The global prevalence of tooth decay in children remains a major problem despite continuous efforts to improve oral health (Hasslöf et al., 2022, Uribe et al., 2021). Especially in western industrialized nations, a skewed distribution of caries in children can be observed. While the majority of children have good oral health, a small proportion of children yields a high caries burden (Dimitrova et al., 2000, Nunes et al., 2014). In addition to the known caries risk factors such as sugary diet, poor oral hygiene and socio-economic background, basic research on caries development is being intensified to find contributing factors for increased susceptibility to caries (Grychtol et al., 2015, Hertel et al., 2019, Hertel et al., 2022, Schulz et al., 2020).

The basis for caries formation is the adsorption of proteins, glycoproteins and macromolecules from saliva and sulcus fluids to the tooth surface, the formation of the acquired pellicle (C. Hannig & Hannig, 2009; M. Hannig & Joiner, 2005). The pellicle acts as a mediator between the tooth surface and the oral environment and has ambivalent properties. It mainly serves as a protective layer through a variety of protective components, but also forms the basis for the receptor-mediated adhesion of bacteria to the tooth surface (C. Hannig et al., 2005; Hertel et al., 2017).

In the search for potential biomarkers responsible for differences in caries susceptibility in children, protective salivary proteins and their abundance in the pellicle are of particular interest. The most important salivary proteins in the context of innate defense mechanisms include lysozyme and lactoferrin. Lysozyme is one of the most important antibacterial enzymes in saliva and pellicle. It is secreted from major and minor salivary glands, gingival crevicular fluid and salivary leukocytes (C. Hannig et al., 2009; Moslemi et al., 2015). The antibacterial property of lysozyme is related to its muramidase activity, whereby the β-1,4 bond between N-acetyl muramic acid and N-acetylglucosamine in the peptidoglycan layer of the bacterial cell wall is hydrolyzed (Laible & Germaine, 1985; Y. B. Wang & Germaine, 1993). This leads to an increased permeability of the bacterial cell membranes and consequently to electrolytic and osmotic changes. As a strongly cationic protein, lysozyme governs the oral microbiome by influencing bacterial aggregation and adhesion and activating bacterial autolysins that damage bacterial cell walls (Rudney & Smith, 1985).

Lactoferrin is a non-enzymatic antibacterial product. It is detected in body fluids such as saliva and tears, and in the secondary granules of polymorphonuclear leukocytes (Berlutti et al., 2011). Lactoferrin has iron-chelating properties that deprive microorganisms of this important element. In its iron-free state, lactoferrin is known as apo-lactoferrin and provides antibacterial properties due to the direct binding of bacteria to lactoferrin and agglutinating Streptococcus mutans (S. mutans) (Moslemi et al., 2015). This allows ease of removal of the agglutinated bacteria from the oral cavity via mechanical action of saliva and the swallowing of the agglutinated bacteria (Berlutti et al., 2011, Jenssen and Hancock, 2009). Due to its antibacterial effect, lactoferrin is commonly used as an ingredient in mouthwashes and toothpastes (Moslemi et al., 2015).

A correlation between the presence of lysozyme and lactoferrin in saliva and caries activity in children has already been investigated (Felizardo et al., 2010, Hao Gao-feng, 2009, Mass et al., 2002). Molslemi et al. found significantly higher salivary lysozyme and also higher lactoferrin levels in caries-free children compared to children with early childhood caries (ECC) (Moslemi et al., 2015).

Another protective salivary protein that has received less attention so far is cystatin S. Cystatin S is a phosphorylated protein that is secreted by the submandibular gland and to a much lesser extent by the parotid and sublingual gland (Koopaie et al., 2021). Cystatin S has four phosphorylation sites that bond with hydroxyapatite and is a component of the pellicle. It plays an important role in the calcium-phosphate balance and in the remineralisation of enamel. It also prevents enamel demineralization by attaching to the enamel surface (Koopaie et al., 2021, Laputková et al., 2018). Cystatin S protects the oral mucosa through its antimicrobial and antiviral activity (Hemadi et al., 2017). A recent study by Koopaie et al. revealed a higher salivary cystatin S level in caries-free children compared to children with ECC (Koopaie et al., 2021) suggesting that higher levels of cystatin S may inhibit proteolytic processes and thereby indirectly protect salivary proteins from proteolysis (Hemadi et al., 2017).

Little is known about the presence of the protective salivary proteins cystatin S, lactoferrin and lysozyme in the pellicle of children. The aim of the study was to quantify for the first time the levels of these proteins in saliva and in the in-situ formed pellicle of children with different caries activity in order to detect them as potential biomarkers for caries.