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Gels 2021, 7, 191. https://doi.org/10.3390/gels7040191 www.mdpi.com/journal/gels
Article
Preparation and In Vitro Characterization of Gels Based on
Bromelain, Whey and Quince Extract
Amalia Mazilu (Moldovan)
1
, Violeta Popescu
1
, Codruta Sarosi
2
, Radu Silaghi Dumitrescu
3
,
Andrea Maria Chisnoiu
4,
*, Marioara Moldovan
2,
*, Laura Silaghi Dumitrescu
2
, Doina Prodan
2
, Rahela Carpa
5
,
Georgiana Florentina Gheorghe
6
and Radu Marcel Chisnoiu
7
1
Physics and Chemistry Department, Technical University of Cluj-Napoca, 28 Memorandumului Str.,
400114 Cluj-Napoca, Romania; amalia.mazilu@gmail.com (A.M.); violeta.popescu@chem.utcluj.ro (V.P.)
2
Department of Polymer Composites, “Raluca Ripan” Institute of Research in Chemistry, Babes-Bolyai
University, 30 Fantanele Str., 400294 Cluj-Napoca, Romania; liana.sarosi@ubbcluj.ro (C.S.);
laura.silaghi@ubbcluj.ro (L.S.D.); doina.prodan@ubbcluj.ro (D.P.)
3
Department of Chemistry, Faculty of Chemistry and Chemical Engineering, 11 Arany Janos Street,
400028 Cluj-Napoca, Romania; rsilaghi@chem.ubbcluj.ro
4
Department of Prosthodontics, “Iuliu Hatieganu” University of Medicine and Pharmacy, 32 Clinicilor
Street, 400006 Cluj-Napoca, Romania
5
Department of Molecular Biology and Biotechnology, Faculty of Biology and Geology, Babeș Bolyai
University, 1 M. Kogălniceanu Street, 400084 Cluj-Napoca, Romania; rahela.carpa@ubbcluj.ro
6
Faculty of Dental Medicine, Carol Davila University of Medicine and Pharmacy, 17-23 Calea Plevnei,
010232 Bucharest, Romania; georgiana.gheorghe@umfcd.ro
7
Department of Odontology, Endodontics and Oral Pathology, “Iuliu Hatieganu” University of Medicine
and Pharmacy, 33 Motilor Street, 400001 Cluj-Napoca, Romania; marcel.chisnoiu@umfcluj.ro
* Correspondence: maria.chisnoiu@umfcluj.ro (A.M.C.); marioara.moldovan@ubbcluj.ro (M.M.)
Abstract: The growing interest in the appearance and color of teeth has led to the emergence of a
wide range of teeth whitening methods, both in dental offices and in patients’ homes. Concerns
about the possible side effects or toxic effects of peroxide-based whitening gels leads to the identi-
fication of alternative whitening methods, based on natural compounds with mild action on tooth
enamel and remineralizing effect. In this context, this study describes the preparation and in vitro
analysis of whitening gels based on natural active agents—bromelain, quince and whey—using or-
ganic (polyacrylate, polyethylene glycol) and/or inorganic (silicate) excipients. Five natural prod-
ucts gels were prepared, containing bromelain extract, quince extract and whey, in various propor-
tions. Two supplementary gels, one containing Lubrizol and another containing SiO
2
, were pre-
pared. All gels were submitted for multiple in vitro analysis such as: SDS-PAGE analysis, UV-vis
and FTIR spectroscopy, SEM microscopy, antibacterial activity on Streptococcus mutans ATCC
25175, Porphyromonas gingivalis ATCC 33277, Enterococcus faecalis ATCC 29212, Escherichia coli
ATCC 25922 and Staphylococcus aureus ATCC 25923. The quince extract sample was the only one
which completely discolored the blue dye on SDS-PAGE analysis. On the UV-vis spectra, the 303
nm band is assigned to an in situ modified form of bromelain. SEM images of gels containing SiO
2
particles show evident marks of these particles, while the rest of the gels containing Lubrizol or
whey are more uniform. Regarding antibacterial tests, the SiO
2
gel samples did not show inhibition
in any strains, but the other tested samples varied in the size of the inhibition diameter depending
on the amicrobial strain tested; the protease activity of bromelain modulates the composition of the
added whey proteins. Bromelain added as a nanoencapsulated assembly better preserves its integ-
rity. The prepared gels showed antibacterial properties.
