Australian Dental Journal 2008; 53: 314–319
SCIENTIFIC ARTICLE
doi: 10.1111/j.1834-7819.2008.00072.x
Protective effect of yogurt extract on dental enamel demineralization in vitro GF Ferrazzano,* T Cantile,* M Quarto, A Ingenito,* L Chianese, F Addeo *University of Naples ‘‘Federico II’’, School of Dentistry, Department of Paediatric Dentistry, Naples, Italy. University of Naples ‘‘Federico II’’, Department of Food Science, Portici, Italy.
ABSTRACT Background: Casein phosphopeptides (CPPs) are phosphorylated casein-derived peptides produced synthetically by proteolytic digestion of as1-, as2- and b-casein. The anticariogenic activity of CPPs is due to their ability to stabilize high levels of amorphous calcium phosphate (ACP) on tooth surface, preventing demineralization and enhancing remineralization of enamel caries. The aim of this study was to test the in vitro ability of natural CPPs (contained in yogurt) to prevent demineralization and promote remineralization of dental enamel. Methods: Eighty human molars were used. After standardizing an in vitro demineralization procedure for producing artificial caries (Group 1: pH 4.8; Group 2: pH 3.97), this procedure was used on teeth, but with the addition of natural CPPs (Group 3: pH 4.8; Group 4: pH 3.97). The effects of these procedures were evaluated by quantitative analysis (change in weight and calcium titration) and qualitative analysis (SEM). Statistical analysis of the results was performed using ANOVA. Results: Statistical analysis showed significant differences in weight changes between the groups with and without natural CPPs. The SEM observation showed the protective effects of natural CPPs. Conclusions: The results demonstrated that CPPs contained in yogurt have an inhibitory effect on demineralization and promote the remineralization of dental enamel. Key words: Bioactive peptides, caries, casein phosphopeptides, prevention, yogurt. Abbreviations and acronyms: ACP = amorphous calcium phosphate; CPP = casein phosphopeptides; CPP-ACP = casein phosphopeptideamorphous calcium phosphate. (Accepted for publication 20 February 2008.)
INTRODUCTION Milk and dairy products have been identified as having anticariogenic activity.1 Petti et al. reported that milk was protective against caries in 6- to 11-year-old children who did not use fluoride, had poor oral hygiene, and had frequent sucrose consumption.2 Głabska et al. found that cheese consumption contributes to a decrease of the dental caries coefficient.3 The cariostatic effect of milk and milk products may be due to their high content of Ca and P ions, the buffer capacity, and the content of casein phosphopeptides (CPPs).4 The CPPs produced by tryptic digestion of casein, a bovine milk protein, have a remarkable ability to bind to amorphous calcium phosphate (ACP) and stabilize it in dental plaque.5 The preventive action of CPPs, in vivo, takes place when there are demineralizating 314
agents (acid pH) that can enhance the release of calcium from the casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) complex, thus increasing the Ca cation concentration and promoting a supersaturation condition that will prevent demineralization and enhance the remineralization of early enamel caries.6 Laboratory, animal and human in situ experiments have demonstrated that synthetic CPP-ACP nanocomplexes contained in mouthrinses and sugar-free chewing gums are anticariogenic.7–9 Furthermore, the addition of 2.0–5.0 g CPP-ACP ⁄ l to milk increases its ability to remineralize enamel subsurface lesions.10 Previously we have shown that, in the presence of synthetic CPPs, acid dissolution of human enamel is reduced by over 50 per cent in vitro.11 The aim of the present study was to test, in vitro, the effectiveness of yogurt extract (chosen for its high ª 2008 Australian Dental Association
Effect of yogurt extract on dental enamel demineralization CPP content) to inhibit demineralization and promote remineralization of early enamel caries.12,13
Table 1. Summary of solutions used Origin of solution
Solution
pH
MATERIALS AND METHODS
Synthetic
Initially, an experimental protocol to standardize a demineralization procedure of dental enamel14 at two different pH levels was established. This step was necessary in order to obtain reproducible dental enamel caries (Groups 1 and 2 as controls). These standardized procedures were later performed in the presence of remineralizing agents naturally present in the yogurt supernatant (Groups 3 and 4).
