175

J. Appl. Glycosci., 53, 175―180 (2006)

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C 2006 The Japanese Society of Applied Glycoscience

Regular Paper

Effects of 1-Kestose and Nystose on the Intestinal

Microorganisms and Immune System in Mice
(Received December 1, 2005; Accepted January 18, 2006)

Noboru Yoshida,1 Wakako Satou,1 Shuko Hata,1 Yasuyuki Takeda,1,＊ Shuichi Onodera,1
Kouichi Ando1 and Norio Shiomi1
1
Graduate School of Dairy Science Research, Rakuno Gakuen University
(582, Bunkyoudai-Midorimachi, Ebetsu 069―8501, Japan)

Abstract: We investigated the effects of the major short chain fructooligosaccharides, 1-kestose and nystose,
on the intestinal microorganisms and on the intestinal and systemic immune responses of mice. Both 1-kestose
and nystose promoted intestinal Lactobacillus number. However, the balance of Lb. reuteri and Lb. intesti-
nalis, the major Lactobacillus species in the mice was not altered. The IgA content in the feces of mice treated
with both 1-kestose and nystose increased from day 4 to day 7 after starting the administration and returned
to the same level of control mice on day 14. Splenocyte responses to Con A, anti-CD3 plus anti-CD28 antibod-
ies and LPS were reduced by 1-kestose and nystose. Nystose lowered IL-2, IFN-γ, IL-12 and IL-4 secretion
from the splenocytes more than 1-kestose. These results suggested that both 1-kestose and nystose can influ-
ence the microorganisms as well as the intestinal and systemic immune responses, but to different degrees.

Key words: fructooligosaccharide, 1-kestose, nystose, intestinal microorganisms, Lactobacillus, immune system

