Milk is a biological fluid providing significant nutrients, immunological protection, and biologically active peptides to both infants and adults (Clare and Swaisgood, 2000). Nutritionists have consistently recommended consumption of several servings of milk and other dairy products on a daily basis for all age groups. Despite this recommendation, fluid milk consumption globally has declined over the past decade. However, consumers have continued to ingest large amounts of dairy products including cheese and yogurt as an alternative supplement to the nutritional benefits of milk. Milk and dairy products are an important source of calcium to attain proper bone development and maintenance of bone mineral density. Likewise, milk and dairy products serve as a source of calcium for tooth development and mineralization (Wise et al., 2002).
An individual's dietary and social patterns are major contributors to one's oral health. The quality of life can be greatly impacted as a result of poor oral health leaving a negative impact on self-esteem, eating ability, and social functioning (Moynihan, 2005). Several oral diseases can be linked back to poor nutrition, and as teeth deteriorate the conditions are exacerbated. Studies (Johansson et al., 1994, Norlen et al., 1993) have shown edentulous individuals are more apt to have inadequate dietary intake (high carbohydrate, high fat, low nutrient density foods) than dentate individuals. Sugars, specifically sucrose, are recognized as being a major contributor to dental caries' etiology. Other social factors such as alcohol and tobacco use, drug abuse, poor hygiene, and poor nutrition are also cited as being major contributory factors to oral diseases.
Saliva secretions supersaturated with calcium phosphate continually moisturize the tooth surface. Saliva serves as a reservoir of minerals (Fig. 7.2) to replenish tooth enamel hydroxyapatite after demoralization by plaque bacteria fermenting dietary sugars to organic acids. If there is a disruption in the calcium and phosphate balance, or other modulators of mineralization are deficient or missing, carious lesions in tooth enamel begin forming eventually leading to dental caries if untreated.
Most foods are noncariogenic, and do not contribute to oral diseases. However, some foods have anticariogenic properties that prevent tooth decay and other oral diseases. Although milk contains the sugar lactose, there is significant research to indicate milk and associated dairy products are anticariogenic. The high buffering capacity of milk is a contributing factor to pH control in the mouth after milk consumption (Mor and McDougall, 1977). Lactose is less fermented by indigenous oral microflora than sucrose only lowering the biofilm microenvironmental pH to around 6.0 compared to pH 5.0 with sucrose (Rugg-Gunn et al., 1985). Most anticaries properties are attributed to presence of calcium, phosphate, and casein, and their modulation of tooth enamel mineralization.
Shaw et al. (1959) first identified milk as an anticariogenic food in 1959 when they reported a reduction in dental caries incidence in rats fed milk and flavored milks. Rat diets were supplemented with milk, chocolate drink, chocolate milk, and a shake-like mixture that contained milk or chocolate milk plus vanilla ice cream. In addition, they also included a group that consumed cheese. All of the groups with milk (except the chocolate drink) demonstrated caries reduction with the cheese variable having best results.
Weiss and Bibby (1966) utilized an in vitro test with extracted teeth and with similar test variables (milk and flavored-milk) to show tooth enamel demineralization was reduced when teeth were exposed to cariogenic substances in an acetate buffer (pH 4.0) system. They concluded casein proteins were rapidly absorbed onto the tooth enamel surface and provide resistance to acid demineralization. Concurrently, Jenkins and Ferguson (1966) studied the effects of milk on acid production in human plaque. Subjects refrained from tooth brushing for three days to allow plaque accumulation on their teeth. Subsequently, milk was orally rinsed, and a microelectrode was used to measure the plaque pH. Milk showed a minimal plaque pH drop. The authors concluded the extent and duration of the pH neutralization would be non-cariogenic compared to a cariogenic positive control (sucrose solution). Bibby et al. (1982) studied how several dairy products and foods impacted acid production and enamel demineralization in an in vitro oral model (Orofax). Human milk was later shown to reduce demineralization and raise plaque pH in a manner similar to bovine milk (Rugg-Gunn et al., 1985). The authors also identified casein as an important component to buffer plaque environmental pH, by showing cheese consumption after drinking sweetened (sucrose) milk minimized pH decline observed with control variables.
The role of milk minerals was also investigated about this same time (Harper et al., 1986). Casein-free milk mineral concentrates were prepared from whey, and tested in rat models to study enamel mineralization and demineralization culminating in dental caries formation. The mineral concentrates contained varying levels of calcium, phosphate, and some residual whey proteins, and were used to study anticariogenicity in rats fed a high (20%) sucrose diet. Although all mineral concentrate diets studied reduced buccal caries, the concentrate with highest calcium (22.4 g/100 g) and phosphate (38.3 g/100 g) was most effective in anticariogenicity of smooth surface caries formation. An explanation for these results was not given.