Keywords: bromelain; whey; whitening gel
Citation: Mazilu, A.; Popescu, V.;
Sarosi, C.; Dumitrescu, R.S.;
Chisnoiu, A.M.; Moldovan, M.;
Dumitrescu, L.S.; Prodan, D.; Carpa,
R.; Gheorghe, G.F.; Chisnoiu, R.M..
Preparation and in Vitro Characteri-
zation of Gels Based on Bromelain,
Whey and Quince Extract. Gels 2021,
7, 191. https://doi.org/10.3390/
gels7040191
Academic Editor: Ashleigh Fletcher
Received: 21 September 2021
Accepted: 28 October 2021
Published: 30 October 2021
Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional
claims in published maps and institu-
tional affiliations.
Copyright: © 2021 by the authors. Li-
censee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and con-
ditions of the Creative Commons At-
tribution (CC BY) license (https://cre-
ativecommons.org/licenses/by/4.0/).
Gels 2021, 7, 191 2 of 13
1. Introduction
The appearance and color of the teeth are a concern for a growing number of people
seeking alternatives to chemical-based dental treatment. Recently, there has been a signif-
icant development of teeth whitening products “without a prescription” and without the
involvement of professionals [1]. Dental staining can result from the accumulation of mol-
ecules with chromophore/chromogenic groups (generally conjugated or even aromatic
unsaturated systems) on the surface of dental structures [2]. The adhesion of chromo-
phores to teeth is due to several types of forces, generally supramolecular—especially van
der Waals. Factors related to eating habits, such as excessive coffee or tea consumption,
smoking, and exposure to chemicals, are associated with dental staining. Teeth whitening
products are traditionally based on hydrogen peroxide or carbamide peroxide gels (hy-
drogen peroxide and urea) [3]. These peroxide-based products release free radicals that
attack chromogens, being able to degrade larger molecules into smaller molecules that can
be easily removed from dental tissu es, thus promoting a certain degree of teeth whitening.
Despite their effectiveness, peroxide-based products can cause severe abrasion of dental
structures and are toxic/corrosive to any tissue in general. However, new studies
highlighted that a low percentage of hydrogen peroxide, together with new nanofillers,
allows for maintenance of enamel structure [4]. In this context, attempts are being made
to develop whitening products with enzymatic action, which in some cases seem to be just
as effective in removing extrinsic stains. Active ingredients derived from natural products
have been shown to have potential antibacterial effects without causing abrasiveness,
which can be a good alternative to those based on peroxides [5].
Bromelain is an enzyme extract with protease activity, which is found mainly in the
pineapple plant (Ananas comosus) of the genus Bromeliaceae [6]. This extract can be
obtained from both the stem and the fruit of the pineapple plant and contains as the main
component a mixture of glycosylated proteolytic sulfhydryl enzymes [7–11]. The
bromelain strain possesses different biochemical properties and compositions compared
to fruit bromelain [12], the latter containing several thiol endopeptidases and also
compounds such as peroxidases, acid phosphatase, glycoproteins, carbohydrates and
organic complexed Ca2+ [6,13]. To date, eight active proteolytic components have been
isolated from bromelain [14]. Proteinases are considered to be the most active fraction,
comprising ~2% of total proteins; occasionally, the term bromelain is also used to describe
only the two dominant proteases in this extract [15]. Bromelain works in a pH range of 4.5
to 9.5 [16]. Bromelain is one of the most widely investigated proteolytic enzymes/extracts
for practical and industrial applications, due to its ease of extraction and low cost given
by the relatively affordable raw material. Because of its protease activity, bromelain has
been cited for its anti-edematous, fibrinolytic, anticancer, anti-inflammatory,
antimicrobial, anticoagulant, and antithrombotic medical applications—generally due to
the ability of bromelain to degrade connective proteins from inflamed tissues or tissues,
or circulating proteins in the blood (such as those involved in coagulation), or proteins in
pathogens (leading to the death of those agents—hence antimicrobial activity) [17].
