nutrients
ArticleEffects of Euglena gracilis EOD-1 Ingestion on
Salivary IgA Reactivity and Health-Related Qualityof Life in Humans
Ken-ichi Ishibashi 1, * , Machiko Nishioka 2 , Nobuteru Onaka 2 , Madoka Takahashi 2 ,Daisuke Yamanaka 1 , Yoshiyuki Adachi 1 and Naohito Ohno 1
1

2*

Laboratory for Immunopharmacology of Microbial Products, School of Pharmacy, Tokyo University of
Pharmacy and Life Sciences, Hachioji, Tokyo 192-0392, Japan; ymnkd@toyaku.ac.jp (D.Y.);
adachiyo@toyaku.ac.jp (Y.A.); ohnonao@toyaku.ac.jp (N.O.)
Kobelco Eco-Solutions Co., Ltd., Kobe, Hyogo 651-2241, Japan; m.nishioka@kobelco-eco.co.jp (M.N.);
n.onaka@kobelco-eco.co.jp (N.O.); md.takahashi@kobelco-eco.co.jp (M.T.)
Correspondence: ishiken@toyaku.ac.jp; Tel.: +81-426-76-5570

Received: 29 March 2019; Accepted: 20 May 2019; Published: 22 May 2019


Abstract: Euglena gracilis EOD-1, a microalgal strain known for high yields of the β-1, 3-glucan
paramylon, is suggested to function as a dietary fiber and enhance immunity. Here, we aimed toinvestigate the effects of E. gracilis EOD-1 biomass (EOD1BM) ingestion on immunoglobulin A (IgA)
antibody titers in saliva, its reactivity, and the health-related quality of life (QOL) in humans. Reacting
human immunoglobulin preparations and saliva with paramylon granules revealed the presence ofanti-paramylon antibodies in the blood and saliva. We conducted a placebo-controlled, double-blind,crossover study involving 13 healthy subjects who ingested the placebo or EOD1BM for 4 weeks.
Saliva was collected from each subject before and after ingestion, and IgA titers and E. gracilis EOD-1
paramylon (EOD1PM) reactivity were compared. In the EOD1BM Ingestion group, the anti-EOD1PM
IgA content and titer increased after EOD1BM ingestion. No such change was observed in thePlacebo group. Furthermore, the health-related QOL, especially mental health, increased in the
EOD1BM Ingestion group. Thus, EOD1BM ingestion led to the production of paramylon (PM)-specific
IgA antibody and increased salivary IgA antibody titers. We demonstrate that EOD1BM ingestion
enhanced the immunity in the mucosal surface, evoked an antigen-specific response, and increased
the health-related QOL, thereby contributing to health improvement.
Keywords: paramylon; β-1, 3-glucan; antibody; IgA; QOL

1. Introduction
Euglena gracilis, a type of microalgae, contains several nutrients and is well known for the synthesisof paramylon, a polysaccharide component. Therefore, its functionality as a food ingredient has
garnered much attention in recent years, and the immunomodulatory effects of E. gracilis in mice andother animals have been reported [1–3].
The E. gracilis EOD-1 strain can produce high paramylon yields (70–80%) depending on culture
conditions [4]. The potential of oral administration of E. gracilis EOD-1 biomass (EOD1BM) and purified
paramylon (EOD1PM) in improving impaired glucose tolerance and serum lipids has been reported
in diet-induced obesity mouse models [4]. Therefore, EOD1BM has been suggested to function as a
dietary fiber, and studies exploiting its functionality in improving immunity have been suggested.
However, the effect of E. gracilis ingestion on the human immune system remains unknown.
Paramylon is a polysaccharide generated as a characteristic cellular reserve material in Euglena spp.
and is stored as granules. Structurally, it is a linear β-1, 3-glucan arranged in a triple helix; these helices
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accumulate to form crystalline granules. Paramylon has been reported to exhibit immunomodulatory
and anti-allergy effects in animal models [5,6]. Moreover, β-glucans have been reported to show
immunomodulatory and antitumor effects and induce cytokine production [7]. Furthermore, β-glucan
is found abundantly in nature, such as in fungi and algae, and it is known that the structural andphysical properties of β-glucans differ with the source organism. The biological activity of β-glucan is
also known to vary with its structural and physical properties [8].
Studies investigating the host recognition mechanism for β-glucans reported dectin-1 as a β-glucan
receptor involved in the mediation of β-glucan biological activity [9,10]. Furthermore, we previously
reported the occurrence of immunoglobulin (Ig)G, IgM, and IgA anti-β-glucan antibodies as hostrecognition molecules for β-glucan in both human and animal sera [11–13]. The immune complex
formed by the binding of β-glucan and its antibody is considered to evoke an antigen-specificimmunological response.
The biological activity of E. gracilis has been reported in animal models, but its biological activity
in humans remains unclear [4–6]. Therefore, we hypothesize that EOD1BM, which is rich in paramylon(β-1, 3-glucan), shows immunological activities in humans as well. In this study, we focused on
the immunity-enhancing function of EOD1PM in humans. We investigated the reactivity of humanserum and salivary anti-EOD1PM antibody and studied the changes in its titer after EOD-1 ingestion.
In addition, lifestyle aspects such as eating habits affect the body and mind and may influence thequality of life (QOL) of an individual. It has been reported that β-glucan affects the immunologicalresponse and QOL in cancer patients, and that yeast β-glucan improves the mood in stressed humanvolunteers [14,15]. Therefore, EOD1BM may also affect QOL in human volunteers. Health-related QOL
is comprised of the basic elements concerning physical, mental, social life-related, and role-limiting
functions. Therefore, we examined the general effect of E. gracilis EOD-1 ingestion on the overall
health-related QOL of study subjects using the Medical Outcomes Study 36-Item Short Form version 2(SF-36v2) health survey, a scale that measures health-related QOL comprehensively. This study
demonstrates that ingestion of EOD1BM, the microalgal biomass containing β-1, 3-glucan, activatedthe immune response in the mucosal surface and improved the health-related QOL, especially mental
health, in humans. These results highlight the novel effects of E. gracilis EOD-1 and suggest its potential
as a functional food for humans.
2. Materials and Methods
2.1. Materials
EOD1BM and EOD1PM were obtained from Kobelco Eco-Solutions Co., Ltd. (Kobe, Japan).
Solubilized EOD1 paramylon (sEOD1PM) was dissolved in sodium hydroxide and diluted with 0.1 M
carbonate buffer (pH 9.6). Solubilized cell wall glucans from Candida albicans and Aspergillus niger(CSBG and ASBG, respectively) were prepared using the NaClO oxidation method as described
previously [16,17]. Polysaccharide fractions (AgHWE) from Agaricus brasiliensis (Agaricus blazei
Murrill sensu Heinem) were also prepared as described previously [18]. Ovalbumin (OVA) and
Dextran T200 were purchased from Sigma-Aldrich (Saint Louis, MO, USA) and Pharmacia (Uppsala,Sweden), respectively.
2.2. Ingestion Study
2.2.1. Target Population for Analysis
This study was conducted from September to December 2017. Selected subjects were healthy (notundergoing any treatment for ailments), non-smoking men between the ages of 30 and 70 years. The
purpose and content of the study were explained thoroughly via written and oral explanation, and
13 individuals who agreed to participate and provided a written informed consent were enrolled as
our target population for analysis. The sample size was chosen based on previous studies of β-glucans