A disturbing trend is occurring in global beverage consumption. Many consumers have stopped drinking milk as a predominant beverage to consuming soft drinks and other beverages highly sweetened with sucrose and other sweeteners. Sadly, the trend is occurring in young children as well as adults. Data analyzed from the National Health and Nutrition Examination Survey I (NHANES) found caries incidence was positively associated with elevated consumption of soft drinks in 9 to 29 year olds (Ismail et al., 1984). Marshall et al. (2003) reported an increased incidence rate for dental caries in young children (4 to 7 years old) when they consumed higher than median intakes of soft drinks, powdered beverages (sweetened with sugar), and to some extent fruit juice during their 'window of infectivity' (2 to 5 years old). They also showed this age group to have inadequate intakes of other nutrients during the same time period (2 to 5 years old). Consumption of fluid milk higher than the observed median had a neutral association with dental caries in these children. A similar study done by Levy et al. (2003) showed in 5 year old children there was a negative association of dental caries with bovine milk consumption from age 24 to 36 months. However, bovine milk and sugared beverage consumption from 6 weeks to 12 months were positively associated with dental caries in 5 year olds. Levine (2001) reported dairy beverages containing less than 5% added sugars have a negligible or low cariogenic potential in older children (> 16 years old).
Marketers in recent years have encouraged increased milk consumption by offering more flavored milks. Many flavored milks are sweetened with fermentable sugars including sucrose and high fructose corn syrup. Non-nutritive (minimal caloric contribution to the diet) sweeteners are an alternative means to sweeten dairy beverages. Polyols are very effective non-nutritive sweetening agents, and some have shown effectiveness in preventing dental caries. Castillo et al. (2005) studied acceptance of milks sweetened with xylitol or sorbitol by Peruvian children aged 4 to 7 years old. Xylitol (0.042 g/ml) was preferred most, followed by sorbitol (0.042 g/ml), xylitol (0.021 g/ml), and plain milk.
Jensen et al. (2000) studied the role of between meal snacks by adults on tooth enamel mineralization and demineralization. Enamel demineralization and caries progression was observed when apple juice, a cola soft drink, and sweetened (sucrose) yogurt were consumed as snacks. Dairy products were also studied, and found to remineralize tooth enamel after consumption. Cheddar cheese was the most effective dairy product to remineralize tooth enamel, but whole milk was most effective at changing the mineral content of dentin lesions.
As mentioned several times in the previous section, cheese is identified as a potent anticariogenic food. Cheese is a concentrated source of casein proteins, milk fat, calcium, phosphorus, and other minerals. In addition, cheese will contain bioactive peptides released from the casein primary amino acid sequences by enzymatic and microbial digestion as cheese ages.
König (1966) was the first to report cheese anticariogenic properties from a study in rats being fed a highly cariogenic diet. Animals fed Emmental cheese developed fewer and less severe caries than the control group. Rugg-Gunn et al. (1975) fed humans cheese after a very sugary snack and observed a blunted plaque pH decline. Edgar et al. (1982) conducted similar experiments and reported cheddar cheese stimulated saliva flow, and reduced the number of smooth surface (sides of teeth) caries. Morrissey et al. (1984) also conducted similar experiments with aged cheddar cheese and reported few smooth surface caries, but an intermediate number of sulcal caries. Rosen et al. (1984) observed cheddar cheese was cariostatic in rats when fed in conjunction with sucrose. The mechanism of action was not a direct antimicrobial effect on potentially odontopathogenic bacteria. Jensen et al. (1982) studied cheese maturity as a factor in pH buffering. They found aged cheeses (Cheddar, Gouda, blue, Monterey Jack, mozzarella, and Swiss) allowed no to slight pH declines after a meal, whereas young cheeses (Cheddar, cream, feta, and provolone) gave pH minima lower than 5.0.
Most authors from these studies proposed the following mechanism of action. After ingestion, cheese forms a film over the outer tooth surface within the biofilm, and there is resultant saliva flow stimulation. As pH declines after eating, cheese buffers the acidic environment and there is less demineralization. However, Krobicka et al. (1987) fed cheddar cheese to desalivated rats and observed fewer and less severe caries lesions in animals ingesting a cariogenic diet. Therefore, the presence of calcium, phosphate, and possibly bioactive peptides must have a direct role in demineralization and remineralization (Moynihan, 2000).
Early researchers observed that dairy products (milk, casein, caseinates, and cheeses) have anti-caries activity (Schweigert et al., 1946b, Shaw, 1950). Several studies proved casein was an effective anticariogenic substance, but casein's adverse organoleptic properties and the large amount required for efficacy disqualified its use as a food or toothpaste. Acid casein as an active ingredient in toothpaste was effective at reducing dental caries, but was required at very high levels for efficacy (Bavetta and McClure, 1957, Schweigert et al., 1946a). Sodium caseinate solublized in water and fed to rats in a caries model was shown to be anticariogenic (Reynolds and Del Rio, 1984). Sodium caseinate as an ingredient in a chocolate confectionary reduced cariogenicity, but high levels of caseinate (17%) were required to demonstrate an effect and the product was unpalatable (Reynolds and Black, 1987, 1989).
