The main objective was to investigate whether low-molecular-weight fraction of edible mushroom shiitake extract (
Dental caries constitutes a multifactorial disease with a complex origin where the acidogenicity of dental plaque as a consequence may affect dental hard tissues [
Over the years, different approaches have been designed intended to prevent this disease from occurring. Apart from strengthening the tooth mineral using fluoride, pronounced changes in environmental factors such as diet, oral hygiene measures, and the use of antimicrobials have been suggested in order to induce ecological shifts in biofilm composition [
Several possible mechanisms by agents of this kind have been suggested. They include the prevention of bacterial adhesion, a reduction in plaque formation, and interference with the bacterial metabolism. The most commonly used antimicrobial agent is chlorhexidine, a bisbiguanid, with known strong antimicrobial activity [
When it comes to different food products and their constituents, interest has recently focused on an edible mushroom, shiitake (
The hypothesis was that frequent mouth rinses with low-molecular-weight fraction of edible mushroom shiitake extract may reduce plaque metabolic activity, change plaque cariogenic microflora towards a healthier oral flora, and reduce plaque amount. Thus, the aim of the present study was to conduct a short-term clinical trial to determine the
This study was carried out as a double-blind, randomized, placebo-controlled, three-leg, cross-over clinical trial. Two sets of data were obtained—one at the Department of Cariology, University of Gothenburg (GOT) and one at the Department of Preventive Dentistry in collaboration with the Department of Periodontology, Academic Centre for Dentistry Amsterdam (ACTA). Despite being very similar, the two substudies were not identical in study design and the data that were obtained and, for this reason, the protocol cannot be regarded as a multicentre approach. The study was performed within an International EU Sixth Framework Programme Consortium project (NUTRIDENT, FOOD-CT-2006-036210), which was granted in order to identify beverage/food constituents that are able to reduce the risk of dental caries and gingivitis. This study focused on the opportunity to prevent dental caries and inhibit dental plaque formation. The two series were approved by the Ethical Committee at the University of Gothenburg (Dnr 102–09) and by the institutional review board at ACTA (METc VUmc, protocol number BL21480.029-08), respectively.
All volunteers made eight visits to the laboratory (GOT) respective clinic (ACTA), for a first visit, when a clinical examination was carried out and information about the study was given, and for a total of seven subsequent test visits. In all, there were four washout periods. The study started with a two (GOT) and a three-week (ACTA) preexperimental washout period followed by three two-week periods with daily mouth rinsing with the assigned product intermitted with two-week washout periods. The total duration of the study was, therefore, 14 (GOT) or 15 (ACTA) weeks.
At the screening (GOT) or at the first visit (ACTA), a medical questionnaire was completed and the oral health status of the participants was determined by an intraoral examination. Professional oral hygiene for GOT was performed at the start of each test period as well as directly after each test period (prior to washout) and for ACTA before the start of the first washout (before the baseline samples).
At each of the following visits, the subjects underwent the following data collection in the order mentioned: (1) collection of resting and fermented plaque for protein/acid analyses, (2) plaque acidogenicity (only GOT), (3) collection of plaque for microbiological analyses, and (4) assessment of plaque score. At the end of each two-week test period, the subjects were asked to fill in a questionnaire with questions related to the usage and experience of the product used. The investigators involved in plaque sampling and pH measurements were blinded with respect to the treatment allocation of the subjects.
The research population at GOT was made up of students and staff at the Institute of Odontology, as well as individuals in the nearby vicinity recruited via advertisements on bulletin boards. A total of 65 volunteers were screened. For ACTA, recruitment was performed using the existing database (approximately 600 entries being not-dental students) at the Department of Periodontology. The inclusion criteria were healthy adults, possessing at least three premolars/molars in each quadrant, who were able to reduce their plaque pH by at least one pH unit after a mouth rinse with 10% sucrose solution for 1 min (only GOT), no metal fillings in the premolar/molar region (only GOT), and a stimulated saliva secretion rate of > 0.7 mL/min (only GOT). The exclusion criteria were subjects with untreated caries or periodontal disease, wearing partial dentures, wearing orthodontic bands, and the use of antibiotics less than three months prior to the start of the study. All the subjects were given verbal and written information about the study and signed an informed consent form prior to the start of the study.
