Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Anticaries effect of an antioxidant-rich apple concentrate

on enamel in an experimental biofilm-demineralization
model
~ oz-Sandoval1,2
R.A. Giacaman1,2, M.P. Contzen1, J.A. Yuri2,3 and C. Mun
1 Cariology Unit, Department of Oral Rehabilitation, University of Talca, Talca, Chile
2 Interdisciplinary Excellence Research Program on Healthy Aging (PIEI-ES), University of Talca, Talca, Chile
3 Centro de Pomaceas, Facultad de Ciencias Agrarias, University of Talca, Talca, Chile

Keywords
antioxidants, apple, biofilms, dental caries,
polyphenols, Streptococcus mutans.
Correspondence
Rodrigo A. Giacaman, 2 Norte 685, Escuela
de Odontologıa, Talca, Chile.
E-mail: giacaman@utalca.cl
2014/0434: received 28 February 2014,
revised 28 May 2014 and accepted 2 June
2014
doi:10.1111/jam.12561
Abstract
Aims: To assess the anticaries activity of an antioxidant-rich apple concentrate
(ARAC) in an experimental biofilm caries model on enamel.
Methods and Results: A validated caries model with Streptococcus mutans
UA159 biofilms was used. Biofilms were formed on enamel slabs during
5 days. To mimic cariogenic challenges, triplicate slabs were exposed three
times per day for 5 min to 10% sucrose followed by five additional minutes of
exposure to serial dilutions of ARAC in 0
9% NaCl. A triplicate slab exposed
to 10% sucrose followed by 0
9% NaCl served as caries-positive control.
Acidogenicity was estimated by medium pH twice per day. After the
experimental phase, biofilms were recovered to determine biomass, viable
bacteria and intra- and extracellular polysaccharides. Slabs were used to
estimate demineralization by the percentage of surface microhardness loss (%
SHL). Differences among treatments were analysed by ANOVA and Bonferroni
test (P < 0
05). Streptococcus mutans biofilms were exposed to ARAC after a
cariogenic challenge with sucrose-induced lower enamel demineralization than
the positive control. The highest dilution of ARAC at 1 : 100 000 (v/v) showed
the most marked reduction in demineralization of about 57%. Although no
differences were observed in the number of bacterial cells, the intracellular
polysaccharides or in the biomass (P > 0
05), the highest dilution of the apple
concentrate induced significantly lower extracellular polysaccharide formation
by the biofilm.
Conclusions: An apple concentrate in low concentrations appears to have a
potential anticaries activity on enamel. Data suggest a metabolic rather than an
antimicrobial mechanism, but further research is needed.
Significance and Impact of the Study: Phenolic compounds contained in
apple concentrates seem to have anticaries properties that may be effective
even in the presence of sucrose and in very low doses. Nutritional
interventions that do not require rescinding from sucrose might be derived
from these findings.

than one-third of the population at all ages (Murray

Introduction
Dental caries continues to be a major public health prob-
lem and one of the conditions with the highest preva-
lence worldwide (Bagramian et al. 2009), affecting more
et al. 2012). Besides the obvious detriment to oral health,
dental caries compromises quality of life (Scarpelli et al.
2013) and it is the main oral cause of disability-adjusted
life years (DALYs) (Murray et al. 2012). Given the high