In addition to the clinical approach, bromelain has been used in various industries,
including the food industry [13], such as breweries [18], meat processing and tenderizing,
textile and cosmetics [19]. An advantage for some industrial applications is that the
optimum temperature of bromelain is ∼50–70 ◦C [20].
Toothpastes based on proteolytic enzymes have proven their effectiveness by
removing tooth stains, this being done with reduced roughness [5]. Indeed, for dental
applications—such as toothpaste or whitening solutions—the main candidates as
alternatives to peroxides are cysteine-protease enzymes, such as papain and bromelain,
described as active agents with whitening potential [5,20–23]. Proteases disrupt or remove
the portion of protein in the film layer that forms on the surface of the teeth, thus removing
the pigments that are bound to them. Muchow et al. [21] conducted a study on the
whitening effect of whitening gels containing proteolytic enzymes (bromelain or papain)
on bovine enamel. The enamel was stained in coffee solution for 1 week and measured
Gels 2021, 7, 191 3 of 13
spectrophotometrically for color evaluation before and after whitening using a proteolytic
enzymes-based gel and a commercial bleaching gel with 20% carbamide peroxide for
comparison. The materials were applied once a week, three times a day, for 4 weeks.
Bromelain and papain gels were effective in discoloring stained tooth enamel, even
though their effectiveness was less than that of the carbamide peroxide product [21].
Whey, as a by-product of milk processing, contains a mixture of α-lactalbumin (15–
20%), β-lactoglobulin (55–60%), and other proteins such as bovine serum albumin,
lactoperoxidase, imogloblobulin, lactoferrin, phospholipoproteins, as well as enzymes,
constituting a natural reservoir of bioactive peptides with physiological and antimicrobial
properties, the release of which requires the hydrolysis of precursor molecules by
digestive proteases or by fermentation with proteolytic microorganisms [24,25]. Whey
proteins have been proposed as possible ingredients in teeth whitening products.
Another possible source of bleaching agents is plant extracts. Organic acids, such as
malic acid, can promote the degradation of bacterial plaque and therefore help to remove
colored compounds from them [26]. Herbal based products for dental hygiene have been
also proved to have anti-inflammatory properties [27].
The present study reports the preparation and characterization of gels based on
natural products—bromelain extract, quince extract, whey with potential applications in
oral microbiome control for gingival or oral treatments and/or tooth whitening.
2. Results and Discussion
2.1. SDS-PAGE Analysis
Figure 1 shows SDS-PAGE data on the gels as an analysis specific to the protein
material, as most of the analyzed gels contain such material (bromelain and/or whey).
Thus, all samples G1–G7 were analyzed, except the G6 gel with SiO
2
which does not have
organic matter. Bromelain is detectable both in the control sample and in the gels in which
it was added as such (G1–G3) or added in the form of nanocapsules (G4), with a band of
about 24.5 kDa according to the calculated molecular weight, but also with a less intense
one at about 10 kDa. The G5 gel, which does not contain bromelain (and no whey, so no
protein), shows no detectable SDS-PAGE signal. It can be noted that the most intense
bromelain band is in the G1 gel. Interestingly, although the G3 gel contains more
bromelain than the others, here the enzyme signal is weaker than in the G1 gel. This result
can be interpreted as being due to the partial degradation of the enzyme in reaction with
the whey present in sample G3 in the largest amount of the five gels.
(a) (b)
Figure 1. Formula of polyacrylic acid (a) and polyethylene glycol (b).
The whey reference sample clearly contains two intense bands at about 20 kDa and
15 kDa, respectively. The band at ~20 kDa is completely absent in the G2-G4 gels, which
contain whey in two different concentrations. In gels G3 and G4, the whey band from ~15
kDa is also very weak, while in gel G2 it is intensified. These data can be interpreted as
suggesting the partial (but not total) degradation of whey proteins in those gels where
bromelain is also present, generating smaller peptide chains, the whitening potential of
which is expected to be higher than that of intact whey.
