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TitleInfluence of chewing force on salivary stress

markers as indicator of mental stress

Author(s)

AlternativeSoeda, R; Tasaka, A; Sakurai, K

Journal Journal of oral rehabilitation, 39(4): 261-269

URL http://hdl.handle.net/10130/2841

Right

This is the pre-peer reviewed version of the

following article: J Oral Rehabil. 2012

Apr;39(4):261-9, which has been published in final

form at http://dx.doi.org/10.1111/j.1365-

2842.2011.02264.x

1

Influence of Chewing Force on Salivary Stress Markers as Indicator of

Mental Stress

Ryohei SOEDA, Akinori TASAKA, Kaoru SAKURAI

Department of Removable Prosthodontics and Gerodontology, Tokyo Dental College, Chiba,

Japan

Running title: Influence of Chewing Force on Mental Stress

Keywords: chewing force, mental stress, cortisol, alpha-amylase, secretory immunoglobulin A

2

Correspondence: Dr. Ryohei SOEDA

Department of Removable Prosthodontics and Gerodontology, Tokyo Dental College,

1-2-2 Masago, Mihama-ku, Chiba City, Chiba, 261-8502, Japan

Phone: +81-43-270-3933/Fax: +81-43-270-3935

E-mail address: [email protected]

3

SUMMARY

The aim of the present study was to investigate the influence of chewing force on salivary stress

markers (alpha-amylase activity, salivary cortisol level, and secretory immunoglobulin A

secretion rate) as indicators of mental stress. Participants comprised 20 healthy men. The first set

of saliva specimens (S1) was collected at immediately after a 20-minute rest to evaluate stress

markers. As stress loading, the participants were required to perform arithmetic calculations for

20 min, after which the second set of saliva specimens (S2) was collected. Each participant was

then required to chew a piece of tasteless gum for 10 min, after which the third set of saliva

specimens (S3) was collected. After a 20-minute rest, the fourth set of saliva specimens (S4) was

collected. Weak, habitual, and strong chewing forces were assigned. Change rates of stress

markers between S2 and S3, and S2 and S4 were calculated. A significant difference was

observed in the change rate of cortisol levels between S2 and S3. Cortisol level decreased more

under strong chewing than under weak chewing. No significant differences were observed in the

change rate of amylase activity or s-IgA secretion rate among the 3 chewing forces. The results

suggest that differences in chewing force influence the salivary cortisol level of the 3 stress

markers, and that a strong chewing force induces a greater reduction in mental stress than a weak

one.

4

Introduction

Many reports have been published on mental stress reduction by chewing as reflected in

changes in sympathetic, endocrine, and immune system markers in saliva. Nakajo et al. reported

that alpha－amylase activity, a sympathetic nervous system stress marker, decreased with

chewing in a mentally stressful environment (1). Tahara et al. （2） and Scholey et al. （3）

reported that salivary cortisol levels, an endocrine system stress marker, decreased with chewing

after mental stress loading. Furthermore, Ishiyama et al. reported that s－IgA levels, an immune

system stress marker, decreased with chewing (4).

In terms of masticatory movement parameters, Tasaka et al. suggested that a fast chewing rate

induced a greater reduction in mental stress than a slow one (5). No studies, however, have

investigated the influence of other masticatory movement parameters such as chewing force,

chewing number, and chewing time on reduction of mental stress. Peter A et al. reported a

correlation between the mean amplitude electromyogram (EMG) of the masticatory muscle and

chewing force (6). Ruf S et al. reported that the mean amplitude EMG of the masticatory muscle

during chewing increased under mental stress (7). Niwa M et al. reported that chewing increased

activity in the prefrontal cortex, which is involved in stress control (8).

5

We hypothesized that chewing force as a masticatory movement parameter induced a

reduction in mental stress. The aim of the present study was to investigate the influence of

chewing force on salivary stress markers (sympathetic system alpha-amylase activity, endocrine

system salivary cortisol level, and immune system s-IgA secretion rate) as indicators of mental

stress.

Materials and methods

Participants

Twenty healthy men of between 23 and 30 years of age (average, 25.5 years) were recruited from

students and staff at Tokyo Dental College. All participants had complete natural dentition,

excluding the third molars, and were without subjective or objective abnormalities of the

stomatognathic system. None had any medical history of mental illness. All were non-smokers.

Written informed consent was obtained from all participants. The study was approved by the

Ethics Committee of Tokyo Dental College (#218).

