.period.
Smallamountsof formic
wereSCFAs
only detected
in fivepost-intervention
subjects; ad therefore,
the
study
period.
Smallamountsofformic
acid
were
only
detected
infivesubjects;
adtherefore,
formic acid was excluded from the analysis. (a) The proportion of eight SCFAs was higher in the RDSW
formicacidinwas
excluded
from theanalysis. (a)The proportionof eight
SCFAs
wasacid
higherin thegroup
thanthe control
group.
In particular,a significant
difference
in only
aceticbetween
RDSW
group
than
in
the
controlgroup.
In
particular,asignificant
differencein
only
aceticacid
the two groups was found. (b) The proportion of the participants by sex. Significant differences
betweenthetwogroupswasfound.
(b)
The
proportionoftheparticipantsbysex.
Significant
between men and women were observed in the RDSW group as follows: acetic acid responders in
differences
betweenmen
women were acidobservedin theinRDSW
as follows:
aceticacidmen,
and isobutyricacid
andand3-methylbutanoic
responders
women.group
Hatchedbar, RDSW
group;
responders
in men,andM,isobutyricacid and* 3-methylbutanoic
acid responders in women. Hatchedblackbar, control
group;
men; W, women;
p < 0.05.
bar, RDSW group; black bar, control group; M, men; W, women; * p < 0.05.

3.3. Analysis of Urinary Isoflavones3.3. Analysis of Urinary Isoflavones
Samples from one subject from each group were excluded because of an insufficient volume ofurine (Figure
Consequently,
from48 subjects
from thebecause
RDSW group(mean age: volume
48 years;ofSamples1).fromone subjectsamples
from eachgroupwere excludedof an insufficient
range:
27–72 years;
18 men andsamples
30 women)48 subjects
fromthe control
group age:
(mean
age:
urine (Figure1). Consequently,
from and48 subjects
from theRDSWgroup (mean
48 years;
44range:years;27–72
range:years;
22–7118
years;men
27 women)wereincluded
thecontrol
urinarygroupisoflavone
analysis.
men21and
30andwomen)
and 48subjects
frominthe(meanage: 44
Daidzein
was not
detectedurine
two subjects
the RDSW
group,
andisoflavone
genistein was
notyears; range:
22–71
years;in
21themen
andfrom
27 women)were inincludedin theurinary
analysis.
detected
in
the
urine
from
one
subjectfromeachgroup.
No
daidzeinorgenistein
weredetectedinDaidzein was not detected in the urine from two subjects in the RDSW group, and genistein wasthe
not
urine
from inone
subjectgroup.
rate ofurinary
daidzein
andin
detectedtheurine fromfromtheoneRDSW
subjectfrom The
eachoverall
group.detection
No daidzein
orbothgenistein
weredetectedgenistein97%(93/96).
detectedin theurine
fromdetection
19 and 17rate
subjects
inurinary
the RDSW
andthe urinewas
fromone
subjectEquol
from wasthe RDSW
group.
The
overallof bothdaidzeincontrolgroups, wasrespectively.
In thecurrent
equol
wasurine
identified
in and
38% 17of subjects in(36/96).
and genistein
97% (93/96).
Equol
wasstudy,
detectedin thefrom 19
the RDSW
detection
rates
of urinary isoflavones
arestudy,
summarizedin Table
3. If urinary
isoflavones
were
andThecontrolgroups,respectively.
In the current
equol wasidentified
in 38% ofsubjects (36/96).
not detected
in a sample,
value wasconsidered
The averagechangesin the concentrationsofThe detection
ratestheof urinary
isoflavones
arezero.
summarizedin Table
3. If urinary
isoflavones weretheisoflavonesthroughintervention
were compared
betweentwo groups.
corrected
notthreedetected
in a sample,
thethevalue
was consideredzero. The
averagethechangesin the The
concentrations
daidzeinand genisteinconcentrationsin thewere
RDSW
group (1.44
and 1.05,of the three
isoflavonesthrough theincreased
intervention
compared
betweenthe respectively)
two groups.butThedecreasedinthecontrolgroup(–0.14
and–0.39,
respectively;
Figure5a).
Considerable
amountsofbothcorrected daidzein and genistein concentrations increased in the RDSW group (1.44 and 1.05,
isoflavones
were
detectedin theinRDSW
group. Equol
were respectively;
evaluated in 16
subjects
respectively)
but
decreased
the control
group concentrations(–0.14 and –0.39,
Figure5a).
fromeach group,
following
an eliminationof threeand onesubjects
fromthe Equol
RDSWconcentrations
and control
Considerable
amountsof bothisoflavones weredetectedin theRDSWgroup.
groups,respectively,
duesubjects
to the lackof eachdetectionof daidzein
at aneitherpoint throughout
were evaluated
in 16
from
group,
following
eliminationof three theandintervention
one subjects(Tableincreasedconcentrations
observedin both(0.42
andfrom 3).
theOverall,
RDSW andcontrolaveragegroups,equol
respectively,
due to werethe lackof detectionof groups
daidzein
at either
0.18
in theRDSW andgroups,respectively;
Figure5b). Theequol equol
productionin the RDSW
point
throughout
thecontrol
intervention(Table3). Overall,increasedaverageconcentrations
were
group
was inat bothleast groups
two times
higherthan
in the control
group;
however,the difference
observed(0.42
and 0.18
inthatthe RDSW
and control
groups,respectively;
Figurewas5b).notThestatisticallysignificant.
equol productionin the RDSW group was at least two times higher than that in the control group;
however, the difference was not statistically significant.

