Potential of microbial-derived biosurfactants for oral applications–a systematic review

Background Biosurfactants are amphiphilic compounds produced by various microorganisms. Current research evaluates diverse types of biosurfactants against a range of oral pathogens. Objectives This systematic review aims to explore the potential of microbial-derived biosurfactants for oral applications. Methodology A systematic literature search was performed utilizing PubMed-MEDLINE, Scopus, and Web of Science databases with designated keywords. The results were registered in the PROSPERO database and conducted following the PRISMA checklist. Criteria for eligibility, guided by the PICOS framework, were established for both inclusion and exclusion criteria. The QUIN tool was used to assess the bias risk for in vitro dentistry studies. Results Among the initial 357 findings, ten studies were selected for further analysis. The outcomes of this systematic review reveal that both crude and purified forms of biosurfactants exhibit antimicrobial and antibiofilm properties against various oral pathogens. Noteworthy applications of biosurfactants in oral products include mouthwash, toothpaste, and implant coating. Conclusion Biosurfactants have garnered considerable interest and demonstrated their potential for application in oral health. This is attributed to their surface-active properties, antiadhesive activity, biodegradability, and antimicrobial effectiveness against a variety of oral microorganisms, including bacteria and fungi.


Introduction
Biosurfactants have recently attracted attention in biomedical research [1].The demand for innovative solutions that prioritize eco-friendly and biobased polymeric surfactants is steadily rising.This growing concern is driven by the need for biodegradability and sustainability, which has prompted the development of technologies utilizing microbial sources [2,3].Biosurfactants or microbial-derived surfactants are surfactants that are produced by various microorganisms [4].It has amphiphilic properties characterized by a hydrophilic head region (either polar or non-polar) and a hydrophobic tail region (such as lipid or fatty acid) [5,6].Biosurfactants have numerous advantages over chemical surfactants, including being less toxic, having a higher biodegradability, being environmentally friendly, having a higher foaming capability, being highly selective, and having specific activity at extreme pH, temperature, and salinity [7,8].They also bear the designations "eco-friendly", "sustainable", "bio-based", or "green" materials [9].
Biosurfactants are classified into two classes based on their molecular weight: low molecular weight (LMW) and high molecular weight (HMW) [10].Glycolipids and lipopeptides are examples of low molecular weight biosurfactants, such as rhamnolipids and surfactin, while phospholipids, lipoprotein, and emulsan are examples of high molecular weight biosurfactants [11,12].However, the market for commercially available biosurfactants is quite limited, with only a few options, such as surfactin, sophorolipids, and rhamnolipids [7].
Biosurfactants have been recognized as having a wide range of potential applications in various industries, including agriculture, food, cosmetics, pharmaceuticals, and petroleum [13][14][15].Numerous studies have been conducted on biosurfactants and their prospective applications in environmental and biomedical fields as antimicrobials, antiadhesive/antibiofilm agents, antivirals, immune modulators, anticancer, wound-healing promoting agents, and drug delivery agents [16][17][18].Biosurfactants also have the potential to be used in oral and dental infections [13].The essential role of biosurfactant properties, including their ability to inhibit microorganisms and modify surface energy, has been well-established in controlling the formation and proliferation of biofilm [19][20][21].
The oral cavity comprises a diverse array of bacteria and fungi, commonly referred to as oral flora, which contribute to forming a complex oral ecosystem [22,23].
They also contribute to the formation of an oral biofilm.Biofilm infections cause the majority of oral and dental pathogenic infections [24].Biofilms are organized aggregates of microorganisms living in an extracellular polymeric matrix microbially produced and irreversibly attached to non-living or living surfaces [25].Biofilm formation occurs in several common steps: initial contact/attachment to a surface, followed by micro colonization, maturation and formation of biofilm structures, and finally, biofilm detachment/dispersion [14,15].One of the most investigated biosurfactants is rhamnolipids.Abdollahi et al. reported that Rhamnolipids can reduce the adhesion of Streptococcus mutans on polystyrene surfaces and disrupt its preformed biofilm [26].
Similarly, Elshikh et al. also found that rhamnolipids from non-pathogenic Burkholderia thailandensis E264 revealed potent abilities to eradicate mature biofilm of some oral pathogens (Streptococcus oralis, Actinomyces naeslundii, Neisseria mucosa, and Streptococcus sanguinis) [27].The complexity and diversity of this mature biofilm consist of numerous microenvironments [28] and can resistant to antimicrobial agents than planktonic cells [29].This investigation has indicated that the utilization of biosurfactants for oral health applications is still in its initial phases.However, the available literature in this domain holds promise and is continually advancing.Hence, this systematic review aims to explore the potential of microbial-derived biosurfactants for oral applications.