Gels 2021, 7, 191 4 of 13
The quince extract sample did not show detectable protein material in SDS-PAGE;
interestingly, this sample completely discolored the blue dye (Coomassie Blue) used in
preparing the sample for SDS-PAGE, which can be interpreted as further evidence of the
excellent potential as a material for discoloration in the experimental gels in the present
study (Figure 2).
Figure 2. SDS-PAGE analysis of experimental gels, together with control samples (Whey, Br = bro-
melain, Gut. = Quince extract; each control sample was diluted to the concentrations present in the
gels—with variant 0.2 for buffalo whey) and known molecular weight references. The intensities of
the signals marked on the gel are calculated with the Gel analyzer program (GelAnalyzer 19.1,
www.gelanalyzer.com by Istvan Lazar Jr., PhD and Istvan Lazar Sr., PhD, CSc).
2.2. UV-Vis Spectra
Figure 3 shows the UV-vis spectra of gels and key materials in solution. There is an
intense UV absorbance in G1-G3 gels, which is very similar between the three samples,
which can be explained by the presence of polymers (silicate and polyethylene glycol in
gels G1 and G2, polyacrylate, in G3) [28].
Notably, the maximum of 303 nm can be explained by neither of the polymers cited
(whose absorbance is well below 300 nm), nor by the protein material: it is observed that
the spectrum of bromelain and the spectrum of whey, from control samples prepared at
equivalent concentrations those in whey, are much less intense than the spectra of G1–G3
and, importantly, have the maximum typical of protein material at 280 nm, not at 303 nm.
However, given that G1–G3 gels have almost identical UV-vis spectra, it should be noted
that their only common ingredient is bromelain. Lubrizol gel (G5), which does not contain
bromelain, has negligible absorbance (and essentially represents the spectrum of the in-
gredient Lubrizol, based on polyacrylate and peroxide). Interestingly, the G4 gel, which
has bromelain captive in nanocapsules, has an intermediate spectrum between that of G5
gel (without bromelain) and that of G1–G3 gels (with free bromelain). Based on these ob-
servations, the 303 nm band is assigned to an in situ modified form of bromelain.
Gels 2021, 7, 191 5 of 13
Figure 3. UV-vis spectra of gels and key materials in compositions.
2.3. IR Spectra
Figure 4 shows the FT-IR spectra of polyethylene glycol, aerosil, and bromelain gel
samples. Wet aerosil-based gel samples show intense absorption maxima around 3711 and
3032 cm−1 and 1645 cm−1. In wet samples, the absorption maxima due to the vibrations of
the OH bonds in the water around 3386 cm−1 and 1647 cm−1 are especially noticeable; these
maxima decrease in intensity following the evaporation of water, increasing the intensity
of the maxima corresponding to the absorption bands due to the −CH2 group from 2928
cm−1. The broad band remaining after evaporation of water at large wave numbers (3711
and 303 cm−1) is due to the contributions of several components of the gel, so they can be
attributed to the vibrations of Si-O bonds in aerosil, OH vibrations in polyethylene glycol
and vibration amid A form whey. The maximum of this band underwent a shift to higher
wave numbers after drying.
4000 3500 3000 2500 2000 1500 1000 500
0.0
0.5
1.0
1.5
2.0
Absorbance [u.a.]
Wavenumber [cm
-1
]
G2 dried
G2 gel
G1 dried
G1 gel
3418.18
3418.12
3386.19
3386.19
2868.88
2864.53
2876.86
2903.43
1666.66
1663.04
1642.72
1647.08
1062.29
1057.94
1081.88
1081.88
4000 3500 3000 2500 2000 1500 1000 500
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
1536
1536
2928 2847
1647
1642
1642
1699
1699
Absorbance [a.u.]
Wavenumber [cm
-1
]
G5 dried
G4 dried
G3 dried
G5 gel
G4 gel
G3 gel 1699
1712
1714
2928 2849 1540
1536
Figure 4. FT-IR spectra of experimental gel samples G1–G4 (according to Table 1).
