© INRA, EDP Sciences, 2009

1. INTRODUCTION

In the recent decades, the consumption of acidic beverages has increased in many countries, which is thought to be responsible for the demineralisation of the hard tissue of enamel and dentin [8]. According to one study, 20–60% of children and adolescents have dental erosion and this has been attributed to the increasing consumption of acidic drinks [3]. Many attempts have been made to modify drinks to reduce the erosive potential, though some of these modifications may change the flavour, texture and pH [5]. For example, fluoride might even cause adverse effects if added to different drinks, leading the total uptake to reach high or overdose levels [3].

In this respect, milk peptides have also been investigated for their protective potential against dental erosion. Caseinophosphopeptide (CPP) is a peptide derived from casein by the action of trypsin. Results indicate that it is able to suppress demineralisation and/or improve remineralisation, and therefore, it would be useful if added to acidic drinks [9, 14]. Glycomacropeptide (GMP), a casein-derived peptide, has also received much attention because of its biological activities, including the reduction of dental caries. During cheese making, rennet (chymosin) cleaves κ-casein in bovine milk at Phe105-Met106 amino acids, producing two separate parts, para κ-casein and GMP. Para κ-casein is the N-terminal and consists of amino acids 1–105. The GMP, also called caseinomacropeptide, is the C-terminal and includes the rest of the amino acids (105–169). Para κ-casein remains with the curd and GMP is released into the whey [4].

One study concluded that GMP prevents adherence of bacteria to tooth surfaces [17]. Furthermore, GMP can be used to produce “high protein-reduced sugar” drinks, which are beneficial to general health in terms of providing the body with nutrients and helping it fight viral and bacterial infections [6].

The aim of this study was to investigate the protective effect of GMP with or without chemical modification, against dental erosion in comparison with CPP.

2. MATERIALS AND METHODS

2.1. Materials

Sodium caseinate (Product No. C8654), bovine trypsin (E.C.3.4.21.4; Product No. T8642), crystalline hydroxyapatite (HA) suspension (Type 1; 25% solids, Product No. H0252), the BCA (Bicinchoninic Acid kit) for protein determination and Dowex 50 (H⁺) ion exchanger resin (Product No. 422096) were obtained from Sigma Chemical Co. (Poole, Dorset, UK). Commercial CPP (CPPc) and commercial GMP (CGMP-10) were provided by Arla Foods Ingredients amba (Surry, UK). All the chemicals used were of analytical grade.

2.2. Methods

2.2.1. Preparation of HA

Six grams of original HA suspension was mixed with 240 mL of water and centrifuged for 10 min at 3000× g. The supernatant was discarded and washed again to remove soluble phosphate. Washed HA was resuspended in 108 mL of distilled water. This suspension was placed on the magnetic stirrer and while it was being stirred continuously, 1100 μL of suspension (20 mg dry weight) was withdrawn and added to a series of 1.5 mL eppendorfs, arranged in four groups of 15 and four groups of 9. After pipetting (n = 96), HA samples were centrifuged at 3000× g for 10 min and then the supernatant was discarded and HA was ready to use [9].

2.2.2. Preparation of CPP

Sodium caseinate (10 g) was dissolved in 100 mL of 0.05 mol·L⁻¹ Tris-HCl buffer, pH of 7.5 and 50 °C. This was followed by the addition of 12.5 mg of trypsin and incubated at 37 °C for 2 h. The reaction was terminated by heating to 90 °C for 5 min. The solution was cooled and pH was adjusted to 4.5 using 1 mol·L⁻¹ HCl, after stirring for 10 min it was centrifuged for 30 min at 3000× g. The pH of the supernatant was adjusted to 7.0 by adding 2 mol·L⁻¹ NaOH. To the final solution, calcium chloride (0.8 g) was added followed by an equivalent volume of 95% aq. ethanol to precipitate the CPP fractions. After 1 h, the solution was centrifuged for 30 min at 3000× g. The supernatant was removed and the pellet was washed and resuspended in 30 mL of 50% (v/v) aq. ethanol. The washed solution was removed by centrifugation (10 min, 3000× g). The pellet was spread on a plastic dish and allowed to dry at room temperature and designated laboratory prepared CPP (CPPlp) [9, 10].

2.2.3. Calcium reduction of peptides

One gram of each peptide (GMP, CPPc and CPPlp) was dissolved in 10 mL of distilled water and added to 24 g of washed Dowex 50 ion-exchange resin. The suspension was stirred for 40 min and left for 10 min to precipitate the resin. The supernatant was removed and centrifuged for clarification, for 5 min at 3000× g. Then, 20 mL of water was added to the resin and stirred for 2 min and the supernatant was removed as above. The pH of the supernatant was adjusted to 7.0 with 1 mol·L⁻¹ NH₄OH.