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Table 3. Detection rates of urinary isoflavones in the two groups.
Table 3. Detection rates of urinary isoflavones in the two groups.

RDSW(Rate, % RDSW
Total)
Daidzein Daidzein(Rate, % Total)

46 */48
46(96)*/48 (96)#/48 (98)
47(98)
47 # /48
Daidzein and Genistein
46/48 (96)Daidzein and Genistein
46/48 (96)Equol
19 †/48 (40)

Genistein Genistein

Mineral Water

Total

Mineral Water
Total(Rate, % Total)(Rate, % Total)(Rate, % Total) (Rate, % Total)
48**/48**/48(100)(100)
94/96 (98)48
94/96 (98)
47/48
(98)
94/96
(98)
47/48 (98)94/96 (98)
47/48 (98)93/96 (97)47/48 (98)93/96 (97)17 ††/48 (35)36/96 (38)

Equolthree subjects in whose
36/96 (38)the19 samples/48 (40) daidzein was17detected/48 (35)* Including
at either point throughout
intervention(pre-inorwhose
post-intervention)of the wasstudy.
** Including
onepoint
subject
in whosethe
samples* Including
three subjects
samples daidzein
detected
at eitherthroughout
intervention(pre- or post-intervention)of theatstudy.
** Includingin whosewasordetecteddaidzein was detected
either point
throughonethe subject
interventionof thesamples
study. # daidzein
No genisteindaidzeinat either# No genistein or daidzein were detected in one subject. † Including
point throughinterventionof the †study.
were thedetectedin one subject.
Including
three subjects in whose samples equol was detected at either†† Including
three subjects
whose samples
equol wasofdetected
at eitherpoint throughthe interventionof the study.
†† Including
pointinthroughthe intervention
the study.
one subject
in whose samples
equol wasone subject in whose samples equol was detected at either point through the intervention of the study.
detected at either point through the intervention of the study.
††

The concentration of equol

The concentration of daidzein and genistein†

5. The differencesin the inchange
in the biomarker
urinary biomarker
concentrations
throughtheFigure 5.Figure
The differencesin the change
the urinary
concentrations
throughthe intervention
intervention (average). Daidzein and genistein (a) and equol concentrations (b). (a) Data for 48(average). Daidzein and genistein (a) and equol concentrations (b). (a) Data for 48 subjects fromsubjects from each group were analyzed. Daidzein and genistein concentrations were corrected foreach group were analyzed. Daidzein and genistein concentrations were corrected for creatinine
creatinine content, and are expressed as mg/g-Cre. (b) Data for 16 subjects from each group were
content, analyzed.
and are Equol
expressed
as mg/g-Cre.
(b) Data
16 subjects
eachgroup aswere
analyzed.
concentrations
were corrected
forfordaidzeincontent,fromand areexpressed
mg/mg
Equol concentrations
wereforblackdaidzeincontent,
are expressed
as error).
mg/mg daidzein.
daidzein. Hatchedbar, corrected
RDSW group;bar, control
group;andbar depicts
SE (standard
Hatched bar, RDSW group; black bar, control group; bar depicts SE (standard error).