Search strategy
This systematic review followed the guidelines outlined in the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) statement [30].Two independent reviewers (K.Z and T.N) conducted a comprehensive search in three electronic databases: Scopus, Web of Science, and PubMed MEDLINE, utilizing the keywords described in Table 1.The titles and abstracts of the studies identified during the search were independently reviewed by both researchers (K.Z and T.N), and any discrepancies were resolved through discussion.Subsequently, the studies that met the inclusion and exclusion criteria were thoroughly examined.The search process included specific limitations on language, study design, and publication year.The complete search strategy employed in the Scopus, Web of Science, and PubMed MEDLINE databases can be found in Fig. 1.

Study selection
A total of 357 articles were retrieved from the search conducted in three electronic databases using the specified keywords.Two independent reviewers (K.Z and T.N) conducted the selection process, reviewing the complete Table 1 Keywords used in searching for the appropriate article Keywords ("biosurfactant" OR "microbial surfactant") AND ("oral pathogen" OR "oral bacteria" OR "oral disease" OR "oral application") AND ("in vitro" OR "experimental study" OR "quasi-experimental study") list of articles and identifying potentially relevant papers based on title and abstract screening.Subsequently, the full texts of these selected articles were thoroughly examined to determine their eligibility based on the inclusion and exclusion criteria.Only articles published in English within the past 10 years and in journals categorized as Q1 and Q2 were included in the analysis as shown in Table 2. Papers in Q3 and Q4 are omitted due to the suboptimal clarity and quality of the images presented in the journal.This could potentially challenge the process of analysis.In disagreements, the reviewers engaged in discussions until a consensus was reached.

Eligibility criteria
This systematic review has been registered with the National Institute for Health Research PROSPERO, International Prospective Register of Systematic Reviews, under the registration number CRD42023426727.The eligibility criteria for each type of study were determined based on specific characteristics, including the use of PICOS (Problem/Population, Intervention, Comparison, Objective, Study design), as outlined in Table 2.
The risk of bias will be evaluated using the Quality Assessment Tool for In Vitro Studies (QUIN tool) to  assess the quality of the included studies.The QUIN tool is a standardized approach that enables researchers to assess the risk of bias in individual in vitro studies, ensuring consistency in evaluating the risk of bias in in vitro studies included in systematic reviews and meta-analyses.This tool has been evaluated for content validity and consists of 12 criteria.Each of these criteria was assigned a score as follows: adequately specified = 2 points, inadequately specified = 1 point, not specified = 0 points, and not applicable (N/A) = criteria excluded from the calculation.The scores for these 12 criteria were then summed to derive a total score for a specific in vitro study.These cumulative scores were subsequently employed to categorize the in vitro study into one of three risk levels: high (< 50%), medium (50-70%), or low risk (> 70%).This categorization was determined using the formula: Final score = (Total score x 100) / (2 x number of criteria applicable) [31].

Data extraction
The

Qualitative study
The searches conducted in Scopus, Web of Science, and PubMed MEDLINE using the specified keywords yielded 246, 44, and 67 results, respectively.In total, 357 articles were collected and organized using a reference manager (EndNote).After manually removing 18 duplicate articles, 339 selected articles remained.Out of these, 274 articles were excluded as they focused on different compounds or chemical surfactants, leaving 65 articles for further consideration.From the remaining 65 articles, 55 were subsequently excluded, resulting in a final selection of 10 articles that met the inclusion criteria, as shown in Table 3.These selected articles were published between 2016 and 2020 [27,[32][33][34][35][36][37][38][39][40].Detailed reasons for exclusion can be found in Fig. 1.

Study characteristics
The articles in this systematic review focus on in vitro studies investigating the efficacy of various biosurfactants.The biosurfactants analyzed in this review are predominantly rhamnolipids [27,35,38,39], followed by surfactin [33,37,40], lipopeptide [32], and sophorolipid  [35], which are comparatively less explored in the selected studies.Other studies in this systematic review did not specify the specific type of biosurfactant utilized in their research [34,36], as shown in Table 4.

