Effects of Probiotic Lactic Acid Bacteria on Oral Pathobionts and on Oral Health: A Review

Abstract

The human oral cavity contains a variety of habitats, all of which are colonized by microorganisms. Oral bacteria form multi-genera communities, exhibiting adhesive properties, both to oral tissue surfaces and to each other. In certain conditions, these properties represent the first step towards the development of oral diseases. The oral microbiome undergoes changes in its composition, which can alter the balance between health and disease and is dynamically interconnected with the host. Probiotics with a targeted effect on the oral cavity can successfully compete with pathobionts and increase the presence of beneficial bacteria, thus contributing positively mainly to the prevention of oral diseases. The application of probiotics to maintain balance of oral microbiota has been a subject of intensive research. Oral health products containing lactic acid bacteria represent a modern approach to prevent or reduce the level of infections in the oral cavity. The application of these products is an alternative and promising way to prevent diseases through competitive interactions of beneficial microorganisms with pathobionts.

1. Introduction

In recent decades, there has been a significant increase in scientific activity focused on the study of probiotic microorganisms. A number of scientific studies have established that many probiotic strains from the lactic acid bacteria (LAB) group possess beneficial effects in the prevention and treatment of infectious diseases in the oral cavity. In the course of increasing research, the beneficial properties of probiotic strains to interact with the oral microbiota and maintain a healthy microbial balance have been established [1].

LAB include a wide variety of representatives that have become part of human life throughout their long probiotic history. They are included in our daily diets in the form of naturally containing products, food products fermented with starter cultures, and various food supplements with probiotic application. LAB also naturally colonizes the human body, contributing in various ways to its health [2].

The human oral microbiome plays an important role in the microbial community and health. Since the oral cavity is the starting point of the gastrointestinal tract (GIT), the development of oral diseases can also contribute to the occurrence of systemic diseases. Therefore, much scientific research is directed towards studying the processes of inhibiting pathobionts and maintaining health in the oral cavity [3].

The level of oral hygiene is of utmost importance and has a significant impact on the composition and condition of the oral microbiome. Maintaining good oral health affects the ability of a person to adapt to physiological changes throughout their life and to maintain their dentition and oral cavity through independent self-care. In recent years, scientific research has expanded its focus on biological strategies to restore microbial balance in the oral cavity. Probiotics with a targeted effect on the oral cavity can exert beneficial effects through multiple mechanisms and successfully compete with potentially pathobiontic commensal microorganisms to increase the presence of beneficial bacteria. With these effects they can positively contribute to the prevention of oral diseases, including caries, gingivitis, periodontitis, and halitosis, as well as their therapy when they occur [4].

An increased focus of research on strains and delivery methods is taking place. Various strains, specifically Lacticaseibacillus rhamnosus GG, Lacticaseibacillus paracasei Shirota, Lacticaseibacillus paracasei, Limosilactobacillus reuteri, Streptococcus salivarius K12 and M18, Bifidobacterium animalis subsp. lactis BB-12, etc., are well known for their specific probiotic traits. The delivery systems for oral probiotics are diverse and include tablets, lozenges, chewing gum, yoghurt, fermented milk, ice cream, etc. The efficacy of oral probiotic products depends not only on the incorporated strain but also on the delivery vehicle, strain viability, dosage, and duration of administration [5].

Therefore, the study of LAB as natural antagonists of various oral pathobionts, as well as their beneficial functional properties, continues to be of significant interest to modern science. Also, studies of their probiotic potential are essential for their inclusion in various food supplements in order to develop effective functional products. This review focuses on descriptive representation of oral microbiota, analyzation of the impact of LAB on reducing the counts of potential pathobiontic commensals, effectiveness of probiotics in preventing different oral diseases and maintaining oral equilibrium, exploration of oral probiotic mechanisms of action, review on in vitro evaluations and clinical trials of commonly used probiotic strains in oral health products, application of probiotic strains in different product vehicles, and highlighting possible future research directions on next-generation probiotics and innovative delivery systems.

2. Microbiota in the Oral Cavity

The human oral microbiota is formed from all microorganisms that are found on or in the oral cavity and its adjacent extensions to the esophagus. It is not uniform and varies according to anatomical and physiological conditions [6,7]. The development of microorganisms depends on factors such as temperature, acidity, redox potential, availability of nutrients, water content, structural morphology of the oral cavity, saliva flow and the presence of antimicrobial compounds. Each of these factors selectively affect the oral ecosystem and help maintain the balance of microbial populations [8].

The oral cavity is one of the most studied microbiomes to date, with a total of 836 taxa with at least one reference genome and a total genome count of over 8170 (

Table 1. Summary of the eHOMD data at phylum level.

Phylum	Taxon	Genome	16S rRNA Refseq
Methanobacteriota	1	5	4
Actinomycetota	133	1463	728
Bacillota	269	3342	2653
Bacteroidota	133	865	877
Chlamydiota	1	10	5
Chlorobiota	3	3	3
Chloroflexota	3	5	6
Cyanobacteriota	1	7	3
Eremiobacterota	1	0	0
Fusobacteriota	40	431	662
Mycoplasmatota	12	41	32
Patescibacteria	37	108	70
Pseudomonadota	139	1799	1417
Spirochaetota	53	70	112
Synergistota	8	17	20
Verrucomicrobiota	2	11	8
Total	836	8177	6600

). According to data described in the Expanded Human Oral Microbiome Database (eHOMD), it is composed of 818 microbial species from 253 genera in 16 bacterial and archaeal phyla. Of all the species in this database, 49% are officially named, 21% are unnamed but cultivated, and 29% are known only as uncultivated phylotypes (https://www.homd.org/, accessed on 11 February 2026).

The main bacterial representatives present in a healthy oral cavity are: Gram-positive cocci of the Streptococcus, Peptostreptococcus, Abiotrophia, and Stomatococcus genera, rod-shaped representatives of the Lactobacillaceae family and the Actinomyces, Bifidobacterium, Corynebacterium, Cutibacterium, Eubacterium, Pseudoramibacter, and Rothia genera; Gram-negative cocci of the Neisseria, Veillonella, and Moraxella genera and rod-shaped representatives of the Fusobacterium, Prevotella, Campylobacter, Capnocytophaga, Leptotrichia, Treponema, Desulfobacter, Desulfovibrio, Eikenella, Haemophilus, Wolinella, Selemonas, and Simonsiella genera [9,10]. The main species that colonize the oral environment are representatives predominantly belonging to the Candida genus, as well as the Cryptococcus, Aspergillus, Fusarium, Cladosporium, Saccharomycetales, and Aureobasidium genera [11].

The gingival pocket is one of the places where microorganisms from the external environment develop primarily. The site is rich in nutrients, has a low redox potential and is colonized mainly by obligate anaerobic rod-shaped bacteria. It is assumed that microorganisms in the gingival pocket are interconnected with the composition of supragingival plaque due to the frequent occurrence of rod-shaped representatives of Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella denticola, Prevotella loescheii, Prevotella melaninogenica, and Prevotella intermedia [8]. The most common bacterial representatives in the gingival pocket are Streptococcus sanguinis, Streptococcus mitis, Streptococcus intermedius, Streptococcus mobillorum, Streptococcus constellatus, Lacticaseibacillus casei, Lactobacillus acidophilus, Staphylococcus epidermidis, Micrococcus spp., Mycoplasma spp., Trichomonas tenax, Entamoeba gingivalis, Veillonella parvula, Peptostreptococcus micros, Eubacterium lentum, Cutibacterium acnes, Actinomyces viscosus, Actinomyces odontolyticus, Actinomyces naeslundii, Catonella spp., Johnsonella spp., Rothia dentocariosa, Prevotella oralis, P. denticola, Capnocytophaga gingivalis, Capnocytophaga ochracea, Bacteroides melaninogenicus, Fusobacterium nucleatum, Campylobacter sputorum, Selenomonas sputigena, Treponema spp., Eikenella corrodens, Wolinella spp., Leptotrichia spp., and Granulicatella spp. [8,12].