Natural
1 2 3 4
4.8 3.97 4.8 3.97
Remineralizing agent
Yogurt supernatant Yogurt supernatant
Demineralizing agent Lactic Lactic Lactic Lactic
acid acid acid acid
in a demineralizing solution containing 50 ml of 0.1 M lactic acid and 0.02% carboxymethylcellulose (Akzo Nobel, Netherlands) (pH 4.8, T 37C).15 The 20 specimens from Group 2 underwent the same treatment as Group 1, but at a pH value of 3.97.
Preparation of enamel specimens Eighty human teeth, extracted for orthodontic reasons or impaction, were cleaned with sterile gauze imbibed in distilled water and stored at 4C in sterile containers with 5% formalin solution. The roots were then cut by mean of a diamond disk assembled on a laboratory handpiece (NSK IS-65, A3311968, Japan) and the crowns polished with pumice dust and non-fluoride toothpaste, using a circular brush with nylon bristles mounted on a dental handpiece (KaVo INTRAmatic LUX3 20 LH, D481631, Germany). They were then rinsed in distilled water. From each crown, three enamel areas (6 · 4 mm2 each) were isolated using an acid-resistant varnish (Fig 1). The 80 specimens were divided into four groups, which subsequently underwent four different chemical treatments (Table 1). Demineralizing systems The 20 specimens from Group 1 were immersed for 96 hours (with one change of solution after 48 hours)
Preparation of CPP additives The specimens from Groups 3 and 4 were treated with a remineralizing solution containing a probioticenriched yogurt (Bifidobacterium Bb-12, L. acidophilus), chosen for its high CPP content.16 A fraction enriched in CPPs was prepared as whey resulting after the centrifugation (CL40R, Thermo Electron, Waltham, USA) of yogurt at 4000 g at 25C for 10 minutes. After three rounds of centrifugation, the supernatant was collected. The yogurt was separated into two fractions by centrifugation; with this procedure the insoluble fraction precipitates at the bottom of the test tube, whereas the soluble fraction containing the CPPs remains in suspension (supernatant). All the Group 3 specimens underwent the same treatment as Group 1 (50 ml of 0.1 M lactic acid and 0.02% carboxymethylcellulose), but with the addition of 50 ml of yogurt supernatant, for four days at 37C, with a change of solution after two days. The pH of the solution was adjusted to 4.8. The 20 specimens from Group 4 underwent the same treatment as Group 3, but at a pH value of 3.97 (the pH of the yogurt). Assessment methods
Fig 1. Isolated specimen. ª 2008 Australian Dental Association
After four days, all specimens were rinsed in distilled water and dried with a jet of warm air for three seconds. The effects of these procedures were evaluated by quantitative and qualitative analysis. The aim of the quantitative analysis was to assess the specimens’ weight changes and the calcium concentration of the solutions after the chemical treatments. Weight changes were measured by a digital analytical scale with a sensitivity of 0.01 mg (Gibertini Electronics, Novate, Milan, Italy). All the specimens were weighed before (t0) and after (t96) the treatments to assess hydroxyapatite weight content. The amount of calcium released into the solution was determined by compleximetric titration with 315
GF Ferrazzano et al. EDTA-Na2 (disodium ethylenediamine tetraacetate) using Eriochrome Black T as an indicator. The calcium concentration in solution was calculated before (t0) and after the treatments (t96). The results obtained were submitted to statistic elaboration through the use of the ANOVA system, programme SPSS 10.0. Furthermore, five specimens from each group were dehydrated in a graded series of ethanol, critical point dried (SPC-900 ⁄ EX, The Bomar Co., Tacoma, Washington, USA), mounted in stubs, sputter-coated with gold (E 306, Edwards, UK) and observed by scanning electron microscope (Stereoscan 250 MK3, Cambridge, UK) (qualitative analysis). RESULTS Statistical analysis showed that the mean weight change of the specimens from Group 1 was )3.28 ± (SD) 2.20 mg. The mean change in the calcium released in solution 1 was 0.63 ± (SD) 0.37 mg ⁄ 50 ml. Figure 2 shows an SEM image of the surface micromorphology of the artificial lesions created in the Group 1 specimens. The mean weight change in the specimens from Group 2 was )16.50 ± (SD) 5.88 mg. The mean change in the calcium released in solution 2 was 2.31 ± (SD) 0.57 mg ⁄ 50 ml. Figure 3 shows an SEM image of the surface micromorphology of a specimen from Group 2. The mean weight change in Group 3 specimens was 2.73 ± (SD) 1.95 mg. The mean change in the calcium released in solution 3 was )0.69 ± (SD) 3.06 mg ⁄ 50 ml. Figure 4 shows an SEM image of the surface micromorphology of a specimen from Group 3. The mean weight change of Group 4 was )1.27 ± (SD) 2.59 mg. The mean change in the calcium released in solution 4 was 1.69 ± (SD) 2.38 mg ⁄ 50 ml. Figure 5 shows an SEM image of the surface micromorphology of a specimen from Group 4.
