Antibacterial Efficacy of Theobroma cacao Mixtures on the Elimination of Streptococcus mutans and Candida albicans

Abstract

Objectives The aim of the study was to compare the antibacterial efficacy of mixtures based on Theobroma cacao (ThC), ThC plus erythritol, and ThC + erythritol + Psidium guajava (PsG) on Streptococcus mutans and Candida albicans in vitro.

Materials and Methods Pure cacao extract was used through desiccation in alcohol, and guava leaves were macerated in 96% alcohol. Tests were conducted on four groups: pure ThC, ThC sweetened with erythritol, ThC sweetened with erythritol and PsG, and chlorhexidine as a control on strains of S. mutans and C. albicans, at concentrations of 100, 75, 50, and 25%, respectively, evaluated at 24 and 48 hours.

Statistical Analysis ANOVA and Bonferroni tests were performed to compare the time and efficacy on S. mutans and C. albicans with a 95% confidence level. Additionally, a linear regression analysis was conducted using SPSS and InfoStat software.

Results Pure ThC at 100% had greater antibacterial activity than other mixtures against S. mutans, with inhibition halos of 15.34 and 15.16 mm at 24 and 48 hours, respectively, which was almost similar to that observed with 0.12% chlorhexidine (15.7 mm). Furthermore, for C. albicans, pure ThC and ThC + erythritol + PsG at 100% had halos of 10.36 and 10.18 mm, respectively, at both 24 and 48 hours, which were significantly superior to chlorhexidine. Among these mixtures, pure ThC at 100% was the most effective against S. mutans, and pure ThC and ThC + erythritol + PsG mixture at 100% showed antibacterial effectiveness against C. albicans.

Conclusion The 100% concentration mixtures were effective against S. mutans and C. albicans, highlighting the effectiveness of the pure cacao extract. Additionally, the mixture with erythritol and guava extract would be useful as a treatment against C. albicans and also showed similar effectiveness to chlorhexidine against S. mutans.

Keywords

Streptococcus mutans - Candida albicans - Theobroma cacao - antibacterial agent - in vitro

Introduction

Infectious diseases represent a critical health problem and are one of the main causes of morbidity and mortality worldwide. The emergence of microorganism resistance to some synthetic antibiotics, along with toxicity during prolonged treatment, makes it necessary to continue the search for new antimicrobial and antifungal substances that are effective against Streptococcus mutans and particularly for Candida albicans, a pathogenic fungus frequently implicated in life-threatening infections in humans, especially in patients with compromised immune systems.[1] [2] These fungal diseases have increased in frequency and clinical importance due to the rise in the use of potent immunosuppressive drugs in transplants, cancer therapy, and the emergence of viral infections that cause immunodeficiency (human immunodeficiency virus [HIV]).[3]

Oral thrush or candidiasis of the oral mucosa (CMB) is one of the most common diseases of the oral mucosa and, undoubtedly, the most prevalent fungal infection in this location. The severity of the fungal infection largely depends on the host's conditions, as the onset of the disease occurs when the physiological balance parameters that maintain homeostasis of the oral environment are disturbed.[4]

Dental plaque is caused by bacteria such as S. mutans, which, in addition to fermenting glucose, produces a glucan capsule (a polymer of sugars) that acts as a sort of adhesive, allowing it to stick to teeth, continue consuming glucose, and generate more acid. It also incorporates unsaturated fatty acids into its plasma membrane to restore the acidic environment it generates.[5] According to information published by the World Health Organization (WHO), approximately 60 to 90% of schoolchildren have dental caries. Currently, it is known that the microbial ecosystem of the oral cavity related to caries is diverse; these microorganisms and others can be found in dentin, enamel, and saliva.[6] Previous research, such as that by Beckett,[7] has proven that cacao, or Theobroma cacao, possesses numerous medicinal properties ranging from improving nutritional status to its use as a preventive and adjunct agent for oral health. Bodero[8] highlights the need for existing studies aimed at counteracting these problems through the use of plant species and their application in dental practice to reach all sectors.