Experiment schedule

6

In consideration of the influence of circadian rhythm on salivary stress marker levels, the

experiments were performed between 2:00 PM and 7:00 PM. The participants were required to

refrain from eating, drinking (alchohol, caffeine), taking drug and exercising within 2 hr prior to

commencement of the experiments (9, 10) and were allowed 20 min in the experimental room to

rest and familiarize themselves with the environment before testing. Immediately after this, the

first set of saliva specimens (S1) was collected. As stress loading, participants were required to

perform arithmetic calculations (addition, subtraction, multiplication, division) for 20 min, after

which the second set of saliva specimens (S2) was collected. Each participant was then required

to chew a piece of tasteless gum base weighing 1.0 g (Lotte, Saitama, Japan) for 10 min. The

hardness of the gum base was 6.4×103 Pa・s (soft type). After that, the third set of saliva

specimens (S3) was collected. After a 20-min rest, the fourth set of saliva specimens (S4) was

collected, completing the experiment (Fig. 1). The schedule of mental stress loading and rests

was determined based on previous reports (2, 5).

Weak, habitual, and strong chewing forces were applied. Participants were told to chew softly

for weak chewing, and strongly for strong chewing. Conscious chewing does not cause

discomfort or fatigue of the masticatory muscle. Therefore, a non-chewing condition was not

applied to avoid excessive stress.

7

The habitual chewing rate of each participant was determined with an electronic metronome.

The participants were instructed to maintain the same posture throughout the experiment. Each

participant was assigned all 3 chewing forces, each on a different days, also left the interval more

than a day. The 3 chewing forces were assigned in random order.

8

Measurements

In this study, salivary cortisol, alpha-amylase and s-IgA levels were selected as markers of

increase in stress. The cortisol and s-IgA saliva specimens were collected using a saliva sampling

device (Salivette, Sarsted, Rommelsdorf, Germany), keeping cotton rolls in the oral cavity for 2

min. In the laboratory, the samples were centrifuged at 3000 rpm for 20 min in a refrigerated

centrifuge. The supernatant of the collected whole saliva was frozen at -20. Salivary cortisol

levels (μg/dl) were determined with a radioimmunoassay kit (GammaCoat, Diasorin, Stillwater,

OK, USA) in accordance with the manufacturer’s instructions. The s-IgA levels (μg/dl) were

determined with an enzyme immunoassay kit (Poseidon2, Aloka, Tokyo, Japan). Alpha-amylase

saliva specimens were collected with an amylase monitor chip (Salivary amylase monitor chip,

Nipro, Osaka, Japan). The monitor chip was immersed in whole saliva under the tongue of the

participant for 30 sec.

Alpha-amylase activity (kU/l) was analyzed with a salivary amylase monitor (Salivary Amylase

Monitor, Nipro, Osaka, Japan). The s-IgA level (μg/dl) was corrected to the s-IgA secretion rate

(μg/min) by saliva flow rate for 2 min (dl). This was because, while salivary cortisol level and

alpha-amylase activity are not influenced by saliva flow rate, s-IgA level is (11-16).

9

This was because, while salivary cortisol level and alpha-amylase activity are not influenced by

saliva flow rate (11-14). S-IgA level is influenced (15, 16).

An EMG recording system (Muscle Tester ME3000P, Mega Electronics, Kuopio, Finland),

with a built-in 12-bit A/D converter was used to monitor chewing rates and determine

myoelectrical activity in the masseter muscle. For EMG of muscle activity, bipolar surface

electrodes (Blue Sensor P-00-S, Medicotest, Olstykke, Denmark) were positioned parallel to the

main direction of the muscle fibers on the maximal bulk of the bilateral masseter muscle, which

was determined by palpation while the participants clenched intermittently. Interelectrode

distance was 25 mm centre-to-centre. Reference (grounding) electrodes with integrated

preamplifiers were attached behind the ear. Prior to attachment of the electrodes and bioelectrical

measurement, the skin was thoroughly cleansed with a specific skin cleansing gel (Skin Pure,

Nihon Kohden, Tokyo, Japan) and ethanol-soaked gauze. Skin impedance between the electrodes

was lower than 8 kΩ. Sampling frequency was 1000 Hz at a sampling period of 0.1 sec.

The daily precision of the EMG was corrected by the mean amplitude EMG of the masseter

muscle under non-chewing conditions.

