J. of Kyushu Univ. of Health and Welfare. 21： 67〜72，2020

67

Analysis of specific antibodies and immune biomarkers in healthy
subjects after consumption of Sophy β-glucan (β-1,3-1,6 glucan) as ahealth food supplement
Nobunao IKEWAKI, Tohru SONODA*, Yasushi MIYAZAWA**, Masayuki CHIKAMORI **Abstract
In this clinical study, we investigated changes in the levels of β-1,3-1,6 glucan (BG)-, lipopolysaccharide(LPS)-, and ovalbumin (OVA)-specific antibodies (IgG, IgA2, and IgM), interleukin-6 (IL-6), soluble CD44(sCD44), and granulocyte-colony stimulating factor (G-CSF) in the sera and saliva of 15 healthy subjects
after consuming commercially produced Sophy β-glucan (main ingredient: BG) for 1-2 weeks as a healthfood supplement. The results showed significant increases in BG-specific IgG (9.51 ± 4.37%) and IgM(16.51 ± 8.87%), but not in BG-specific IgA2. We observed no changes in LPS- and OVA-specific IgG,IgA2, or IgM between before and after consumption of the supplement. Clear increases in serum IL-6
(39.37 ± 16.81%), sCD44 (15.88 ± 8.39%), and G-CSF (15.04 ± 8.43%) were also observed after consuming
Sophy β-glucan. Increases in IL-6 (8.33 ± 3.82%), BG-IgA2 (11.27 ± 4.63%), and sCD44 (14.12 ± 9.54%) in
the subjects’ saliva were also observed after consuming Sophy β-glucan for 1 week. Taken together, ourdata suggest that the consumption of Sophy β-glucan clearly enhanced the BG-specific immune network
system in the whole body.
Key words ：β-1,3-1,6 glucan (BG), BG-specific antibody, immune biomarker, immune network system

Introduction

latest culture technology as a health food supplement(Sophy β-glucan), which contains about ten-fold theGlucans are mainly found in extracts of some species

quantity of BG in mushroom extracts.

of mushrooms and in certain microbes, such as black

It has recently been reported that human and animal

yeast, and they have several unique immunologicalsera contain BG-specific antibodies 4), and we haveactivities. In particular, β-glucans have been shown toreported finding an immunological relationshipactivate cytotoxic activity against cancer cells

between BG-specific antibodies and immune

accompanied by increased production of interleukin-2

biomarkers, including various cytokines and soluble-(IL-2), IL-6, IL-12, interferon-γ (IFN-γ), and tumor

form molecules 5). However, the rates of changes or

necrosis factor-α (TNF-α) 1, 2). These findings strongly

kinetics of these BG-specific antibodies and otherindicate that β-glucans enhance the immune system

immune biomarkers after consuming Sophy β-glucan

underlying the activation of lymphocytes, monocytes,(main ingredient: BG) as a health food supplement had

macrophages, granulocytes, and natural killer (NK)never been investigated.

cells via the β-glucan receptor, Dectin-1 .
3)

In this clinical study we investigated changes in theWe established and succeeded in purifying β-1,3-1,6levels of BG-specific antibodies (IgG, IgA2, and IgM)

glucan (BG) produced by the black yeast Aureobasidium

and other immune biomarkers, IL-6, soluble-form CD44

pullulans (A. pullulans ) strain AFO-202 by using the(sCD44), and granulocyte-colony stimulating factor

Department of Medical Life Science, Kyushu University of Health and Welfare School of Medical Life Science, Institute of Immunology, Junsei
Educational Institution, 1714-1 Yoshino-machi, Nobeoka-city, Miyazaki, 882-8508 Japan*Department of Occupational Therapy, Kyushu University of Health and Welfare School of Health Science, Institute of Immunology, Junsei
Educational Institution, 1714-1 Yoshino-machi, Nobeoka-city, Miyazaki, 882-8508 Japan**Chikamori Hospital, 1-1-16 Okawasuji, Kochi-city, Kochi, 780-8522 Japan68

九州保健福祉大学研究紀要 21： 67〜72，2020(G-CSF), in the sera and saliva of 15 healthy subjects

gargle and rinse their mouth (oral cavity) 15 times withafter consuming Sophy β-glucan.

water, and about 40 min later a saliva specimen wascollected in a 15-mL sterile tube for 2 min. All serum

Materials and Methods

and saliva samples were centrifuged at 3000 rpm for 30
min, and the supernatants were harvested and stored atReagents
Sophy β-glucan was produced by Sophy Co. (Kochicity, Kochi) from A. pullulans strain AFO-202, by using-80°
C until assayed.