Fructooligosaccharides (FOSs) are short- and medium-
chains of fructose in which two to nine fructosyl units are
bound by β-2,1-glycosidic linkages and a glucose mole-
cule is present at the end of the chain joined by an α-1,2-
glycosidic bond. Fructooligosaccharides occur in various
plants such as onions, asparagus, artichokes and garlic.1)
Short-chain FOSs (sc-FOSs), such as 1-kestose, nystose
and 1F-β-fructofuranosylnystose can also be enzymatically
synthesized from sucrose.2,3)
Fructooligosaccharides are prebiotics that have been
studied in detail and they are widely used as a food ingre-
dient.4―6) A commercially available FOS product comprises
a mixture of sc-FOSs.4) The primary physiological func-
tion of FOSs is the preferential stimulation of colonic Bi-
fidobacteria. The effect has been confirmed in experimen-
tal animal models and in humans.7―10) Other physiological
functions of FOSs include the improvement of calcium
bioavailability, the modulation of lipid metabolism and
the inhibition of potential pathogen metabolism.5,11,12) These
functions are mainly due to the indigestibility13) and selec-
tive fermentability of FOSs in the gastrointestinal sys-
tem.14)
Studies have recently focused more on the immuno-
modulating effect of oligosaccharides.15,16) Nakamura et
al .17) reported that FOSs enhance the IgA response in the
intestine of infant mice and Hosono et al .18) also showed
that the amounts of fecal IgA and of IgA secretion by
Peyer’ s Patch cells are increased in FOS-treated mice.
Furthermore, Nicole et al .19) and Pierre et al .20) demon-
strated that dietary FOSs influence gut-associated lym-
phoid tissue.
Although beneficial colonic bacteria proliferation and
＊
Corresponding author (Tel. +81―11―388―4820, Fax. +81―11―387―
5848, E-mail: takeda@rakuno.ac.jp).
short-chain fatty acid production in the gut are thought to
be involved in the action mechanism of FOSs on the im-
mune system, details have not been fully established. Fur-
thermore, whether the prebiotic and immunomodulating
activities differ among FOSs in vivo and in vitro remains
obscure.
We investigated the effects of 1-kestose and nystose,
which are major components of a dietary FOS-product, on
intestinal microorganisms and on the splenocyte response
to mitogenic stimulation in mice.
MATERIALS AND METHODS
Animals. Female 6-week-old BALB／c mice obtained
from Charles River Japan (Yokohama, Japan) were main-
tained at 22―24° C and around 50% relative humidity un-
der a 12 h light-dark cycle. A commercial diet (CE-2,
Clea Japan, Tokyo, Japan) and water were available ad
libitum. The mice were separated by body weight into
three experimental groups consisting of five animals. The
experimental protocol was approved by the Animal Ex-
periment Committee of Rakuno Gakuen University and
the animals were managed in accordance with the Guide
for the Care and Use of Laboratory Animals of the same
institution.
Bacterial strains. Lactobacillus reuteri JCM 1081,
Lactobacillus murinus JCM 1717T and Lactobacillus in-
testinalis JCM 7548T purchased from the Japan Collection
of Microorganisms were cultured at 37° C in MRS me-
dium for DNA extraction.
Preparation and administration of 1-kestose and
nystose. Crystalline 1-kestose and nystose were prepared
by the method of Takeda et al .3) The FOSs were dis-
solved in sterilized distilled water at a concentration of 10
mg／mL and orally administered to mice via an intubation
 176 J. Appl. Glycosci., Vol. 53, No. 3 (2006)
needle at a dose of 100 mg／kg／day for 14 days.
Fecal sample preparation and measurement of micro-
organisms and total IgA content. Fresh fecal samples
were suspended in 99 volumes of sterilized saline (w／v)
and diluted × 10−2. This solution was serially diluted to
10−8 and 0.1-mL aliquots were spread onto agar plates.
We determined the numbers of Lactobacillus, E. coli and
Bifidobacterium using LBS agar, MacConkey agar and
MRS agar containing 0.3% LiCl, 0.01% neomycin sulfate
and 0.0015% nalidixic acid (MRS-LNN agar),21) respec-
tively. LBS and MRS-LNN agar plates were incubated at
37° C under anaerobic conditions with AnaeroPack (Mi-
tsubishi Gas Chemical Co., Inc. Tokyo) for 48 and 72 h,
respectively. MacConkey agar plates were incubated for
24 h at 37° C under aerobic conditions. We visibly distin-
guished E. coli as red colonies. Supernatants of the di-
luted samples were stored at ―80° C. Amounts of total IgA
in fecal samples were assayed using a mouse IgA ELISA
kit (Bethel Laboratories, Montgomery, TX, USA).
Analysis of fecal Lactobacillus by Denaturing Gradi-
ent Gel Electrophoresis (DGGE). Fecal suspensions
!
were centrifuged at 14,000 to obtain pelleted microor-
ganisms from which DNA was extracted and purified us-
ing a DNeasy tissue kit (Qiagen, Tokyo, Japan). The
DNA was amplified using the polymerase chain reaction
(PCR) with the Lactobacillus species-specific primers,
Lac1 and Lac2 with GC-clamp (Lac2-GC), according to
the program described by Walter et al .22) We also per-
formed DGGE according to Walter et al .23)
Preparation of spleen cells. Three mice in each group
were randomly selected and killed by cutting the carotid
artery under ether anesthesia. Excised spleens were pooled
and mechanically dissociated into single cell suspensions
on pairs of glass slides in RPMI-1640 medium. Erythro-
cytes were removed with ammonium chloride and then
cell suspensions were passed through a stainless mesh and
washed three times with the medium. Cell viability was
assessed by Trypan blue dye exclusion.
Assay of mitogenic response of spleen cells. Spleen
cells (106／mL) were incubated with mitogen in 0.2 mL of
RPMI-1640 medium containing 10% fetal bovine serum,
100 U／mL of penicillin, and 100 µg／mL of streptomycin
in flat-bottomed 96-well microplates for 3 days at 37° C cillus intestinalis were the marker strains as described by
under 5% CO2 in air. The Mitogens included plates pre-
Table 1. Effects of orally administered 1-kestose and nystose on bacteria number in feces.
Group
0 4
Bifidobacterium
Control 8.33±0.12
1-Kestose 8.39±0.39
Nystose 8.23±0.14
Lactobacillus
Control 8.09±0.35
1-Kestose 8.62±0.59
Nystose 8.30±0.31
E. coli
Control 6.09±0.24
1-Kestose 5.87±0.31
Nystose 6.01±0.16
Each value is the mean of log10 number per gram of feces ± SD, (n=5).＊(p<0.05) and
from value of control group determined by Dunnett’
s multiple comparison test.
coated with anti-CD3 antibody (CBL1317, Cymbus
Biotechnology, Hants, UK) plus 1 µg／mL of anti-CD28
antibody (sc-12727, Santa Cruz Biotechnology, Santa
Cruz, CA, USA, anti-CD3／CD28), concanavalin A (Con
A, 1 µg／mL; C-7275, Sigma, St. Louis, MO, USA) and
lipopolysaccharide (LPS, 5 µg／mL) from E. coli 0111:B4
(L-2630, Sigma). Three hours before the end of the cul-
ture, MTT solution (25 µL of 5µg／mL) was added to the
wells. The plates were centrifuged, the supernatant was
removed and 100 µL of 2-propanol was added to each
well. The absorbance of each well at 570 nm (A570) was
then measured using a microplate reader. The result is
shown as a stimulation index (S.I.) calculated by the fol-
lowing formula: S.I.=A570 of cell lysate with the mitogen／
A570 of cell lysate without the mitogen.
Assay of cytokine production. We measured the
amounts of IL-2, IL-4, IL-12 and IFN-γ in the superna-
tants of spleen cell cultures stimulated with anti-CD3／
CD28 or Con A for 2 days using a sandwich ELISA kit
(Biosource International, Camarillo, CA, USA).
Statistical analysis. The data were analyzed by one-
way analysis of variance followed by Dunnett’ s multiple
comparison test.
RESULTS
Effects of 1-kestose and nystose on fecal micro-
organisms.
The weight of all animals similarly increased (data not
shown), but the numbers of fecal bacteria differed (Ta-
ble 1). The administration of 1-kestose and nystose sig-
nificantly increased the numbers of Lactobacillus on days
4 and 7, and on days 4 and 14, respectively. The number
of Bifidobacterium did not significantly change with 1-
kestose or nystose administration under our experimental
conditions. The number of E. coli was significantly re-
duced by 1-kestose and nystose administration on day 14
and on day 4, respectively.
To determine whether 1-kestose and nystose could also
qualitatively affect intestinal lactobacilli, we performed
PCR-DGGE analyses using species-specific primers (Fig. 1).
Lactobacillus reuteri, Lactobacillus murinus and Lactoba-
Mitsuoka.24) Each Lactobacillus species was distinguish-
Days
7 14
8.25±0.15 8.04±0.25 8.11±0.16
8.11±0.46 7.69±0.24 8.24±0.22
8.00±0.28 8.16±0.29 7.91±0.26
8.39±0.14 8.66±0.26 8.66±0.10
9.03±0.21＊＊ 9.03±0.24＊ 8.86±0.26
8.84±0.12＊＊ 8.90±0.06 8.94±0.06＊
6.12±0.14 5.95±0.23 5.97±0.07
5.98±0.40 5.71±0.32 5.48±0.39＊
5.43±0.14＊＊ 5.66±0.11 5.83±0.12
＊＊
(p<0.01) represent the significant differences
 Prebiotic and Immunomodulating Activities of sc-FOSs 177

