F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.152800.3 Research Article Articles 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 3; peer review: 2 approved, 2 approved with reservations, 1 not approved]

 Angela Thalia Conceptualization Formal Analysis Investigation Visualization Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0009-0000-3692-545X 1 Wahyuni Siti Project Administration Supervision Validation Writing – Review & Editing a 2 Halim Susanna Funding Acquisition Methodology Project Administration Resources Writing – Review & Editing https://orcid.org/0009-0000-3692-545X 3 Dental Undergraduate Study Program, Faculty of Dentistry, University of Sumatera Utara, Medan, North Sumatra, Indonesia Department of Prosthodontics, University of Sumatera Utara, Medan, North Sumatra, Indonesia Faculty of Medicine, Dentistry and Health Sciences, Prima Indonesia University, Medan, North Sumatra, Indonesia siti.wahyuni@usu.ac.id

No competing interests were disclosed.

 25 3 2025 2024 13 933 24 2 2025 Copyright: © 2025 Angela T et al. 2025 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 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.

 heat polymerized acrylic resin avocado seed extract mono-species biofilm polymicrobial biofilm denture plaque MBIC The author(s) declared that no grants were involved in supporting this work. 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.

 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).

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 Inhibition Value ( % ) = [ ( Control OD − Sample OD ) ÷ Control OD ] × 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).

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

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 <sub>50</sub> determination of avocado seed extract and its effect on the mono-species biofilms of <italic toggle="yes">C. albicans</italic>, <italic toggle="yes">C. glabrata</italic>, <italic toggle="yes">A. odontolyticus</italic>, <italic toggle="yes">S. gordonii</italic>, <italic toggle="yes">S. aureus</italic>

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.

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).

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 <sub>50</sub> 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.

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).

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.

SEM results of 5% avocado seed extract (left) and 15% avocado seed extract (right). Discussion The MBIC <sub>50</sub> of avocado seed extract and its effect on the mono-species biofilms of <italic toggle="yes">C. albicans</italic>, <italic toggle="yes">C. glabrata</italic>, <italic toggle="yes">A. odontolyticus</italic>, <italic toggle="yes">S. gordonii</italic>, <italic toggle="yes">S. aureus</italic>

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 <sub>50</sub> 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.

 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)

