Avocados were obtained from Brastagi, Karo Regency, North Sumatra Province, Indonesia. The avocado fruit used in this research has been determined by the Medanense Herbarium Plant Systematics Laboratory (MEDA) at the University of Sumatera Utara with letter number 1835/MEDA/2023.

This research used in vitro experimental methods with post-test only control group design. The sample used in this research was HPA resin in the shape of a disc with a diameter of 10 mm and a thickness of 2 mm ( Figure 1). The number of samples used in this study was determined using Federer formula, hence the number of samples for each group was 4 samples. There were 6 treatment groups in this study which were avocado seed extract groups of 5%, 10%, 15%, 20%, as well as the positive control group (alkaline peroxide) and the negative control group (aquadest). As this research was conducted on mono-species biofilms: Candida albicans, Candida glabrata, Streptococcus gordonii, Actinomyces odontolyticus, Staphylococcus aureus, and on polymicrobial biofilm which is a combination of the five microorganisms, the final total amount sample that would be used in this research was 144 samples (n=144).

A disc-shaped brass metal master models with a diameter of 10 mm and a thickness of 2 mm were made to be used as a research sample mould. The dental cuvette, which had been smeared with Vaseline, was poured with a type II blue dental stone gypsum mixture made with a ratio of 300 g of gypsum: 90 mL of water to fill the bottom cuvette while being shaken with a vibrator so that no bubbles were trapped in the mixture. The master models, which had been smeared with vaseline, were then placed in the dough in a cuvette, avoiding the surface of the master models being flat with the gypsum surface. The gypsum was left to harden for ± 30-45 minutes and then smeared with vaseline. The upper cuvette was attached to the lower cuvette and filled with the same gypsum mixture as described previously. After the plaster had hardened, the cuvette was opened and the master models were taken out to obtain a mould.

The surface of the mould was smeared thinly with cold mould seal and left for 10 minutes. The HPA resin mixture was prepared with a weight ratio of 2.5:1. When it reached the dough stage, the acrylic resin mixture was put into the mould, then covered with plastic cellophane along with the top cuvette. The cuvette was pressed slowly with a hydraulic press until the pressure reached 1000 psi. Excess dough was cleaned with dental lecron, then the cuvette was closed and pressed again with a pressure of 2200 psi. The cuvette was reopened and cleaned of excess acrylic resin mixture. The cuvette was closed again and locked with the cuvette bolts, then left for 30 minutes. The cuvette was inserted into a water bath filled with aquadest, then the temperature and time were set at 70°C for 90 minutes, then at 100°C for 30 minutes. After 30 minutes, the cuvette was left in the water bath until the water reached room temperature for the cuvette cooling process. The samples were removed from the cuvette, then the sharp parts and plaster residue were trimmed with a fraser bur and sand paper.

Avocado seeds were extracted using a maceration technique. The avocado seeds would be cut into slices which would be dried in a drying cabinet at a temperature of ±40°C for about 24 hours, then coarsely grounded and blended until they became a fine powder. The avocado seed powder was put into a vessel and poured with 70% ethanol solvent with a ratio of 1:10 (10 g: 100 mL), then stirred until evenly mixed and left for 1×24 hours protected from light while stirring periodically every 6 hours so that the solution was evenly mixed. The solution was filtered until macerate I was obtained and the remaining filtered dregs were subjected to a second maceration process. The results of macerate I and II would be mixed and transferred into a closed vessel, then left in a cool place protected from light for 2×24 hours. The extract was concentrated using a rotary evaporator at a temperature of ±50°C to evaporate the solvent until a thick extract was obtained. The thick extract was then made into a concentration of 5%, 10%, 15%, 20%.

The thick extract of avocado seeds was sent to the Pharmaceutical Biology Laboratory, University of Sumatera Utara, Medan for phytochemical examination and quantitative testing of phytochemical compounds.