846 Journal of Applied Microbiology 117, 846--853 © 2014 The Society for Applied Microbiology
R.A. Giacaman et al.
prevalence and its negative consequences for the popula-
tion, new approaches to cope with the disease are
required. As caries restorative treatment is expensive and
dental health care has usually low coverage among the
population in most countries, treatment strategies must
focus on finding novel preventive approaches.
Widely accepted in the dental field, fluoridated agents
have been highly effective in preventing caries (Jones et al.
2005). Furthermore, fluorides are perhaps the dental ther-
apy more strongly based on evidence (Ten Cate 2012). Yet,
functional foods, nutraceutics and prebiotics from natural
origin are being increasingly considered within the medical
field as “healthier” approaches to treat diseases, and their
consumption is rapidly increasing (Ozen et al. 2012).
Hence, growing interest in the dental field in finding natu-
ral agents with anticaries properties, different to fluoride, is
perceived. Results from the scarce published studies, how-
ever, are far from being conclusive (Rethman et al. 2011).
Containing a wide range of molecules from different
origin and structure, an anticaries effect of various natu-
ral products has been reported (Jeon et al. 2011). Among
the substances with putative anticaries effect, polyphenol
antioxidants contained in fruits and vegetables have been
claimed as potentially active against the disease (Ferrazz-
ano et al. 2011). Phenolic compounds are substances
belonging to a heterogeneous group, from simple to
highly polymerized molecules, including flavonoids
(quercetin and kaempferol), phenolic acids (chlorogenic
acid and caffeic acid) and carotenoids (lutein and zeaxan-
thin). Health benefit from phenolic consumption derives
not from one particular component, but from the syner-
gistic activity of the bioactive compounds and other
nutrients contained in fruits, vegetables, whole grains and
other plant foods (Liu 2013). Besides a reported associa-
tion between the consumption of fruit and vegetables
with a reduced rate of coronary heart disease (Dauchet
et al. 2006), phenolic compounds in natural products
have been investigated as potential anticaries agents (Yoo
et al. 2011). Indeed, these molecules have shown to
reduce caries-associated bacterial growth (Matsumoto
et al. 2004) and biofilm formation (Yamanaka et al.
2004). An inhibitory effect of the enzymatic activity of
glucosyltransferases, a key virulent mechanism in cario-
genic bacteria, has been proposed as the responsible
mechanism (Nakahara et al. 1993). In this context, apple
contains a high concentration of polyphenol antioxidants
(Wu et al. 2004). Regardless of the cultivar, the antioxi-
dant capacity of the apple is mostly due to quercetin, epi-
catechin, and procyanidin B2 and in a less extent to
vitamin C, phloretin and chlorogenic acid (Lee et al.
2003). It has been reported that the antioxidant capacity
of the apple is substantially higher in the skin than in the
core (Yuri et al. 2009).
Journal of Applied Microbiology 117, 846--853 © 2014 The Society for Applied Microbiology
Anticariogenicity of an apple concentrate
Although some studies suggest an anticaries effect from
polyphenols (Ferrazzano et al. 2011) and also from apple-
derived polyphenols (Yanagida et al. 2000), most of them
have been performed through microbiological assays
against cariogenic bacteria. It is less clear that this effect
could be evidenced when a relevant caries model with cari-
ogenic biofilms and dental tissues is assayed, nonetheless.
With that purpose in mind, we conducted the present
report whose aim was to evaluate the anticaries effect of
an apple concentrate on dental enamel in a validated bio-
film-based caries model (Giacaman et al. 2012).
Materials and methods
Experimental design
A previously validated caries model with biofilms of the
cariogenic Streptococcus mutans (Strep. mutans) UA159
was used (Ccahuana-Vasquez and Cury 2010). This
model allows assessing the effect of putative anticaries
substances in both biofilm formation and the hard tissue
demineralization inhibition upon several cariogenic chal-
lenges. Strep. mutans biofilms were formed on bovine
enamel slabs for 5 days. Biofilms/slabs were exposed three
times per day to a solution of 10% sucrose (w/v) for
5 min to create cariogenic conditions followed by five
additional minutes of exposure to an experimental anti-
oxidant-rich apple concentrate in three serial dilutions of
1 : 1, 1 : 1000 and 1 : 100 000 (v/v). The dilutions used
were based on previous experiments showing effect at
very high dilutions. A group of slabs was exposed to 10%
sucrose followed by 0
9% NaCl (positive caries-control)
and another to 0
9% NaCl followed by 0
9% NaCl (nega-
tive caries-control). Culture medium was replenished
twice per day, and the pH of the spent medium was mea-
sured to evaluate acidogenicity by the biofilm. After the
5 days of the experimental phase, biofilms were recovered
to assess total biomass, viable bacteria, soluble proteins
and three fractions of polysaccharide production. Enamel
demineralization was estimated by surface Knoop
microhardness (SH) loss. Two independent experiments
were carried out, each in triplicate (n = 6).
Apple concentrate elaboration
An apple concentrate produced by the Centro de
Pomaceas from the University of Talca was provided for