Statistical analysis

10

Alpha-amylase activity at S1 was regarded as signifying the baseline relaxed state. Participants

showing a lower alpha-amylase activity at S2 than at S1were regarded as being uninfluenced by

the mental stress loading applied in this study and excluded from the statistical analysis. Changes

in salivary stress markers (salivary cortisol level, alpha-amylase activity and s-IgA secretion rate)

between S2 and S3, and S2 and S4 were determined and compared. The mean amplitude and

integrated EMG of the masseter muscle over 10 min was calculated. Changes in salivary stress

markers and the mean amplitude and integrated EMG of the masseter muscle among the 3

chewing forces were compared using a one-way repeated measures analysis of variance

(ANOVA) and the post-hoc Bonferroni test at a significance level of 5% using statistical

software (SPSS 11.0J; SPSS, Chicago, IL, USA).

Results

Figure 2 shows changes in salivary cortisol level with time. Change in the salivary cortisol

level was expressed as a change rate, as individual differences exist in cortisol levels. The change

in the salivary cortisol level between S2 and S3 was significantly smaller with strong chewing

than with weak chewing. Only the change rate in salivary cortisol level with weak chewing

increased, suggesting an increase in mental stress. The change in salivary cortisol level between

11

S2 and S4 decreased under all chewing conditions, but no significant difference was observed

among the 3 chewing forces (Fig. 3).

Figure 4 shows change in alpha-amylase activity with time. The mean change rates of alpha-

amylase activity between S2 and S3, and S2 and S4 decreased with strong chewing and increased

with weak chewing, but no significant difference was observed among the 3 chewing forces

(Figs. 5).

Figure 6 shows change in s-IgA secretion rate with time. The change in the s-IgA secretion

rate between S2 and S3 decreased under all chewing conditions. No significant difference was

observed among the 3 chewing forces. Change in the s-IgA secretion rate between S2 and S4

decreased with weak and habitual chewing and increased with strong chewing. No significant

differences were observed among the 3 chewing forces (Fig. 7).

The mean amplitude EMG of the masseter muscle showed significant differences among the 3

chewing rates (Fig. 8). Comparison of the mean amplitude EMG revealed that strong chewing

comprised 169% of habitual chewing, and weak chewing 25%. The integrated EMG of the

masseter muscle revealed significant differences among the 3 chewing rates (Fig. 9).

Discussion

12

Individual differences were observed in the level of chewing force required to fatigue each

participant in a pilot experiment. Therefore, chewing conditions were determined by the

participants themselves to avoid physical stress. The participants were instructed to adjust the

chewing force consciously. Mean amplitude EMG is considered to be associated with chewing

force. Significant differences in the mean amplitude EMG of the masseter muscle were observed

under all chewing conditions, thus confirming differences in chewing force and the validity of

setting different chewing forces as an experimental condition. Although chewing force can be

adjusted by changing the hardness of the gum base, this method was not applied in the present

study, as occlusal force differs among individuals and recognition of softness and change in gum

base hardness in itself may cause physical stress.

In the pilot study, change in cortisol level, alpha-amylase activity, and s-IgA secretion rate in

saliva after mental stress loading was compared between with and without chewing. The results

showed that stress markers in saliva were lower under chewing than non-chewing conditions.

This was consistent with the results of earlier reports (1-4). In the present study, taking stress into

consideration, the change rate was compared without applying a non-chewing condition (there

was a 10-min rest without chewing after stress loading).

13

The change rate in salivary cortisol level between S2 and S3 showed a significantly greater

decrease with strong chewing compared with weak chewing. This suggests that strong chewing

reduces mental stress more than weak chewing.

Stress includes both mental and physical stress. In the present study, arithmetic calculation

(addition, subtraction, multiplication, and division) was used to apply mental stress. It was

confirmed that alpha-amylase activity was higher at S2 (after mental stress loading) than at S1

(before mental stress loading). It has been reported that alpha-amylase, a sympathetic system

stress marker, reacts rapidly, and is therefore considered to be an effective mental stress marker

(17). Four of 20 participants in this study showed lower alpha-amylase activity at S2 than at S1,

and these were excluded from the statistical analysis.

Salivary cortisol level was endocrine system stress marker. In this study all participants were

male. Because, female participants need to control estradiol cycle (18). If it were not controlled ,

daily varience of cortisol level was large.

Salivary cortisol level did not increase at S2 compared with at S1 with weak chewing. We

believe that this was because salivary cortisol level increases at S1 before stress loading due to

stress anticipation ( 19, 20), and it takes 5-20 minutes for stress-induced blood cortisol to appear

14

in saliva (12, 21). S-IgA secretion rate and amylase activity are increase immediately after stress

loading.