 References Ferro KJ : The glossary of prosthodontic terms (GPT-9). J. Prosthet. Dent. 2017;117(5S):C1–e105. 10.1016/j.prosdent.2016.12.001 Kangsudarmanto Y Rachmadi P Arya WI : Perbandingan perubahan warna heat cured acrylic basis gigi tiruan yang direndam dalam klorhesidin dan effervescent ( alkaline peroxide). DENTINO. 2014;2(2):205–9. 2337-5310. Fadriyanti O Alamsyah Y Rabianti D : Evaluasi pemakaian denture adhesive pada gigi tiruan lengkap resin akrilik: Scoping review. Menara Ilmu. 2022;16(2):55–62. 10.31869/mi.v16i2.3289 Zarb G Hobkirk JA Eckert SE : Prosthodontic treatment for edentulous patients. 13th ed. St. Louis: Elsevier Mosby;2013;133–134. Bohra PK Ganesh PR Reddy MM : Colour stability of heat and cold cure acrylic resins. J. Clin. Diagn. Res. 2015;9(1):ZC12–ZC15. 25738078 10.7860/JCDR/2015/11620.5400 PMC4347169 Rashid AA : Temperature effect on the hardness of different types of resin denture base materials. MDJ. 2013;10(1):69–76. 10.32828/mdj.v10i1.186 Jain V Babu J Ahuja S : Comparison of fungal biofilm formation on three contemporary denture base materials. Int. J. Exp. Dent. 2015;4(2):104–108. 10.5005/jp-journals-10029-1106 O’Donnell LE Robertson D Nile CJ : The oral microbiome of denture wearers is influenced by levels of natural dentition. PLoS One. 2015;10(9):e0137717. 26368937 10.1371/journal.pone.0137717 PMC4569385 Patel M : Oral cavity and Candida albicans: Colonisation to the development of infection. Pathogens. 2022;11(335):6–8. 10.3390/pathogens110303355 Coulthwaite L Verran J : Potential pathogenic aspects of denture plaque. Br. J. Biomed. Sci. 2007;64(4):180–189. 18236742 10.1080/09674845.2007.11732784 Shi B Wu T McLean J : The denture-associated oral microbiome in health and stomatitis. mSphere. 2016;1(6):e00215–e00216. 28066812 10.1128/mSphere.00215-16 PMC5196032 Morse DJ Wilson MJ Wei X : Denture-associated biofilm infection in three-dimensional oral mucosal tissue models. J. Med. Microbiol. 2018;67:364–375. 29458673 10.1099/jmm.0.000677 PMC5882079 Thieme L Hartung A Tramm K : MBEC versus MBIC: The lack of differentiation between biofilm reducing and inhibitory effects as a current problem in biofilm methodology. Biol. Proced. Online. 2019;21(18):15–18. 31528123 10.1186/s12575-019-0106-0 PMC6743098 Kumari KS Dash P Subudhi E : Antimicrobial resistance: A dentists’ prospective. Indian J. Med. Forensic Med. Toxicol. 2020;14(4):8456–8460. 10.37506/ijfmt.v14i4.13018 Ponde NO Lortal L Ramage G : Candida albicans biofilms and polymicrobial interactions. Crit. Rev. Microbiol. 2021;47(1):91–111. 33482069 10.1080/1040841X.2020.1843400 PMC7903066 Peracini A Regis RR Souza RF : Alkaline peroxides versus sodium hypochlorite for removing denture biofilm: A crossover randomized trial. Braz. Dent. J. 2016;27(6):700–704. 27982182 10.1590/0103-6440201600913 Oussama M Ahmad H : Materials and methods for cleaning dentures- A review. Int. J. Dent. Clin. 2014;6(2):19–20. Reference Source Lestari R Sukamto LA Aprilianti P : Selection of avocado plants based on fruit characters, fat content, and continual harvest along the year in west java-indonesia. Int. J. Adv. Sci. Eng. Inf. Technol. 2016;6(1):77–83. 10.18517/ijaseit.6.1.621 Domínguez MP Araus K Bonert P : The avocado and its waste: An approach of fuel potential application. Lefebvre G Jiménez E Cabañas B , editors. Environment, energy and climate change II: Energies from new resources and the climate change. Switzerland: Springer Cham;2016;199–223. 10.1007/698_2014_291 Setyawan HY Sukardi S Puriwangi CA : Phytochemicals properties of avocado seeds: A review. IOP Conf. Ser.: Earth Environ. Sci. 2021;733(1):012090–012097. 10.1088/1755-1315/733/1/012090 Bahru TB Tadele ZH Ajebe EG : A review on avocado seed: Functionality, composition, antioxidant and antimicrobial properties. Chem. Sci. Int. J. 2019;27(2):1–10. 10.9734/CSJI/2019/v27i230112 Anggraini V Masfufatun M : Efektivitas kombinasi ekstrak daun sirih merah ( Piper crocatum) dan ekstrak biji alpukat ( Persea americana) dalam menghambat pertumbuhan Candida albicans. Jurnal Kimia Riset. 2017;2(2):86–92. 10.20473/jkr.v2i2.6196 Thalib B Nahar CL : Efektivitas antibakteri ekstrak biji alpukat ( Persea americana Mill.) terhadap Streptococcus mutans. Makassar Dent. J. 2018;7(1):26–29. 10.35856/mdj.v7i1.12 Hamzah H Hertiani T Pratiwi SUT : The inhibition activity of tannin on the formation of mono-spesies and polymicrobial biofilm Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans. Trad. Med. J. 2019;24(2):110–118. 10.35856/mdj.v7i1.12 Mundada Y Gajare S Jankar A : Flexural strength of heat polymerized acrylic resin required with different reinforcing materials. Int. J. Health Sci. 2022;6(S7):2389–2396. 10.53730/ijhs.v6nS7.11867 Abubakar AR Haque M : Preparation of medicinal plants: Basic extraction and fractionation procedures for experimental purposes. J. Pharm. Bioallied Sci. 2020;12(1):1–10. 32801594 10.4103/jpbs.JPBS_175_19 PMC7398001 Morse DJ Wilson MJ Wei X : Modulation of Candida albicans virulence in in vitro biofilms by oral bacteria. Lett. Appl. Microbiol. 2019;68:337–343. 30825340 10.1111/lam.13145 PMC6849710 Nadeem SG Shafiq A Hakim ST : Effect of growth media, pH, and temperature on yeast to hyphal transition in Candida albicans. Open J. Med. Microbiol. 2013;03:185–192. 10.4236/ojmm.2013.33028 Atriwali T Azeem K Husain MF : Mechanistic understanding of Candida albicans biofilm formation and approaches for its inhibition. Front. Microbiol. 2021;12:638609. 33995297 10.3389/fmicb.2021.638609 PMC8121174 Wulansari S Mintarjo DF : Efek ekstrak etanol biji alpukat ( Persea americana) terhadap biofilm Candida albicans. JKGT. 2023;5(1):239–243. 10.25105/jkgt.v5i1.17178 Hassan Y Chew SY Than LTL : Candida glabrata: Pathogenicity and resistance mechanisms for adaptation and survival. J. Fungi. 2021;7(667):1–18. 34436206 10.3390/jof7080667 PMC8398317 Vale-Silva L Sanglard D : Tipping the balance both ways: Drug resistance and virulence in Candida glabrata. FEMS Yeast Res. 2015;15(fov025):1–8. 25979690 10.1093/femsyr/fov025 Farahyar S Zaini F Kordbacheh P : Expression of efflux pumps and fatty acid activator one genes in azole resistant Candida glabrata isolated from immunoccompromised patients. Acta Med. Iran. 2016;54(7):459–464. 27424018 Yu S Chang Y Chen Y : Deletion of ADA2 increases antifungal drug susceptibility and virulence in Candida glabrata. Antimicrob. Agents Chemother. 2018;62(3):e01924–e01917. 29311082 10.1128/AAC.01924-17 PMC5826168 Wolff A Rodloff AC Vielkind P : Antimicrobial susceptibility of clinical oral isolates of Actinomyces spp. Microorganisms. 2022;10(125):1–11. 