the experiments. Phenolic content of the concentrate was
of 20 g CAE per 100 g of lyophilized product, with an
ORAC value of 180 000 lm TE per 100 g (CAE: chloro-
genic acid equivalents and TE: Trolox equivalents). Given
the high concentration of phenolic compounds, the
concentrate was named antioxidant-rich apple concentrate
847
Anticariogenicity of an apple concentrate
(ARAC). The ARAC is under licensing process, including
its elaboration methodology (N/Ref: 628-BP). Phenolic
concentration was determined by the Folin- Ciocalteu
method (Coseteng and Lee 1987). In brief, 0
1 ml of
ARAC was mixed with 0
5 ml of a phenolic reagent
(Merck, Darmstadt, Germany) and incubated for 5 min.
0
5 ml of a 10% Na2CO3 solution (w/v) was added and
incubated for 15 min at room temperature. A calibration
curve was prepared with chlorogenic acid, and the optical
density (OD) was measured at 640 nm by a spectropho-
tometer. Total phenol concentration was expressed as
CAE* g 1 of lyophilized product. Antioxidant capacity of
ARAC was assessed by ORAC values, as described
elsewhere (Huang et al. 2002), with modifications. Briefly,
a free radical is required to bind a fluorescent molecule
subsequently forming a non-fluorescent complex. The
presence of the antioxidant inhibits binding of the free
radical to the fluorescent compound. Hence, if the sample
contains a high content of antioxidants, a longer time will
take place for the fluorescence to disappear.
Dental enamel samples and Streptococcus mutans
biofilms
Bovine incisors were disinfected with a 5% NaOCl solu-
tion and stored in 0
9% NaCl (w/v) until use, avoiding
storage times longer than 30 days. Enamel slabs of
7 9 4 9 1 mm were obtained using diamond discs with
a low-speed handpiece and Soflex polishing discs
(3 mol l 1, St. Paul, MN). To obtain an initial value for
Knoop SHi, three indentations were performed with a
microindenter with a microhardness tester (402 MVD,
Wolpert Wilson Instruments, Norwood, MA) at 50 g for
5 s. Mean baseline SHi of all the slabs was calculated
(336
85  6
93), and only those with SHi within a 10%
variation were included (n = 30). Thus, bias from includ-
ing slabs with too different SHi was avoided. Slabs were
sterilized with ethylene oxide (Thomas et al. 2007). To
create an acquired pellicle-like structure on the enamel
surface to emulate the in vivo situation and also to facili-
tate bacterial adhesion, slabs were immersed in ultrafil-
tered (0
22 lm) pooled human saliva for 30 min with a
protease inhibitor cocktail (Koo et al. 2003). Initially
measured, saliva-coated and sterile slabs were placed into
a 24-well cell culture plate suspended by sterile wire.
Streptococcus mutans biofilms were formed on the
enamel slabs, following a previously reported protocol
(Ccahuana-Vasquez and Cury 2010). In brief, enamel
slabs were inoculated with Strep. mutans cultures (OD
0
8 at 600 nm) and 1% sucrose-containing medium to
form the adherent biofilm (Koo et al. 2003) and incu-
bated for 8 h. Biofilms were allowed to mature on the
slabs in BHI medium supplemented with 0
1 m mol l 1
848 Journal of Applied Microbiology 117, 846--853 © 2014 The Society for Applied Microbiology
R.A. Giacaman et al.
glucose for 24 h. Once mature, biofilms underwent the
experimental treatments, as described below.
Treatments
Slabs with the mature biofilms were exposed to the
experimental substances for 5 min three times per day, as
described in the Experimental Design section, at defined
times (8:30, 12:30 and 16:30). Experimental exposure
were: 10% sucrose + ARAC 1 : 1; 10% sucrose + ARAC
1 : 1000; and 10% sucrose + ARAC 1 : 100 000. Tripli-
cates exposed to 10% sucrose + 0
9% NaCl and to 0
9%
NaCl + 0
9% NaCl were used as positive and negative
caries controls, respectively. After each exposure, biofilms
were washed with 0
9% NaCl and repositioned in the
plate. Culture medium was replenished twice a day,
before the first and after the last exposure. The exposure
cycles were repeated to complete 5 days, a known time to
induce enamel demineralization (Ccahuana-Vasquez and
Cury 2010).
Biofilm acidogenicity
To verify acidogenicity of the biofilm, culture medium pH
was measured in the wells. A microelectrode (HI 1083B,
Hanna instruments, Woonsocket, RI) coupled to a
portable pH meter (HI 9126-02, Hanna instruments) was
used to register the pH. Spent culture medium generated
by the biofilm was read with the electrode twice per day,
before each medium change (Ccahuana-Vasquez and Cury
2010).
Enamel demineralization
Once the experimental phase was completed, slabs were
washed three times with 0
9% NaCl and biofilms were
detached from the slabs by shaking the slabs for 30 s with a
vortex mixer in a tube containing 1 ml 0
9% NaCl, making
a visual confirmation that no biofilm was retained on the
slabs. Final indentations were performed at 100 lm from
those performed before the experimental phase to obtain a
final SHf reading (kg mm 2) in a triplicate row. Mean val-
ues from the initial and final measurements were used to
obtain the percentage of SH loss (%SHL) calculated by the
formula: (SHi SHf) 9 100 / SHi. SH has been exten-
sively used as a reliable methodology to evaluate deminer-
alization (Zero 1995), and it has been validated for enamel
caries (Cury et al. 2000).
Biofilm analysis
Using the recovered biofilm from the previous step, a
biofilm suspension was formed and divided into different
R.A. Giacaman et al. Anticariogenicity of an apple concentrate

