archives of oral biology 55 (2010) 607–612

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The effects of peroxidase on the enzymatic and candidacidal

activities of lysozyme

Jeong-Yun Lee a, Yoon-Young Kim a, Ji-Youn Chang a, Moon-Soo Park b, Hong-Seop Kho a,*
a
Dept. of Oral Medicine and Oral Diagnosis, School of Dentistry and Dental Research Institute, Seoul National University, Yunkeun-Dong 28,
Chongro-Ku, Seoul 110-749, Republic of Korea
b
Dept. of Oral Medicine and Diagnosis, College of Dentistry, Kangnung-Wonju National University, Kangnung, Republic of Korea

article info abstract

Article history: Objective: To investigate the effects of peroxidase or the peroxidase system on the enzy-
Accepted 6 June 2010 matic and candidacidal activities of lysozyme.
Design: The effects of peroxidase on lysozyme were examined by incubating hen egg-white
Keywords: lysozyme (HEWL) with bovine lactoperoxidase (bLPO). The influence of the peroxidase
Lysozyme system on lysozyme was examined by the subsequent addition of potassium thiocyanate
Peroxidase and hydrogen peroxide. Lysozyme activity was determined by the turbidity measurement of
Candidacidal a Micrococcus lysodeikticus substrate suspension. Candidacidal activity was determined by
comparing the colony forming units of Candida albicans ATCC 10231, ATCC 18804, and ATCC
11006. The Wilcoxon signed rank test was used to analyze the effects of variables.
Results: bLPO at physiological concentrations enhanced the enzymatic activity of HEWL and
its effect was dependent on bLPO concentration. The enhancement of enzymatic activity of
HEWL by bLPO was affected by pH and ionic strength. The addition of potassium thiocyanate
and hydrogen peroxide did not lead to an additional enhancement of the enzymatic activity
of HEWL, as compared with bLPO alone. HEWL displayed candidacidal activity in all 3 strains
of C. ablicans. The addition of bLPO alone did not affect the candidacidal activity of HEWL, but
the bLPO system enhanced candidacidal activity of HEWL in all 3 strains of C. ablicans.
Conclusions: bLPO enhanced the enzymatic activity of HEWL, but the bLPO system did not
show additional enhancement of the enzymatic activity of HEWL. The addition of bLPO did
not affect the candidacidal activity of HEWL, but the bLPO system did enhance the
candidacidal activity of HEWL.
# 2010 Elsevier Ltd. All rights reserved.

1. Introduction
Many of the antimicrobial defense systems in saliva are
common to all exocrine secretions such as tears, milk, and
seminal, vaginal, and gastrointestinal fluids. Especially lyso-
zyme and peroxidases which are the main oral innate defense
factors are present in measurable concentrations in all of
these secretions. These antimicrobial agents are mainly
synthesized in, and secreted via, the major or minor salivary
glands. A smaller amount enters the oral cavity from tissue
fluid or polymorphonuclear leukocytes from the gingival
crevicular fluid.1–4
The antimicrobial activity of lysozyme occurs through a
muramidase-dependent mode and a cationic-dependent or
structure-related bactericidal mechanism.5,6 Peroxidase pro-
vides its antimicrobial activity and protection of oral tissues
from oxygen toxicity through the oxidation of SCN and
consumption of H2O2.3,4 Adsorbed lysozyme and peroxidase