The microorganisms used in this study were cultured and maintained under the following conditions. Candida albicans ATCC ^® 24433 ^TM and Candida glabrata ATCC ^® 90030 ^TM were each cultured on Sabouraud Dextrose Agar (SDA) with yeast nitrogen base supplemented with 100 mmol L ⁻¹ glucose and cultured at 37°C under aerobic conditions for 24 hours. Actinomyces odontolyticus ATCC ^® 10558 ^TM was cultured on fastidious anaerobe agar with 5% (v/v) defibrinated bovine blood at 37°C under anaerobic conditions for 24 hours. Streptococcus gordonii ATCC ^® 10558 ^TM was cultured on blood agar with 5% (v/v) defibrinated bovine blood at 37°C under aerobic conditions for 24 hours. Staphylococcus aureus ATCC ^® 25923 ^TM was cultured on blood agar with 5% (v/v) defibrinated bovine blood and incubated at 37°C under aerobic conditions for 24 hours.

Sterile HPA resin discs were preconditioned for 24 hours by immersion in artificial saliva. The density of the microorganism cultures must be adjusted using a densitometer following the 0.5 McFarland standard, namely 1.5 × 10 ⁸ CFU/mL for bacterial suspensions ( A. odontolyticus, S. gordonii, S. aureus) and 1.0 McFarland standard, namely 3.0 × 10 ⁸ CFU/mL for fungal suspensions ( C. albicans and C. glabrata). The preconditioned discs were then placed aseptically into 24-microplates, and 100 μL of standardized microorganisms were added to each surface of the disc. Biofilm preparations carried out were mono-species biofilms for each microorganism studied ( C. albicans, C. glabrata, A. odontolyticus, S. gordonii, S. aureus) and polymicrobial biofilms which were a combination of the five microorganisms studied. Sterile Dulbecco’s Modified Eagle Medium (DMEM) (supplemented with 50 mmol L-1 L-glutamine per liter) was added to a final volume of 2 mL in each plate. Culture media discs in 24-wells microplates were shaken on an orbital shaker for 30 minutes to homogenize the media and culture solutions, then incubated at 37°C for 24 hours.

HPA resin discs which had been grown by mono-species and polymicrobial biofilms would be treated with immersion in avocado seed extract of 5%, 10%, 15%, 20%, as well as positive control (alkaline peroxide) and negative control (aquadest) for 8 hours at room temperature. The discs were then cleaned with distilled water, then put into a test tube together with 5 mL of Mueller Hinton broth and each shaken with a vortex mixer for 1 minute. A total of 100 μL of test solution was taken from the dilution and added into 96-wells microplates with a repetition of three times. Microplates were incubated at 37°C for 24 hours. After incubation, the microplates were cleaned with distilled water and patted vigorously on a lab mat to remove as much distilled water as possible. As much as 125 μL of 1% crystal violet solution was added to each microplate to colour the formed biofilm and left for 15 minutes. The crystal violet solution was discarded, then cleaned with distilled water and patted hard on a lab mat. The stained biofilm plates were allowed to dry until the remaining water in the microplates evaporated, then 150 μL of 95% ethanol was added to each plate and left for 10 minutes. The absorption value (OD) reading was carried out with a microplate reader at a wavelength of 595 nm and the results were calculated using the percentage inhibition value formula of which Control OD was defined as negative control absorption value and Sample OD was defined as test sample absorption value. ²⁴ Inhibition Value ( % ) = [ ( Control OD − Sample OD ) ÷ Control OD ] × 100

The treated sample which had an inhibition value of at least 50% of biofilm formation could be considered as the Minimum Biofilm Inhibitory Concentration (MBIC ₅₀). ²⁴

Univariate analysis was carried out to determine the average (mean) and standard deviation of the inhibition values for immersion of heat polymerized acrylic resin discs in each group. The conversion of absorption value to inhibition value in percentage is counted using the percentage inhibition value formula that had been coded in Excel 2021 software. The normality test was carried out using the Shapiro-Wilk test (p>0.05) and the homogeneity test was carried out using the Levene test (p>0.05). Data analysis was carried out using one-way ANOVA, which could be accompanied by Welch ANOVA on non-homogeneous data, to determine the effect of treatment in each group. Data were analyzed with IBS SPSS Statistics (RRID: SCR_016479) v.22.0 software and presented in tabulation and graphic form as mean and standard deviation. Significant differences were defined at p<0.05.