There were reported about circadian rhythm. In cortisol level, circadian variability is low after

3:00 PM (22). In amylase activity, circadian variability is low between 4:00 and 5:00 PM (23). In

s-IgA secretion rate, circadian variability is low afternoon (24). Considering all of stress marker’s

condition, experimental schedule is severe. In this study, the experiment is performed between

2:00PM and 7:00PM.

And then, there were reported about stress marker reaction time. In cortisol levels, it takes 5-

20 minutes for increase after stress loading (12, 21). In amylase activity, it takes 1 minute for

increase after stress loading (25). In s-IgA secretion rate, immediately increase after stress

loading (26). Considering all of stress marker’s character, saliva was collected immediately after

task and each task was set enough long time in this study.

The cortisol-involving hypothalamic-pituitary-adrenal axis (HPA), the amylase-involving

sympatho-adrenal system (SAS), and the s-IgA-involving immune system react to mental stress.

However, it was reported that the s-IgA reaction under acute mental stress reflects SAS stress

reaction (27, 28). The s-IgA secretion rate was reported to increase by acute stress and decrease

by chronic stress (29, 30). In this study, it was anticipated that s-IgA secretion rate would

15

increase with acute stress and decrease with chewing. Previous reports have shown that mental

stress is reduced by chewing, as evidenced by a decrease in alpha-amylase activity, a sympathetic

nerve marker (1). A number of studies have reported that chewing activates the sympathetic

nerves (4, 31, 32). Meanwhile, other reports have demonstrated that chewing activates the

parasympathetic nerves (33). In this study, the results showed that using sympathetic nerve

activation as a marker of stress reduction was not consistent. Together with the results of the

earlier studies mentioned above, this suggests that the reaction changes under different chewing

conditions. Although we set the hardness of the sample food, chewing time, and chewing rate,

the change rate of alpha-amylase activity varied widely. It is considered difficult to evaluate

mental stress reduction based on SAS-related alpha-amylase and s-IgA activity. On the other

hand, change in HPA cortisol level showed no relationship with sympathetic nerve activity, and,

therefore, no correlation with change in alpha-amylase or s-IgA level (34, 35). This suggests that

chewing force specifically affects not the SAS, but the HPA stress reaction.

The integrated EMG of the bilateral masseter muscles (workload) showed a significant

difference between weak, habitual, and strong chewing conditions, suggesting that the workload

changed under different chewing forces. The relationship between the integrated EMG and

salivary cortisol level change rate was analyzed. A moderate negative correlation was observed

16

in the change rate of the salivary cortisol level and the integrated EMG between S2 and S3 (Fig.

13). The results showed that an increase in chewing force increased the workload and released

mental stress. Tasaka et al. reported that there was a correlation between the number of chewing

cycles and mental stress reduction (r = 0.49). This suggests that conscious strong chewing that

does not cause fatigue, and increasing the number of chewing cycles are effective for stress

reduction.

Conclusion

The results of the present study showed that chewing force affected an HPA stress marker,

salivary cortisol level, and that a strong chewing force was more effective than a weak one in

reducing mental stress.

Acknowledgments

We are grateful to the participants for their kind cooperation in this study. We would also like to

thank Dr Mutsumi Takagiwa (Associate Professor, Mathematics Laboratory, Tokyo Dental

College) for his help with the statistical analysis and Associate Professor Jeremy Williams,

Tokyo Dental College, for his assistance with the English of the manuscript. Lotte Co., Ltd.

17

(Saitama, Japan) are also acknowledged for their kind gifts of the test foods. This work was

supported by a grant for the Promotion and Mutual Aid Corporation for Private Schools of Japan.

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Figure legend

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Fig1

Experimental schedule . S was saliva collection timing .

Fig2

Salivary cortisol level under each condition. These were compared between saliva collection

timing in each condition using a one-way repeated measures analysis of variance (ANOVA) and

the post-hoc Bonferroni test at a significance level of 5%.

Fig3

Changes in salivary cortisol level between after stress loading (S2) and after chewing (S3), after

stress loading (S2) and after rest (S4). These were compared using a one-way repeated measures

analysis of variance (ANOVA) and the post-hoc Bonferroni test at a significance level of 5%.

Fig4

Alpha-amylase activity under each condition. These were compared between saliva collection



Fig5

24

Changes in alpha-amylase activity between after stress loading (S2) and after chewing (S3), after



Fig6

The s-IgA secretion rate under each condition. These were compared between saliva collection



Fig7

Changes in s-IgA secretion rate between after stress loading (S2) and after chewing (S3), after



Fig8

Mean amplitude EMG of masseter muscle for 10 minutes chewing. These were compared using a

one-way repeated measures analysis of variance (ANOVA) and the post-hoc Bonferroni test at a

significance level of 5%.

