Comparative biocompatibility and antimicrobial studies of sorbic acid derivates

Article abstract of DOI:10.1016/j.ejps.2019.105162

Nowadays, the sorbates are the third largest group of antimicrobial preservatives in food and pharmaceutical industries, following the parabens and benzoates whose safety is questioned by recent publications. A disadvantage of sorbates is their pH dependence, as their antimicrobial effect is greatly reduced in alkaline environment. The main, widely used sorbate derivatives are sorbic acid and potassium sorbate, no sorbic acid esters are involved in current industrial application. We aimed to test whether the esters of sorbic acid are capable to extend the antimicrobial spectrum of the original molecule while maintaining its advantageous biocompatibility profile. A comparative biocompatibility study of different derivatives (sorbic acid, potassium sorbate, isopropyl sorbate and ethyl sorbate) was carried out. In vitro cell viability assays of MTT (2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide), Neutral Red (3-amino-7-dimethylamino-2-methylphenazine hydrochloride) and flow cytometry with propidium iodide and annexin were performed on Caco-2 cells. In case of in vivo toxicity study, G. mellonella larvae were injected with different concentrations of the test compounds. Time-kill tests were executed on reference strains of C. albicans, E. coli, and S. aureus. According to the MTT-assay, the IC₅₀ values were the following: ethyl sorbate, sorbic acid <0.045% ^w/_w, isopropyl sorbate 0.32% ^w/_w, potassium sorbate >0.75% ^w/_w, while Neutral Red values were >0.75% ^w/_w for the esters and potassium sorbate and 0.66% ^w/_w for sorbic acid. Flow cytometry results indicated the higher cell damage in case of isopropyl sorbate. However, the cytotoxic results of isopropyl sorbate, in vivo toxicity study on G. mellonella larvae did not show significant mortality. It was found, that the antimicrobial properties of isopropyl sorbate were outstanding compared to sorbic acid and potassium sorbate. These results indicate, that the use of sorbate esters can be advantageous, hence, further toxicity studies are needed to prove their safety.

Full text of DOI:10.1016/j.ejps.2019.105162

Journal Pre-proof
Comparative biocompatibility and antimicrobial studies of sorbic acid
derivates
´
´ ´
´
´
´
Daniel Nemes , Renato Kovacs , Fruzsina Nagy , Zoltan Toth ,
´
´
´
Pal Herczegh , Aniko Borbas , Viktor Kelemen ,
´
´
Walter P. Pfliegler , Istvan Rebenku , Peter B. Hajdu ,
´
´
´
´
Palma Feher , Zoltan Ujhelyi , Ferenc Fenyvesi , Judit Varadi ,
´
´
´
´
Miklos Vecsernyes , Ildiko Bacskay
PII:
S0928-0987(19)30435-X
DOI:
Reference:
https://doi.org/10.1016/j.ejps.2019.105162
PHASCI 105162
To appear in:
European Journal of Pharmaceutical Sciences
Received date:
Revised date:
Accepted date:
15 July 2019
24 October 2019
18 November 2019
´
´ ´
´
´
´
Please cite this article as: Daniel Nemes , Renato Kovacs , Fruzsina Nagy , Zoltan Toth ,
´
´
´
´
Pal Herczegh ,
Aniko Borbas ,
Viktor Kelemen ,
Walter P. Pfliegler ,
Ferenc Fenyvesi ,
Istvan Rebenku ,
´
´
´
´
´
Judit Varadi ,
Peter B. Hajdu ,
Palma Feher ,
Zoltan Ujhelyi ,
´
´
´
´
Miklos Vecsernyes , Ildiko Bacskay , Comparative biocompatibility and antimicrobial stud-
ies of sorbic acid derivates, European Journal of Pharmaceutical Sciences (2019), doi:
https://doi.org/10.1016/j.ejps.2019.105162
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier B.V.

Comparative biocompatibility and antimicrobial studies of
sorbic acid derivates
Dániel Nemes1, Renátó Kovács2, Fruzsina Nagy2, Zoltán Tóth2, Pál Herczegh3, Anikó
Borbás3, Viktor Kelemen3, Walter P. Pfliegler4, István Rebenku5, Péter B. Hajdu5,
Pálma Fehér1, Zoltán Ujhelyi1, Ferenc Fenyvesi1, Judit Váradi1, Miklós Vecsernyés1,
Ildikó Bácskay1,*
1
Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Debrecen, Debrecen 4032, Hungary
Department of Medical Microbiology, Faculty of Medicine, University of Debrecen, Debrecen 4032, Hungary
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Debrecen, Debrecen 4032, Hungary
Department of Biotechnology and Microbiology, Faculty of Science and Technology, University of Debrecen, Debrecen
2
3
4
4032, Hungary
5
Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Debrecen 4032, Hungary
* Correspondence: Ildikó Bácskay, bacskay.ildiko@pharm.unideb.hu
Abstract: Nowadays, the sorbates are the third largest group of antimicrobial preservatives
in food and pharmaceutical industries, following the parabens and benzoates whose safety is
questioned by recent publications. A disadvantage of sorbates is their pH dependence, as
their antimicrobial effect is greatly reduced in alkaline environment. The main, widely used
sorbate derivatives are sorbic acid and potassium sorbate, no sorbic acid esters are involved
in current industrial application. We aimed to test whether the esters of sorbic acid are
capable to extend the antimicrobial spectrum of the original molecule while maintaining its
advantageous biocompatibility profile. A comparative biocompatibility study of different
derivatives (sorbic acid, potassium sorbate, isopropyl sorbate and ethyl sorbate) was carried
out. In vitro cell viability assays of MTT (2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-
tetrazolium bromide), Neutral Red (3-amino-7-dimethylamino-2-methylphenazine
hydrochloride) and flow cytometry with propidium iodide and annexin were performed on
Caco-2 cells. In case of in vivo toxicity study, Galleria mellonella larvae were injected with
different concentrations of the test compounds. Time-kill tests were executed on reference
strains of C. albicans, E. coli, and S. aureus. According to the MTT-assay, the IC50 values
w
w
were the following: ethyl sorbate, sorbic acid <0.045% /w, isopropyl sorbate 0.32% /w,
potassium sorbate >0.75% w/w, while Neutral Red values were >0.75% w/w for the esters and
potassium sorbate and 0.66% w/w for sorbic acid. Flow cytometry results indicated the higher
cell damage in case of isopropyl sorbate. However, the cytotoxic results of isopropyl sorbate,
in vivo toxicity study on Galleria mellonella larvae did not show significant mortality. It was
found, that the antimicrobial properties of isopropyl sorbate were outstanding compared to
sorbic acid and potassium sorbate. These results indicate, that the use of sorbate esters can be
advantageous, hence, further toxicity studies are needed to prove their safety.
Keywords: sorbates; preservatives; biocompatibility; galleria mellonella; caco-2 cells;
antimicrobials;
1. Introduction
Many commonly used excipients are presented in pharmaceutical and food industries.
One such jointly used group of compounds are the antimicrobial preservatives. As both liquid,
oral pharmaceutical preparations and certain beverages and drinks can be opened and closed
multiple times until their expiration date, every interaction with the outer environment risks
the contamination of the product. The alkyl esters of 4-hydroxybenzoic acid, the parabens are