35056574 10.3390/microorganisms10010125 PMC8779083 Steininger C Willinger B : Resistance patterns in clinical isolates of pathogenic Actinomyces species. J. Antimicrob. Chemother. 2016;71:422–427. 10.1093/jac/dkv347 Calosa BT Sugiaman VK Pranata N : Comparison of antibacterial activity of both seeds and leaves ethanol extract of avocado ( Persea americana Mill.) against Streptococcus mutans. MDJ. 2023;12(1):38–42. 10.35856/mdj.v12i1.629 Liu S Sun Y Liu Y : Genomic and phenotypic characterization of Streptococcus mutans isolates suggets key gene clusters in regulating its interaction with Streptococcus gordonii. Front. Microbiol. 13:945108. 36033899 10.3389/fmicb.2022.945108 PMC9416482 Campos J Pires MF Sousa M : Unveiling the relevance of the oral cavity as a Staphylococcus aureus colonization site and potential source of antimicrobial resistance. Pathogens. 2023;12(765):1–10. 37375455 10.3390/pathogens12060765 PMC10304336 Montelongo-Jauregui D Lopez-Ribot JL : Candida interactions with the oral bacterial microbiota. J. Fungi. 2018;4(122):1–15. 30400279 10.3390/jof4040122 PMC6308928 Santosa CM Rosyadi I Arifianto D : Kajian kliniko-patologik dan antimikroba ekstrak biji alpukat ( Persea americana Mill.). Jurnal Sain Veteriner. 2019;37(2):160–165. 10.22146/jsv.40445 Morelli VG Oliveira VC Vasconcelos GLL : Effect of effervescent tablets on removable partial denture hygiene. Am. J. Dent. 2023;36(2):75–80. 37076296 O’Brien TJ Figueroa W Welch M : Decreased efficacy of antimicrobial agents in polymicrobial environment. ISME J. 2022;16:1694–1704. 35304578 10.1038/s41396-022-01218-7 PMC9213441 Kart D Tavernier S Acker H : Activity of disinfectants against multispecies biofilms formed by Staphylococcus aureus, Candida albicans, and Psedomonas aeruginosa. Biofouling. 2014;30(3):377–383. 24579656 10.1080/08927014.2013.878333 Anju VT Busi S Imchen M : Polymicrobial infections and biofilms: Clinical significance and eradication strategies. Antibiotics. 2022;11(1731):1–31. 36551388 10.3390/antibiotics11121731 PMC9774821 Olson ML Jayaraman A Kao KC : Relative abundances of Candida albicans and glabrata in in vitro coculture biofilms impact and formation. Appl. Environ. Microbiol. 2018;84(8):e02769–e02717. 29427422 10.1128/AEM.02769-17 PMC5881068 Brunke S Hube B : Two unlike cousins: Candida albicans and C. glabrata infection strategies. Cell. Microbiol. 2013;15(5):701–708. 23253282 10.1111/cmi.12091 PMC3654559 Bernard C Girardot M Imbert C : Candida albicans interaction with gram-positive bacteria within interkingdom biofilms. J. Mycol. Médicale. 2020;30(1):1–8. 31771904 10.1016/j.mycmed.2019.100909 Diaz PI Strausbaugh LD Dongari-Bagtzoglou : Fungal-bacterial interactions and their relevance to oral health: linking the clinic and the bench. Front. Cell. Infect. Microbiol. 2014;4(101):1–6. 25120959 10.3389/fcimb.2014.00101 PMC4114182 Hu Y Niu Y Ye X : Staphylococcus aureus synergized with Candida albicans to increase the pathogenesis and drug resistance in cutaneous abscess and peritonitis murine models. Pathogens. 2021;10(1036):1–17. 34451500 10.3390/pathogens10081036 PMC8398722 Guo Y Wei C Liu C : Inhibitory effects of oral Actinomyces on the proliferation, virulence and biofilm formation of Candida albicans. Arch. Oral Biol. 2015;60:1368–1374. 26143096 10.1016/j.archoralbio.2015.06.015 Uruén C Chopo-Ecuin G Tommassen J : Biofilms as promoters of bacterial antibiotic resistance and tolerance. Antibiotics. 2021;10(3):1–36. 33374551 10.3390/antibiotics10010003 PMC7822488 Batoni G Maisetta G Esin S : Therapeutic potential of antimicrobial peptides in polymicrobial biofilm-associated infections. Int. J. Mol. Sci. 2021;22(482):1–24. 33418930 10.3390/ijms22020482 PMC7825036 Vinha AF Moreira J Barreira SVP : Physicochemical parameters, phytochemical composition and antioxidant activity of algarvian avocado ( Persea americana Mill.). J. Agric. Sci. 2013;5(12):100–109. 10.5539/jas.v5n12p100 Gutiérrez-Venegas G Gόmez-Mora JA Meraz-Rodríguez MA : Effect of flavonoids on antimicrobial activity of microorganisms present in dental plaque. Heliyon. 2019;5:e03013. 10.1016/j.heliyon.2019.e03013 Matilla-Cuenca L Gil C Cuesta S : Antibiofilm activity of flavonoids on staphylococcal biofilms through targeting BAP amyloids. Nat. Res. 2020;10(18968):18912–18968. 33144670 10.1038/s41598-020-75929-2 PMC7641273 Dennis Nurliza C Savitri W : Antibacterial effect of ethanol extract of the avocado seed ( Persea americana Mill.) as an alternative root canal irrigants againts Porphyromonas gingivalis ( in vitro). Int. J. App. Dent. Sci. 2017;3(1):89–93. 2394-7497. Villanueva X Zhen L Ares JN : Effect of chemical modifications of tannins on their antimicrobial and antibiofilm effect against Gram-negative and Gram-positive bacteria. Front. Microbiol. 2023;13(987164):1–15. 36687646 10.3389/fmicb.2022.987164 PMC9853077 Yan Y Li X Zhang C : Research progress on antibacterial activities and mechanisms of natural alkaloids: A review. Antibiotics. 2021;10(318):1–30. 33808601 10.3390/antibiotics10030318 PMC8003525 Brahim MAS Fadli M Markouk M : Synergistic antimicrobial and antioxidant activity of saponins-rich extracts from paronychia argentea and Spergularia marginata. European J. Med. Plants. 2015;7(4):193–204. 10.9734/EJMP/2015/16597 Monte J Abreu AC Borges A : Antimicrobial activity of selected phytochemicals against Escherichia coli and Staphylococcus aureus and their biofilms. Pathogens. 2014;3:473–498. 25437810 10.3390/pathogens3020473 PMC4243457 Tamfu AN Ceylan O Cârâc G : Antibiofilm and anti-quorum sensing potential of cycloartane-type triterpene acids from Cameroonian grassland propolis: Phenolic profile and antioxidant activity of crude extract. Molecules. 2022;27(4872):1–19. 35956824 10.3390/molecules27154872 PMC9369644 Kaypetch R Rudrakanjana P Tuangam P : Effects of two novel denture cleansers on mutispesies microbial biofilms, stain removal and the denture surface: An in vitro study. BMC Oral Health. 2023;23(852):1–12. 37951865 10.1186/s12903-023-03535-5 PMC10640750 Lucena-Ferreira SC Cavalcanti IMG Cury AAB : Efficacy of denture cleansers in reducing microbial counts from removable partial dentures: A short-term clinical evaluation. Braz. Dent. J. 2013;24(4):353–356. 24173255 10.1590/0103-6440201302183 Angela T Halim S : Avocado seed extract on inhibiting mono-species and polymicrobial biofilm.[Dataset]. figshare. 2024. 10.6084/m9.figshare.25996006 10.5256/f1000research.178613.r372991 Reviewer response for version 3 Hegde Veena 1 Referee https://orcid.org/0000-0003-4856-7797 Manipal Academy of Higher Education, Manipal, Karnataka, India

