Elsevier

Journal of Dentistry

Volume 103, December 2020, 103506
Journal of Dentistry

Addition of CPP-ACP to yogurt inhibits enamel subsurface demineralization

https://doi.org/10.1016/j.jdent.2020.103506Get rights and content

Abstract

Objectives

The aim of this study was to investigate the capacity of an approved food additive with anticariogenic properties, casein phosphopeptide-amorphous calcium phosphate (CPP-ACP), when added to a processed, sugar-containing yogurt with added lactic-acid bacteria (probiotics), to prevent demineralization of enamel subsurface lesions in vitro.

Methods

Enamel subsurface demineralised lesions were created in thirty extracted human third molars. These were then exposed to artificial saliva (AS) with: 1) Danone yogurt alone; 2) Danone yogurt with 0.2 % w/w CPP-ACP; or 3) Danone yogurt with 0.5 % w/w CPP-ACP at 37 °C for two weeks. The yogurt/AS was replaced with fresh preparations each day. At the completion of each treatment the enamel slabs were embedded, sectioned and analyzed using transverse microradiography to measure changes in enamel lesion depths and subsurface mineral content. Yogurt samples were analysed for soluble calcium (Ca) and inorganic phosphate (Pi) levels and pH.

Results

Yogurt alone demineralized enamel subsurface lesions to produce significantly larger lesions. However, the addition of 0.2 % CPP-ACP to the yogurt resulted in significant reduction in demineralization compared with yogurt alone (p < 0.0001). The addition of 0.5 % CPP-ACP to the yogurt produced a net remineralization effect with a significant increase in lesion mineral content (p < 0.0001). The addition of CPP-ACP resulted in a significant (p < 0.0001) dose-related increase in Ca, Pi and pH.

Conclusions

The addition of CPP-ACP to a commercial yogurt exhibited a dose related protective effect with 0.5 % CPP-ACP producing remineralization of existing enamel subsurface lesions under the in vitro experimental conditions.

Clinical significance

The results of this study suggest that some processed yogurts with added sugar could result in enamel demineralization when frequently consumed by individuals with poor oral hygiene. The addition of CPP-ACP to these yogurts may help prevent demineralization and promote enamel subsurface lesion remineralization, and therefore, make them safer for teeth.

Introduction

Yogurt is a cultured milk product obtained by fermentation of lactose through the action of specific lactic acid bacterial cultures added to milk. The typical starter cultures used in yogurt manufacture are Lactobacillus bulgaricus and Streptococcus thermophilus. In addition, other lactobacilli (e.g. Lactobacillus lactis) and bifidobacteria (e.g. Bifidobacterium lactis) are sometimes added during or after the culturing process. The lactic acid produced by the culture coagulates the milk protein and produces the characteristic sour flavor. To improve the organoleptic properties of the yogurt some commercial products contain many additives including sucrose. Yogurt has become increasingly popular in recent years, buoyed by its perceived health benefits, flavor and appeal as a convenient snack. It is an excellent source of protein, minerals (calcium, potassium, zinc, phosphorus, magnesium) and numerous vitamins (vitamin A, riboflavin, vitamin B5, and vitamin B12).

Milk and dairy products are normally considered as having low cariogenic potential even though they can contain fermentable carbohydrate (lactose) and this is attributed to the presence of anticariogenic components [1], [2] such as their high content of calcium and phosphate ions, their buffering capacity, and the content of casein phosphopeptides (CPPs) [3], [4]. The balance of these protective factors to the sugar/acids is critical in determining whether the product may put oral health at risk. This is exemplified by some reports on the potential cariogenic/erosive properties of low pH, processed yogurt products, particularly those with added sugar. A comparative study by Ravishankar et al. evaluated the effect of consuming different dairy products on calcium, phosphorus and pH levels of human dental plaque [5]. The authors concluded that yogurt products without added sugar (sucrose) were unlikely to be cariogenic based on plaque pH changes and increased calcium and phosphorus concentrations. However, Moeiny et al. assessed the effect of plain and fruit-containing yogurt consumption, as well as that of a 10 % sucrose solution, on plaque pH [6] and found that the lowest pH value was recorded after fruit yogurt consumption followed by plain yogurt and then the 10 % sucrose solution [6]. The authors concluded that both the fruit and plain yogurts could be considered potentially cariogenic since plaque pH decreased to below the critical pH for enamel demineralization. This finding suggests that highly processed yogurts may be harmful to teeth and therefore there is a need for further studies to determine the potential cariogenicity/erosive potential of processed yogurt products, particularly those with added sugar.

Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) is a remineralization technology based on the protective factors of dairy products [4], [7], [8], [9]. The CPP released from milk casein by tryptic digestion contain the sequence -Ser(P)-Ser(P)-Ser(P)-Glu-Glu- which stabilizes supersaturated solutions of calcium and phosphate ions to produce CPP-amorphous calcium phosphate nanocomplexes (CPP-ACP) [10], [11]. Through this mechanism, the CPP have a remarkable ability to stabilize calcium and phosphate ions in solution and act as a delivery vehicle to substantially increase the level of calcium and phosphate in dental plaque [12]. The CPP are biomimetics of the salivary protein statherin which contains the sequence Asp-Ser(P)-Ser(P)-Glu-Glu-, however due to the greater content of phosphoseryl residues [Ser(P)] in the sequence –Ser(P)-Ile/Leu-Ser(P)-Ser(P)-Ser(P)-Glu-Glu- of the CPP, they are superior to statherin in their ability to stabilize and deliver calcium and phosphate ions [9]. This greater remineralization potential translates to enhanced clinical efficacy in prevention and reversal of early stages of dental caries and erosion [9], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29].

CPP-ACP, unlike fluoride, can be added to sugar-containing foods and therefore has commercial potential as an additive to foods as well as to toothpastes and mouthwashes for the control of dental caries and erosion [4]. CPP-ACP is GRAS affirmed (Generally Recognized as Safe) and has negligible effects on taste [30].

The aim of this study was to evaluate the ability of CPP-ACP added to a processed yogurt to inhibit enamel subsurface demineralization and promote enamel subsurface remineralization, in vitro. The null hypothesis for the study was that no significant difference in the changes to lesion depth and enamel mineral content would be detected after addition of CPP-ACP to the yogurt.

Section snippets

Yogurt, artificial saliva and CPP-ACP

Danone Activia Vanilla Yogurt (Danone Murray Goulburn Pty Ltd) was purchased from Woolworths (Australia). The listed ingredients of Danone Activia Vanilla Yogurt were: whole milk; sugar; water; milk solids; thickener (1442); lemon juice concentrate; live lactic acid cultures (Bifidobacterium lactis CNCM I-2494, Streptococcus thermophilus, Lactococcus lactis and Lactobacillus bulgaricus); natural flavour; stabilizer (410); vanilla beans (0.012 %); natural colour (160a). More information on the

Results

The pH of each yogurt/AS slurry and the soluble Ca and Pi levels for each of the three yogurt samples is shown in Table 2. The addition of CPP-ACP at 0.2 % and 0.5 % produced a significant (p < 0.0001) dose-related increase of yogurt pH and soluble Ca and Pi levels (Table 2). Representative microradiographs of enamel subsurface lesions before and after exposure to the three yogurt slurries are presented in Fig. 1. There was significant further demineralization of the enamel lesions after

Discussion

This study investigated the effect of processed yogurt in artificial saliva, in the absence and presence of CPP-ACP, on demineralization/remineralization of enamel subsurface lesions, in vitro. The results demonstrated that the yogurt alone in artificial saliva produced significant enamel subsurface demineralization under the experimental conditions of this study. In fact, the TMR images showed a lesion typical of a progressing caries lesion for the yogurt alone treatment (Fig. 1). The

Conclusions

The processed yogurt tested in this study produced significant enamel subsurface demineralization and this was attributed to its high content of lactic acid, sucrose and live lactic acid producing bacteria. The addition of CPP-ACP to the yogurt not only significantly hindered enamel subsurface demineralization but also significantly remineralized enamel subsurface lesions. A dose related protective effect of the added CPP-ACP was observed, with the 0.5 % CPP-ACP completely inhibiting

CRediT authorship contribution statement

Peiyan Shen: Conceptualization, Investigation, Methodology, Formal analysis, Project administration, Supervision, Methodology, Writing - original draft. James R. Fernando: Conceptualization, Methodology, Writing - review & editing. Glenn D. Walker: Formal analysis, Investigation, Writing - review & editing. Yi Yuan: Investigation, Methodology, Writing - review & editing. Coralie Reynolds: Investigation, Methodology, Project administration, Writing - review & editing. Eric C. Reynolds:

Declaration of Competing Interest

The authors declare that they have no conflict of interest.

Acknowledgements

This study was funded by the Australian Government Department of Industry, Innovation and Science Grant ID 20080108. The funding organisation had no role in the study design, implementation, analysis or interpretation of the data. The excellent technical assistance of David Stanton in measuring soluble calcium and phosphate levels of the yogurts is gratefully acknowledged.

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