The SEM procedure was carried out at the USU Integrated Research Laboratory, Medan. HPA resin disc samples that had been preconditioned with artificial saliva were then grown with polymicrobial biofilm according to the previous biofilm formation procedure and given a soaking treatment in avocado seed extract. HPA resin disc samples that had biofilm grown on were cleaned with distilled water three times and fixed with 2.5% (w/v) glutaraldehyde in cacodylate buffer for about 6 hours. The wet sample was then coated with a thin layer of gold to make the sample conductive. Sample reading using SEM was carried out with a voltage of 5 kV.

The denture base subjects’ research was approved on 27 ^th February 2024 and performed according to the ethical standards by the Health Research Ethics Committee of the University of Sumatera Utara, Indonesia as stated in letter number 166/KEPK/USU/2024.

The following are the results of avocado identification by the Medanense Herbarium, University of Sumatera Utara.

Kingdom: Plantae

Division: Spermatophyta

Class: Dicotyledoneae

Order: Laurales

Family: Lauraceae

Genus: Persea

Species: Persea americana Mill.

Local Name: Avocado Seed

The phytochemical test on the ethanol extract of avocado seeds was done using specific reagents to determine the presence of secondary metabolite compounds which were alkaloids, flavonoids, glycosides, saponin, tannin, triterpenoids/steroids ( Table 1). The test showed positive results of all the tested secondary metabolite compounds and none negative results.

The secondary metabolite compounds existing in the avocado seed ethanol extract could be further assessed by doing a quantitative analysis to determine the amount of the secondary metabolites in the sample extract which were flavonoids, phenol, saponin, and alkaloids ( Table 2). The analysis showed a total amount of phenol (66,8157 mgGAE/g extract), total amount of flavonoids (4,0888 mgQE/g extract), total percentage of saponin (1,59%), and total percentage of alkaloids (1,22%).

Each sample in each group was repeated three times to obtain three absorption values (OD) which then using univariate analysis, the mean and standard deviation were obtained. The obtained absorption value was calculated using the percentage inhibition value formula with an inhibition value of 50% as a parameter for determining MBIC ₅₀ ( Figure 2). Based on calculations, MBIC ₅₀ of avocado seed extract in mono-species C. albicans biofilm was 5% avocado seed extract. MBIC ₅₀ avocado seed extract in mono-species C. glabrata biofilm was 5% avocado seed extract. MBIC ₅₀ avocado seed extract in mono-species A. odontolyticus biofilm was 15% avocado seed extract. MBIC ₅₀ avocado seed extract in mono-species S. gordonii biofilm was 15% avocado seed extract. MBIC ₅₀ avocado seed extract in mono-species S. aureus biofilms was 10% avocado seed extract.

The sample was tested for normality and a value of p>0.05 was obtained, hence the data were normally distributed. Then, a homogeneity test was carried out by which the mono-species C. albicans biofilm sample obtained a value of p<0.001 (p<0.05), the mono-species A. odontolyticus biofilm sample obtained a value of p=0.002 (p<0.05), and the mono-species S. gordonii biofilm sample obtained a value of p≤0.001 (p<0.05) so the data were not homogeneous, while the mono-species C. glabrata biofilm sample obtained a value of p=0.054 (p>0.05) and the mono-species S. aureus biofilm sample obtained a value of p=0.116 (p>0.05) so the data were homogeneous. Data which was normally distributed and homogeneous was tested using one-way ANOVA and data which was normally distributed but not homogeneous was analysed using Welch ANOVA. This study found that mono-species biofilm samples of C. albicans, C. glabrata, A. odontolyticus, S. gordonii, S. aureus obtained a value of p≤0.001 (p<0.05) which indicated a significant effect of 5%, 10%, 15%, 20% avocado seed extract in inhibiting mono-species biofilms of C. albicans, C. glabrata, A. odontolyticus, S. gordonii, S. aureus ( Table 3).

