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Science & Research

DENTAL TRIBUNE

Asia Pacific Edition

In Vitro Studies of Adsorption of Milk Proteins onto Tooth Enamel
Tove G. Devold, Morten Rykke, Esben S. Sørensen, Brian Christensen, Jan Karlsen & Gerd E. Vegarud, Norway Introduction
Milk and dairy products play an important role in the daily diet of people all over the world. The proteins of milk are appreciated for their high biological value. Milk proteins can be divided into two groups; i.e., caseins and whey proteins, based on differences in solubility at their isoelectric points (the pH value at which an amphiphilic molecule carries no net charge). The caseins make up approximately 80% of the protein fraction in bovine milk, and consist of four different gene products (␣s1-, ␣s2-, ␤- and ␬-casein). In milk the caseins exist in large spherical aggregates of several thousand casein molecules, known as casein micelles. The intermolecular forces holding the micelles together are mainly hydrophobic interactions and calcium phosphate bridges. Indeed, approximately 60% of the total content of these minerals in milk, known as colloidal calcium phosphate, is associated with the micelles. In their natural environment the micelles are stabilized against aggregation by a negative surface charge. When milk is acidified and the pH value approaches the isoelectric point of the casein fraction (pH 4.6), the micelles aggregate. If the acidification is brought about by direct addition of acid, the aggregated micelles form a precipitate that can easily be separated from the remaining whey fraction by filtration or centrifugation. In the production of fermented milk products such as yoghurt and sour milk products, lactic acid bacteria metabolize lactose into organic acids (mainly lactic acid). Compared to the direct acidification described above, this process is slow, and the casein micelles aggregate into a loosely connected network being responsible for the increased viscosity of fermented milk. The whey protein fraction of milk is a heterogeneous group of proteins. The most abundant protein species are ␤-lactoglobulin, ␣-lactalbumin, serum albumin, immunoglobulins and lactoferrin. These proteins differ from the casein fraction in that they maintain their solubility at their isoelectric point. Several milk proteins of different mammalian species are known to be bioactive and to positively influence various aspects of human health, including reducing the risk of caries (Korhonen and Pihlanto, 2006). Substantial scientific evidence from human and animal studies supports the conclusion that milk exerts anti-cariogenic or cariostatic properties, and that diets including milk and dairy products reduce the risk of dental caries (Vaccasmith, Vanwuyckhuyse, Tabak & Bowen, 1994; Guggenheim, Schmid, Aeschlimann, Berrocal & Neeser, 1999; Grenby, Andrews, Mistry & Williams, 2001; RuggGunn, 2001; Warner, Kanekanian & Andrews, 2001). Indeed, it seems that many of the reported anti-cariogenic properties of milk proteins including lubrication, digestion, regulation of the oral microflora, remineralization and film formation on oral surfaces are similar to those exerted by saliva proteins (for review see Amerongen & Veerman, 2002). Moreover, phosphopeptides released during proteolysis of casein may aid in remineralisation of enamel surfaces (Reynolds et al., 1999). In vitro and in vivo experiments have indicated a selective adsorption of different protein species onto hydroxyapatite (HA) surfaces and enamel surfaces in the oral cavity (Rykke & Sonju, 1991; Rølla & Rykke, 1994; Lamkin, Arancillo & Oppenheim, 1996; Guggenheim, Giertsen, Schupbach & Shapiro, 2001). The formation of this protein film, normally referred to as the acquired enamel pellicle, is of major importance in maintaining proper oral health. Based on scanning electron microscopy studies, the surface morphology of the acquired enamel pellicle has been described as being predominantly globular or with a dotted, raspberry-like structure (Lie, 1977; Busscher, Uyen, Stokroos & Jongebloed, 1989; Rølla & Rykke, 1994). Interestingly, morphological features similar to those observed in acquired enamel pellicle have been observed when enamel specimens were incubated in skimmed milk, and some of the beneficial properties of milk proteins for oral health and reduced risk of caries may directly relate to their ability to adsorb to tooth enamel surfaces in the oral cavity. However, to date no information regarding the pellicle forming abilities of proteins in different fluid milk products is available. The following article describes the study of the adsorption of milk proteins onto tooth enamel and how the matrix (eg, sweet versus fermented milk products) in which they are present may affect the adsorption in terms of amount of protein and the protein species found in the pellicle. In brief: the enamel specimens—freshly extracted third molars—were rinsed according to standard procedures prior to use. “Milk protein pellicles” were prepared in model systems by incubation of enamel specimens in different fluid milk products for 2 hours. The enamel specimens were either used as such or pre-incubated in saliva to form acquired enamel pellicle to simulate the conditions in the oral cavity. Sweet and fermented commercial dairy products, skimmed milk (pH 6.7) and yoghurt (pH 4.2) and their acidified and neutralized counterpart, ie, milk acidified by direct addition of acid (pH 4.2) and neutralized yoghurt (pH 6.7), respectively, were used to study how product acidity may affect protein adsorption. Throughout this article we will use the term in vitro “name of product (pH of product) protein pellicles” to describe the protein films that were formed on enamel surfaces in our model system. Thus, “skimmed milk (pH 7.6) protein pellicle” and “skimmed milk (pH 4.2) protein pellicle” refer to pellicle films formed by incubation of enamel specimens in skimmed milk (pH 6.7) and acidified skimmed milk (pH 4.2), respectively. After the incubation period, the enamel specimen were rinsed and “milk protein pellicles” were isolated. Their protein composition was investigated by the use of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were identified either by their pattern of migration in SDS-PAGE compared to purified protein standards or by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) fingerprinting. MALDI-TOF-MS fingerprinting has become the method of choice for many proteomic applications, including identification of unknown protein species in samples of different biological origin. ified “milk protein pellicles” indicated that the pH value of the incubation medium was a major factor in determining protein adsorption. Neutral “milk protein pellicles” formed by incubation of enamel specimens in skim milk (pH 6.7) and in yoghurt (pH 6.7) mainly consisted of caseins. At neutral pH casein micelles carry a net negative charge and may adsorb onto hydroxyapatite through calcium bridging (Sienkiewicz, Mohamed & Lipke, 1988). The acidic “milk protein pellicles”, skimmed milk (pH 4.2) pellicle and yoghurt (pH 4.2) pellicle were markedly different from their neutral counterparts. At low pH close to their isoelectric point, caseins were almost unable to adsorb. The most abundant proteins in “skimmed milk (pH 4.2) pellicle” were high molecular weight whey protein species such as bovine serum albumin and lactoferrin. Low molecular weight proteins, ␣-lactalbumin and ␤-lactoglobulin samples, were present in smaller amounts. One of the more remarkable results in this study was that virtually no proteins in yoghurt were able to adsorb onto enamel surfaces. This may be explained in part by denaturation of BSA and Lf during the high heat treatment normally employed in yoghurt manufacture. However, this heat treatment did not affect adsorption behaviour of caseins in the neutralized yoghurt (pH 6.7) samples. Pre-incubation of enamel specimens in saliva did not inhibit or affect milk pellicle formation. Our results revealed that the acquired enamel pellicle formed by incubation of enamel specimens in saliva mainly consisted of low molecular weight protein species. When these specimens were re-incubated in different milk products, none of the saliva pellicle components could be detected in the “milk pellicle”. These results may indicate that saliva pellicle proteins are desorbed during prolonged exposure in milk products. These pre-incubated specimens were used to simulate in vivo conditions, and our results showed that milk proteins were able to adsorb onto the acquired enamel pellicle normally covering the tooth surfaces in the oral cavity. The clinical significance of these differences in adsorption behaviour of sweet and fermented milk products as observed in our study is not yet

