Comparison of the use of tablets containing a cysteine protease actinidin and tongue brushes for reduction of the bacterial load on the tongue

Comparison of the use of tablets containing a cysteine protease actinidin and tongue brushes for reduction of the bacterial load on the tongue

Naho Mugita

, Kazuya Takahashi

and Yutaka Komasa

1Graduate School of Dentistry (Geriatric Dentistry) and²Department of Geriatric Dentistry, Osaka Dental University, 8-1 Kuzuhahanazono-cho, Hirakata-shi, Osaka 573-1121, Japan

Tongue coating as an oral biofilm is responsible for 60% of all halitosis cases. It also in­

terferes with gustatory perception, and is associated with periodontal disease and pneu­

monia. As it is difficult to remove, the development of new cleaning methods is being promoted. We studied tablets containing actinidin, a cysteine protease, which has been reported to effectively remove this coating on the tongue. As tablets placed in the oral cavity are usually abraded by the tongue and palate to promote their dissolution, their ef­

fect in the elderly is likely to be reduced because of decreased motor function of the tongue. However, there is no literature discussing this effect in the elderly or comparing tablets with tongue brushes. To evaluate the efficacy of tablets containing actinidin on reducing the bacterial load on the tongue, we compared the effect of tablets, tongue brushes, and a combination of both on reduction of the bacterial load on the tongue.

Tongue cleaning was performed for elderly individuals requiring assistance or care (the elderly group) and healthy young adults (the young group) using three methods : two tablets containing actinidin (the tablet method), brushing with a tongue brush (the brush­

ing method), and the combined use of both (the combined method). The bacterial load on the tongue dorsum was measured before treatment, immediately after treatment, and at 1 and 2 hours after treatment. With the brushing method, neither the elderly nor young groups showed significant changes in the bacterial load until 2 hours after treatment. In contrast, a marked decrease in the bacterial load was observed in both groups immedi­

ately after treatment using the tablets, and immediately after and at 2 hours after treat­

ment using the combined method. The tablets were particularly effective for the young group, and decreases in the bacterial load were observed immediately and at 1 hour after treatment when they were administered alone or in combination with the tongue brush.

Furthermore, in the young group, the bacterial load immediately after and at 1 hour after treatment varied between the tablet and brushing methods.

In contrast, in the elderly group, although the bacterial load was significantly reduced with the tablet method, this decrease was not observed when treatment was combined with the tongue brush. In both groups, the decrease in the bacterial load was more marked even at 2 hours after treatment when the combined use was compared with the brushing method alone. These results demonstrated that tablets containing cysteine pro­

tease are more effective than tongue brushes for reduction of the bacterial load, and that the effect lasts longer when the two methods are combined. (J Osaka Dent Univ 2016 ; 50 : 93­101)

Key words : Actinidin ; Cysteine protease ; Tablet ; Bacterial load ; Tongue

INTRODUCTION

Volatile sulfides are considered responsible for physiologic halitosis,

and 60% of the cases are caused by the putrefying activity of bacteria in the tongue coating. The tongue coating is the main cause of physiologic halitosis.

Comprising a large number of papillae, the dorsal surface of the tongue is rough. The tongue surface, with its numerous pits, is an ideal niche for bacterial attachment and proliferation, reducing the effect of cleaning and consequently allowing the development of an ex­

tensive oral biofilm.

In the elderly, the tongue coat­

ing tends to be particularly thick and difficult to re­

move. Increased tongue coating levels may influ­

ence gustatory perception. Winkler noted an en­

hanced gustatory perception in elderly patients (particularly those using dentures) after removal of the thick plaque layer on their tongue.

In addition, as a correlation has also been reported between bacteria in the tongue coating and the frequency of fever in the elderly,

not only young individuals who are concerned about halitosis, but also elderly who have decreased tongue function require the re­

moval of the tongue coating.

Currently, mechanical cleaning methods, such as using toothbrushes or tongue brushes, which are generally used to remove the tongue coating, are somewhat effective.

On the other hand, problems such as excessively abrasion of the mucous mem­

brane of the tongue and triggering on the gag re­

flex, have been noted. Quirynen et al. conducted a questionnaire survey after tongue scraping, and re­

ported that some respondents had experienced vomiting.

Considering these points, we focused on methods to chemically remove the tongue coating.