Cacao has been studied by traditional medicine, but there are no products or studies promoting its use for oral health, despite its numerous antimicrobial components.[9] Similarly, for 21st-century dentistry, preventive action through the use of sweets represents a new alternative. There are various initiatives involving the use of xylitol, but few studies propose the use of erythritol, which has therapeutic components and active antimicrobial substances.[10]

Additionally, this study incorporated Psidium guajava in the ThC + erythritol + PsG mixture due to its potential antimicrobial effect, attributed to its phytochemical composition and previously reported in vitro and in vivo activities.[11] Furthermore, P. guajava has been shown to inhibit glucose transport in in vitro studies,[12] which may be relevant for assessing its antibacterial activity in this study. These findings suggest the potential application of these compounds in the development of industrially formulated products in the future.

In this context, given the challenge of developing tooth-friendly products using natural ingredients such as cacao, as well as sweeteners that cannot be fermented by oral bacteria, the present research aims to compare the antibacterial efficacy of cacao seeds plus erythritol on S. mutans and C. albicans. The goal is to provide information about the potential benefits for oral health.

Materials and Methods

This in vitro experimental study was conducted with 100 Petri dishes, 50 with blood agar for the growth of S. mutans ATCC 35668 strains and 50 with Sabouraud agar for the growth of C. albicans ATCC 10231 strains. Each plate was used to evaluate four treatments, which constituted each experimental unit (GE1 to GE3) along with a positive control treatment (GE4), as detailed below:

• 50 Petri dishes with S. mutans strains:

  • GE1: Pure T. cacao (100, 75, 50, and 25%).

  • GE2: T. cacao + erythritol (100, 75, 50, and 25%).

  • GE3: T. cacao + erythritol + P. guajava (100, 75, 50, and 25%).

  • GE4: Chlorhexidine 0.12% (control).

• 50 Petri dishes with C. albicans strains:

  • GE1: Pure T. cacao (100, 75, 50, and 25%).

  • GE2: T. cacao + erythritol (100, 75, 50, and 25%).

  • GE3: T. cacao + erythritol + P. guajava (100, 75, 50, and 25%).

  • GE4: Chlorhexidine 0.12% (control).

Process of Preparing the Ethanolic Extract of Cacao Seed (Theobroma cacao) at 100%

The process began by selecting and collecting the cacao fruit. The fruit was cut open, and the seeds were extracted and then dried. Next, the best-quality seeds were selected based on their organoleptic characteristics. The selected seeds were then roasted slowly to prevent burning. Afterward, the seeds were peeled by rubbing them together, thus removing all the shells. The peeled seeds were then crushed and stored in a glass container, covered with 96° ethanol, filling the container to the top of the seeds. The container was sealed airtight and left to macerate for 7 days. After the 7 days, the mixture was filtered three times to ensure a 100% pure liquid extract, which was then bottled in a transparent glass container.

Process of Preparing the Ethanolic Extract of Guava Leaves (Psidium guajava) at 100%

The process began by selecting guava leaves from their optimal growth habitat, which were collected in the early morning hours when there was no sunlight. The leaves in good condition were selected, based on their organoleptic characteristics.

The selected leaves were cleaned and disinfected through three processes: washing with a jet of water, soaking for 5 minutes, and disinfecting with 70° ethanol. Afterward, the leaves were placed in craft paper trays, arranged in a continuous manner, with each leaf separated from the others. They were kept at room temperature for 7 days to dehydrate, allowing them to lose all their water content. Once the leaves were dry, they were crushed and placed in a glass container, then covered with 96° ethanol until it reached the level of the leaves, and sealed airtight, leaving them to macerate for 7 days. After that, the mixture was filtered three times to ensure a 100% pure liquid extract, which was then bottled in a transparent glass container.

To remove the alcohol from both obtained extracts, evaporation at room temperature was used. This process concentrates the compounds of interest by eliminating the solvent and is characterized by greater stability compared with ethanolic solutions. For storage, the extract was placed in dark, airtight containers and refrigerated to prevent the degradation of light- and time-sensitive compounds. The entire procedure was performed in a well-ventilated, contaminant-free environment.

