The effect of soaking heat-polymerized acrylic resin denture base in avocado seed extract ( Persea americana Mill.) on the inhibition of denture-plaque microorganisms biofilm growth

Version Changes

Revised. Amendments from Version 2

In this revised version of the manuscript, there are adjustments in the abstract including the addition of research objective included in the background. In the method section, we have added additional explanation on ensuring the sterility of HPA resin. In the result section, we have added additional result of the post hoc test on the mono-species and polymicrobial biofilms which further complete data can be accessed from the link on the data availability section. In the discussion section, any redundant information which has been explained on the previous section has been omitted. Moreover, we have added a separate conclusion section and study limitation section, as well as added a brief explanation of proper microorganism handling and disposal in the ethical consideration section.

Abstract

Background

Heat polymerized acrylic (HPA) resins are known to have high porosity that contributes to increased surface roughness and microcrack formation in stress areas. This facilitates the attachment and growth of polymicrobial biofilms contributing to increased antimicrobial resistance. This research aims to study avocado seeds effect on denture-plaque microorganism mono-species and polymicrobial biofilm on HPA resin.

Methods

This study uses 144 samples (n=144), namely HPA resin discs covered with mono-species and polymicrobial biofilms. The discs are soaked for 8 hours in the 5%, 10%, 15%, 20% avocado seed extract, positive control (alkaline peroxide), and negative control (aquadest). Each disc is shaken with a vortex mixer for 1 minute, and 100 μL is added into 96-well microplates with three times repetition and incubated for 24 hours. A microtiter plate biofilm formation assay is then conducted. The inhibition values are determined from the percentage inhibition value formula which requires absorption values from a microplate reader (595 nm). The research data are analyzed using a univariant test, and a one-way ANOVA test, accompanied by Welch ANOVA on non-homogenous data.

Results

In this research, it is found that the MBIC ₅₀ of avocado seed extract against the mono-species of C. albicans (5%), C. glabrata (5%), A. odontolyticus (15%), S. gordonii (15%), S. aureus (10%), while against polymicrobial was 20%. There is a significant effect of soaking HPA resin in avocado seed extract on the inhibition of mono-species and polymicrobial biofilms with a value of p<0.001 (p<0.05).

Conclusion

The MBIC ₅₀ of avocado seed extract in polymicrobial biofilm group is higher than that in the mono-species biofilm groups. Hence, 20% avocado seed extract is concluded as the effective concentration to inhibit denture-plaque microorganism biofilm.

Introduction

The denture base is a part of the denture which rests on the supporting tissue and serves as a place for the arrangement of tooth elements. ¹ Denture base materials vary greatly, but the most commonly used and popular material is polymethyl methacrylate acrylic resin (PMMA) with more than 95% of fabricated denture bases are made from acrylic resin. ^{2 , 3} Acrylic resin itself has various types, one of which is heat polymerized acrylic resin. ⁴ Heat polymerized acrylic (HPA) resin has better strength properties and a higher degree of polymerization, less residual monomer, and more stable colour. ^{5 , 6} However, it still has limitations, some of which have porous properties and high surface roughness which can increase the attachment of fungal and bacterial biofilms. ⁴

Colonization in a biofilm requires strong attachment of oral microorganisms by integrating into the salivary pellicle to form plaque on the denture material. Surface roughness and surface free energy are two factors that can promote plaque development. ⁷ Surface roughness of acrylic resin can be reduced by adequate polishing. However, this cannot prevent the build-up of plaque on the denture due to the presence of microporosity in the acrylic resin which cannot be completely avoided. ⁴ This area of porosity becomes an environment which can protect microorganisms in the biofilm. ⁷ In addition, the abiotic surface of the denture causes less exposure of the denture biofilm to the host immune system so that microorganisms can grow without hindrance and have sufficient time to develop into plaque with varying compositions. ⁸

O’Donnell et al. (2015) stated that the composition and diversity of dental plaque was different from denture plaque. Denture plaque in the oral cavity is found to be colonized by Candida spp. against the denture surface which co-aggregated with bacteria in the oral cavity. ⁸ As many as 60% to 100% of denture wearers are found to carry Candida in the oral cavity in higher quantities compared to those who did not wear dentures. ^{8 , 9} The commonly found Candida species in denture plaque is Candida albicans. Another Candida species that is found in denture plaque and increases with age is Candida glabrata. Together with C. albicans, these two fungal species can form more pathogenic and invasive biofilms and increase the severity of denture stomatitis. ⁸ Several studies have found that denture plaque compared to dental plaque has a higher proportion of obligate anaerobic Actinomyces spp., a low proportion of Gram-negative rods, and the common presence of Staphylococcus aureus. ¹⁰ Shi et al. (2016) found that the genus of bacteria which was most commonly found on both surfaces of denture teeth and remaining natural teeth was the genus Actinomyces, followed by Streptococcus, Veillonella, Capnocytophaga, Neisseria, Prevotella, and Corynebacterium. ¹¹ Based on the genus mentioned, the bacterial species in this study were Actinomyces odontolyticus, Staphylococcus aureus, and Streptococcus gordonii.