Table 1. Diameter of inhibition areas (mm) of the tested gel samples.
Sample G2 G3 G6 G5
Bacteria Bromelain Gel Bromelain Gel SiO2 Gel Lubrizol Gel
Streptococcus mutans 0 0 0 11
Porphyromonas gingivalis 9 11 0 10
Enterococcus faecalis 8 12 0 11
Escherichia coli 0 9 0 0
Staphylococcus aureus 7 11 0 10
Dehydrated aerosil has only two absorption peaks at 1630 cm−1 and 3700 cm−1, but
one may also note the presence of a wide intense band due to -OH vibrations from aerosils
in the range of 3300–3700 cm−1, SiO-H at 3409 cm−1, Si-O-Si at 1130, a band that appears to
be displaced both in the spectrum of wet samples and in the case of dry ones, and with a
higher intensity. The characteristic bands of PEG depend on the degree of polymerization,
0
0.2
0.4
0.6
0.8
1
200 300 400 500 600 700
A
λ(nm)
G1
G2
G3
G4
G5
Whey
Bromelain
Extract
Gels 2021, 7, 191 6 of 13
and can be located at 3441 cm
−1
in the case of OH stretching vibrations, 2878 cm
−1
for CH
elongation, 1464 cm
−1
and 1343 cm
−1
for CH deformation and 1094 cm
−1
OH and COH.
For gels G5 and G6, the IR spectra feature the characteristic peaks of water and pos-
sibly of the amide groups, because the vibrations of the bands amide A and amide 1 are
located in the same absorption range. Thus, in their case an intense band is observed with
a maximum around 3700–3000 cm
−1
due to the vibrations of the OH group in water, com-
pared to 3404 cm
−1
, a value that corresponds to the absorption band of pure liquid water,
due to symmetric stretching vibrations (v1), asymmetric stretching (v3) and bending de-
formation (v2) of OH, for all analyzed samples. An asymmetric absorption band can be
observed in the spectra of wet gels around 1642 cm
−1
. This band can be attributed to the
deformation vibrations (υ2) of the O-H bonds in the water, which can be located at 1643
cm
−1
in the case of pure water at 25 °C. This band overlaps with the Amide I absorption
band, due to vibrations of C=O groups and hydrogen bonds coupled with COO- from
whey and bromelain and vibrations C=O from polyacrylic acid.
2.4. SEM Microscopy
In Figure 5, SEM images of samples G1, G2 and G6 and the incorporated aerosil par-
ticles may be observed, while the rest of the gels containing Lubrizol or whey are much
more uniform. Gels that do not contain aerosil, but that contain the product with the en-
zymatic action of bromelain (3 and 4), are expected to be much more effective. These, in
addition to a satisfactory whitening, are expected to produce a reduced abrasion of the
surface compared to the rest of the gels, leading to fewer morphological changes on the
enamel surface.
Figure 5. SEM images for (left to right): (a) G1; (b) G2, (c) G3; (d) G4 (a) G1; (e) G5 (f) G6.
2.5. Antimicrobial Testing of Bleaching Gels
After the end of the incubation period at 37 °C, the zones of inhibition (mm) in the
tested microbial strains were determined. It was observed that the gel sample with SiO
2
(G6) did not show inhibition in all strains, but the other samples tested varied in the size
of the inhibition diameter depending on the amicrobial strain tested (Table 1).
With the bacterial strain Streptococcus mutans ATCC 25175, high inhibition was ob-
served in the G5 sample (inhibition = 11 mm). Samples G2 and G3 (bromelain based gel)
did not show any inhibition (Figure 6).
Gels 2021, 7, 191 7 of 13
Figure 6. Streptococcus mutans ATCC 25175 (a) zero moment (b) inhibition after the incubation
period.
With the bacterial strain Porphyromonas gingivalis ATCC 33277, inhibition was ob-
served at (9–13 mm) in all samples tested, with differences depending on the type of sam-
ple. The lowest value of inhibition was recorded in the G2 sample (bromelain based gel)
(9 mm) (Figure 7).