Laboratory Experimentation Procedure

The procedures were reviewed and supervised by the Morphological Sciences Laboratory of the University of Huánuco. The strains used to test the antibacterial and antifungal activity of T. cacao and P. guajava sweetened with erythritol were S. mutans ATCC35668 and C. albicans ATCCR 10231.

Preparation Method for Culture Media

1. Agar preparation: Fifty plates of Sabouraud dextrose chloramphenicol (CAF) agar and 50 plates of Trypticase soy sheep blood agar from the commercial supplier “Andina Médica Filial Perú” were used.

2. Microorganism activation: The activation of S. mutans ATCC 35668 and C. albicans ATCCR 10231 was done as follows: The strains were taken out of refrigeration, where they had been stored until use, and were left at room temperature. The lyophilized strains were rehydrated in Brain Heart Infusion (BHI) Broth C5141. The cultures were incubated in an oven at 37°C for 48 hours under microaerophilic conditions suitable for their survival. The strain was replicated on a blood agar plate to verify its viability. Finally, a 0.5 McFarland bacterial suspension was prepared, representing a microorganism concentration of 1.5 × 10⁸. To correlate the absorbance measurement with the concentration of microorganisms, a tube with sterile BHI broth was included, and this reading was subtracted from the other readings to eliminate the absorbance from the BHI broth.

3. Inoculation of microorganisms on agar plates: A bacterial suspension of the S. mutans inoculum was prepared at 0.5 McFarland, corresponding to a microorganism concentration of 1.5 × 10⁸ CFU/mL, and streak plating was performed using a swab on each blood agar plate. For C. albicans, a 0.65 McFarland suspension was prepared, and streak plating was performed using a swab on each Sabouraud agar plate. The plates were then left undisturbed for 3 minutes to allow proper adherence of the microorganisms to the agar.

Microbiological Analysis

Microbiological analysis with S. mutans: Dilutions of pure T. cacao, T. cacao sweetened with erythritol, and T. cacao sweetened with erythritol and P. guajava were prepared at concentrations of 100, 75, 50, and 25%, along with 0.12% chlorhexidine and distilled water. These were applied to 5-cm-diameter filter paper disks and placed equidistantly on the plates containing the medium inoculated with S. mutans for analysis.

Microbiological analysis with C. albicans: Similarly, dilutions of pure T. cacao, T. cacao sweetened with erythritol, and T. cacao sweetened with erythritol and P. guajava were prepared at concentrations of 100, 75, 50, and 25%, along with 0.12% chlorhexidine and distilled water. These were applied to 5-cm-diameter filter paper disks and placed equidistantly on the plates containing the medium inoculated with C. albicans for analysis. In both cases, the plates were incubated at 37°C for 24 and 48 hours. The inhibition zone of S. mutans bacterial growth and C. albicans growth was measured in millimeters at the specified times.

Experimental protocol: Strains of S. mutans ATCC 35668 and C. albicans ATCCR 10231 were manipulated for the respective study. The experiment was conducted using pure cacao extract processed and obtained from the cacao fruits (T. cacao) of the “Fino de Aroma” variety, and pure guava extract processed and obtained from the leaves of guava fruits (P. guajava) from Tingo María – Huánuco, Peru. Erythritol, 0.12% chlorhexidine, and distilled water were also used. The necessary replicates were performed for each observation. Resistance: halo < 8 mm; intermediate sensitivity: 8 ≤ halo ≤ 14 mm; highly sensitive: halo > 14 mm.[9]

Statistical Analysis

The means, standard deviations, and maximum and minimum values for each treatment were reported. To evaluate the statistical differences when comparing the time and effectiveness of the different treatments on S. mutans and C. albicans, as well as the halo diameters by concentration, an analysis of variance (ANOVA) was conducted, followed by a Bonferroni multiple range test to determine which treatments showed statistical differences at a 95% confidence level. Additionally, a scatter plot analysis was conducted to examine the relationship and dependency between cacao concentration in the treatments and the inhibition halo size, using polynomial and exponential fitting. The coefficient of determination (R ²) was used to evaluate the goodness of fit. These analyses were performed using the statistical software Origin, MS Excel, SPSS, and InfoStat.