2.2.4. Assessment of protective effect of the peptides at different pH

The HA samples were prepared as described in two series of four groups. The first group was used for GMP, GMP-ca, CPPc and CPPc-ca with control and the second group for CPPlp and CPPlp-ca (-ca: calcium-reduced peptide) with control to investigate the protective effect of the peptides at different pH levels 3, 3.5, 4 and 4.5. The concentration of all peptides was adjusted to 5 mg·mL⁻¹ using the BCA method. From the first group, the four subgroups were treated with GMP and GMP-ca, CPPc and CPPc-ca, respectively. The final subgroup was kept as control. All the samples were resuspended and mixed thoroughly with the peptide solutions for 20 min with intermittent stirring to permit maximum binding to the HA, then centrifuged at 3000× g, for 10 min. The supernatant was discarded and HA pellet was rinsed with 1 mL of distilled water to remove excess unbound peptide to HA particles and it was removed by centrifuging at 3000× g, for 10 min. To the treated HA and control, 1 mL of 0.1 mol·L⁻¹ citric buffer at a pH of 3, was added. The mixture was resuspended and allowed to stand for 10 min, stirred once during the standing time and centrifuged (3000× g, 10 min). The supernatants were rapidly decanted off into clean tubes for calcium and phosphate analysis. All the remaining groups were treated similarly but at different pH levels.

2.2.5. Assessment of calcium and phosphate in erosive solution

Calcium was measured by atomic absorption spectroscopy to measure the calcium content of the erosive sample. Different concentrations of standard calcium were prepared from calcium ion solution of 1000 mg·mL⁻¹ (2.5, 5, 7.5, 10, 15, 20) and absorbance was measured at 422.7 nm. Samples were diluted with distilled deionised water into appropriate concentration [8].

The level of phosphate in the erosive solution was measured with colorimetric determination using the method described by Allen [2].

3. RESULTS

Hydroxyapatite suspension was used as a substitute for teeth mineral. The amount of calcium and phosphate loss from HA was used to evaluate the protective effect of the peptides against acid dissolution-erosion in comparison with the control.

3.1. Protective effect of intact peptides at different pH values

After each experiment, the calcium and the phosphate content of the citric buffer was measured and compared with the control. At a pH of 3, the HA treated with GMP showed a protective effect of 38.5% and its effect increased to 47.5% with increasing pH. The CPPc showed around 14% less protection than GMP at different pH values, and CPPlp showed less protection than GMP at pH 3 but its effect increased with increasing pH. The protection of CPPlp was the highest at a pH of 4.5 (Fig. 1).

Figure 1. Protective effect of the peptides against calcium and phosphate dissolution from HA. GMP, glycomacropeptide; CPPc, commercial caseinophosphopeptide and CPPlp, laboratory prepared caseinophosphopeptide.

3.2. Protective effect of calcium-reduced peptides at different pH levels

The calcium content of each peptide sample at the same protein concentration (5 mg·mL⁻¹) was measured, which was 78 mg·L⁻¹ in GMP, 19 mg·L⁻¹ in CPPc and 1157 mg·L⁻¹ in CPPlp. The calcium content of each peptide was measured after calcium reduction, and the calcium content of both GMP and CPPlp was reduced by 80%, and that of CPPc was reduced by 13%.

The protective effect of GMP-ca and CPPc-ca after reduction of the calcium contents was similar, apart from that at a pH of 4.5, where the protective effects were reduced by 31% and 26%, respectively. On the other hand, CPPlp-ca showed minimal reduction in protection at all pH levels. The data in Figure 2 compare the protection effect of reduced peptides with the original samples at different pH levels.

Figure 2. Protective effect of calcium-reduced peptides against calcium and phosphate dissolution from HA. GMP, glycomacropeptide; CPPc, commercial caseinophosphopeptide; CPPlp, laboratory prepared caseinophosphopeptide and -ca, calcium-reduced peptide.

4. DISCUSSION

The extent of tooth erosion is determined by the number, duration of acid attacks and the pH of acidic solution [14, 19]. The role of GMP/CPP is to coat the HA particles to suppress demineralisation and to enhance remineralisation during an acid attack. It has been suggested that CPP might replace the calcium lost from the enamel by providing fast remineralisation and also provide a “supersaturated” calcium-rich environment [14, 15]. It has been reported that CPP could localise the amorphous calcium phosphate on tooth surfaces.

The GMP inhibits the adhesion of cariogenic bacteria such as Streptococcus spp. mutans, sanguis and sobrinus to oral surfaces [4, 7, 12–14, 18], and it can modify the composition of plaque bacteria to control its acid production and in turn reduce the demineralisation of enamel and promote remineralisation [1, 11, 16]. The GMP is a source of N-acetyl-necromantic acid [17] and one study showed that dietary intake of GMP can increase the sialic acid content of saliva, with effects on its viscosity and protective function [20].

According to the results of this study, GMP demonstrated a higher mineral protective effect than CPP at lower pH levels. However, the mineral protective effect of all three peptides increased with the increasing pH of the erosive solution. Finally, since the reduction in the calcium content of the peptides can reduce their protective effect, a state of calcium saturation may be responsible for a least part of the protection effect of the peptides. Therefore, we concluded that GMP and CPP can have the ability to reduce the dental erosion.

Acknowledgments

The authors thank Amir. H Abedi for his dentistry view and Arla Food Ingredient is greatly appreciated for supplying the GMP and CPP for this study.