In this study, three absorption values (OD) of polymicrobial biofilm samples were obtained from which the mean and standard deviation were obtained using univariate analysis. Using the percentage inhibition value formula, the inhibition values were obtained for each group of avocado seed extract of 5%, 10%, 15%, 20%, and the positive control (alkaline peroxide) where the 50% inhibition value was set as a parameter for determining MBIC ₅₀ ( Figure 3). Hence, the MBIC ₅₀ avocado seed extract in polymicrobial biofilm was 20% avocado seed extract.

The sample was tested for normality and a value of p >0.05 was obtained, hence the data was normally distributed. Then, a homogeneity test was carried out by which the polymicrobial biofilm samples obtained a value of p=0.006 (p<0.05) so that the data was not homogeneous. Data that were normally distributed but not homogeneous were analysed using the Welch ANOVA. This study found that the polymicrobial samples obtained a value of p≤0.001 (p<0.05) which indicated a significant effect of soaking in 5%, 10%, 15%, 20% avocado seed extract in inhibiting polymicrobial biofilm ( Table 4).

Based on the Integrated Laboratory Test Results Report of the University of Sumatera Utara with the number 113/UN5.4.6.K/KPM/2024, the results of SEM tests carried out on HPA resin discs with polymicrobial biofilm which had been soaked with avocado seed extract could be detected and clearly seen in Figure 4 below. SEM results showed that there were microorganisms growing on the HPA resin disc. The soaking in 5% avocado seed extract showed a denser formation of biofilm compared to soaking in 15% avocado seed extract.

Candida albicans has the pathogenic ability in the form of morphogenesis, which is the ability to transition C. albicans from a unicellular yeast form to a pathogenic filament form (pseudohyphae or hyphae) reversibly. ^{28 , 29} C. albicans yeast cells will adhere to the denture surface via A1s1-8p adhesin, then proliferate to form microcolonies which become the basal layer of the biofilm and produce extracellular matrix (ECM). As the biofilm matures, there is an increase in biomass with the presence of yeast cells, hyphae and pseudohyphae encapsulated in the extracellular matrix. These hyphae are fundamental and important components in supporting the structural integrity of the biofilm and provide a means of attachment for additional yeast cells, pseudohyphae, other hyphae, and bacteria due to their ability to express specific adhesins such as Hwp1p and Hyr1p. ¹⁵ These hyphae are also capable of damaging epithelial cells and destabilizing membranes through induced calcium ion influx and release of lactate dehydrogenase. ⁹ In this study, the MBIC ₅₀ of avocado seed extract in mono-species C. albicans biofilm was 5% avocado seed extract with an inhibition value of 76.09 ± 3.21%. This is in accordance with previous research by Wulandari et al. (2023) who tested the effect of avocado seed extract on C. albicans biofilms. Using the inhibition percentage formula, the lowest concentration of avocado seed extract tested, which is a concentration of 3.13%, was able to inhibit C. albicans biofilms incubated for 24 hours by 75.37%. ³⁰

Candida glabrata is the second most frequently isolated cause of candidiasis and is often found together with C. albicans in the form of co-isolation in which C. glabrata budding yeast is found attached to C. albicans hyphae. ³¹ There was still little to none research done on the effect of avocado seed extract on C. glabrata. This study found that the MBIC ₅₀ of avocado seed extract in mono-species C. glabrata biofilm was 5% avocado seed extract with an inhibition value of 52.41 ± 9.64%. When compared with the C. albicans inhibition value, there was a decrease in the biofilm inhibition activity of avocado seed extract against C. glabrata. This is due to the higher antifungal resistance in C. glabrata than in C. albicans, and the rapid ability of C. glabrata to develop resistance to currently used antifungal agents. ³² Farahyar et al. (2016) found that C. glabrata had Candida drug-resistant (CgCDR) genes CgCDR1 and CgCDR2, and Fatty Acid Activator 1 (FAA1) which was positively regulated twice as much in resistant strains. ³³ Yu et al. (2018) found that another factor that played an important role in the antifungal tolerance and cell wall integrity of C. glabrata is ADA2 which was mediated by the ERG6 gene. ³⁴