Results

• Antibacterial Effect of Theobroma cacao (ThC), ThC with Erythritol, and ThC + Erythritol +  Psidium guajava (PsG) on Streptococcus mutans

Initially, an analysis of the means was conducted, and with a 100% concentration of pure ThC, it was observed that it showed similar effectiveness to 0.12% chlorhexidine. Additionally, when comparing the means in each group, we observed that the 0.12% chlorhexidine solution was slightly superior to T. cacao (pure ThC; [Fig. 1]). Moreover, the mean of treatment 1 (pure ThC) falls within the lower and upper limits of treatment 4 (0.12% chlorhexidine), and the mean of treatment 4 (0.12% chlorhexidine) falls within the lower and upper limits of treatment 1 (pure ThC), suggesting that both solutions have a similar antibacterial effect ([Table 1]). Through the ANOVA analysis for the 100% concentration, it was observed that there is indeed a statistically significant difference between the groups evaluated (p = 0.000). Therefore, a Bonferroni analysis was performed, where it was observed that there is a significant difference among the four groups at different concentrations. Once again, it was observed that at 100% concentration, pure ThC demonstrated similar effectiveness to 0.12% chlorhexidine (control group) and greater overall effectiveness compared with the other three groups at lower concentrations ([Fig. 2]). Hence, it is deduced that ThC possesses significant antibacterial effectiveness against S. mutans when used without additives.

Fig. 1 Blood agar (Streptococcus mutans) and Sabouraud agar (Candida albicans). (A) Sheep blood agar with tryptic soy base showing S. mutans growth at 24 hours. (B) Sheep blood agar with tryptic soy base showing S. mutans growth with inhibition disks at 48 hours. (C) Sabouraud dextrose agar chloramphenicol (CAF) showing C. albicans growth at 24 hours. (D) Sabouraud dextrose agar CAF showing C. albicans growth at 48 hours with inhibition disk and inhibition halo.

Table 1 Bacterial growth inhibition halos of Streptococcus mutans ATCC 35668 reading at 100% in the different experimental treatments

Solutions	N	Mean	SD	Lower limit	Upper limit
T1. ThC puro	50	15.34	5.69	13.72	16.95
T2. ThC + erythritol	50	6.98	4.90	5.58	8.37
T3. ThC + erythritol + PsG	50	9.18	6.02	7.46	10.89
T4. Chlorhexidine 0.12% (control)	50	15.70	3.13	14.81	16.59
ANOVA
Difference between groups		p = 0.000

Abbreviations: ANOVA, analysis of variance; N, number of Petri dishes used; ThC + erythritol + PsG, Theobroma cacao + erythritol+ Psidium guajava; ThC + erythritol, Theobroma cacao + erythritol; ThC, Theobroma cacao; SD, standard deviation.

Source: The authors.

Note: 95% confidence interval.

Fig. 2 Box and whisker plot comparison of the antibacterial effect on Streptococcus mutans versus treatment concentration. Different lowercase letters indicate statistically significant differences at a 95% significance level according to Bonferroni's multiple range test. A comparison of treatments by cacao concentration levels and their mixtures (100, 75, 50, and 25%) is shown using a box and whisker plot, evaluated based on the inhibition halo diameter. According to the analysis of variance (ANOVA), these treatments exhibit highly significant statistical differences (p < 0.001; see [Table 1]). The determination of differences between treatments, conducted through Bonferroni's multiple range test, indicates statistically significant differences among them (p < 0.05), represented by lowercase letters. The 100% cacao mixture exhibits the highest values and the greatest dispersion in halo diameter compared with the other treatments. Additionally, only the 100% cacao treatment shows similarity to the positive control (0.12% chlorhexidine). The other 100% cacao-based mixtures demonstrate statistically significant differences but with a reduced effect in terms of halo diameter. The remaining mixtures (75, 50, and 25%) exhibit a similar behavior, with the positive effect of 0.12% chlorhexidine standing out.