Oral mucosa­derived detached epithelial cells and bacteria account for the majority of tongue coating components.

Proteases are effective at removing such coating.

For example, Yoshi­

matsu et al. confirmed the effectiveness of tablets containing the kiwifruit­derived cysteine protease actinidin for removing the tongue coating and re­

ducing halitosis.

However, there is no literature comparing the tongue­cleaning effect of such tab­

lets with conventional mechanical cleaning meth­

ods.

Furthermore, the elderly, defined as those requir­

ing care based on the Long­term Care Insurance System in Japan, tend to face age­related reduc­

tions in motor function of the tongue as well as la­

ryngeal and hyoid elevation, leading to difficulty in bringing the tongue dorsum into contact with the palate.

As tablets are moved between the ton­

gue and palate in the oral cavity, sufficient effect may not be obtained when they are used by indi­

viduals requiring care, who are likely to have de­

creased tongue muscle strength. We studied the ef­

fect of tablets and tongue brushes for cleaning the tongue dorsum of elderly individuals requiring as­

sistance or care, and healthy young adults.

MATERIALS AND METHODS Subjects

We studied 10 elderly individuals requiring assis­

tance or care (the elderly group : 2 males and 8 fe­

males with a mean age of 83±5 years) and 10 healthy young adults (the young group : 7 males and 3 females aged 28±2 years) using the follow­

ing exclusion criteria : the absence of tongue coat­

ing ; their difficulty in performing oral care inde­

pendently ; their inability to provide responses ; a reduced cognitive level ; and an increased risk of improperly ingesting the tablets. The cognitive level was evaluated using a Japanese scale to assess the degree of independence in daily activities for the elderly with dementia. Subjects were included who had cognitive levels corresponding to the cate­

gories from “independent” (without dementia) to

“IIb” (mild level of dementia).

Study design

A 3­group, 3­treatment crossover (orthogonal Latin square) study was conducted, with a 1­week wash­

out period (Table 1). To examine the effects of

daily tongue cleaning, all subjects were instructed

to perform tongue and oral cleaning independently,

as they usually did, until noon on the measurement

day. Considering the stability of oral bacteria, the

first baseline measurement (T

) of the bacterial load

on the tongue dorsum was performed more than 30 minutes after lunch. Subsequently, tongue cleaning was performed for the subjects using three meth­

ods : use of two tablets (BREO EX

; Ezaki Glico, Osaka, Japan) containing the cysteine protease ac­

tinidin (the tablet method), brushing the tongue with a tongue brush (Mini Moa Brush

; Oral Care, To­

kyo, Japan) (the brushing method) ; and a combi­

nation of the two (the combined method).

When using the tablet method, the subjects were given one tablet and instructed to suck, rather than chew it, moving the dorsal surface of their tongues until it dissolved naturally. The second tablet was administered in the same manner. The second (T

) measurement was performed immediately after confirming that the second tablet had completely dissolved. When using the brushing method, the area for tongue dorsum cleaning was vertically di­

vided into three sections in accordance with the size of the brush. The brush was moved back and forth along the long axis of the tongue in the cen­

tral, right and left sections, in that order. The exam­

iner repeated this procedure, abrading the tongue while applying a force of 50 to 60 g, 10 times. After treatment, the subjects gargled twice, and, subse­

quently, the second (T

) measurement was per­

formed.

When the two methods were combined, the cleaning was done using the tongue brush after ad­

ministration of the two tablets, followed by subse­

quent measurements.

In short, when using the tablet or brushing me­

thod, the bacterial load was measured four times : before treatment (T

), immediately after treatment (T

), and at 1 (T

) and 2 (T

) hours after treatment.

When using the combined method, it was meas­

ured 5 times : before treatment (T

), after tablet ad­

ministration (T

) and after use of the tongue brush

(T

), and at 1 (T

) and 2 (T

) hours after treatment.

The study was conducted with the approval of the director of the study facility and Osaka Dental Uni­

versity Medical Ethics Committee (Approval num­

ber : 110797).

