The Effect of Dairy Probiotic Beverages on Oral Health

THE EFFECT OF DAIRY PROBIOTIC BEVERAGES ON ORAL HEALTH

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Marcela Baraúna Magno⁎, Patricia Nadelman⁎, Thayse Caroline de Abreu Brandi⁎, Matheus Melo Pithon†, Andréa Fonseca-Gonçalves⁎, Adriano Gomes da Cruz‡, Lucianne Cople Maia⁎ *

Department of Pediatric Dentistry and Orthodontics, School of Dentistry, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil † Department of Health, School of Dentistry, Universidade Estadual do Sudoeste da Bahia (UESB), Bahia, Brazil ‡Food Department, Instituto Federal de Educação, Ciência e Tecnologia do Rio de Janeiro (IFRJ), Rio de Janeiro, Brazil

15.1 Introduction Functional beverages are used to provide specific health benefits beyond those of general nutrition. They are defined as nonalcoholic drinks containing special ingredients, including herbs, vitamins, minerals, amino acids, raw fruit, or other added vegetable ingredients (US Functional Beverages Market: A Young Market with Growing Popularity). Probiotics can be included within these functional products, as they are considered living microorganisms safe for human consumption that when ingested in sufficient quantities result in beneficial effects on human health (Wu and Wei, 2002). Probiotics compete with other microorganisms for adhesion sites and/or nutrients, inactivate toxins, and their receptors, modify the environment, produce bacteriocins that inhibit pathogenic bacteria, provide bioactive or regulatory metabolites, and prevent colonization by other microorganisms (Ishihara et  al., 1985; de Vrese and Schrezenmeir, 2008; Grudianov et  al., 2002; Tsubura et  al., 2009; Reid et  al., 2003; Gill et al., 2001). In this way, probiotics inhibit some pathogens and influence the host’s adaptive immune response (Arunachalam et al., 2000; Diaz-Ropero et al., 2007). There are several vehicles for probiotic administration, such as ice cream, capsules, chewing gum, and Milk-based Beverages. https://doi.org/10.1016/B978-0-12-815504-2.00015-3 © 2019 Elsevier Inc. All rights reserved.

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cheese; however, milk, fermented milk, and yogurt are the most common (McGee, 2003). Furthermore, dairy foods provide health benefits due to the presence of nutrients that are vital for the health and homeostasis of the human body. These nutrients include calcium, potassium (Muehlhoff et al., 2013), vitamins, and protein (Wendling and Weschenfelder, 2013). The most commonly used probiotics for the oral environment are Lactobacillus and Bifidobacterium species (Meurman and Stamatova, 2007). New strategies to prevent oral diseases are based on the inhibitory effect of these probiotics on the growth of pathogenic agents (Reid et al., 2003). This is interesting, as the oral cavity has a large microflora, including microorganisms that cause diseases such as dental caries, gingivitis, periodontitis, candidiasis, and halitosis (Baehni and Takeuchi, 2003). Moreover, saliva has immunological components that can be modulated by probiotics (Harbige et al., 2016; Yamamoto et al., 2017). Considering that differences between the intrinsic quality of saliva and individual oral environment vary based on eating habits, probiotics have been consumed for therapeutic and prophylactic reasons (Karuppaiah et al., 2013; Staab et al., 2009; Slawik et al., 2011; Ishikawa et al., 2011; Parra and Martínez, 2007; He et al., 2008; de Vrese et al., 2011; Tongtawee et al., 2015). It is worth emphasizing that the incorporation of probiotics into the diet can be associated with a positive impact on quality of life and contribute to the prevention/control of various oral diseases, thus improving the population’s health (Laleman et al., 2014; Mendonça et al., 2012). In this regard, the present chapter aims to present and discuss an updated viewpoint of the effects of probiotic dairy drinks on oral health, describing the relationship among the consumption of these functional beverages and the main oral diseases, oral microbiota, and salivary immune components.

15.2  Dairy Probiotic Beverages as Functional Foods The philosophy that food can be health promoting beyond its nutritional value is gaining acceptance. The known disease prevention aspects of nutrition have led to a new science, “functional food science.” Functional foods, first introduced in Japan, can be described as foods or food ingredients that may provide health benefits and prevent diseases (Van den Driessche and Veereman-Wauters, 2002). Functional beverage consumption is included among the desirable food habits that may contribute to overall health and disease prevention (Wu and Wei, 2002). These beverages, with health-promoting properties, are nonalcoholic drinks containing special nutrients and/or ingredients,

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such as vitamins, herbs, minerals, amino acids, raw fruit, vegetables, and probiotics (US Functional Beverages Market: A Young Market with Growing Popularity). Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host (Hill et al., 2014). Lactic acid bacteria (LAB; Firmicutes), Bifidobacteria (Actinobacteria), yeasts (fungi), and molds (fungi) are predominantly found in fermented dairy, meat, cereal, vegetables, and other fermented foods and beverages (De Roos and De Vuyst, 2017). The most studied and widely used vehicle for the administration of probiotics is dairy products, which includes milk and its processed derivatives such as cheese, yogurt, curd, ice cream, and kefir. The factories that produce these foods are characterized by the handling of milk, a highly perishable product, which must be kept under surveillance and analyzed correctly during all steps of the cold chain until its delivery to the consumer (McGee, 2003). Most commonly used milk in dairy production is cow’s milk, although milk from other mammals such as goats, sheep and in some countries, buffalo, camel, yak, and mare (among other animals) is also used (Mattila-Sandholm and Saarela, 2003). The most popular probiotic foods are produced by the dairy industry because fermented dairy products have been shown to be the most efficient delivery vehicle for live probiotics (Araújo et  al., 2002). The main dairy probiotic drinks that have been studied are milk, fermented milk (Çaglar et al., 2008), and yogurt (Fuller, 1991). These beverages also contain proteins, fats, lactose, minerals, and vitamins and are considered nutritious (Yin and Yang, 2016). Several benefits are attributed to the consumption of beverages containing probiotic microorganisms. The health benefits are directly related to the mechanism of action of probiotics, which includes: influence of host gut microbiota and pathogenic bacteria, improvement in specific enzymatic activities, the production/induction of antibacterial/bacteriocin-like and defensin substances, competitive exclusion of pathogenic bacteria, improvement in intestinal barrier function, modulation of host immune functions, intestinal carcinogenesis and cholesterol uptake, alteration of local pH, competing for nutrients, and forming physical barriers (Ishihara et  al., 1985; Reid et al., 2003). The scientific and popular interest concerning the effects of probiotics on health has increased due to beneficial pharmacological properties demonstrated in clinical studies such as the treatment of ulcerative colitis (Ishikawa et  al., 2011), treatment of lactose intolerance (Parra and Martínez, 2007; He et al., 2008), treatment of diarrhea (de Vrese et  al., 2011), and antimicrobial activity (Tongtawee et  al., 2015) among others.

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The oral cavity is a very broad ecosystem containing hundreds of bacterial species. Therefore, probiotics have recently been introduced in dentistry for the prevention or treatment of biofilm-dependent oral disease. Probiotics have shown to be useful in the prevention and treatment of tooth caries, periodontal diseases, oral soft-tissue lesions, candidiasis, and halitosis (Gungor et al., 2015).