• Antibacterial Effect of Theobroma cacao (ThC), ThC with Erythritol, and ThC + Erythritol +  Psidium guajava (PsG) on Candida albicans

Similarly, the effect on C. albicans was analyzed. When observing the means of each solution, it was found that treatment 1 (pure ThC) had a mean of 10.36 mm, similar to treatment 3 (ThC + erythritol + PsG) with a mean of 10.18 mm, while treatment 2 (ThC + erythritol) presented a slightly lower mean of 9.8 mm compared with the other two ([Fig. 1]). Upon analyzing these values in relation to the upper and lower limits, it was observed that the mean of treatment 1 (pure ThC) and treatment 2 (ThC + erythritol) fell within the lower and upper limits of treatment 3 (ThC + erythritol + PsG); likewise, the mean of treatment 3 (ThC + erythritol + PsG) was within the lower and upper limits of treatments 1 (pure ThC) and 2 (ThC + erythritol). This descriptively shows that the three treatments—1 (pure ThC), 2 (ThC + erythritol), and 3 (ThC + erythritol + PsG)—are apparently similar at 100% concentration ([Table 2]).

Table 2 Bacterial growth inhibition halos of Candida albicans ATCCR 10231 strain; 100% reading across the different experimental treatments

Solutions	N	Mean	SD	Lower limit	Upper limit
T1. ThC puro	50	10.3600	4.42516	9.1024	11.6176
T2. ThC + erythritol	50	9.8000	4.39852	8.5500	11.0500
T3. ThC + erythritol + PsG	50	10.1800	3.86871	9.0805	11.2795
T4. Chlorhexidine 0.12% (control)	50	1.9200	2.43143	1.2290	2.6110
ANOVA
Difference between groups		p = 0.000

Abbreviations: ANOVA, analysis of variance; N, number of Petri dishes used; ThC + erythritol + PsG, Theobroma cacao + erythritol+ Psidium guajava; ThC + erythritol, Theobroma cacao + erythritol; ThC, Theobroma cacao; SD, standard deviation.

Source: The authors.

Note: 95% confidence interval.

Additionally, through the ANOVA analysis for the 100% concentration, a statistically significant difference was between the groups evaluated (p = 0.000). A Bonferroni analysis was then performed, which showed significant differences among the four groups at various concentrations. At 100% concentration, pure ThC and the mixture of ThC + erythritol + PsG exhibited greater effectiveness than 0.12% chlorhexidine, as well as higher effectiveness compared with lower concentrations. This suggests that pure ThC and the combination with erythritol and PsG exhibit intermediate sensitivity in terms of effectiveness against C. albicans ([Fig. 3]).

Fig. 3 Box and whisker plot comparison of the antifungal effect on Candida albicans versus treatment concentration. Different lowercase letters indicate statistically significant differences at a 95% significance level according to Bonferroni's multiple range test. The box and whisker plot, which evaluates the antifungal effect on C. albicans, shows significant differences according to ANOVA (p < 0.001; see [Table 2]). Bonferroni's multiple range test indicates significant differences among treatments, represented by different lowercase letters. Among the 100% concentration group, Theobroma cacao and T. cacao combined with erythritol stand out due to their higher values and greater dispersion in the inhibition halo diameter. The various mixtures within the 100% concentration group do not show significant differences among themselves but do exhibit a clear distinction from the negative control (0.12% chlorhexidine). In the 75% concentration group, significant differences are observed, with the highest mean corresponding to pure T. cacao extract. The mixtures of T. cacao with erythritol and T. cacao with erythritol plus Psidium guajava exhibit similar means. These three groups show statistically significant differences compared with 0.12% chlorhexidine. Within the 50% concentration group, T. cacao demonstrates the highest response effect in halo diameter, showing some similarity with the T. cacao plus erythritol mixture, but clear differences from the T. cacao + erythritol + P. guajava mixture. Chlorhexidine at 0.12% in this group also shows significant differences compared with the other mixtures. In the 25% concentration group, the lowest mean values are observed compared with the other concentration groups. Within this group, the highest mean corresponds to T. cacao at 25%, which exhibits some similarity to the T. cacao + erythritol mixture. The latter, in turn, is similar to the T. cacao + erythritol + P. guajava mixture and the control. These last two mixtures are comparable to each other but distinct from the rest of the group. In all groups, the control treatment (0.12% chlorhexidine) showed significant differences compared with the other mixtures. Additionally, the mixtures themselves were distinct from one another; however, the 100 and 25% extracts were similar, as were the 75 and 50% extracts.