aliquots to evaluate the following biofilm properties: bio-
mass, viable micro-organisms (Ccahuana-Vasquez and
Cury 2010) and intra- and extracellular polysaccharides
(Aires et al. 2008), all of which are succinctly described:
Biomass
A 200 ll aliquot from the biofilm suspension was trans-
ferred to a preweighted tube and incubated with 100%
ethanol at 20°C for 15 min, centrifuged (10 min at
5000 g and 4°C), and the resulting pellet was washed
with 500 ll of 75% ethanol. After a second centrifuga-
tion, the pellet was dried for 24 h in a desiccator. Bio-
mass was calculated by subtracting the final weight to the
initial weight of the empty tube. Biomass dry weight of
the biofilm was expressed as mg per ml of biofilm sus-
pension (Koo et al. 2003).
Viable bacteria in the biofilm
Serial dilutions from the biofilm suspension in 0
9%
NaCl (v/v) were drop-plated on BHI agar plates in dupli-
cate. Plates were incubated anaerobically for 24 h at
37°C, and colonies were enumerated from the dilution
that allowed visualization of isolated colonies. Counting
was corrected by the dilution factor and expressed as
CFU mg 1 of biofilm dry weight (Aires et al. 2008).
Intra- and extracellular polysaccharides
Three different polysaccharide fractions were assessed
from the original biofilm suspension: soluble (SEPS) and
insoluble (IEPS) extracellular and intracellular (IPS) poly-
saccharides (Aires et al. 2008). Suspension was centri-
fuged (10 000 g for 5 min at 4°C) to obtain SEPS from
the supernatant, and the resulting pellet was exposed to
200 ll of 1M NaOH, homogenized and centrifuged to
obtain IEPS from the supernatant (Cury et al. 1997). IPS
were obtained from the pellet of the previous extraction.
Pellet was incubated with 200 ll 1 mol l 1 NaOH for
15 min at 100°C, centrifuged (10 000 g for 5 min at
4°C), and the polysaccharide concentration was measured
from the supernatant. SEPS, IEPS and IPS fractions were
treated with 3 volumes of cold 100% ethanol and incu-
bated for 30 min at 20°C. Samples were immediately
centrifuged, and the resulting pellet was washed with cold
70% ethanol and centrifuged again. Total carbohydrate
concentration from each fraction was estimated by the
sulphuric phenol method (Dubois et al. 1951). Results
were standardized by biofilm dry weight and expressed as
percentage of the different fractions of polysaccharides by
mg of biomass.
Biofilm soluble proteins
Fifty microlitre aliquots of the biofilm suspension were
treated with 2 mol l 1 NaOH and incubated for 15 min
to 100°C. Samples were centrifuged at 10 000 g at 4°C
for 10 min, and the supernatant was used to assess the
total protein concentration of the biofilm by Lowry’s
method (Lowry et al. 1951) with a commercial kit (DC
Protein Assay Kit, Bio-Rad, Hercules, CA). Results
were expressed as micrograms of protein by milligram of
biomass.
Statistical analysis
Parametric distribution of the data was verified by the
Kolmogorov–Smirnov test. An ANOVA test was carried out
to detect differences among all the experimental groups
in each of the dependent variables under study. A Bonfer-
roni post hoc test served to detect statistical difference