Fig9

25

Integrated EMG of masseer muscle for 10 minutes chewing. These were compared using a one-

way repeated measures analysis of variance (ANOVA) and the post-hoc Bonferroni test at a

significance level of 5%.

Fig10

Correlation between change in salivary cortisol levels between S2 and S3 and integrated EMG of

masseter muscle for 10 minutes. This was compared using Spearmans’ correlation.

20 20 2010

Rest RestChewingStress-loading

S1 S3S2 S4

Saliva collection

Time2 2 2 2

: Chewinga. Weak chewingb. Habitual chewingc. Strong chewing

S

min

Fig1 Experimental schedule

0

0.1

0.2

0.3

0.4

0.5n=16

: ± SDNS

Weak chewing Habitual chewing Strong chewing

Saliv

ary

corti

soll

evel

μg/dl

Fig2

S1 S3 S4 S2

Salivary cortisol level under each condition

S1 S3 S4 S2 S1 S3 S4 S2

‐100

‐50

0

50

100*

Change in salivary cortisol level between after stress loading (S2) and after rest (S4)

Cha

nge

in sa

livar

y co

rtiso

llev

el

Weak chewing

Habitual chewing

Strong chewing

n = 16: ± SD: p<0.05

*

‐100

‐50

0

50

100

%

Cha

nge

in sa

livar

y co

rtiso

llev

el

n = 16: ± SD

NS

Weak chewing

Habitual chewing

Strong chewing

Fig3Change in salivary cortisol level between after stress loading (S2) and after chewing (S3)

%

n=16: ±SD: p<0.05

0

20

40

60

80

100

120


Alp

ha-a

myl

ase

activ

itykU/l

Fig4Alpha-amylase activity under each condition

S1 S3 S4 S2 S1 S3 S4 S2 S1 S3 S4 S2

**

* *

*

‐100

‐50

0

50

100

150

%

Cha

nge

in a

lpha

-am

ylas

e ac

tivity

n = 16: ± SD

NS

Fig5

Weak chewing

Habitual chewing

Strong chewing

-100

-50

0

50

100

150

%

Cha

nge

in a

lpha

-am

ylas

e ac

tivity

n = 16: ± SD

NS

Weak chewing

Habitual chewing

Strong chewing

Change in alpha-amylase activity between after stress loading (S2) and after chewing (S3)

Change in alpha-amylase activity between after stress loading (S2) and after rest (S4)

0

100

200

300

400

500

600

700

800


Fig6

n=16: ± SD: p<0.05

S-Ig

Ase

rect

ion

rate

μg/min

s-IgA secretion rate under each condition

S1 S3 S4 S2 S1 S3 S4 S2 S1 S3 S4 S2

** *

**

*

*

-300

-200

-100

0

100

200

300

400

%

Cha

nge

in s-

IgA

sere

ctio

nra

te

n = 16: ± SD

NS

Fig7

Weak chewing

Habitual chewing

Strong chewing

-300

-200

-100

0

100

200

300

400

%

Cha

nge

in s-

IgA

sere

ctio

nra

te

n = 16: ± SD

NS

Weak chewing

Habitual chewing

Strong chewing

Change in s-IgA secretion rate between after stress loading (S2) and after chewing (S3)

Change in s-IgA secretion rate between after stress loading (S2) and after rest (S4)

0

0.1

0.2

0.3

0.4M

ean

ampl

itude

EM

G o

f mas

sete

rmV


** *

n = 16: ± SD: p<0.05*

Fig8 Mean amplitude EMG of masseter muscle over 10 min chewing

0

10

20

30

40

n = 16: ± SD: p<0.05

mVsM

ean

inte

grat

ed E

MG

of m

asse

ter

** *


*

Fig9 Integrated EMG of masseter muscle over 10 min chewing

-100

-80

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-20

0

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0 5 10 15 20 25 30 35 40 45 50

Mean integrated EMG of masseter over 10 min

S2-S

3 C

hang

e in

saliv

ary

corti

sol l

evel

s

r=-0.420P<0.01n=16

Fig10 Correlation between change in salivary cortisol levels between S2 and S3 and integrated EMG of masseter muscle over 10 min

: Weak chewing: Habitual chewing: Strong chewing

mVs

%Pearson’s correlation coefficient

influence of chewing force on salivary stress journal...

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