15.3  Process of Dental Biofilm Formation and Biofilm-Dependent Oral Disease Biofilm is defined as a population or community of bacteria living in organized structures at an interface between a solid and a liquid surface (Fejerskov, 2004). Dental biofilm is described as a relatively indefinable microbial community associated with the tooth surface or any hard non-shedding material (Simon-Soro and Mira, 2015). A biofilm formed on teeth, implants, or dental material surfaces is called a dental plaque or dental biofilm. These microbial communities are complex and dynamic structures that accumulate through the sequential and ordered colonization of multiple oral bacteria (Hojo et al., 2009). When basic oral hygiene is poor, with (Fig. 15.1) or without orthodontic appliances (brackets, bands, wires, and other attachments), when there is an excess or lack of restorative material (over contours

Fig. 15.1  Patient with an orthodontic appliance, poor hygiene, and biofilm accumulation.

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and gaps), ill-adapted prostheses and dental implants with exposed thread, areas of undisturbed biofilm will be created. The accumulation of dental biofilms, which are accompanied by a change in bacterial composition, form the main aetiological factors for the majority of the buccal illnesses such as dental caries, gingivitis, periodontitis, and others (Baehni and Takeuchi, 2003).

15.3.1 Biofilm Formation Process The formation of a biofilm in the oral cavity is a multistage process. Soon after teeth, implants, and dental materials are cleansed a thin film derived from salivary proteins (called an acquired pellicle) covers these surfaces. Oral bacteria colonize this surface by adhering and co-aggregating to the pellicle, leading to the formation of multilayered cell clusters in the polymer matrix. The initial colonizers are streptococci (Streptococcus viridans, Streptococcus mitis, Streptococcus oralis) (Hojo et al., 2009). The bacteria that are unable to bind directly to the tooth surface may bind via receptors to the cell surface of early colonizers. Coaggregation is a specific cell-to-cell reaction that occurs between different bacterial species and is an important mechanism leading to bacterial colonization and dental biofilm formation. The bacteria multiply and co-aggregate with partner species. However, the co-aggregation process can occur between more than three bacterial species, connecting species that are not co-aggregation partners. Fusobacterium nucleatum can co-aggregate with many oral bacteria, such as streptococci and obligate anaerobes. Thus, this species is a key component in dental biofilms, bridging the early and the late colonizers. Co-aggregation among oral bacteria goes beyond physicochemical mechanisms and enables metabolic communication and genetic exchange, as each bacterium can easily access a neighboring bacterial cell and its metabolites. Oral bacteria present in dental biofilms provide their metabolites as energy sources for other members (Hojo et al., 2009). While cooperative interactions exist in dental biofilms, antagonistic interactions too exist, with bacteria competing for nutrients. Some bacterial species are able to produce substances that inhibit the growth of other closely related bacterial species or strains, thereby selecting their neighbors and promoting the establishment of a community with specific bacterial species. These substances are called bacteriocins (Hojo et al., 2009). As observed by Hojo et al. (2009), the mature biofilm has a “circulatory system,” behaving as a complex microorganism. In collaboration with each other, oral bacteria may confront oxygen, host immunity, and antimicrobial agents as a united barrier through

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dental biofilm formation. In this process, the bacterial composition of the biofilm often changes from a scant biofilm dominated by Gram-positive bacteria (usually observed in healthy individuals) to an increased number of Gram-negative anaerobic bacteria (usually found in periodontitis) (Hojo et al., 2009). Finally, it is important to remember that not only bacterial interactions but also bacterial-fungal associations can promote virulence in disease-associated biofilm bacterial composition (Harriott and Noverr, 2011). This disease, also called dysbiosis, results from imbalances, often through external factors (as disease or medicaments), in the microbial communities that normally colonize the oral cavity, as well as other parts of the body (Cho and Blaser, 2012). Hundreds of bacterial species inhabit the oral ecosystem and most are considered commensals. The majority of oral diseases have been clearly identified as polymicrobial, including gingivitis, periodontitis, halitosis, and root canal infections (Rôças and Siqueira, 2012). However, the pathogenic species are also frequently found in healthy subjects, although at lower levels than in individuals with disease (Human Microbiome Project Consortium Structure, 2012). Low-level detection of pathogenic microorganisms in healthy individuals strongly suggests that they cannot be considered infectious organisms and are thus better described as pathobionts (Ayres et al., 2012). These organisms are resident bacteria with the potential to cause disease and under homeostatic conditions, the immune system does not have an active response against them. Knowing that oral disease-associated bacterial biofilm composition varies at different stages of disease progression, initial hypotheses regarding the microbiology of oral diseases gave way to ecology-based propositions where the disease is seen as the output of a skewed microbial community arising due to environmental changes. Thus, most oral diseases are now viewed as a dysbiosis (Simon-Soro and Mira, 2015).

15.3.2  Probiotics and Dental Biofilm Oral hygiene maintenance is directly related to the control of biofilms in the oral cavity. Mechanical removal of supragingival plaque, through tooth brushing and dental floss use, is the most effective tool to prevent most oral diseases (Slot et al., 2012) and an indispensable daily chore for every individual. Three factors determine whether disease will develop in an individual: a susceptible host, the presence of pathobionts, and the reduction or absence of the so-called beneficial bacteria (Salvi and Lang, 2005). In relation to the third aetiological factor‚ the use of probiotics has been proposed as an alternative preventive tool. The introduction of strains could theoretically modify key microbial interactions

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in the oral biofilm, favoring a healthy, balanced ecosystem, as observed in some studies of dental plaque and probiotic dairy products (Karuppaiah et al., 2013; Staab et al., 2009; Slawik et al., 2011). Lactobacillus is largely used as probiotic microorganism when studying the association of probiotics and plaque index, as this microorganism can co-aggregate with F. nucleatum, modulating the composition of oral biofilms (Koll-Klais et al., 2005). The mechanisms of action of probiotics in dental biofilm are not yet clearly described; however, some hypotheses are suggested in Table 15.1.

15.3.3  Dairy Products and Biofilm Quantity Some studies have observed a significant increase in biofilm accumulation with the use of a commercial fermented milk drink containing Lactobacillus casei Shirota (Yakult®) for over a 4- and 8-week period.