Dental biofilm forms on the surface of the teeth and is composed of microorganisms that produce a complex matrix composed of their extracellular products and salivary components. Bacteria isolated from the plaque are mainly Gram-positive facultative anaerobic representatives of the Streptococcus and Actinomyces genera. Bacteria of the Veillonella, Haemophilus, and Bacterioides genera are usually isolated from the deeper biofilm layers. Anaerobic microorganisms, including Lactobacillaceae, Cutibacterium spp., Prevotella buccalis, P. oralis, Actinomyces israelii, A. naeslundii, A. viscosus, A. odontolyticus, Fusobacterium spp., Leptotrichia spp., R. dentocariosa, Peptostreptococcus, Nocardia spp., and Veillonella spp., and aerobic representatives, including Streptococcus mutans, S. sanguinis, Aggregatibacter actinomycetemcomitans, Neisseria spp., and Mycoplasma spp., can be isolated from the surface of the teeth [8].

The tongue can serve as a reservoir for microorganisms involved in the pathogenesis of periodontal diseases. S. salivarius can be frequently isolated but also other aerobic representatives, including S. mitis, S. sanguinis, Staphylococcus spp., Corynebacterium spp., Candida, Enterobacteriaceae, Neisseriaceae, and Micrococcus spp., and anaerobic ones, including C. sputorum, B. melaninogenicus, P. oralis, Cutibacterium, Actinomyces, Peptococcus, Peptostreptococcus, and Veillonella [8,13].

Saliva has the potential to remineralize hard dental tissues and exert a suppressive effect on the development of caries due to the presence of unsaturated ions of phosphates, fluorine and calcium. The presence of glycoprotein mucin plays the role of lubricating oral surfaces and forms a protective barrier against the external environment. Mucin is one of the factors through which the aggregation of bacteria on the tissues in the oral cavity occurs. Representatives, including S. mitis, S. sanguinis, Streptococcus gordonii, S. mutans, S. salivarius, Streptococcus milleri, Streptococcus mitior, A. actinomycetemcomitans, Escherichia coli, Pseudomonas aeruginosa, Lactobacillaceae, Actinomyces spp., and Veillonella spp., can be found in saliva [14].

3. Microbial Balance and Diseases in the Oral Cavity

The oral microbiome can alter the balance between health and disease, and the specific activities of microbial communities are expressed in their participation in physiological, metabolic and immunological functions [15,16]. The oral cavity contains a complex mixture of fluids, mainly saliva and gingival fluid, which play an important role in maintaining a healthy oral environment [17,18,19]. At a neutral pH, a balance is maintained between the minerals of tooth enamel and saliva. When bacteria produce organic acids the acidity in the oral cavity increases, reaching values under pH 5.5, and tooth demineralization begins. Salivary components can neutralize the acidity to a certain extent and reduce the rate of demineralization, thus delaying or preventing the formation of caries. This buffering capacity is achieved through the presence of bicarbonate, phosphate and protein buffers [20]. Maintaining 37 °C in the oral cavity and salivary pH between 6.5 and 7.5 provide a stable habitat for bacterial growth [21]. Bacteria, together with saliva, have a direct impact on the formation and growth of dental biofilm. Salivary components are the main source of nutrients for microorganisms and are necessary for the development of a balanced microbiome. Many components, including secretory immunoglobulin A, lysozyme, lactoferrin, lactoperoxidase, statherin and histatins, directly and indirectly regulate the oral microbiome, maintaining it in a balanced state [22]. Enzymes that help regulate the microbiome balance are immobilized in the acquired enamel pellicle, forming an active structure [23,24]. The individually structured pellicle activates and supports the adhesive ability of bacteria to non-detachable hard surfaces through various interactions. Saliva not only helps maintain an environment that allows the biofilm to thrive but also modulates its layers through enzymes, glycoproteins and minerals, which, in turn, control the formation of the biofilm and its activity [22,25].

Upon initial contact with the oral cavity, bacteria interact with saliva, which promotes their absorption on the dental surface and, through their multiplication, the biofilm matrix starts to form. The first bacteria to attach to dental tissues are species of the Streptococcus genus, as they are part of the commensal microflora and characterize the biofilm formed by them, associated with healthy teeth and gums. These microorganisms exert their beneficial effects by significantly hindering the colonization of potentially pathobiontic species [17,26]. However, disruption of the oral ecosystem leads to dysbiosis, with carbohydrate-fermenting Gram-positive species becoming predominant, leading to demineralization of dental tissues, which, in turn, leads to oral pathological processes [6,27].

Oral dysbiosis is an imbalance in the oral microbial community. It is associated with an increase in pathogenic bacteria and a decrease in beneficial bacteria alike and can contribute to the development of oral and systemic diseases [28]. Dysbiosis can occur rapidly if salivary flow is disrupted, as saliva supplies components of the innate and adaptive immune response that are vital for maintaining homeostasis in the oral cavity. Some bacterial species possess intrinsic resistance to host defense peptides, while others can modulate their susceptibility to environmental stimuli. There is also significant interstrain variation in their susceptibility to defense peptides within the same species [29,30].

It is generally accepted that bacteria historically considered as potential oral pathobionts can be found in low numbers in healthy sites, with oral diseases occurring as a consequence of a detrimental alteration in the natural balance of the oral microbiota rather than as a result of exogenous infection. During a dysbiotic state, these disease-associated bacteria can grow to higher proportions than under healthy conditions, where they are normally commensal residents of the dental biofilm [31].

Oral Pathobionts and Prevalent Oral Diseases

Maintaining good oral health affects the ability of an individual to adapt to physiological changes throughout their life and maintain their dentition and oral cavity through independent self-care. Although largely preventable, oral diseases are widespread throughout life and have significant negative effects on people and are a global public health problem [32,33,34,35].

Oral pathobionts are commensal microorganisms that can induce diseases only under specific genetic or environmental changes in the oral cavity. The differences between them and opportunistic pathogens are that the latter lead to acute infections and are mostly acquired from external sources. Commensals can alter into pathobionts when influenced by endogenous or exogenous factors that can cause oral dysbiosis and ultimately leading to pathological conditions. The pathobiont-state transition can be influenced by different factors, including microbial dysbiosis, antibiotic resistance, impaired immune system, and genetic mutations [36].