able in this PCR-DGGE analysis (Fig. 1, lane 1). Two
amplicons corresponding to Lactobacillus reuteri and
Lactobacillus intestinalis, were detected in all fecal sam-
ples. The intensity of the two amplicons obtained from fe-
ces on days 7 and 14 was similar to that determined on
day 0. These results suggest that 1-kestose and nystose in-
fluenced the intestinal Lactobacillus quantitatively but not
qualitatively.
IgA contents in feces of mice treated with 1-kestose
and nystose.
We examined the effect of 1-kestose and nystose on the
fecal IgA content (Fig. 2). The total IgA content in feces
of mice treated with 1-kestose or nystose increased from
day 4 and showed a significantly higher level as com-
pared with control mice on day 7. But it decreased to
nearly the same level as that of control mice on day 14.

Mitogenic response of splenocytes from mice treated

with 1-kestose and nystose.
To determine the effects of 1-kestose and nystose on
the systemic immune response in vivo, we examined
whether the FOSs could alter the proliferation of spleno-
cytes against several mitogenic stimuli on day 7. Both 1-
kestose and nystose lowered the proliferative responses of
splenocytes against anti-CD3／CD28 and Con A, which
are T-cell mitogens, and against LPS, which is a B-cell
mitogen (Figs. 3A, B, C).