The presence of these three bacteria in dentures can increase the virulence of C. albicans thereby increasing damage and invasion of mucosal tissue which increases the risk of denture stomatitis. Morse et al. (2018) found a significant increase in tissue damage from mixed Candida and bacterial biofilms where the composition of the biofilm was broadly the composition of denture plaque. ^{8 , 12} The difference between biofilms and planktonic bacteria or fungi is that biofilms are a community of microbial cells enveloped in a matrix, while planktonic bacteria or fungi do not have this matrix layer. The presence of matrix can cause failure of treatment with antimicrobial agents, relapse of infection, and increased mortality. ¹³ Penetration of antimicrobial agents can be complicated due to the formation of extracellular polysaccharides (EPS) which reduce the permeability of the biofilm thereby protecting microorganisms in the deepest layers of the biofilm from antimicrobial agents, minor mechanical stress, and host immune response. ^{14 , 15} To determine the inhibitory effect of an antimicrobial agent on biofilm formation, it can be done using the Minimum Inhibitory Biofilm Concentration (MBIC), which is almost the same as the MIC. The difference between the two is that MBIC is defined as the lowest concentration of an antimicrobial agent at which there is no time-dependent increase in the average number of cells capable of surviving in the biofilm. Meanwhile, MIC is defined as the lowest concentration of an antimicrobial agent against planktonic microorganisms. ¹³

To prevent the accumulation of denture plaque, adequate and routine denture cleaning needs to be done. Denture cleaning can be done chemically using alkaline peroxide type denture cleaning agent. However, alkaline peroxide is found not to show stable biofilm cleaning efficacy with previous studies showing that alkaline peroxide is not effective in cleaning biofilm and was only effective in cleaning new plaque. ^{16 , 17} Therefore, it is necessary to develop a denture cleanser product in solution preparation with natural ingredients that have antimicrobial effects which can effectively clean denture plaque. One example of natural ingredient that can be used as an antimicrobial and antibiofilm agent is avocado seeds.

Avocados ( Persea americana Mill.) are one of the most popular types of fruit among Indonesian and are widely used as food ingredients (salads, sandwiches, cakes) and drinks (juice, ice cream), cosmetic ingredients, medicines and ornamental plants. ¹⁸ However, avocado seeds have no practical use and have not been utilized optimally so they tend to be an organic waste. ¹⁹ Avocado seed can actually be used as an antimicrobial agent because of the higher amounts of phytochemical components contained in avocado seed, namely flavonoids, tannins, saponins, and alkaloids, than in avocado skin and pulp, which are 64% in seed, 23% in skin, and 13% in pulp. ^{20 , 21} The inhibitory effects of avocado seed extract has been studied. Anggraini et al. (2017) studied the inhibition zone of avocado seed extract at concentrations of 10%, 20%, 40%, 80%, 100% on the growth of C. albicans, and found that the 10% concentration was the most effective concentration in inhibiting C. albicans. ²² Another study by Talib et al. (2018) tested the effectiveness of avocado seed extract in inhibiting Streptococcus mutans at concentrations of 2%, 4%, 6%, 8%, 10% and found that the most effective concentration was 10%. ²³

However, most studies using avocado seed extract are carried out on planktonic bacteria or fungi, that is different from denture plaque in the patient’s oral cavity which is a polymicrobial biofilm that tends to be more resistant to antimicrobial agents. Unlike planktonic bacteria and fungi, polymicrobial biofilm has a protective layer, extracellular polysaccharide (EPS) formation, which decrease the permeability of the biofilm from antimicrobial agents. Moreover, the various microorganisms in the biofilm also build a communication mechanism called quorum sensing which enables the alteration of virulence gene expressions, the production and utilization of varied nutrition effectively through metabolic consortium, and even the ability to slow their growth in the presence of antimicrobial agents in the biofilm thereby suggesting a better resistance of polymicrobial biofilms towards antimicrobial agents than planktonic microorganism’s resistance. ^{14 , 15 , 24} This can be seen in a study by Hamzah et al. (2019) who found an increase in the minimum biofilm inhibitory concentration of tannin in polymicrobial biofilms ( Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans) when compared to the concentration in mono-species biofilms. The minimum biofilm inhibitory concentration of tannin in mid-phase polymicrobial biofilms (24 hours) was 1%, while the minimum inhibitory concentration of tannins in mono-species biofilms (24 hours) varied widely, namely E. coli (0.125%), S. aureus (0.5%), P. aeruginosa (0.25%), C. albicans (0.5%). ²⁴ Hence, this study aims to determine the effect of avocado seed extract ( Persea americana Mill.) with concentrations of 5%, 10%, 15%, 20% on denture-plaque microorganisms, which were Candida albicans, Candida glabrata, Actinomyces odontolyticus, Streptococcus gordonii, and Staphylococcus aureus, in the form of mono-species and polymicrobial biofilms on HPA resin through determining their MBIC ₅₀.