Dental caries is the local destruction of hard dental tissues by acidic by-products of bacterial fermentation of free sugar compounds. It is manifested through two stages. The first is initiation, where bacterial acids dissolve minerals from the tooth enamel, causing white spots. This stage is reversible through the normal process of remineralization and with good oral hygiene. The second is progression, which is a continued breakdown of enamel and then dentin, forming a physical tooth cavity. This stage is irreversible and requires mechanical intervention to prevent further destruction and devitalization of teeth [37]. Under normal physiological conditions, the processes of demineralization and remineralization in the supragingival biofilm are in a balanced state and are unlikely to have a detrimental effect on the teeth. The process of caries formation is dynamic, with periods of de- and remineralization being disrupted by tooth-related fluctuations in the pH values of the dental biofilm. The lower the acidity, the higher the tendency to degrade the components of the hard tissues. The bacteria in the biofilm metabolize the sugars and increase the acidity through their products. Over time, the pH values return to neutral due to the good buffering capacity of saliva and the additional dissolved mineral content from the tooth surfaces [38,39,40]. Scientific data shows that oral diseases are not caused by a single microorganism but are polymicrobial in nature. Microorganisms associated with the formation of dental caries are S. mutans, representatives of the Cutibacterium, Neisseria, and Selenomonas genera, as well as Candida albicans [41,42].

Periodontal diseases are chronic inflammatory conditions affecting the soft tissues surrounding and supporting the teeth. Periodontal disease initially manifests as gingivitis, a reversible inflammation of the periodontal soft tissues, which results in bleeding and swelling of the gums. If left untreated, a greater number of anaerobic organisms begin to colonize the deeper periodontal pockets, including P. gingivalis and A. actinomycetemcomitans, which progresses gingivitis to periodontitis and triggers the host inflammatory responses [43,44,45]. A wide range of microorganisms from the Bacteroidetes, Spirochaetes, and Synergistetes phyla, from the Clostridia, Erysipelotrichia, and Negativicutes class, from the Fusobacterium and Prevotella genera, as well as species including P. gingivalis, Treponema denticola, Tannerella forsythia, Filifactor alocis, A. actinomycetemcomitans, and Parvimonas micra, as well as archaea, including Methanobrevibacter oralis, Methanosarcina mazeii, and Methanobacterium curvum/congolense are responsible for the occurrence of periodontal diseases [46,47,48,49].

Oral candidiasis is one of the most common fungal infections caused by Candida species. They are present in the oral cavity as a commensal microorganism under balanced oral conditions but, when an imbalance in the microbiota occurs, different species, most often C. albicans, start to predominate and cause infections [50]. As a polymorphic fungus, it adheres to epithelial cells and transitions from yeast to a hyphal form in order to enhance tissue penetration. Further accumulation leads to formation of white-colored dense biofilms that can withstand antifungal metabolites and immune defenses, leading to further soft tissue damage [51]. Oral candidiasis can vary in its entity, including pseudomembranous candidiasis (thrush), hyperplastic or atrophic (denture) candidiasis, linear gingival erythema, median rhomboid glossitis, and angular cheilitis [52]. In addition to C. albicans being the most ubiquitous in oral yeast infections, species including Candida glabrata, Candida tropicalis, Candida pseudotropicalis, Candida parapsilosis, Candida krusei, and Candida stellatoidea are also present in the oral cavity and can be a part of the disease etiology [52,53].

Halitosis or oral malodor is a disease characterized by an unpleasant odor emanating from the oral cavity. Its main cause is the presence of volatile sulfur compounds, including hydrogen sulfide, dimethyl sulfide, and methyl mercaptan, as a result of the prevalence of anaerobic oral Gram-negative bacteria. Extra-oral causes of halitosis include gastrointestinal diseases, respiratory tract infections, as well as systemic disorders. The intra-oral causes are mainly associated with microbial degradation of sulfur- and non-sulfur-containing amino acids derived from proteins in the course of other oral diseases or poor oral hygiene [54,55]. Several bacterial representatives are known to produce volatile sulfur compounds in the oral cavity, including T. denticola, P. intermedia, P. loescheii, P. gingivalis, P. endodontalis, Bacteroides loescheii, Centipeda periodontii, E. corrodens, F. nucleatum, Bacteroides forsythus, T. forsythyia, Selenomonas and Eubacterium [54].

4. Role of Oral Probiotic Microorganisms and Criteria for Their Selection

In the dynamic ecosystem of the oral cavity, the microbiome is heterogeneous and diverse, and therefore more opportunities arise to cause imbalances leading to the manifestation of oral diseases. Conventional treatment of these diseases includes mechanical removal of bacterial plaque and antimicrobial therapy with antibiotic drugs. Due to the limited efficacy of antibiotic substances, arising from the emergence of antibiotic resistance in microorganisms, it is necessary to explore other alternative treatment options and, in this context, probiotic microorganisms can play a significant role.

A significant number of strains of the Lactobacillaceae family, Bifidobacterium, Streptococcus, Enterococcus, Saccharomyces, and Bacillus genera have a long history of safe application and have been subject to extensive monitoring and evaluation, so it can be concluded that there are no serious safety concerns for their application in foods and dietary supplements. Their mechanism of action is expressed through non-specific, species-specific and strain-specific mechanisms [56,57].

In recent years, the application of probiotics to maintain ecological balance and the effectiveness of normalizing the oral microbiota has been the subject of intensive research [58,59,60,61,62]. Regulation of the composition of microbiota provides an opportunity to influence the development and implementation of mucosal and systemic immunity. Probiotic microorganisms reduce the damaging effects caused by pathobionts by naturally colonizing the oral cavity. Changing the active composition of an infected tissue area from an environment rich in inflammatory cytokines to a more benign one through colonization of beneficial microorganisms contributes to systemic health in general. The most commonly used species in oral probiotic products are bacterial representatives, including Lactobacillus delbrueckii subsp. bulgaricus, L. casei, L. acidophilus, Lactiplantibacillus plantarum, Ligilactobacillus salivarius, Lactobacillus helveticus, Streptococcus thermophilus, Enterococcus faecalis, and Enterococcus faecium, as well as the yeast representative Saccharomyces boulardii. With the advancement of research and application of oral probiotics, there has been a shift from their use solely as an adjunct to dental treatments to a more established routine therapeutic strategy [5].

Probiotic treatments are widely used in diseases of the intestinal tissues but are not used in oral diseases due to the still limited scientific evidence of their beneficial effects. Probiotics with a targeted effect on the oral cavity may cause chemical and physical alterations to the oral microbiota [63]. Research suggests that probiotics possess expressed adhesive properties to oral tissues, higher than pathobionts, and can successfully compete for adhesion surfaces and increase the presence of beneficial bacteria, thus contributing positively mainly to the prevention of oral diseases [63,64,65,66]. For oral probiotics to be effective, they must be able to maintain the environmental conditions in the oral cavity. Through aggregation, co-aggregation and formation of new biofilms, probiotics adhere to and colonize oral tissues in order to compete for adhesion sites, nutrients, and growth factors, thus inhibiting the transformation of commensals to pathobionts [67].

An important matter to be mentioned is the correlation of some lactobacilli in the further development of caries [68,69]. Their metabolic pathways lead to the production of acidic compounds which may increase the risk of caries [70]. While being identified at lesion sites, the initiation of the disease starts with the accumulation of pathobionts, including S. mutans, which, in low pH conditions, increases the demineralization time and starts the destruction of the enamel. After this, the dentin structure is presented to and favored by a more widespread variety of microorganisms. But, according to many studies, it can be claimed that the consumption of probiotics does not cause the initiation of caries [63]. It can be suggested that oral species of the Lactobacillaceae family display a niche specificity. Based on the 16S gene sequence [71] and the 73 core genes [72], phylogenetic relationships between dominant oral species belong to different clades or phylogroups. This indicates that caries-associated lactobacilli do not arise from a recent common ancestor but their adaptation to an existing lesion niche is independent in different lineages [73].