the most commonly used group of pharmaceutical preservatives. However, recent studies
indicated that they could actively promote the proliferation of estrogen dependent cell lines
[Roszak et al., 2017]. Their interaction with human endocrine system was also described
[Nishihama et al., 2016]. These results question their safe use and numerous governments
limit the utilization of these materials [European Commission Regulation (EU) No
1004/2014]. Accordingly, preservatives performing favourable biocompatibility profiles
combined with reliable antimicrobial activity may replace parabens in food, pharmaceutical
and cosmetics industry.
2,4-hexadienoic acid, better known as sorbic acid and its potassium salt are alternatives of
parabens. They are already widespread throughout the food and pharmaceutical industries,
their application is well-established. Originally extracted from rowanberry, sorbic acid can
also be found in various other plants [Shabir et al., 2011; Esquivel-Ferriño et al., 2012],
although nowadays it is synthetically produced for commercial purposes. Aqueous solutions,
o/w emulsions, suspensions, gels or any other product with high water content in
pharmaceutical or food industries can be preserved by these compounds considering the
chemical or physical interactions. Sorbic acid can be applied in more lipophilic environment
such as ointments as well. Nowadays, potassium sorbate and sorbic acid with the sole purpose
of antimicrobial preservation in concentration range of 0.1-0.2% are widely used in
pharmaceutical industry [Rowe et al., 2009]. Generally, they are accepted to be safe for
human use, although toxicity and biocompatibility data profiles are incomplete and
fragmentary. Rats, fed by the ten times of acceptable daily intake (ADI) for 60 days
developed medium levels of toxicity [Abo-EL-Sooud et al., 2018], while the liver tissue of
mice, fed with lower concentrations of potassium sorbate, than ADI showed no elevation in
inflammatory genes [Raposa et al., 2016]. Other publications revealed low ciliary toxicity in
rabbits [Wang et al. 2012] and improved growth performance in swine through the increase of
IGF-I [Lou et al., 2011]. Also, the inhibition of gastrointestinal endoproteinases was reported
[Esimbekova et al., 2017]. Cell line investigations include low toxicity on HL7702 hepatocyte
cells and high toxicity in acidic conditions on Dunaliella tertiocleta [Chen et al., 2017], and
no toxicity on human primary nasal ciliary epithelial cells compared to benzalkonium-
chloride [Jiao et al., 2014; Ho et al., 2008] and low genotoxicity on human lymphocytes
[Mamur et al., 2010]. These various data show, that sorbates do not express serious toxicity
and if the regulatory concentrations are not exceeded, the human health risk is minimal
[Mpountoukas et al., 2008]. Acceptable human daily intake of sorbates (sorbic acid and
potassium sorbate) is a maximum of 25 mg/kg but the official regulations vary in different
countries [Dehghan et al., 2018].
The antimicrobial action of sorbates is not well understood, yet it is considered to be
basically based on the intracellular acidification of microbes [Bagar et al., 2009; Plumridge et
al., 2004]. After penetrating the cell membrane, at the pH level of the cytosol, as a weak
carboxylic acid, it releases a proton, which acidifies the cytosol, thus leading to the disruption
of catabolic pathways [Mira et al., 2010]. One possible resistance mechanism is the
preventive acidification of cytosol and the adaption to it, in order to decrease the uptake of
sorbic acid [Stratford et al., 2014]. Another method is the decarboxylation of sorbic acid to
1,3-pentadiene [Plumridge et al., 2008; Plumridge et al., 2010]. It was also proved, that with
the increase of extracellular pH, the antimicrobial action of sorbates decreases, as only the
nonionized form can enter the cells [Wang et al., 2018]. Also, there are signaling pathways,
that sense the intracellular presence of sorbate ion and upregulates certain specific defense
mechanisms [Kim et al., 2019] and these mechanisms does not provide general resistance
against all weak acids [Creamer et al., 2017]. Sorbates can also effectively reduce bacterial
biofilm formation [Al-Ahmad et al., 2008; Arzweiler et al., 2008;]. Cellular stress caused by
sorbates can also result in increased toxin production [Fodil et al., 2018]. In theory the

antimicrobial effect is increased, if a more lipophilic compound enters the cell more easily. In
this case, the ester derivatives of sorbic acid act as a prodrug, as further enzymatic activation
is needed, to release the carboxylic group [Larsen et Johnson, 2019].
Based on these previous studies, our aim was to test the alkyl esters of sorbic acid
compared to potassium sorbate and sorbic acid. Antimicrobial properties of the tested
Figure 1. Substances involved in our experiments
substances (Figure 1.) were studied on frequent pathogens, C. albicans, S. aureus and E. coli
with time-kill method.
Meanwhile, cytocompatibility was assessed by MTT (2-(4,5-dimethyl-2-thiazolyl)-3,5-
diphenyl-2H-tetrazolium bromide) and Neutral Red (3-amino-7-dimethylamino-2-
methylphenazine hydrochloride) assays on Caco-2 human colon adenocarcinoma cell line and
Galleria mellonella larve survivability tests. Caco-2 cells can model the susceptibility of the
gastrointestinal tract as they morphologically represent the intestinal epithelium [Mao et al.,
2016; Medrano-Padial et al., 2019]. MTT and Neutral Red assays are rapid cytotoxicity
methods which complement each other, because their mechanisms of action are different
[Fotakis et Timbrell 2006]. Galleria mellonella larvae is a recent, emerging method for in
vivo toxicity testing. [Maguire et al., 2016]
2. Materials and methods
2.1 Materials
Ph. Eur. 9. quality sorbic acid was purchased from Hungaropharma (Budapest, Hungary).
Potassium sorbate and Neutral Red (3-amino-7-dimethylamino-2-methylphenazine
hydrochloride) was obtained from Alfa Aesar (Karlsruhe, Germany) and ethyl-sorbate from TCI
(Zwijndrecht, Belgium). The MTT (2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazolium
bromide)) dye, Dulbecco’s Modified Eagle’s Medium with high glucose and L-glutamin
(DMEM), phosphate buffered saline (PBS), trypsin from porcine, ethylene-diamine-tetra-acetic
acid (EDTA), heat-inactivated fetal bovine serum (FBS), Roswell Park Memorial Institute-1640
(RPMI-1640) and Mueller-Hinton broth, sorbic chloride and propidium iodide were purchased
from Sigma-Aldrich (Budapest, Hungary). Non-essential amino acids solution and penicillin-
streptomycin mix, GlutaMax™ supplement, cell culture flasks and Annexin V, Alexa Fluor™
647 conjugate were obtained from Thermo-Fisher (Darmstadt, Germany). Propan-2-ol,
pyridine, dichloromethane were purchased from Molar Chemicals (Halásztelek, Hungary).
2.2. Cell culture