Methods

Study design

This research uses in vitro experimental methods with post-test only control group design. The sample used in this research is 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 is determined using Federer formula, hence the number of samples for each group is 4 samples. There are 6 treatment groups in this study which are 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 is 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 will be used in this research was 144 samples (n=144).

Figure 1. Shape and size of heat polymerized acrylic resin disc.

Preparation of heat polymerized acrylic resin disc samples

A disc-shaped brass metal master models with a diameter of 10 mm and a thickness of 2 mm are made to be used as a research sample mould. The dental cuvette, which has been smeared with Vaseline, is 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 are trapped in the mixture. The master models, which have been smeared with vaseline, are then placed in the dough in a cuvette, avoiding the surface of the master models being flat with the gypsum surface. The gypsum is left to harden for ± 30-45 minutes and then smeared with vaseline. The upper cuvette is attached to the lower cuvette and filled with the same gypsum mixture as described previously. After the plaster has hardened, the cuvette is opened and the master models are taken out to obtain a mould. ²⁵

The surface of the mould is smeared thinly with cold mould seal and left for 10 minutes. The HPA resin mixture is prepared with a weight ratio of 2.5:1. When it reaches the dough stage, the acrylic resin mixture is put into the mould, then covered with plastic cellophane along with the top cuvette. The cuvette is pressed slowly with a hydraulic press until the pressure reaches 1000 psi. Excess dough is cleaned with dental lecron, then the cuvette is closed and pressed again with a pressure of 2200 psi. The cuvette is reopened and cleaned of excess acrylic resin mixture. The cuvette is closed again and locked with the cuvette bolts, then left for 30 minutes. The cuvette is inserted into a water bath filled with aquadest, then the temperature and time are set at 70°C for 90 minutes, then at 100°C for 30 minutes. After 30 minutes, the cuvette is left in the water bath until the water reached room temperature for the cuvette cooling process. The samples are removed from the cuvette, then the sharp parts and plaster residue are trimmed with a fraser bur and sand paper. ²⁵

Avocado seed extract procedure and tests

Avocado sample

Avocados are obtained from Berastagi, 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.

Avocado seed extraction procedure

Avocado seeds are extracted using a maceration technique. The avocado seeds will be cut into slices which will be dried in a drying cabinet at a temperature of ±40°C for about 24 hours, then coarsely grounded and blended until they become a fine powder. The avocado seed powder is 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 is evenly mixed. The solution is filtered until macerate I is obtained and the remaining filtered dregs are subjected to a second maceration process. The results of macerate I and II will be mixed and transferred into a closed vessel, then left in a cool place protected from light for 2×24 hours. The extract is concentrated using a rotary evaporator at a temperature of ±50°C to evaporate the solvent until a thick extract is obtained. The thick extract is diluted with aquadest to be made into a concentration of 5%, 10%, 15%, 20%. ²⁶

Phytochemical examination and quantitative test of phytochemical compounds of avocado seed extract

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

Formation of denture plaques on heat polymerized acrylic resin disc samples

Microorganisms and culture conditions

The microorganisms used in this study are cultured and maintained under the following conditions. Candida albicans ATCC ^® 24433 ^TM and Candida glabrata ATCC ^® 90030 ^TM are 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 ^® 17982 ^TM is 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 is 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 is cultured on blood agar with 5% (v/v) defibrinated bovine blood and incubated at 37°C under aerobic conditions for 24 hours. ²⁷

Biofilm formation on heat polymerized acrylic resin disc

HPA resin discs are first sterilized on both sides, each for 30 minutes, using ultraviolet germicidal irradiation technique which employs ultraviolet light or more specifically UVC. In our study, we sterilize the HPA resin discs using the UV lights from the biosafety cabinets. Sterile HPA resin discs are preconditioned for 24 hours by immersion in artificial saliva which are kept inside a refrigerator to preserve the artificial saliva from being spoiled or contaminated. After the immersion period, the HPA resin discs still immersed in artificial saliva are sterilized again using ultraviolet germicidal irradiation technique. 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 are then placed aseptically into 24-microplates, and 100 μL of standardized microorganisms are added to each surface of the disc. Biofilm preparations carried out are mono-species biofilms for each microorganism studied ( C. albicans, C. glabrata, A. odontolyticus, S. gordonii, S. aureus), and polymicrobial biofilms which are a combination of the five microorganisms studied, resulting in a total of 500 μL on each polymicrobial biofilms disc. Sterile Dulbecco’s Modified Eagle Medium (DMEM) (supplemented with 50 mmol L-1 L-glutamine per liter) is added to a final volume of 2 mL in each plate. Culture media discs in 24-wells microplates are shaken on an orbital shaker for 30 minutes to homogenize the media and culture solutions, then incubated at 37°C for 24 hours. ²⁷