Outcome measures
In this systematic review, all included studies employ diverse methodologies to examine the effects of various biosurfactants on different oral microorganisms.These evaluations encompass an array of characteristics associated with each biosurfactant, including surface tension, Critical Micelle Concentration (CMC), and physicochemical characterization.Additionally, antimicrobial activity, antibiofilm activity, antioxidant activity, and other factors are assessed.Some studies also utilize imaging techniques to provide visual clarity to their findings.Distilled water containing 5% sugarcane molasses, 5% waste frying oil, and 3% corn steep liquor Janek T. et al. [37] Bacillus subtilis #309 10 g l -1 of sucrose (POCH, Gliwice, Poland), 10 g l -1 of NaCl (POCH), 2 g l -1 of NH 4 NO 3 (Chempur, Poland), 5 g l -1 of Na 2 HPO 4 (POCH), 2 g l -1 of KH 2 PO 4 (POCH), and 0. The outcomes of these studies suggest that the identified biosurfactants hold promise as potential ingredients in various oral-related applications, such as toothpaste [32] and mouthwash [36].The comprehensive overview of the methods employed and outcomes obtained in the studies included in this systematic review is presented in Table 5.

Bibliometric analyses
A sum of 357 research articles and reviews were encompassed in the study.Figure 2 illustrates the distribution and the total of citations of these publications over ten years.The peak year for publications was 2021, with 64 articles published, while the year with the fewest publications was 2015, with only 12 articles published.Citation counts fluctuated between 707 citations in 2013 and only 6 in 2023 since the publication in 2023 is still in progress.The year with the most substantial citations was 2017, with a remarkable 1360 citations.
A total of 37 keywords were extracted from the ten selected articles as seen in Fig. 3.The three most frequently used keywords were "biosurfactants, " "Candida albicans, " and "biofilms, " with respective total link strengths of 27, 19, and 16.Cluster analysis was performed on the co-occurrence of keywords from the ten selected articles, resulting in seven clusters.Cluster 1, comprising 8 keywords, included terms such as "antibiofilm coating, " "cytotoxicity, " "fungal-bacterial biofilm, " "mixed biofilm, " and more.Cluster 2 mainly focused on terms related to "Candida albicans, " "hypha-specific genes, " "morphogenesis, " "Streptococcus mutans, " and others.Cluster 3 primarily concentrated on "biofilm inhibition, " "minimum inhibitory concentration, " "oral bacteria, " and other related terms.Cluster 4 consists of 4 terms such as "antimicrobial activity, " "biosurfactants, " and more.Clusters 5, 6, and 7 consist of 4, 4, and 3 terms respectively.Analyzing the keyword co-occurrences, it becomes evident that numerous in vitro experiments involving biosurfactants focused on evaluating their effectiveness against Candida albicans for inhibiting biofilm formation.Notably, surfactin emerged as the predominant type of biosurfactant utilized in these studies.
A total of 243 journals contributed to the collection of enrolled publications.Table 6 presents the top 10 journals that extensively covered the subject of "potential biosurfactant for oral application." The Journal of Applied Microbiology emerged as the leading article regarding productivity, having produced the highest number of publications on this topic.Furthermore, it was also identified as the most influential journal, with the highest number of citations per paper to the subject matter.