* Corresponding author. Tel.: +82 2 2072 3989; fax: +82 2 744 9135.
E-mail address: hkho@snu.ac.kr (H.-S. Kho).
0003–9969/$ – see front matter # 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.archoralbio.2010.06.001
608 archives of oral biology 55 (2010) 607–612
molecules incorporated as pellicle components display bacte-
ricidal activity and reduce the adherence of Streptococcus
mutans to the hydroxyapatite surface.7–9 These antimicrobials,
either alone or in combination with other antimicrobial
molecules, have been incorporated in oral health care
products to restore the antimicrobial capacity of saliva.10
It is well-known that many antimicrobial proteins in saliva
interact in vitro with each other. The interactions result in
additive, synergistic, or inhibitory effects on mutans strepto-
cocci, lactobacilli, or fungi.2,11,12 For example, such interactions
have been reported between sIgA and peroxidase,13 lactoferrin
and peroxidase,14,15 lactoferrin and lysozyme,16 and lysozyme
and histatins.17 Although these observations are from in vitro
experiments, it is likely that such concerted effects exist also in
vivo in mixed saliva or in oral health care products, where all the
components are simultaneously present.
Although many reports have investigated interactions
between various kinds of antimicrobials present in saliva,
there is no information about the interactions between
lysozyme and peroxidase in the aspects of enzymatic and
antifungal activities. In the present study, we have investigated
the effects of peroxidase or the peroxidase system on the
enzymatic and candidacidal activities of lysozyme.
2. Materials and methods
2.1. Lysozyme and peroxidase
Hen egg-white lysozyme (HEWL) and bovine lactoperoxidase
(bLPO) (Sigma–Aldrich Chemical Co., St Louis, MO, USA)
dissolved in simulated salivary buffer (SSB, 0.021 M Na2HPO4/
NaH2PO4, pH 7.0, containing 36 mM NaCl and 0.96 mM CaCl2)18
with phenylmethylsulfonylfluoride (PMSF, at a final concentra-
tion of 1.0 mM) served as sources of peroxidase and lysozyme,
respectively. A preliminary experiment showed that PMSF did
not affect the enzymatic activities of lysozyme.
2.2. Measurement of the enzymatic activity of lysozyme
Lysozyme activity was determined by the turbidimetric
method.19 Samples were placed in a lyophilized cell suspension
of Micrococcus lysodeikticus ATCC 4698, starting at OD450 = 0.65–
0.70, so that the lysozyme could degrade the bacterial substrate.
The lysozyme activity was expressed as units/mL.
2.3. Candidacidal assay
Candida albicans ATCC 10231, ATCC 18804, and ATCC 11006
were used in the experiments. One colony of C. albicans was
inoculated into 10 mL RPMI 1640 medium and incubated with
shaking for 24 h at 37 8C. Cells were then harvested, washed,
and resuspended to a concentration of 1  105 cells per mL in
0.01 M phosphate buffer (pH 7.4). For the determination of
candidacidal activity, 20 mL of cell suspension was added to
40 mL of experimental samples in sterile tubes. The samples
were incubated at 37 8C for 1.5 h and mixed every 15 min. At
the end of the incubation, samples were diluted 10 times, and
50 mL (167 cells) of the diluted cells was plated on Sabouraud
dextrose agar plates in triplicate and grown overnight at 37 8C.
Candidacidal activity was determined by comparing the
number of colonies on experimental plates with that on
control plates. The percent loss of cell viability (1 minus the
ratio of the number of colonies on test plate to that on control
plate) was also calculated.
2.4. Influence of peroxidase on the enzymatic and
candidacidal activities of lysozyme
The effects of peroxidase on the enzymatic activity of
lysozyme were examined by incubating 500 mL of bLPO (at a
final concentration of 12.5 mg/mL) with 500 mL of HEWL (at a
final concentration of 10.0 mg/mL) for 10 min at room
temperature. The incubated mixture was placed in a suspen-
sion of M. lysodeikticus and an incubated mixture of buffer with
HEWL was used as a control. Either an incubated mixture of
bLPO with buffer, or an incubated buffer alone was used as a
blank. The dose effect of peroxidase on lysozyme activity was
examined using different concentrations of bLPO (3.13, 6.25,
12.5, and 25.0 mg/mL). The effects of peroxidase on the
enzymatic activity of lysozyme were also examined at
different levels of pH (from pH 4 to 9) and at different levels
of ionic strength (from 0 to 150 mM). The experiment was
duplicated and performed 12 times.
The effects of peroxidase on the candidacidal activity of
lysozyme were determined by comparing the number of
colonies on experimental plates (a mixture of HEWL with
bLPO, at final concentrations of 10.0 and 12.5 mg/mL, respec-
tively) with control plates (a mixture of HEWL with buffer). An
incubated mixture of bLPO with buffer and an incubated buffer
alone were also used as samples. The experiment was
triplicated and performed 8 times.
2.5. Influence of the peroxidase system on the enzymatic
and candidacidal activities of lysozyme
The effect of the peroxidase system on the enzymatic activity
of lysozyme was examined by comparing the enzymatic