Determination of minimum biofilm inhibitory concentration (MBIC ₅₀) using microtiter plate biofilm formation test

HPA resin discs which have been grown by mono-species and polymicrobial biofilms will 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, away from direct sunlight. The preparation of the positive control, alkaline peroxide, is done according the manufacture procedure and along with the negative control, aquadest is sterilized using ultraviolet germicidal irradiation technique. The discs are 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 is taken from the dilution and added into 96-wells microplates with a repetition of three times. Microplates are incubated at 37°C for 24 hours. After incubation, the microplates are 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 is added to each microplate to colour the formed biofilm and left for 15 minutes. The crystal violet solution is discarded, then cleaned with distilled water and patted hard on a lab mat. The stained biofilm plates are allowed to dry until the remaining water in the microplates evaporated, then 150 μL of 95% ethanol is added to each plate and left for 10 minutes. The absorption value (OD) reading is carried out with a microplate reader at a wavelength of 595 nm and the results are calculated using the percentage inhibition value formula of which Control OD is defined as negative control (aqaudest) absorption value and Sample OD is defined as test sample (avocado seed extracts and alkaline peroxide) absorption value. ²⁴

$$\text{Inhibition Value}(\%)=[(\text{Control}\mathrm{OD}-\text{Sample}\mathrm{OD})÷\text{Control}\mathrm{OD}]\times 100$$

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

Statistics analysis

Univariate analysis is 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 is carried out using the Shapiro-Wilk test (p>0.05) and the homogeneity test is carried out using the Levene test (p>0.05). Data analysis is carried out using one-way ANOVA, which can be accompanied by Welch ANOVA on non-homogeneous data, to determine the effect of treatment in each group. Data are 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 are defined at p<0.05.

Scanning electron microscopy (SEM)

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

Ethical approval

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.

Results

In this study, there are 6 treatment groups consisting of samples of HPA resin discs soaked in avocado seed extract 5%, 10%, 15%, 20%, as well as a positive control (alkaline peroxide) and a negative control (aquadest). The HPA resin disc samples are grown with mono-species biofilms of C. albicans, C. glabrata, A. odontolyticus, S. gordonii, S. aureus and polymicrobial biofilms so that the number of samples in this study is 144 samples (n=144).

Determination of avocado fruit plants

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

Phytochemical test results of avocado seed ethanol extract

The phytochemical test on the ethanol extract of avocado seeds is done using specific reagents to determine the presence of secondary metabolite compounds which are alkaloids, flavonoids, glycosides, saponin, tannin, triterpenoids/steroids ( Table 1).

Table 1. Phytochemical test of avocado seed ethanol extract.

No.	Secondary metabolites	Reagents	Results
1	Alkaloids	Dragendorff	+
		Bouchardat	+
		Mayer	+
2	Flavonoids	Mg Powder + Amyl Alcohol + HCl p	+
3	Glycosides	Molisch + H 2SO 4	+
4	Saponin	Hot water/shaken	+
5	Tannin	FeCl 3	+
6	Triterpenoids/Steroids	Lieberman-Burchard	+

Quantitative analysis results for phytochemical compounds of avocado seed ethanol extract

The secondary metabolite compounds existing in the avocado seed ethanol extract can be further assessed by doing a quantitative analysis to determine the amount of the secondary metabolites in the sample extract which are flavonoids, phenol, saponin, and alkaloids ( Table 2). The analysis shows the highest total amount of phenol and the lowest total amount of alkaloids. This might be caused by the extraction technique used in the research which is the maceration technique, a method that is very suitable for secondary metabolite compounds that are sensitive to heat, such as polyphenolic compounds, especially flavonoids. Extracting the avocado seed with maceration technique can cause the discovery of high levels of flavonoids and polyphenols. ²⁶

Table 2. Quantitative analysis for phytochemical compounds of avocado seed ethanol extract.