Table 15.1  Possible Mechanisms of Action of Probiotics in Dental Plaque Ishihara et al. (1985)

Grudianov et al. (2002); Tsubura et al. (2009) Reid et al. (2003)

Arunachalam et al. (2000); Gill et al. (2001); Diaz-Ropero et al. (2007)

de Vrese and Schrezenmeir (2008)

de Vrese and Schrezenmeir (2008)

Probiotics may affect the oral ecology by specifically preventing or modulating the adherence of other bacteria and by modifying the protein composition of the salivary pellicle or reducing the pH, such that microorganisms cannot form a dental plaque Probiotics can alter the composition of dental biofilm by colonizing microbial biofilms, co-aggregating and competing with pathogenic bacteria, subsequently replacing or reducing their numbers Probiotics can produce antimicrobial substances such as organic acids, hydrogen peroxide, and bacteriocins, resulting in the inhibition of pathogenic bacteria Promoting a beneficial host response through modulating immunological parameters. For example, enhanced phagocytic activity and improvements in natural killer (NK) cell activity and in the percentage of NK cells in the peripheral blood of humans, as well as exerting immunostimulatory and anti-inflammatory responses Interaction with pathogenic bacteria (genetic changes, or by producing bioactive or regulatory metabolites) resulting in the reduction or death of pathogenic bacteria Probiotics can also exert effects either by modulating immunological parameters, epithelial permeability, and bacterial translocation or by producing bioactive or regulatory metabolites

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However, it is important to highlight that participants were advised not to brush their teeth for 4 days or more. There is no doubt that after these periods of refraining from mechanical and chemical plaque control, plaque accumulates. Despite this, the observed biofilm increase was more pronounced in the probiotic group. It could be hypothesized that this effect was due to the increased availability of carbohydrates for the oral microorganisms in the tested probiotic milk drink, perhaps followed by a shift in the composition of the oral microflora. It is also important to highlight that a higher plaque score was associated with a reduction in plaque-induced gingival inflammation (Staab et al., 2009; Slawik et al., 2011). So, in general, dairy products appear to be associated with increased biofilm accumulation; however, when associated with probiotics this does not interfere with other oral health indices related to gingival health or dental caries, suggesting that the microbial composition of the biofilm is beneficially altered (Karuppaiah et al., 2013; Staab et al., 2009; Slawik et al., 2011). Although not related to beverages, some reports concerning dental plaque and dairy probiotics used a short-term daily ingestion of probiotics delivered in the diet via curd and showed subsequently reduced levels of biofilm (Karuppaiah et al., 2013).

15.4  Dental Caries 15.4.1  Definition and Aetiopathology For decades, dental caries has been defined as a multifactorial disease, depending on at least three essential factors: (1) the host, represented by the individual and his components such as the oral cavity, teeth, salivary flow, oral hygiene habits, etc.; (2) the microbiota, represented by the oral microorganisms; and (3) the substrate, represented by the individual’s diet, characterized by frequency of consumption of fermented carbohydrates—mainly sucrose, which is the most cariogenic (Keyes, 1960) Actually, dental caries is considered a dysbiosis and can be defined as a biofilm sugar-dependent disease that affects both temporary and permanent teeth. In summary, the resident microorganisms in the oral cavity form biofilms on teeth (Fejerskov, 2004). As mentioned before, biofilms are biological communities presenting a high degree of organization, where bacteria form structured, coordinated, and functional communities. Biofilm bacteria live in microcolonies encapsulated in a matrix of extracellular polymeric substances (Davey and O'toole, 2000). The dental biofilm is formed by microorganisms adhered to dental surfaces and the resident flora in the oral cavity form biofilms on teeth (Fejerskov, 2004).

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Of the oral microorganisms, species involved in the caries process such as Streptococcus mutans, Streptococcus sobrinus and Lactobacillus are frequently studied due to their ability to rapidly make acid from dietary carbohydrates (acidogenic microorganisms), capacity to tolerate low pH (aciduric microorganisms), and ability to produce extracellular and intracellular polysaccharides, which aid the adherence to dental surfaces and serve as a nutrient reserve, respectively (Simon-Soro and Mira, 2015). However, biofilm accumulation alone is not enough for caries lesion progression. In this process, sugars are fundamental for the disease progression (Sheiham and James, 2015). After sugar ingestion, oral sucrose-dependent microorganisms are prone to ferment it. As a consequence of this fermentation process, acids are produced, which lower the biofilm fluid to around pH 5.0 or below (Cury et  al., 2016). The acid pH produced not only provokes dissolution of the underlying dental minerals but also selects the most cariogenic bacteria in the biofilm (Marsh, 2006). These factors contribute to the beginning and progression of dental caries, since the acidic oral environment caused by bacterial fermentation, disturb the Ca2+, (PO4)3−, and F− ions that are important for maintaining the mineral equilibrium between the tooth and the oral environment. Low pH and the concentration of these ions are critical factors in the loss and gain of minerals in the saliva/biofilm/teeth environment. The acid from sucrose fermentation binds to the Ca2+ and (PO4)3− causing dental demineralization and consequently, a caries lesion develops (Buckley et al., 2014). Dietary fermentable sugars (glucose, sucrose, and fructose) are involved in the establishment of dental caries and are considered the primary factors responsible for biochemical and physiological changes in dental biofilm (Stephan, 1944; Bowen et al., 1966). In summary, there is no dental caries without sugars, since they are responsible for the chain of causality. Among these dietary sugars, sucrose is the most cariogenic because, in addition to being easily fermented into acids, it is the only carbohydrate that changes the matrix of the biofilm in a more cariogenic matrix (Paes Leme et al., 2006). Frequent long-term carbohydrate consumption increases the proportions of S. mutans, S. sobrinus, and Lactobacillus, with a concomitant decrease in levels of Streptococcus sanguinis and other oral streptococci (De Stoppelaar et al., 1970; Dennis et al., 1975). These bacteria catabolize dietary sucrose and convert it into polysaccharides. Many of the polysaccharides are hydrolyzed and subsequently used for growth during periods when no readily fermentable dietary carbohydrates are present in the oral cavity (Staat et al., 1975). Some hypotheses for caries development are illustrated in Table 15.2 (Paes Leme et al., 2006).

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Table 15.2  Some Hypothesis for Caries Development (Paes Leme et al., 2006) (1) Constant low pH values attained in the biofilm matrix, due to persistent sucrose fermentation, would dissolve mineral reservoirs or avoid their storage (2) Enamel could take up these ions Ca+2, (PO4)−3, and F− from dental biofilm fluid (3) The low pH values caused by sucrose fermentation in biofilms would release the reservoir of ions bound to bacterial cell walls (4) Low bacterial density due to high insoluble extracellular polysaccharide content could result in fewer binding sites for these ions Ca+2, (PO4)−3, and F− (5) Low concentrations of specific ion-binding proteins could result in fewer mineral reservoirs in biofilms formed in the presence of sucrose

Clinically, caries signs may present in two forms: active white spot lesions (non-cavitated lesions with enamel mineral loss) (Fig.  15.2) (Denis et  al., 2013) and cavitated lesions [lesions with mineral and structural loss reaching enamel, dentin, or cementum (root structure)] (Fig. 15.3) (Fontana, 2015).

Fig. 15.2  Patient with dental caries (active white spot lesions) after orthodontic appliance removal due to poor oral hygiene and biofilm accumulation.

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Fig. 15.3  Patient with cavitated caries lesions.