Figure 7. Porphyromonas gingivalis ATCC 33277 (a) zero moment (b) inhibition after the incubation
period.
With the bacterial strain Enterococcus Faecalis ATCC 29212, inhibition was observed
in all samples tested except G6. There was also a slight difference between the types of
samples (8–13 mm). Sample G3 showed a slightly higher inhibition compared to the other
samples (Figure 8).
Figure 8. Enterococcus faecalis ATCC 29212 (a) zero moment (b) inhibition after the incubation
period.
Gels 2021, 7, 191 8 of 13
With the Escherichia coli strain ATCC 25922, a rather low inhibition was observed
and only in the G3 samples (9 mm) (Figure 9).
Figure 9. Escherichia coli ATCC 25922 (a) zero moment (b) inhibition after the incubation period.
With the bacterial strain Staphylococcus aureus ATCC 25923, an inhibition was ob-
served in all three gels, and in this case the Gel 3 sample showed a slightly higher inhibi-
tion compared to the other samples (Figure 10).
Figure 10. Staphylococcus aureus ATCC 25923 (a) zero moment (b) inhibition after the incubation
period.
Based on the analyses reported here, one can emphasize the fact that UV-vis data in
the solution can be interpreted as evidence of a transformation undergone by bromelain
in G1-G4 gels. Such a transformation leaves the protein intact and active (as seen in the
following sections from the fact that it is detectable in SDS-PAGE and, moreover, that the
SDS-PAGE also shows evidence of its action by degrading whey proteins). The only
known example to date that draws a parallel with the data in Figure 3 (bromelain spec-
trum intensified by an order of magnitude and with the maximum shifted above 300 nm)
is that of bromelain exposed to transitional metal ions under denaturing conditions [29].
The conditions mentioned (iron, aerobic, denaturant) are ideal both for the formation of
complexes with a charge transfer character from metal to ligand (characterized by signif-
icantly higher absorbance than the side chains of proteinogenic amino acids) and for the
oxidation in the Fenton regime of aromatic amino acids in the protein. In contrast, the
mere denaturation of the protein would not explain the increase in absorbance by an order
of magnitude [30].
It should also be noted that at high concentrations of polymers such as polyethylene
glycol, proteins may be partially denatured; such concentrations are found in the gels
Gels 2021, 7, 191 9 of 13
analyzed in the present experiments (PEG + Aerosil in gels G1 and G2, polyacrylate in gels
G3 and G4) [9]. Under these conditions, it can be interpreted that in gels G1-G4 bromelain
is partially degraded and may form complexes with traces of transitional metals in the
bromelain or whey preparation. Degradation, according to the UV-vis spectrum, would
be lower in the G4 gel.
This interpretation is also consistent with the SDS-PAGE data in Figure 2; their whey
proteins are partially degraded in gel G2 (which has free bromelain and partially de-
graded according to the UV-vis spectrum) but essentially invisible (so more hydrolyzed
later) in the G4 gel, where bromelain is (nano) encapsulated. For gel G3, the same trend is
preserved, but it is only visible in the quantitative analysis of SDS-PAGE data in Figure 2.
Thus, nanoencapsulation would allow the bromelain in the gels to have a longer lifespan.
This can be seen as an advantage of the ingredient, although the partial degradation of
bromelain in G1-G3 gels may itself provide an advantage by reducing selectivity (at the
denaturation/partial degradation of the active site), which could increase the spectrum of
protease action of bromelain in gels.
Displacement of the IR spectrum absorption bands from those of pure PEG and aer-
osil demonstrates the existence of strong interactions between these compounds, probably
due to the formation of hydrogen bonds. It seems that due to the presence of bromelain,
the bands play a less important role, probably destroying the three-dimensional structure
due to the appearance of stronger hydrogen bonds of C = O • • • OH, which are formed
between polyethylene glycol and bromelain. By replacing the C = O • • • NH bonds in
bromelain, bromelain interactions with aerosil are also possible. On the other hand, the
low concentration of bromelain in 2% whey explains the lack of clear maximum absorp-
tion of it.