No.	Analysis	Total	Unit
1	Total Flavonoids	4,0888	mgQE/g extract
2	Total Phenol	66,8157	mgGAE/g extract
3	Total Saponin	1,59	%
4	Total Alkaloids	1,22	%

The MBIC ₅₀ determination of avocado seed extract and its effect on the mono-species biofilms of C. albicans, C. glabrata, A. odontolyticus, S. gordonii, S. aureus

Each sample in each group is repeated three times to obtain three absorption values (OD) which then using univariate analysis, the mean and standard deviation are obtained. The obtained absorption value is calculated using the percentage inhibition value formula with an inhibition value of 50% as a parameter for determining MBIC ₅₀ ( Table 3). Based on calculations, MBIC ₅₀ of avocado seed extract in mono-species C. albicans biofilm is 5% avocado seed extract. MBIC ₅₀ avocado seed extract in mono-species C. glabrata biofilm is 5% avocado seed extract. MBIC ₅₀ avocado seed extract in mono-species A. odontolyticus biofilm is 15% avocado seed extract. MBIC ₅₀ avocado seed extract in mono-species S. gordonii biofilm is 15% avocado seed extract. MBIC ₅₀ avocado seed extract in mono-species S. aureus biofilms is 10% avocado seed extract.

Table 3. Inhibition value of the mono-species biofilm growth soaked in avocado seed extract and alkaline peroxide.

Mono-species	Inhibition value (%)
	Avocado Seed Extract				Positive control	Negative control
	5%	10%	15%	20%
Candida albicans	76.09 ± 3.21 *	69.33 ± 8.57	77.80 ± 6.58	63.29 ± 7.85	66.43 ± 28.29	0.00 ± 0.00
Candida glabrata	52.41 ± 9.64 *	59.99 ± 7.55	58.55 ± 12.52	60.75 ± 15.80	67.24 ± 15.41	0.00 ± 0.00
Actinomyces odontolyticus	29.42 ± 5.21	40.50 ± 5.65	50.88 ± 7.31 *	52.58 ± 15.38	54.25 ± 13.87	0.00 ± 0.00
Streptococcus gordonii	38.52 ± 5.04	42.95 ± 3.61	51.16 ± 4.74 *	52.79 ± 4.26	69.79 ± 22.20	0.00 ± 0.00
Staphylococcus aureus	45.30 ± 4.05	51.63 ± 5.05 *	52.79 ± 5.05	56.15 ± 4.98	61.38 ± 8.54	0.00 ± 0.00

^*= MBIC ₅₀, inhibition value are in mean and standard deviation.

The sample is tested for normality and a value of p>0.05 is obtained, hence the data were normally distributed. Then, a homogeneity test is carried out by which the mono-species C. albicans biofilm sample obtains a value of p<0.001 (p<0.05), the mono-species A. odontolyticus biofilm sample obtains a value of p=0.002 (p<0.05), and the mono-species S. gordonii biofilm sample obtains a value of p≤0.001 (p<0.05) so the data are not homogeneous, while the mono-species C. glabrata biofilm sample obtains a value of p=0.054 (p>0.05) and the mono-species S. aureus biofilm sample obtains a value of p=0.116 (p>0.05) so the data are homogeneous. Data which is normally distributed and homogeneous is tested using one-way ANOVA and data which is normally distributed but not homogeneous is analysed using Welch ANOVA. This study finds that mono-species biofilm samples of C. albicans, C. glabrata, A. odontolyticus, S. gordonii, S. aureus obtains a value of p≤0.001 (p<0.05). This shows that there is 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 4).

Table 4. One-way ANOVA and Welch ANOVA of the treatment groups against mono-species biofilms.

Treatment groups	Absorption values	p
Mono-species C. albicans Biofilm
Avocado Seed Extract of 5%	0.2949 ± 0.0397	<0.001 *
Avocado Seed Extract of 10%	0.3782 ± 0.1057
Avocado Seed Extract of 15%	0.2738 ± 0.0812
Avocado Seed Extract of 20%	0.4527 ± 0.0968
Positive control (+) Alkaline Peroxide	0.4140 ± 0.3489
Negative control (-) Aquadest	1.2332 ± 0.1059
Mono-species C. glabrata Biofilm
Avocado Seed Extract of 5%	1.0400 ± 0.2107	<0.001 *
Avocado Seed Extract of 10%	0.8743 ± 0.1651
Avocado Seed Extract of 15%	0.9057 ± 0.2735
Avocado Seed Extract of 20%	0.8576 ± 0.3452
Positive control (+) Alkaline Peroxide	0.7160 ± 0.3368
Negative control (-) Aquadest	2.0627 ± 0.2138
Mono-species A. odontolyticus Biofilm
Avocado Seed Extract of 5%	1.4803 ± 0.1093	<0.001 *
Avocado Seed Extract of 10%	1.2479 ± 0.1185
Avocado Seed Extract of 15%	1.0302 ± 0.1534
Avocado Seed Extract of 20%	0.9946 ± 0.3225
Positive control (+) Alkaline Peroxide	0.9595 ± 0.2909
Negative control (-) Aquadest	2.0972 ± 0.1524
Mono-species S. gordonii Biofilm
Avocado Seed Extract of 5%	1.0829 ± 0.0887	<0.001 *
Avocado Seed Extract of 10%	1.0048 ± 0.0635
Avocado Seed Extract of 15%	0.8602 ± 0.0834
Avocado Seed Extract of 20%	0.8316 ± 0.0751
Positive control (+) Alkaline Peroxide	0.5322 ± 0.3910
Negative control (-) Aquadest	1.7614 ± 0.0895
Mono-species S. aureus Biofilm
Avocado Seed Extract of 5%	0.9012 ± 0.0666	<0.001 *
Avocado Seed Extract of 10%	0.7968 ± 0.0832
Avocado Seed Extract of 15%	0.7779 ± 0.0832
Avocado Seed Extract of 20%	0.7225 ± 0.0820
Positive control (+) Alkaline Peroxide	0.6362 ± 0.1407
Negative control (-) Aquadest	1.6475 ± 0.0884