Measurements

The bacterial load was measured using a simple oral bacterial load meter (Bacterial Counter

, Panasonic Healthcare, Tokyo, Japan). Sterile cot­

ton swabs, attached to the Bacterial Counter, were reciprocated 5 times to abrade the tongue surface for collection of specimens from the point of inter­

section of the line between both first molars and the midline of the tongue to approximately 1 cm for­

ward, while applying a force of 20 g. This proce­

dure was only done by two researchers to avoid procedural errors. The cotton swabs were soaked in 5 mL of sterile distilled water attached to the Bacterial Counter before bacterial detection.

Statistical analysis

The logarithm of the bacterial load for each treat­

ment method, measurement time, and age, were determined. The distribution was normal in all cases using the Shapiro­Wilk test. An analysis of variance was performed, adopting a split­plot three­

factorial design, in which repeated measures analy­

sis of variance was performed based on the treat­

ment method and measurement time, and an un­

paired test was conducted based on age. As signifi­

cant interactions were observed, two­way repeated measures analysis of variance was performed for all factors. Among these, only those showing sig­

nificant differences were examined through multiple comparisons using Bonferroni’s method. The signifi­

cance level was set at a reject rate of 5% (＜5%).

We used IBM

SPSS

Statistics version 22, (IBM

,

Table 1 Treatment protocol (Williams’ Latin square 3×3)

Group Treatment

1 : 3 young and 3 elderly adults Tablet Tongue brush Tablet and tongue brush 2 : 4 young and 3 elderly adults Tongue brush Tablet and tongue brush Tablet

3 : 3 young and 4 elderly adults Tablet and tongue brush Tablet Tongue brush

Armonk, NY, USA) as statistical analysis software.

RESULTS

Bacterial load before treatment and carry-over effects

There were no significant differences at T

in the bacterial load among the three treatment methods in the elderly or young groups (data not shown), confirming that there were no carry­over effects in this crossover study (Fig. 1).

Comparison based on the measurement time among the treatment methods and age groups Regardless of the age, the bacterial load was mark­

edly lower when using the combined method com­

pared with the brushing method at T

, T

, and T

(Fig. 1). In contrast, when using the tablets com­

pared with the brushing method, the values were significantly lower at T

in both age groups, and at T

in the young group. Furthermore, there were significant differences between the age groups, re­

gardless of the treatment method or measurement time.

Comparison based on the measurement time For the young group (Fig. 2), compared with the values at T

, the bacterial load was markedly lower at T

and T

with tablets, and at T

, T

and T

with the combined method. In contrast, there were no significant differences at any measurement time with brushing. For the elderly group (Fig. 3), com­

pared with the values at T

, the bacterial load was significantly lower at T

with tablets, and at T

with the combined treatment. Furthermore, the values with tablets were markedly higher at T

than at T

. With brushing, the values were significantly lower at T

than at T

.

Changes over time with the combined method When the bacterial load with the combined method was monitored over time, the values were markedly lower at T

, T

and T

than at T

in the young group, while this decrease was observed only at T

and T

in the elderly group (Fig. 4).

Fig. 1 Comparison of the number of bacteria on the tongue for various treatment methods and age groups. The elderly had higher bacterial loads than the young for all time periods. On the other hand, the bacterial load with the combined treatment was markedly lower than with brushing at T1, T1hand T2h. In contrast, the values of bacterial load with tablets alone were significantly lower than with brushing at T1in both age groups, and at T1hin the young group (*p＜0.05).

Fig. 2 Number of bacteria on the tongues of the young healthy adults. Compared with the values at T0, the bacterial load was markedly reduced at T1and T1hwith tablets, and at T1, T1hand T2hwith combined treat­

ment. In contrast, there were no significant differences at any measurement times with brushing treatment.

Fig. 3 Number of bacteria on the tongue of elderly receiving nursing care. Compared with the values at T0, the bacterial load was significantly reduced at T1with tablets, and at T2hwith the combined treatment.

Furthermore, with tablets, the values were markedly higher at T2hthan at T1. With the brushing method, the values were significantly lower at T2hthan at T1.

Fig. 4 Number of bacteria on the tongue with the combined treatment. The bacterial load with the com­

bined treatment was markedly lower at T1’, T1hand T2hcompared with that at T0in the young group, while this decrease was observed only at T1’and T2hin the elderly group.

DISCUSSION

Experimental methods

Ito et al. reported that nylon brushes effectively re- move the tongue coating, while reducing care- related pain.