Sample size calculations were made by using the results of plaque acidogenicity relating to the effect of Meridol mouthwash (AmF-SnF2) on the amount of lactate in sucrose-fermenting plaque [
Apart from the specific instructions given to participants in GOT/ACTA for each test period, the volunteers were asked to refrain from any oral hygiene procedures during the last 72 hours (GOT) and 48 hours (ACTA), respectively, prior to each visit, as well as eating/drinking during the last two hours prior to the test. A toothpaste containing 1450 ppm F as NaF was distributed to all subjects to be used twice daily throughout the entire study: Pepsodent Super Fluor, Unilever Sverige AB, Stockholm, Sweden (GOT) and Prodent, Sara Lee, the Netherlands (ACTA), respectively.
The following three products were tested: (1) shiitake (low-molecular-weight fraction of shiitake mushroom (
The volunteers were asked to rinse with the assigned solution twice daily. On each rinsing occasion, they were instructed to rinse vigorously with 10 mL (1/2 of the volume of the vial) for 30 sec, after which they expectorated the solution. A second identical rinsing procedure with the remaining 10 mL was repeated directly after the first one. The total daily exposure was, therefore, 40 mL for 120 sec. No food or drink intake was allowed for at least one hour after the rinse. To standardise the sampling procedure after two weeks’ use of the mouthwash, all the volunteers were asked to rinse exactly three hours before the visit on day 14. No food or drink intake was allowed for at least one hour after the rinse.
In GOT, changes in plaque acidogenicity were measured before and after a mouth rinse with 10% sucrose using the microtouch method [
Two plaque samples were collected for the protein and acid anion profile, before (resting) and 10 min after the start of rinsing (fermented). The collection of resting plaque was carried out on the buccal surface of the right upper second molar using a sterile carver (GOT) and Teflon spatula (ACTA), respectively. The volunteers then rinsed for 2 min (ACTA) or 1 min (GOT) with 10 mL of 10% sucrose (w/v) solution. Fermented plaque, collected 10 min after the start of the sucrose rinse, was collected from the contralateral buccal surface (left second upper molar). For GOT, the fermented plaque sample was collected at the same time point as the pH measurements.
The plaque was transferred to a precooled Eppendorf tube containing 50
In GOT, a stimulated saliva sample was collected by chewing on a piece of paraffin for 5 min. The saliva sample was within one hour handled at the laboratory for microbial analyses. The samples were dispersed on a Whirlimixer, diluted in 10-fold stages in a potassium phosphate buffer and plated in duplicate on MSB agar (mutans streptococci), MS agar (total streptococci), Rogosa SL agar (lactobacilli), blood agar (total viable count). After being incubated in its respective atmosphere, the number of colony-forming units (CFU) was counted. The number of mutans streptococci was identified by their characteristic colony morphology on the MSB agar.
At ACTA, all visible plaque was collected from a buccal surface of the upper first molar using a Teflon spatula. Plaque was put into sterile Eppendorf tubes and kept on ice until stored at −80°C. Samples were sent on dry ice to the Department of Microbial Diseases (UCL Eastman Dental Institute, University College, London, UK) for analyses of microbiological composition. The numbers of
The plaque score was in GOT calculated using the Turesky modification of the Quigley-Hein index (TQHPI-index) [
At the end of each test period, the volunteers were requested to complete a questionnaire with a Visual Analogue Scale (VAS) with a total of nine questions related to their experience of using the assigned mouth rinse solution. They marked their answer on a 100 mm line with the negative extreme on the left and the positive extreme on the right.