Tryptic digestion of caseinate enhances the proteins' ability to modulate enamel mineralization in a human oral caries model (Reynolds, 1987). Analysis of human dental plaque samples found elevated concentrations of casein peptides, calcium, and phosphorus. It was concluded these peptides were caseinophosphopeptides (CPP) derived from specific tryptic activity on as1-, as2-, and b-caseins. This prompted investigators to focus on casein peptides in subsequent research.
Dental caries prevention by milk-derived bioactive peptides is a complex physical and chemical sequence of cascading events. In general, bioactive peptides with anticariogenic activity have multiple functions to prevent dental lesions including bacterial inhibition; competitive exclusion to enamel binding sites, improved buffering capacity in the pellicle surrounding teeth, reduced enamel demineralization, and enamel remineralization. The interaction with salivary secretions is not well understood, but appears to be important for some of these prophylactic events.
Peptides identified to sequester calcium and other minerals, hence acting as biocarriers, are called phosphopeptides. The term was first given to casein-derived phosphorylated peptides that enhance vitamin D-independent bone calcification in rachitic infants (Mellander, 1950). Embedded within casein primary sequences are motifs of phosphopeptides that sequester calcium and other minerals (Fig. 7.6). Milk micelles contain physiologically significant amounts of calcium and phosphorus. The vast majority of bovine caseins exist in a phosphorylated form, with individual variants possessing as little as one phosphorylated residue (k-casein) and as much as 13 for others (as2-casein). Phosphorus in milk is bound via monoester linkages to casein serine residues. The calcium and inorganic phosphate residues associated with bovine milk caseins are greater than expected from the physico-chemical solubility of calcium phosphate in milk (Kitts, 2005). The presence of phosphorus and calcium bound to casein helps to maintain thermodynamically stable casein micelles in fluid milk.
Tryptic digestion of caseins either in vitro or in vivo yields phosphoseryl peptides that sequester divalent metal ions (Kitts and Yuan, 1992). Caseinophosphopeptides (CPPs) can be found in the stomach and duodenum after milk ingestion (Chabance et al., 1998). Casein phosphopeptides released from casein molecules are resistant to further proteolytic breakdown in the intestinal tract. Further evidence for intact passage through the upper gastrointestinal tract to the distal ileum was confirmed by identifying CPPs in ileostomy fluid (Meisel et al., 2003). In addition to in vivo proteolysis of as1-CN
Amino acid residue
Fig. 7.6 Position of caseinophosphopeptide motifs embedded in major casein primary sequences. The motifs contain a common sequence within them of SerP-Serp-SerP-
milk casein to release CPPs, lactic acid bacteria possess proteolytic enzymes that release CPPs during cheese ripening. CPPs are found as natural constituents in Comté, Cheddar, and Grana Padano cheeses (Pellegrino et al., 1997, Roudot-Algaron et al., 1994, Singh et al., 1997).
A major physiological role for CPPs is their ability to sequester minerals. The phosphate residues, corresponding to about 30% of the phosphorus content in milk, are present as monoesters of serine and occur mainly as clusters in the primary sequence of caseins, especially as1-, as2-, and b-caseins (Silva and Malcata, 2005). Of note, it is interesting most CPPs have a common sequence of three phosphoseryl residues followed by two glutamic acid residues (SerP-SerP-SerP-Glu-Glu) (Meisel, 1997). This high concentration of negative charges is responsible for CPPs' resistance to further digestion in the digestive tract (Clare and Swaisgood, 2000, Reynolds et al., 1994). Furthermore, phosphate groups and negatively charged side chains are binding sites for divalent minerals such as calcium, magnesium, and iron (Meisel, 1998), and renders them more bioavailable (Hansen et al., 1996, Peres et al., 1999). This mineral binding ability is important for CPPs' role in tooth mineralization.
The antioxidant potential of proteins derived from milk and dairy products is known (Allen and Wrieden, 1982). Casein has antioxidant activity at concentrations comparable to bovine fluid milk sources, but whey is not as effective at similar concentrations (Allen and Wrieden, 1982). Antioxidant activity of milk proteins is due to sequestering of iron and copper metals by phosphoseryl residues located on the casein micelle surface. Another possible mechanism is that whey proteins donate hydrogen to reduce free radicals (Colbert and Decker, 1991), and free sulfhydral groups from cysteine are effective at inhibiting lipid auto-oxidation (Taylor and Richardson, 1980a, 1980b). Buttermilk powder was shown to have antioxidant properties in a model lipid emulsion system (Wong and Kitts, 2003). A stronger affinity was noted for ferrous than ferric ions.