Measurement of BG-, LPS-, and OVA-specificantibodies in the serum and saliva samples

the latest biological culture and preparation techniques,
and it is currently available commercially as a health

EIA plates (Sumitomo Co., Tokyo) were coated withfood supplement. The super-purified BG derived from

1 µg/mL of BG, LPS, and OVA in carbonate bicarbonate

Sophy β-glucan described above that was used as anbuffer (0.010 M NaCO3, 0.035 M NaHCO3, pH9.6) for 24

antigen in this study was kindly provided by the Sophy

hr at 4°
C. The wells were washed 4 times withCo. Enzyme immunoassay (EIA) kits for IL-6 andphosphate-buffered saline (PBS) containing 0.05%

sCD44 were purchased from Diaclone Laboratories Co.

Tween-20 (PBST) and blocked with PBST containing(USA). An EIA kit for G-CSF was purchased from IBL

2% BSA (BSA-PBST) for 60 min at room temperature,

Laboratories Co. (Fujioka, Gunma). Lipopolysaccharide

then washed 3 times with PBST. The sera (x1000(LPS) and ovalbumin (OVA) were purchased from

dilution for IgG, x50 dilution for IgA2 and x500 dilution

Sigma Co. (USA). Horseradish peroxidase (HRPOD)-for IgM) and the saliva (x5 dilution for IgA2) were thenconjugated rabbit anti-human IgG, HRPOD-conjugatedadded to each well, and the plate was incubated for 60rabbit anti-human IgM and HRPOD-conjugated mouse

min at room temperature with shaking. The wells

anti-human IgA2 monoclonal antibody (mAb) werewere then washed 5 times with PBST, and after

purchased from MBL Co. (Nagoya).

adding HRPOD-conjugated rabbit anti-human IgG(x10000 dilution), HRPOD-conjugated mouse anti-human

Ethics statement

IgA2 mAb (x3000 dilution), or HRPOD conjugatedrabbit anti-human IgM (x5000 dilution) to each well, theThe study protocol was approved by the institutional

plate was incubated for 60 min at room temperature

review boards (IRBs) of Kyushu University of Healthwith shaking. The wells were then washed 10 times

and Welfare and Chikamori Hospital, and the IRB

with PBST, and after adding substrate chromogen

numbers were 18-024 and 161, respectively. Informed(TMB; Cosmo Bio Co.) to each well, the plate wasconsent was obtained from all of the subjects prior toincubated for 20 min at room temperature with gentle

their participation in this study.

shaking. The reaction was stopped by the addition of0.5 M-HCl, and optical density (O.D.) was read at 450

Consumption of Sophy β-glucan as a healthfood supplement and preparation of serumand saliva samples
Fifteen healthy subjects with no abnormalities of thenm with a multichannel EIA-microplate reader(TOSHO Co.). The experiment was repeated 5 times.

Measurement of IL-6, sCD44, and G-CSF in
the serum and saliva samples

oral cavity (5 males, age 38.2 ± 9.5 yr; 10 females, age
28.7 ± 5.2 yr) in Chikamori Hospital (Kochi-city, Kochi)

The levels of IL-6, sCD44, and G-CSF in the serum

consumed Sophy β-glucan, 40 mg/day for 1-2 weeks, asand saliva samples were measured with EIA kits. Eacha health food supplement. Blood collection and serumof the measurements was repeated 3 times.

preparation were performed according to the standard
methods. To collect saliva, subjects were asked toNobunao IKEWAKI, Tohru SONODA, Yasushi MIYAZAWA, Masayuki CHIKAMORI：β-1,3-1,6 glucan-specific antibodies and immune biomarkers

Statistical analysis

69OVA-specific IgG, IgA2, and IgM after consuming
Sophy β-glucan, suggested that the BG-specific immune

Data were expressed as the mean ± standard deviation

reaction was effectively appeared in the whole body.

(SD). The data were statistically analyzed by performing

IL-6 is a cytokine produced by T-cells, B-cells,

the unpaired t -test. A P value <0.05 was considered

monocytes, fibroblasts, endothelial cells, etc., and plays

evidence of a statistically significant difference.

an important role in both natural and acquired immune
responses, in particularly by regulating the growth andResults and Discussion

m a t u r a t i o n o f B - c e l l s t o p r o d u c e a n t i b o d i e s 6) .
Interestingly, the results of this study showed increased

First, we examined the changes in BG-, LPS-, OVA-

IL-6 in both serum and saliva after consuming Sophy β

specific IgG, IgA2, and IgM and the immune biomarkers-glucan. Thus, it is likely that the increases inIL-6, sCD44, and G-CSF in the serum and saliva samples

BG-specific IgG and IgM in the sera and BG-specificof the 15 healthy subjects after consuming Sophy

IgA2 in the saliva were directly or indirectly influenced

β-glucan for 1-2 weeks.

by IL-6 action.