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3 of 14in humans
Subjects
whoSubjects
were deemed
not suitablein the ingestionsuch
glucans[19–21].
in humans[19–21].
who were
deemedfor
notconsideration
suitable for considerationin test,the ingestion
as thosewho
wereunabletotakemeasurements
atanappropriate
time,
were
excluded,leavingatest, such as those who were unable to take measurements at an appropriate time, were excluded,
totalleavingof seven
subjects
for analysis.
of the subjects
excludedwas
becauseof a because
BMI overFiveovera totalof seven
subjects One
for analysis.
One ofwas
the subjects
excluded
of30.
a BMI
subjects
wereexcludedbecause
they
took
the
test
and
appearedforsamplecollectionattimes
different
30. Five subjects were excluded because they took the test and appeared for sample collection at times
fromdifferent
those forthe thosebaseline
This test wasThis
approved
byapproved
the Kenshokai
Reviewfromfor measurements.
the baseline measurements.
test wasby theEthics
Kenshokai
EthicsCommittee
20170915-1,
Approval Date:
15 September
Review (ApprovalCommitteeNumber:(Approval
Number: 20170915-1,
Approval
Date: 152017).
September 2017).
2.2.2.
TestTest
Method
2.2.2.
MethodThe Thetest test
waswasdesigned
andandperformed
as a asdouble-blindcrossoverstudy(Figure1). Thebaseline
designed
performed
a double-blindcrossoverstudy(Figure1). Thebaseline
waswasmeasured,andapre-ingestion
examination
at
0
weeks
(0
w)
was
performed
whereinsalivameasured, and a pre-ingestion examination at 0 weeks (0 w) was performed whereinsalivasampleswerecollected
andandthe subjects
werewereadministered
severalteststests
and anda questionnaire-based
sampleswerecollected
the subjects
administered
severala questionnaire-based
surveyregardingtheirtheir
feelings
associatedwithwiththe usage.
Afterinitialexamination,subjects
wereweresurveyregardingfeelings
associatedthe usage.
Afterinitialexamination,subjects
given
the
test
food
(EOD1BM)oraplacebo(cornstarch)with
aningestionamountof
500
mg/daygiven the test food (EOD1BM) or a placebo (cornstarch) with an ingestion amount of 500 mg/day andandwerewererequested
requestedto
tocontinue
continueingesting
ingestingfor
foraaperiod
periodofof44weeks.
weeks.After
After4 4weeks
weeksofofingestion
ingestion(4(4w),w),they
theywere
wereagain
againsubjectedsubjectedtotothe
thesamesametests
testsandandquestionnairequestionnaireused
usedatat0 0w.
w.Next,Next,a a4-week
4-weekperiod
periodofofnonnon-ingestion(washout
period)wasinstituted,
andtheexaminations
wererepeated(8w).
Subjectswere
ingestion (washout period) was instituted, and the examinations were repeated (8 w). Subjects
werethenthenprovided
providedwith
withaatest
testfood
fooddifferent
differentfrom
from thatthat received
received at
at 00 ww andand were
were requested
requested to
to ingestingest itit forfor 44 weeks.
Subjects
were
once
again
tested
after
the
lapse
of
these
4
weeks
(12
w).
weeks. Subjects were once again tested after the lapse of these 4 weeks (12 w).

Figure1. Testdesign.
w: weeks.
EOD1BM:
EuglenagracilisEOD-1biomass.
Figure1. Testdesign.
w: weeks.
EOD1BM:
EuglenagracilisEOD-1biomass.

2.2.3. Saliva Collection
2.2.3. Saliva Collection
Saliva was collected from subjects before the meal and before caffeine intake at a fixed time in the
Saliva was collected from subjects before the meal and before caffeine intake at a fixed time inmorning in consideration of its daily fluctuation. Saliva samples were collected for 1 min using Salivettethe morning in consideration of its daily fluctuation. Saliva samples were◦ collected for 1 min using(Sarstedt K.K., Tokyo, Japan). The collected saliva samples were stored at −20 C until further use.
Salivette (Sarstedt K.K., Tokyo, Japan). The collected saliva samples were stored at −20 °C until
further use.

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4 of 142.2.4. Health-Related QOL Scale Measurement
We used the SF-36v2 [22–24] health survey, which is a QOL questionnaire based on universally
applicable health-related concepts. For each of the 36 questions, a five-point scale was used for the
replies, and the test was analyzed based on the following eight multi-item dimensions of health:physical functioning (PF), role limitations owing to physical problems (RP), body pain (BP), general
health perception (GH), energy/vitality (VT), social functioning (SF), role limitations owing to emotional
problems (RE), and mental health (MH). Next, the physical, mental, and social aspects of the component
summary scores were calculated.
2.3. Flow Cytometry Analysis for the Reactivity of Anti-EOD1PM Antibody
EOD1PM suspension was blocked with 0.5% sodium caseinate in phosphate-buffered saline
containing 0.05% Tween-20 (PBST pH 7.4; FUJIFILM Wako Pure Chemical, Tokyo, Japan) at 4 ◦ C
for 60 min. After centrifugation and removal of supernatants, EOD1PM was reacted with a humanimmunoglobulin preparation, polyglobin N (Bayer, Berlin, Germany), or pooled saliva at 4 ◦ C for 60 min.
The reacted EOD1PM granules were washed with fluorescence activated cell sorter (FACS) buffer (2%calf serum, 0.1% sodium azide, PBS) and incubated with fluorescein isothiocyanate (FITC)-conjugatedanti-human IgG or biotin-conjugated anti-human IgA and FITC-conjugated streptavidin (1:200;
BioLegend, Santiago, CA, USA) at 4 ◦ C for 60 min and analyzed using a flow cytometer (FACSCalibur,
Becton Dickinson, Franklin Lakes, NJ, USA). The histogram shows count data of 1 × 104 EOD1PM.
Next, the specificity of the anti-EOD1PM antibody was tested. EOD1PM particles were blockedwith 0.5% sodium caseinate in PBST. Polyglobin N or pooled human saliva were mixed with 100 µg/mL
competitive soluble antigen, namely sEOD1PM, CSBG, or OVA. The mixture was incubated at
4 ◦ C for 60 min. The reacted EOD1PM granules were washed with FACS buffer and incubatedwith FITC-conjugated anti-human IgG or biotin-conjugated anti-human IgA and FITC-conjugatedstreptavidin (1:200; BioLegend) at 4 ◦ C for 60 min and analyzed using a flow cytometer. The histogramshows count data of 1 × 104 EOD1PM.
2.4. ELISA for Anti-B Glucan Antibody in Serum and Saliva
A 96-well microplate was coated with sEOD1PM (25 µg/mL) in 0.1 M carbonate buffer (pH 9.6)
by incubation at 4 ◦ C overnight. The plate was washed with PBST and blocked with 0.5% sodium
caseinate in PBST at 37 ◦ C for 60 min. The plate was washed with PBST and incubated with humansera (Sigma-Aldrich) or saliva at 37 ◦ C for 60 min. The plate was then washed with PBST and incubatedwith peroxidase-conjugated anti-human IgG, IgM, or IgA (1:2000; Sigma-Aldrich) in PBST containing
0.5% sodium caseinate and developed with a TMB substrate system (KPL, Gaithersburg, MD, USA).
Color development was stopped with 1 N phosphoric acid, and optical density was measured at
450 nm with the absorbance reader MTP-450 (CORONA Electric, Ibaraki, Japan).
For testing the specificity of the anti-sEOD1PM antibody, an ELISA plate was coated withsEOD1PM (25 µg/mL in carbonate buffer) and blocked with 0.5% sodium caseinate in PBST before
use. Human sera or pooled human saliva were mixed with serially diluted competitive soluble
antigen, namely sEOD1PM, CSBG, OVA, AgHWE, ASBG, or dextran, and then applied to the ELISA
plate. The amount of plate-bound Ig was determined using peroxidase-conjugated anti-human IgG or
IgA (1:2000).
2.5. Statistical Analyses
The Mann–Whitney test was used for comparisons of results from the placebo and EOD1BMgroups. The Wilcoxon signed-rank test was used for comparisons of results obtained before and afteringestion. These statistical analyses were performed using MedCalc statistical software version 11.6.1.0(Acacialaan, Ostend, Belgium).