Fig. 1. DGGE analysis of PCR products obtained with primer pair

Lac 1 and Lac 2-GC of fecal samples from mice treated
with water (control, lanes 2, 3, 4), 1-kestose (lanes 5, 6, 7)
and nystose (lanes 8, 9, 10) on days 0 (lanes 2, 5, 8), 7
(lanes 3, 6, 9) and 14 (lanes 4, 7, 10).
Fragment a, b and c represent PCR products of Lactobacillus in-
testinalis JCM 7548T, Lactobacillus murinus JCM 1717T and Lacto-
bacillus reuteri JCM 1081, respectively.

Fig. 3. Effects of orally administered 1-kestose and nystose on

Fig. 2. Effects of orally administered 1-kestose and nystose on a
total IgA content in feces.
Amount of fecal IgA from mice treated with water (control, ○),
1-kestose (●) or nystose (▲) was measured by ELISA. Results are
expressed as means ± SD, (n=5). ＊(p<0.05) and ＊＊(p<0.01) repre-
sent significant differences from value of control group determined
by Dunnett’ s multiple comparison test.
mitogenic responses of mouse splenocytes.
Splenocytes were prepared from mice treated with 1-kestose,
nystose or water (control) and cultured with anti-CD3／anti-CD28
antibodies (A), Con A (B) or LPS (C). Results are expressed as
means of four cultures ± SD. ＊(p<0.05) and ＊＊(p<0.01) represent
significant differences from value of control group determined by
Dunnett’ s multiple comparison test.

Table 2. Effects of orally administered 1-kestose and nystose on cytokine production from mouse splenocytes stimulated with Con A or
anti-CD3／anti-CD28 antibodies.

IL-2 (pg／mL) IFN-γ (pg／mL) IL-12 (pg／mL) IL-4(pg／mL)

Group Anti- Anti- Anti- Anti-
Con A Con A Con A Con A
CD3／CD28 CD3／CD28 CD3／CD28 CD3／CD28

Control 256.3±12.3 125.9±22.1 128.3±26.7 ND 83.4±14.6 ND ND 33.0±13.1

1-Kestose 207.0±18.4＊＊ 99.8±14.9 162.1±23.5＊ ND 94.9± 7.2 ND ND 25.5± 4.9
Nystose 136.3±23.8＊＊ 86.0±17.6＊ 43.6±15.7＊＊ ND 66.6± 6.5＊ ND ND 19.2± 2.9＊