activities of HEWL (at a final concentration of 10.0 mg/mL), an
incubated mixture of HEWL with bLPO (at a final concentra-
tion of 12.5 mg/mL), and an incubated mixture of HEWL with
the peroxidase system (at final concentrations of 12.5 mg/mL
bLPO, 1.0 mM KSCN, and 50 mM H2O2). Either an incubated
mixture of the peroxidase system with buffer, an incubated
mixture of peroxidase with buffer, or an incubated buffer
alone was used as a blank. The experiment was duplicated
and performed 12 times.
First, the candidacidal effects of the peroxidase system
were examined by comparing the number of colonies on
experimental plates (at final concentrations of 12.5 mg/mL
bLPO, 1.0 mM KSCN, and 50 mM H2O2) with control plates (a
mixture of bLPO with buffer or an incubated buffer alone). The
experiment was triplicated and performed 8 times. Secondly,
the effects of the peroxidase system on the candidacidal
activity of lysozyme were determined by comparing the
number of colonies on experimental plates (at final concen-
trations of 10.0 mg/mL HEWL, 12.5 mg/mL bLPO, 1.0 mM KSCN,
and 50 mM H2O2) with control plates (a mixture of HEWL and
bLPO, a mixture of HEWL with buffer, and an incubated buffer
alone). The experiment was triplicated and performed 8 times.
 archives of oral biology 55 (2010) 607–612
[(Fig._1)TD$IG]
Fig. 1 – Effects of peroxidase on the enzymatic activity of
lysozyme according to peroxidase concentration. The
enzymatic activity of hen egg-white lysozyme (HEWL) was
measured following the pre-incubation of HEWL (10.0 mg/
mL) with different levels of bovine lactoperoxidase (bLPO
at concentrations of 3.13, 6.25, 12.5, and 25.0 mg/mL).
Enhancement % compared with its control was
determined. The assay was performed 12 times.
2.6. Binding of peroxidase to lysozyme
Each microtiter well was coated with 100 mL of HEWL (100 mg/
mL) dissolved with SSB supplemented with PMSF. The plate was
then incubated for 1 h at 37 8C. The wells were aspirated and
washed 3 times with 200 mL of phosphate buffered saline with
0.02% Tween 20. Unoccupied binding sites were blocked with 1%
bovine serum albumin in 300 mL for 30 min at 37 8C. Volumes of
100 mL of bLPO, dissolved in blocking buffer and serially diluted
from 500 to 7.8 mg/mL, were dispensed into each well and the
plate was incubated for 1 h at 37 8C. The wells were then
aspirated and washed 3 times. Volumes of 100 mL of sheep anti-
bLPO horseradish peroxidase (Abcam Inc., Cambridge, MA, USA)
diluted 1:2000 with blocking buffer were added to each well and
the plate was incubated for 1 h at 37 8C. After three washes,
100 mL of 3,30 ,5,50 -tetramethylbenzidine substrate solution was
added to each well. Reactions were terminated by adding 100 mL
of 1N HCl. Absorbance values were determined at 450 nm. The
mean absorbencies from wells with no HEWL and no bLPO were
subtracted from experimental values for background correc-
tion. All experiments were duplicated and performed 5 times.
2.7. Statistics
The Wilcoxon signed rank test was used to analyze the effects
of variables, as compared with their controls. P-values less
than 0.05 were considered statistically significant.
3. Results
3.1. Influence of peroxidase on the enzymatic and
candidacidal activities of lysozyme
bLPO enhanced the enzymatic activity of HEWL, and its effects
were statistically significant at 6.25 mg/mL (P < 0.05), 12.5 mg/
mL (P < 0.01), and 25.0 mg/mL (P < 0.05). The percentage of
609
[(Fig._2)TD$IG]
Fig. 2 – Effects of peroxidase on the enzymatic activity of
lysozyme according to pH. Bovine lactoperoxidase (bLPO)
enhanced the enzymatic activity of hen egg-white
lysozyme (HEWL) at pH values of 5, 6, and 7. The
enzymatic activity of HEWL was measured following the
pre-incubation of HEWL (10.0 mg/mL) with bLPO (12.5 mg/
mL) for each different pH. The assay was performed 12
times. *P < 0.05 and **P < 0.01.
enhancement was the highest at 12.5 mg/mL (12.4  10.6%) in
which the enzymatic activities of HEWL and a mixture of
HEWL and bLPO were 227.5  38.8 and 255.3  46.1 units/mL,
respectively (Fig. 1). The enhancement of enzymatic activity of
HEWL by bLPO was affected by pH and ionic strength. The
effects of enhancement occurred at pH 5 (P < 0.05), pH 6
(P < 0.05), and pH 7 (P < 0.01). The enzymatic activity of HEWL
was decreased at pH values of 4 and 8, and the effects of
enhancement were also decreased (Fig. 2). The enzymatic
activity of HEWL was decreased by increasing the level of ionic
strength. The enhancement occurred in deionized distilled
water (0 mM) (P < 0.01) and at an ionic strength of 50 mM
[(Fig._3)TD$IG]
Fig. 3 – Effects of peroxidase on the enzymatic activity of
lysozyme according to ionic strength. Bovine
lactoperoxidase (bLPO) enhanced the enzymatic activity of
hen egg-white lysozyme (HEWL) at low ionic strength
conditions (0 and 50 mM). The enzymatic activity of HEWL
was measured following the pre-incubation of HEWL
(10.0 mg/mL) with bLPO (12.5 mg/mL). The assay was
performed 12 times. X-axis values refer to ionic strength of
NaCl. *P < 0.05 and **P < 0.01.
610 archives of oral biology 55 (2010) 607–612