Likewise, the ability to sequester divalent cations also presents an opportunity for CPPs to act as antioxidants (Diaz et al., 2003). Activities of CPP and less defined casein hydrolysates have antioxidant properties in a muscle food system (Diaz and Decker, 2004). Another research group reported CPP antioxidant activity in aqueous and emulsion model systems (Taylor and Richardson, 1980a). Antioxidation activity may also have a physiological role in humans in reducing inflammatory agents produced in response to reactive oxygen species. Calcium enriched CPP stimulates release of the anti-inflammatory, interleukin-6 (IL-6) in a human gastrointestinal cell culture line (Kitts and Naknamura, 2006). This CPP bioactivity may be important in reducing inflammatory response elicited during periodontal disease.
Phosphoseryl peptides execute important functions at the organic-inorganic interface in the bioprocesses of biomineralization and calcium phosphate stabilization for bones and teeth. Disruption of biomineralization processes leads to consequences in bone and tooth hypomineralization or hypermineralization. Insights into molecular dynamics of these processes are beginning to assist scientists and clinicians in understanding how to design products that lead to applications in the biomineralization arena to repair damaged teeth and bones.
Caseinophosphopeptides released from casein tryptic digests account for approximately 10% by weight of the total casein protein. The major tryptic CPP are released from b-casein (f 1-25), as1-casein (f59-79), and as2-casein (f 1-21 and f46-70) (Fig. 7.6). All of these peptides contain three continuous phosphoserine residues capable of sequestering their own equal weight in amorphous calcium phosphate and forming colloidal nanocomplexes (Cross et al., 2005). The amount of calcium bound and calcium phosphate stabilized is influenced by peptide net charge, length, primary sequence, and peptide conformational folds and shapes. In general, longer peptides bind more calcium. In the presence of phosphate ions, CPP binds additional calcium above and beyond that bound in the absence of phosphate. This reaction appears to be pH dependent, and more calcium and phosphate are bound as pH increases (Cross et al., 2005). More detailed information is written on the molecular conformation changes and stabilization of calcium and phosphate by CPP (Cross et al., 2006, Cross et al., 2004, Laila et al., 2005).
Tryptic digests of casein significantly reduced enamel subsurface demineralization in a human in situ caries model (Reynolds, 1987). In that study, the authors speculated CPP was incorporated into dental plaque, and serves several purposes in anticariogenicity including a role in mineralization, buffering acids produced by plaque bacteria, and amino acid transport. In a subsequent study, CPP purified from a tryptic digest of sodium caseinate was used to confirm utility of CPP in mineralization of the tooth's surface of rats (Reynolds et al., 1995). A dephosphorylated peptide with the same amino acid sequence as CPP showed no anticariogenicity. Results from this study also indicated specific amino acid residues and/or conformational specificity, such as in the longer as1- and b-peptides, are required to increase the level of mineralization (Wikiel et al., 1994). Further studies in humans using in situ caries models (Reynolds, 1995, Reynolds, 1997) showed CPP was effective in reducing frequency and severity of dental caries lesions. The mechanism proposed is CPP stabilizes calcium phosphate as amorphous calcium phosphate (ACP). The multiple phosphorylserine residues in CPP bind ACP to form size-limiting nanocomplexes in a metastable solution. In turn, the nanoclusters' growth is controlled to a critical size required for nucleation and phase transformation at the enamel surface. Enamel subsurface caries lesions are also remineralized by CPP-ACP nanocomplexes (Reynolds et al., 1999).
The bioactivity of CPP can be compared to the saliva protein statherin. Milk casein and statherin have been mapped to the q arm of human chromosome 4 (Laila et al., 2005), and it is speculated the salivary protein ancestral gene evolved from the caseins (Kawasaki and Weiss, 2003). Both proteins control crystal nucleation and growth of hydroxyapatite in the tooth microenvironment (Stayton et al., 2003). Secondary sequence predictions of the N-terminus of statherin suggest a-helix formation (Gururaja and Levine, 1996). However, NMR spectral data for as1-casein (f 59-79) CPP does not indicate either a-helical or b-strand conformation (Cross et al., 2004). Despite this difference, both peptides adopt conformations that allow specific amino acid sequences to interact with calcium ions to control nucleation and/or biomineral growth. In the case of CPP, glutamyl and phosphoserine side chains form nanocomplexes with calcium (Cross et al., 2004), whereas statherin structures calcium on the phosphorylated serines and carboxylate-containing aspartic and glutamic acid residues (Stayton et al., 2003).