Moreover, the lack of vibrations due to the amide groups in the case of the sample
that does not contain whey determined the appearance of a more symmetrical absorption
band in the case of the gel based on polyacrylic acid. The shape of the absorption strips
suggests that an important contribution to the vibrations of the wet gel strips is due to the
amide groups in the whey. The bands underwent a shift from the vibrations of the OH
groups in the water, from 1643 to 1647 cm−1, due to the interactions with the molecules of
the whey proteins.
In the case of dry samples, there is a shift in the absorption characteristic peaks of the
C=O groups of polyacrylic acid for all samples, suggesting that the decrease in water con-
centration caused significant changes in the C = O vibrations of polyacrylic acid, which
become dominant. In the spectrum of samples that contain whey, there is an increase in
the intensity of the bands due to the CH2 groups which can be attributed to the amide
group B due to the asymmetric stretching vibrations of the CH2 group around 2928 cm−1,
bands that are less defined in the case of sample G5, which contains only Lubrizol. At
smaller wave numbers, a band around 2880 cm−1 can be noticed in the case of dry G3 and
G4 samples the absorption band of the CH3 group, due to the symmetrical stretching vi-
brations in the protein. Because bromelain and whey are protein-based, the main absorp-
tion bands of the two compounds overlap.
As early as 1999 [30], bromelain has been shown to act as an antibacterial agent by
inhibiting the growth of intestinal bacteria, such as Escherichia coli, by helping to stop the
production of enterotoxins by these bacteria. In 2014, the antibacterial effect against strong
periodontal pathogens was studied [31], showing that this enzyme also demonstrates an
anthelmintic effect against gastrointestinal nematodes, such as Heligmosomoides polygyrus,
Trichoderma viride and Trichurismuris [32]. Following antimicrobial tests on gels enriched
with bromelain, it can be stated that they have different antimicrobial activity depending
on the type of extract and depending on the bacterial strain tested. No inhibition was rec-
orded in the SiO2 gel sample, which means that in the gel samples tested only the active
compounds in them acted on the bacteria and not the solvent.
Currently, bromelain is administered for many clinical applications due to its thera-
peutic effects in the treatment of inflammation and soft tissue lesions. The studied gels
Gels 2021, 7, 191 10 of 13
can be used for tooth whitening gels and other gingival or oral treatments. More evidence
is necessary to establish the efficiency and safety of these products for use in dentistry.
3. Conclusions
Beyond ensuring the desired physical properties for application as whitening gels,
the excipients modulate and allow the preservation of the integrity and/or properties of
natural ingredients. The protease activity of bromelain modulates the composition of the
added whey proteins. Bromelain added as a nano encapsulated assembly better preserves
its integrity, showing different antimicrobial activity depending on the type of extract and
depending on the bacterial strain tested. Health professionals can use bromelain gel for
its therapeutic effects in oral injuries of soft tissues, and they can explore its properties as
a whitening tool for dental enamel.
4. Materials and Methods
4.1. Preparation of Experimental Gels
Bromelain-based gels were obtained using polyethylene glycol (PEG 400) or aerosil
(SiO2 medical products; Sigma Aldrich) and Carbopol (Lubrizol Advanced Materials, Inc.,
Belgium) as thickeners. Under the trade name aerosil, amorphous nanometric SiO2 pow-
ders of the “pyrogenic silica”, “fumed silica” or “fume silica” type are generally available.
This powder, used as a thickening agent, is obtained by pyrolysis of silicon tetrachloride
at temperatures above 1500°C. The carbomers used as thickeners are high molecular
weight polymers of acrylic acid. As an alternative thickening agent, polyethylene glycol
was also used—a polyether type polymer with the structural formula H−(O-CH2-CH2)n-
OH.
The lyophilized whey used at this stage is a by-product resulting from milk pro-
cessing, resulting from the separation of the cheese. For a better dispersion of bromelain,
it was dissolved in water prior to adding the gels.
The chemical composition of the developed gels is shown in Table 2.
Table 2. Chemical composition of the developed experimental gels.