Table 1 – Effects of peroxidase on the candidacidal activity of lysozyme.

C. albicans strain Control HEWL bLPO HEWL + bLPO Significance
(Group I) (Group II) (Group III) (Group IV) between groups
*
ATCC 10231 CFU 152.2  29.3 119.4  31.1 133.0  32.0 114.4  25.6 (I, II) (I, IV) (II, III)
*
% killing – 21.7  13.7 12.4  14.8 23.6  18.4 (II, III)
*
ATCC 18804 CFU 126.4  23.5 105.4  16.2 105.6  12.5 112.2  26.7 (I, II)
% killing – 14.6  17.5 13.3  23.0 9.8  20.1
*
ATCC 11006 CFU 153.2  20.6 118.0  19.6 142.4  17.5 128.2  16.6 (I, II) (I, IV) (II, III) (III, IV)
*
% killing – 22.6  10.7 6.2  12.7 15.8  9.8 (II, III) (III, IV)

HEWL at 10.0 mg/mL displayed candidacidal activity. The addition of bLPO (12.5 mg/mL) did not affect the candidacidal activity of HEWL. The
assay was performed 8 times.
HEWL, hen egg-white lysozyme; bLPO, bovine lactoperoxidase; CFU, colony forming unit.
*
P < 0.05.

Table 2 – Candidacidal activity of the peroxidase system.

C. albicans strain Control bLPO bLPO system Significance
(Group I) (Group II) (Group III) between groups
*
ATCC 10231 CFU 144.5  14.8 149.1  14.3 129.2  13.1 (I, III) (II, III)
*
% killing – 4.1  14.7 10.3  7.2 (II, III)
*
ATCC 18804 CFU 127.8  23.9 119.9  32.8 92.1  26.8 (I, III)
% killing – 2.8  37.4 28.6  11.7
*
ATCC 11006 CFU 154.6  38.9 144.1  34.8 105.3  32.4 (I, III) (II, III)
*
% killing – 4.4  20.2 27.3  28.8 (II, III)

bLPO at 12.5 mg/mL itself did not show candidacidal activity. The bLPO system (12.5 mg/mL bLPO, 1.0 mM KSCN, and 50 mM H2O2) displayed
candidacidal activity. The assay was performed 8 times.
HEWL, hen egg-white lysozyme; bLPO, bovine lactoperoxidase; CFU, colony forming unit.
*
P < 0.05.