Fig 3. Demineralized enamel surface at pH 3.97 (1000·).
Fig 4. Remineralized enamel surface at pH 4.8 and supernatant of yogurt (1000·).
Fig 5. Remineralized enamel surface at pH 3.97 and supernatant of yogurt (1000·).
Fig 2. Demineralized enamel surface at pH 4.8 (3000·). 316
Analysis of the results obtained after chemical treatment shows that artificial demineralization in the presence of natural ‘‘protective’’ factors (yogurt supernatant) provides significantly lower weight and calcium loss. ª 2008 Australian Dental Association
Effect of yogurt extract on dental enamel demineralization 4.00 calcium changes (mg/50ml)
weight changes (mg)
8.00
4.00
0.00
-4.00
-8.00 group 1
3.00
2.00
1.00
0.00
group 3
solution 2
solution 1
Fig 6. Significance of weight changes for Groups 1 and 3.
Fig 8. Significance of calcium changes for Solutions 1 and 2.
The mean difference in weight changes between Groups 1 and 3 was statistically significant (F = 83.76; p < 0.001) (Fig 6). Furthermore, SEM observation of the specimens from Group 1 shows substantial lesions, where the surface micromorphology looks ‘‘etched’’ (Fig 2). The SEM images of the specimens from Group 3 did not show any macroscopic erosive lesions (Fig 4). When these images were compared with the ones from Group 1, it was clear that the CPPs contained in yogurt played a positive role. The quantitative and qualitative analyses of the results obtained through artificial demineralization, in the presence of a dairy-derived remineralizing factor, clearly demonstrated the protective effects of natural CPPs on early dental enamel caries. The encouraging results obtained in Group 3 could pave the way for the development of preventive measures against dental caries based on the use of food-derived active ingredients (yogurt, cheese) that offer the advantage of being completely biocompatible, easy to obtain, non-toxic and inexpensive. When Group 2 was compared against Group 4, it was clear that the difference between the means of the weight changes was statistically significant (F = 112.25; p < 0.001) (Fig 7). In Group 2, the conspicuous weight
loss was due to the greater acidity of the solution which caused a considerable loss of substance. By contrast, the Group 4 specimens lost less weight, despite the fact that they were exposed to the same pH as Group 2. The SEM images of the specimens from Group 2 showed deep and irregular erosions, and the surface appeared disarranged and heterogeneous (Fig 3). The SEM images of the Group 4 specimens showed slightly irregular surfaces characterized by microporosities, due to the acid pH (Fig 5). These results are consistent with CPPs’ ability to inhibit the enamel demineralization. Analysis of the results of the calcium released in solution confirms what has been reported to date. A comparison between the concentration of the calcium released in solution 1 and the one in solution 2 clearly revealed that the difference between the means of the calcium content was statistically significant (F = 123.25; p < 0.001) (Fig 8). However, a comparison between the calcium concentration found in solution 3 and the one in solution 4 showed that the difference between the means of the changes of calcium content was not statistically significant (F = 7.57; p < 0.001) (Fig 9). These results are consistent with the previous data concerning weight loss. Therefore, it can be stated that 6.00 calcium changes (mg/50ml)
weight changes (mg)
5.00
-6.25
-17.50
-28.75
-40.00
3.00
0.00
-3.00
-6.00 group 2
group 4
Fig 7. Significance of weight changes for Groups 2 and 4. ª 2008 Australian Dental Association
solution 4
solution 3
Fig 9. Significance of calcium changes for Solutions 3 and 4. 317