4. Discussion

4. Discussion

In the current study, we evaluated the effect of prolonged intake of RDSW on the gut health inhumans,as assessedusing
a questionnaire
andofthechanges infecal ofandurinaryonbiomarker
In thecurrent
study, weevaluatedthe effectprolonged
intakeRDSW
the gut healthconcentrations of microbial metabolites both pre- and post-intervention. Drinking RDSW was morein humans, as assessed using a questionnaire and the changes in fecal and urinary biomarker
beneficial than drinking conventional mineral water, significantly increased the concentrations of fiveconcentrationsof microbial metabolites both pre- and post-intervention. Drinking RDSW was more
SCFAs, and relieved constipation. These changes support the use of RDSW as a beverage that
beneficial
than drinking
mineral water, significantly increased the concentrations of fivepromotes
intestinalconventionaland systemic health.
SCFAs, and relieved
Thesechanges
supportthe use forof RDSW
aslifea beverage
promotes
Moderateconstipation.
and balanced foodandliquid intake
is essential
a healthy
in all age thatgroups,cansystemic
eliminate health.
the risk factors or prevent the onset of disease. The quality of processed and naturalintestinalandandfoods, andincludingocean-related
products,
is gaining
publicTheinocean,Moderatebalanced
food and liquid
intakeis essential
for aattention.
healthy life
all ageoccupyinggroups, and canapproximately 70% of the Earth surface, supports all life on Earth as a source of food and energy. We
eliminate the risk factors or prevent the onset of disease. The quality of processed and natural foods,
previously reported that food components and certain natural products from the Kochi Prefecture
including ocean-related products, is gaining public attention. The ocean, occupying approximately
70% of the Earth surface, supports all life on Earth as a source of food and energy. We previously
reported that food components and certain natural products from the Kochi Prefecture (Japan), such as
Chinese chive [37] and RDSW produced from the DSW in the Muroto promontory, provide important
health benefits [38]. In particular, RDSW, a mineral-rich drinking water, exerts potentially beneficial
effects on human health, as demonstrated by previous clinical studies [1–17].

Nutrients 2020, 12, 2646

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In the current study, we showed that drinking RDSW effectively increases the amount offecal SCFAs post-intervention. The concentrations of five SCFAs (succinic, lactic, acetic, isobutyric,and 3-methylbutanoic acids) significantly increased post-intervention in the RDSW group but not in
the mineral water group. An increased concentration of propionic acid was observed without statisticalsignificance. The proportion of the eight SCFAs in the RDSW group was higher than in control group.
The proportion of acetic acid was significantly different between the two groups. A sex gap in theparticipants was observed in acetic acid, isobutyric acid, and 3-methylbutanoic acid in the RDSW
group. SCFAs widely affect the interplay between the host and gut microbiota, impacting humanhealth [39]. The effect of drinking RDSW depends on the gut microbial environment, indicating that
RDSW contributes to the maintenance of the gut ecosystem. The multiple logistic regression analysis
revealed that drinking RDSW significantly affected two SCFAs (acetic acid and 3-methylbutanoic
acids). Acetate is the predominant fecal SCFA, followed by propionate and butyrate [40,41]. This study
showed that drinking RDSW but not mineral water increased the fecal amounts of acetate andpropionate. Acetate promotes the defense function of the host epithelial cells against pathogenic
infection, i.e., inhibition of the translocation of toxins [42]. Propionate stimulates the release of theanorectic gut hormone peptide YY and glucagon-like peptide-1 by human colonic cells. These hormones
reduce appetite and prevent long-term weight gain in humans, helping lead to a healthy life [43].
SCFAs generated by fermentation of dietary fibers by the gut microbiota, particularly acetate andpropionate, have been implicated in a variety of beneficial effects on the host, including trophic andanti-inflammatory effects on the gut [44]. This study demonstrated that drinking RDSW increased
the concentrations of six among nine SCFAs, indicating that RDSW seems to induce the balanced
production of SCFAs. SCFAs positively impact human health and prevent a variety of disorders
acting via chemoattractant receptors expressed in diverse organs and cells, even in the neurons in
the brain [45]. It is generally accepted that SCFAs act as an energy source and stimulate the colon
epithelium, which facilitates the peristaltic motion. In the current study, constipation was alleviated toa significantly greater extent in the RDSW group that was accompanied by increased SCFA production
than in the mineral water group. Alleviation of constipation probably led to decreased concentrationsof phenol and sIgA in the RDSW group. At the very least, the increased SCFA levels might be involvedin the diverse beneficial effects of RDSW mentioned above. However, the amounts of fecal SCFAs
do not always reflect their amounts absorbed by the body. Direct comparison of the amounts of
SCFAs absorbed by the body in the two groups would clarify the effectiveness of the RDSW treatment.
This study was restricted by duration and budget. Thus, a large-scale clinical study would provide
more details about the effects of RDSW.
Most Fabaceae contain copious amounts of isoflavones, such as genistein and daidzein.
Aglycone-type isoflavones produced by the gut microbiota exert physiological effects on various aspectsof health, including cancer, osteoporosis, cardiovascular diseases, and climacteric symptoms [46–57].
Equol is produced from daidzein by specific members of the gut microbiota. In western populations,the equol producers are estimated to be 20–30% of the total compared with 40–60% in Asian countries
depending on the existence of specific gut bacteria and the intake of soy foods [25,58]. In the current
study, the proportion of equol producers was 38% (36/96), similar to data from previous Japanesestudies [58–60]. After RDSW intake, the urinary levels of equol increased, with the change being at least
two times higher than in the mineral water intake group. The increases in the two other isoflavones(daidzein and genistein) were observed in only RDSW group and showed the same tendency as equol,
without statistical significance.
In general, consumption of soy foods rich in daidzein does not lead to a conversion of anequol non-producer to an equol producer [61,62]. However, an equol non-producer can convert
to an equol producer after consuming soy foods for more than four months [63,64], indicating theexistence of unidentified bacteria that convert daidzein to equol even in the gut of equol non-producers.
Iino et al. [58] identified novel bacteria that contribute to equol production and demonstrated thatsome equol non-producers that harbor such bacteria are converted to equol producers after a shortNutrients 2020, 12, 2646