solubilized antigen to the immunoglobulin was investigated. sEOD1PM and CSBG addition bothsuppressed the peak shift and antibody binding; however, OVA addition did not result in such
suppression (Figure 2b). These results indicate that the anti-EOD1PM antibody is β-glucan–specific,
and cross-reacts with the Candida-derived β-glucan.
To understandof theNutrients
2019, 11, 1144the binding of a mucosal antibody secreted in response to exposure
5 of 14mucosal surface to foreign antigens, we investigated the anti-EOD1PM IgA antibody in the saliva
samples obtained from the study subjects. The addition of saliva shifted the peak in a concentration3. Results
dependent matter, indicating the presence of anti-EOD1PM IgA antibody in the saliva (Figure 3).
Furthermore,experiments
based toonParamylon
competitive
reactions with the addition of
3.1. Bindingantibodyof Human specificity
Serum and Saliva
Immunoglobulin
Granulessolubilized antigens revealed that the addition of these antigens suppressed the binding; these results
First, to investigate the binding of human immunoglobulins with paramylon granules, weare consistentwiththose withobtainedfor the serum
antibody.from
These
findings
indicatethat their
the antiincubated the
granulesimmunoglobulin
preparations
human
sera andanalyzed
EOD1PM
IgA
antibody
inthesalivais
also
β-glucan-specificandcross-reacts
withCandida-derived
binding using flow cytometry. Immunoglobulin addition resulted in a concentration-dependent shift
β-glucan.
in the peak, revealing the binding of human serum immunoglobulin to EOD1PM (Figure 2a).

Figure2. Bindingof humanimmunoglobulin
Euglenagracilis
gracilisEOD-1
EOD-1 paramylon
paramylon (EOD1PM).
Figure2. Bindingof humanimmunoglobulin
to toEuglena(EOD1PM). (a)(a)
Reactivityoftheanti-EOD1PM
antibody.
EOD1PM
particles
wereblocked,reactedwith
polyglobin
Reactivity of the anti-EOD1PM antibody. EOD1PM particles were blocked, reacted withpolyglobin
N, and analyzed by flow cytometry; (b) Specificity of the anti-EOD1PM antibody. EOD1PM particles
N, and analyzed by flow cytometry; (b) Specificity of the anti-EOD1PM antibody. EOD1PM particles
were blocked, reacted with mixture of polyglobin N and competitive soluble antigen, and analyzed
were blocked, reacted with mixture of polyglobin N and competitive soluble antigen, and analyzed
using flow cytometry. CSBG: solubilized cell wall glucan from Candida albicans; OVA: ovalbumin;usingflow cytometry.
CSBG:
sEOD1PM:solubilized
EOD1solubilized
paramylon. cell wall glucan from Candida albicans; OVA: ovalbumin;sEOD1PM: solubilized EOD1 paramylon.
Next, to examine the specificity of this anti-EOD1PM antibody, sEOD1PM, CSBG, or OVA weremixed with the immunoglobulin preparation, and the competitive binding of EOD1PM granules
and solubilized antigen to the immunoglobulin was investigated. sEOD1PM and CSBG addition
both suppressed the peak shift and antibody binding; however, OVA addition did not result in such
suppression (Figure 2b). These results indicate that the anti-EOD1PM antibody is β-glucan–specific,
and cross-reacts with the Candida-derived β-glucan.
To understand the binding of a mucosal antibody secreted in response to exposure of the mucosal
surface to foreign antigens, we investigated the anti-EOD1PM IgA antibody in the saliva samples
obtained from the study subjects. The addition of saliva shifted the peak in a concentration-dependent
matter, indicating the presence of anti-EOD1PM IgA antibody in the saliva (Figure 3). Furthermore,
antibody specificity experiments based on competitive reactions with the addition of solubilized
antigens revealed that the addition of these antigens suppressed the binding; these results areNutrients 2019, 11, 1144

6 of 14consistent with those obtained for the serum antibody. These findings indicate that the anti-EOD1PM
Nutrients
11, x FORPEER
REVIEWIgA 2019,
antibodyin thesalivais also β-glucan-specific and cross-reacts with Candida-derived β-glucan. 6 of 14

Figure3. Binding
humansaliva
salivato
to Euglena
Euglena gracilis(EOD1PM).
(a) ReactivityofFigure3. Binding
of of
human
gracilisEOD-1
EOD-1paramylon
paramylon(EOD1PM).
(a) Reactivity
ofsalivary anti-EOD1PM immunoglobulin A (IgA) antibody. EOD1PM particles were blocked, reacted
salivary anti-EOD1PM immunoglobulin A (IgA) antibody. EOD1PM particles were blocked, reactedwith pooled human saliva, and analyzed by flow cytometry. (b) Specificity of salivary anti-EOD1PM
with pooled human saliva, and analyzed by flow cytometry. (b) Specificity of salivary anti-EOD1PM
IgA antibody. EOD1PM particles were blocked, reacted with a mixture of pooled human saliva and
IgA100antibody.
EOD1PM particles
were blocked,
reactedwithmixture of pooled human saliva andµg/mL competitive
soluble antigen,and analyzed
using
flowacytometry.
100 µg/mL competitive soluble antigen, and analyzed using flow cytometry.
3.2. Anti-EOD1PM Antibody Reactivity to Solubilized EOD1PM in ELISA

3.2. Anti-EOD1PM
Antibody
Reactivity toantibody
Solubilizedin ELISA
To further examineanti-paramylon
in a EOD1PM
more quantifiable
and simple way, sEOD1PM wassolidified onto ELISA plates, treated with different dilutions of pooled human sera, and the reactivities
To further examine anti-paramylon antibody in a more quantifiable and simple way, sEOD1PMof detection antibodies of different classes (IgG, IgM, IgA) were analyzed. Anti-EOD1PM antibodies
was solidified onto ELISA plates, treated with different dilutions of pooled human sera, and theof each class, IgG, IgM, and IgA, were detected, and their binding was concentration-dependent
reactivitiesof detection antibodies of different classes (IgG, IgM, IgA) were analyzed. Anti-EOD1PM(Figure 4a). Antibody titer analysis revealed that IgG showed the highest titer and reactivity, followed
antibodies
of eachclass,solubilized
IgG, IgM,antigen-based
and IgA, weredetected,
andwastheirbinding
wastheconcentrationby IgA and
IgM. Next,
competitive
ELISA
used
to examine
binding
dependent(Figure4a).
Antibody
titer
analysis
revealedthat
IgG
showedthehighesttiter andspecificity of IgG. sEOD1PM addition suppressed the binding of the solidified sEOD1PM, indicating
reactivity,
followedby IgA and IgM.
Next,
solubilized
antigen-based
competitive ELISA was used tothe presenceof EOD1PM-specificIgG
in humansera (Figure
4b).