We used the Mini Moa tongue brush in this study. The Bacterial Counter is a simple de- vice that was recently developed to conveniently examine the bacterial load in the oral cavity. The device automatically measures the bacterial load by the mechanism of electrophoresis of specimens collected using sterile cotton swabs in sterile dis- tilled water. The values obtained are highly accu- rate, and show a significant correlation with those obtained using quantitative polymerase chain re- actin (PCR) after collecting specimens from the tongue in the same manner using swabs, as indi- cated by Tashiro et al.

The device is also light and portable, enabling researchers to take it to care facilities. The results can be obtained immediately after each measurement for specimens collected from the subjects. Specimen collection was per- formed using the method of Tashiro et al.

Comparison of the bacterial load at the baseline between the age groups

The bacterial load at T

did not markedly vary among the treatment methods in either group. As this confirms that there had been no carry-over ef- fects in this crossover study, the inclusion criteria for each age group are likely to have been appro- priate. On the other hand, the bacterial load at T

was significantly higher in the elderly than in the young group. Winkler et al.

reported that the tongue coating tends to be greater in the elderly, suggesting that the bacterial load increases with the level of tongue coating.

Effects of the brushing method on the bacterial load

We examined the effect of tongue cleaning from before treatment to up to 2 hours after as a short- term trial. Tashiro et al.

noted that in general there was a temporary increase in the bacterial load in the oral cavity immediately after oral care. Reports

on the bacterial load on the tongue dorsum after treatment vary widely. While Gilmore and Bhaskar

reported that tongue cleaning reduced the bacterial load, Van der Velden et al.

and Menon et al.

noted that there were no significant differences in the colony forming unit (CFU) when they examined specimens collected immediately after tongue cleaning using swabs.

In the study by Hotta et al.,

the bacterial load at T

was unchanged or greater than the value at T

in both groups when using the brushing method. This is consistent with reports by Van der Velden

and Menon.

In another study, the appropriate force when using tongue brushes was determined to be 50 to 100 g. In our study, tongue cleaning was per- formed by only two researchers who uniformly ap- plied a force of approximately 60 g during the pro- cedure after calibration using a meter. The results highlighted the fact that the bacterial load is not re- duced by abrading the tongue using brushes for tongue cleaning, even with application of an appro- priate force.

Quirynen et al.

conducted experiments to exam- ine long-term changes in the bacterial load on the dorsum of the tongue after cleaning. They found that although tongue cleaning for two weeks de- creased the level of tongue coating, it did not much change the load. They observed that the complex surface properties of the tongue dorsum may have prevented tongue brushes from reaching deep into the grooves. Similarly, the higher bacterial loads that we observed after tongue cleaning using a tongue brush may be indicative of how difficult it is to remove bacteria with only mild abrasion of the tongue surface. Instead, such brushing may possi- bly have resulted in pushing the bacteria deeper into the tongue coating.

When time-dependent changes were observed

beginning immediately after treatment, there were

significant differences in the values at T

and T

in

the elderly group. With time, bacteria and debris

that had surfaced as a result of cleaning using a

tongue brush may have been washed away and re-

moved from the tongue dorsum by the saliva and

tongue movements for up to 2 hours after treat-

ment. However, significant decreases in the bacte- rial load were not observed at any measurement time compared with T

with the brushing method. In short, cleaning using tongue brushes may require several hours or days to visually affect removal of the tongue coating and reduce halitosis. It is not likely to significantly reduce the bacterial load.

Effect of tablets only and the combined method on bacterial load

The tablets were effective immediately after admini- stration. The bacterial load at T

was markedly lower than at T

. This was also true for the com- bined method in the young group. This decrease at T

was also observed in the elderly group with the tablets although there was no significant difference with the combined method. The difference in the bacterial load immediately after treatment between the chemical and mechanical treatment methods may have been associated with variations in the actions of the tablets and tongue brushes in terms of bacterial removal.

Biofilms are composed of bacterial cells and ex- tracellular polymeric substances (EPS). EPS ac- count for 50 to 90% of all organic matter contained in biofilms, and they protect bacterial cells from physical stimulation when attached to their sur- faces. EPS comprise polysaccharides, DNA, and proteins.

The tablets used contain the kiwi-derived cysteine protease actinidin with a low degree of substrate specificity, which is thought to effectively degrade proteins and detached epithelial cells, which are in the EPS and tongue coating. Com- pared with such tablets, mechanical cleaning meth- ods, including the brushing method, may be ineffec- tive for complex three-dimensional structures, rep- resented by the tongue surface.