15.4.2 Salivary IgA and Caries Saliva plays an important role in maintaining the equilibrium of the oral ecosystem, which is essential for dental caries control. Saliva is able to maintain the oral microbiota balance because it contains a variety of proteins. The advantages of these proteins are as follows: (1) they are essential constituents of the acquired pellicle; (2) they encourage bacterial aggregation; (3) they are a source of food for certain bacteria; (4) they possess antimicrobial activity due to some of them being capable of modifying bacterial metabolism; and (5) they have the ability to adhere to the surface of the tooth (Bagherian and Asadikaram, 2012). Immunoglobulins are soluble proteins and important components of the immune response. They are present in saliva and other body secretions such as serum, milk, and tears. Different types of immunoglobulins are found in saliva (IgG, IgM, IgE, IgD), with secretory immunoglobulin A (s-IgA) being the predominant type (Rúpolo et  al., 1998). Studies have shown that saliva displays altered protein levels (including immunoglobulins) under oral and systemic pathological conditions (Akram et al., 2016; Rangbulla et al., 2017). Previous studies have shown that people with dental caries present higher s-IgA concentrations when compared with people who are caries-free (Pal et al., 2013; Hagh et al., 2013). A direct relationship has also been found between higher levels of s-IgA and the number of lesions on carious surfaces (Chopra et al., 2012). In a 1-year prospective study, Parisotto et  al. (2011) found high concentrations of total s-IgA in children with dental caries and preschoolers with a lower baseline level of salivary IgA antibody reactive to S. mutans had a 7.5-fold greater risk of developing caries during the

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period of the study (Parisotto et al., 2011). This result was confirmed in a systematic review and meta-analysis, where higher levels of s-IgA were found in the caries-active group. This finding demonstrates that IgA is associated with the immune response to the caries disease. IgA may increase in response to the exposition of dental caries, or cariogenic microorganisms, as a form of a protective mechanism against caries attack. In addition, specific antibodies could play a role in oral/ bacterial homeostasis (Fidalgo et al., 2014). There are some mechanisms of action related to s-IgA such as it prevents the adherence of cariogenic microorganisms to hard surfaces of the tooth, inhibits the activity of glucosyltransferases (enzymes responsible for producing insoluble extracellular polysaccharides from sucrose degradation in S. mutans), neutralizes viruses and toxins, inactivates enzymes, excludes antigens in saliva, and prevents activities that may aid cariogenic microorganism colonization (Smith and Mattos-Graner, 2008; Law et al., 2007).

15.4.3  Probiotics and Dental Caries New approaches to improve oral health aimed at modulating inflammation and reducing the amount of biofilm or microorganisms have been researched and developed. One of the novel strategies to combat dental biofilm diseases is the use of probiotics, particularly strains belonging to the genera Lactobacillus and Bifidobacterium, due to their capacity to adhere to and colonize various surfaces of the oral cavity (Meurman and Stamatova, 2007). To have a beneficial effect in limiting or preventing dental caries, a probiotic must be able to adhere to dental surfaces and integrate into the bacterial communities making up the dental biofilm. It must also compete with the cariogenic bacteria; thus, preventing their proliferation and instituting a healthy oral colonization (Bonifait et al., 2009). In addition, some probiotics can modify the cariogenicity of S. mutans when they coexist within the microbial consortium of the biofilm (Fernández et al., 2015). Another probiotic modality for preventing and controlling dental caries could be bacterial co-aggregation (the aggregation relationship between microorganisms of the same species or between different species) favoring their adhesion to dental surfaces. Some probiotic strains tested in an in vitro study co-aggregated with oral pathogens and S. mutans showed a higher capability to co-aggregate with probiotic strains rather than pathogens. Among probiotic microorganisms, Lactobacillus acidophilus demonstrated the best co-aggregation (Twetman and Stecksén-Blicks, 2008). Various efforts have been made to affect the prevalence and cariogenic properties of S. mutans and Lactobacillus due to their essential

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role in caries development (Meurman and Stamatova, 2007). Several randomized clinical trials have been performed testing probiotic administration to reduce cariogenic bacteria counts in saliva or plaque (Laleman et  al., 2014). These clinical studies were conducted after positive results from in vitro studies showed that probiotic strains suppress and decrease the growth of cariogenic bacteria (Söderling et al., 2011; Keller et al., 2011; Haukioja et al., 2008). This anticariogenic effect is associated with the probiotics capacity to modify the protein composition of the salivary pellicle and form an artificial biofilm on hydroxyapatite (the enamel mineral), which specifically prevents the adherence of S. mutans (Bizzini et al., 2012). Therefore, probiotics are administered to maintain or restore the natural microflora against pathogen proliferation associated with the development of major oral diseases. The effect of probiotics on dental caries and its related risk factors has been evaluated in several experimental studies. The studies used different probiotic strains, including Lactobacillus rhamnosus GG, L. casei, Lactobacillus reuteri, Lactobacillus plantarum, L. brevis CD2, Bifidobacterium spp., etc. These probiotic strains are used to reduce S. mutans and Lactobacilli counts in saliva and/or biofilm, control plaque pH, and reverse root caries lesions (Laleman and Teughels, 2015). Research is still being carried out on probiotic strains to identify and characterize species that can compete or antagonize streptococci and other bacteria with cariogenic potential. There are several types of studies (such as in  vitro and in situ studies, randomized or non-­ randomized clinical trials, reviews, and meta-analyses) available in the literature dealing with the inhibitory effect of probiotics on cariogenic microorganism growth; however, these studies used different species and vehicles since these points have not been standardized (Bizzini, et al., 2012; Çaglar et al., 2015; Laleman et al., 2014; Söderling et al., 2011; Flichy-Fernández et al., 2010; Lodi et al., 2010).

15.4.4  Probiotic Dairy Beverage Products and Dental Caries Metabolism of sugars by probiotics should result in low acid production. The advantage of incorporation of probiotics into dairy products lies in their capacity to neutralize acidic conditions (Bonifait et al., 2009). Therefore, adding probiotics to dairy products could be a useful strategy for the prevention and/or treatment of major oral diseases including dental caries. Due to the beneficial effects of probiotics on human health, these bacteria have been added to several foods (Hempel et al., 2011), which are considered vehicles for administration (Rad et  al., 2016). In this sense, dairy beverage products could be considered a probiotic food

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of interest, due to their natural casein, calcium and phosphate contents, and being safe for consumption (Levine, 2001; Petti et al., 2001; Caglar et al., 2005). In regard to the use of probiotic bacteria in milk products, the high protein content and milk fat levels allow greater protection for bacteria against passage through the gastrointestinal tract (including the oral cavity). The high level of protein is also a favorable feature for the production of fermented milk, since it confers nutrient availability for microbial growth, leading to a reduction in fermentation time without directly impacting the manufacturing process (Balthazar et al., 2016). Therefore, studies have been carried out to test the oral effect of probiotics contained in dairy products. Probiotic ice cream has demonstrated a positive effect by significantly reducing S. mutans numbers compared to control products (Ashwin et al., 2015; Nagarajappa et al., 2015). In addition, yogurt is a potential carrier of probiotics and may influence the cariogenic potential and colonization of the oral cavity (Twetman and Stecksén-Blicks, 2008) by demonstrably reducing S. mutans numbers (Caglar et al., 2005; Ghasemi et al., 2017). In addition, kefir drink (Ghasempour et al., 2014), which contains probiotics, has also exhibited good results in the reduction of salivary bacteria counts and can thus be exploited for the prevention of enamel demineralization as a long-term, cost-effective resource (Srivastava et al., 2016). Studies have shown promising results for plain milk containing probiotics in the prevention, control, and treatment of dental caries. Milk containing L. rhamnosus significantly reduced the caries risk by reducing S. mutans counts (Nase et al., 2001; Teanpaisan and Piwat, 2013), significantly reversed the soft and leathery texture of primary root caries lesions in elderly people (Petersson et al., 2011), and has also been shown to prevent the development of new caries with cavities or without cavities (Stecksén-Blicks et al., 2009; Rodriguez et al., 2016).