The mean ± SD of all clinical parameters and individuals as calculated. For plaque pH, the mean of the values for the left and right side was collected. From each pH curve, the area under the curve (AUC5.7 and AUC6.2), minimum-pH, and maximum-pH decrease was calculated. For the plaque score, the mean score for each tooth was first calculated, after which the mean score for the whole dentition was calculated. Protein content was expressed in
In GOT, two-way analysis of variance, ANOVA, was used to test the significance of differences between the seven test occasions (after each test period and the washout periods). When ANOVA rejected the multisample hypothesis of equal means, multiple comparison testing was performed using Fisher’s PLSD.
At ACTA, a paired
All 30 and 35 individuals, respectively, completed the study, apart from the final washout period for one subject in GOT. The mean age of the volunteers was
The most pronounced metabolic activity for the sucrose rinse at the end of the three test periods was found after rinsing with the placebo, and the least attenuated pH fall was found for the positive control (AmF-SnF2), while the active compound (shiitake) resulted in an intermediate position (Figure
The changes in dental plaque pH up to 45 min after a mouth rinse with 10% sucrose for 1 min. The rinse was carried out after two weeks use of a mouth rinse with a shiitake mushroom extract, a placebo, or positive control (AmF-SnF2). Mean values for 30 subjects. The standard deviation for some of the time points is shown.
There was no difference between shiitake and placebo in plaque protein mass, while the positive control (AmF-SnF2) resulted in significantly less plaque protein than the test or placebo rinse (
The profiles of acetate, lactate, and minor acids (propionate, formate, succinate, and butyrate) for the resting and fermented plaque gave similar values for GOT and ACTA (Figure
Amount of acetate, lactate, and minor acids (propionate, formate, succinate, and butyrate) in resting (presucrose) and fermented (postsucrose) dental plaque. After baseline, after the three legs of crossover (shiitake, placebo, positive control [AmF-SnF2]) and after three washout periods (washout shiitake, washout placebo, and washout positive control) are all shown. Data are shown separately for volunteers in Gothenburg (GOT) and Amsterdam (ACTA). Mean values ± SD for 30 (GOT) and 35 (ACTA) subjects, respectively. Due to the high standard deviation when analysing lactate in fermented plaque for five of the test sessions in GOT, the
All the microbial data are presented in Table
Number of salivary and plaque microorganisms and proportions of microorganisms after baseline, the three test periods (shiitake, placebo, positive control (AmF-SnF2; Pos Ctrl)) and three washout periods (washout shiitake, washout placebo, and washout positive control) for GOT (
City/microorganisms | Test session | ||||||
Baseline | Shiitake | Washout | Placebo | Washout | Pos. Ctrl. | Washout | |
shiitake | placebo | Pos. Ctrl. | |||||
Mutans streptococci (log CFU/mL) | |||||||
Lactobacilli (log CFU/mL) | |||||||
Total streptococci (log CFU/mL) | |||||||
Total oral flora (log CFU/mL) | |||||||
Total streptococci/total flora (%) | |||||||
Mutans streptococci/total streptococci (%) | |||||||
Universal probe counts (log10 CFU) | |||||||
1Statistically significantly different from shiitake group (GOT
2Statistically significantly different from placebo group (ACTA
3Statistically significantly different from shiitake and placebo groups (GOT
For plaque samples from ACTA analysed by PCR assays,
The plaque scores are shown in Tables
Quigley-Hein plaque index score (Turesky modification 1970) (mean ± SD) after baseline, the three legs of the crossover (shiitake, placebo, positive control (AmF-SnF2; Pos Ctrl)) and three washout periods (washout shiitake, washout plaque, and washout positive control) for GOT (
City | Test session | ||||||
Baseline | Shiitake | Washout | Placebo | Washout | Pos. Ctrl. | Washout | |
shiitake | placebo | Pos. Ctrl. | |||||
1Statistically significantly different from the placebo test period (
Silness & Löe plaque index score (as modified by Danser et al. [
Shiitake | Placebo | Positive control | |
---|---|---|---|
Plaque score before test period | | | |
Plaque score after test period | | | |
Plaque score after washout | | | |
The mean difference in plaque score before and after test period | |
1Statistically significantly different from the respective plaque score obtained before the test period (
2Statistically significant plaque score reduction compared to other test periods (
Taste experience was, when marked from very poor to very good, described as significantly worse by the volunteers after the shiitake test period (GOT