6.50
a a a a
6.00
b

c
pH

5.50 b
b
c
b
5.00
c c
4.50
8 24 32 48 56 72 80 96 104 120
Time (h)

Figure 1 Acidogenicity elicited from the Streptococcus mutans biofilms formed on enamel in response to sucrose and dilutions of the apple con-
centrate. Plot shows mean pH registered in the spent culture medium and the error bars represent the standard deviation (SD) of the mean for
each experimental condition (as indicated). Measurements were performed after 24 h of biofilm formation, twice per day, at defined times (as
indicated) in two independent experiments, each in triplicate. Medium changes are represented by the arrows. Different letters represent signifi-
cant differences between treatments (P < 0
05). ( ) Sucrose + 0.9% NaCl; ( ) Sucrose + 1 : 1 ARAC; ( ) Sucrose + 1 : 1000 ARAC; ( )
Sucrose + 1 : 100 000 ARAC; ( ) Nacl+Nacl

Journal of Applied Microbiology 117, 846--853 © 2014 The Society for Applied Microbiology 849
Anticariogenicity of an apple concentrate R.A. Giacaman et al.

between each pair of experimental group. The SPSS 15.0

3
53E + 10  1
52E + 10a

0
9% NaCl + 0
9% NaCl
statistical software, (IBM Corporation, Somers, NY) was

72
9b
used to manipulate the data, setting a 95% confidence

0
3b

0
1b
0
6b
0
8b
Table 1 Biofilm traits after exposure to sucrose followed by dilutions of an apple concentrate. Mean  SD, n = 6. Different letters represent statistically significant differences between
level.






0
29
219
12
0
26
0
91
1
09
Results
Acidogenicity elicited by the biofilm exposed to the
ARAC was evaluated by medium pH measurements per-

10% Sucrose + 1 : 100 000 ARAC

formed twice a day during the entire length of the
experiment. No differences among the groups were

5
68E + 10  4
17E + 10a

detected during the first 80 h (P > 0
05). From that time

187
5ab
point on, however, slabs/biofilms exposed to the 1 : 1

0
5ab

0
6ab

1
7ab
1
0b
dilution of the ARAC showed similar behaviour than the
caries-positive control (P > 0
05) and lower pH






0
92
375
16
1
07
2
19
2
61
(P = 0
0001) than the rest of the experimental dilutions
(Fig. 1). While the group exposed to sucrose followed by
the 1 : 1000 dilution of the ARAC showed a slightly
lower pH than the critical demineralizing pH of enamel

10% Sucrose + 1 : 1000 ARAC

of 5
5, samples exposed to the 1 : 100 000 dilution of
the ARAC failed to reach the critical pH at any time

6
54E + 10  2
44E + 10a

SEPS: soluble extracellular polysaccharides, IEPS: insoluble extracellular polysaccharides, IPS: intracellular polysaccharides.
point (Fig. 1).

167
6ab
0
5ab

0
6ab
1
4ab
Table 1 depicts the characteristics of the biofilms of

1
5a
Strep. mutans retrieved from the slabs after the experi-






mental phase. When compared with the caries-positive

0
92
432
95
1
55
3
57
4
21
control, none of the tested dilutions of the ARAC
induced changes in the number of CFU ml 1 of
Strep. mutans from the biofilms (P < 0
05). Despite a
dose-dependent trend to decrease, sucrose exposure fol-
10% Sucrose + 1 : 1 ARAC

4
41E + 10  3
43E + 10a

lowed by the apple concentrate did not show a significant

reduction of either the biofilm biomass, soluble proteins, 228
1a
0
8a

0
6a
1
3a
1
3a
SEPS or IPS at any of the dilution assayed (P > 0
05)
compared with the positive control. Exposure of the bio-





1
50
613
09
1
92
4
83
4
46
film to sucrose and to the highest dilution (1 : 100 000)
of the ARAC, however, induced a reduction of the IEPS
from the Strep. mutans biofilm (P = 0
013).
Slabs and the biofilms exposed to sucrose and to the
10% Sucrose + 0
9% NaCl

2
54E + 10  1
03E + 10a

1 : 1000 and to the 1 : 100 000 dilutions showed less

enamel demineralization (%SHL) than the 1 : 1 dilution
107
6a

(P = 0
003 and P = 0
0001, respectively), which did not

0
5a

0
2a
1
0a
1
0a

differ from the caries-positive control (P = 1
00) (Fig. 2).