Risk of bias and quality assessment
Two independent reviewers (K.Z and T.N) assessed the risk of bias in this study using the Quality Assessment Tool For In Vitro Studies (QUIN) Tool [31].The risk of bias in each study can be found in Table 7.
Five articles were classified as having a low risk of bias [32,34,35,38,39], while another five were categorized as having a medium risk [27,33,36,37,40].Consequently, all the articles included in this review met or exceeded 50% of the assessed criteria.
A recent review focusing on the utilization of biosurfactants in oral hygiene applications noted that most of the examined biosurfactants for oral-related purposes belong to the lipopeptides or lipoproteins category.This investigation has revealed that the use of biosurfactants in oral health is in its nascent stage.Nevertheless, the published research in this field is promising and shows ongoing development [41].
The oral cavity continuously hosts oral microflora, which play a vital role in maintaining oral health.Disruptions in this equilibrium, whether caused by host factors or external influences, can create binding sites that opportunistic oral pathogens exploit, allowing them to dominate the oral cavity [66,67].Biosurfactants also play a role in quorum sensing and serve as antimicrobial agents involved in microbial competition [68,69].It is also crucial to uphold oral hygiene by consistently employing oral care products, such as toothpaste and mouthwash.These habits can effectively manage plaque development and suppress the proliferation of bacteria linked to dental diseases [70,71].
One of the properties that need to be included in the toothpaste formula is good foaming ability since it allows the dentifrice to distribute evenly throughout the mouth during brushing and make thorough contact with tooth surfaces [72,73].This is typically accomplished by using a surface-active agent [74].Incorporating biosurfactants into a toothpaste formulation can substantially diminish the need for chemical surfactants.Formulas containing 2. -Formula BIO-1: emulsifier with biosurfactant 0.5 g; sodium alginate 1 g; calcium carbonate 4 g; sodium chloride 1.5 g; sodium fluoride 0.5 g; glycerin 4 g -Formula BIO-2: emulsifier with biosurfactant 0.5 g; calcium carbonate 1.5 g; sodium chloride 1.5 g; sodium fluoride 0.5 g; glycerin 4 g 3. The desiccation loss of the biosurfactant-formulated toothpaste was between 22 and 30%.Biosurfactant-based toothpaste presented lower foaming ability (33%).The spreading ability test was 20 mm for BIO-1, indicating a low value equal to 16.5 mm.The water activity of BIO-1 and BIO-2 ranged from 0.22 to 0.28.4. The formula BIO-1 and the commercial toothpaste had the same ability to clean stains.Formula 2 (BIO-2) showed a change in the color of eggs from yellow to brown.Due to the heterogeneity of formula 1, its high pH value, low spreading ability, and low cleaning efficiency, formula 1 was used for further experiments.5. BIO-1 was very effective against the tested microorganisms except E. coli.The inhibition diameter was observed against Enterobacter sp (22 mm) and Salmonella typhinirium (20 mm), and Listeria monocytogenes (12.67 mm).BIO-1 was more effective than commercial toothpaste and SDS in inhibiting Listeria monocytogenes, Klebsiella pneumoniae, and Salmonella typhinirium.6.The spreading power of all formulas did not change during storage.The foaming ability of BIO-1 was not stable.There was a decrease in the pH value of all formulas, except the biosurfactantbased toothpaste, and an increase in the water activity value of all formulated toothpaste.growth inhibition for all microorganisms tested except for S. mutans (around 60%).Using 0.2 mg/ml of LSL caused 90% biofilm inhibition against all the organisms used.9. Pre-coating experiment using 0.2 mg/ml LSL prevented more than 80% biofilm formation of all microorganisms investigated, except S. mutans, which required a higher concentration of 0.4 mg/ ml to achieve 40% growth inhibition.10.Biofilm disruption assessment demonstrated excellent potency of rhamnolipids and LSLs to restrict developing biosurfactants.11.Increasing concentrations of rhamnolipids mixture (0.5-5-50) mg/ml and LSL (0.25-2.5-25) mg/ml directly related to increasing dye concentrations in the bacterial cells.1.The crude mixture of biosurfactants was characterized by preparative reversed-phase high-performance liquid chromatography (RP-HPLC).
3. SF and its metal(II) complexes inhibited biofilm formation in a dose-dependent manner when these compounds were added to C. albicans cells after a short initial adhesion period.SF at 1 mM (960 mg/ml) caused 85% inhibition.This effect was enhanced when metal (II)-SF complexes were used.4. SF and its metal(II) complexes inhibited biofilm biomass production.The mature biofilm was estimated to be 38% when SF was added at the concentration of 1 mM (960 mg/ml).In addition, Ca(II)-SF, Mg(II)-SF, Cu(II)-SF and Zn(II)-SF (1 mM) reduced mature biofilms by 78%, 79%, 72% and 69%, respectively.5.The cells treated with SF and metal(II)-SF complexes showed attenuated fluorescence density of HWP1-GFP, suggesting that the compounds had an impact on the expression of hypha-related genes.
6.The expression of the hypha-specific genes for planktonic and biofilm-forming cells were downregulated after exposure to SF and Mg(II)-SF.All metal(II)-SF complexes significantly altered the expression of hypha-specific and biofilm-related genes.7. The relative CSH of untreated C. albicans cells was 0.67, and the CSH underwent a reduction in response to SF concentration.The best results were obtained for Mg(II)-SF, where the CSH was reduced to 0.23, 0.12, and 0.01 with exposure to 0.5, 0.75, and 1 mM of Mg(II)-SF, respectively.Tambone E. et al. [ 1.The supernatant of crude biosurfactant was extracted three times with ethyl acetate, and the composition of the raw extract was confirmed by mass spectrometry analysis.
2. R89BS coating more effectively inhibited biofilm biomass than cell metabolic activity and viability.Quantitatively, R89BS-coated TDs demonstrated the highest ability to reduce biofilm formation at 24 h, with inhibitions of biofilm biomass, cell metabolic activity, and cell viability exceeding 90%.After 48 h, biofilm biomass and cell viability were inhibited by 36% and 29%, respectively, while metabolic activity showed a less pronounced effect with a 14% inhibition.
3. Cell viability of hOBs (Human primary osteoblasts) decreased below 70% at R89BS concentrations exceeding 50 µg/mL.Concentrations equal to or lower than 50 µg/mL showed no interference with hOBs growth, maintaining cell viability above 80%.No cytotoxic effects were observed on hOBs cultured in the eluate from R89BS-coated titanium.