Bacteria are thought to play an important role in the formation of denture plaque considering that denture plaque can contain 10 ¹¹ microbes per milligram. ⁸ Actinomyces is a genus commonly found in denture plaque with a large proportion which can be caused by the ability of C. albicans biofilms to provide a positive anaerobic environment to some anaerobic bacteria so that Actinomyces which is an anaerobic bacteria can easily grow in oxygen-rich areas. ^{10 , 29} However, the clinical significance of Actinomyces spp. still needs to be proven and the available data regarding the antimicrobial susceptibility of Actinomyces is still limited with the susceptibility method which has not been standardized. In this study, the MBIC ₅₀ of avocado seed extract against the mono-species Actinomyces odontolyticus biofilm was high, namely 15% avocado seed extract with an inhibition value of 50.88 ± 7.31%. This can be explained by several studies which had found the existence of antimicrobial agent resistance or antibiotic resistance in A. odontolyticus. Wolff et al. (2022) found an A. odontolyticus isolate that showed multi-drug resistance (MDR) to benzylpenicillin, meropenem, moxifloxacin, and daptomycin. ³⁵ Steininger et al. (2016) tested the susceptibility of Actinomyces spp. taken from 387 patients over a 7-year period and found that Actinomyces spp. was susceptible to β-lactam antimicrobial agents with and without β-lactamase inhibitors and there was an A. odontolyticus isolate that was resistant to tetracycline. ³⁶

Shi et al. (2016) found that S. gordonii colonized denture teeth in healthy denture users at a significantly higher rate. ¹¹ In this study, the MBIC ₅₀ of avocado seed extract in mono-species S. gordonii biofilm was 15% avocado seed extract with an inhibition value of 51.16 ± 4.74%. There was still no research done on the effect of avocado seed extract on S. gordonii, but most researches on S. mutans had been carried out, where these researches focused on treating caries and dental plaque rather than denture plaque. Calosa et al. (2023) found that the minimum inhibitory level of avocado seed extract against S. mutans as seen from the sample absorbance was 12.5%. ³⁷ S. gordonii usually competes with S. mutans where S. gordonii metabolically produces hydrogen peroxide which is able to inhibit the growth of S. mutans, and produces alkaline ammonia which is able to mitigate acidity on the tooth surface. The presence of S. mutans in plaque is strongly and positively associated with caries while S. gordonii is negatively associated with caries. ³⁸ Considering the antagonistic relationship between S. mutans and S. gordonii, the presence of S. gordonii in denture plaque minimizes the presence of S. mutans. ³⁷ Further research into the effects of avocado seeds on S. gordonii needs to be carried out.

S. aureus is often associated with higher amount in the elderly, seriously ill patients, individuals with low salivary secretion, and denture wearers. ³⁹ S. aureus is also commonly found in patients with oral infections associated with Candida albicans, such as denture stomatitis and angular cheilitis, due to the nature of S. aureus which tends to attach more easily to the hyphal phase of C albicans compared to abiotic surfaces. ⁴⁰ In this study, the MBIC ₅₀ of avocado seed extract in mono-species S. aureus biofilm was 10% avocado seed extract with a value of inhibition was 51.63 ± 5.05%. Research on the effect of avocado seed extract on S. aureus that has been carried out has found the Minimum Inhibitory Concentration (MIC) of avocado seed extract, but not the MBIC. Santosa et al. (2019) using the zone of inhibition test concluded that avocado seed extract was effective in inhibiting multi-resistant S. aureus at a concentration of 6.25%. ⁴¹