15.4.5  Probiotic Dairy Beverage Products and Salivary pH Considering the acidic nature of dairy products and high carbohydrate content, it is a concern whether these vehicles and probiotics may drop salivary and biofilm pH below critical levels and foster dental caries development. However, based on the results of some studies, there is still controversy regarding the benefits of dairy beverages containing probiotics and dental caries control and development. In an in  vivo study, Shakovetz et al. (2013) found positive changes in saliva and pH in children after probiotic yogurt consumption. In contrast, Ghasempour et al. (2014) did not find any significant difference between pH values of saliva before and after consumption of probiotic yogurt for the same 2-week time period (Ghasempour et al., 2014).

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15.4.6  Probiotic Dairy Beverage Products and Salivary IgA The use of milk containing Enterococcus faecium probiotics significantly increased the salivary s-IgA levels in underweight and normal bodyweight children when compared to milk alone. Thus, probiotics can influence the humoral immune response and bodyweight of preschool children (Surono et  al., 2011). Harbige et  al. (2016) also reported that daily ingestion of a probiotic drink containing L. casei strain Shirota increased salivary IgA1 and IgA2 concentrations in healthy adults. Similar results were observed in another randomized clinical trial involving elderly persons consuming yogurt fermented with Lactobacillus bulgaricus over a 12-week period. Yogurt intake positively affected the IgA flow rate of saliva (Yamamoto et al., 2017). It can be speculated that the intake of milk or yogurt with probiotics activates the gut mucosal immune system and increases the expression of cytokines in the gut. As a result, salivary IgA secretion might be accelerated by the increase in IgA-positive B-cells mediated via cytokines in the gut. In addition, the B-cell isotype switch to IgA production is controlled by transforming growth factor (TGF)-β; therefore, it is possible that L. casei strain Shirota induces the production of IgA1 and IgA2 via TGF-β induction (Harbige et al., 2016). Thus, probiotics hold great promise for stimulating the total salivary s-IgA level over dairy products alone. The increase in secretory IgA level is important to protect the mucosal and tooth surface from diseases, independent of age.

15.5  Periodontal Disease 15.5.1  Definition and Aetiopathology "Peri" means around and "odontal" refers to teeth. Periodontal diseases are infections of the structures around the teeth. These include the gums, the cementum (layer that covers the root), the periodontal ligament, and the alveolar bone around the root (Lindhe, 2009). Periodontal disease is classified into two types: gingivitis and periodontitis (Bonifait et al., 2009). While gingivitis is the earlier stage, characterized by inflammation limited to the gums (Fig. 15.4), periodontitis occurs when untreated gingivitis progresses to the loss of the gingiva, bone, and ligament (Fig. 15.5). This is the more severe form of the disease, being progressive, destructive, and eventually leading to tooth loss (Houle and Grenier, 2003). Periodontitis is more prevalent in adults but may also occur in children and adolescents. Advanced forms of periodontitis that result in severe loss of supporting structures and substantial tooth loss affect

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Fig. 15.4  Patient with gingivitis.

Fig. 15.5  Patient with periodontitis.

10%–15% of the population globally. This estimated prevalence range includes both severe aggressive periodontitis, which primarily affects adolescents or young adults and severe chronic periodontitis, which primarily affects adults and whose prevalence increases with age in all populations (Lindhe, 2009). Gingivitis and periodontitis are initiated and sustained by the microorganisms of the dental biofilm (Darveau, 2010). Furthermore, no single pathogen causes the disease alone; it is initiated as a result of dysbiosis (Feres et al., 2016). In a single person, the microbial biofilm includes around 150 species and up to 800 different species have been identified in human dental

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biofilms (Lourenco et al., 2014). Pathogens including Gram-negative anaerobic bacteria, spirochetes, and even viruses can be identified in periodontal disease onset. However, the main pathogenic agents associated with the diseases are Aggregatibacter actinomycetemcomitans (AA), Porphyromonas gingivalis (PG), Treponema denticola, and Tannerella forsythia (Bonifait et al., 2009). Among these microorganisms, AA and PG are regarded as the main aetiological agents of early and refractory forms of periodontal disease (Slots, 2004). According to the presence of a variety of virulence characteristics, periodontal pathogenic bacteria can colonize the subgingival sites, escape the host’s defence system, and cause tissue damage. The persistence of the host’s immune response also constitutes a determining factor in the progression of the disease (Houle and Grenier, 2003). Although periodontal disease aetiology is related to the biofilm microorganisms (Kinane et al., 2006), several risk factors for periodontal disease have been established: (1) cigarette smoking, (2) diabetes mellitus, (3) socioeconomic and demographic variables, (4) psychosocial factors (e.g., stress), (5) genetic predispositions, (6) hormonal characteristics (e.g., pregnancy), and (6) impaired immunity (Bouchard et al., 2016).

15.5.2  Probiotics and Periodontal Diseases The temporary use of antibiotics or antiseptics, either locally or systemically, does not really improve the long-term effect of periodontal therapy (Quirynen et al., 2002). Thus, the focus on the reducing pathogenic bacteria has begun. Pathogenic bacteria are one of the major aetiological factors for periodontal inflammation (Teughels et al., 2011). Consequently, increasing the number of beneficial microorganisms via the ingestion of probiotics might be a strategy of great interest for the prevention of gingivitis and periodontitis. Probiotic microorganisms do not act exclusively by affecting the microbiota. They can also exert effects either by modulating immunological parameters, epithelial permeability, and bacterial translocation, or by producing bioactive or regulatory metabolites (de Vrese and Schrezenmeir, 2008). The latter effects are interesting for periodontal health since the host significantly mediates the destruction of the periodontium and bacterial challenge determines this damage (Sanz et al., 2005). Therefore, probiotics may not only suppress the rise of endogenous pathogens or prevent infection with exogenous pathogens but might also protect individuals through the promotion of a beneficial host response (Roberts and Darveau, 2002). Unfortunately, evidence available in the literature, through randomized clinical trials focused on the effect of probiotics in periodontal disease, demonstrated large heterogeneity due to the variability in the probiotic strains used, the probiotic concentration, the d ­ elivery

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vehicle, the clinical characteristics of the tested population, etc. (Teughels et al., 2011; Yanine et al., 2013). Although there are limitations, these studies reported reductions in total anaerobic bacteria, increases in total aerobic bacteria, reductions in black-pigmented bacteria, reductions in AA numbers, reductions in PG numbers, and reductions in T. forsythia. Moreover, the literature indicates that the effects of probiotic bacteria on the periodontal condition display benefits on the plaque index and gingival index (Teughels et al., 2011; Yanine et al., 2013). There are a few studies in the literature testing the effect of dairy products containing probiotics in the prevention or treatment of periodontal disease (Staab et  al., 2009; Lexner et  al., 2010; Karuppaiah et al., 2013; Caglar, 2014). The periodontal microflora has been studied a little deeper. Staab et  al. (2009) evaluated whether gingiva and plaque criteria were altered in individuals consuming commercial fermented milk containing L. casei Shirota strains. The results demonstrated no difference in the plaque index, interproximal plaque index, or papilla bleeding index between the probiotic and control groups. In the test group, elastase activity and MMP-3 levels were significantly lower after the intake of the probiotic milk drink than the control group.