Table 5 (continued)
biosurfactants demonstrated the ability to generate foam, suggesting that biosurfactants serve effectively as detergents in toothpaste [75,76].This finding aligns with the work of Das et al., who substituted SLS with biosurfactants from Nocardiopsis VITSISB in toothpaste [77].Biosurfactants sourced from Bacillus subtilis SPB1 (HQ392822) in toothpaste formulations also reported can exhibit favorable characteristics, including strong foaming capabilities, effective stain removal properties on eggshells, an alkaline pH conducive to neutralizing acidic biofilms and demonstrates potent antimicrobial activity against the tested microorganisms [32].Biosurfactants derived from Pseudomonas aeruginosa UCP 0992 (PB) and Candida bombicola URM 3718 (CB) combined with chitosan also exhibited significantly lower toxicity compared to commercial mouthwash products.These findings underscore the safety and effectiveness of natural product-based mouthwashes as a viable alternative for controlling oral microorganisms, providing a healthier option than commercially available mouthwashes [36].Biofilm formation is related to all microbiological and chronic illnesses, particularly in oral and dental diseases, and is used by microorganisms to shield themselves from a hazardous environment [78,79].In normal physiological conditions, dental biofilm development involves the formation of a protein-rich acquired pellicle on dental surfaces, followed by the coaggregation and co-adhesion of various initial colonizers, such as Streptococci and members of the Actinomyces family [80][81][82].Bridging colonizers such as Fusobacterium also contributes to this process by facilitating co-adhesion and coaggregation [83].Typically, these biofilms consist predominantly of Gram-positive facultative anaerobes.However, inadequate hygiene can lead to an elevated percentage of Gram-negative species (e.g., Porphyromonas spp., T. forsythia, Treponema denticola, and A.
Recent advances in biofilm physiology have allowed researchers to learn more about bacterial biofilm inhibition [85].There are two main inhibitory techniques, which are centered on the development of new antibiofilm chemicals and the development of biofilm-resistant surfaces [86].Biosurfactants are the most promising choices for bacterial biofilm inhibition [5,87,88].In heterogeneous systems, biosurfactants tend to aggregate at phase boundaries or interfaces, similar to how organic molecules in the aqueous phase immobilize at solid interfaces [89].This aggregation forms a conditioning film, altering the surface properties such as surface energy and wettability and influencing the adhesion properties of microorganisms [90].
Moreover, they can disrupt membranes, causing cell lysis by increasing membrane permeability, which leads to the leakage of cellular metabolites.This disruption can occur through changes in the physical membrane structure or by altering protein conformations that affect critical membrane functions like transport and energy generation.The role of biosurfactant as an anti-biofilm agent can be seen in Fig. 5 [91].
Rhamnolipids derived from the non-pathogenic Burkholderia thailandensis E264 strain (ATCC 700,388) exhibit notable antibiofilm properties when tested in co-incubation experiments, pre-coated surface applications, and the disruption of immature biofilm against Fig. 3 The co-occurrence of keywords from the ten selected articles.The proximity of two nodes in the graph indicates a higher number of co-occurrences between the corresponding keywords Fig. 2 The total count of articles published and citations within ten years oral bacteria biofilms [27].In vitro studies demonstrated that rhamnolipids can prevent and disrupt oral pathogen biofilms by increasing the permeability of oral pathogens in planktonic and oral biofilm states [35].Rhamnolipids are reported to have the potential to