Finally, the scatter plot was created, which showed that the higher the concentration of the treatment, the greater its effectiveness. Additionally, a stronger effect was observed on S. mutans compared with C. albicans, suggesting that pure ThC also demonstrates effectiveness against this strain ([Fig. 4]). Moreover, it should be noted that no statistically significant differences were observed between the 24- and 48-hour time points, according to the Kruskal–Wallis test.

Fig. 4 Scatter plot. Relationship between halo diameter and cacao concentration, polynomial, and exponential fit. A regression plot illustrates the relationship between cacao concentration (percentage) and the inhibition halo diameter (millimeters). The effect of cacao concentration on both microorganisms shows a significant increase in halo diameter as cacao concentration increases. This effect is more pronounced for Streptococcus mutans, where the halo growth is greater. Additionally, a well-fitted polynomial function (R ² = 0.9985) confirms the strong relationship between cacao concentration and the inhibition halo. Meanwhile, the response of Candida albicans is lower than that of S. mutans, but it follows a similar trend, demonstrating a good fit to an exponential function (R ² = 0.996).

Discussion

This study provides evidence of the effect of T. cacao (ThC) extract, which exhibited inhibition zones with diameters greater than 10 mm for both S. mutans and C. albicans at 100% concentration. It also showed that when erythritol and PsG were added, there was a reduction in the inhibitory effect on the strains. These results suggest that the ThC solution was more effective in inhibiting both strains, showing better results against S. mutans.

One of the main findings of the study demonstrates that the pure cocoa extract, ThC at 100%, has an inhibitory effect on the growth of S. mutans colonies, forming an inhibition zone of 15.34 mm at 24 hours and 15.16 mm at 48 hours. This result is similar to the one obtained in Correa Jiménez's[9] study, which demonstrated the inhibitory effect of chocolate sweetened with xylitol, where the average inhibition zone at 24 hours was 16.5 mm. However, it should be noted that in our study, the use of erythritol and guava leaf extract did not exhibit an agonistic effect on T. cacao (ThC). As a result, the treatment with the best bacteriostatic effect was the one using 100% pure chocolate. Therefore, it can be concluded that guava leaf extract and erythritol do not enhance the formation of the inhibition halo, and their bacteriostatic effect remains undetermined. Thus, it can be affirmed that guava and erythritol do not enhance the formation of the inhibition zone, and their bacteriostatic effect remains indeterminate. This result could be explained by the study by Mandava et al,[13] where they investigated the anticaries effect of various medicinal plants, including guava. They demonstrated an inhibitory effect on the glucosyltransferase of S. mutans, an enzyme responsible for the virulence of this bacterium, as it mediates the production of glucans, which allows the cohesion and maturation of the dental biofilm. It is likely that guava has a better effect when used alone and not with additives.