The selection of probiotic strains requires a systematic approach using an incremental strategy. In many cases, evaluated strains are narrowed down through sequential series of tests. Finishing this procedure yields a selection of strains which present the highest number of functional properties. The selected strains are then proceeded through product incorporation and clinical trials [74]. Important criteria for effectiveness include capacity to survive oral conditions (salivary flow, pH fluctuations, and lysozyme), adhesion ability to epithelial cells (binding to mucin) and further colonization, production of metabolites with antimicrobial activity (organic acids, bacteriocins, and H₂O₂), physical competition with oral pathobionts, and safety assessment (taxonomic identification; absence of virulence factors, toxicity, and transferable antibiotic resistance genes) [75,76]. In addition, host-associated functional properties should also be considered, including anti-cancer and immunostimulatory activities [74]. The beneficial effects of oral probiotic LAB strains are reported to involve antimicrobial, anti-mutagenic, anti-cariogenic, immunomodulating, and antioxidant activities. These probiotic features are reported as species- and even strain-dependent [76]. Emphasis in strain-related traits should be considered, as different strains within the same species can have entirely different effects. Some can exhibit beneficial properties, while others possess insignificant or no effects at all [59]. It must be noted that, when presented in the environment of the oral cavity, probiotic strains should not actively ferment sugars. This is important in order to avoid lowering pH and enamel demineralization, inhibiting the organization of the extracellular matrix in the dental biofilm, limiting the cytotoxic productivity of pathobiontic bacteria, and favorably changing biochemical parameters affecting the biofilm (buffer capacity and salivary components) [77,78].

5. Specific Mechanisms of Probiotic Activity of Lactic Acid Bacteria in the Oral Cavity

LAB are a dominant bacterial group that is widely distributed in nature, being present in dairy, plant and meat fermented foods, in the microbiome of the GIT and urogenital tract (UGT) of humans and animals, but are also naturally present in water, soil and plants [79]. This group of microorganisms is well known for its ability to produce lactic acid as the main end product of their anaerobic metabolism, as well as for the synthesis of a wide range of metabolic products, including exopolysaccharides (EPSs), polyols, organic acids, aromatic compounds, bacteriocins, enzymes, and vitamins, which contribute to the nutritional, aromatic and technological properties of various fermented food products, as well as maintaining the health of the human organism [80,81,82]. As a result of these properties, LAB are widely used as probiotics, starter cultures, and for biosynthesis of various biological compounds due to their adaptive metabolism [83,84,85]. Other important applications include their use as bioprotective agents, contributing to the inhibition of various pathobiontic microorganisms [86], as well as recombinant vectors for transfer of proteins and DNA vaccines as preventive and therapeutic drugs [87].

The spectrum of species belonging to the Lactobacillaceae family is a subject of intensive research interest and widespread application due to the established properties to maintain the health of the human organism [88]. Their selection as probiotic microorganisms is due to the expression of many vital properties, including the ability to adhere to mucosal tissues, high tolerance to acids, the ability to withstand low pH values, to exhibit resistance to antibiotics, antimicrobial and antagonistic activity, production of EPSs, etc. [89,90,91].

LAB-containing probiotic products represent a modern approach to prevent or reduce the level of infectious diseases caused by pathobiontic microorganisms in the oral cavity [92]. Scientific studies have established positive effects of probiotic microorganisms through interaction with host tissues, synthesis of metabolic products and competitiveness [93].

5.1. Production of Antimicrobial Metabolic Compounds

As a result of the fermentative type of metabolism in LAB, a variety of organic acids are synthesized, which is an important metabolic feature of probiotics. They mainly produce lactic acid but also acetic, citric, propionic, 3-hydroxypropionic, succinic, formic, 3-phenyllactic, gamma-aminobutyric, and 2-hydroxyisocaproic acids [94,95]. The synthesized organic acids can affect the functions of the bacterial cell wall, cytoplasmic membrane and specific metabolism, which can lead to cell devitalization of potentially pathobiontic microorganisms (Figure 1) [96]. Although possessing antimicrobial effects, the produced acids have an ambiguous nature. They lower pH in the oral cavity, which leads to acidogenicity in the oral environment, thus contributing to increased potential of initiating caries [97]. Huang et al. investigated the antimicrobial activity of acetic, propionic and formic acids, establishing inhibition to varying degrees of some of the common oral pathobionts, including S. mutans, S. gordonii, S. sanguinis, P. gingivalis, F. nucleatum, A. actinomycetemcomitans, and C. albicans [98]. Studies show that, even though acetic acid production is the lowest compared to other organic acids, it has a higher proportion of non-dissociating acid, therefore possessing a higher antimicrobial effect than lactic acid at certain pH levels [94].

The synthesis of hydrogen peroxide (H₂O₂) is a well-known property of several bacterial species associated with the human oral microbiota [99]. These representatives do not possess production mechanisms for H₂O₂-scavenging enzymes, including catalase and NADH peroxidase, and cannot biotransform it by themselves. Synthesis and accumulation of H₂O₂ is due to exposure to molecular oxygen of anaerobic LAB by oxidizing lactic acid, NADH, and pyruvate [100]. H₂O₂ is characterized by antibacterial activity, and, depending on various factors, it can exhibit a bacteriostatic or bactericidal effect (Figure 1) [101]. In direct co-cultivations of LAB, including Lactobacillus gasseri, W. cibaria, L. salivarius, and L. fermentum, with several periodontal pathobionts, including S. mutans, S. sanguinis, Streptococcus sobrinus, F. nucleatum, P. intermedia, P. gingivalis, and Porphyromonas catoniae, definite inhibitory activity on the number of viable cells was indicated [102,103].

Bacteriocins are metabolic products composed of short-chain peptides, polypeptides, proteins and protein complexes with antibacterial activity, which are synthesized by bacteria [104]. The main action of bacteriocins is to inhibit the development and reproduction of a variety of bacteria, through mechanisms of suppressing cell wall synthesis, increasing cell membrane permeability, suppressing nucleic acid and protein synthesis (Figure 1) [105]. Bacteriocins produced by Gram-positive bacteria are divided into several classes [106]. In a study by Messaoudi et al., the antimicrobial activity of bacteriocin produced by oral isolate of L. salivarius BGHO1 against S. mutans, S. pneumoniae, S. aureus, and E. faecalis was established [107]. Gautam and Sharma established the bacteriocin-synthesizing potential of Levilactobacillus spicheri G2, with the production of bacteriocin, exhibiting high activity against S. mutans, S. aureus, and Bacillus cereus [108]. In a study by da Silva et al., the authors established the antimicrobial activity of bacteriocin-like inhibitory substances (BLIS) produced by L. plantarum ST16 Pa, Lactococcus lactis CECT-4434, B. animalis subsp. lactis BL 04, and Lactococcus lactis subsp. lactis 27 against S. mutans, E. coli, S. aureus, Listeria innocua, and Carnobacterium maltaromaticum [109].