Caco-2 (COlon adenoCArcinoma) cell line was obtained from the European Collection of
Cell Cultures (ECACC, No. 86010202). Cells were grown in Nunc™ EasyFlask™ (Thermo-
Fisher, Darmstadt, Germany) surface-treated plastic cell culture flasks in Dulbecco's Modified
Eagle's Medium, supplemented with 3,7 g/l NaHCO3, 10% (v/v) heat-inactivated fetal bovine
serum (FBS), 1% (v/v) non-essential amino acids solution, 0.584 g/L L-glutamine, 4.5 g/L D-
glucose, 100 IU/mL penicillin, and 100 µg/mL streptomycin at 37°C in an atmosphere of 5%
CO2. The cells were routinely maintained by regular passaging and glutamine was
supplemented by GlutaMax™. The cells used for cytotoxic experiments were between
passage numbers 20 and 40.
2.3. Cell viability tests
The cytotoxic effects of the various solutions were evaluated using the MTT and Neutral Red
methods. Caco-2 cells in complete medium were seeded on 96-well plates at a final density of
10.000 cells/well. After 7 days, the medium was removed, and the cells were incubated for 30
minutes with the test solutions. In case of MTT-assay, the samples were removed, and a 5
mg/mL MTT solution (MTT salt solved in PBS) was added to each well. The plates were
incubated for 3 hours, then the MTT solution was removed and 0.1 mL of a solution of
isopropanol – 1 M hydrochloride acid (25:1) was added to each well to dissolve the formed
formazan crystals. In case of Netural Red assay, the test solutions were removed and a 33,3
mg/mL NR solution (NR solved in cell culture medium) was added to each well. The cells
were incubated for 2 hours then, the NR solution was removed and 0.1 mL of a solution of
isopropanol – 1 M hydrochloride acid (25:1) was added to each well to dissolve the cells. The
absorbance compounds were measured at 565 nm for MTT-assay and 540 nm for NR-assay.
We used empty wells of the plate as reference and all the measurements were carried out with
a Thermo-Fisher Multiskan Go (Thermo-Fisher, USA) microplate reader. Cell viability was
expressed as a percent of the cell viability of the untreated control cells, which were incubated
with PBS for 30 minutes.
2.4. Galleria mellonella larvae survivability tests
Larvae of the sixth developmental stage of G. mellonella were obtained from Bugs World Inc.
(Budapest, Hungary). Larvae were at 10 °C and in a dark environment prior to use. Larvae
size was between 2 and 3 centimetres and they showed no sign of melanisation. For each
treatment, 20 healthy larvae were placed in sterile vented Petri dishes. The test compounds
were dissolved in PBS 20 μl of each sample was injected into the G. mellonella haemocoel
through the last pro-leg using a 29G needle. The injected larvae were incubated at 30 °C for
96 hours in dark environment. For the assessment of larval viability, larvae were gently
probed with a blunt-ended needle and if no response was observed, the larvae were considered
to be dead. Viability was observed at 24 h, 48 h, 72 h, and 96 h.
2.5. In vitro time-kill antimicrobial tests
In killing studies, we tested Escherichia coli (American Type Culture Collection® 25922™),
Staphylococcus aureus (ATCC® 43300™) and Candida albicans (ATCC® 10231™)
reference strains. The activity of sorbates was determined against C. albicans and bacterium
strains in RPMI-1640 and Mueller-Hinton broth at 0.045%, 0.09%, 0.18%, 0.375%, 0.75%
w/w concentrations using a starting inoculum of 1x105 cells/mL and 1x106-107 cells/mL,
respectively, in a final volume of 5 mL, pH set to 7 [Nagy et al., 2019]. In case of C. albicans,
aliquots of 100 µl were removed after 0, 4, 8, 12 and 24 h of incubation, tenfold serial
dilutions were prepared, and samples of dilutions (4x30 µl) were plated onto a single
Sabouraud dextrose agar plate and incubated at 35°C for 48 h. In case of E. coli and S. aureus,
aliquots of 100 µl were removed after 0, 2, 4, 6, 8, 10, 12 and 24 h of incubation, tenfold
serial dilutions were prepared, and samples of dilutions (4x30 µl) were plated onto a single