Competing interests: No competing interests were disclosed.

 17 6 2026 Copyright: © 2026 Hegde V 2026 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve

Do not have any comment.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Yes

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

Dental materials related to prosthodontics

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

 10.5256/f1000research.173600.r361435 Reviewer response for version 2 Hegde Veena 1 Referee https://orcid.org/0000-0003-4856-7797 Manipal Academy of Higher Education, Manipal, Karnataka, India

Competing interests: No competing interests were disclosed.

 14 2 2025 Copyright: © 2025 Hegde V 2025 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve

The effect of immersing Heat polymerized acrylic resin denture base in different concentration of avocado seeds extract is done stystematically to determine the minimum biofilm inhibitory concentration.

Difference in minimum biofilm inhibitory concentration of avocado extract against polymicrobial biofilm and  monospecies biofilm is well justified.

Overall, this is a clear, concise, and well-written manuscript.

Strengths of the Study:

Innovative Use of Natural Extracts:

The study explores the antimicrobial properties of avocado seed extract, a novel approach within the field of denture care. This investigation aligns with increasing interest in natural products as alternatives to conventional antimicrobial agents, potentially benefiting oral health practices .

Comprehensive Testing Across Multiple Concentrations:

It systematically examines a variety of concentrations (5%, 10%, 15%, and 20%), providing a clear understanding of the dose-response relationship regarding biofilm inhibition. This thorough assessment is vital for identifying the most effective concentration for practical applications ..

Significant Results with Clinical Relevance:

The study's findings indicate that a 20% concentration of avocado seed extract can inhibit more than 50% of polymicrobial biofilm growth, suggesting a promising natural alternative for denture hygiene. This has important implications for improving oral health among denture wearers .

Implications for Future Research:

The research offers a foundation for further studies into the phytochemical constituents of avocado seed extract, which could lead to the identification of specific antimicrobial compounds. This paves the way for more targeted and effective natural products in dental care.

 Weaknesses of the Study:

Extraction Method Concerns:

The use of 70% ethanol as a solvent in the extraction process may confound the results, as the observed antimicrobial effects might result from the ethanol rather than the avocado seed compounds alone. This raises questions about the specificity of the extract's efficacy .

Limited Representation of Real-World Conditions:

Conducting the study in vitro means the findings may not fully translate to the complexities of the oral cavity in actual denture wearers, where additional factors (such as saliva, diet, and oral microbiome dynamics) play significant roles in biofilm formation .

Understanding the spectrum of microbial inhibition would provide a better assessment of the extract’s antibacterial profile .

Summary

The study presents a promising exploration of avocado seed extract's antimicrobial properties for denture care, showcasing innovative methods and significant findings that could enhance oral health. While there are notable strengths, including its systematic approach and robust statistical analysis, addressing the extraction method, real-world applicability, and microbial profiling would enhance the study's overall impact and relevance. Balancing these strengths and weaknesses will contribute to informed discussions regarding the potential use of natural remedies in dental hygiene practices.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Yes

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

Dental materials related to prosthodontics

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Angela Thalia Department of Dentistry, University of Sumatera Utara, Medan, North Sumatra, Indonesia

Competing interests: We do not have any competing interests.

 23 2 2025

We would like to express our gratitude for the reviewer’s approval of our research and for the mentioned strengths and weaknesses of our study.

 10.5256/f1000research.173600.r359394 Reviewer response for version 2 Yacob Norlela 1 Referee Universiti Sains Islam Malaysia, Nilai, Malaysia

Competing interests: No competing interests were disclosed.

 7 2 2025 Copyright: © 2025 Yacob N 2025 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

Abstract does not contain objective of study. please add.

Result in abstract is confusing:  There was 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).- which one is correct?

Introduction was good.

Methodology was satisfactory.

Result are satisfactory. 

Table 3. asteriks mean what? need to explain further

Result in Table 6. should have post hoc test that shows which concentration does have significant different. 

Figure 2. The image does not self-explanatory. please improve the label and what the findings.

discussion was good. 

Where is the conclusion? it would be good to recommend the clinical application of this avocado solution as denture cleanser.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Partly

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

micorbial adherence, denture microbiome, digtial denture, 3D printing

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Angela Thalia Department of Dentistry, University of Sumatera Utara, Medan, North Sumatra, Indonesia

Competing interests: We do not have any competing interests.

 23 2 2025

First and foremost, we would like to thank the reviewers for the reviews given to improve the content and writing of the articles. We have made the changes accordingly in the new version and we would also like to humbly respond to some of the reviews given.