1
88
622
92
2
07
5
26
4
62

Discussion
As an attractive disease-prevention approach, natural
products are increasingly receiving more attention in the
Proteins (lg mg 1 biomass)
SEPS (% per mg biomass)
IEPS (% per mg biomass)

dental field. In fact, during the decade of the eighties a

IPS (% per mg biomass)
(CFU mg 1 biomass)
treatments (P < 0
05)

mean of 15
6 articles were retrieved in PubMed with the

terms “Caries” and “Natural products”, compared with a
Viable bacteria

mean of 38
9 in the first decade of the two thousands

Biomass (mg)

and 59
3 in the present decade. Natural products usually

contain high content of biologically active components
with antioxidant properties. In particular, apple has high

850 Journal of Applied Microbiology 117, 846--853 © 2014 The Society for Applied Microbiology
R.A. Giacaman et al.
%SHL
Figure 2 Enamel demineralization induced by
the cariogenic challenge followed by dilutions
of an antioxidant-rich apple concentrate
(ARAC). Demineralization was assessed by
surface hardness loss (%SHL) induced by
10% sucrose followed by each ARAC dilution
tested (as indicated) after 5 days. Data were
obtained from two independent experiments,
each in triplicate. Bars represent mean%SHL
of the slabs. Error bars indicate SD. Different
letters represent significant differences
between treatments (P < 0
05).
content of antioxidant polyphenols, and it is also highly
consumed by the population (Yuri et al. 2009).
The results of this study strongly suggest a potential anti-
caries effect of an apple concentrate in very low concen-
trations presented to the cariogenic biofilm after sucrose
exposure.
wCaries antagonistic activity of antioxidants polyphenols
have been attributed to three main mechanisms (Jeon et al.
2011): (i) Direct antimicrobial effect by killing the respon-
sible bacteria, (ii) Inhibition of extracellular polysaccharide
production and (iii) Impairment of bacterial adhesion.
Our findings suggest that the anticaries effect is metabolic
rather than antibacterial. Indeed, the ARAC does not seem
to affect the number of viable bacteria (Table 1). The setup
of the caries model used here rules out the mechanism of
interfering on bacterial adhesion. Actually, Strep. mutans
biofilms were already formed and mature once exposed to
the experimental substances. Instead, the ARAC seems to
affect extracellular polysaccharide production, in an inverse
dose-dependent manner. These polysaccharides are
responsible for 40% of the composition of the dental bio-
film, and they are one of the main virulence factors of the
bacterial consortium, as they allow bacterial cell adhesion
to the acquired pellicle, serve as scaffold for biofilm matu-
ration and increase the porosity of the structure allowing
sugar diffusion within the biofilm (Paes Leme et al. 2006).
The fact that IEPS production was affected by the ARAC
may explain why there was less biomass in the biofilms
without compromising the number of viable Strep. mutans
cells (Table 1). Extracellular polysaccharide formation is
mediated by the enzymatic activity of glycosyltransferases
(GTFs) produced by Strep. mutans. GTFs establish natural
glycosidic linkages from simple sugars to form complex
molecules. Polyphenols may act as inhibiting GTFs causing
a reduction in extracellular polysaccharides, as observed
here. Consistently, a study showed that procyanidins
Journal of Applied Microbiology 117, 846--853 © 2014 The Society for Applied Microbiology
Anticariogenicity of an apple concentrate
60
a
50 a
40
b
b
30
c
20
10
0
Sucrose + 0·9% Sucrose + Sucrose + Sucrose + NaCl + NaCl
NaCl 1:1 1 : 1000 1 : 100 000
ARAC ARAC ARAC
Treatment
contained in apples as a mixture of condensed tannins
inhibited the activity of GTFs (Yanagida et al. 2000). GTFs
are one of the main virulence factors of cariogenic bacteria,
as they are used to make the biofilm thicker and less
susceptible to natural protective factors in the oral environ-
ment (Schilling and Bowen 1992). Unlike this study, the
authors used commercial polyphenols. Arguably, the ARAC
could have acted in a similar manner to procyanidins.
The inverse relation of the effect and the dose on poly-
saccharide production also holds true for the acidogenici-
ty and enamel demineralization (Figs 1 and 2). The
highest dilutions of the ARAC induced the most signifi-
cant reduction on tissue demineralization and higher pH
in the culture medium. Consistent with our findings, a
study tested an extract of Psidium cattleianum leaves on
Strep. mutans (Brighenti et al. 2008). While high concen-
trations resulted in bacterial killing, low concentrations