examine the binding specificity of IgG. sEOD1PM addition suppressed the binding of the solidified
sEOD1PM, indicating the presence of EOD1PM-specific IgG in human sera (Figure 4b).
Next, to further investigate the binding of different classes of salivary anti-EOD1PM antibodies,a dilution series of pooled human saliva was created against sEOD1PM and detection antibodies
against the different antibody classes were used to determine the titer values. The IgA antibodies
showed a concentration-dependent reactivity, but no reactivity was observed for IgG and IgM
antibodies (Figure 5a). Solubilized antigen-based competitive ELISA for salivary antibodies revealed
that addition of dextran (α-1, 6-glucan), AgHWE (primarily comprised of β-1, 6-glucan), and OVA
did not suppress anti-EOD1PM antibody binding (Figure 5b). However, the addition of sEOD1PM,
CSBG, and ASBG did suppress antibody binding. These results indicate that the salivary antiEOD1PM antibody is specific for β-1, 3-glucan. In addition, the anti-EOD1PM antibody titers in thesaliva collected from 13 healthy human volunteers were analyzed. Anti-EOD1PM IgA was detectedNutrients 2019, 11, 1144
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7 of 14

Figure4. Class
antibodyagainst
againstEuglena
Euglenagracilis
gracilis EOD-1
EOD-1 paramylon
paramylon (EOD1PM)
andFigure4. Classof ofantibody(EOD1PM)ininhuman
humansera
sera
andspecific reactivities. (a) Reactivity of anti-sEOD1PM immunoglobulin (Ig) G, IgM, and IgA antibody in
specific reactivities. (a) Reactivity of anti-sEOD1PM immunoglobulin (Ig) G, IgM, and IgA antibody
human sera. An ELISA plate was coated with solubilized EOD1PM (sEOD1PM) and blocked. Sera werein human sera. An ELISA plate was coated with solubilized EOD1PM (sEOD1PM) and blocked. Sera
diluted, and the amount of plate-bound Ig was determined using the detection antibody; (b) Specificity
were diluted, and the amount of plate-bound Ig was determined using the detection antibody; (b)of the anti- sEOD1PM IgG antibody in human sera. An ELISA plate was coated with sEOD1PM andSpecificity of the anti- sEOD1PM IgG antibody in human sera. An ELISA plate was coated withblocked. Human sera were mixed with serially diluted competitive soluble antigen and then appliedsEOD1PMand blocked.
Human
sera
mixed Ig
with
serially
dilutedcompetitive
solubleantigento the ELISA
plate. The
amountof wereplate-boundwasdeterminedusing
the detection
antibody.
andAgHWE:
then applied
to the ELISA
plate.
amountof plate-boundIg was determined
using
the
Polysaccharide
fractions
fromTheAgaricusbrasiliensis;
ASBG: solubilized
cell wall glucan
fromdetection
antibody.
AgHWE:
Polysaccharide
fractionsfrom
Agaricusbrasiliensis;
ASBG:
solubilized
Aspergillus niger.
cell wall glucan from Aspergillus niger.

Next, to further investigate the binding of different classes of salivary anti-EOD1PM antibodies,a dilution series of pooled human saliva was created against sEOD1PM and detection antibodies against
the different antibody classes were used to determine the titer values. The IgA antibodies showeda concentration-dependent reactivity, but no reactivity was observed for IgG and IgM antibodies(Figure 5a). Solubilized antigen-based competitive ELISA for salivary antibodies revealed that additionof dextran (α-1, 6-glucan), AgHWE (primarily comprised of β-1, 6-glucan), and OVA did not suppress
anti-EOD1PM antibody binding (Figure 5b). However, the addition of sEOD1PM, CSBG, and ASBG
did suppress antibody binding. These results indicate that the salivary anti-EOD1PM antibody is
specific for β-1, 3-glucan. In addition, the anti-EOD1PM antibody titers in the saliva collected from13 healthy human volunteers were analyzed. Anti-EOD1PM IgA was detected in the saliva of eachhealthy volunteer, and different titer values were observed for each volunteer (Figure 6).

were diluted, and the amount of plate-bound Ig was determined using the detection antibody; (b)Specificity of the anti- sEOD1PM IgG antibody in human sera. An ELISA plate was coated withsEOD1PM and blocked. Human sera were mixed with serially diluted competitive soluble antigen
and then applied to the ELISA plate. The amount of plate-bound Ig was determined using thedetection antibody. AgHWE: Polysaccharide fractions from Agaricus brasiliensis; ASBG: solubilized
Nutrients 2019, 11, 1144
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FigureReactivityof antibody
againstEuglenagracilis
EOD-1
paramylon(EOD1PM)in humanFigure5. 5.
Reactivityof antibody
againstEuglenagracilis
EOD-1
paramylon(EOD1PM)in humansaliva.
(a) Reactivityof humananti-sEOD1PM
IgG, IgM,
and IgA
antibody.
ELISA
plate
(a)saliva.
Reactivityof humansalivarysalivaryanti-sEOD1PM
IgG, IgM,
and IgA
antibody.
An An
ELISA
plate
was
was
coatedwithsEOD1PMandblocked.
Salivasampleswerediluted,andtheamountofplate-boundcoated with sEOD1PM and blocked. Saliva samples were diluted, and the amount of plate-bound Ig
Ig determined
was determinedusing
detection
antibody.(b)(b)Specificity
Specificity ofof thewasusing
thethedetection
antibody.
the human
humansalivary
salivaryanti-sEOD1PM
anti-sEOD1PM
IgA
antibody.
An
ELISA
plate
was
coatedwithsEOD1PMandblocked.
Human
was
mixed
IgA antibody. An ELISA plate was coated with sEOD1PM and blocked. Humansaliva
saliva
was
mixed
with
serially
dilutedcompetitive
solubleantigenandthenapplied
to
the
ELISA
plate.
The
amountof ofwith serially diluted competitive soluble antigen and then applied to the ELISA plate. The amountplate-boundIg
wasdetermined
using
the
detection
antibody.
plate-bound Ig was determined using the detection antibody.

Figure6. Reactivityof antibody
againstEuglenagracilis
EOD-1
paramylon(EOD1PM)in salivaof healthy
Figure6. Reactivityof antibody
againstEuglenagracilis
EOD-1
paramylon(EOD1PM)in salivaofhumanvolunteers.
An
ELISA
plate
was
coatedwithsEOD1PMandblocked.
Humansalivasamples
healthy human volunteers. An ELISA plate was coated with sEOD1PM and blocked. Human saliva(n samples= 13) wereandthe amountplate-boundIg was determinedusing peroxidase-conjugated(n =diluted,13) werediluted,and theofamountof plate-boundIg was determinedusing peroxidaseanti-human
IgA
antibody.
Eachcolumnrepresents
data
from
one
individual.
conjugated anti-human IgA antibody. Each column represents data from one individual.