Abstract does not contain objective of study. please add.

Response: We have added the objective of the study on the last sentence of the abstract background.

Result in abstract is confusing:  There was 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) - which one is correct?

Response: Both are correct. The result from our ANOVA test specifically showed a value of p<0.001. The value in brackets were for further emphasis that the p value is below 0.05, which 0.05 is the most commonly set standard of significant levels. A p-value below 0.05 means there was a significant effect of soaking HPA resin in avocado seed extract on the inhibition of mono-species and polymicrobial biofilms

Introduction was good. Methodology was satisfactory. Result are satisfactory. Discussion was good.

Response: We would like to express our gratitude for the reviewer’s approval of the sections mentioned.

Table 3. asteriks mean what? need to explain further

Response: We have added the meaning of the asterisk right below the table

Result in Table 6. should have post hoc test that shows which concentration does have significant different

Response: We have done the post hoc test, however we could not insert the whole data in the journal as they were too many data to be included. Despite that, we have added a brief explanation of the post hoc test results in the result section, and we have inserted the full complete data which can be accessed from the link in the data availability section.

Figure 2. The image does not self-explanatory. please improve the label and what the findings.

Response: We have further explained the image (Figure 2)

Where is the conclusion? it would be good to recommend the clinical application of this avocado solution as denture cleanser.

Response: We have added the conclusion Section.

 10.5256/f1000research.173600.r336000 Reviewer response for version 2 Mohammed Nada Z. 1 Referee https://orcid.org/0000-0002-2107-7010 Hamdoon Suhad M. 2 Co-referee Department of Prosthodontics, College of Dentistry, University of Mosul (Ringgold ID: 108489), Mosul, Nineveh Governorate, Iraq Department of Basic Dental Sciences, College of Dentistry, University of Mosul (Ringgold ID: 108489), Mosul, Nineveh, Iraq

Competing interests: No competing interests were disclosed.

 21 11 2024 Copyright: © 2024 Mohammed NZ and Hamdoon SM 2024 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve

The authors do the requested corrections.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Partly

Are all the source data underlying the results available to ensure full reproducibility?

Partly

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

Prosthetic dentistry, dental materials, Microbiology

We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Angela Thalia Department of Dentistry, University of Sumatera Utara, Medan, North Sumatra, Indonesia

Competing interests: We do not have any competing interests.

 23 2 2025

We would like to express our gratitude for the reviewer’s approval of our research and for the previous review that has been given which has helped us improve our research paper.

 10.5256/f1000research.167602.r320505 Reviewer response for version 1 Newaskar Prabha S 1 Referee https://orcid.org/0000-0002-5893-3458 Department of Prosthodontics, Pravara Institute of Medical Sciences, Ahmednagar, Maharashtra, India

Competing interests: No competing interests were disclosed.

 29 10 2024 Copyright: © 2024 Newaskar PS 2024 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

The current in-vitro study is based on denture plaque microorganism inhibition by avocado seed extract on heat-cured denture base resin. The author considered the 5%, 10%, 15% & 20% concentrations of the same extract and two groups of controls. for the study. and concluded that a 20% concentration was more effective than other concentrations. The following are suggestions for improvement of the current manuscript:

1. Abstract: Ensure that the abstract concisely summarizes the key findings. The methodology, key results, and conclusion can be streamlined further to highlight the core contributions without too much technical detail.

2. Methodology: Include any steps taken to mitigate bias or ensure reproducibility. For instance, if there were any controls (such as positive and negative controls), clearly define how they were handled to ensure the integrity of the experiment. Specify more details about the process of ensuring the sterility of HPA resin disks or provide a brief explanation of the significance of the preparation technique.

3. Result: Add more visual aids (tables, charts, or graphs) to clarify results. This will make it easier for readers to interpret the data. provide context to the statistical findings. Instead of just reporting p-values, discuss the biological or practical significance of these results.

4. Discussion: Ensure that each point is directly tied back to the research question. Avoid over-explaining background information already provided in the introduction. Provide a more detailed comparison between your findings and previous studies. Highlight areas where your results diverge or align with other research to emphasize the novelty or contribution of your work.

5. Conclusion: You may consider discussing the potential limitations of your study (e.g., the in vitro setting) and suggest avenues for future research.

6. Reference & citations: Double-check all citations to ensure they adhere to the journal's guidelines (e.g., proper format, all sources accounted for). Ensure recent and relevant literature is cited, particularly on the antimicrobial activity of avocado seed extracts.

7. Ethical consideration: If the study involves the handling of microorganisms, briefly outline the safety measures taken to ensure proper disposal and handling, in compliance with biosafety standards.

8. Language & grammar: Revise for grammatical consistency. For instance, avoid shifting tenses within the same section. Avoid redundancy. For example, once the MBIC50 is defined, you do not need to explain it repeatedly in different sections.

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

Yes

Are all the source data underlying the results available to ensure full reproducibility?

Partly

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

Prosthodontics, Dental materials, Implants, Maxillofacial prosthodontics, Systematic reviews, Fixed prosthodontics, Removable prosthodontics.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Angela Thalia Department of Dentistry, University of Sumatera Utara, Medan, North Sumatra, Indonesia

Competing interests: We do not have any competing interests.

 23 2 2025

First and foremost, we would like to thank the reviewers for the reviews given to improve the content and writing of the articles. We would like to apologize for the long update of the article new version and we are very grateful for your patience. We have made the changes accordingly in the new version and we would also like to humbly respond to some of the reviews given.

Abstract: Ensure that the abstract concisely summarizes the key findings. The methodology, key results, and conclusion can be streamlined further to highlight the core contributions without too much technical detail.