This study found a significant effect of soaking in avocado seed extract ( Persea americana Mill.) concentrations of 5%, 10%, 15%, 20%, as well as the positive control of alkaline peroxide in inhibiting the growth of denture plaque microorganisms on HPA resin discs in the form of C. albicans, C. glabrata, A. odontolyticus, S. gordonii, and S. aureus mono-species biofilms, each with a value of p≤0.001 (p<0.05). If the inhibition value of avocado seed extract was compared with the inhibition value of the positive control alkaline peroxide, only the MBIC ₅₀ inhibition value of avocado seed extract on mono-species C. albicans biofilm (76.09 ± 3.21%) was found to be higher than the inhibition value of the positive control (66, 43 ± 28.29%). In other mono-species biofilms, such as C. glabrata, A. odontolyticus, S. gordonii, S. aureus, the inhibition value of avocado seed extract was lower than the inhibition value of the positive control. Morelli et al. (2023) stated that effervescent tablets showed good antimicrobial activity against C. glabrata, S. mutans, and S. aureus on a cobalt-chromium surface. However, none of these peroxide-based solutions showed a reduction in C. albicans biofilms or substantially eliminated aggregated biofilms. ⁴²

From the research results, the MBIC ₅₀ of avocado seed extract against polymicrobial biofilm in this study was 20% avocado seed extract with an inhibition value of 50.81 ± 8.32%. When compared with MBIC ₅₀ in mono-species biofilms, it was found that polymicrobial biofilm required a higher concentration of avocado seed extract. This is in accordance with research results which state that polymicrobial biofilms have higher resistance to antimicrobial agents compared to mono-species biofilms. O’Brien et al. (2022) who tested three clinically relevant antimicrobial agents namely colistin, fusidic acid, and fluconazole against polymicrobial populations containing P. aeruginosa, S. aureus, and C. albicans found a higher antimicrobial agent resistance in polymicrobial biofilm compared to mono-species biofilms. These researchers found that there was a decrease in antimicrobial activity against target microorganisms in polymicrobial cultures compared to mono-species cultures. ⁴³ However, Kart et al. (2014) stated that polymicrobial biofilms did not always have higher resistance compared to mono-species biofilms as its susceptibility to antimicrobial agents depends on the nature of the microbial species present and the disinfectant used. ⁴⁴

In polymicrobial biofilms, the interactions between microbes are very complex, some of which include cooperative and antagonistic interactions. Synergism between species in polymicrobial biofilms can produce effects on growth enhancement, antimicrobial resistance, virulence, and greater exopolysaccharide production compared to individual species alone. ⁴⁵ C. albicans and C. glabrata are often found together in the form of co-isolates that cause increased pathogenicity of both species. ⁴⁶ This is due to the ability of C. albicans to damage host tissue which can be exploited by C. glabrata to reach deeper tissues. C. glabrata itself has very high antifungal resistance capabilities, and is able to modify the maturation of macrophage phagosomes so that they can hide inside macrophages from the host immune system so C. glabrata can produce infections that are much more severe and require quite complicated treatment. ⁴⁷ Other microorganisms that were found to have a very synergistic interaction were S. gordonii and C. albicans. S. gordonii was found to be capable of promoting filamentation and increasing fungal biofilm formation. Higher biomass was also found in polymicrobial biofilms formed by C. albicans and S. gordonii. ⁴⁸ Diaz et al. (2014) showed an increase in the ability of oral streptococci to form biofilms on abiotic surfaces in the presence of C. albicans. ⁴⁹ This is caused by C. albicans adhesins which facilitate the interaction of bacterial species, such as Als1p, Als2p, Als3p, Hwp1p. ²⁸ On the other hand, these bacteria is able to influence the local environment of C. albicans by altering nutrient supply and carbon dioxide levels thereby favouring C. albicans’ hyphal transition and virulence. ⁴⁹ The interaction of the two species causes increased resistance to antimicrobial agents. ⁴⁸ The relationship between S. aureus and C. albicans has also been studied extensively where C. albicans can increase S. aureus resistance to vancomycin by 100-fold due to the production of the cell wall component β-1,3-glucan. These compounds were identified as matrix constituents that provide bacteria with increased drug tolerance. In addition, the production of farnesol and prostaglandin E2 by C. albicans can increase S. aureus biofilm formation. ⁵⁰