Exopolysaccharides (EPSs) are macromolecules which are secondary metabolic products of oral LAB and are associated with their probiotic functions. EPSs are synthesized as extracellular products and play a role as postbiotic components, providing health benefits to humans through modulation of the immune system, antimicrobial, antitumor, antioxidant and anti-inflammatory properties and modulation of the microbiome [110]. Some of the main bioactive EPSs from LAB include dextran, levan, inulin, and reuteran [111]. The production of EPSs by bacterial cells supports their normal growth in the presence of stress factors, such as changes in pH, dehydration, osmotic stress, antibiotics, bacteriophages, and lysozymes [112]. One of the main properties is their role as adhesins, promoting the adhesion of LAB to epithelial cells during colonization, and they are particularly important in the formation and accumulation of biofilms. These properties improve the survival and persistent presence of LAB in the human body [113]. Also, promoting their adhesion to mucosal tissue and the formation of biofilms may complement their antimicrobial and antibiofilm properties (Figure 1) [114]. Both antagonistic and antibiofilm activity have been established by a newly isolated oral LAB group against S. mutans and C. albicans [115]. The antimicrobial properties of EPSs produced by Bifidobacterium bifidum WBIN03 and L. plantarum R315 strains have been studied by Li et al. and Mahdhi et al. The authors reported that, at different concentrations, EPSs exhibit inhibitory effects against C. albicans, E. coli, B. cereus, S. aureus, and P. aeruginosa [116,117]. Allonsius et al. studied EPSs from L. rhamnosus GG and reported that, in a purified state, they were able to inhibit the formation of hyphae and adhesion of Candida through coaggregation, competition for binding sites, and immunomodulation of epithelial cells [118]. The antibiofilm activity of dextran produced by Weissella confusa was investigated by Rosca et al. The authors reported activity against the formation and pre-formed biofilms of C. albicans SC5314 [119].

LAB can produce substances with antioxidant properties that are safe and can have a variety of beneficial effects on the human oral cavity [92,120]. Antioxidants can avoid, reduce or interrupt oxidative damage and are widely used in pharmaceutical and food industries [121]. As a main function, antioxidants are able to neutralize free radicals and are closely related to health effects and can potentially prevent the development of many diseases [122,123]. It has been studied that LAB produce antioxidative substances that can neutralize reactive oxygen species (ROS), upregulate host antioxidant enzymes, downregulate enzymes related to ROS synthesis, and regulate the signaling pathway for antioxidant production in the host (Figure 1) [124]. Various oral LAB strains, including L. plantarum, Latilactobacillus sakei, L. gasseri, S. salivarius, and E. faecium, have been reported to demonstrate hydroxyl radical scavenging activities [123].

As different metabolites are produced from LAB, they are released in the oral environment and connect with saliva. Saliva by itself contains a wide variety of organic and inorganic components and plays crucial roles in oral health. Secreted metabolites can not only possess effects against pathobionts but could change the properties of saliva. Metabolites can affect salivary flow, buffering capacity, and pH fluctuations. This can lead to either balancing the oral environment and contributing to a healthy oral cavity or disrupt the equilibrium and cause dysbiosis and disease initiation [125,126].

5.2. Formation and Activity of Oral Biofilms

Oral biofilms are comprised of complex microbial communities that adhere to the hard and soft tissues in the oral cavity and are constantly influenced by mastication, periodical nutrient intake, and host immune responses, thus creating a dynamic microenvironment [127,128]. Microorganisms in these biofilms are embedded in a highly organized matrix, comprised of water, EPSs, proteins, lipids, inorganic ions, and extracellular DNA with different functions. EPSs stabilize the biofilm and facilitate microbial adhesion and aggregation; extracellular proteins promote structural integrity, enzymatic activity, and processing of nutrients; lipids enhance biofilm hydrophobicity and physical barrier functions; inorganic ions contribute to cross-link matrix components and regulate the demineralization–remineralization process; extracellular DNA from lysed cells maintain structural cohesion, enhance antimicrobial resistance, and trigger host immune responses (Figure 2) [129].

Microorganisms in the oral cavity form complexly structured biofilms, comprised of multiple layers of different species occupying distinct ecological niches. This multispecies biofilm architecture enables interspecies interactions and competition [130]. Within this structure, microorganisms arrange into spatial organizations to form multilayered communities. This structuring creates microenvironments with different nutrient gradients, oxygen levels, and metabolic activities. Reduction in local oxygen tension by the early colonizers, including Streptococcus spp., creates anaerobic niches for obligate anaerobes. Although probiotic LAB are mostly anaerobic, this decrease in oxygen levels makes a suitable environment for potential pathobiontic species implicated in periodontal diseases and can contribute to maturation and pathobiont-abundant oral biofilms [129]. The architectural complexity of biofilms provides physical stability and increases resistance to antibiotics and immune defenses, as well as influencing bacterial gene expression and metabolism. This promotes interspecies interactions and, in most cases, transforms commensal communities into pathobiontic with increased pathogenicity, evasion of immune responses, and pro-inflammatory activity [129]. Similar biofilm structures present a critical target for management of biofilm-associated oral diseases [131].

Initially, oral biofilms start to form from pioneer microorganisms that adhere to the salivary pellicle. This structure is comprised of proteins and carbohydrates that selectively bind oral microorganisms and is mediated by adhesion factors in the form of adhesins and biofilm-associated proteins [127]. Adhesion can be influenced by factors, including temperature, pH, nutrient content, and mechanical breakdown from the salivary flow [132]. Following the initial adhesion, structural and compositional changes occur. The early colonizers proliferate but new microbial species are also incorporated, increasing complexity and heterogeneity of the biofilm. Bacteria produce extracellular polymeric substances to form a matrix, which provide structural stability and promote interspecies interactions, including nutrient sharing and metabolic cross-feeding, thereby increasing microbial diversity [114,133]. As biofilms mature and become more diverse, their architecture and protective matrix increase the resistance to antimicrobial agents. A mature oral biofilm is structurally resilient, with metabolically active microbial communities that can endure environmental stress and support diverse microbial populations [134]. The stability is maintained through synergistic interactions between the matrix, microbial metabolism, and intercellular signaling pathways, as well as providing a physical barrier with antimicrobial functions [114,135].

The formation of bacterial biofilms is controlled by the quorum sensing (QS) system. With an increase in cell density, it allows bacteria to secrete chemical signal molecules that are used for intra/interspecific communication to coordinate group behavior through gene regulation [136]. Specific signals can activate physiological and biochemical reactions, including formation of biofilms, accumulation of extracellular matrix, production of secondary metabolites, and the QS system itself [137]. Bacteria in the oral cavity utilize QS to respond to environments continuously, to coordinate the expression of genes and coordinate colony behavior based on the density of their current population [136]. This interspecific communication is achieved through the LuxS/AI-2 signaling system, which is a universal QS system of autoinducer-2 (AI-2). In LAB small peptides are more prevalent as signaling molecules of the auto-inducing peptide (AIP) signaling system [138]. AIPs are cyclic thiodepsipeptide QS molecules synthesized by Gram-positive bacteria, which can regulate secondary metabolite production, including bacteriocins, and impact the composition of the microbiome [139].

Oral biofilms have a dual function regarding oral health. Under certain conditions, pathobionts can become dominant and biofilms can transition into a dysbiotic state, promoting oral infections and contributing to systemic diseases [140]. But oral biofilms are not intrinsically pathobiontic in nature and function as dynamic ecological communities essential for maintaining oral homeostasis and microbial balance. In a healthy host state, oral biofilms are dominated by commensal bacteria that act as a barrier against external pathogens and stimuli. This stable oral ecosystem can modulate host immune responses, promote immune tolerance, and prevent excessive inflammation. In this oral eubiotic state, balanced microbial interactions maintain tissue integrity and can prevent the occurrence of microbial dysbiosis [141,142]. Probiotic species from the Lactobacillaceae family can contribute to oral health by producing antimicrobial compounds, downregulating virulence genes, and preventing the adhesion of oral pathobionts, including S. mutans [142,143,144].