R: We have tried to streamline the conclusion section of the abstract. However, for the methodology and results, we couldn’t do much especially with the previous version review that suggest us to add the name of the test and the statistical tests done on this study. We hope the small changes in the abstract could be sufficient in this revised manuscript.

Methodology: Include any steps taken to mitigate bias or ensure reproducibility. For instance, if there were any controls (such as positive and negative controls), clearly define how they were handled to ensure the integrity of the experiment. Specify more details about the process of ensuring the sterility of HPA resin disks or provide a brief explanation of the significance of the preparation technique.

R: We have added additional explanation on the process of HPA resin disks sterility and the handling of the controls.

Result: Add more visual aids (tables, charts, or graphs) to clarify results. This will make it easier for readers to interpret the data. provide context to the statistical findings. Instead of just reporting p-values, discuss the biological or practical significance of these results.

R: We couldn’t add more visual aids as we have already included the tables in the result section, in which each table is marked with asterisk to signify certain attention to data. We have also added more data interpretation according to our statistical findings.

Discussion: Ensure that each point is directly tied back to the research question. Avoid over-explaining background information already provided in the introduction. Provide a more detailed comparison between your findings and previous studies. Highlight areas where your results diverge or align with other research to emphasize the novelty or contribution of your work.

R: We have ensured our point of discussion has referred back to the research purpose and deleted any over-explaining background information.

Conclusion: You may consider discussing the potential limitations of your study (e.g., the in vitro setting) and suggest avenues for future research.

R: We have added conclusion section and a separate section for the study limitation in the revised manuscript.

Reference & citations: Double-check all citations to ensure they adhere to the journal's guidelines (e.g., proper format, all sources accounted for). Ensure recent and relevant literature is cited, particularly on the antimicrobial activity of avocado seed extracts.

R: We have double-checked our reference and citations.

Ethical consideration: If the study involves the handling of microorganisms, briefly outline the safety measures taken to ensure proper disposal and handling, in compliance with biosafety standards.

R: We have added the brief outline of microorganism proper disposal and handling.

Language & grammar: Revise for grammatical consistency. For instance, avoid shifting tenses within the same section. Avoid redundancy. For example, once the MBIC50 is defined, you do not need to explain it repeatedly in different sections.

R: We have revised our grammatical errors and avoided redundancy.

10.5256/f1000research.167602.r320506 Reviewer response for version 1 Abd-Ellah Mervat E. 1 2 Referee Department of Prosthodontics, Faculty of Dentistry, Alexandria University, Alexandria, Alexandria Governorate, Egypt Prothodontics, Alexandria University Faculty of Dentistry, Alexandria, Alexandria Governorate, Egypt

Competing interests: No competing interests were disclosed.

 4 10 2024 Copyright: © 2024 Abd-Ellah ME 2024 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation reject

The work being reviewed is entitled “ The effect of soaking heat-polymerized acrylic resin denture base in avocado seed extract on the inhibition of denture-plaque microorganisms biofilm growth”  

This study aimed to evaluate the effect of avocado seeds on denture-plaque microorganism biofilm on Heat polymerized acrylic (HPA) resins. It was concluded that 20% avocado seed extract was effective in inhibiting polymicrobial biofilm because it was able to inhibit more than 50% polymicrobial biofilm. 

There are certain limitations for approving this study;

- First, it was mentioned that “The avocado seed powder was put into a vessel and poured with 70% ethanol solvent” then may be the antimicrobial effect from the ethanol not the avocado seed powder and that makes the idea of the study not sound.

- Second, the work partly cite the current literature as it does not justify the use of the used concentrations of  avocado seed extract clearly. It was mentioned that “Anggraini et al. (2017 [Ref - 1]) 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 [Ref - 2]) 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 ” It was already stated that 10% is the most effective concentration, then it was useless to test the same concentration that was used before. This is an unnecessary repetition. Moreover, it was concluded in the current study that 20% avocado seed extract was effective in inhibiting polymicrobial biofilm but it was not justified way the difference in percentage of avocado seed extract from the literature that stated that 10 % was the most effective concentration in inhibiting  C. albicans

- Third, it was mentioned that “Sterile HPA resin discs were preconditioned for 24 hours by immersion in artificial saliva.” You did not say how the process of sterilization happened. Please clarify.

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.

Are all the source data underlying the results available to ensure full reproducibility?

No source data required

Is the study design appropriate and is the work technically sound?

No

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

prosthodontics

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

References : EFEKTIVITAS KOMBINASI EKSTRAK DAUN SIRIH MERAH (Piper Crocatum) DAN EKSTRAK BIJI ALPUKAT (Persea americana) DALAM MENGHAMBAT PERTUMBUHAN Candida albicans. Jurnal Kimia Riset .2017;2(2) : 10.20473/jkr.v2i2.6196 10.20473/jkr.v2i2.6196 : Efektivitas antibakteri ekstrak biji alpukat (Persea americana Mill.) terhadap Streptococcus mutans (Antibacterial effectiveness of avocado seed (Persea americana Mill.) extract on Streptococcus mutans). Makassar Dental Journal .2018;7(1) : 10.35856/mdj.v7i1.12 10.35856/mdj.v7i1.12 Angela Thalia Department of Dentistry, University of Sumatera Utara, Medan, North Sumatra, Indonesia

Competing interests: No competing interests were disclosed.

 18 10 2024

First and foremost, we would like to thank the reviewers for the reviews given to improve the content and writing of the articles. We have made the changes accordingly in the new version and we would also like to humbly respond to some of the reviews given.

Reviewer comments and Angela et al. responses (R.)

First, it was mentioned that “The avocado seed powder was put into a vessel and poured with 70% ethanol solvent” then may be the antimicrobial effect from the ethanol not the avocado seed powder and that makes the idea of the study not sound.