Summary
The aim of this investigation was to study in vitro adsorption of milk proteins onto tooth enamel. In vitro “milk protein pellicles” were formed on enamel specimens incubated in fluid milk products: skimmed milk (pH 6.7), acidified skimmed milk (pH 4.2), yoghurt and neutralized yoghurt (pH 6.7). The enamel specimens were used as such or pre-inscubated in saliva. Neutral “milk protein pellicles” (skimmed milk and neutralized yoghurt) mainly consisted of caseins. At pH values close to their isoelectric point caseins remained unabsorbed, and lactoferrin and bovine serum albumin were the most abundant protein species in the acidified “skimmed milk pellicle”. Virtually no proteins in yoghurt were able to adsorb onto enamel. Pre-incubation of enamel specimens in saliva did not inhibit or affect “milk pellicle” formation indicating that in vivo adsorption of milk proteins may occur on tooth surfaces in the oral cavity and have implications in oral hygiene.

clear. Caries progression has been reported to be markedly different between subjects consuming yoghurt (sweetened, strawberry jam) and those consuming skimmed milk as a snack between meals. In the group of yoghurt consumers, caries progression increased whereas in subjects who consumed skimmed milk, a decrease was observed (Jensen, Donly & Wefel, 2000). These results may be explained by the observations made in our investigation, however, future studies are needed to fully answer this question.

Conclusion
These in vitro studies demonstrated that the formation of “milk protein pellicles” is a selective adsorption process strongly depending on the product in which the enamel samples were incubated. The differences in protein profiles of neutral and acidified “milk protein pellicles” indicated that the pH value of the incubation medium was a major factor in determining protein adsorption. Neutral “milk protein pellicles” (skimmed milk and neutralized yoghurt) mainly consisted of caseins. At pH values close to their isoelectric point, caseins remained unabsorbed, and lactoferrin and bovine serum albumin were the most abundant protein species in the acidified “skimmed milk pellicle”. Virtually no proteins in

Results & Discussion
These in vitro studies demonstrated that the formation of “milk protein pellicles” is a selective adsorption process, and that the ability of a protein to adsorb onto tooth enamel strongly depended on the product in which the enamel samples were incubated. The differences in protein composition of neutral and acid-

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