As shown in Figure 1, clear increases in BG-specificCD44 is expressed on lymphocytes, macrophages,

IgG (9.51 ± 4.37%) and IgM (16.51 ± 8.87%), but not in

granulocytes, fibroblasts, endothelial cells, NK cells,

IgA2 (1.12 ± 0.67%), were observed in the sera of theetc., and it plays an important role in stimulating

healthy subjects after consuming Sophy β-glucan, but noNK-cell and lymphokine-activated killer cell (LAK)changes in LPS- or OVA-specific IgG, IgA2, or IgM werefunctions underlying IFN-γ production. The marked

seen in their sera under the same conditions. Theincreases in sCD44 in both sera and saliva after

BG-specific IgG and IgM were statistically significant (Pc o n s u m i n g S o p h y β- g l u c a n s u g g e s t t h a t t h e< 0.01) compared with the percentage increases in LPS-

enhancement of NK-cell and LAK- cell functions in thespecific IgG and IgM and OVA-specific IgG and IgM,

whole body 7).

respectively.

G-CSF is a glycoprotein that stimulates the bone

Next, we also examined the changes in IL-6, sCD44,

marrow to produce granulocytes (neutrophils) andand G-CSF in the sera after consuming Sophy β-glucan.

release them into the bloodstream, and it prolongs theFigure 2 shows that there were marked increases insurvival and stimulates the proliferation andIL-6 (39.37 ± 16.81%), sCD44 (15.88 ± 8.39%), and G-CSFdifferentiation, and function of neutrophil precursors and(15.04 ± 8.43%) in their sera. We also examined themature neutrophils 8). In this study we observed anchanges in some immune biomarkers in saliva after

increase in G-CSF production in the sera after

consuming Sophy β-glucan for 1 week, and clear

consuming Sophy β-glucan. Sophy β-glucan consumption

increases in IL-6 (8.33 ± 3.82%), sCD44 (14.12 ± 9.54%),

probably stimulates the proliferation and differentiation

and BG-IgA2 (11.27 ± 4.63%) were observed.

of neutrophils in the immune response and protects

In this study increases in BG-specific IgG and IgMagainst microbial infection.

were observed in the sera after consuming Sophy

Although the exact mechanism(s) underlying theβ-glucan in comparison with before consumption, andincrease and immunological roles of BG-specificthe increases in BG-specific IgM were marked. It is wellantibodies and immune biomarkers IL-6, sCD44, andknown that IgM is the first antibody to appear as partG-CSF in the sera and saliva of healthy subjects afterof the immunological response. It may be surmised that

consuming Sophy β-glucan remains unclear, we are

the increase in BG-specific IgM after consuming

vigorously investigating the mechanism(s) by using

Sophy β-glucan plays an important role in somemolecular immunological techniques. Since β-glucan has

immunobiological response network. Although we did

been reported to stimulate intestinal immunity 9), it is

not establish a placebo group in this study because ofpossible that the action of intestinal bacteria flora

the short period of consumption of Sophy β-glucan, thepromotes the increase in BG-specific antibodies andfact that there were no significant increases in LPS-,

some immune biomarkers after Sophy β-glucan

70

九州保健福祉大学研究紀要 21： 67〜72，2020

consumption, and more effectively regulates theinduced cooperative oligomerization of Dectin-1

BG-related immune network system in the whole bodyC-type lectin-like domain. Glycobiology. 28:612-623,

as a biological response modifier (BRM). Further2018.

analyses at the cellular and molecular levels in an invitro model are needed to identify the exact4Ikewaki, K., Sonoda, T., Azuma, K.: A study of antimechanism(s) responsible for the increase in BG-specificβ-glucan antibody in human serum. J. Kyushu Univ.

antibodies and some immune biomarkers afterH. W. 16: 115-119, 2015.

consuming Sophy β-glucan.
5

Acknowledgements

Ikewaki, N., Sonoda,T., Miyazawa, Y., et al.: Serumlevels of β-1,3-1,6 glucan-specific antibodies andimmune biomarkers in normal individuals. J.

We wish to express our gratitude to T. Onaka andKyushu Univ. H. W. 19: 87-94, 2018.

Y. Ikeue of Sophy Co. (Kochi-city, Kochi) for preparing
and providing us with β-1,3-1,6 glucan.

6Shen, P., Fillatreau, S.: Antibody-independentfunctions of B cells: a focus on cytokines. Nat. Rev.

Disclosure
None of the authors has any conflicts of interestImmunol. 15: 441-451, 2015.
7(COI) to disclose.

Sun, J., Law, G.P., McKallip, R.J. : Role of CD44 inlymphokine-activated killer cell-mediated killing ofmelanoma. Cancer Immunol. Immunother. 61: 323-References
12Ikewaki, N., Fujii, N., Onaka, T., et al.: Immunological334, 2012.
8Pahnke, S., Egeland, T., Halter, J., et al.: Current use

actions of Sophy beta-glucan (beta-1,3-1,6-glucan),of biosimilar G-CSF for haematopoietic stem cell

currently available commercially as a health foodmobilisation. Bone Marrow Transplant. 54:858-866,supplement. Microbiol. Immunol. 51:861-873, 2007.