R: We would like to explain the use of 70% ethanol solvent when it was poured inside the vessel containing the avocado seed powder. According to Abubakar et al. (2020), solvent is also known as menstruum which is defined as a liquid chosen for an effective extraction process. 1 The solvent used in this study is ethanol which is a polar solvent that is used to extract polar compounds such as flavonoids and polyphenols. The reason we are using 70% ethanol (water-ethanol) instead of water only is that according to Plaskova et al. (2023), all the bioactive molecules from the plant material cannot be extracted using only single solvent, instead binary solvent mixture such as water and organic solvent are recommended. The study claimed that water-ethanol solvent shows a much better extraction of polyphenols than any single solvent. 2 Another study that supported this claim is a study by Munthe et al. (2023) which concluded that 70% ethanolic extract of avocado seeds using maceration technique showed a strong antioxidant activity. 3

In our study, at the end of extraction process, the filtered liquid avocado seed extract underwent evaporation on a rotary evaporator which removed the solvents used for extracting the avocado seeds, leaving behind a very thick extract. Plaskova et al. (2023) did mentioned that some residual solvents might remain unintentionally, however the level of the remaining solvent is limited. The study has also presented a table showcasing the maximum limitation of the solvent which can be further read in the published paper. 2 The thick extract that our study has gained are then diluted using aquadest, hence the negative control of our study is also aquadest because it can be said that through the evaporation process, the ethanol solvent has little to no effect in contributing to the antimicrobial effect.

References

Abubakar AR, Haque M. Preparation of medicinal plants: basic extraction and fractionation procedures for experimental purposes. J Pharm Bioallied Sci. 2020;12(1):1-10. doi: 10.4103/jpbs.JPBS_175_19

Plaskova A, Mlcek. New insights of the application of water or ethanol-water plant extract rich in active compounds in food. Front Nutr. 2023;10:1118761. doi: 10.3389/fnut.2023.1118761

Munthe SWN, Riskianto R, Juvi D, Novia J. Antioxidant, total phenolic, and total flavonoid of 70% ethanol extract of avocado seeds ( Persea americana Mill.). Pharmacogn J. 2023;15(4):599-605. doi: 10.5530/pj.2023.15.126

Second, the work partly cite the current literature as it does not justify the use of the used concentrations of avocado seed extract clearly. It was mentioned that “Anggraini et al. (2017 [Ref - 1]) 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 [Ref - 2]) 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” It was already stated that 10% is the most effective concentration, then it was useless to test the same concentration that was used before. This is an unnecessary repetition. Moreover, it was concluded in the current study that 20% avocado seed extract was effective in inhibiting polymicrobial biofilm but it was not justified way the difference in percentage of avocado seed extract from the literature that stated that 10% was the most effective concentration in inhibiting C. albicans.

R: We would like to explain the reason why we included those previous studies in our Introduction section and why we did our research based on the concentrations that the previous studies have concluded. Denture plaque that is formed in the denture wearers appliances is not a mono-species biofilm but a polymicrobial one.  O’Donnell et al. (2015) declared that denture plaque consists of Candida spp. which co-aggregates with bacteria. 1 The previous studies we included in our paper are only done on planktonic bacteria and fungal, not polymicrobial biofilm. Meanwhile a study by Hamzah et al. (2019) has found that a much higher concentration is needed to inhibit polymicrobial biofilm than mono-species biofilms. Not to mention, the previous studies we included our papers are done on C. albicans and S. mutans while our paper actually studies the effect of avocado seed on C. albicans, C. glabrata, A. odontolyticus, S. gordonii, S. aureus, both mono-species and polymicrobial biofilms . Hence, our study still used the previous established effective concentration (10%) with varieties that centered on the concentration from previous studies (5%, 15%, 20%). 2 We also still studied the effect of avocado seed on the mono-species biofilms to not only know which concentration can be determined as the MBIC 50 on each mono-species biofilms, but also to see whether there is truly a difference between the MBIC 50 of each mono-species biofilm and the polymicrobial biofilm. The result of our study, which summary can be seen on our Abstract section, has shown that MBIC 50 for each mono-species are different from the previous studies’ concentration. This shows that our study did not do any unnecessary repetition.

On a side note, we have added further explanation on the research gap in our introduction section. We hope it could add more clearance on the matter. We truly appreciate the feedback given by the reviewer.

References

O’Donnell LE, Robertson D, Nile CJ, et al.: The oral microbiome of denture wearers is influenced by levels of natural dentition. PLoS One. 2015;10(9):e0137717. 26368937 10.1371/journal.pone.0137717 PMC4569385

Hamzah H, Hertiani T, Pratiwi SUT, et al.: The inhibition activity of tannin on the formation of mono-spesies and polymicrobial biofilm Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans. Trad. Med. J. 2019;24(2):110–118. 10.35856/mdj.v7i1.12

Third, it was mentioned that “Sterile HPA resin discs were preconditioned for 24 hours by immersion in artificial saliva.” You did not say how the process of sterilization happened. Please clarify.

R: We have clarified the sterilization process of the HPA resin discs in the Methods section.

10.5256/f1000research.167602.r320510 Reviewer response for version 1 Mohammed Nada Z. 1 Referee https://orcid.org/0000-0002-2107-7010 Hamdoon Suhad M. 2 Co-referee Department of Prosthodontics, College of Dentistry, University of Mosul (Ringgold ID: 108489), Mosul, Nineveh Governorate, Iraq Department of Basic Dental Sciences, College of Dentistry, University of Mosul (Ringgold ID: 108489), Mosul, Nineveh, Iraq

Competing interests: No competing interests were disclosed.

 3 10 2024 Copyright: © 2024 Mohammed NZ and Hamdoon SM 2024 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

Abstract

- The Methods are concise, it needs to be more informative, add the name of tests used.