Splenocytes were cultured at 37°

C with 5% CO2 for 48 h. Amounts of cytokines in the supernatants were measured by ELISA. Results
are expressed as means of four cultures ± SD. ＊(p＜0.05) and ＊＊(p＜0.01) represent significant differences from value of the control
group determined by Dunnett’ s multiple comparison test. ND: not detected.
 178 J. Appl. Glycosci., Vol. 53, No. 3 (2006)
Cytokine production from splenocytes.
We confirmed the suppressive effect of 1-kestose and
nystose on the splenocyte response as follows. We stimu-
lated cells with anti-CD3／CD28 or Con A and then mea-
sured the production of several cytokines that are involved
in T-cell proliferation and immune response modulation
(Table 2). Both 1-kestose and nystose significantly re-
duced IL-2 and IL-4 production. The production of IFN-γ
was obviously reduced by nystose, but slightly enhanced
by 1-kestose. IL-12 production was significantly reduced
by administration of nystose but not influenced by 1-
kestose.
DISCUSSION
Nystose and 1-kestose are the main constituents of a
dietary FOS-product, which is a characterized prebiotic.
Many investigators have clarified the biological activities
of FOSs, but mostly in a mixture. Here, we determined
the effects of both 1-kestose and nystose on the intestinal
microorganisms and on the immune cell response of mice.
When studying their biological activities, higher doses of
FOSs are generally applied to rodent models than in hu-
mans. Buddington et al .10) reported that 4 g of neosugar／
day per person alters the fecal flora. On the other hand,
the effects of a diet containing 25―100 g FOSs／kg on
mouse intestinal immunity have been determined.17―19)
However, our preliminary examination found that 20―100
mg／kg of 1-kestose per day altered the Con A response
of mouse splenocytes (data not shown). We therefore used
a dose of 100 mg／kg／day of FOS and found that the im-
mune cell response and the intestinal microorganisms
were indeed affected.
A dietary FOS product containing 1-kestose and nys-
tose dominantly increases intestinal Lactobacillus in
mice18) and rats.25) Our observations suggested that both
the FOSs had Lactobacillus-promoting activity. Our pre-
liminary experiments showed that 1-kestose and nystose
were fermentable by Lb. intestinalis and Lb. reuteri iso-
lates obtained from mouse feces (data not shown). If the
various intestinal Lactobacillus species can utilize FOSs
differently, then the Lactobacillus flora might change.
However, neither 1-kestose nor nystose altered the fecal
Lactobacillus ratio as assessed by PCR-DGGE using a
Lactobacillus-specific primer. The abilities of Lb. intesti-
nalis and Lb. reuteri in the mouse intestinal tract to util-
ize 1-kestose and nystose were essentially identical.
A dietary FOS-product can stimulate intestinal IgA se-
cretion. We confirmed that 1-kestose and nystose can en-
hance IgA secretion as the level increased in the middle
of our FOS administration schedule. Our results were very
similar to those of Nakamura et al .17) and of Hosono et
al .,18) although the dose of the FOS in our study was
much lower than theirs. Hosono et al .18) suggested that
stimulation of intestinal IgA secretion with FOSs corre-
lates with an increased proportion of intestinal Gram-
positive bacteria such as Lactobacillus. In our result the
enhancement of IgA content by 1-kestose and nystose was
shown from day 4 to 7 correlating with the increase of
Lactobacillus. The enhancement of IgA content by 1-
kestose and nystose, however, was not maintained on day
14. The feces showed slight diarrhea on day 14. Dilution
of the fecal IgA content by the diarrhea would be partly
influenced by the diminishment of the effect of 1-kestose
and nystose. Additionally, the unresponsiveness towards
the resident microflora was thought to be important to
permit their successful colonization.26) The enhancement of
the intestinal immune responses by the commensal micro-
organisms, such as Lactobacillus might not last for a long
time.
We found that both nystose and 1-kestose reduced the
splenocyte mitogenic response and Th1- (IL-2, IFN-γ and
IL-12) and Th2- (IL-4) oriented cytokine secretion. The
reduction of splenocyte proliferation with T-cell mitogens
is thought to be due to the decrease of IL-2 secretion.
Hosono et al .18) reported that a dietary FOS-product sup-
plementation could suppress Th-2 type antibody in the
sera, but not Th-1 type antibody. The dose amount and
period, and the type of FOS sample might account for the
difference between our results and theirs. Although our
results are not fully consistent with their observation, it
was suggested that both 1-kestose and nystose can prob-
ably modulate the systemic immune response to suppress
allergic and inflammatory reactions.
Interestingly, nystose suppressed cytokine secretion
more than 1-kestose whereas the Lactobacillus-promoting
activities of the FOSs did not significantly differ. Anti-
CD3 antibody plus anti-CD28 antibody can stimulate T-
cells directly to secrete IL-2, whereas Con A stimulation
requires some accessory cells, such as dendritic cells or
macrophages for IL-2 secretion from T-cells.27,28) Nystose
administration could suppress cytokine secretion from
splenocytes with both types of stimulation. IL-12 is one
of the Th1-oriented cytokines secreted mainly from den-
dritic cells and macrophages with various types of stimu-
lation.29) Nystose, but not 1-kestose, significantly reduced
IL-12 secretion, hence nystose administration can influ-
ence the function of dendritic cells／macrophages more
strongly than 1-kestose. Since IL-12 is a potent IFN-γ in-
ducer against various cells,29) the decrease of IFN-γ secre-
tion by nystose administration may be involved in the
down-regulated IL-12 secretion. Since nystose has a
higher suppressive activity for cytokine secretion, the in-
crease of the ratio of nystose in a dietary FOS-product
might enhance the immunomodulating activity.
Hosono et al .18) suggested that the increase of Gram-
positive bacteria, such as Lactobacillus, induced by a di-
etary FOS-product can affect not only the intestinal im-
mune response but also the systemic one. It would be
likely that the effects of 1-kestose and nystose on the sys-
temic immune response are strongly concerned with the
increase of Lactobacillus. But, the reason why the sup-
pressive effect of nystose is higher than that of 1-kestose
can’ t be explained by only the Lactobacillus number.
Zdunczyk et al .30) reported that a nystose-rich FOS prepa-
ration increased volatile fatty acid production, which is a
byproduct of fermentation by Gram-positive bacteria, in
the caecum of rats, when compared with a 1-kestose-rich
preparation. Although the effect of the two FOS prepara-
tions on the microflora or the immune system was not de-
scribed in their report, it is mostly probable that 1-kestose
and nystose show different activities on the microflora.
 Prebiotic and Immunomodulating Activities of sc-FOSs
Additional studies are required to determine which intesti-
nal microbes other than Lactobacillus and Bifidobacte-
rium are involved in the systemic immunomodulating ac-
tivity of 1-kestose and nystose, and whether the FOSs can
directly influence the immune cells.
Recent clinical and experimental data suggest that in-
flammatory bowel diseases can be prevented by inulin and
FOSs.31) Many types of cytokines are involved in the in-
flammatory bowel reaction in experimental models, such
as the dextran sulfate colitis or trinitrobenzene sulfonic
acid models.32,33) Strober et al .34) noted that IFN-γ and
TGF-β play critical roles in proinflammatory and in anti-
inflammatory reactions, respectively, and that the inhibi-
tion of Th1-oriented cytokines such as IFN-γ and／or IL-
12 might be central to the treatment of inflammatory
bowel disease. Cherbut et al .35) reported that FOSs reduce
intestinal inflammatory activity mainly by increasing the
amount of intestinal lactic acid bacteria. Although further
studies are still required to understand the anti-inflam-
matory activity of FOSs, their ability to reduce cytokine
secretion might be important to prevent the development
of bowel inflammation.
In conclusion, 1-kestose and nystose, which are major
components of a dietary FOS-product, can modulate the
intestinal microorganism flora through mechanisms such
as increasing Lactobacillus and／or decreasing E. coli, of
IgA production in the intestinal tract, and suppressing cy-
tokine production. However, these components have dif-
ferent degrees of effectiveness. The results of the present
study revealed more details of the biological activities of
1-kestose and nystose, and also indicated that the ratios of
the FOSs can be changed to improve their activities.
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1 1