The result obtained with the 100% T. cacao extract is similar to the control, where 0.12% chlorhexidine was used. These findings align with the review by Fideles et al[14] and the study by Osawa et al[15] that found that cocoa bean husk extract exhibited inhibitory activity on glycosyltransferase enzymes and antibacterial effects against S. mutans cultures. In this study, chromatographic analyses identified several bioactive compounds present in the cocoa bean husk that may be associated with antiglycosyltransferase activity, such as polyphenols (epicatechins), and antibacterial effects against S. mutans, such as fatty acids, particularly oleic and linoleic acids. Subsequent studies with similar results demonstrated that cocoa husk extract reduced the viability and adhesion of S. mutans in vitro, as well as acid production and the synthesis of insoluble polysaccharides in bacterial cultures. According to Matsumoto et al,[16] Smullen et al,[17] and Ooshima et al,[6] a significant reduction in the number of viable S. mutans cells was observed in human biofilms collected from children aged 4 to 15 years after in vitro exposure of these biofilms to cocoa husk extract for 1 hour. All these findings support the results obtained through the antibacterial susceptibility assay using the disk diffusion method.

A second important finding of the study was regarding C. albicans: the 100% pure cacao extract formed an inhibition zone of 10.36 mm, demonstrating antifungal activity, which was not found in the control group with 0.12% chlorhexidine, where the inhibition zone was 1.92 mm. For this strain, when the cacao extract was mixed with erythritol and 100% guava leaf extract, an inhibition zone of 10.18 mm was observed, suggesting that they have similar effects. These results could be corroborated with those obtained by García and Maibely, who demonstrated that the ethanolic extract of guava leaves has a similar effect to ketoconazole, forming an inhibition zone of 16.53 mm, which was slightly higher than that obtained in our study. It is worth noting that our study used a combined approach, unlike García and Maibely's[18] study, where guava was used without additives, demonstrating that guava leaves have fungistatic potential.

Regarding the extraction procedure, the cacao extract was obtained by collecting seeds that were subjected to a drying process, then selecting the best-quality seeds, roasting, and soaking them in 96% ethanol for 7 days, resulting in a pure extract, which was filtered three times to ensure its quality. In the case of guava, the leaves were collected in the morning to maximize the active compounds, primarily those related to the content of bioactive compounds and the quality of the extracts obtained, as reported by Qian and Nihorimbere.[19] The leaves were then selected, washed with distilled water, disinfected with 70% ethanol, and stored in kraft paper to dehydrate for 7 days. Afterward, they were crushed and macerated in 96% ethanol, and after 7 days, the extract was filtered. This same process was followed by de Assis Braga et al,[20] where the maceration process was left for 10 days. After this period, the ethanolic liquid was drained and the process was repeated twice more when complete leaf discoloration was observed. The ethanolic liquid was then mixed, and ethanol was removed by rotary evaporation under low pressure (80 mbar) at 40°C. The remaining liquid extract was further dried at 40°C until a crude paste-like extract was obtained.

The 100% T. cacao extract demonstrated a well-marked effectiveness, represented by the inhibition zones formed in this study. The concentrations evaluated were 100, 75, 50, and 25%, with readings taken at 24 and 48 hours for both S. mutans and C. albicans. The most interesting results were obtained at the 100% concentration, where bacteriostatic activity against S. mutans was observed with 100% pure T. cacao, and fungistatic activity with the mixture of pure T. cacao, erythritol, and guava leaf extract. It is important to highlight the inclusion of erythritol in these mixtures, as, like xylitol, it has cariogenic inhibitory activity. Kõljalg et al,[10] in their study on the synergistic effect of xylitol and erythritol, found these two polyol solutions inhibit the biofilms of S. mutans, reducing dental plaque growth.

Finally, the relevance of this study should be emphasized, as interesting inhibitory results of T. cacao (ThC) on Streptococcus were observed, one of the most important and relevant microorganisms in the field of dentistry.

Conclusion

Based on the results, it is concluded that the 100% concentration mixtures of ThC are apparently the most effective against S. mutans and C. albicans. Additionally, it is suggested that pure cacao extract has inhibitory activity against S. mutans similar to the potential of 0.12% chlorhexidine. On the other hand, the solution of pure cacao extract with erythritol and 100% guava leaf extract shows superior inhibitory activity against C. albicans compared with S. mutans, suggesting that guava leaves possess interesting antibacterial and antifungal effects.

Conflict of Interest

None declared.

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Publication History

Article published online:
22 April 2025