^*Significant, absorption values are in mean and standard deviation.

Since ANOVA test showed significant results, posthoc test using LSD (( Least Significant Difference) test for homogenous data, or Games-Howell test for non-homogenous data is done. All mono-species biofilms groups show that there is a significant difference between the MBIC ₅₀ avocado seed extract as well as the positive control and the negative control with a value of p<0.001 (p<0.05).

The absorption values of the negative control are the control value of each mono-species biofilms, while the absorption values of avocado seed extract and positive control are the treatment sample value. The lower the absorption value of the treatment, the higher the inhibition value of the treatment. On the mono-species C. albicans biofilm, the MBIC ₅₀ (5% avocado seed extract) absorption value is lower than that of the positive control, indicating a higher inhibition value. The difference of inhibition value is found slightly higher on the MBIC ₅₀ avocado seed which inhibits roughly 10% more than that of positive control. Eventhough the posthoc test shows that there is no significant difference between the MBIC ₅₀ avocado seed and the positive control with p=0,840 (p>0.05), avocado seed extract still shows a great potential of its practical implications as an antibiofilm agent towards C. albicans biofilm that is on par with the positive control (alkaline peroxide).

On the other hand, the C. glabrata, A. odontolyticus, S. aureus, S.gordonii biofilms’ MBIC ₅₀ show higher absorption values than that of the positive control, which means lower inhibition values of these biofilms compared to the positive control inhibition value. The posthoc test on C. glabrata biofilm and S. aureus biofilm groups show that there is a significant difference between the MBIC ₅₀ avocado seed extract and the positive control, respectively p=0,004 (p<0.05) and p≤0.001 (p<0.05) which might indicate limited practical applications of avocado seed extract on the biofilms. Meanwhile, the posthoc test on A. odontolyticus biofilm and S. gordonii biofilm show that there is no significant difference between the MBIC ₅₀ avocado seed extract and the positive control, respectively p=0.973 (p>0.05) and p=0.117 (p>0.05) which might indicate a potential of avocado seed extract’s practical applications as antibiofilm agents towards A. odontolyticus and S. gordonii biofilms.

Evenso, the highest concentration of avocado seed extract in this study (20% avocado seed extract) still shows a great inhibition on the mono-species biofilms, especially towards C. albicans, A. odontolyticus and S. aureus biofilms, in comparison with the positive control. Posthoc test also shows that there is no significant difference between 20% avocado seed extract and positive control on all of the studied mono-species biofilms (p<0.05).

The MBIC ₅₀ determination of avocado seed extract and its effect on polymicrobial biofilm

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

Table 5. Inhibition value of the polymicrobial biofilm growth soaked in avocado seed extract and alkaline peroxide.

Polymicrobial biofilm	Inhibition value (%)
	Avocado Seed Extract				Positive control	Negative control
	5%	10%	15%	20%
	31.94 ± 11.53	36.32 ± 12.24	45.36 ± 5.70	50.81 ± 8.32 *	63.10 ± 28.26	0.00 ± 0.00

^*= MBIC ₅₀, inhibition values are in mean and standard deviation.

The sample is tested for normality and a value of p >0.05 was obtained, hence the data is normally distributed. Then, a homogeneity test is carried out by which the polymicrobial biofilm samples obtain a value of p=0.006 (p<0.05) so that the data is not homogeneous. Data that are normally distributed but not homogeneous are analysed using the Welch ANOVA. This study finds that the polymicrobial samples obtain a value of p≤0.001 (p<0.05) This show that there is a significant effect of soaking in 5%, 10%, 15%, 20% avocado seed extract in inhibiting polymicrobial biofilm ( Table 6).

Table 6. Welch ANOVA of the treatment groups against polymicrobial biofilm.