Commonly mediated by a biofilm is dental caries. The ability to inhibit the formation and growth of pathobiontic biofilms in the oral cavity is a crucial property of probiotic microorganisms. Some species, including L. fermentum, L. rhamnosus, L. plantarum, L. casei, L. acidophilus, L. brevis, L. delbrueckii, L. salivarius, Lactiplantibacillus pentosus, Latilactobacillus curvatus, S. salivarius, Streptococcus oralis, Bifidobacterium lactis, Bifidobacterium longum, and W. confusa, have been reported to exert antibiofilm activity against oral pathobionts [115,145]. Probiotic microorganisms, including S. salivarius K12, L. casei ATCC 393, L. reuteri ATCC 23272, L. plantarum ATCC 14917, and L. salivarius ATCC 11741, can modulate microbial composition and behavior by downregulating S. mutans glucosyltransferase genes (gtfB, gtfC, and gtfD), which suppresses cariogenic EPS synthesis and biofilm formation and stability [146,147,148]. LAB biofilms have also been shown to possess antifungal activity. An investigation involving L. plantarum, Lactobacillus crispatus, Limosilactobacillus vaginalis, and L. gasseri biofilms tested against C. albicans, C. tropicalis, C. glabrata, Candida lusitaniae, C. parapsilosis, and C. krusei clinical isolates. The results indicated that biofilm cell-free supernatants exerted higher fungistatic activity against all Candida representatives compared to planktonic cell-free supernatants [149]. Probiotics are also capable of inhibiting the yeast–hyphae transition of C. albicans, which is a crucial step in its pathogenesis. A combination of L. helveticus CBS N116411, L. plantarum SD5870, and S. salivarius DSM 14685 can significantly downregulate the expression of transition genes, including EFG1, SAP5, ALS3 and HWP1 [150].

The adhesion capability of LAB is the first step for determining their probiotic properties. The attachment to hard and soft oral tissues is determined by the physicochemical characteristics of the bacterial surface. Initial adhesion and biofilm formation are controlled by different genes, and adhesion is an important prerequisite for biofilm formation [151]. An important characteristic of probiotic microorganisms is the ability to outcompete potential oral pathobionts for adhesion sites and colonization [152]. A successful colonization of S. salivarius M18 showed anticaries activity as indicated by a reduction in plaque scores and S. mutans levels [153]. L. brevis KCCM 202399 showed antimicrobial and antibiofilm effects against S. mutans KCTC 5458 by reducing its self-aggregation, cell surface hydrophobicity, and EPS production [154].

Aggregation is very important regarding the formation of biofilms and is related to cell adherence properties. This trait directly involves the ability of LAB to survive and persist in the oral cavity. Co-aggregation is an advantageous property of LAB as it allows them to form a physical barrier that impedes surface attachment and colonization of potential pathobionts [155] and has been observed between microbial species in the oral microbiome’s composition. LAB, which can co-aggregate with oral pathobionts, may exert important host defense mechanisms against the development of infections [156]. LAB are reported to exhibit specific co-aggregative properties with oral pathobionts, including S. mutans and C. albicans in vitro [115,157,158].

6. In Vitro and Clinical Trials Evidence and Application of Lactic Acid Bacteria in Delivery Systems

6.1. In Vitro and Clinical Research Viewpoint

The prophylactic and therapeutic potential of probiotic microorganisms has been widely studied and has led to promising results in serious oral diseases, including dental caries and periodontitis [159,160,161]. Despite numerous studies and developments, questions remain about which probiotic species or strains are most effective against the different oral diseases, as well as for each population or individual [162]. In addition, questions remain about what carriers and at what dose a probiotic microorganism can be administered, as well as the frequency of its administration.

A number of authors have conducted studies with various probiotic strains of oral origin, establishing in vitro and clinical outcomes towards the prevention and therapy of various oral diseases, inhibition of oral pathobionts, as well as clinical indicators.

Table 2. Effects of studied probiotics on the development of different pathobionts and on different conditions of the oral cavity.

Type of Study	Probiotic Strain/s	Assessment	Results	Reference
in vitro	S. thermophilus, L. delbrueckii subsp. bulgaricus	Individual antagonistic evaluation of collection of LAB strains in planktonic form	Selective activity against S. mutans and presumed bactericidal effect of LAB in vivo	[163]
	L. rhamnosus ATCC 9595	Gingival stromal stem cells pretreatment with LAB strain and stimulation with P. gingivalis ATCC33277	Modulation of inflammatory signals by the LAB strain and induction of CXCL8 chemokine secretion to initiate rapid and effective immune response	[164]
	L. plantarum 44,048 and NC8	Antimicrobial evaluation of cell-free supernatants and two-peptide bacteriocin PLNC8 αβ	Growth inhibition of P. gingivalis from supernatants and PLNC8 αβ bacteriocins could prevent its colonization and subsequent pathogenicity	[165]
	L. salivarius BGHO1, Lactobacillus delbrueckii subsp. lactis BGHO99	Antimicrobial and antagonistic evaluation and pronase E bacteriocin signaling	Qualitative bacteriocin production and antagonistic activity against S. mutans and Streptococcus pneumoniae but not against C. albicans	[166]
	L. rhamnosus DSM16605, B. longum DSM 24706	Antagonistic and antibiofilm evaluation	Antagonistic activity against S. mutans and A. actinomycetemcomitans and efficacy in reduction of biofilm accumulation	[167]
	Lactobacillus crispatus, L. paracasei, L. gasseri, L. plantarum, L. rhamnosus strains	Preventive and treatment evaluation of inhibitory properties	Better expressed inhibition in the preventive rather than the treatment model, as well as decreasing the growth of S. mutans and A. actinomycetemcomitans	[168]
	S. salivarius AMBR074, AMBR075, AMBR024, and AMBR158	Antagonistic evaluation of direct interactions and flow cytometric evaluation of cell-free supernatants	Qualitative antagonistic properties and quantitative inhibitory activity against P. intermedia, P. gingivalis, and F. nucleatum	[169]
	Lactobacillus johnsonii MT4	Inhibition of planktonic and biofilm growth evaluation	Planktonic growth inhibition and reduction in metabolic activity of biofilms from C. albicans in a dose–response pattern	[170]
	L. crispatus, L. gasseri, Limosilactobacillus fermentum, L. salivarius	Adhesion to oral mucosal cells, salivary-coated hydroxyapatite and antibacterial evaluation	Expression of adherence to oral mucosal cells and salivary-coated hydroxyapatite, as well as antibacterial activity against A. actinomycetemcomitans and A. naeslundii	[171]
	L. salivarius CP3365	Inhibitory viability evaluation of heat-killed LAB cells	Significant decrease in viable cell count of P. gingivalis and F. nucleatum	[172]
Clinical	L. reuteri ATCC 55730 and ATCC PTA 5289	Double-blind, placebo-controlled trial with 42 participants consuming chewing gum for 2 weeks	Improved bleeding on probing, significant decrease in gingival crevicular fluid volume, tumor necrosis factor-α, interleukin-8, and interleukin-1-β, but unchanged levels of interleukin-6 and -10 during and after the trial	[173]
	L. salivarius WB21	Open-label comparative trial with 64 volunteers consuming an oral tablet a single time and short-term administration trial with 8 volunteers consuming oral tablets for 2 weeks	Open-label comparative trial—decrease in S. mutans levels, significant increase in LAB levels, no change in salivary flow and salivary pH, significant increase in salivary buffering capacity. Short-term administration trial—significant decrease in S. mutans levels after the trial.	[174]
	L. paracasei SD1	Randomized, double-blinded, placebo-controlled trial with 60 participants consuming milk for 12 months	Increase in salivary human neutrophil peptides 1–3, significant decrease in S. mutans counts and caries increment during and after the trial	[175]
	L. acidophilus strain	Double-blind parallel randomized trial with 60 volunteers consuming curd for 7 days	Significant increase in salivary pH and significant reduction in S. mutans counts after the trial	[176]
	L. reuteri ATCC PTA 5289 and DSM 17938	Randomized, double-blind, placebo-controlled trial with 39 participants consuming a single-time drop and lozenges for 12 weeks	No effect from the probiotic drops were observed. Significant decrease in probing pocket depth, gingival recession, clinical attachment level, full-mouth plaque and bleeding scores after 12 and 24 weeks. No influence in A. actinomycetemcomitans, F. nucleatum, P. gingivalis, and P. intermedia levels was observed.	[177]
	B. animalis subsp. lactis HN019	Randomized placebo-controlled trial with 30 participants consuming lozenges for 30 days	Decrease in plaque index and marginal gingival bleeding was observed and higher expression of βdefensin-3, toll-like receptor 4, and cluster of differentiation-4 during and after the trial	[178]
	Weissella cibaria CMU	Randomized, double-blind, placebo-controlled trial with 92 participants consuming tablets for 8 weeks	Improved bleeding on probing was observed, no change in probing depth, gingival index and plaque index, and significant change in the F. nucleatum levels during and after the trial	[179]
	L. casei strain	Randomized placebo-controlled trial with 41 participants consuming Swiss cheese for 28 days	Decrease in the total number of S. mutans, A. actinomycetemcomitans, and P. gingivalis during and after the trial	[180]
	W. cibaria CMU	Randomized, double-blind, placebo-controlled trial with 92 participants consuming tablets for 8 weeks	Significant reduction in subjective halitosis and improvement in quality of life of oral health after the trial	[181]
	L. salivarius CP3365	Randomized, placebo-controlled trial with 64 participants consuming oral tablets for 8 weeks	Plaque control record, bleeding on probing, and probing pocket depth parameters were decreased during the trial but no changes in P. gingivalis and F. nucleatum abundance	[172]

