SCI ENTI F I C ARTI CL E

Australian Dental Journal 2008; 53: 314319

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 a
s1
-, a
s2
- 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 remineral-
ization 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 signicant 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 phosphopeptide-
amorphous calcium phosphate.
(Accepted for publication 20 February 2008.)
INTRODUCTION
Milk and dairy products have been identied 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
Gabska et al. found that cheese consumption contrib-
utes 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
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 super-
saturation 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 nanocom-
plexes contained in mouthrinses and sugar-free chewing
gums are anticariogenic.
79
Furthermore, the addition
of 2.05.0 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
314 2008 Australian Dental Association
CPP content) to inhibit demineralization and promote
remineralization of early enamel caries.
12,13
MATERIALS AND METHODS
Initially, an experimental protocol to standardize a
demineralization procedure of dental enamel
14
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).
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-uoride
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 mm
2
each) were isolated using an
acid-resistant varnish (Fig 1). The 80 specimens were
divided into four groups, which subsequently under-
went 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)
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 CPP additives
The specimens from Groups 3 and 4 were treated with
a remineralizing solution containing a probiotic-
enrichedyogurt (BidobacteriumBb-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 frac-
tion 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
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
(t
0
) and after (t
96
) the treatments to assess hydroxy-
apatite weight content.
The amount of calcium released into the solution
was determined by compleximetric titration with Fig 1. Isolated specimen.
Table 1. Summary of solutions used
Origin of
solution
Solution pH Remineralizing
agent
Demineralizing
agent
Synthetic 1 4.8 Lactic acid
2 3.97 Lactic acid
Natural 3 4.8 Yogurt supernatant Lactic acid
4 3.97 Yogurt supernatant Lactic acid
2008 Australian Dental Association 315
Effect of yogurt extract on dental enamel demineralization
EDTA-Na
2
(disodium ethylenediamine tetraacetate)
using Eriochrome Black T as an indicator. The calcium
concentration in solution was calculated before (t
0
) and
after the treatments (t
96
). The results obtained were
submitted to statistic elaboration through the use of the
ANOVA system, programme SPSS 10.0.
Furthermore, ve specimens from each group were
dehydrated in a graded series of ethanol, critical point
dried (SPC-900 EX, The Bomar Co., Tacoma, Wash-
ington, 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 articial lesions created in the Group 1 speci-
mens.
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.
Analysis of the results obtained after chemical
treatment shows that articial demineralization in the
presence of natural protective factors (yogurt super-
natant) provides signicantly lower weight and calcium
loss. Fig 2. Demineralized enamel surface at pH 4.8 (3000).
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).
316 2008 Australian Dental Association
GF Ferrazzano et al.
The mean difference in weight changes between
Groups 1 and 3 was statistically signicant (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 articial demin-
eralization, in the presence of a dairy-derived reminer-
alizing 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 signicant (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 microporos-
ities, 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 conrms 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
calciumcontent was statistically signicant (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 signicant (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
8.00
4.00
0.00
w
e
i
g
h
t

c
h
a
n
g
e
s

(
m
g
)
-4.00
-8.00
group 1 group 3
Fig 6. Signicance of weight changes for Groups 1 and 3.
5.00
-6.25
-17.50
-28.75
w
e
i
g
h
t

c
h
a
n
g
e
s

(
m
g
)
-40.00
group 2 group 4
Fig 7. Signicance of weight changes for Groups 2 and 4.
4.00
3.00
2.00
1.00
0.00
c
a
l
c
i
u
m

c
h
a
n
g
e
s

(
m
g
/
5
0
m
l
)
solution 2 solution 1
Fig 8. Signicance of calcium changes for Solutions 1 and 2.
6.00
3.00
0.00
-3.00
c
a
l
c
i
u
m