Antagonistic interactions are a type of competitive interaction where one species will inhibit the growth of another species by producing a variety of secondary metabolites that can inhibit or kill competing species so that the biofilm architecture can be disrupted. ⁴⁵ Guo et al. (2015) found an inhibitory effect of A. odontolyticus on proliferation, adhesion, metabolic enzyme activity, hypha formation, and biofilm development of C. albicans. Actinomyces was found to produce many metabolites with antifungal activity, including lincomycin and geldanamycin. ⁵¹ However, another study by Morse et al. (2019) showed opposite results and found that polymicrobial biofilms of S. sanguinis, S. gordonii, A. odontolyticus, and A. viscosus were able to increase the number of C. albicans hyphae. ⁵²

In this study, there was a significant effect of soaking in avocado seed extract ( Persea americana Mill.) concentrations of 5%, 10%, 15%, 20%, as well as the positive control of alkaline peroxide in inhibiting the growth of polymicrobial biofilm on HPA resin discs with a value of p≤0.001 (p<0.05). The results of this analysis were also supported by SEM results which showed a much sparse biofilm on HPA resin discs soaked in 15% avocado seed compared to those soaked in 5% avocado seed extract. This showed that avocado seed extract had the ability to damage the mucus layer of polymicrobial biofilms. Polymicrobial biofilms are highly structured associations of microorganisms encased in an extracellular matrix (ECM) which attached to biotic or abiotic surfaces. One of the advantages of biofilms to the microorganisms within them is the presence of collective recalcitrant which is defined as the ability of pathogenic biofilms to survive in the presence of high concentrations of antibiotics. Cells in biofilms were found to be 10-1000 times more resistant to various antimicrobial agents than their planktonic forms. ⁵³ Polymicrobial biofilms were found to have tolerance to antimicrobial agents and increased virulence due to an extracellular matrix (ECM) containing abundant extracellular polymeric substances (EPS) to protect all microbial cells from various dangers. ⁵⁴ The presence of extracellular matrix (ECM) can influence pH, oxygen concentration, and nutrient availability in the deepest layers of the biofilm. In addition, ECM can limit the penetration of antimicrobial agents and cause the accumulation of antibiotic-degrading enzymes. ⁴⁴ Therefore, increasing the permeability of polymicrobial biofilms is one of the targets of antimicrobial agents to inhibit the microorganisms within them.

In the phytochemical tests that have been carried out, flavonoid, tannin, alkaloid, saponin, triterpenoid and polyphenol class compounds were found present in avocado seed extract. Followed by quantitative tests of phytochemical compounds, it was found that the total flavonoid content in avocado seed extract was 4.0888 mgQE/g, the total phenol content was 66.8157 mgGAE/g, the total alkaloid content was 1.22%, and the total saponin content was 1. 59%. Vinha et al. (2013) found higher levels of flavonoids and total phenolics in avocado seeds compared to avocado flesh and skin. ⁵⁵ The extraction technique used in the research was the maceration technique, which is a method that is very suitable for secondary metabolite compounds that are sensitive to heat, such as polyphenolic compounds, especially flavonoids, causing the discovery of high levels of flavonoids and polyphenols. ²⁶ Flavonoids and tannins are a family of polyphenolic components that are widely distributed in Kingdom Plantae. ⁵⁶ Flavonoids were found to have an antibiofilm effect by penetrating the biofilm layer and inhibiting bacterial growth and attachment. surface. The presence of hydrophilic parts of the chemical structure of flavonoids, including glycoside and hydroxy groups, could increase penetration of the biofilm structure and increase antibiofilm activity. ²⁷ Matilla-Cuenca et al. (2020) found that the antibiofilm activity of flavonoids which could inhibit S. aureus biofilm formation was specifically mediated by Bap. ⁵⁷ Tannins were also found to influence the gene expression of virulent factors such as biofilm, enzymes, adhesins, motility and toxins, and act as quorum sensing inhibitors. ⁵⁸ Villanueva et al. (2023) found that all unmodified natural tannins had broad spectrum activity due to their ability to exhibit very significant anti-biofilm activity against Gram-positive and Gram-negative bacteria at least at a concentration of 150 mg/L. ⁵⁹