inhibit the growth of oral bacteria and the formation of biofilms by A. actinomycetemcomitans Y4, making them a promising candidate for a novel oral drug to combat localized invasive periodontitis [40].Surfactin was also reported to promote the antimicrobial activity of terpinen-4-ol (TP) against S. mutans, the causal agent of tooth decay, and can inhibit microbial pathogens' growth and adhesion when combined with TP [33].Lipopeptide biosurfactant demonstrates potent antimicrobial and anti-biofilm properties against Enterococcus faecalis grown in dentin specimens.It shows promise both as a standalone root canal irrigation solution and as an adjunct prior to the use of NaOCl in root canal treatments [92].
On implant applications, R89BS (biosurfactant extracted from P. aeruginosa 89) coating demonstrated effectiveness in reducing mixed biofilms of C. albicans and S. aureus on titanium surfaces, making it a promising approach for preventing microbial colonization on dental implants [38].The same coating was applied to three different commercial implant surfaces, and the identical coating yielded a remarkable biomass inhibition exceeding 90% for S. aureus and reaching as high as 78% for S. epidermidis within 24 h [39].
In terms of the dose, some papers reported biosurfactants showed dose-dependent characteristics.Biosurfactants obtained from Lactobacillus acidophilus DDS-1, Lactobacillus rhamnosus ATCC 53,103, and Lactobacillus paracasei B21060 exhibited substantial inhibition of adhesion and biofilm formation on titanium surfaces by S. mutans and S. oralis in a dose-dependent manner.This was evident from the significant reduction in cfu/ml values and biomass production [34].Elshikh M. et al. also reported that higher rhamnolipid concentrations can increase the permeabilization effects on both the grampositive and gram-negative bacteria used in their study [27].Both Surfactin C -15 (SF) and metal(II)-SF complexes demonstrated a concentration-dependent inhibition of biofilm formation and a reduction in the metabolic   activity of mature biofilms that led to a decrease in the mRNA expression of hypha-specific genes (e.g., HWP1, ALS1, ALS3, ECE1, and SAP4) without causing significant growth inhibition of C. albicans [37].Lipopeptide biosurfactant (F7) extracted from Bacillus clausii also demonstrated dose-dependent against S. mutans, E. faecalis, and C. albicans.Higher F7 biosurfactant concentrations showed greater inhibition percentages [93].The limitation of this systematic review is that numerous studies do not provide sufficient evidence regarding the thorough purity or comprehensive characterization of the active biosurfactant fractions they employ.This issue is exemplified by the research conducted by Tahmourespour et al. (2011) [94], Tahmourespour, Salehi, and Kas-raKermanshahi (2011) [95], and Salehi et al. (2014) [96], Savabi et al. (2014) [97], during their investigation of the gene expression of gtfB, gtfC, and ftf in S. mutans which directly involved in the formation of biofilm matrices.Notably, these studies are pioneering efforts as they represent the first instances of exploring the gene expression of oral-related bacteria following treatment with biosurfactants.Other limitations include the relatively short timeframe covered by this systematic review.The choice of this timeframe was motivated by the need to present the most current research papers exploring biosurfactants' use in oral applications.Interpreting the results of in vitro studies presents challenges due to variations in the methods used for material preparation and microbial exposure across different studies.This is crucial because data comparison becomes arduous without standardized methods, and drawing meaningful conclusions and extrapolating findings becomes problematic.Deviating from these recommendations in experiments may limit the applicability of the results.