Mueller-Hinton plate and incubated at 35°C for 48 h. Tests were carried out in duplicates and
mean values were presented. In any give concentration, were results differed from each other
more than 5%, a third experiment was carried out.
2.6. Synthesis of isopropyl sorbate
Isopropyl sorbate was synthetized in situ for our experiments. To a stirred solution of
isopropyl alcohol (11.7 mL, 0.15 mmol) in dry dichloromethane (100 mL) under inert argon
atmosphere and cooled to 0°C, 2.0 equivalent (24.2 mL, 0.3 mmol) of dry pyridine and 1.0
equivalent (20 mL, 0.15 mmol) of sorbic chloride was added. The reaction was stirred at room
temperature overnight. After completion, 2 mL of water was added, and the reaction mixture
was stirred for 1 hour. Then, the reaction mixture was diluted with dichloromethane (300 mL)
and was washed with saturated solution of NaHSO4 twice, and Na2CO3 twice as well. The
organic layer was then separated, dried over MgSO4, filtered and distilled under vacuum to
give isopropyl sorbate (9 g, 40%), yellow, fruity smell liquid.
2.7. Flow cytometry measurements
For the flow cytometry measurements, a BD FACSArray (BD Biosciences, Germany) flow
cytometer were used. 5x3 million Caco-2 cells were harvested from cell culture flasks with
w
trypsin-EDTA solution and were treated with 0.75% /w solutions of the tested compounds,
dissolved in cell culture media. After 30 minutes, the cells were centrifuged, the culture media
was removed and the cells were gently washed with cold PBS and centrifuged again.
Supernatant was removed and with annexin-binding puffer, 1x106 cells/mL cell suspension
was created. 100 µl of this suspension was treated with 5 µl of Alexa Fluror™ 647 and 1 µl of
100 µg/mL propidium iodide solution. The cell suspension was stained for 15 minutes on ice
then immediately analysed with the flow cytometer. The propidium iodide were excited with
the 532 nm laser line and detected between 564-606 (yellow parameter). The Alexa Fluor™
647 were excited with the 635 nm laser line and detected between 653-669 nm (red
parameter). The evaluation was made with FCS Express 6 (De Novo Software, USA). On the
FSC SSC scatterplot the non-cellular events were excluded. On FSC-A-FSC-W scatterplot the
duplets were excluded. The remaining events (8000-10.000) were analysed on a propidium
iodide-Alexa Fluor 647 scatterplot, the quadrant gates were determined on non-labelled
samples. The double positive cells regarded as necrotic/late apoptotic cells. The annexin V
positive population was regarded as early apoptotic, the double negative population regarded
as viable cells.
2.8. Statistical analysis
All data were analysed using GraphPad Prism (version 6; GrapPad Software, San Diego,
California, USA). In case of MTT-assay and NR-assay results, the data was presented as
means ± SEM. Each cell viability value represents the mean of twelve independent, parallel
wells, with the highest and lowest absorbance values were excluded when calculating the
mean. After that, at each concentration, the means of different solutions where compared with
Kruskal-Wallis test followed by Dunn’s test when all solutions were compared to each other.
Previously, all data groups were analysed with Shapiro-Wilk test for Gaussian distribution
and Bartlett's test for equal variances. In each case we used significance level p < 0.05. In vivo
survival curves of Galleria mellonella larvae were plotted according to the Kaplan-Meier
analysis, the survival curves were compared with Mantel-Cox log-rank test, GraphPad’s
Logrank test for trend and Gehan-Breslow-Wilcoxon test. Table 1. shows the statistical
analysis of the results of the MTT and NR assays. Flow cytometry tests were carried out as
triplicates.
3. Results

3.1. Cell viability tests
Preservatives have high concentrations in the pharmaceutical product, to ensure the absolute
inhibition of microbial growth. However, they are diluted in the stomach and later parts of the
gastrointestinal tract. In order to compare both the antimicrobial and the biocompatibility
w
tests, all compounds were tested in a wide range, setting 0.75% /w as maximum value and
halving the concentration of every further solution. According to the regulation of Hungarian
pharmaceutical compounding formulation, the maximum applied dose of sorbic acid (only
w
slightly soluble in water, but moderately in hot water) and potassium sorbate is 1% /w. and
tolerable according to Hungarian regulations. Also, according to the European regulations,
sorbic acid and potassium sorbate as food additives can be used from 0.02% (200 ppm) to
0.5% (5000 ppm) [Commission Regulation (EU) No 1129/2011]. We aimed to investigate the
biocompatibility and antimicrobial properties of the tested compounds above this approved
range in order to get a more detailed view of such properties. Therefore, our concentrations
w
were 0.045%, 0.09%, 0.18%, 0.375% and 0.75% /w which cover the whole range of
application. All of the sorbates for cytotoxicity tests were all diluted in PBS.
MTT assay (Figure 2.) showed a dose-dependent toxicity of sorbates, where potassium
sorbate was the least toxic compound, followed by isopropyl sorbate and ethyl sorbate, while
sorbic acid had the lowest cell viability results. However, at the highest concentration,
isopropyl sorbate, ethyl sorbate and the sorbic acid caused nearly total cell death. Calculated
IC50 values are <0.045% w/w for ethyl sorbate and sorbic acid, 0.32% w/w for isopropyl sorbate
and >0.75% w/w for potassium sorbate.
Figure 2. Cytotoxicity of sorbates measured by MTT assay. Cell viability expressed as the percentage of the
absorbance of the untreated control cells. Data expressed as mean ± SEM, n = 12.
Cell viability of the test samples at 0.045%, 0.09%, 0.18%, 0.375% and 0.75% (w/w)%:
Sorbic acid: 3.6% ±0.18%; 4.3% ±0.1%; 3.8% ±0.1%; 5.1% ±0.3%; 4.0%±0.3%
Potassium sorbate: 99.8% ±1.5%; 96.6% ±2.1%; 91.9% ±2%; 89.3% ±2%; 54.7% ±2.2%
Ethyl sorbate: 17.0% ±2.2%; 22.6% ±3.3%; 19.9% ±4.3%; 10.7% ±4.3%; 4.8% ±1.2%
Isopropyl sorbate: 59.1% ±1.7%; 62.1% ±3.7%; 57.4% ±3.5%; 47.2% ±2.6%; 3.8% ±1.6%