Alkaloids have been found to damage bacterial cell membranes, inhibit efflux pumps, inhibit ATP synthesis which affects the metabolic processes of microorganisms, damage DNA/RNA molecules or inhibit DNA thereby preventing the expression of virulent genes, and inhibit FtsZ protein synthesis by participating in the diaphragm formation and forming a ring structure in division sites to control the division process and growth of bacterial cells. ⁶⁰ Saponin can reduce the surface tension of bacterial cell walls and damage cell permeability so that saponin can diffuse into the cell and bind to the cytoplasmic membrane which can lead to cell lysis. ⁵⁸ This activity can facilitate the influx of antimicrobial agents to the deeper layers of the polymicrobial biofilm. Brahim et al. (2015) found that the combination of saponin extract with fluconazole showed good synergism against C. albicans, C. parapsolosis, C. krusei, and C. glabrata. ⁶¹ Monte et al. (2014) showed the potential of saponins in controlling the shape of plankton and biofilms of E. coli and S. aureus. ⁶² Triterpenoids with more polar groups such as hydroxyl, carboxyl and carbonyl have anti-biofilm activity due to their hydrophilic nature so they are able to penetrate the exopolysaccharide polymeric matrix in bacterial biofilms and has an effect on bacterial cells in the biofilm, and shows anti-quorum sensing activity. ⁶³

The inhibition value of the positive control alkaline peroxide against polymicrobial biofilm was found to be higher (63.10 ± 28.26%) than the MBIC ₅₀ inhibition value of 20% avocado seed extract (50.81 ± 8.32%). This shows that alkaline peroxide has a good anti-biofilm effect. Kaypetch et al. (2023) found that acrylic resin soaked in alkaline peroxide for more than 3 hours could efficiently penetrate and inhibit multispecies denture biofilm with an effect comparable to immersion in 0.5% NaClO for 10 minutes. ⁶⁴ Research by Lucena-Ferreira et al. (2013) found that daily use of alkaline peroxide could improve denture cleanliness by reducing total microorganisms and total Streptococcus, but had no effect on the Candida spp. population. ⁶⁵ This is contrary to research by Li et al. (2010) who examined the effect of alkaline peroxide on C. albicans biofilms mixed with microorganisms taken from human saliva samples that were conditioned in cases of denture stomatitis and found that alkaline peroxide was able to reduce the viability of Candida growing on the surface of acrylic resin by 3-4 times. ⁶⁶ However, MBIC ₅₀ avocado seed extract has been declared effective in inhibiting polymicrobial biofilm with an inhibition value exceeding 50% so that 20% avocado seed extract has the potential to be applied clinically as a natural denture cleanser.

Several limitations have been found in this study. First, the diversity and composition of microorganisms in the polymicrobial biofilm in this study is a broad generalization of the diversity and composition of denture plaque in denture wearers. Second, the research was carried out in vitro, which means that all research variables were under the control of the researcher, which cannot be used to represent the condition of the oral cavity in patients using dentures that can be influenced by factors such as age, gender, habits, and so on. Third, this research can only tell how much of the biofilm biomass that can be inhibited with avocado seed extract, but cannot know what microorganisms are inhibited in the polymicrobial biofilm.