15.6  Oral Candidiasis 15.6.1  Definition and Aetiopathology Candidiasis is an infection caused by commensal and opportunistic species of the yeast Candida, usually Candida albicans, which is a normal inhabitant of the oral cavity (Meurman and Stamatova, 2007). Other species have also been observed in the infection: Candida glabrata, Candida tropicalis, Candida parapsilosis, Candida kefyr, Candida dubliniensis, Candida lusitaniae, Candida krusei, and Candida guilliermondii (Hoare et al., 2017). Oral candidiasis, which is attributed in part to dysbiosis, accounts for a major proportion of fungal infections found in the oral cavity (Coronado-Castellote and Jimenez-Soriano, 2013). Candida spp. biofilm formation and infection is a multistage process comprising (1) adherence to the surface, (2) colonization, proliferation, and invasion, and (3) detachment of biofilm cells to promote colonization and infection of sites. In the case of C. albicans, biofilm formation has been reported to be associated with the upregulation of genes involved in adherence (such as agglutinin-like sequence genes and hyphal wall protein 1; Hwp1) and also those genes involved in amino acid biosynthesis and metabolism (Nobile and Johnson, 2015). Some local contributory factors that may promote Candida overgrowth include wearing a removable prosthesis (Fig. 15.6), poor oral

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Fig. 15.6  Oral candidiasis due to the use of dental prosthesis (A) patient with dental prosthesis and (B) patient without dental prosthesis.

Fig. 15.7  Oral candidiasis in baby due to the consumption of acidic foods (A) superior arch and (B) inferior arch.

hygiene, certain foods (Fig. 15.7), tobacco use, and hyposalivation. Saliva seems to be the key element in the control of Candida overgrowth, as it has components such as soluble IgA and mucins that bind and clear the fungi from the oral cavity, as well as histatin 5 and calprotectin, which have potent antifungal activities (Salvatori et al., 2016). The incidence of this infection has increased significantly, especially in the elderly people (Williams and Lewis, 2011) since oral dysbiosis mostly results from impaired immunity, medication use (such as broad-spectrum antibiotics and immunosuppressive agents), systemic disease (diabetes and malignancies), age (children or elderly), and AIDS (Ai et al., 2017). Affected individuals can present different clinical forms of the primary oral candidiasis including (1) acute pseudomembranous candidiasis; (2) chronic erythematous candidiasis; (3) acute erythematous candidiasis; and (4) chronic hyperplasic candidiasis (Hoare et al., 2017).

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However, the presence of these species in the oral cavity alone is not sufficient for disease onset. Oral candidiasis development is mostly determined by conditions that compromise the systemic immune response such as organ transplantation, HIV infection, chemotherapy, radiotherapy, and advanced age (Lalla et al., 2013). The symptoms include white, curd-like patches in the mouth or throat and the disorder is frequently characterized by local discomfort, such as burning pain and altered taste sensation (Ai et al., 2017). More serious infections, when the infection spreads through the bloodstream or upper gastrointestinal tract in immune-compromised patients, can lead to the possibility of morbidity and mortality (Akpan and Morgan, 2002).

15.6.2 Prevention Understanding the processes involved in candida spp. biofilm formation and growth makes the adequate prevention of this infection possible and enables the identification of possible therapeutic targets. Systemic and local antifungal agents have proven to be successful in preventing mucosal and invasive fungal infections. However, antifungal drugs can manifest some adverse effects, such as hepatic and renal toxicity, nausea, vomiting, and diarrhea (Oliver et al., 2004). Furthermore, the increased number of resistant strains and antifungal prophylaxis remains problematic (Sardi et al., 2011; Sardi et al., 2013). In immunosuppressed patients, elderly individuals or those wearing dentures, oral candidiasis frequently recurs or becomes recurrent. Thus, agents with low toxicity or no side effects that are effective against Candida are needed (Nittayananta, 2016; Pfaller, 2012).

15.6.3  Probiotics and Oral Candidiasis Several in vitro studies have shown that probiotics may affect the virulence potential of C. albicans. A recent meta-analysis found that probiotic agents were an effective means for decreasing the prevalence of high counts of oral yeasts in elderly individuals (Ai et  al., 2017). Clinical trials reported positive effects for probiotic consumption in decreasing the risk of developing oral candidiasis. Salivary levels of yeast in elderly subjects have been shown to decrease compared to basal levels after probiotic intake (Hatakka et al., 2007; Kraft-Bodi et al., 2015; Mendonça et al., 2012), together with a significant increase in anti-Candida IgA levels (Mendonça et al., 2012). In patients diagnosed with oral candidiasis, the local administration of a mixture of Bifidobacterium longum, L. bulgaricus, and Streptococcus thermophilus was shown to improve oral pain and reduce the prevalence of Candida spp. compared to conventional antifungal therapies (Li et al., 2014). Moreover, in asymptomatic denture wearers, oral Candida spp.

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yeast detection was reduced in the probiotic group compared to the placebo group (Ishikawa et al., 2015).

15.6.4  Probiotic Dairy Products and Oral Candidiasis The association between candidiasis and dairy products, in general, has been tested. Probiotic cheese significantly reduced the risk of high yeast counts and the risk of hyposalivation compared to the control group (Hatakka et  al., 2007). In addition, another cheese containing different probiotic bacteria (L. acidophilus NCFM and L. rhamnosus Lr-32) was also tested and presented positive result against Candida infection (Miyazima et al., 2017). On the other hand, probiotic microorganisms (Lactobacillus rhamnosus GG and Lactobacillus rhamnosus LC) contained in a cheese did not significantly reduce yeast compared to a cheese without probiotics (Akpan and Morgan, 2002). Although promising results have been observed with the use of dairy products, the association between candidiasis and dairy beverages lacks controlled studies. Yakult LBz (L. casei and Bifidobacterium breve) consumption over a 30-day period, reduced the prevalence of Candida spp. in the oral cavity of 42 healthy individuals. Furthermore, the secretory immune response against Candida spp. was increased in this population, suggesting that this beverage can control oral candidiasis (Mendonça et al., 2012).

15.7 Halitosis 15.7.1  Definition and Aetiopathology Bad breath or halitosis is an unpleasant odor exhaled from the mouth, nasal cavity, or facial and pharyngeal sinuses. Halitosis was first reported several years ago. Hippocrates mentioned that any girl should have pleasant breath, making sure always to wash her mouth with wine, anise, and dill seeds (Gani et al., 2012). This condition afflicts up to 31.8% of adolescents and adults (Silva et al., 2017b) to various degrees and can be serious enough to cause personal embarrassment. In Germany, 20% of patients complain of oral malodor (Struch et  al., 2008), while in Italy that number is between 50% and 60% (Aimetti et al., 2015). Many factors may cause halitosis, including (1) reduction in salivary flux, (2) consumption of particular foods, (3) metabolic disorders, (4) respiratory tract infections, but in most cases, it is associated with an imbalance of the commensal microflora of the oral cavity (Scully and Greenman, 2008).