of the extract inhibited Strep. mutans acid production
and reduced expression of proteins involved in acid
metabolism. As the elaboration of the concentrate implies
water elimination, sugars become highly concentrated.
Undiluted ARAC, therefore, shows a cariogenic effect.
Thus, the cariogenic effect of the less diluted ARAC
darkens the anticaries effect due to its sugar content.
Hence, a highly diluted ARAC (1 : 1000 or 1 : 100 000)
possesses a very low sugar content, and little amounts of
the concentrate can exert its anticaries activity. As a
matter of fact, the most efficient anticaries effect was
observed with the highest dilutions. The latter is probably
attributable to the very high content of phenolic com-
pounds in the ARAC, which remain active even in the
highest dilutions.
Other natural sources of polyphenols with a putative
anticaries activity have been reported. Cranberries, tea
(Yoo et al. 2011) and cacao bean husk extract (Matsumoto
et al. 2004) have been described as having anti-Strep.
851
Anticariogenicity of an apple concentrate
mutans properties. Acid production inhibition in the
same micro-organism has been demonstrated by Green
tea polyphenols (Hirasawa et al. 2006). Likewise, a GTFs
inhibition has been reported from Oolong tea, with the
subsequent reduction in polysaccharide production
(Nakahara et al. 1993). Furthermore, red wine polyphe-
nols have shown to reduce bacterial adhesion in vitro
(Furiga et al. 2008), and remineralizing properties have
been claimed in grape extracts (Xie et al. 2008) and galla
chinese (Chu et al. 2007). Although we could not verify
an antibacterial effect of ARAC, the large variability
observed in bacterial counting could hide some effect on
Strep. mutans viability. To decrease the variability, there-
fore, techniques used for bacterial counting should be
refined in further investigations.
Most of the studies available focus on microbiological
assays with natural products to state the anticaries poten-
tial of polyphenols and natural products (Ferrazzano
et al. 2011). We used here an in vitro caries approach
with cariogenic biofilms and not planktonic cells. This is
a relevant model, as it resembles the oral environment by
using a Strep. mutans biofilm formed on dental enamel,
intermittently exposed to a metobolizable substrate. Like
in the oral situation, sucrose exposures results in a pH-
cycling system with pH drops with sugar exposure and
pH recovery with culture medium change. Cariogenic
challenges used here were designed having in mind food
consumption during main meals. Yet, study design limi-
tations must be acknowledged. We modelled caries using
a single-species biofilm of Strep. mutans. The oral biofilm
comprises a metabolically active and organized consor-
tium of hundreds of bacterial species, nonetheless (Marsh
2010). Moreover, the model used here does not include
saliva. Saliva has important anticaries properties that may
modulate the mere effect of the nutrients presented to
the oral biofilm (Stookey 2008).
Although polyphenols appear as interesting potential
anticaries agents from their anti- Strep. mutans effect, this
is insufficient to support a clinical effect. Evidence show-
ing a reduction on clinical caries incidence is missing and
requires more research (Petti and Scully 2009). Despite
the limitations of this in vitro investigation, it uses a rele-
vant anticaries model that may well serve as proof-of-
principle of a caries-inhibitory effect of a natural apple
concentrate. Further in vivo studies must be conducted to
actually replicate the complexity of the oral environment,
including the complete dental biofilm and the presence of
saliva to confirm these findings.
Interestingly, the anticaries effect observed here occurs
in the presence of sucrose (Figs 1 and 2). Changing peo-
ple’s dietary habits is usually a challenging task. If our
results are confirmed in clinical studies, potential applica-
tions of these findings would not require dietary modifica-
852 Journal of Applied Microbiology 117, 846--853 © 2014 The Society for Applied Microbiology
R.A. Giacaman et al.
tions to achieve the desired goal. Hence, an advantageous
approach compared with other type of caries-preventive
interventions may be found.
Acknowledgements
Authors thank Amalia Neira for the preparation of the
apple concentrate and assistance with technical aspects of
the exposures to the caries model. This study has been
partly funded by the Chilean Government Grants: Fonde-
cyt 11100005 to RAG and Fondef AF0I1022 to JAY.
Conflict of Interest
None to declare.
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