Gels/Composition
(g/10 g Gel) PEG400 SiO2 Quince
Extract Bromelain Whey Nanoencapsulated
Bromelain Lubrizol H2O
G1 2.216 1.222 7.042 0.2 - - - -
G2 2.216 1.222 - 0.2 0.2 - - 6
G3 - - - 0.2 0.5 - 1 11.5
G4 - - - - 0.2 2 1 14
G5 (Carbopol) - - - - - - 0.25 10
G6 (SiO2) 1.22 10
G7 (Bromelaina) 0.2 10
4.2. SDS-PAGE Analysis
Gel electrophoresis under denaturing conditions (SDS-PAGE) was performed for the
experimental gels in order to examine the stability of whey proteins and bromelain, fol-
lowing an electrophoresis and quantitative analysis protocol previously described and
applied to milk and whey [21].
4.3. UV-Vis Spectra
Alternatively, the gels were dissolved in distilled water and measured in solution
using a Cary 50 UV-vis apparatus (Varian, Inc., Foster City, CA, USA), and Lambda 25
(PerkinElmer Singapore) spectrophotometers. The wavelengths ranged from 200 to 700
nm.
Gels 2021, 7, 191 11 of 13
4.4. Infrared Absorption Spectra
Infrared absorption spectra of the gels were determined using a Jasco-FTIR 610 FTIR
spectrometer, with an attenuated reflection device (ATR), for wet gel samples placed on
the ATR window. Due to the high content of water for obtaining absorption spectra for
the main compounds of the gels, the samples were dried in an oven at a temperature of
30 °C. The resolution was 2 cm−1 in the range of 4000–500 cm−1.
4.5. SEM microscopy
The surface morphology of gels was analyzed using the scanning electron micro-
scope SEM (Inspect S, FEICompany), at different magnifications.
4.6. Antimicrobial Testing of Bleaching Gels
The microorganisms tested in this study were: Streptococcus mutans ATCC 25175,
Porphyromonas gingivalis ATCC 33277, Enterococcus faecalis ATCC 29212, Escherichia
coli ATCC 25922 and Staphylococcus aureus ATCC 25923, from the collection of the La-
boratory of Microbiology, Faculty of Biology and Geology, UBB, Cluj.
Each bacterial strain was grown for 24 h on a Nutrient Agar medium. A dilution of
0.5 McFarland in sterile saline was then made from each strain. From these dilutions, each
Petri dish is inoculated with a sterile swab soaked in the 0.5 McFarland microbial suspen-
sion and spread over the entire surface of the solid culture medium (Mueller Hinton-Ox-
oid), after which the dishes were incubated for 20 min at 37 °C. Then, with a micropipette
and sterile cut tips, wells were made in the solid culture medium with a 6 mm diameter
each. Subsequently, in each well, the control samples and the samples with the prepared
test gels were added with a syringe. Incubation was performed for 24 h at 37 °C. The
reading was made by measuring the diameter of the inhibition zone: the larger the diam-
eter of the inhibition zone, the greater the sensitivity of the bacterium to the respective
antibacterial substances.
Author Contributions: Conceptualization, M.M. and A.M.; methodology, M.M., A.M. and R.M.C.;
software, A.M.C.; validation, V.P., C.S. and R.S.D.; formal analysis, A.M.C., A.M., M.M. and D.P.;
investigation, V.P., A.M.C., A.M., R.C., and G.F.G.; resources, C.S. and G.F.G.; data curation, L.S.D.
and R.C.; writing—original draft preparation, M.M., A.M., R.M.C. and L.S.D.; writing—review and
editing, M.M., A.M., A.M.C. and V.P.; visualization, L.S.D.; supervision, M.M. and V.P.; project ad-
ministration, M.M.; funding acquisition, V.P., D.P. and R.S.D. All authors have read and agreed to
the published version of the manuscript.
Funding: This research was funded by Romanian National Authority for Scientific Research and
Innovation, UEFISCDI (http://uefiscdi.gov.ro, 20 June 2021) Project 334 PED/2020 program PN-III-
P2-2.1-PED-2019-2953
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
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