Kelompok	Absorption Values of Polymicrobial Biofilm	p
Avocado Seed Extract of 5%	1.3573 ± 0.2300	<0.001 *
Avocado Seed Extract of 10%	1.2699 ± 0.2442
Avocado Seed Extract of 15%	1.0897 ± 0.1137
Avocado Seed Extract of 20%	0.9810 ± 0.1658
Positive control (+) Alkaline Peroxide	0.7359 ± 0.5637
Negative control (-) Aquadest	1.9942 ± 0.6417

^*Significant, absorption values are in mean and standard deviation.

Since ANOVA test showed significant results, posthoc test using Games-Howell test for non-homogenous data is done. The polymicrobial biofilm group shows that there is a significant difference between the MBIC ₅₀ of avocado seed extract and the negative control with the value of p=0.002 (p<0.05).

The MBIC ₅₀ of avocado seed extract on polymicrobial biofilm has higher absorption values than that of positive control, indicating a lower inhibition value. There is quite a difference of inhibition value with positive control inhibited about 13% more than that of the MBIC ₅₀ avocado seed extract which happens to be the highest concentration in this study. However, the posthoc test shows that there is no significant difference between avocado seed extract MBIC ₅₀ and the positive control with p=0.702 (p>0.05), which indicates a good potential of avocado seeds that can be studied further.

Scanning electron microscopy (SEM) results of polymicrobial biofilm on HPA resin discs soaked in avocado seed extract

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 have been soaked with avocado seed extract can be detected and clearly seen in Figure 2 above. SEM results showed that there are microorganisms growing on the HPA resin disc. The soaking in 5% avocado seed extract shows a denser formation of biofilm compared to soaking in 15% avocado seed extract. The biofilm in 5% avocado seed extract shows more clusters of tightly binded microorganisms meanwhile the biofilm in 15% avocado seed extract shows less clusters and more loosely binded microorganisms, indicating a penetration of extracellular matrix (ECM) and breakdown of extracellular polymeric substances (EPS) from 15% avocado seed extract.

Figure 2. SEM results of 5% avocado seed extract (left) and 15% avocado seed extract (right).

Discussion

The MBIC ₅₀ of avocado seed extract and its effect on the mono-species biofilms of C. albicans, C. glabrata, A. odontolyticus, S. gordonii, S. aureus

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 is 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. The lowest concentration of avocado seed extract tested in the study, which was a concentration of 3.13%, was able to inhibit C. albicans biofilms incubated for 24 hours by 75.37%. ³⁰ These results indicate that avocado seed extract is effective in inhibiting C. albicans, considering that the lowest concentration of avocado seed extract has already shown a greater inhibition value than the standard inhibition value.

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 According to the researcher’s knowledge, there is still little to none research done on the effect of avocado seed extract on C. glabrata. This study finds that the MBIC ₅₀ of avocado seed extract in mono-species C. glabrata biofilm is 5% avocado seed extract with an inhibition value of 52.41 ± 9.64%. When compared with the C. albicans inhibition value, there is 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 is 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 is 15% avocado seed extract with an inhibition value of 51.16 ± 4.74%. There is still no research done on the effect of avocado seed extract on S. gordonii, but most researches on S. mutans have been carried out where these researches focus 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 is 10% avocado seed extract with a value of inhibition is 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 has 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 is 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%) is 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 is lower than the inhibition value of the positive control. This can be explained by the findings of Morelli et al. (2023), which 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

The MBIC ₅₀ of avocado seed extract and its effect on polymicrobial biofilm

From the research results, the MBIC ₅₀ of avocado seed extract against polymicrobial biofilm in this study is 20% avocado seed extract with an inhibition value of 50.81 ± 8.32%. When compared with MBIC ₅₀ in mono-species biofilms, it is 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 are found to have a very synergistic interaction are S. gordonii and C. albicans. S. gordonii is found to be capable of promoting filamentation and increasing fungal biofilm formation. Higher biomass is 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. ²⁷

In this study, there is 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 are also supported by SEM results which show a much sparse biofilm on HPA resin discs soaked in 15% avocado seed compared to those soaked in 5% avocado seed extract. This shows that avocado seed extract has 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 are found to be 10-1000 times more resistant to various antimicrobial agents than their planktonic forms. ⁵² Polymicrobial biofilms are found to have tolerance to antimicrobial agents and increased virulence due to an ECM containing abundant EPS to protect all microbial cells from various dangers. ⁵³ The presence of 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 are found present in avocado seed extract. Followed by quantitative tests of phytochemical compounds, it is found that the total flavonoid content in avocado seed extract is 4.0888 mgQE/g, the total phenol content is 66.8157 mgGAE/g, the total alkaloid content is 1.22%, and the total saponin content is 1. 59%. The result is aligned with the study of Vinha et al. (2013) which found higher levels of flavonoids and total phenolics in avocado seeds compared to avocado flesh and skin. ⁵⁴ Flavonoids and tannins are a family of polyphenolic components that are widely distributed in Kingdom Plantae. ⁵⁵ Flavonoids are 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, can increase penetration of the biofilm structure and increase antibiofilm activity. Matilla-Cuenca et al. (2020) found that the antibiofilm activity of flavonoids which could inhibit S. aureus biofilm formation was specifically mediated by Bap. ⁵⁶ Tannins are also found to influence the gene expression of virulent factors such as biofilm, enzymes, adhesins, motility and toxins, and act as quorum sensing inhibitors. ⁵⁷ 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. ⁵⁸