The lower concentrations of sorbates had only a minor impact on the viability of Caco-2
cells measured by Neutral Red assay (Figure 3.). Meanwhile, 0.375% and 0.75% drastically
increased the toxicity of the test substances. Compared to the results of MTT assay, sorbic
acid was the most toxic compound in this experiment too. Calculated IC50 values are above
0.75% w/w for the esters and potassium sorbate and 0.66% w/w for sorbic acid.
Figure 3. Cytotoxicity of sorbates measured by Neutral Red assay. Cell viability expressed as the percentage of
the absorbance of the untreated control cells. Data expressed as mean ± SEM, n = 12.
Cell viability of the test samples at 0.045%, 0.09%, 0.18%, 0.375% and 0.75% (w/w)%:
Sorbic acid: 100% ±1%; 100% ±1.4%; 100% ±1.6%; 79.7% ±2.9%; 40.8%±3.4%
Potassium sorbate: 100% ±1.1%; 100% ±1.6%; 100% ±1.9%; 100% ±0.5%; 98.2% ±2%
Ethyl sorbate: 99.6% ±0.4%; 94.9% ±0.7%; 91.7% ±0.9%; 89.9% ±0.9%; 59.8% ±3.3%
Isopropyl sorbate: 96.7% ±2.1%; 95.5% ±2%; 93% ±1%; 91.6% ±1%; 55.4% ±2.5%
3.2. In vivo toxicity tests
Galleria mellonella larvae were injected with 20 µl of the four test substances, dissolved
in PBS. Throughout the 4 days of the experiment, their viability was observed every 24 hours.
Two concentrations of sorbates were used, 0.18% and 0.018% w/w. Each group consisted of 20
healthy larvae. Only a minor number of specimens died during the experiment and overall, the
larvae showed no sign of melanisation or increased mortality. (Figure 4.) According to the
statistical analysis, no curves were significantly different from PBS control and from each
other.
Figure 4. Survival curve of Galleria mellonella larvae. Larvae were injected with 20 µl of test samples, each
group had 20 larvae in it.
Death events of the experiment:
24 h: 0
48 h: 0
72 h: 1 - 0.18% isopropyl sorbate; 1 – 0.018% isopropyl sorbate
96 h: 2 – 0.018% sorbic acid; 1 – 0.018% ethyl sorbate; 2 – 0.18% isopropyl sorbate; 2 – 0.018% isopropyl
sorbate
3.3 In vitro antimicrobial time-kill experiments

Time-kill tests were carried out, in order to study the antimicrobial effect of sorbates.
Candida albicans, Escherichia coli and Staphylococcus aureus were inoculated in RPMI-
w
1640 or Mueller-Hinton broth at 0.045%, 0.09%, 0.18%, 0.375%, 0.75% /w concentrations of
the different compounds. At given timepoints, 100 µl of aliquots were plated on agar plates
and counted. Killing activity was determined by a threshold of 99.9% (log10 CFU=2.24)
extermination of initial CFU.
In case of the lowest concentration, Candida albicans (Figure 5. A-D) was resistant to
w
every tested compound. At 0.09% /w concentration isopropyl sorbate (Figure 5. D) had a
w
slight fungistatic effect, inhibiting the further growth of fungal cells. At 0.18% /w, isopropyl
sorbate terminated all pathogens after 12 hours. No other tested substance had any effect on
C. albicans at these concentrations. Potassium sorbate (Figure 5. B) had fungistatic effect at
w
0.375% /w and above, while sorbic acid (Figure 5. A) and ethyl sorbate (Figure 5. C) could
prevent the germination only at the highest tested concentration. Meanwhile, isopropyl
w
sorbate had an increased killing effect above 0.18% /w, as both higher concentrations
identically eliminated all cells after 8 hours.
Figure 5. A-D Antimicrobial effect of sorbates on C. albicans.
Staphylococcus aureus was totally resistant to potassium sorbate, ethyl sorbate and sorbic
acid (Figure 6. A-C) as the inoculum size increased with time in case of every concentration.
As such, S. aureus was the least sensitive organism in our experiment. Isopropyl sorbate
(Figure 6. D) had a bacteriostatic effect at 0.375% w/w concentration and above.

Figure 6. A-D Antimicrobial effect of sorbates on S. aureus.
The growth of Escherichia coli was heavily affected by isopropyl sorbate (Figure 7. D),
as after twelve hours, no antimicrobial activity could be detected at 0.375% w/w concentration
w
and above. 0.18% /w concentration of isopropyl sorbate was bacteriostatic. Sorbic acid and
potassium sorbate (Figure 7. A, B) were totally ineffective against this species. Meanwhile the
w
results of ethyl sorbate (Figure 7. C) are contradictory, as 0.375% /w had a stable static
effect, while 0.75% w/w proved to be ineffective.
Figure 7. A-D Antimicrobial effect of sorbates on E. coli.
3.4. Flow cytometry measurements
w
Caco-2 cells were treated with 0.75% /w solutions of the tested substances for 30
minutes and stained with propidium iodide and annexin V. Figures 8 A-E shows the results
the distribution of the gated cells. The double positive cells regarded as necrotic/late apoptotic
cells, the annexin V positive population was regarded as early apoptotic, the double negative
population regarded as viable cells. Propidium iodide negative and annexin positive cells were
negligible. Isopropyl sorbate had increased cytotoxic effect, compared to the other compounds
which had increased dead cell percentage than the untreated control.
Figure 8 A-D. Flow cytometric measurement of Caco-2 cells, treated with 0.75% w/w solutions of the test
compounds, stained with propidium iodide (PI) and annexin (A). Data is represented as mean of triplicates. Mean
percentage distribution of cells between the upper left (PI+, A-), upper right (PI+, A+), lower left (PI-, A-) and
lower right (PI-, A+) quadrant, ± SEM:
control: 1.4% ±0.1%, 18.1% ±1.2%, 80.3% ±1.3%, 0.3% ±0.0%