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period of isoflavone consumption. These studies highlighted the possibility that compounds such as
isoflavones can stimulate the abundance and/or activity of unidentified bacteria that participate inequol production. In the current study, we did not observe conversions to equol production evenafter 12 weeks of RDSW ingestion. Further investigations are needed to identify new bacteria that
contribute to equol production using next-generation sequencing of feces. However, drinking RDSW
obviously increased urinary equol levels, indicating that it enhanced the activity of gut bacteria thatparticipate in equol production. The creation of aglycone-type isoflavone, which is substantiallyabsorbed by the body and functions physiologically [65], requires β-glucosidase activity. We cannot
exclude the possibility that RDSW influenced the activity of β-glucosidase in the gut microbiota and/or
gut environment to produce aglycone-type isoflavone. The differences in the mineral concentrations
between RDSW (hard water) and mineral water (soft water) reflect the differences observed in thisstudy. Thus, a clinical study with medium-harness water (approximately 100 hardness) would provide
more insight into the human gut ecosystem with respect to mineral concentrations.
Throughout the study, we strictly monitored the dietary habits of the participants to ascertain
common lifestyle, without consumption of unusual foods, supplements, or medicines. Taken together,
the findings of the current study indicated that RDSW intake fundamentally promotes the induction ofthese metabolites on the gut environment. The habit of eating soy foods and RDSW is a promisingdietary approach to effectively maintaining human health by supporting the gut ecosystem.
5. Conclusions
The presented clinical study revealed that RDSW intake effectively relieves constipation andincreases fecal SCFA levels and urinary isoflavone levels, including equol levels. These observations
indicated that drinking RDSW potentially improves the gut ecosystem and these beneficial effects
extend to human health.
Author Contributions: H.T. designed and developed the protocol of this clinical study and wrote the manuscript;
S.I., K.T. and S.T. coordinated the clinical trials; K.H., Y.Y. and R.T. contributed to measurement of samples anddata analyses; W.O., A.K. (Asako Kimura), J.M.I. and A.K. (Ayami Kaneko) supported the clinical trials anddata analyses; S.S. statistically analyzed the data. 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 fundedby the Ministry of Health, Labour and Welfare in Japan. This work was partially supported by JSPS KAKENHI
Grant Number 20K07857.
Acknowledgments: We thank the Kochi government office and the Muroto city office for supporting thisindustry–academia–government collaboration project.
Conflicts of Interest: The authors declare no conflict of interest.

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2.

3.
4.
5.

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