-You should add a concise information about the statistical analysis that used in the study.

Introduction

-The introduction is informative but lacks a clear statement of the research gap and aim of the research.

Methods

-The methodology section is detailed but could benefit from sequential numbering of the subheadings for better organization

- There are many paragraphs without reference ; you should add the reference.

- In Avocado seed extraction procedure ; Chromatography also should be carried out to identified the extract composition.

- Mention the name of  medium used to culture Actinomyces odontolyticus on fastidious anaerobe agar.

- In Biofilm formation on heat polymerized acrylic resin disc ; How much the amount of polymicrobial biofilm was added.

- Inhibition Value%=[(ControlOD_SampleOD)÷-ControlOD]×100; clarify if the subtracted control was the negative or positive ?

-Statistical analysis required more clarification  as mentioned by adding table for the inhibition value for each extract treatment group obtained by the inhibition formula to be compared directly  

Result

-The results are presented clearly; but it could be improved; the results display need to be more consistent  without excessively repeating the data. Display the main finding and explain the reason leads to this result

-Please insert table contain the inhibition value obtained by the inhibition formula

Discussion

-The authors should enrich the discussion by the analysis and interpreting the obtained results and by comparing the obtained results with what reported in other papers.

-The limitations of the studies should be presented.

-The clinical relevance should be further highlighted.

Tables

-The MBIC50 of avocado seed extract and its effect on polymicrobial biofilm; the 50.81_8.32%. value must be placed in table.

References

There are too many references. Remove the outdated and unnecessary references .

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Partly

Are all the source data underlying the results available to ensure full reproducibility?

Partly

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

Prosthetic dentistry, dental materials, Microbiology

We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above.

Angela Thalia Department of Dentistry, University of Sumatera Utara, Medan, North Sumatra, Indonesia

Competing interests: No competing interests were disclosed.

 18 10 2024

First and foremost, we would like to thank the reviewers for providing necessary reviews to improve the content and writing of the articles. We have made changes in the new version of the article according to the reviews given.

Reviewer comments and Angela et al. responses (R.)

Abstract

1. The Methods are concise, it needs to be more informative, add the name of tests used.

2. You should add a concise information about the statistical analysis that used in the study

R: We have included the name of the test (microtiter biofilm plate assay) and the statistical analysis used in the study as concise as possible.

Introduction

The introduction is informative but lacks a clear statement of the research gap and aim of the research.

R: We have given a much clearer statements on the research gap and the research aim which can be seen on the last paragraph of the introduction section.

 Methods

1. The methodology section is detailed but could benefit from sequential numbering of the subheadings for better organization

R: We have added sequential numbering on the methods section.

2. There are many paragraphs without reference; you should add the reference.

R: We have added necessary references on the methods section.

3. In Avocado seed extraction procedure; Chromatography also should be carried out to identified the extract composition.

R: We do agree that the extract can use Chromatography to further identify the extract composition. In our research location, the only available Chromatography is Gas Chromatography Mass Spectrometry (GC-MS). However, we decided not to do the test during our research period as we only wanted to know the presence of flavonoids, polyphenols, alkaloids, and saponin in our avocado seed extract as many research have shown their antimicrobial properties in order to make sure that we fulfilled the purpose of our study which is to know the effect of avocado seed extract on denture plaque microorganisms grown on HPA resin. The two tests we have done, phytochemical examination and its quantitative tests, have shown the existence of those compound and specified their amount in the extract. Hence, we directly continue our research without doing Chromatography.

4. Mention the name of medium used to culture Actinomyces odontolyticus on fastidious anaerobe agar.

R: We have previously written the name of the medium used to culture A. odontolyticus  which is fastidious anaerobe agar which we supplemented with 5% (v/v) defibrinated bovine blood.

5. In Biofilm formation on heat polymerized acrylic resin disc; How much the amount of polymicrobial biofilm was added.

R: We have specified the amount of polymicrobial biofilm added which can be seen on the second subheading “Biofilm formation on heat polymerized acrylic resin disc”

6. Inhibition Value%=[(ControlOD_SampleOD)÷-ControlOD]×100; clarify if the subtracted control was the negative or positive?

R: We confirmed that the ControlOD for the inhibition value formula is positive, not negative.

7. Statistical analysis required more clarification as mentioned by adding table for the inhibition value for each extract treatment group obtained by the inhibition formula to be compared directly

R:  We have added the inhibition value table on the Result section.

Result

1. The results are presented clearly; but it could be improved; the results display need to be more consistent without excessively repeating the data. Display the main finding and explain the reason leads to this result

R: We have removed the repetitive sentences.

2. Please insert table contain the inhibition value obtained by the inhibition formula

R: We have replaced the figures of inhibition value into tables (Table 3 dan Table 5).

Discussion

1. The authors should enrich the discussion by the analysis and interpreting the obtained results and by comparing the obtained results with what reported in other papers.

R: We would like to apologize for our paper shortcoming. We have previously included as much other paper results as possible, however there is still limited data that can be used as comparison, especially for the microorganisms that we studied in this paper, such as C. glabrata, A. odontolyticus, S. gordonii.

2. The limitations of the studies should be presented.

R: We have presented the limitations of the study and made a separate section for it.

3. The clinical relevance should be further highlighted.

R: We have highlighted the clinical relevance which is written on the last sentence in the discussion section, above the study limitation.

Tables

The MBIC50 of avocado seed extract and its effect on polymicrobial biofilm; the 50.81_8.32%. value must be placed in table.

R: We have added it to Table 5 in the results section.

References

There are too many references. Remove the outdated and unnecessary references

R: We have removed the outdated and unnecessary references.