References

1. Aimutis W.R., Bioactive properties of milk proteins with particular focus on anticariogenesis, J. Nutr. 134 (2004) 989–995.
2. Allen R.J., The estimation of phosphorous, Biochem. 34 (1940) 858–864.
3. Attin T., Meyer K., Hellwig E., Buchalla W., Lennon A.M., Effect of mineral supplements to citric acid on enamel erosion, Arch. Oral Biol. 48 (2003) 753–759 [CrossRef] [PubMed].
4. Brody E.P., Biological activities of bovine glycomacropeptides, Brit. J. Nutr. 84 (2000) S39–S46.
5. Chunmuang S., Jitpukdeebodintra S., Chuenarrom C., Benjakul P., Effect of xylitol and fluoride on enamel erosion in vitro, J. Oral Sci. 49 (2007) 293–297 [CrossRef] [PubMed].
6. Etzel M.R., Manufacture and use of dairy protein fractions, J. Nutr. 134 (2004) 996–1002.
7. Guggenheim B., Schmid R., Aeschlimann J.M., Berrocal R., Neeser J.R., Powdered milk micellar casein prevents oral colonization by Streptococcus sobrinus and dental caries in rat: a basis for the caries-protective effect of dairy products, Caries Res. 33 (1999) 446–454 [CrossRef] [PubMed].
8. Hara A.T., Zero D.T., Analysis of the erosive potential of calcium- containing acidic beverages, Eur. J. Oral Sci. 116 (2008) 60–65 [PubMed].
9. Kanekanian A.D,Williams R.J.H., Brownsell V.L., Andrews A.T., Caseinophosphopeptides and dental protection: concentration and pH studies, Food Chem. 107 (2008) 1015–1021 [CrossRef].
10. McDonagh D., FitzGerald R.J., Production of caseinophosphopeptides (CPPs) from sodium caseinate using a range of commercial protease preparations, Int. Dairy J. 8 (1998) 39–45 [CrossRef].
11. Mikkelsen T.L., Rasmussen E., Olsen A., Barkholt V., Frøkiær H., Immunogenicity of -casein and glycomacropeptide, J. Dairy Sci. 89 (2006) 824–830 [PubMed].
12. Neeser J.R., Chambaz A., Del Vedovo S., Prigent M.J., Guggenheim B., Specific and nonspecific inhibition of adhesion of oral actinomyces and Streptococci to erythrocytes and polystyrene by caseinoglycomacropeptide derivatives, Inf. Immun. 56 (1988) 3201–3208.
13. Neeser J.R., Golliard M., Woltz A., Rouvet M., Gillmann M.L., Guggenheim B., In vitro modulation of oral bacterial adhesion to saliva-coated hydroxyapatite beads by milk casein derivatives, Oral Microbiol. Immunol. 9 (1994) 193–201 [CrossRef] [PubMed].
14. Oshiro M., Yamaguchi K., Takamizawa T., Inage H., Watanabe T., Irokawa A., Ando S., Miyazaki M., Effect of CPP-ACP paste on tooth mineralization: an FE-SEM study, J. Oral Sci. 49 (2007) 115–120 [CrossRef] [PubMed].
15. Reynolds E.C., Remineralization of enamel subsurface lesions by casein phosphopeptide stabilized calcium phosphate solutions, J. Dent. Res. 76 (1997) 1587–1595 [CrossRef] [PubMed].
16. Schupbach P., Neeser J.R., Golliard M., Rouvet M., Guggenheim B., Incorporation of caseinoglycomacropeptide and caseinophosphopeptide into the salivary pellicle inhibits adherence of mutans Streptococci, J. Dent. Res. 77 (1996) 1779–1788.
17. Thoma-Worringer C., Sorensen J.S., Lopez-Fandino R., Health effects and technological features of caseinomacropeptide, Int. Dairy J. 16 (2006) 1324–1333 [CrossRef].
18. Vacca Smith A.M., Bowen W.H., The effects of milk and -casein on salivary pellicle formed on hydroxyapatite discs in situ, Caries Res. 34 (2000) 88–93 [CrossRef] [PubMed].
19. Vanuspong W., Eisenburger M., Addy M., Cervical tooth wear and sensitivity: erosion, softening and rehardening of dentin, effects of pH, time and ultrasonication, J. Clin. Periodontol. 29 (2002) 351–357 [CrossRef] [PubMed].
20. Wang B., Staples A., Sun Y., Karim M., Brand-Miller J., Effect of dietary sialic acid supplementation on saliva content in piglets, Asia Pacific J. Clin. Nutr. 13 (2004) S75.

All Figures

	Figure 1. Protective effect of the peptides against calcium and phosphate dissolution from HA. GMP, glycomacropeptide; CPPc, commercial caseinophosphopeptide and CPPlp, laboratory prepared caseinophosphopeptide.
In the text

	Figure 2. Protective effect of calcium-reduced peptides against calcium and phosphate dissolution from HA. GMP, glycomacropeptide; CPPc, commercial caseinophosphopeptide; CPPlp, laboratory prepared caseinophosphopeptide and -ca, calcium-reduced peptide.
In the text