2019.

Jin, Y., Li, P., Wang, F. : β-glucans as potential

9Kamiya, T., Tang, C., Kadoki, M., et al.: β-Glucans in

immunoadjuvants: A review on the adjuvanticity,

food modify colonic microflora by inducing

structure-activity relationship and receptor

antimicrobial protein, calprotectin, in a Dectin-1-recognition properties. Vaccine. 36:5235-5244, 2018.

induced-IL-17F-dependent manner. MucosalImmunol. 11:763-773, 2018.

3Dulal, H.P., Adachi, Y., Ohno, N., et al.: β-Glucan-

Nobunao IKEWAKI, Tohru SONODA, Yasushi MIYAZAWA, Masayuki CHIKAMORI：β-1,3-1,6 glucan-specific antibodies and immune biomarkers

Figure 1. Measurement of BG-, LPS-, and OVA-specificIgG, IgA2 and IgM in the sera of 15 healthy subjects
after consuming Sophy β-glucan. The measurements
were performed using an EIA system (See Materialsand Methods for details). Each measurement wasrepeated 4 times. The percentage increases in eachsubject were calculated as follows. Percent increase(%) = {δOD value after consumption - δOD value before
consumption)/δOD value before consumption} x 100,and the average values for the entire population (n =15) were then calculated.
* P < 0.01 (BG-specific IgG vs. LPS-specific IgG or OVAspecific IgG), ** P < 0.01 (BG-specific IgM vs. LPSspecific IgM or OVA-specific IgM).

Figure 2. Measurement of IL-6, sCD44, and G-CSF in
the sera of 15 healthy subjects after consuming Sophy
β-glucan. The measurements were performed using anEIA system (See Materials and Methods for details).
Each measurement was repeated 4 times. The
percentage increases in each subject were calculated
as follows. Percent increase (%) = {amount afterconsumption - amount before consumption)/amount
before consumption} x 100, and the average values forthe entire population (n = 15) were then calculated.

71

Figure 3. Measurement of IL-6, sCD44 and BG-IgA2 inthe saliva of 15 healthy subjects after consuming
Sophy β-glucan. The measurements were performed
using an EIA system (See Materials and Methods fordetails). Each measurement was repeated 4 times. The
percentage increases in each subject were calculated
as follows. Percent increase (%) = {amount or δOD value
after consumption - amount or δOD value beforeconsumption)/amount or δOD value before consumption}
x 100, and the average values for the entire population(n = 15) were then calculated.

72

九州保健福祉大学研究紀要 21： 67〜72，2020

Sophy β-glucan（β-1,3-1,6 glucan）摂取後の特異抗体と免疫バイオマーカーの解析
池脇 信直, 園田 徹*, 宮澤 靖**, 近森 正幸**
九州保健福祉大学生命医科学部生命医科学科・順正学園免疫学研究所 〒882-8508 宮崎県延岡市吉野町1714-1*九州保健福祉大学保健科学部作業療法学科・順正学園免疫学研究所 〒882-8508 宮崎県延岡市吉野町1714-1**近森病院 〒780-8522 高知県高知市大川筋1-1-16

要旨
Sophy β-glucan（β-1,3-1,6 glucan）摂取後の健康人血清中および唾液中のβ-1,3-1,6 glucan（BG）特異抗体と免疫バイオマーカーの動態を酵素抗体法で検討した。その結果、Sophy β-glucan 摂取 2 週間後、摂取前に対して血清中に BG 特異 IgG(9.51 ± 4.37%) と IgM(16.51 ± 8.87%) の増加が認められた。一方、
lipopolysaccharide (LPS) および ovalbumin (OVA) 特異 IgG、IgA2 および IgM の増加は認められなかった。
さらに、Sophy β-glucan 摂取 2 週間後、摂取前に対して血清中に IL-6(39.37 ± 16.81%)、sCD44(15.88 ±8.39%)、G-CSF(15.04 ± 8.43%) の増加が認められた。また、Sophy β-glucan 摂取 1 週間後、摂取前に対して唾液中に IL-6(8.33 ± 3.82%)、BG-IgA2(11.27 ± 4.63%)、sCD44(14.12 ± 9.54%) の増加が認められた。以上の結果は、Sophy β-glucan を摂取後、血清中または唾液中の BG 特異 IgG、IgA2 および IgM、さらにIL-6、sCD44、G-CSF が増加することから BG 特異的な生体免疫システムが増強されたことがわかった。
キーワード：β-1,3-1,6 glucan(BG)、BG 特異抗体、免疫バイオマーカー、免疫ネットワークシステム