sorbic acid: 1.9% ±0.1%, 29.3% ±0.4%, 68.2% ±0.4%, 0.5% ±0.0%
potassium sorbate: 4% ±0.2%, 27.9% ±2.1%, 67.5% ±2.2%, 0.5% ±0.1%
ethyl sorbate: 6.5% ±0.1%, 28.5% ±0.3%, 64.3% ±0.5%, 0.6% ±0.0%
isopropyl sorbate: 1.9% ±0.1%, 67.8% ±0.4%, 27.3% ±0.4%, 3% ±0.1%
4. Discussion
2,4-hexadienoic acid, as known as sorbic acid, is widely used as an antimicrobial
preservative for food, cosmetic and pharmaceutical industry. Its mechanism of action is stated
to be based on the diffusion through the cell membrane and intracellular acidification of the
targeted microbe [Stratford et al., 2013]. As the sorbates can only enter the cell in unionized
form, low pH greatly enhances their action, as they can be mostly found in that state at such
conditions [Abu-Ghazaleh, Bayan M. 2010]. If the pH of a given product cannot be adjusted
to acidic range, due to its stability, the effect of sorbates is reduced [Wang et al., 2018]. The
alkyl esters or sorbic acid might be the solution for the pH-dependency issue. Thus, ethyl and
isopropyl sorbates were involved in our study.
Tzatzarakis et al. [Tzatzarakis et al., 2000; Charvalos et al., 2001] previously formulated
different polyvinylpyrrolidone based polymers, to which sorbic acid was covalently bonded,
and tested it against several fungi species. The inhibitory concentrations were promising, yet,
no toxicity data is available, connected to the newly formed compounds. Moreover, they were
not tested against bacterial strains either. Narasimhan et al. synthesized 42 different sorbic
acid esters and analysed their antimicrobial potential [Narasimhan et al., 2007]. This
publication suggested, that the increase of lipophilicity enhances the antibacterial and
antifungal actions of the given compound. However, a disadvantage of these derivatives was
the poor water solubility which limits their application in water-based systems. Our test
substances were two esters with short alkyl chains, performing moderate water solubility.
The literature revealed, that all tested compounds are generally well tolerated. Qu et al. [Qu et
al., 2019] reported, that potassium sorbate had an IC50 value of 1.25 g/L after 24 h of
incubation on HepG2 human liver cell line measured by MTT, while HUVEC cell line
showed an 659.96ꢀµM IC50 value of after 24 h of incubation measured by MTT
[Mohammadzadeh-Agdash et al., 2018]. These results match our findings (Figure 2.), as after
30 minutes of incubation, 0.18% potassium sorbate concentration lowered the cell viability to
91.9%. The cell viability difference between potassium sorbate and sorbic acid might be
explained by the acidifying nature of the latter.
Smith et al. measured the cytotoxicity of potassium sorbate on Balb/C 3T3 clone A31
embryonic mouse cells with Neutral Red and found that it was toxic only in extremely high
concentrations, far over the generally applied concentrations [Smith et Alexander 2005]. Our
results (Figure 3.) well correlates with this, as only the highest concentrations decreased cell
viability. The high correlation of Neutral Red and MTT cytotoxitcy tests was reported
[Fotakis et Timbrell 2006]. However, the differences between the assays in our experiments,
were based on the acidification of the cytosol of Caco-2 cells. As the change of intracellular
pH disrupted the metabolism of the cell, the enzymatic conversion of MTT is highly
decreased [Berridge et al., 2005], but the lysosomal staining by Neutral Red was not inhibited
[Elliott et Auersperg 1993]. Another possible explanation of the cytotoxicity profile
differences of sorbates is their binding to proteins, as it was proved that relatively similar
molecules as carboxylic acids have various binding sites [Mohammadzadeh-Agdash et al.,
2018].
Flow cytometry measurements revealed that compared to the control, potassium sorbate,
sorbic acid and ethyl sorbate could increase the amount of propidium iodide and annexin
positive cells with 10%. However, isopropyl sorbate was significantly more cytotoxic (68%

compared to the 28% of other tested substances), than any other derivatives. We suspected
that this can be explained by the non-pH dependent mechanism of action and the higher
membrane permeability of the isopropyl sorbate, which greatly exceeds the less lipophilic
ethyl sorbate. MTT and NR assays did not certify that difference, as both substances showed
similar cytotoxic effects. However, it was proved that the minimum change in the length of
alkyl chain greatly modifies the biological activity and membrane passage in the case of
salicylic acid derivatives [Li et al., 2019]. Further investigation is needed to explain
particularly the modification of sorbate esters membrane permeability with different lengths
of alkyl chains.
The use of Galleria mellonella larvae as a biocompatibility model organism is relatively
new. However, Maguire et al. found that the correlation between LD50 values observed on this
species and the results of previous rat feeding toxicity and cytotoxicity results was linear
[Maguire et al., 2016]. Several recent publications concluded, that the use of Galleria
mellonellae larvae not just complemented to cell culture studies in toxicity experiments,
[Allegra et al., 2018] [Bombarda et al., 2019] might be a good substitute of rodent model
systems [Ignasiak et Maxwell 2017], thus the prediction of human toxicity of tested
compounds can be greatly enhanced. In our experiments, there was no significant difference
between the mortality of different treated groups (Figure 4), the larvae showed no sign of
toxicity. As the injectable liquid volume is limited and 0.18% is a higher concentration, than
sorbates are generally used at, we found that the further increase of the dose in not necessary.
Our results match the findings of Narasimhan et al. who reported, that isopropyl sorbate
was significantly more active against S. aureus, E. coli and C. albicans, than ethyl sorbate,
which exceeded the original molecule only against E. coli and C. albicans [Narasimhan et al.,
2007]. In our experiment, MIC value was reached neither in the case of sorbic acid, nor with
potassium sorbate (Figure 5-7 a, b) against the tested microbes. In many previous
publications, it was found, that, the efficacy of sorbic acid and potassium sorbate decreases
with the elevation of pH [Lues et Theron 2012; Hwang et al., 2015]. Wang et al. found, that
w
potassium sorbate had a MIC value of 0.4 /w % against E. coli and S. aureus at pH 5, but 1.6
and 3.2 w/w% if the pH was adjusted to 7 [Wang et al., 2018]. Isopropyl sorbate could actively
kill E. coli (Figure 7. D) and C. albicans (Figure 5. D) cells and inhibit the growth of S.
aureus (Figure 6. D) at pH 7, which is a remarkable feat compared to other sorbates.
Lipophilicity and long-term acidification of the cytosol are critical in the antimicrobial
mechanism of weak acids [Ullah et al., 2012] and we suspect, that isopropyl sorbate could
more effectively pass through the cell membranes without the need of specific proteins [Peter
W. Piper 2011], than the other tested compounds. Bacterial esterases are known to be part of
antibiotic resistance in several species and thus [Egorov et al., 2018], they could possibly
cleave the sorbate esters, as known in the case of parabens [Valkova et al., 2003].
Two generally accepted and applied preservatives, sorbic acid and potassium sorbate and
two lipophilic sorbate derivates were tested on human colorectal cells, Galleria mellonella
larvae and various pathogens in order to test their biocompatibility and antimicrobial
properties. Further studies are needed, to specifically describe, the mechanism of action of
sorbate esters, whether they have antimicrobial action on their own, or they act as prodrugs
and can only be effective after enzymatic conversion to sorbic acid. While ethyl sorbate had
no significant inhibitory activity against the tested bacteria and fungi, isopropyl sorbate
demonstrated a significant bactericide and fungicide potential. As there is only one methyl
group difference between the ethyl sorbate, which had limited effect against the tested
microbes based on our experiments, an antimicrobial study with more sorbate esters would be
able to clarify the lipophilicity-antimicrobial action correlations. Our results indicate, that the
more lipophilic sorbate derivates could be promising antimicrobial preservatives, but their
low water solubility can limit their application. In order to properly assess the safety

biocompatibility profiles of these compounds, beside our study, different in vitro and in vivo
genotoxicity and toxicity studies are also required including vertebrates and human cell lines.