Whey proteins from cottage cheese manufacture or acid casein precipitation were less effective than CPP in precluding Ca and phosphate solubilization from hydroxyapatite (Warner et al., 2001). Proteose peptone fractions 3 and 5 were shown to inhibit hydroxyapatite demineralization in vitro (Grenby et al., 2001). Both proteose peptone fractions are embedded within b-casein and are liberated by plasmin hydrolysis (Eigel and Keenan, 1979, Eigel, 1981). Plasmin is an endogenous milk protease. Proteose peptone fractions would elute with whey proteins after casein insolubilization. Although whey proteins may not prevent enamel demineralization, it has been suggested whey may exert a topical anticariogenic effect by acting as a buffer (Reynolds and Del Rio, 1984).
Several patents have been issued to produce and utilize CPP as anticariogenic compounds (Han and Shin, 1998, Reynolds, 1991, 2002, 2004). Others have utilized this technology to develop commercial products for prophylactic treatment of dental caries. For example, Reynolds et al. (1992) patented a dentifrice composition (toothpaste) containing CPP to prevent demoralization that leads to gingivitis and dental caries. Later, Reynolds (1993) was issued a United States patent for specific CPP cation complexes that when formulated into mouthwash, toothpaste, lozenge, tablet, foodstuff, beverage, or other pharmaceutical compositions incorporates into plaque. Once CPP-cations are incorporated, they are resistant to endogenous phosphatase or peptidase activity and inhibit calculus activity. Reynolds (1999) extended this technology further by incorporating a phosphatase or peptidase inhibitor into dentifrice formulations containing CPP. Caseinate calcium fluorophosphates combined with CPP were patented (Bannister, 2003) for use in confectionary-like food forms to prevent gingivitis and halitosis. Sodium bicarbonate can be incorporated into chewing gums with CPP-AMP to provide dental health benefits (Luo and Wong, 2005). Dixon and Kaminski (2005) were awarded a patent for creating stable oral compositions containing CPP-AMP nanocomplexes in combination with a fluoride ion source, and a calcium chelator. Han (2006) invented an interproximal toothbrush coated with CPP that is recoated after each use by storing the brush in an accompanying storage and sealing unit.
Experimental evidence is available for efficacy of these patented formulations. For example, Shen et al. (2001) reported a sugar-free gum containing CPP-AMP nanocomplexes remineralized enamel subsurface lesions in a human in situ model system. Reynolds et al. (2003) reported chewing gum containing CPP-ACP produced higher levels of enamel remineralization than other calcium sources. In a follow-up study (Iijima et al., 2004), they demonstrated tooth enamel remineralized by CPP-AMP was more resistant to an acid challenge than control (chewing gum without CPP-AMP) remineralized enamel. A mouthwash that contained CPP-ACP significantly increased plaque calcium and inorganic phosphate levels. Sugar-free lozenges are a suitable composition to deliver CPP-ACP and promote enamel remineralization (Cai et al., 2003). They observed a dose-related response in a human in situ model system. Glass-ionomer cement (GIC) is used to chemically adhere restorations to tooth tissues. The incorporation of CPP-ACP into GIC allows slow release of ions into dentin to provide further protection during acid challenges without any negative effects to compressive strength, net setting time, or microtensile bond strength to the dentin (Mazzaoui et al., 2003). Lennon et al. (2006) reported amine fluoride gel (12,500 ppm fluoride) was more effective at protecting enamel demineralization after acid challenge than CPP-AMP, NaF (250 ppm), or a combination of CPP-AMP and NaF.
Tooth surfaces are colonized by a variety of bacterial species that hydrolyze dietary carbohydrates to organic acids (Figs 7.2 and 7.3). This commensal relationship establishes a complex microbial ecosystem composed of aerobic, microaerophilic, and anaerobic microorganisms. These bacteria are integral components of plaque. Some of them are opportunistic cariogenic pathogens that demineralize tooth surfaces and form lesions that develop into dental caries. Plaque composition is a complex mixture of viable and dead bacterial cells, carbohydrate glucans, proteins, and a variety of minerals (Margolis and Moreno, 1994).
In the oral microbial ecosystem, bacteria must adhere to a surface for colonization. There are over 400 different indigenous microbial species in the human oral cavity (Moore and Moore, 1994). Different bacterial species colonize the tooth's surface than found in the gingival crevice. Tooth surface bacteria interact with saliva, host diet constituents, and growth factors to colonize. Bacteria usually identified on the tooth surface are Streptococcus mutans, S. sanguis, S. sobrinus, S. gordonii, and S. oralis. This bacterial group is usually referred to as mutans streptococci because of their ability to secrete polysaccharides that add to the tooth's biofilm layer. Researchers frequently isolate Lactobacillus acidophilus, L. gasseri, and L. fermentum from teeth of healthy individuals, and predominantly L. plantarum from periodontal patients (Koll-Klais et al., 2005).