(P < 0.05). The effects of enhancement disappeared at high
ionic strength conditions of 100 and 150 mM (Fig. 3).
HEWL at a concentration of 10.0 mg/mL displayed candi-
dacidal activity in all 3 kinds of C. ablicans strains. bLPO did not
show candidacidal activity and did not affect the candidacidal
activity of HEWL (Table 1).
3.2. Influence of the peroxidase system on the enzymatic
and candidacidal activities of lysozyme
The enzymatic activities of HEWL, a mixture of HEWL and
bLPO, and a mixture of HEWL and the bLPO system were
222.8  26.0, 278.2  29.0, and 278.3  32.5 units/mL, respec-
tively. The bLPO system showed enhancement of the
enzymatic activities of HEWL (P < 0.01), but the addition of
KSCN or H2O2 did not lead to additional enhancement of the
enzymatic activity of HEWL compared with bLPO alone.
The bLPO system displayed candidacidal activity in all 3
kinds of C. albicans strains (P < 0.05) (Table 2). The bLPO system
also boosted the candidacidal activity to HEWL in all 3 kinds of
C. ablicans strains (P < 0.05) (Table 3).
3.3. Binding of peroxidase to lysozyme
Fig. 4 shows that bLPO binds to HEWL-coated on a microtiter
plate. Absorbance values reflect the amount of bLPO which

Table 3 – Effects of the peroxidase system on the candidacidal activity of lysozyme.

C. albicans strain Control HEWL HEWL + bLPO HEWL +bLPO Significance between groups
(Group I) (Group II) (Group III) system (Group IV)
*
ATCC 10231 CFU 135.6  38.0 112.1  45.5 106.1  37.8 97.1  44.2 (I, II) (I, III) (I, IV) (II, IV)
*
% killing – 18.9  12.4 21.4  17.3 29.3  18.3 (II, IV)
*
ATCC 18804 CFU 129.4  28.1 89.7  18.3 100.5  28.3 75.6  18.4 (I, II) (I, III) (I, IV) (II, IV) (III, IV)
*
% killing – 29.6  11.2 23.2  6.9 41.0  10.3 (II, IV) (III, IV)
*
ATCC 11006 CFU 156.1  32.6 132.9  16.1 140.2  34.9 117.8  19.6 (I, II) (I, III) (I, IV) (II, IV) (III, IV)
*
% killing – 13.5  9.4 10.4  9.4 23.1  12.8 (II, IV) (III, IV)