The scientific approach and study design of this paper are based on the results of previous studies performed within the Nutrident project. The study was planned as a consequence of the initial chemical characterization of the shiitake mushroom [
The main finding in this study is that rinsing twice daily with a natural food extract may reduce the metabolic activity of the dental biofilm. Although not evaluated in the present study, a reduction of this kind may result in the long term in a lower degree of demineralisation. This is supported by recent data where a subfraction of shiitake showed a strong inhibiting effect on dentine demineralisation when evaluated in an environment using saliva-derived microcosms [
The antimicrobial, antiadhesive, and antiplaque properties of polyphenol-rich beverages have previously been demonstrated [
The interpretation of the acid anion profiles of the resting and fermented plaque is complicated. Although a corresponding pattern when comparing the data with the results from the plaque-pH measurements would have seemed logical, it was difficult to obtain a clear and consistent picture from the current data. This may be related to the low “
Microbiological analyses included both the total cell count and bacteria related to periodontal diseases, dental caries, and oral health. Only minor differences in both the salivary levels (GOT) and the plaque levels (ACTA) of oral microorganisms were found between the different visits. The numerically lowest salivary number of mutans streptococci in comparison to the total number of streptococci was found for shiitake. For plaque microflora, significantly reduced proportions of microorganisms were only found for the Gram-negative organism
While GOT found a significant reduction in plaque score when comparing shiitake with placebo, no such difference was found for ACTA. However, a reduction in dental plaque deposition has also been found when evaluating the active compound against gingivitis- and periodontitis-related variables [
The subjects reported a less favourable outcome for the different questions related to taste for the shiitake extract mouth rinse. All the subjects gave an assurance that they had followed the given instructions. However, following the reported negative reaction to the taste of the shiitake mouth rinse by a large number of the volunteers, one cannot exclude that this may have had a negative impact on compliance. As a consequence, some of the subjects may not have rinsed with the active compound according to instructions and that they may have rinsed their mouth with water shortly after using the active substance cannot be excluded. In order to secure the regular use of potential future products, it is important that this aspect is also considered seriously, as this factor alone may determine whether or not an oral health product is used. Aspects related to food safety also need to be taken into account.
Functional foods have not been introduced in order to replace traditional caries-prevention strategies but instead to add another tool to offer patients at higher risk. The positive finding of reduced plaque fermentation activity indicates that there is an opportunity to add one more strategy to the palette of preventive methods. It is not surprising that a stronger effect was found for this variable and that only a limited effect was found for several of the other variables. The metabolic activity of the dental biofilm is the end result of a large number of biological and biochemical caries-related factors. As shown by previous laboratory work [
The limited effect on several dental biofilm properties seen in the present study may indicate that frequent exposure for a longer period is needed. One important factor is believed to be the contact time between the active compound and the different oral properties. The repeated rinsing with
The main finding of this study is that frequent mouth rinses with a natural food extract (shiitake mushroom) may reduce the metabolic activity of the dental biofilm. Only a limited effect on other dental plaque properties related to the caries disease was found and not to the same extent as the positive control.
The research leading to these results has received funding from the European Union’s Sixth Framework Programme (FP6) under the contract FOOD-CT-2006-036210 (project NUTRIDENT). Sincere thanks to Ann-Charlotte Börjesson and Ann-Britt Lundberg, Department of Cariology, University of Gothenburg, for technical support. The clinical and logistic support of Nienke Hennequin-Hoenderdos, Department of Periodontology, Academic Centre for Dentistry Amsterdam, is gratefully acknowledged.