GF Ferrazzano et al. the calcium increase in solution is directly proportional to the decrease in pH, the protective effect of natural CPPs is valid at a low pH value, and the remineralizing treatment can determine the transport of calcium from the solution to the tooth. DISCUSSION The data from this study showed that yogurt extract was protective against dental caries. This finding is in agreement with other reports demonstrating an association between dairy products consumption and reduction of dental caries.17–19 Several mechanisms by which dairy products may reduce enamel demineralization have been proposed. Levine proposed three mechanisms.20 Firstly, milk proteins may be adsorbed onto the enamel surface and may impede enamel demineralization; secondly, milk fat could be adsorbed onto the enamel surface and may have a protective role; and thirdly, milk enzymes may have a role in reducing the growth of acidogenic plaque bacteria.20 Herod also reported that milk and cheese could reduce the effects of metabolic acids, and could help restore the enamel that is lost during eating.21 From this study, it was postulated that protective mechanisms could involve buffering, salivary stimulation, reduction of bacterial adhesion, reduction of enamel demineralization, and ⁄ or promotion of remineralization by casein and ionizable Ca and P.21 Constituents in dairy products which may exert a direct effect on the tooth surface include calcium, phosphorus and CPPs. In particular, milk and milk products release calcium and phosphorus and, increases in their concentrations in dental plaque, inhibit demineralization and favour remineralization by a common-ion effect.19 CPPs, containing the sequence Ser(P)-Ser(P)-Ser-(P)Glu-Glu, stabilize nanoclusters of ACP in metastable solution. These multiple phosphoseryl residues of the CPPs bind to form nanoclusters of ACP in supersaturated solutions, preventing growth to the critical size required for phase transformations. CPP-ACP localize ACP in dental plaque, which buffers the free calcium and phosphate ion activities, helping to maintain a state of supersaturation with respect to tooth enamel, depressing demineralization and enhancing remineralization.6 Since milk and yogurt have similar components, it can be supposed that the mechanisms by which yogurt may have protective effect against dental caries are the same as milk. Variables that play a part during processing of milk are temperature, duration of heat exposure, exposure to light, and storage conditions. The final composition of yogurt is influenced by the species and strains of bacteria used in the fermentation, 318
the source and type of milk solids that may be added before fermentation, and the temperature and duration of the fermentation process. The protein content of yogurt is generally higher than that of milk because of the addition of non-fat dry milk during processing and concentration, which increases the protein content of the final product. In addition, yogurt is an excellent source of calcium and phosphorus. Fermentation has little effect on the mineral content of milk and therefore the total mineral content remains unaltered in the yogurt. Furthermore, because of the lower pH of yogurt compared with that of milk, calcium is present in yogurt mostly in an ionic form.22 Interestingly, the concentration of CPPs in yogurt is higher than that in milk due to the proteolytic activity of micro-organisms contained in yogurt.23 Consumption of probiotic products, such as yogurt containing live micro-organisms, can improve oral health status. Recent studies and results from randomized controlled trials have shown that yogurt, containing L. reuteri and Bifidobacterium DN-173 010, may reduce the levels of selected caries-associated microorganisms in saliva.24,25 In conclusion, results from this study indicate that CPPs in yogurt may have a remineralizing action in vitro when associated with a demineralizing agent. This preventive effectiveness is not due to the fact that the dental enamel is strengthened, but to the inhibition of demineralization. Although CPPs do not represent a treatment method, they nevertheless provide valid prevention against early demineralization of enamel when protective physiological mechanisms are insufficient. Future studies should focus on in vivo and epidemiological effects of yogurt consumption in reducing or eliminating dental caries. REFERENCES 1. Aimutis WR. Bioactive properties of milk proteins with particular focus on anticariogenesis. J Nutr 2004;134:989S–995S. 2. Petti S, Simonetti R, Simonetti D’Arca A. The effect of milk and sucrose consumption on caries in 6- to 11-year-old Italian schoolchildren. Eur J Epidemiol 1997;13:659–664. 