The bLPO system (12.5 mg/mL bLPO, 1.0 mM KSCN, and 50 mM H2O2) enhanced the candidacidal activity of HEWL at 10.0 mg/mL. The assay was
performed 8 times.
HEWL, hen egg-white lysozyme; bLPO, bovine lactoperoxidase; CFU, colony forming unit.
*
P < 0.05.
 archives of oral biology 55 (2010) 607–612
[(Fig._4)TD$IG]
Fig. 4 – Binding of bovine lactoperoxidase (bLPO) to hen
egg-white lysozyme (HEWL) immobilized on a microtiter
plate. Absorbance values reflect the amount of bLPO which
bound to HEWL. The assay was performed 5 times.
bound to HEWL. The amount of bLPO binding to HEWL was
increased by increasing the amount of bLPO in a certain range.
4. Discussion
Oral antimicrobial factors work in the same milieu – saliva,
gingival fluid, and dental plaque – at the same time, and may
interact with each other.2 Previous studies suggested that
many salivary antimicrobial agents interact with each other,
in most cases in a synergistic or additive way.2,11,12 Almost all
previous studies have focused on the bacteriostatic and
bactericidal effects on cariogenic or periopathogenic micro-
organisms. Lysozyme was reported to enhance the inhibitory
effects of the peroxidase system on glucose metabolism of S.
mutans.20 Lysozyme and lactoferrin showed synergistic anti-
staphylococcal properties21 and additive bactericidal effects
against S. mutans.16 The additive effects of lysozyme and
histatins on coaggregation between Porphyromonas gingivalis
and Streptococcus mitis were also suggested.17 Regarding
peroxidase, IgA was suggested to augment the antimicrobial
effect of the lactoperoxidase system against S. mutans.13
Lactoferrin and lactoperoxidase system showed an additive
inhibitory effect on the viability of S. mutans.15
These antimicrobials have been actually incorporated in oral
health care products to restore the antimicrobial capacity of
saliva especially for the patients with dry mouth whose
susceptibility for developing candidiasis is increased.10 The
antifungal activity of lysozyme22,23 and the peroxidase sys-
tem24–26 have been reported. The synergic activity of lysozyme
and antimycotics has also been reported.27,28 However, the
possibility of additive antifungal effects of lysozyme and
peroxidase has not been reported, nor the interaction in the
aspects of enzymatic activity.
Our results showed that bLPO enhanced the enzymatic
activity of HEWL, and this effect was dependent on the
concentration of bLPO in certain ranges. The concentrations of
bLPO and HEWL were within the physiological ranges of
salivary peroxidase and lysozyme in human whole saliva. The
increase of lysozyme activity by peroxidase was not further
affected by the complete peroxidase system composed by the
611
subsequent addition of KSCN and hydrogen peroxide. The
enhancement of enzymatic activity of HEWL by bLPO was
displayed at pH values of 5, 6, and 7 and at low ionic strength
conditions. Thus, increase of lysozyme activity by peroxidase
may occur in unstimulated whole saliva. The assay showed
the possibility of direct binding of bLPO to HEWL. The cationic
nature of both innate antimicrobials and the effects of pH and
ionic strength suggest that the interaction between lysozyme
and peroxidase is ionic in nature.
Although the precise mechanism by which lysozyme
interacts with the fungus is not clear, several mechanisms
have been suggested: direct binding of cationic lysozyme and
yeast cell-wall mannans followed by an influence on yeast
viability,22,29 the activation or de-regulation of autolytic
enzymes,22,29 the interaction of lysozyme with other non-
substrate fungal components followed by resultant interfer-
ence with ion transfer and de-regulation of the influx and
efflux of cellular constituents,22 the enzymatic hydrolysis of
N-glycosidic bonds that link polysaccharides and structural
proteins of the yeast cell wall,30,31 and the modulation of
secreted aspartyl proteinase (Sap), a virulence factor of C.
albicans.23
Although it has been reported that C. albicans is sensitive to
HOSCN/OSCN,25 the precise mechanism by which the
peroxidase system interacts with the fungus has not been
reported. All components of the peroxidase system are needed
for its antifungal effects,26,32 and its antifungal activity is
affected by relative proportions of its components and
phosphate concentration.25 It has also been suggested that
the direct binding of peroxidase to cell-wall mannans is
important for candidacidal activity.24
Our results showed that HEWL displayed candidacidal
activity, but an increase of lysozyme activity by bLPO did not
lead to significant changes of candidacidal activity. The bLPO
system added candidacidal activity to HEWL in all 3 kinds of C.
ablicans strains. Therefore, our data indicated that lysozyme
and the peroxidase system exert a synergic candidacidal
activity through different mechanisms, either suggested or as
yet unknown.
There have been attempts to enhance or restore saliva’s
own antimicrobial capacity by commercially available oral
health care products. This idea would add physiological
salivary antimicrobial agents into the mouth that lacks
saliva-mediated protection, as seen in patients with dry
mouth. The antimicrobial host proteins most widely used in
these products are lysozyme, lactoperoxidase, and lactofer-
rin.10 Although the lack of appropriate control makes it
impossible to rule out the placebo effect, some studies have
shown that Biotene1 alone or with Oralbalance1 gel
containing salivary antimicrobial agents relieve subjective
symptoms in patients with dry mouth.33,34 It is not certain
that the results obtained in vitro can be extended to in vivo
scenarios. However, lysozyme might enhance the inhibitory
effects of the peroxidase system on S. mutans in these
products.20 bLPO might increase the enzymatic activity of
HEWL, and the bLPO system might increase candidacidal
activity of lysozyme in these products. In addition, salivary
lysozyme and peroxidase in the residual saliva of patients
with dry mouth could interact with each other and with
these antimicrobials in the products.
612 archives of oral biology 55 (2010) 607–612
Collectively, despite the in vitro nature of our study, the
results of the present study provide valuable information on the
influence of peroxidase or the peroxidase system on lysozyme
in the aspects of enzymatic and candidacidal activities in oral
health care products and potentially in the oral cavity.
Acknowledgements
Funding: This work was supported by the Korea Science &
Engineering Foundation (KOSEF) grant funded by Korea
government (R13-2008-008-01001-0) through the Oromaxillo-
facial Dysfunction Research Center for the Elderly (ODRCE) at
Seoul National University.
Competing interests: None declared.
Ethical approval: Not required.
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