Tablets are likely to be effective for both chemi- cal removal by enzymes and mechanical cleaning by the rough surface of the tablet. Motor functions are important in the latter mechanism, which occurs through abrasion by the tongue and palate. Sugi- moto et al. examined the ingredients in tablets and their effect in removing the tongue coating. They concluded that chemical cleaning is more effective

than mechanical cleaning.

We observed significant decreases in the bacterial load in the care-de- pendent elderly who had reduced tongue function.

The effect of the tablets was even greater in young adults, and the bacterial load was reduced 90%.

With the combined method, the effect was differ- ent for the two age groups during the period be- tween T

and T

. While the bacterial load was sig- nificantly lower at T

after tablet administration, compared with that at T

in both groups, it showed a marked increase at T

only in the elderly when the tongue brush was used. Such an age-related difference in the bacterial load after tongue brush use may be explained by two factors. First, there was a higher tongue coating level and bacterial load of the elderly. As the bacterial load on the tongue dorsum was already significantly greater in them compared with the young group at T

, the level of bacteria that had surfaced from the depth as a result of the tongue brushing may have ex- ceeded that removed by the tablet. The second fac- tor is the age-related difference in the motor func- tion of the tongue. The reduced tongue function of the elderly may have interfered with the effective removal by the tablets. In contrast, in the young group, time-dependent changes in the bacterial load were similar between the tablet and combined methods.

Although Nohno et al. reported that the admini-

stration of tablets containing actinidin for 7 days

was effective for reducing halitosis, their effect on

the bacterial load of the tongue dorsum was not ex-

amined.

We found that compared with the values

at T

, the bacterial load was significantly lower at

T

when using the tablet or combined method in

the young group, and at T

when using the com-

bined method in both groups. This indicates that

tablets were effective for reducing the bacterial load

for up to 1 hour after use in the young group, and

that this effect was prolonged for up to 2 hours

when the tongue brush was also used. In short, al-

though the effect of tablets is likely to last for a

shorter period in elderly who have reduced tongue

function, this effect may last for up to 2 hours when

the tongue brush is also used. We concluded that

the time needed for the treatment to be effective may be shortened, and the duration may be pro- longed in accordance with the level of debris re- moved with the tongue brush. Further study is needed to clarify the effect of the tablets for periods longer than 2 hours, and to examine methods of combination treatment to prolong this effect.

CONCLUSION

The effect on bacterial load on the tongue dorsum of the three treatment methods were similar, re- gardless of age. Compared with the tongue brush, the use of tablets was more effective at reducing the value for up to 1 hour after treatment in both the elderly and young groups. Furthermore, their combined use maintained this effect for at least 2 hours. These results indicated the effectiveness of combining tablets and tongue brushes for all age groups. On the other hand, even when using the combined method, the bacterial load was markedly higher at T

than at T

in the elderly group, and the time needed for this value to increase was less than in the young group. There were also differ- ences in the values at T

and T

between the two groups when this method was used. This suggests that the initial level of tongue coating also influ- ences the duration of the effect of the tablets.

Tashiro et al.

discussed methods of aftercare to more effectively reduce the bacterial load. After oral care, a mass of detached bacteria is generally dis- charged from the oral cavity when gargling or swal- lowing, and most of the swallowed bacteria is killed by gastric acid. This emphasizes the importance of collecting detached contaminants, as well as re- moving debris, when performing oral care for the elderly who have difficulty gargling. When using the methods of the present study, it may be possible to further reduce the bacterial load after use of the tongue brush, and prolong the effects of treatment by initially wiping the tongue with gauze, or continu- ously performing tongue cleaning to lower the baseline values.

REFERENCE

1. Dadamio J, Laleman I, Quirynen M. The role of toothpastes in oral malodour management.Monogr Oral Sci 2013; 23: 45-60.

2. Quirynen M, Dadamio J, Van den Velde S, De Smit M, Dekeyser C, Van Tornout M, Vandekerckhove B. Character- istics of 2000 patients who visited a halitosis clinic.J Peri- odontol 2009; 36: 970-975.