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Accumulation of bacteria and food residue at the posterior part and in the furrows of the tongue is considered a major cause of halitosis (Scully et al., 1997). Atopobium parvulum, Eubacterium sulci, and Solobacterium moorei are the dominant species located in the tongue dorsum of people with oral halitosis (Kazor et al., 2003). Furthermore, bacteria associated with gingivitis and periodontitis may produce putrid odors and play a fundamental role in this problem (Silva et  al., 2017a). The periodontal species of microorganisms most often related to oral odor are F. nucleatum, PG, Prevotella intermedia, and T. denticola (Scully and Greenman, 2008). Tongue bacteria and periodontal pathogens causing halitosis are mostly Gram-negative and anaerobic with proteolytic abilities (Scully and Greenman, 2012). More specifically, halitosis results from volatile sulfur compounds such as methyl mercaptan, hydrogen sulfide, and dimethyl sulfide. Additional odorous compounds such as putrescine, indole, skatole, and cadaverine (Kleinberg and Westbay, 1992) are generated during bacterial degradation of food residues, saliva proteins, desquamated cells from the mucous membrane, dental biofilm, leukocytes, and microbial putrefaction (Scully and Greenman, 2008). The periodontal pocket also provides an ideal environment for volatile sulfur compound production. Even extremely low concentrations of volatile sulfur compounds are toxic to periodontal tissues and it is presumed that volatile sulfur compounds can facilitate bacterial invasion into deeper tissues. Accordingly, volatile sulfur compounds not only cause halitosis but are also regarded as a periodontal pathogenic factor (Morita and Wang, 2001). The intensity of clinical halitosis has been associated with intraoral volatile sulfur compound levels and with periodontal health status since periodontal pockets tend to increase in quantity and in depth in severe cases of periodontitis (Stamou et al., 2005). In some patients, oral malodor may originate from extra-oral sources. These oral malodor metabolites can be produced in any part of the body, arrive at the lungs via the blood and be released as gases in the breath (Van den Velde et al., 2007). Therefore, interdisciplinary approaches may be necessary for the management of this type of halitosis.

15.7.2 Classification and Treatments Needs According to the International Society for Breath Odor Research, halitosis can be categorized as genuine halitosis, pseudo-halitosis, and halitophobia. In addition, genuine halitosis has been further subclassified into physiological halitosis in which there is no readily evident disease or pathological condition, and pathological halitosis, which occurs as a result of an infection of the oral tissues (Murata et al., 2002). Table 15.3 illustrates the classification method for halitosis and corresponding treatment needs.

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Table 15.3  Classification of Halitosis and Corresponding Treatment Needs (TN) (Murata et al., 2002) Classification

TN

I-Genuine halitosis A. Physiological halitosis

TN-1

B. Pathological halitosis   (i) Oral

TN-2

  (ii) Extra-oral

TN-3

II-Pseudo-halitosis

TN-4

III-Halitophobia

TN-5

Description Obvious malodor, with intensity beyond perceived socially acceptable level Malodor arises through putrefactive processes within the oral cavity. Neither a specific disease nor a pathological condition capable of causing halitosis is found Origin is mainly the dorsoposterior region of the tongue Temporary halitosis due to dietary factors (e.g., garlic) should be excluded

Halitosis caused by disease, pathological condition, or malfunction of oral tissues Halitosis derived from tongue coating, modified by pathological condition (e.g., periodontal disease, xerostomia) is included in this subdivision Malodor originates from nasal, paranasal, and/or laryngeal regions Malodor originates from pulmonary tract or upper digestive tract Malodor originates from disorders anywhere in the body whereby the odor is blood-borne and emitted via the lungs (e.g., diabetes mellitus, hepatic cirrhosis, uraemia, and internal bleeding) Obvious malodor not perceived by others, although the patient stubbornly complains of its existence Condition is improved by counselling (using literature support, education, and explanation of examination results) and simple oral hygiene measures After treatment for genuine halitosis or pseudo-halitosis, the patient persists in believing that he/she has halitosis No physical or social evidence exists to suggest that halitosis is present

To provide guidelines for clinicians in treating halitosis patients, halitosis treatment needs in dental practice have been categorized into five classes (Table 15.4). The treatment of physiological halitosis (TN-1), oral pathological halitosis (TN-2), and pseudo-halitosis (TN-4) is the responsibility of dentists. However, the treatment of extra-oral pathologic halitosis (TN-3) is within the realm of medical specialists. Halitophobia (TN5) needs to be treated by a psychiatrist, physician, or a psychologist (Yaegaki and Coil, 2000).

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Table 15.4  Treatment Needs (TN) for Halitosis (Yaegaki and Coil, 2000) Category

Description

TN-1

Explanation of halitosis and instructions for oral hygiene (support and reinforcement of a patient's self-care for further improvement of their oral hygiene) Oral prophylaxis, professional cleaning, and treatment for oral diseases, especially periodontal diseases Referral to a physician or a medical specialist Explanation of examination data, further professional instruction, education, and reassurance Referral to a clinical psychologist, a psychiatrist, or other psychology specialist

TN-2 TN-3 TN-4 TN-5

15.7.3  Treatments for Halitosis Related to the Oral Cavity Knowing that the bacteria that cause oral malodor mostly colonize the tongue and gums, the successful management of halitosis appears to rely on the removal or reduction of the total number of bacteria. Consequently, volatile sulfur compound levels and some other foul volatiles are also reduced. Mechanical interventions such as brushing, flossing, and tongue scraping are intended to reduce the quantity of these bacteria, persistent food matter, and cellular debris from the gingiva and tongue (Kuo et al., 2013). In some cases, professional tooth cleaning, and periodontal therapies for the removal of colonization sites of these bacteria are also necessary (Deutscher et al., 2018). However, mechanical cleaning appears to have limited benefits. The bacteria that cause oral malodor return to the same regions shortly after the termination of therapy (Van der Sleen et al., 2010). Chemical products such as mouth rinses are widely used in the treatment of halitosis because they may be more effective in reaching the less accessible parts of the oral cavity, are refreshing, and possess a pleasant odor. Mouth rinses with chlorhexidine have antibacterial activity in supragingival plaque and on the tongue and thus are seen as potentially effective agents in controlling halitosis (Mishra et  al., 2016). However, chlorhexidine cannot be used for long because it causes tooth and tongue staining, some reduction in taste sensation, and has a bad taste (James et al., 2017). To achieve long-term reduction in malodor, either more targeted antimicrobials should be applied that will minimize extermination of

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the beneficial microbiota, or specific replenishment of the microbiota with beneficial bacteria should take place shortly after antimicrobial therapy. Bacteriotherapy can also improve halitosis. The replacement of bacteria implicated in halitosis by colonization of probiotic bacteria into oral tissues may have applications in both the prevention and treatment of oral malodor (Burton et al., 2006).