3.3.3.3.
Influence
IgAAntibody
AntibodyTiter
Titer
InfluenceofofEOD1BM
EOD1BMIngestion
Ingestionon
onSalivary
Salivary Anti-EOD1PM
Anti-EOD1PM IgA
The
concentration
ofofsecretory
The
concentration
secretoryIgA
IgA(s-IgA)(s-IgA)ininthe
thesaliva
salivaandandthethesecretionsecretion rate
rate were
were determined
determined instudy
subjects
after
EOD1BM
ingestion.
An
increaseintheconcentration
andsecretion
rate
salivaryin study subjects after EOD1BM ingestion. An increase in the concentration and secretionofrate
of
s-IgA
was
observed
insubjects
who
ingested
EOD1BM(Table1).
salivary s-IgA was observed in subjects who ingested EOD1BM (Table 1).
Next, we performed sEOD1PM-based ELISA to determine titer changes for EOD1PM-specificsalivary IgA after EOD1BM ingestion. The salivary anti-EOD1PM IgA antibody titer in the EOD1BM
Ingestion group was higher than that in the Placebo group (Figure 7). When OVA was used as thecontrasting antigen, no significant changes were observed in antibody titers before and afteringestion. These findings indicate that EOD1BM ingestion triggers an EOD1PM-specific immune

Next,subjects
we performedsEOD1PM-based
to determine
titer changes forin study
after EOD1BM
ingestion. ELISA
An increasein the concentration
andEOD1PM-specificsecretion rate ofafter
salivaryanti-EOD1PM
IgA antibody
titer in the EOD1BM
salivary IgA
s-IgA
wasEOD1BM
observedingestion.
in subjectsThe
whoingested
EOD1BM (Table
1).
Ingestion
was higher
than that in theELISA
Placebo(FigureWhen OVA
was used as the
Next,group
we performedsEOD1PM-based
to group
determine
titer7).changes
for EOD1PM-specificcontrastingno significant
wereanti-EOD1PM
observed in IgA
antibody
titersafter
salivary
IgA antigen,after EOD1BM
ingestion.changes
The salivary
antibody
titerbefore
in the and
EOD1BM
ingestion.
These
findings
indicatethatinEOD1BM
ingestion
triggers7).anWhen
EOD1PM-specificimmuneIngestiongroup
washigherthan that
the Placebogroup (Figure
OVA was usedas the9 of 14
Nutrients
2019, 11,1144
response in themucosalcontrasting
antigen,no surface.
significant changes were observed in antibody titers before and afteringestion. These findings indicate that EOD1BM ingestion triggers an EOD1PM-specific immune
Table
1. Secretory
immunoglobulin
A (s-IgA)
concentrationand secretionrate
in saliva
beforeTable
Secretory
immunoglobulin
A (s-IgA)
concentrationand secretion
rate insalivabeforeand and
response
in1.themucosalsurface.
the ingestionof Euglena
gracilisEOD-1biomass(EOD1BM).
afterafter
the ingestionof Euglena
gracilisEOD-1biomass(EOD1BM).

Table 1. Secretory immunoglobulin A (s-IgA) concentration
andPostsecretion ratein saliva before andPre
Pre-Post
Pre
Post
Pre-PostRatio
Ratio
after the ingestion of Euglena gracilis EOD-1 biomass (EOD1BM).
s-IgA
concentration
Placebo
31.1
±
10.7
22.9
±
7.6
0.77
±
0.82
Placebo31.1 ± 10.7
22.9 ± 7.6
0.77 ± 0.82
s-IgA concentration†(µg/mL)EOD1BM
23.1±±10.5
27.0
±±4.2
1.41
Post
Pre-Post
Ratio(μg/mL)EOD1BM
23.1Pre
10.5
27.0
4.2
1.41±±0.26
0.26secretion
Placebo
48.6
41.6
0.88
Placebo31.1±±11.110.7
22.9±±±13.5
7.6
0.77
0.82
s-IgAs-IgA
concentration
secretion
raterate
48.6
11.1
41.6
13.5
0.88±±0.280.28††(µg/min)
EOD1BM
34.2
±
16.5
50.9
±
13.0
*
1.91
±
1.29
(μg/mL)EOD1BM
23.1 ± 16.5
10.5
27.0± ±13.0
4.2 *1.41 ± 1.29
0.26(μg/min)
34.2
50.9
1.91
Allsecretion
valuesshown
mean±Placebo±SDSD(n(n == 7).
7). s-IgA:
s-IgA:
signed-ranks-IgA
rateasasmean
48.6Secretory± 11.1 immunoglobulin
41.6 ± 13.5 A. * pA.< *0.05,
0.88± 0.28All
valuesare areshown
Secretory
immunoglobulin
p < Wilcoxon
0.05,
Wilcoxon
test comparing the results before and after ingestion (n = 7). † p < 0.05, Mann–Whitney’s U test comparing† the(μg/min)
EOD1BM
34.2
50.9 (n± 13.01.91± 1.29signed-rank
test
the
results
beforeand ±afteringestion= 7). *† p < 0.05,
Mann–Whitney’s
pre–post
ratioscomparingof the placebo
and
EOD1BM
groups(n16.5= 7).

U testcomparing
the pre–post
and EOD1BMgroups (n = A.
7). * p < 0.05, WilcoxonAllvaluesare shownas mean ±ratios
SD (nof=the7). placebos-IgA: Secretory
immunoglobulin
signed-rank
testperformed
comparing theresults before andafter ingestion(n = 7).
† p <changes
0.05, Mann–Whitney’s
Next, wesEOD1PM-based
ELISA
to determine
titer
for EOD1PM-specificU
testcomparingthepre–post
ratiosoftheplaceboand
EOD1BM
groups(n=
7).
salivary IgA after EOD1BM ingestion. The salivary anti-EOD1PM IgA antibody titer in the EOD1BM

Ingestion group was higher than that in the Placebo group (Figure 7). When OVA was used as thecontrasting antigen, no significant changes were observed in antibody titers before and after ingestion.
These findings indicate that EOD1BM ingestion triggers an EOD1PM-specific immune response in the
Nutrients 2019,11, x FOR PEER REVIEW
9 of 14mucosalsurface.

Figure 7. Changes in anti-Euglena gracilis EOD-1 paramylon (EOD1PM) antibody titer after EOD1BM
Figure 7. Changes
in anti-Euglena
gracilis
EOD-1
paramylonby(EOD1PM)antibody
titer after EOD1BM
ingestion.
The anti-EOD1PM
antibody
titer
was measured
ELISA usingsEOD1PM-coatedplatesingestion.
The
anti-EOD1PM
antibody
titer
wasmeasuredby ELISA
using sEOD1PM-coatedplates(n= 7). Therate
of increase in
the titer
wascalculated
using
the following
formula: Increase
in
(n
=
7).
The
rate
of
increase
in
the
titer
was
calculated
using
the
following
formula:Increaseinantianti-EOD1PM antibody titer (%) = ((anti-EOD1PM antibody titer after ingestion—anti-EOD1PM
EOD1PMtiterantibody(%) = ((anti-EOD1PM antibody
after
ingestion—anti-EOD1PM
antibody
beforetiteringestion)/anti-EOD1PM
antibodytiter
titer
beforeingestion) × 100. *: pantibody< 0.05titer
beforeingestion)/anti-EOD1PM
antibody
titer
beforeingestion)×
p < 0.05(Mann–Whitney(Mann–Whitney U test, n = 7). (a) Changes in anti-EOD1PM antibody 100.
titer *:after
EOD1BM
ingestion.
U test,n = 7).
(a) Changes
in anti-EOD1PM
antibodyingestion.
titer after EOD1BM ingestion. (b) Changes in(b)
Changesin anti-OVA
antibody
titer after EOD1BM
anti-OVA antibody titer after EOD1BM ingestion.