c
h
a
n
g
e
s

(
m
g
/
5
0
m
l
)
-6.00
solution 4 solution 3
Fig 9. Signicance of calcium changes for Solutions 3 and 4.
2008 Australian Dental Association 317
Effect of yogurt extract on dental enamel demineralization
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 nding is in
agreement with other reports demonstrating an associ-
ation between dairy products consumption and reduc-
tion of dental caries.
1719
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 demineral-
ization, 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 supersatu-
rated 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 remineral-
ization.
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 nal composition of yogurt is inuenced by the
species and strains of bacteria used in the fermentation,
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 nal product. In addition,
yogurt is an excellent source of calcium and phos-
phorus. 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 con-
trolled trials have shown that yogurt, containing
L. reuteri and Bifidobacterium DN-173 010, may
reduce the levels of selected caries-associated micro-
organisms 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 epide-
miological 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;134:989S995S.
2. Petti S, Simonetti R, Simonetti DArca A. The effect of milk and
sucrose consumption on caries in 6- to 11-year-old Italian
schoolchildren. Eur J Epidemiol 1997;13:659664.
3. Gabska 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;58:6975.
4. McDougall WA. Effect of milk on enamel demineralization and
remineralization in vitro. Caries Res 1977;11:166172.
5. Cai F, Shen P, Morgan MV, Reynolds EC. Remineralization of
enamel subsurface lesions in situ by sugar-free lozenges contain-
ing casein phosphopeptide-amorphous calcium phosphate. Aust
Dent J 2003;48:240243.
6. Cross KJ, Huq NL, Palamara JE, Perich J W, Reynolds EC.
Physicochemical characterization of casein phosphopeptide-
amorphous calcium phosphate nanocomplexes. J Biol Chem
2005;280:1536215369.
318 2008 Australian Dental Association
GF Ferrazzano et al.
7. Shen P, Cai F, Nowicki A, Vincent J, Reynolds EC. Remineral-
ization of enamel subsurface lesions by sugar-free chewing
gum containing casein phosphopeptide-amorphous calcium
phosphate. J Dent Res 2001;80:20662070.
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;82:206211.
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 phosphopeptide-
amorphous calcium phosphate. Caries Res 2004;38:551556.
10. Walker G, Cai F, Shen P, et al. Increased remineralization of
tooth enamel by milk containing added casein phosphopeptide-
amorphous calcium phosphate. J Dairy Res 2006;73:7478.
11. Ferrazzano GF, Troianiello S, Sangianantoni G, Ingenito A.
[New strategies in dental caries prevention: experimental study
on casein phosphopeptides.] Prevenzione odontostomatologica
2005;4:1521.
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;61:365374.
13. Ferranti P, Barone F, Chianese L, et al. Phosphopeptides from
Grana Padano cheese: nature, origin and changes during ripening.
J Dairy Res 1997;64:601615.
14. White DJ, Faller RV, Bowman WD. Demineralization and rem-
ineralization evaluation techniquesadded considerations. J Dent
Res 1992;71 Spec No:929933.
15. Iijima Y, Takagi O, Ruben J, Arends J. In vitro remineralization
of in vivo and in vitro formed enamel lesions. Caries Res
1999;33:206213.
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 Tex-
ture of Fermented Milk. 22 June 2002. Kolding, Denmark. IDF,
2002:290301.
17. Merritt J, Qi F, Shi W. Milk helps build strong teeth and pro-
motes oral health. J Calif Dent Assoc 2006;34:361366.
18. Petridou E, Athanassouli T, Panagopoulos H, Revinthi K. Soci-
odemographic and dietary factors in relation to dental health
among Greek adolescents. Community Dent Oral Epidemiol
1996;24:307311.
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;66:3841.
20. Levine RS. Milk, avoured 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;36:120125.
22. Adolfsson O, Meydani SN, Russell RM. Yogurt and gut function.
Am J Clin Nutr 2004;80:245256.
23. Rasic JL, Kurmann JA. Bidobacteria and their role. Micro-
biological, 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 Bidobacterium DN-173 010 on
salivary mutans streptococci and lactobacilli in young adults.
Acta Odontol Scand 2005;63:317320.
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;95:219223.
Address for correspondence:
Gianmaria Fabrizio Ferrazzano
Via Cuoco no. 5
80100 Naples
Italy
Email: gianmariafabrizio@yahoo.it
2008 Australian Dental Association 319
Effect of yogurt extract on dental enamel demineralization