Dental caries etiology is quite complex and still being extensively studied. Although plaque is an important component in lesion development, other factors such as the host's oral microflora and the microbial ecosystem in the lesion interact to progress decay (Figs 7.2 and 7.3). The organisms discussed above are frequently isolated from carious lesions. Furthermore, if genes responsible for mutans production are deleted from streptococci then lesions do not form in model systems. Therefore presence of these organisms in dental plaque indirectly indicates they could be an important factor in caries formation. Total elimination of mutans streptococci in the complex oral environment would not totally eliminate caries formation, but it would be at a significantly reduced level (Bowden, 2000).
Bacteria found in gingival crevicular fluid depend on environmental and host conditions to colonize. Most of these organisms are strict anaerobes, and capable of eliciting a host inflammatory response if their population becomes too large. Anaerobic bacteria such as Porphymonas gingivalis, spirochetes (such as Treponema species), Bacteroides, Campylobacter, and Fusobacterium are frequently isolated from gingival crevicular space. These bacteria are opportunistic pathogens and will cause gingivitis that could lead to the more serious periodontitis.
Saliva is a multi-function biological fluid that controls and stabilizes the oral ecosystem. The primary composition of saliva is mucinous glycoproteins, which coats the teeth. This results in an acellular insoluble membranous layer referred to as the salivary pellicle. The pellicle is strongly adhered to the enamel surface and has numerous functions (Bowen and Li, 1997, Tabak and Bowen, 1989). Saliva also contains secondary components important for mineral modulation (statherin), immune response (immunoglobulins), food digestion (amylase), and plaque formation (glucosyltransferases) (Lijemark and Bloomquist, 1996). Saliva is not a separate oral microbial ecosystem. Instead, it acts as a diluent to deliver bacteria to most surfaces in the oral cavity from the tongue, dental plaque, mucosal surfaces, and ingested food.
Nesser et al. (1994) studied milk casein derivative's ability to inhibit odontopathogenic bacteria by preventing bacterial adhesion to a simulated tooth surface. Sodium caseinate, CPP, and glycomacropeptide (GMP) inhibited adherence of oral bacteria to saliva-coated hydroxyapatite beads (S-HA). The potential dental pathogens, S. sobrinus OMZ 176 and S. sanguis OMZ 9 were competitively excluded from S-HA beads by casein derivatives. However the more favorable oral organism, Actinomyces viscosus Ny 1, adhered to S-HA beads. In addition, caseinate, CPP, and GMP were able to bind directly to cell walls of examined cariogenic bacterial strains. Anticariogenicity by these proteins and peptides was accomplished by selectively inhibiting streptococcal adhesion to teeth. Furthermore, microbial composition of dental plaque was modulated to favor establishment of less cariogenic species such as oral actinomyces.
Milk and individual caseins (a-, b-, and k-caseins) were studied to determine adherence of S. mutans to saliva-coated hydroxyapatite discs (sHA) (Vacca-Smith et al., 1994). Milk inhibited in vitro adherence of S. mutans GS-5. Individual caseins were also examined. No effect on streptococcal adherence was observed when a- or b-casein was incubated with sHA. However, k-casein inhibited adherence of S. mutans GS-5. Inhibitory properties were credited to a 40,000 dalton glycoprotein.
Micellar casein was shown to prevent oral colonization in rats by S. sobrinus OMZ 176, and to promote colonization by A. viscosus Ny1 (Guggenheim et al., 1999). Sodium caseinate was not as effective as micellar casein at inhibiting streptococcal colonization. Whey proteins and soy protein isolate had no effect on microbial colonization.
Inhibition of cariogenic bacterial adherence to the salivary pellicle would protect teeth from developing lesions. Casein peptides, GMP and CPP, were incubated with saliva coated bovine enamel discs to study incorporation into the pellicle (Schupbach et al., 1996). Electron microscopy confirmed both proteins were incorporated into the pellicle in exchange for albumin. The effect of incorporated GMP or CPP on adherence of mutans streptococci was studied. Adherence of S. sobrinus was reduced 49%, 75%, and 81% by GMP, CPP, and GMP+CPP, respectively. Adherence of S. mutans was more efficient with reductions of 64%, 83%, and 84% by GMP, CPP, and GMP+CPP, respectively. This relatively selective inhibition of mutans streptococci could eventually produce a non-cariogenic plaque. Rose (2000) reported CPP-ACP has a greater affinity for calcium than S. mutans R9 cells. As CPP-ACP binds to plaque, it provides a large reserve of calcium for remineralization.