and overview: recent advances in the immunotherapy of in- 1

酪農学園大学大学院酪農学研究科
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36―42 (2003). (069―8501 江別市文京台緑町 582)
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Ehrhardt and M. Neurath: Reciprocal IFN-gamma and TGF-
beta responses regulate the occurrence of mucosal inflamma- スおよびニストースの腸内菌叢および腸内と全身免疫反
tion. Immunol. Today, 18, 61―64 (1997). 応に及ぼす影響について，それらを個別に投与したマウ
35) C. Cherbut, C. Michel and G. Lecannu: The prebiotic charac-
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of TNBS-induced colitis in rats. J. Nutr., 133, 21―27 (2003). 投与はいずれも糞便中の Lactobacillus (Lb) 増殖促進作用
を示した (Table 1)．しかしながら，これらフラクトオリゴ
糖の投与はマウス糞便中に存在した 2 種の Lb. reuteri と
Lb. intestinalis のバランスは変化させなかった (Fig. 1). 1-
ケストースおよびニストースを投与されたマウス糞便中
の IgA 量は投与 4―7 日目にかけて増加し，14 日目にはコ
ントロール群と同等のレベルに戻った (Fig. 2). Con A，抗
CD3＋抗 CD28 抗体および LPS による脾臓リンパ球増殖
反応は，どちらのフラクトオリゴ糖の投与によっても減
少した (Fig. 3)．ニストースの投与は，脾臓リンパ球から
の IL-2，IFN-γ，IL-12 そして IL-4 産生を 1-ケストースよ
りも強く低下させた (Table 2)．これらの結果から 1-ケス
トースおよびニストースは，どちらも腸内細菌および腸
内と全身免疫反応に影響を及ぼすが，両者の効果の程度
は異なることが示唆された．