15.7.4  Probiotics and Halitosis To achieve a long-term reduction in halitosis, specific replenishment of the microbiota with beneficial bacteria needs to occur shortly after mechanical or chemical therapy. Preventing the regrowth of odor-causing organisms by pre-emptive colonization of the oral cavity with non-virulent, commensal microorganisms seems like a reasonable alternative (Faveri et al., 2006). Given that the dorsum of the tongue and periodontal tissues are the origins of most malodor problems, a candidate probiotic to counter this condition should be able to persist within this particular ecosystem. A probiotic strain that efficiently colonizes these surfaces and does not produce odorous metabolic by-products would be highly advantageous. Some possible probiotic species are (Burton et al., 2005; Burton et al., 2006; Jamali et al., 2016; Zupancic et al., 2017): • Weissella cibaria. Its mechanism of action is based on the capacity to co-aggregate with F. nucleatum and adhere to epithelial cells, reducing the levels of volatile sulfide components produced by F. nucleatum. This effect may be due to hydrogen peroxide production by W. cibaria, causing F. nucleatum inhibition (Kang et al., 2006). • Streptococcus salivarius. It is the most numerically predominant microorganism of the tongue microbiota in healthy individuals (Kazor et  al., 2003). This microorganism suppresses volatile sulfide effects by competing with volatile sulfide-producing species of colonization areas (Burton et al., 2005). This species has also only a limited ability to produce volatile sulfur compounds, is unlikely to contribute significantly to oral odor, and is most closely related to S. thermophilus, a bacterium widely used in the dairy food industry (formerly S. salivarius ssp. thermophilus). The bacteriocins produced by S. salivarius strain K12 may prevent regrowth of key microbial participants in the halitosis-associated ecosystem. In addition, the bacteriocins produced by strain K12 are auto-inducible and can cross-stimulate bacteriocin production by other indigenous S. salivarius and related species. Thus, the administration of strain K12 may also boost the production levels of these antibacterial compounds by the host’s existing oral microbiota (Burton et al., 2006; Jamali et al., 2016).

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15.7.5  Probiotic Dairy Products and Halitosis A well-designed in  vitro study showed that four probiotic bacterial species isolated from fresh yogurt inhibit periodontal pathogens. However, the competition between yogurt probiotics and periodontal pathogens depends on the sequence of inoculation. When probiotics were the first to be inoculated, Bifidobacterium inhibited PG, F. nucleatum, A. actinomycetemcomitans, Porphyromonas circumdentaria, and Prevotella nigrescen. L. acidophilus inhibited P. gingivalis, A. actinomycetemcomitans, P. circumdentaria, P. nigrescen, and Peptostreptococcus anaerobius. L. bulgaricus inhibited P. gingivalis, A. actinomycetemcomitans, and P. nigrescen; and S. thermophilus inhibited P. gingivalis, F. nucleatum, and P. nigrescen. However, their antimicrobial properties were reduced when both species (probiotics and periodontal pathogens) were inoculated simultaneously. Thus, bio-yogurt and the probiotics contained therein can inhibit specific periodontal pathogens but have no effect on the protective periodontal bacteria (Zhu et al., 2010). Vestman et al. (2013) isolated the dominant Lactobacillus species detected in the saliva and oral cavity of breastfed and formula-fed infants and evaluated the in vitro probiotic proprieties. Lactobacillus gasseri was the most prevalent Lactobacillus species (88%) and this species inhibited growth of cariogenic species (S. mutans), periodontal pathogens producers of volatile sulfur compounds (F. nucleatum) related to halitosis, yeast (C. albicans), and others (S. sobrinus, Actinomyces naeslundii, Actinomyces oris) in a concentration-dependent manner. In addition, Lactobacilli colonized the oral cavity of breastfed infants significantly more frequently than formula-fed infants. Thus, salivary L. gasseri demonstrated several probiotic traits including attachment to HGEPp.05 human gingival epithelial cells, saliva, and growth inhibition of several oral species (Vestman et al., 2013). A pilot clinical study evaluating the effect of a probiotic milk drink showed that probiotics might have immunomodulatory effects in the gingival region. It is known that periodontopathogens such as P. gingivalis and A. actinomycetemcomitans (periodontal pathogens and producers of volatile sulfur compounds related to halitosis) stimulating the release of factors influencing collagen degradation [e.g., MMP-3 and myeloperoxidase (MPO)] are highly discriminatory biomarkers for site-specific diagnosis of periodontitis. The group that ingested the probiotic milk drink showed a decrease in MMP-3. In addition, MPO activity was not induced by experimental gingivitis in the test group but was induced in the control group, indicating a reduction in ­neutrophil-derived markers (Staab et al., 2009). An in  vitro and placebo-controlled trial involving the administration of yogurt containing L. rhamnosus L8020 to 50 participants showed that bovine milk fermented with L. rhamnosus L8020 reduced the oral

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c­ arriage of S. mutans and four periodontal pathogens: P. gingivalis, P. intermedia, T. forsythia, and Fusobacterium spp. The phenomenon was not observed with the placebo yogurt, suggesting that yogurt with L. rhamnosus L8020 may reduce the risk of dental caries, periodontal disease, and periodontal pathogens related to malodor (Nikawa et al., 2011). Probiotic treatment of halitosis has already achieved promising outcomes. There are several studies confirming the reduction of halitosis in individuals who have used lozenges, tablets, or gums containing probiotics (Burton et al., 2005, 2006; Jamali et al., 2016; Zupancic et al., 2017). However, the supporting data to show the use of probiotics contained in dairy products (especially beverages) in malodor treatment are still deficient and remain poorly explored (Staab et al., 2009). The current results mandate the need for further randomized clinical trials before any specific clinical recommendations can be defined for the use of probiotic-containing dairy products.

15.8 Conclusion There is no doubt about the benefits of dairy probiotics beverages for human health, and its use for this purpose is largely recommended. Because of these beneficial effects, probiotics have been reported as promising agents against some of the major biofilm-dependent oral diseases. However, there is still controversy over their mechanisms of action and effects as protectors and maintainers of oral environment balance, and so their indication on oral health promotion is still inconclusive. In this sense, new in situ and in vivo studies with different dairy probiotics products are suggested to identify and characterize some probiotic strains that may, definitely, have benefits against dental caries, gingival and periodontal diseases, and oral candidiasis. In addition, the promotion of increase in salivary IgA levels, pH values, as well as in halitosis improvement and control are also needed.

Acknowledgments This chapter was partially supported by National Council for Scientific and Technological Development from Brazilian Government (CNPQ) process number 401048/2016-6, Coordination for the Improvement of Higher Education Personnel (CAPES) under scholarship 00.889.834/0001-08, and Foundation for Research Support of the State of Rio de Janeiro (FAPERJ) process number E-26/202.174/2016.

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Further Reading Ahola, A.J., Knuuttila, H.Y., Suomalainen, T., Poussa, T., Ahlstrom, A., Meurman, J.H., Korpela, R., 2002. Short-term consumption of probiotic-containing cheese and its effect on dental caries risk factors. Arch. Oral Biol. 47, 799–804. J. Busetto (2008) U.S. Functional Beverages Market: A Young Market with Growing Popularity (http://www.frost.com/sublib/display-market-insight.do?id=132005699) (Accessed 31 October 2017).

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Costerton, J.W., Montanaro, L., 2005. Biofilm in implant infections: its production and regulation. Int. J. Artif. Organs 28, 1062–1068. Jensen, M.E., Wefel, J.S., 1990. Effects of processed cheese on human plaque pH and demineralization and remineralization. Am. J. Dent. 3, 217–223. Lee, D.K., Park, S.Y., An, H.M., Kim, J.R., Kim, M.J., Lee, S.W., Cha, M.K., Kim, S.A., Chung, M.J., Lee, K.O., Ha, N.J., 2011. Antimicrobial activity of Bifidobacterium spp. isolated from healthy adult Koreans against cariogenic microflora. Arch. Oral Biol. 56, 1047–1054.