3.4. Effect of EOD1BM Ingestion on Health-Related QOL
3.4. Effect of EOD1BM Ingestion on Health-Related QOL
To study the effect of EOD1BM ingestion on health-related QOL in all the analyzed volunteers(n = 7),
usedscientificingestion
scale withreliability
andvalidity.
In the volunteers
EOD1BM
To we
study
theSF-36v2,
effect of aEOD1BM
onverified
health-related
QOL
in allthe analyzed
ingestion
Group,
scores for
all the eight
PF, RP,reliability
BP, GH, VT,
RE, andInMH,(n = 7), we
usedtheSF-36v2,
a scientificscalesubscales,with verified
andSF,
validity.
the increased
EOD1BM
after
EOD1BM
ingestion(Figure8). theIn particular,a statisticallyincreasein theingestion
Group,
the scoresfor alleight subscales,
PF, RP,significant
BP, GH, VT,
SF, RE,wasandobserved
MH, increased
mentalhealth category(Figure8a), 8).
andInconsiderable
was observedin increase
the component
summary
after EOD1BM
ingestion(Figureparticular, aincrease
statisticallysignificant
was observed
in
score
for
both
physicalandmentalhealthcategories(Figure8b).
However,inthePlacebogroup,
nothe mental health category (Figure 8a), and considerable increase was observed in the component
significant
observed
anymentalof the eightsubscales.
Furthermore,
post-ingestion
mentalsummary increasescore forwas
bothphysicalinand
healthcategories(Figure 8b).the
However,in the Placebo
healthsummary
scorein theEOD1BM
Ingestion
significantly
higher thanthatgroup,component
no significant
increase
wasobservedin anyof the groupeight wassubscales.
Furthermore,
the postin
thePlacebogroup.
IntheRole–Social
Component
Summary
Score,
therewasnodifference
betweeningestion mental health component summary score in the EOD1BM Ingestion group was significantly
the
EOD1BM
Ingestion
Placebogroups.
higherthan thatin the andPlacebogroup.
In the Role–Social Component Summary Score, there was nodifference between the EOD1BM Ingestion and Placebo groups.

Nutrients 2019, 11, 1144
Nutrients 2019, 11, x FOR PEER REVIEW

10 of 1410 of 14

Figure
8. Effectof ofingestion
EOD-1biomass
biomass(EOD1BM)on health-related
quality
Figure
8. Effect
ingestionofofEuglena
Euglena gracilis
gracilis EOD-1(EOD1BM)on health-related
qualityofof life(QOL).
(a)Influenceof
EOD1BM
ingestion
onthescoresofthesubscalesofMedicalOutcomes
life (QOL). (a) Influence of EOD1BM ingestion on the scores of the subscales of Medical OutcomesStudy
36-ItemShort
FormversionA questionnairequestionnairesurvey
surveyfrom
SF-36v2
Study
36-ItemShort
Formversion2 2(SF-36v2)(SF-36v2)(n(n== 7).
7). A
from
SF-36v2
waswasadministered
to thesubjects
afteringestion(n(n= =Thescoresadministered
to thesubjects
afterapproximately
approximately 44 weeks
weeks of EOD1BM
EOD1BM ingestion7).7).
The
scoresbeforeafter
EOD1BM
ingestionwere
wereanalyzed
analyzedandand compared.
compared. *:*:pp<<0.05,
Wilcoxon
signed-rank
beforeandandafter
EOD1BM
ingestion
0.05,
Wilcoxon
signed-rank
EffectEOD1BMingestion
ingestionon
on SF-36v2
SF-36v2 component
component summary7).7).
TheThetest test(n =(n7).= 7).
(b)(b)
Effectof of
EOD1BMsummaryscore
score(n(n= =component
summaryscoresPhysicalComponent
Component Summary
Summary (PCS),
Summary
component
summaryscoresofof
Physical(PCS),Mental
MentalComponent
Component
Summary(MCS),
Role–Social
ComponentSummary
Summary (RCS)(RCS) based
beforeandafter(MCS),
andandRole–Social
Component
based ononthe
thesubscale
subscalescoresscoresbeforeandafter
EOD1BM
ingestion
obtainedbytheadministrationofthe
SF-36v2
questionnaire
werecalculated.
TheTheEOD1BM ingestion obtained by the administration of the SF-36v2 questionnaire were calculated.
of change
beforeandafter
EOD1BMingestion
ingestionwas
was calculated
calculated byformula:Pre–Post
rate rateof change
beforeandafter
EOD1BM
bythe
thefollowing
following
formula:Pre–Post
Component summary score ratio = Component summary score after ingestion/Component summary
Component summary score ratio = Component summary score after ingestion/Component summaryscore ratio before ingestion. The changes before and after the ingestion within the group and thedifferences between the two groups were compared. *: p < 0.05, Mann–Whitney U test comparing theplacebo and EOD1BM groups at the pre–post ratio (n = 7).

Nutrients 2019, 11, 1144

11 of 14

4. Discussion
In this study, we aimed to investigate the effects of E. gracilis EOD1BM on the immunomodulationand health-related QOL in humans. We demonstrate the presence of antibodies against paramylon,
a major component of E. gracilis, in human serum and the saliva. Paramylon may be involved in
inducing an immunomodulatory effect. Furthermore, we demonstrated the applicability of solubilized
paramylon-based ELISA for testing and measuring the antibody titers against paramylon. In the
EOD1BM Ingestion group, the anti-EOD1PM titer increased. Furthermore, EOD1BM ingestion
increased the health-related QOL.
Although several studies have reported the effect of paramylon ingestion in animal models, very
few studies investigated the effects of ingestion in humans. Therefore, in this study, we investigatedthe effect of EOD1BM ingestion, with respect to antibody response, in healthy volunteers. EOD1BM
ingestion increased the amount of salivary s-IgA secretion. EOD1BM was able to activate the mucosal
immune system. Furthermore, this increase in salivary anti-EOD1PM antibody suggests the inductionof an EOD1PM-specific immune response. The ingestion amount of EOD1BM was 500 mg/day andwould not cause any problems as a microalgae or dietary fiber [25]. The amount of paramylon
was considered to be 350 mg/day. A previous study reported that yeast glucan supplementationof 250 mg/day reduces upper respiratory tract infection after exercise stress [26]; therefore, it wasconsidered normal and effective. Dectin-1, a β-glucan receptor, was reported to be expressed in theintestinal epithelial cells [27]. These findings indicate that EOD1PM induces an immune response via a
specific recognition mechanism.
The E. gracilis EOD1 strain is known for a high production of the β-glucan paramylon undercertain conditions. Various studies have reported the biological activity of β-glucans [28–30]. We
previously investigated anti-β-glucan antibodies and reported the presence of IgG, IgA, and IgM classantibodies specific to β-1, 3-glucan and β-1, 6-glucan in human sera [13,31]. The present study indicatesthe presence of an antibody response against EOD1PM, and that the antibody is specific to β-1, 3-glucan.
Additionally, the anti-EOD1PM antibody also responded to Candida and Aspergillus-derived β-glucan.
We previously reported that anti-β-glucan antibodies in human sera are generated in response topathogenic fungi and that these antibodies enhance the antifungal activity of human macrophages [11].
Therefore, anti-EOD1PM antibody would enhance the antifungal activity of macrophages. Candida iscommonly present in the oral and vaginal cavities and can cause infections on the mucosal surface,whereas Aspergillus causes bronchial and pulmonary infections. It is considered that anti-β-glucan IgA,
including anti-EOD1PM IgA, which is secreted on the mucosal surface, is also involved in the defense
against infectious agents such as pathogenic fungi. Therefore, the increase in anti-EOD1PM antibody
titer after EOD1BM ingestion suggests that EOD1BM ingestion could potentially enhance the immune
response against such microbial infections.
We used SF-36v2 to examine the influence of EOD1BM ingestion on the health-related QOL of thestudy subjects. We found that EOD1BM ingestion promoted mental and physical health. Previous
studies have indicated that β-glucan ingestion provides a baseline increase in the overall QOL and anincrease in QOL for allergy patients [32,33]. Thus, the paramylon contained in EOD1BM may be acontributing factor in this effect. Furthermore, β-glucan ingestion was reported to affect the intestinal
flora, especially the proportion of Bacteroides [34,35]. The β-glucan ingestion has been reported tocause changes in microflora by the production of antimicrobial peptides via Dectin-1 [34]. This may
also be the case with EOD1BM ingestion. Recent studies have reported a brain-gut connection inwhich the intestinal flora affect the central nervous system via neuroendocrine, enteric, and immune
systems [36]. It was reported that the proportion of Bacteroides fragilis is lower in a mouse model withsome features of autism [37]. With the progress in studies regarding the relation between the intestinal
flora and the mind via the brain–gut connection, it is suggested that the effects of β-glucan ingestion
are mediated via such mechanisms.
In the present study, we demonstrate that EOD1BM ingestion for 4 weeks in healthy 30–70-year-old
volunteers increased the health-related QOL. Furthermore, EOD1BM ingestion increased the production