3. Lang NP, Attstrom R, Lindhe J. Proceedings of the third European workshop on periodontology. Berlin : Quintes- sence, 1999 : 102-129.

4. Winkler S, Garg AK, Mekayarajjananonth T, Bakaeen LG, Khan E. Depressed taste and smell in geriatric patients. J Am Dent Assoc 1999; 130: 1759-1765.

5. Takeshita T, Tomioka M, Shimazaki Y, Matsuyama M, Koy- ano K, Matsuda K, Yamashita Y. Microfloral characterization of the tongue coating and associated risk for pneumonia- related health problems in institutionalized older adults. J Am Geriatr Soc2010; 58: 1050-1057.

6. Yaegaki K, Coil JM, Kamemizu T, Miyazaki H. Tongue brushing and mouth rinsing as basic treatment measures for halitosis.Int Dent J 2002; 52: 192-196.

7. Slot DE, De Geest S, van der Weijden FA, Quirynen M.

Treatment of oral malodour. Medium-term efficacy of me- chanical and/or chemical agents : a systematic review. J Clin Periodontol 2015; 42: 303-316.

8. Quirynen M, Avontroodt P, Soers C, Zhao H, Pauwels M, van Steenberghe D. Impact of tongue cleansers on micro- bial load and taste.J Clin Periodontol 2004; 31: 506-510.

9. Yaegaki K, Sanada K. Volatile sulfur compounds in mouth air from clinically healthy subjects and patients with peri- odontal disease.J Periodont Res1992; 27: 233-238.

10. Kojima K. Clinical studies on the coated tongue. Nihon Koukugeka Gakkai (Jpn J Oral Max Fac Surg) 1985; 1: 1659-1676. (Japanese)

11. Yoshimatsu D, Sugimura S, Ioka S, Shiraishi H, Yonetani S, Yamaga T, Miyazaki H. The effects of tablets including pro- tease on tongue coating removal. Koukueisei Kai Shi (J Dent Hlth) 2006; 56: 37-41. (Japanese)

12. Nohno K, Yamaga T, Kaneko N, Miyazaki H. Tablets con- taining a cysteine protease, actinidine, reduce oral malodor : a crossover study.J Breath Res2012; 6: 1-5.

13. Kays SA, Hind JA, Gangnon RE, Robbins J. Effects of din- ing on tongue endurance and swallowing-related outcomes.

J Speech Lang Hear Res2010; 53: 898-907.

14. Mendes AE, Nascimento L, Mansur LL, Callegaro D, Jacob Filho W. Tongue forces and handgrip strength in normal in- dividuals : association with swallowing.Clinics (Sao Paulo) 2015; 70: 41-45.

15. Ito K, Fukuhara T, Kouchi I, Inoue M. Relation between tongue cleaning effect and type of brush. Nihon Sessho- kuenge Riha Kaishi (Jpn J Dysphasia Rehabilitation) 2009; 13: 77-87. (Japanese)

16. Tashiro H, Tamura F, Hirabayashi M, Hamada R, Yone- yama T, Kikutani T. Development and clinical application of a novel rapid oral bacteria detection apparatus. Nihon Shogaisha Shika Gakkai (J Jpn Soc Disabil Oral Hlth) 2012; 33: 85-89. (Japanese)

17. Gilmore, EL, Bhaskar, SN. Effect of tongue brushing on bacteria and plaque formed in vitro. J Periodontol 1972; 43: 418-422.

18. Van der Velden U, Kippuw N, Petit M, Van Winkelhoff AJ, De Graaf J. Localization of microorganisms on the tongue and the effect of cleaning.J Dent Res1989; 68: 1007.

19. Menon MV, Coykendall AL. Effect of tongue scraping. J Dent Res1994; 73: 1492.

20. Hotta M, Nagai K, Imade S, Sano A, Yamamoto K. The safe use of tongue brush−Stained tongue brushing force in sub- jects−.Nihon Shika Hozon Shi (Jpn J Conserv Dent) 2007;

50: 332-337. (Japanese)

21. Flemming H-C, Wingender J. The biofilm matrix. Nat Rev Microbiol 2010; 8: 623-33.

22. Sugimoto J, Takahashi K and Komasa Y. Effect of a protease-containing tablet with rough surface on the number of bacteria on the tongue.J Osaka Dent Univ 2015; 49: 165-170.