A similar study evaluated GMP and CPP as adhesion inhibitors of oral bacteria using saliva-coated hydroxyapatite beads (sHA) (Nesser et al., 1994). Both casein peptides blocked adhesion of S. sanguis OMZ 9 and S. sobrinus OMZ 176 to sHA. Neither peptide inhibited adhesion of A. viscosus Ny 1 to sHA. S. sanguis OMZ 9 also attaches to human buccal epithelial cells (HBEC). However, adherence is inhibited in the presence of GMP (Nesser et al., 1995). Desialylation of either GMP or HBEC was less effective at inhibiting adhesion of S. sanguis OMZ 9. These results indicate bacterial adhesion to oral epithelial cells is dependent upon sialic acid presence. This is in contrast to tooth enamel adhesion where ionic interactions are important for adherence (Nesser et al., 1995). This exemplifies the multiple mechanisms odontopathogenic bacteria utilize to establish in the oral microecology.
Malkoski et al. (2001) have further fractionated GMP using reversed phase high performance liquid chromatography to identify the peptide fragment responsible for S. mutans growth inhibition. They found the active form to be a non-glycosylated, phosphorylated peptide in residues 138-158. They designated the antimicrobial as kappacin. The non-phosphorylated peptide did not posses inhibitory activity.
Lactoperoxidase in combination with hydrogen peroxide and thiocyanate ion is a powerful bacteriostatic system found in milk, tears, and saliva. Inhibition of mutans streptococci (serotypes a through g) metabolism was studied in a washed cell model system (Thomas et al., 1983). Serotypes (BHT, FA-1, OMZ 176) that produced large amounts of hydrogen peroxide were greater than 90% inhibited. Intermediate hydrogen peroxide producers
(B-13 and Ingbritt) were only inhibited 20 to 50%. No inhibition was shown with low peroxide producing strains (AHT, HS-6, GS-5, LM-7, OMZ 175, and 6715-15). Similar effects were observed on the metabolism of S. mitis, S. sanguis, S. salivarius, and S. mutans grown under aerobic and anaerobic conditions. Glyceraldehyde 3-phosphate dehydrogenase, an important enzyme in glycolytic pathways, was inhibited by hypothiocyanite (Carlsson et al., 1983). A commercially available toothpaste formulated with lactoperoxidase showed elevated levels of hypothiocyanous acid and hypothiocyanite ions in the saliva of patients (Lenander-Lumikari et al., 1993). However, no effect was noted on the oral microflora after 30 days treatment.
Saliva, milk, and tears also contain the bactericidal enzyme lysozyme. Lysozyme hydrolyzes b(1 ^ 4)-glucosidic linkages in bacterial cell wall peptidoglycan to kill susceptible organisms. This enzyme also has several other defenses to inhibit oral pathogens; for example, bacterial autolysins are activated (Labile and Germaine, 1985), bacterial cells are aggregated (Pollock et al., 1987), and adherence (Iacono et al., 1980) and metabolism (Lumikari and Tenovuo, 1991) are inhibited. Synergistic effects of lysozyme and subsequent lactoperoxidase exposure results in total inhibition of glucose metabolism by mutans streptococci (Lenander-Lumikari et al., 1992).
Bovine milk lactoferrin inhibits saliva-induced S. mutans aggregation (Mitoma et al., 2001). Amino acid residues 473 to 538 of lactoferrin are important for inhibition. Oho et al. (2002) subsequently reported lactoferrin peptide (f 473-538) inhibited adherence of S. mutans to sHA beads. Other milk proteins were also tested in this study. Lactoperoxidase and immunoglobulin G showed moderate inhibitory activity. Individual intact casein and whey proteins showed minimal inhibitory activity.
Several patents have been issued utilizing much of the technology discussed above. Micellar casein incorporated into an edible composition inhibits oral colonization of S. sobrinus (Berrocal et al., 1995). Another patent (Berrocal et al., 1998) protects a process to prepare micellar casein with 100 ppm fluoride for use in dentifrices. Two patents (Nesser, 1991a, 1991b) were issued to utilize intact and desialylated GMP in compositions for inhibiting buccal cavity bacteria. Gelatin combined with GMP and a surfactant (sodium lauryl sulfate) was formulated into a toothpaste or chewing gum (Zhang and Gaffar, 1998b). A patent (Zhang and Gaffar, 1998a) discloses a multi-component oral hygiene product for enhanced remineralization that contains GMP in one component and a fluoride ion in a second component. Xylitol and GMP combined in a composition remineralize teeth (Zhang and Gaffar, 2001). A couple of inventions claim periodontitis prevention by reduction of dental plaque and calculus deposition by using GMP and lactoferrin (Braun and Nimmagudda, 2002a and 2002b). Both patents show data for growth inhibition of Actinobacillus haemophilus and S. pyogenes. Valenti and Antonini (1998) formulated a topical protein preparation containing lysozyme. The composition is recommended for therapy of periodontitis and diseases of the teeth and oral cavity.