Conflict of interest
The authors declare no conflict of interest.
Acknowledgments:
The project was financed by the Gedeon Richter’s Talentum Foundation (1103 Budapest,
Gyömrői street 19-21.). The published work was also supported by EFOP-3.6.1-16-2016-
00022 and EFOP-3.6.3-VEKOP-16-2017-00009 projects of the European Union and ÚNKP-
18-3 New National Excellence Program of the Ministry of Human Capacities of Hungary.
The research was co-financed by the Higher Education Institutional Excellence Programme of
The Ministry of Human Capacities in Hungary, within the framework of the Research and
Development on Therapeutic purposes thematic programme of the University of Debrecen
(NKFIH-1150-6/2019).

References
Abo-EL-Sooud, K., Hashem, M., M., Badr, Y., A., Eleiwa, M., M., E., Gab-Allaha, A., Q., Abd-
Elhakim, Y., M., Bahy-EL-Diel, A., 2018. Assessment of hepato-renal damage and
genotoxicity induced by long-term exposure to five permitted food additives in rats.
Environ. Sci. Pollut. Res. Int. 25, 26341–26350. https://doi.org/10.1007/s11356-018-
2665-z
Al-Ahmad, A., Wiedmann-Al-Ahmad, M., Auschill, T., M., Follo, M., Braun, G., Hellwig, E.,
Arweiler, N., B., 2008. Effects of commonly used food preservatives on biofilm formation
of
Streptococcus
mutans
in
vitro.
Arc.
Oral
Biol.
53,
765-772.
https://doi.org/10.1016/j.archoralbio.2008.02.014
Allegra, E., Titball, R., W., Carter, J., Champion, O., L., 2018 Galleria mellonella larvae allow the
discrimination of toxic and non-toxic chemicals. Chemosphere 198, 469-472
https://doi.org/10.1016/j.chemosphere.2018.01.175
Arweiler, N., B., Lenz, R., Sculean, A., Al-Ahmad, A, Hellwig, E, Auschill, T., M., 2008. Effect
of food preservatives on in situ biofilm formation. Clin. Oral Investig. 12, 203-208.
https://doi.org/10.1007/s00784-008-0188-6.
Bagar, T., Altenbach, K., Read, N., D., Benčina, M., 2009. Live-cell imaging and measurement of
intracellular pH in filamentous fungi using a genetically encoded ratiometric probe.
Eukaryot. Cell 8, 703-712. https://doi.org/10.1128/EC.00333-08
Bayan, AG., M, 2010. Inhibition of growth and caseinase production of Pseudomonas aeruginosa
and Escherichia coli 28 by combination of low pH and NaCl, potassium sorbate or
Thymus vulgaris extract. Acta Microbiol. Immunol. Hung. 57, 95-108.
https://doi.org/10.1556/AMicr.57.2010.2.3
Berridge, M., V., Herst, P., M., Tan, A., S., 2005. Tetrazolium dyes as tools in cell biology: new
insights into their cellular reduction. Biotechnol. Annu. Rev. 11, 127-152.
https://doi.org/10.1016/S1387-2656(05)11004-7
Bombarda, G., F., Rosalen, P., L., Paganini, E., R., Garcia, M., AR., Silva, D., R., Lazarini, J., G.,
Freires, I., A., Regasini, L., O., Sardi, J., CO., 2019. Bioactive molecule optimized for
biofilm reduction related to childhood caries. Future Microbiol. 14, 1207-1120.
https://doi.org/10.2217/fmb-2019-0144
Charvalos, E., Tzatzarakis, M., Tsatsakis, A., Petrikkos, G., 2001. Controlled release of water-
soluble polymeric complexes of sorbic acid with antifungal activities. Appl. Microbiol.
Biotechnol. 57, 770-775. https://doi.org/10.1007/s00253-001-0853-z
Chen, HH., Xu, XL., Shang, Y., Jiang, JG., 2017. Comparative toxic effects of butylparaben
sodium, sodium diacetate and potassium sorbate to Dunaliella tertiolecta and HL7702 cells.
Food Funct. 8, 4478-4486. https://doi.org/10.1039/C7FO01102D
Creamer, K., E., Ditmars, F., S., Basting, P., J., Kunka, K., S., Hamdallah, I., N., Bush, S., P.,
Scott, Z., He, A., Penix, S., R., Gonzalez, A., S., Eder, E., K., Camperchioli, D., W., Berndt,
A., Clark, M., W., Rouhier, K., A., Slonczewski, J., L., 2017. Benzoate- and salicylate-
tolerant strains of Escherichia coli K-12 lose antibiotic resistance during laboratory
evolution. Appl. Environ. Microbiol. 83, e02736-16. https://doi.org/10.1128/AEM.02736-
16.
Dehghan, P., Mohammadi, A., Mohammadzadeh-Aghdash, H., Dolatabadi, J., E., N., 2018.
Pharmacokinetic and toxicological aspects of potassium sorbate food additive and its
constituents.
Trends
in
Food
Sci.
&
Tech.
80,
123-130.
https://doi.org/10.1016/j.tifs.2018.07.012
Egorov, A., M., Ulyashova, M., M., Rubtsova, M., Y., 2018. Bacterial enzymes and antibiotic
resistance. Acta Naturae 10, 33-48. PMID: 30713760
Elliott, W., M., Auersperg, N., 1993. Comparison of the neutral red and methylene blue assays to
study
cell
growth
in
culture.
Biotech.
Histochem.
68,
29-35.
https://doi.org/10.3109/10520299309105573