Nutrients 2019, 11, 1144

12 of 14of an antibody with antigen-specific response to paramylon, the major component of EOD1BM, andwas effective in mucosal surface IgA production. These findings suggest that EOD1BM activatesthe immune response in the mucosal surface. Thus, we report a novel effect of E. gracilis EOD-1 inhealthy volunteers, although further studies are warranted to elucidate the underlying mechanism.
The sample size was chosen based on previous studies of β-glucans in humans. The subjects who
were unable to take the measurements at the appropriate time were excluded because the appropriatecomparisons could not be performed. The EOD1 BM Ingestion group showed a statistically significant
effect compared with the Placebo group. However, because the number and diversity of subjects werelimited, further studies with a larger number of more diverse subjects will also be necessary. EOD1BM
ingestion resulted in immunomodulation and increased health-related QOL. Thus, we suggest that
EOD1BM would be a useful functional food for humans.
Author Contributions: Conceptualization, K.-i.I., M.N., N.O. (Nobuteru Onaka), M.T., D.Y., Y.A., N.O.
(Naohito Ohno); methodology, K.-i.I., M.N., N.O. (Nobuteru Onaka), M.T., N.O. (Naohito Ohno); formal
analysis, K.-i.I., M.N., N.O. (Nobuteru Onaka), N.O. (Naohito Ohno); investigation, K.-i.I., M.N., N.O.
(Nobuteru Onaka); resources, M.N., N.O. (Nobuteru Onaka), M.T.; writing—original draft preparation, K.-i.I.,
M.N., N.O. (Nobuteru Onaka); writing—review and editing, D.Y., Y.A., M.T., N.O. (Naohito Ohno); supervision,
M.T., N.O. (Naohito Ohno).
Funding: This study was funded by Kobelco Eco-Solutions Co., Ltd.
Acknowledgments: We would like to thank Editage (www.editage.jp) for English language editing.
Conflicts of Interest: Three of the authors (M.N., N.O. (Nobuteru Onaka) and M.T.) are salaried employees of theKobelco Eco-Solutions Co., Ltd. The remaining authors (K.-i.I., D.Y., Y.A., N.O. (Naohito Ohno)) have no conflictof interest to disclose. The food substance used in this study was provided by Kobelco Eco-Solutions Co., Ltd.,and proper examination was performed by a third-party organization.

References
1.

2.
3.

4.
5.

6.

7.
8.
9.
10.

Nakashima, A.; Suzuki, K.; Asayama, Y.; Konno, M.; Saito, K.; Yamazaki, N.; Takimoto, H. Oral administrationof Euglena gracilis Z and its carbohydrate storage substance provides survival protection against influenza
virus infection in mice. Biochem. Biophys. Res. Commun. 2017, 494, 379–383. [CrossRef] [PubMed]
Levine, R.; Horst, G.; Tonda, R.; Lumpkins, B.; Mathis, G. Evaluation of the effects of feeding dried algae
containing beta-1, 3-glucan on broilers challenged with Eimeria. Poult. Sci. 2018, 97, 3494–3500. [CrossRef]
Bianchi, V.A.; Castro, J.M.; Rocchetta, I.; Conforti, V.; Pascual, M.; Luquet, C.M. Modulating effects oforally supplied Euglena gracilis on the physiological responses of the freshwater mussel Diplodon chilensis,
exposed to sewage water pollution in a Patagonian river (Argentina). Fish Shellfish Immunol. 2016, 51, 17–25.
[CrossRef] [PubMed]
Takahashi, M.; Kawashima, J.; Nishida, N.; Onaka, N. Beta-Glucan Derived from Euglena (Paramylon);
CMC Publishing Co., Ltd.: Tokyo, Japan, 2018; pp. 174–182.
Russo, R.; Barsanti, L.; Evangelista, V.; Frassanito, A.M.; Longo, V.; Pucci, L.; Penno, G.; Gualtieri, P.
Euglena gracilis paramylon activates human lymphocytes by upregulating pro-inflammatory factors.
Food Sci. Nutr. 2017, 5, 205–214. [CrossRef]
Sugiyama, A.; Hata, S.; Suzuki, K.; Yoshida, E.; Nakano, R.; Mitra, S.; Arashida, R.; Asayama, Y.; Yabuta, Y.;
Takeuchi, T. Oral administration of paramylon, a beta-1, 3-d-glucan isolated from Euglena gracilis Z inhibits
development of atopic dermatitis-like skin lesions in NC/Nga mice. J. Vet. Med. Sci. 2010, 72, 755–763.
[CrossRef]
Jin, Y.; Li, P.; Wang, F. β-glucans as potential immunoadjuvants: A review on the adjuvanticity,
structure-activity relationship and receptor recognition properties. Vaccine 2018, 36, 5235–5244. [CrossRef]
Ohno, N. Modulation of host defense systems by beta-glucans. Nihon Saikingaku Zasshi 2000, 55, 527–537.
[CrossRef] [PubMed]
Brown, G.D.; Gordon, S. Immune recognition. A new receptor for beta-glucans. Nature 2001, 413, 36–37.
[CrossRef]
Adachi, Y.; Ohno, N. Contribution of dectin-1 to the recognition of fungal cell wall products and the activationof innate immune response. Nihon Ishinkin Gakkai Zasshi 2006, 47, 185–194. [CrossRef]

Nutrients 2019, 11, 1144

11.

12.
13.

14.

15.
16.

17.
18.
19.

20.

21.

22.
23.
24.
25.
26.

27.
28.
29.

30.

31.