3. Głabska D, Sin´ska B, Remiszewski A. Analysis of the dependence between milk and dairy products consumption, and dental caries observed in group of children and teenagers. Rocz Panstw Zakl Hig 2007;58:69–75. 4. McDougall WA. Effect of milk on enamel demineralization and remineralization in vitro. Caries Res 1977;11:166–172. 5. Cai F, Shen P, Morgan MV, Reynolds EC. Remineralization of enamel subsurface lesions in situ by sugar-free lozenges containing casein phosphopeptide-amorphous calcium phosphate. Aust Dent J 2003;48:240–243. 6. Cross KJ, Huq NL, Palamara JE, Perich J W, Reynolds EC. Physicochemical characterization of casein phosphopeptideamorphous calcium phosphate nanocomplexes. J Biol Chem 2005;280:15362–15369. ª 2008 Australian Dental Association
Effect of yogurt extract on dental enamel demineralization 7. Shen P, Cai F, Nowicki A, Vincent J, Reynolds EC. Remineralization of enamel subsurface lesions by sugar-free chewing gum containing casein phosphopeptide-amorphous calcium phosphate. J Dent Res 2001;80:2066–2070. 8. Reynolds EC, Cai F, Shen P, Walker GD. Retention in plaque and remineralization of enamel lesions by various forms of calcium in a mouthrinse or sugar-free chewing gum. J Dent Res 2003;82:206–211. 9. Iijima Y, Cai F, Shen P, Walker G, Reynolds C, Reynolds EC. Acid resistance of enamel subsurface lesions remineralized by a sugar-free chewing gum containing casein phosphopeptideamorphous calcium phosphate. Caries Res 2004;38:551–556. 10. Walker G, Cai F, Shen P, et al. Increased remineralization of tooth enamel by milk containing added casein phosphopeptideamorphous calcium phosphate. J Dairy Res 2006;73:74–78. 11. Ferrazzano GF, Troianiello S, Sangianantoni G, Ingenito A. [New strategies in dental caries prevention: experimental study on casein phosphopeptides.] Prevenzione odontostomatologica 2005;4:15–21. 12. Addeo F, Chianese L, Sacchi R, Musso SS, Ferranti P, Malorni A. Characterization of the oligopeptides of Parmigiano-Reggiano cheese soluble in 120 g trichloroacetic acid ⁄ l. J Dairy Res 1994;61:365–374. 13. Ferranti P, Barone F, Chianese L, et al. Phosphopeptides from Grana Padano cheese: nature, origin and changes during ripening. J Dairy Res 1997;64:601–615. 14. White DJ, Faller RV, Bowman WD. Demineralization and remineralization evaluation techniques–added considerations. J Dent Res 1992;71 Spec No:929–933. 15. Iijima Y, Takagi O, Ruben J, Arends J. In vitro remineralization of in vivo and in vitro formed enamel lesions. Caries Res 1999;33:206–213. 16. Chianese L, Caira S, Pizzolongo F, et al. Production of a probiotic yogurt with increased levels of bioactive peptides. Proceedings of the International Dairy Federation Seminar on Aroma and Texture of Fermented Milk. 22 June 2002. Kolding, Denmark. IDF, 2002:290–301.
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17. Merritt J, Qi F, Shi W. Milk helps build strong teeth and promotes oral health. J Calif Dent Assoc 2006;34:361–366. 18. Petridou E, Athanassouli T, Panagopoulos H, Revinthi K. Sociodemographic and dietary factors in relation to dental health among Greek adolescents. Community Dent Oral Epidemiol 1996;24:307–311. 19. Silva MF, Burgess RC, Sandham HJ, Jenkins GN. Effects of water-soluble components of cheese on experimental caries in humans. J Dent Res 1987;66:38–41. 20. Levine RS. Milk, flavoured milk products and caries. Br Dent J 2001;191:20. 21. Herod EL. The effect of cheese on dental caries: a review of the literature. Aust Dent J 1991;36:120–125. 22. Adolfsson O, Meydani SN, Russell RM. Yogurt and gut function. Am J Clin Nutr 2004;80:245–256. 23. Rasic´ JL, Kurmann JA. Bifidobacteria and their role. Microbiological, nutritional-physiological, medical and technological aspects and bibliography. Experientia Suppl 1983;39:1– 295. 24. Caglar E, Sandalli N, Twetman S, Kavaloglu S, Ergeneli S, Selvi S. Effect of yogurt with Bifidobacterium DN-173 010 on salivary mutans streptococci and lactobacilli in young adults. Acta Odontol Scand 2005;63:317–320. 25. Nikawa H, Makihira S, Fukushima H, et al. Lactobacillus reuteri in bovine milk fermented decreases the oral carriage of mutans streptococci. Int J Food Microbiol 2004;95:219–223.
Address for correspondence: Gianmaria Fabrizio Ferrazzano Via Cuoco no. 5 80100 Naples Italy Email:
[email protected]
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