serves as exemplary material on research with specific strains and different outcomes from the conducted studies.

6.2. Delivery Systems Viewpoint

Research of probiotic strains incorporated into oral health products provides a modern approach to address oral infections before they occur. Replacement therapy in the form of probiotic products is an alternative and promising way to prevent diseases through competitive interactions of beneficial microorganisms with pathobionts [182]. In recent years, various probiotic strains have been added to a variety of carriers, including pastilles, hard and soft candies, lollipops, chewing gums, lozenges, ice cream and various dairy products, as well as oral hygiene products. The information below will provide exemplary material on different delivery system formulations, applied probiotic strains, and outcomes from the conducted studies.

Lozenges and candies are products that are easily prepared and can deliver multiple substances during their slow dissolution in the oral cavity [183]. During the time they are present in the oral cavity, they can contribute to a local beneficial effect in the prevention or therapeutic manipulation of oral diseases. Therefore, lozenges are a suitable potential carrier of probiotic strains for oral administration [184]. Studied L. reuteri strains ATCC 55730, DSM 17938, and ATCC PTA 5289 in lozenge formulations have been reported to significantly reduce gingival pocket depth on probing, gingival inflammation and bleeding, of saliva-associated S. mutans and C. albicans, and of P. gingivalis, P. intermedia, A. actinomycetemcomitans, F. nucleatum, and T. forsythia in patients with chronic periodontitis [185,186,187,188]. Lozenges containing L. brevis CD2 strain have been reported to reduce the development of severe forms of oral mucositis, dental biofilm accumulation, gingival inflammation, and gingival pocket depth on probing in aggressive periodontitis [189,190]. Prolonged use of candy with L. paracasei DSMZ16671 has been reported to reduce the levels of saliva-associated S. mutans [191]. Lozenges containing S. salivarius M18 have been reported to possess inhibitory activity against S. mutans, S. mitis, S. sobrinus and Rothia mucilagenosa, as well as plaque index reduction [153]. Long-term application of lozenge formulations containing L. rhamnosus GG and B. lactis BB-12 has been reported to reduce gingival and plaque indices [192].

Tablets are a commonly used delivery vehicle for probiotics and have been used in numerous scientific studies [157]. They contain components that have undergone freeze-drying and machine tableting or molding processes. Long-term intake of freeze-dried tablets containing inactivated L. paracasei 111 and 141 strains has been reported to decrease the depth of the gingival pocket on probing, inhibition of the development of periodontal diseases, as well as improvement of the condition of oral halitosis [157]. Authors have reported significant reductions in S. mutans, F. nucleatum, P. gingivalis, P. intermedia, and A. actinomycetemcomitans levels with long-term use of probiotic tablets containing L. reuteri ATCC 55730, ATCC PTA 5289, and DSM-17938 [193,194]. Other authors have reported a significant improvement in plaque index, decrease in the concentration of volatile sulfur compounds, and reduced gingival pocket depth on probing in patients with an increased risk for periodontal diseases and halitosis after prolonged intake of tablets with added L. salivarius WB21 [195,196].

Chewing gum is a widely used product for oral hygiene. Many products containing adjuvants such as xylitol, sorbitol, and chlorhexidine have been used in studies as antibiofilm agents and to increase pH and salivary flow [197]. The addition of probiotic microorganisms to the composition of various chewing gum bases to reduce the presence of oral pathobionts provides new ways to prevent infections of the oral cavity [198]. A significant reduction in saliva-associated S. mutans with prolonged use of probiotic chewing gum and mastic gum with added L. reuteri ATCC 55730 and ATCC PTA 5289 has been reported [198,199]. Long-term use of probiotic gum and toothpaste with added L. reuteri SGL 01, L. salivarius SGL 03, and L. plantarum SGL 07 strains has been reported to reduce F. nucleatum and P. intermedia concentrations [200].

Dairy products are some of the most widely consumed products worldwide [201]. Numerous types of dairy products are supplemented with additional probiotic strains and are easily available and suitable for all ages [182]. Ice cream is a food product with significant nutritional value, well digestible by the human body, and a suitable carrier for delivering probiotic microorganisms [202,203]. B. lactis BB-12 and L. acidophilus LA-5 has been reported to successfully colonize upon long-term intake of probiotic ice-cream-based product, as well as significantly inhibit saliva-associated S. mutans [203,204,205,206]. Prolonged intake of probiotic milk with added L. rhamnosus GG has been reported to reduce the risk of caries development [207]. Long-term consumption of cheese formulations with added L. rhamnosus GG, LC 705 and Propionibacterium freudenreichii subsp. shermanii JS has been reported to reduce the presence of S. mutans and Candida in the oral cavity [208,209]. Long-term consumption of milk with added L. rhamnosus strains has been reported to reduce the risk of caries development, as well as the levels of saliva-associated S. mutans [210,211]. Long-term consumption of milk and milk powder with L. paracasei SD1 has been reported to decrease S. mutans levels and increase the presence of the probiotic strain in the oral cavity [212,213]. Consumption of B. lactis BB-12 incorporated into cottage cheese has been reported to decrease S. mutans presence and increase salivary pH values [182].