13 of 14Ishibashi, K.; Yoshida, M.; Nakabayashi, I.; Shinohara, H.; Miura, N.N.; Adachi, Y.; Ohno, N. Role ofanti-beta-glucan antibody in host defense against fungi. FEMS Immunol. Med. Microbiol. 2005, 44, 99–109.
[CrossRef]
Ishibashi, K.; Dogasaki, C.; Motoi, M.; Miura, N.; Adachi, Y.; Ohno, N. Anti-fungal cell wall β-glucan
antibody in animal sera. Nihon Ishinkin Gakkai Zasshi 2010, 51, 99–107. [CrossRef] [PubMed]
Ishibashi, K.; Morita, M.; Motoi, M.; Liu, Y.; Miura, N.N.; Adachi, Y.; Ohno, N. Analysis of the titer andreactivity of antibody/ies against fungal cell wall beta-glucans in human sera. Int. J. Med. Mushrooms 2013,15, 115–126. [CrossRef] [PubMed]
Ostadrahimi, A.; Esfahani, A.; Jafarabadi, M.A.; Ziaei, J.E.; Movassaghpourakbari, A.; Farrin, N. Effectof Beta glucan on quality of life in women with breast cancer undergoing chemotherapy: A randomized
double-blind placebo-controlled clinical trial. Adv. Pharm. Bull. 2014, 4, 471–477. [CrossRef] [PubMed]
Talbott, S.M.; Talbott, J.A. Baker’s yeast beta-glucan supplement reduces upper respiratory symptoms andimproves mood state in stressed women. J. Am. Coll. Nutr. 2012, 31, 295–300. [CrossRef]
Ohno, N.; Uchiyama, M.; Tsuzuki, A.; Tokunaka, K.; Miura, N.N.; Adachi, Y.; Aizawa, M.W.; Tamura, H.;
Tanaka, S.; Yadomae, T. Solubilization of yeast cell-wall β-(1→3)-d-glucan by sodium hypochlorite oxidationand dimethyl sulfoxide extraction. Carbohydr. Res. 1999, 316, 161–172. [CrossRef]
Ishibashi, K.; Miura, N.N.; Adachi, Y.; Tamura, H.; Tanaka, S.; Ohno, N. The solubilization and biologicalactivities of Aspergillus β-(1→3)-d-glucan. FEMS Immunol. Med. Microbiol. 2004, 42, 155–166. [CrossRef]
Ohno, N.; Furukawa, M.; Miura, N.N.; Adachi, Y.; Motoi, M.; Yadomae, T. Antitumor β-glucan from thecultured fruit body of Agaricus blazei. Biol. Pharm. Bull. 2001, 24, 820–828. [CrossRef]
Ojiri, Y.; Endoh, H.; Okumoto, T.; Atsuta, K.; Yoshinari, O.; Moriyama, H. Randomized, double-blind,placebo-controlled, crossover study to evaluate the effects of beta-1,3/1,6 glucan on stress associated withdaily lifestyle in healthy subjects. Funct. Foods Health Dis. 2015, 5, 145–154. [CrossRef]
Keogh, G.F.; Cooper, G.J.; Mulvey, T.B.; McArdle, B.H.; Coles, G.D.; Monro, J.A.; Poppitt, S.D. Randomized
controlled crossover study of the effect of a highly beta-glucan-enriched barley on cardiovascular diseaserisk factors in mildly hypercholesterolemic men. Am. J. Clin. Nutr. 2003, 78, 711–718. [CrossRef] [PubMed]
Liu, Y.; Fukuwatari, Y.; Okumura, K.; Takeda, K.; Ishibashi, K.; Furukawa, M.; Ohno, N.; Mori, K.; Gao, M.;
Motoi, M. Immunomodulating activity of Agaricus brasiliensis KA21 in mice and in human volunteers.
Evid.-Based Complement. Altern. Med. 2008, 5, 205–219. [CrossRef]
Fukuhara, S.; Ware, J.E., Jr.; Kosinski, M.; Wada, S.; Gandek, B. Psychometric and clinical tests of validity of
the Japanese SF-36 Health Survey. J. Clin. Epidemiol. 1998, 51, 1045–1053. [CrossRef]
Fukuhara, S.; Bito, S.; Green, J.; Hsiao, A.; Kurokawa, K. Translation, adaptation, and validation of the SF-36
Health Survey for use in Japan. J. Clin. Epidemiol. 1998, 51, 1037–1044. [CrossRef]
Fukuhara, S.; Suzukamo, Y. Manual of SF-36v2 Japanese Version; Institute for Health Outcomes and Process
Evaluation Research: Kyoto, Japan, 2004; pp. 7–145.
De Jesus Raposo, M.F.; de Morais, A.M. Microalgae for the prevention of cardiovascular disease and stroke.
Life Sci. 2015, 125, 32–41. [CrossRef]
McFarlin, B.K.; Carpenter, K.C.; Davidson, T.; McFarlin, M.A. Baker’s yeast beta glucan supplementation
increases salivary IgA and decreases cold/flu symptomatic days after intense exercise. J. Diet. Suppl. 2013, 10,
171–183. [CrossRef]
Cohen-Kedar, S.; Baram, L.; Elad, H.; Brazowski, E.; Guzner-Gur, H.; Dotan, I. Human intestinal epithelial
cells respond to β-glucans via Dectin-1 and Syk. Eur. J. Immunol. 2014, 44, 3729–3740. [CrossRef]
Jesenak, M.; Urbancikova, I.; Banovcin, P. Respiratory tract infections and the role of biologically active
polysaccharides in their management and prevention. Nutrients 2017, 9, 779. [CrossRef]
Borchani, C.; Fonteyn, F.; Jamin, G.; Destain, J.; Willems, L.; Paquot, M.; Blecker, C.; Thonart, P. Structuralcharacterization, technological functionality, and physiological aspects of fungal β-d-glucans. Crit. Rev. Food
Sci. Nutr. 2016, 56, 1746–1752. [CrossRef]
Ishimoto, Y.; Ishibashi, K.; Yamanaka, D.; Adachi, Y.; Kanzaki, K.; Okita, K.; Iwakura, Y.; Ohno, N. Modulationof an innate immune response by soluble yeast β-glucan prepared by a heat degradation method. Int. J.
Biol. Macromol. 2017, 104, 367–376. [CrossRef]
Masuzawa, S.; Yoshida, M.; Ishibashi, K.I.; Saito, N.; Akashi, M.; Yoshikawa, N.; Suzuki, T.; Nameda, S.;
Miura, N.N.; Adachi, Y.; et al. Solubilized candida cell wall β-glucan, CSBG, is an epitope of natural humanantibody. Drug Dev. Res. 2003, 58, 179–189. [CrossRef]

Nutrients 2019, 11, 1144

32.

33.
34.

35.
36.
37.

14 of 14

Feldman, S.; Schwartz, H.I.; Kalman, D.S.; Mayers, A.; Kohrman, H.M.; Clemens, R.; Krieger, D.R. Randomized
phase II clinical trials of Wellmune WGP® for immune support during cold and flu season. J. Appl. Res.
2009, 9, 30–43.
Talbott, S.M.; Talbott, J.A.; Talbott, T.L.; Dingler, E. β-Glucan supplementation, allergy symptoms, and qualityof life in self-described ragweed allergy sufferers. Food Sci. Nutr. 2013, 1, 90–101. [CrossRef]
Kamiya, T.; Tang, C.; Kadoki, M.; Oshima, K.; Hattori, M.; Saijo, S.; Adachi, Y.; Ohno, N.; Iwakura, Y.
β-Glucans in food modify colonic microflora by inducing antimicrobial protein, calprotectin, in a
Dectin-1-induced-IL-17F-dependent manner. Mucosal Immunol. 2018, 11, 763–773. [CrossRef]
Nguyen, S.G.; Kim, J.; Guevarra, R.B.; Lee, J.H.; Kim, E.; Kim, S.I.; Unno, T. Laminarin favorably modulates
gut microbiota in mice fed a high-fat diet. Food Funct. 2016, 7, 4193–4201. [CrossRef]
Bruce-Keller, A.J.; Salbaum, J.M.; Berthoud, H.R. Harnessing Gut Microbes for Mental Health: Getting from
Here to There. Biol. Psychiatry 2018, 83, 214–223. [CrossRef]
Hsiao, E.Y.; McBride, S.W.; Hsien, S.; Sharon, G.; Hyde, E.R.; McCue, T.; Codelli, J.A.; Chow, J.; Reisman, S.E.;
Petrosino, J.F.; et al. Microbiota modulate behavioral and physiological abnormalities associated withneurodevelopmental disorders. Cell 2013, 155, 1451–1463. [CrossRef]© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open accessarticle distributed under the terms and conditions of the Creative Commons Attribution(CC BY) license (http://creativecommons.org/licenses/by/4.0/).