7.3.4 Effect of dairy components on other oral diseases Oral mucositis is inflammation of oral mucosa from chemotherapeutic agents or ionizing radiation (Sonis, 1998). Oral mucositis typically manifests as erythema or ulcerations. This condition is sometimes called stomatitis, and is a major dose-limiting side effect of chemotherapy. Severity of oral mucositis is partially determined by epithelial cell turnover (Sonis and Sonis, 1979). Older patients (>60 years old) have a lower incidence of oral mucositis than pediatric patients (Sonis, 1998).
Clarke et al. (2002) reported a biologically active extract containing bovine whey proteins prevented oral mucositis in hamsters undergoing chemotherapy. They termed this extract whey growth factor extract - A (WGFE-A), and demonstrated the preparation contained mitogens that stimulate cells of mesenchymal origin and inhibit epithelial cell growth in culture (Belford et al., 1997). The bioactive properties of WGFE-A include anti-proliferative, anti-apoptotic, and antimicrobial.
Naturally occurring growth factors contained in whey were formulated into a mouthwash (PV701) and tested in human patients undergoing chemotherapy (Prince et al., 2005). Growth factors previously identified from whey preparations include insulin-like growth factor, platelet-derived growth factor, transforming growth factor b, fibroblast growth factor, and epidermal growth factor (Belford et al., 1997, Rogers et al., 1996). In addition, other bioactive compounds were reportedly present with bacteriostatic properties that would inhibit opportunistic infectious agents. PV701 mouthwash was used six times per day by patients with lymphoma starting six days before chemotherapy began, and continuing for five days after completion. Incidence and severity of oral mucositis was reduced in patients receiving PV701 compared to a placebo (Prince et al., 2005). No adverse effects were reported with PV701 in this clinical trial.
7.3.5 Fermented foods and probiotics anticariogenicity
The word 'probiotic' is translated from the Greek language and means 'for life'. A more modern definition for probiotics is living organisms that upon ingestion in certain numbers exert health benefits beyond basic nutrition (Aimutis, 2002). Many others have defined probiotics as live microbial food supplements that beneficially affect the host by improving its intestinal microbial balance. Thus they create a healthy gut environment. Most human probiotic products contain species of lactic acid bacteria, especially species of Lactobacillus or Bifidobacterium. Probiotics are beneficial to human health by demonstrating such diverse activities as regulation of the intestinal microbial balance, improvement of stool consistency, pathogen antagonism to prevent diarrhea, stimulation of immune response, and anti-cancer activity (Meurman, 2005, Sanders, 1999).
A randomized, double-blind, placebo-controlled intervention study was used to determine if milk containing Lactobacillus rhamnosus GG administered to kindergarten children reduced their caries risk and initial caries development (Näse, 2001). Children consumed probiotic or normal milk 5 days a week for 7 months in their day-care facilities. Children consuming probiotic milk had less dental caries than the control group. In addition, the probiotic strains reduced numbers of mutans streptococci.
Cornelli et al. (2002) screened 23 dairy microorganisms for antagonistic behavior against cariogenic bacteria. They identified two S. lactis and two Lactococcus lactis strains that adhered to saliva-coated hydroxyapatite beads. S. thermophilus NCC 1561 and L. lactis ssp. lactis NCC2211 were incorporated into a biofilm mimicking dental plaque. S. oralis 0MZ607, a cariogenic strain, was inhibited from growing in the plaque by L. lactis NCC2211.
Cheese containing L. rhamnosus GG and L. rhamnosus LC 705 was consumed by 74 subjects in a randomized, double-blind, controlled study with two parallel groups (Ahola et al., 2002). Subjects ate 15 g of cheese five times a day preferably after meals or snacks. No statistically significant difference was observed between control and test groups in S. mutans counts after the intervention period, but during the post-treatment phase there was a significantly greater reduction in the intervention group compared to control. Mutans counts decreased 20% and yeast counts 27% in all subjects, regardless of treatment. This further confirms the beneficial effects of cheese to prevent dental caries.
Strains must be thoroughly screened for selection as a potential oral probiotic. Some species of lactobacilli are thought to be cariogenic. In a study by Matsumoto et al. (2005), L. salivarius LS1952R established itself in the oral cavity of rats and induced a significant level of dental caries. They confirmed L. salivarius could adhere to saliva-coated hydroxyapatite beads. Montalto et al. (2004) also showed patients receiving an oral administration of lactobacilli probiotic for 45 days had significantly higher lactobacilli counts in their saliva than placebo. S. mutans population was not significantly affected. Therefore, patients ingesting probiotic products should have their dental health closely monitored. L. rhamnosus GG reportedly does not colonize in the oral cavity (Yli-Knuuttila et al., 2006), despite its benefits mentioned earlier.
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