Other model products, including sachets, oil drops, freeze-dried capsules, etc., are also being formulated and investigated as suitable vessels for probiotics. Controlled continuous drinking of water through straws with immobilized L. reuteri ATCC 55730 has been reported to significantly reduce S. mutans levels [193]. Freeze-dried L. rhamnosus, Bifidobacterium sp., and Bacillus coagulans in powdered form has been reported to reduce the levels of saliva-associated S. mutans during administration [214]. Long-term oral intake of probiotic drops containing refined oils and freeze-dried L. reuteri ATCC 55730 has been reported to reduce the incidence of caries and improve the gingival index [215]. After long-term use of capsules containing freeze-dried L. acidophillus HS101, L. rhamnosus HS111, and B. bifidum has been reported to significantly inhibit the presence of Candida sp. in patients without clinical symptoms [216]. Long-term application of drops with refined oils, a prebiotic and added L. reuteri ATCC 55730, L. rhamnosus ATCC 15820, and Bifidobacterium longum subsp. infantis ATCC 15697 strains has reported a decrease in saliva-associated S. mutans [217]. Mouth rinsing with water-dissolving capsules containing L. salivarius and L. reuteri has been reported to improve plaque and gingival indices [218]. Long-term use of sachets containing L. rhamnosus SP1 has been reported to improve periodontal health in patients with chronic periodontitis [219]. Long-term application of freeze-dried inactivated cells of L. salivarius CECT 5713 dissolved in water and used as a mouthwash has been reported to reduce the concentration of S. mutans in saliva [220].

6.3. Critical Analysis

Oral probiotic formulations show promising results in inhibiting oral pathobionts, balancing the oral microbiome, and improving oral health conditions. However, evaluations from studies reveal several limitations, inconsistencies, and non-regulatory oversight.

Many studies apply a variety of probiotic strains, different dosages in the product formulations, as well as numerous delivery vehicles. Probiotic efficacy is highly strain-specific, but trials often use different strains or combinations, hindering the comparison of results across studies. Furthermore, there is an absence of standardized dosages, expressed in colony-forming units (CFU) counts and, in some cases, these are not reported. This methodological inconsistency complicates the meta-analyzation and comparison of effectiveness across results. Data from trials indicate that applied probiotics have transient colonization in the oral cavity. Their inability to permanently colonize in the oral environment requires continuous intake for the specific effects to persist. Most studies evaluate achieved results over a short time which is insufficient to determine sustained health benefits and potential side effects remain understudied. Many randomized controlled trials are also unreliable, involving too few participants to make solid conclusions regarding efficacy. This makes it difficult to distinguish the probiotic effect from the placebo [5,59,221,222,223,224].

In order to improve these limitations, trials should be standardized considering dosages, defined delivery methods, and long-term studies. Studies are needed to determine how long benefits last after discontinuation and the safety of long-term consumption. Relying on clinical markers, including gingival index, plaque index, etc., provides insufficient data on how the oral environment changes after probiotic application. Molecular methods, including 16S rRNA sequencing, RAPD, and metagenomic analyses should be implemented to measure how probiotics transform the oral microbiome. Quality control of products to improve regulations or standards should also be considered. Analysis on CFU counts should be accurately provided, in order to ensure viability at the time of consumption [5,59,221,222,223,224].

7. Challenges and Future Perspectives in Oral Probiotics Development

The evaluation of probiotic candidates, formulation of suitable carrier matrix, and sustainability present many difficulties. Assessment of new isolates often narrows down suitable candidates for specific application in the oral cavity as many effects are strain-specific [225]. On the other hand, development of new formulations as a delivery system encounters challenges and key points should be taken into account, including selection and integration of the composition components, initial stability of the matrix base during preparation, long-term stability of the product with and without the addition of probiotic strains, viability of the strains after incorporation, cross-contamination with undesirable microorganisms, and assuring minimum permissible resident microbiota of non-heat-treatable ingredients.

Another aspect is maintaining the probiotic strains viable in the final product. This mainly refers to strains that are susceptible to technological processing and long-term storage. Some strains may decrease in viable cell counts in a short period of time, leading to the inability of colonizing in the oral cavity. Colonization and persistence in the oral cavity are another important matter. Many LAB often possess lower affinity to hard dental surfaces and/or are transient, often affected by salivary flow [226]. Also, for some strains (e.g., L. acidophilus and L. casei) the production of excess lactic acid is probable, lowering oral pH and increasing the risk of caries initiation [96]. Other limitations also apply, as constant intake of certain probiotic product could lead to undesirable and unpredictable changes in the oral microbial populations, although research on the long-term ecological impacts in the oral cavity is scarce [225].

Future concern regarding the application of exogenous probiotics is their foreign nature to the immune system of the host that, in rare cases, could increase the chance of oral disease development. A promising approach is the incorporation of autoprobiotics into oral products. It involves isolation of microorganisms from the oral cavity of the host and developing personalized products to restore balance of the oral microbiota. This approach is considered as a modern direction with better efficacy in periodontal disease treatment, including reducing gum inflammation and pathobiont counts in periodontal pockets, but needs more research [227,228].

The majority of research on conventional probiotic microorganisms continues to develop and expand across scientific and industrial fields. The knowledge on established benefits of their direct physical interactions and metabolic activity can shift research to look at non-conventional and next-generation microorganisms as promising probiotic candidates. Several bacterial species have already been identified as potential probiotic candidates, including Akkermansia muciniphila, Bacteroides thetaiotaomicron, Christensenella minuta, and Faecalibacterium prausnitzii, as well as yeast species from the genera Debaryomyces, Hanseniaspora, and Pichia. These microorganisms are currently undergoing research and development and have demonstrated the capacity to exert beneficial effects regarding different systemic disorders [229,230]. Research on these bacterial and yeast representatives for probiotic characteristics aimed at the oral cavity and against oral pathobionts could be a promising scientific contribution.

Besides the traditional product formulations, non-conventional probiotic delivery systems are gaining attention in order to surmount limitations [231]. Innovative methods for delivery systems preparation are already being studied and incorporate different EPSs, including gum arabic and starch-based biopolymers, which are easily soluble in the GIT and could also be promising for oral health products [232]. Technological processes, including spray-drying and freeze-drying rely on the addition of protectants to ensure cell viability. The implementation of natural products, like fruits in the form of juices and pulps with the addition of prebiotic components, could increase the development of better and more stable delivery systems [230,233].

8. Conclusions

Microbiological and biotechnological research on microbial interactions in the oral microbiome and evidence on developed oral probiotic products show promising results. The composition of oral microbiota shows the importance of maintaining an ecological balance, to prevent transition of oral commensal microorganisms to pathobionts. Probiotic LAB are well studied and beneficial effects have been established in the prevention and treatment of oral diseases. Outcomes from in vitro and clinical studies with different delivery vehicles suggest oversight on strain-specific traits, rather than the application of certain species of probiotics. Although short-term benefits have been widely observed, the persistence of these effects is still understudied, suggesting longer periods of application and standardization among studies. Overall, probiotic product development is consistent and further optimization of the addition of probiotic microorganisms, vehicle formulations, and trials will effectively guide their successive application.