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. ⁵⁹ 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. ⁵⁷ 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. ⁶⁰ Monte et al. (2014) showed the potential of saponins in controlling the shape of plankton and biofilms of E. coli and S. aureus. ⁶¹ 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. ⁶²

The inhibition value of the positive control alkaline peroxide against polymicrobial biofilm is 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. ⁶³ 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. ⁶⁴ However, MBIC ₅₀ avocado seed extract has been shown effective in inhibiting polymicrobial biofilm with an inhibition value slightly exceeding 50%.

Conclusion

As avocado seed is a natural waste product which can cause significant environmental consequences if not managed properly, repurposing it as a composition of natural denture cleanser might not only resolve its environmental issues but also potentially lower the cost of the current denture cleanser and might show a much better biocompatibility with denture users than the current ones. Not to mention, avocado seed has larger amounts of antioxidants compared to its pulp and skin, which made avocado seed as one of the natural products that needs to be further studied. This research has shown that there is a difference of the avocado seed extract MBIC ₅₀ between mono-species and polymicrobial denture plaque microorganism biofilms. The MBIC ₅₀ on each studied mono-species are C. albicans (5%), C. glabrata (5%), A. odontolyticus (15%), S. gordonii (15%), S. aureus (10%). On the other hand, the MBIC ₅₀ on polymicrobial biofilm showed a slightly higher concentration which was 20%, suggesting that polymicrobial biofilm might have higher resistance to the given extract than the mono-species biofilms. However, the MBIC ₅₀ of avocado seed extract on both mono-species and polymicrobial biofilms have been shown to have significant effect in inhibiting those biofilms, which suggests that avocado seed extract, specifically at a concentration of 20%, has the potential to be applied clinically as a natural denture cleanser. Further studies involving the effect of avocado seed extract on the physical, mechanical, and chemical traits of HPA, such as colour stability, surface roughness, tensile strength, and so on, should be carried in order to further ensure that avocado seed extract can be clinically applied as denture cleanser.

Study limitations

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. However, the microorganisms studied are the more commonly found denture plaque microorganisms and since avocado seed extract could inhibit these microorganisms, it shows that avocado seed could be one of the natural wastes that can be further studied and potentially used as natural denture cleaning agent. 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. Hence, further studies involving humans or denture users should be conducted. Third, this research can only tell how much of the biofilm biomass that can be inhibited with avocado seed extract, but cannot know which microorganisms are inhibited in the polymicrobial biofilm. Nonetheless, considering the different diversity and composition of each denture users, it might not be as crucial to know the type of microorganisms inhibited as the reduction of the denture plaque itself. Lastly, further studies need to be done in order to determine which phytochemical component of the avocado seed extract that are effective in inhibiting denture plaque microorganisms and to gain more comparison data.

Ethical considerations

This study did not include any human participants or animal. Handling of microorganisms were all done in a Biosafety Cabinet, and disposal of any clinical samples or disposable tools which had been in contact with the microorganisms were placed in a specialized disposable containers that had a 1:10 (10%) bleach dilution.

Funding Statement

The author(s) declared that no grants were involved in supporting this work.

Data availability

Figshare: Avocado Seed Extract on Inhibiting Mono-species and Polymicrobial Biofilm. https://doi.org/10.6084/m9.figshare.25996006. ⁶⁵

This project contains the following underlying data:

• •Ethical Clearance No. 166KEPKUSU2024. pdf
• •Determination of Avocado Fruit Plants. pdf
• •Phytochemical Test Results of Avocado Seed Ethanol Extract. pdf
• •Quantitative Analysis for Phytochemical Compounds. pdf
• •Research Data of Mono-species C. albicans Biofilm. docx
• •Research Data of Mono-species C. glabrata Biofilm. docx
• •Research Data of Mono-species A. odontolyticus Biofilm. docx
• •Research Data of Mono-species S. gordonii Biofilm. docx
• •Research Data of Mono-species S.aureus Biofilm. docx
• •Research Data of Polymcrobial Biofilm. docx

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0)