Conclusion
This systematic review suggests that biosurfactants hold significant promise for oral applications.Their properties, such as antimicrobial and antibiofilm activity against both gram-positive bacteria, gram-negative bacteria, and fungi, the ability to form stable or metastable microemulsions, and their capacity to enhance the bioavailability of hydrophobic compounds, make biosurfactants attractive candidates for use in cosmetic or therapeutic oral hygiene products, as well as oral-related medical devices.Utilizing biosurfactants alone or combined with other antimicrobial or chemotherapeutic agents presents a promising strategy for preventing and combating microbial infections, biofilm formation, and proliferation.

Perspectives and future directions
Biosurfactants have recently gained attention within the scientific community as a promising oral application addition to the next generation.However, to fully realize the potential biosurfactants, substantial efforts are required to improve the quality of research in this area.Enhancing research quality may help attract skeptical industrial collaborators.When attributing bioactivity to biosurfactants, it is crucial to use high-purity biosurfactants.It is also crucial to emphasize that their multifaceted properties can interact and potentially lead to side effects in various applications, necessitating thorough investigation.At the same time, the commercial utilization of biosurfactants is becoming increasingly pertinent and essential to mitigate the environmental impact associated with conventional synthetic surfactants.Nevertheless, challenges related to cost-effectiveness and availability of biosurfactants for potential applications still require resolution.

Fig. 1
Fig. 1 The outline of the article screening procedure in the PRISMA flowchart

Table 2
The criteria for eligibility based on the PICOS framework

Table 3
Articles Selected for Inclusion in this Systematic Review

Table 4
Different types of biosurfactants in this systematic review 70% for N. mucosa and A. naeslundii, and 83% for S. sanguinis.A low rhamnolipid concentration of 0.19 mg/ml produced a significant inhibition of around 65% for S. sanguinis, 0.03 mg/ml of around 80% for S. oral and A. naeslundii, and 50% for N. mucosa.6.For all the organisms investigated, a direct proportionality exists between the increase in rhamnolipid concentration experienced by the cells and the intracellular accumulation of bisbenzimide.7.There was an increasing leakage of UV-absorbing material from the cells with increasing rhamnolipid concentration to all the

Table 5
Overview of the methods employed and outcomes of studies included in this systematic review Inhibition was observed when surfactin was mixed with TP against S. aureus, P. gingivalis, and E. coli, while no inhibition was detected for surfactin against all microorganisms.2. MIC of surfactin ranged from 22 µg/mL to 700 µg/mL, with no inhibition for P.gingivalis.3. Combination index for surfactin and TP were synergism for S. mutans and P.gingivalis, slight synergism for C. albicans and E. coli, additive for S. aureus, and antagonism for P. aeruginosa, S. salivarius, and S. mitis.4. The micellar solution of surfactin and TP showed very high enhancement in antiadhesion activity for two oral pathogens (C.albicans and P. gingivalis).

Table 5
(continued) 7. MR 0.2 mg/ml eliminated preformed biofilms of S. oralis, S. sanguinis, and A. naeslundii, while N. mucosa and S. mutans biofilms were inhibited by 0.4 and 1.0 mg/ml of MR.Biofilms of S. oralis, S. sanguinis, N. mucosa, and A. naeslundii were inhibited with 0.2 mg/ ml LSLs, while they required 1.0 mg/ml to reduce the biofilm of S. mutans.8. Using as little as 0.2 mg/ml, MR resulted in more than 80-90%

Table 5
(continued)The biosurfactant PB was the most effective against S. aureus, E. coli, and S. salivarius (MIC: 20 µg/mL), while PB and CB had similar effects on S. mutans (MIC: 20 µg/mL).All biosurfactants exhibited uniform effects on C. albicans and L. acidophilus (MIC: 40 µg/ mL).Combining biosurfactants with chitosan reduced MIC for all microorganisms.When combined with peppermint essential oils, the MIC for C. albicans either increased (CB + POE and BB + POE: 30 µg/mL) or remained the same (PB + POE: 20 µg/mL).The low MIC combination for L. acidophilus (20 µg/mL) was peppermint essential oil with PB.For S. mutans, only CB + POE maintained the biosurfactant's MIC, with reductions observed in all other combinations.
4. The combinations of the CB and PB with chitosan demonstrated an additive effect on the majority of microorganisms tested and an indifferent effect on E. coli and C. albicans.The combinations of CB and BB with the peppermint essential oil exhibited an additive effect only on the gram-negative bacterium E. coli and an indifferent effect on the other microorganisms tested.5.The results demonstrate that the test mouthwashes were classified as non-toxic to the fibroblast line, with cell inhibition rates lower than 20%.However, the mouthwash containing the biosurfactant extracted from C. bombicola + peppermint essential oil + chitosan exhibited moderate toxicity to the macrophage line (66% inhibition).

Table 6
Top ten journals with the highest number of publications on oral application of biosurfactants between 2013 and 2023

Table 7
Quality assessment of the included studies according to the QUIN tool for in vitro studies