Esimbekova, E., N., Asanova, A., A., Deeva, A., A., Kratasyuk, V., A., 2017. Inhibition effect of
food preservatives on endoproteinases. Food Chem. 235, 294-297.
https://doi.org/10.1016/j.foodchem.2017.05.059.
Esquivel-Ferriño, P., C., Favela-Hernández, J., M., J., Garza-González, E., Waksman, N., Ríos,
M., Y., Camacho-Corona, M., R. 2012. Antimycobacterial activity of constituents from
Foeniculum vulgare var. dulce grown in Mexico. Molecules 17, 8471-8482.
https://doi.org/10.3390/molecules17078471
European Commission Regulation (EU) No 1004/2014 of 18 September 2014 amending Annex
V to Regulation (EC) No 1223/2009 of the European Parliament and of the Council on
cosmetic
products.
https://eur-lex.europa.eu/legal-
content/EN/TXT/?uri=CELEX%3A32014R1004
European Commission Regulation (EU) No 1129/2011 of 11 November 2011 amending Annex
II to Regulation (EC) No 1333/2008 of the European Parliament and of the Council by
establishing a Union list of food additives. https://op.europa.eu/en/publication-detail/-
/publication/28cb4a37-b40e-11e3-86f9-01aa75ed71a1/language-en
Fodil, S., Delgado, J., Varvaro, L., Yaseen, T., Rodríguez, A., 2018. Effect of potassium sorbate
(E‐ 202) and the antifungal PgAFP protein on Aspergillus carbonarius growth and
ochratoxin A production in raisin simulating media. J. Sci. Food Agric. 98, 5785-5794.
https://doi.org/10.1002/jsfa.9128.
Fotakis, G., Timbrell, J., A., 2006. In vitro cytotoxicity assays: Comparison of LDH, neutral red,
MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride.
Toxicol. Lett. 160, 171-177. https://doi.org/10.1016/j.toxlet.2005.07.001
Ho, CY., Wu, MC., Lan, MY., Tan, CT., Yang, AH., 2008. In vitro effects of preservatives in
nasal sprays on human nasal epithelial cells. Am. J. Rhinol. 22, 125-129.
https://doi.org/10.2500/ajr.2008.22.3154
Hwang, CA., Huang, L., Juneja, V., 2015. Effect of acidified sorbate solutions on the lag-phase
durations and growth rates of listeria monocytogenes on meat surfaces. J. Food. Prot. 78,
1154-1160. https://doi.org/10.4315/0362-028X.JFP-14-408
Ignasiak, K., Maxwell, A., 2017. Galleria mellonella (greater wax moth) larvae as a model for
antibiotic susceptibility testing and acute toxicity trials. BMC Res. Notes. 10, 428
https://doi.org/10.1186/s13104-017-2757-8
Jiao, J., Meng, N., Zhang, L., 2014. The effect of topical corticosteroids, topical antihistamines,
and preservatives on human ciliary beat frequency. ORL. 76, 127-136.
https://doi.org/10.1159/000363575
Kim, M., S., Cho, K., H., Park, K., H., Jang, J., Hahn, JS., 2019. Activation of Haa1 and War1
transcription factors by differential binding of weak acid anions in Saccharomyces
cerevisiae. Nucleic Acids Res. 47, 1211-1224. https://doi.org/10.1093/nar/gky1188.
Larsen, E., M., Johnson, R., J., 2019. Microbial esterases and ester prodrugs: An unlikely marriage
for
combating
antibiotic
resistance.
Drug
Dev.
Res.
80,
33-47.
https://doi.org/10.1002/ddr.21468
Li, L., Tavallaie, M., S., Xie, F., Xia, Y., Liang, Y., Jiang, F., Fu, L., 2019. Identification of lipid-
like salicylic acid-based derivatives as potent and membrane-permeable PTP1B inhibitors.
Bioorg. Chem. 93, 103296 https://doi.org/10.1016/j.bioorg.2019.103296
Lou, ZF., Fang, XL., Shu, G., Wang, SB., Zhu, XT., Gao, P., Chen, LL., Chen, CY., Xi, QY.,
Zhang, YL., Jiang, QY., 2011. Sorbic acid improves growth performance and regulates
insulin-like growth factor system gene expression in swine. J. Anim. Sci. 89, 2356-2364.
https://doi.org/10.2527/jas.2010-3677
Lues, J., F., R., Theron, M., M., 2012. Comparing organic acids and salt derivatives as
antimicrobials against selected poultry-borne Listeria monocytogenes strains in vitro.
Foodborne Pathog. Dis. 9, 1126-1129 https://doi.org/10.1089/fpd.2012.1220.

Maguire, R., Duggan, O., Kavanagh, K., 2016. Evaluation of Galleria mellonella larvae as an in
vivo model for assessing the relative toxicity of food preservative agents. Cell. Biol.
Toxicol. 32, 209-216. https://doi.org/10.1007/s10565-016-9329-x
Mamur, S., Yüzbaşıoğlu, D., Ünal, F., Yilmaz, S., 2010. Does potassium sorbate induce genotoxic
or mutagenic effects in lymphocytes?. Toxicol. In Vitro 24, 790-794.
https://doi.org/10.1016/j.tiv.2009.12.021
Mao, X., Nguyen, T., H., D., Lin, M., Mustapha, A., 2016. Engineered nanoparticles as potential
food contaminants and their toxicity to Caco‐ 2 cells. J. Food Sci. 81, 2107-2113.
https://doi.org/10.1111/1750-3841.13387
Medrano-Padial, C., Puerto, M., Moreno, F., J., Richard, T., Cantos-Villar, E., Pichardo, S., 2019.
In vitro toxicity assessment of stilbene extract for its potential use as antioxidant in the
wine industry. antioxidants, 8, 467. https://doi.org/10.3390/antiox8100467
Mira, N., P., Teixeira, M., C., Sá-Correia, I., 2010. Adaptive response and tolerance to weak acids
in Saccharomyces cerevisiae:
a
genome-wide view. OMICS. 14, 525-540.
https://doi.org/10.1089/omi.2010.0072
Mohammadzadeh-Agdash, H., Sohrabi, Y., Mohammadi, A., Shanehbandi, D., Dehghan, P.,
Dolatabadi, J., E., N., 2018. Safety assessment of sodium acetate, sodium diacetate and
potassium
sorbate
food
additives.
Food
Chem.
257,
211-215.
https://doi.org/10.1016/j.foodchem.2018.03.020
Mohammadzadeh-Agdash, H., Akbari, N., Esazadeh, K., Dolatabadi, J., E., N., 2019. Molecular
and technical aspects on the interaction of serum albumin with multifunctional food
preservatives Food Chem. 293, 491-498. https://doi.org/10.1016/j.foodchem.2019.04.119
Mpountoukas, P., Vantarakis, A., Sivridis, E., Lialiaris, T., 2008. Cytogenetic study in cultured
human lymphocytes treated with three commonly used preservatives. Food Chem. Toxicol.
46, 2390-2393. https://doi.org/10.1016/j.fct.2008.03.021
Nagy, F., Tóth, Z., Bozó, A., Czeglédi, A., Rebenku, I., Majoros, L., Kovács, R., 2019.
Fluconazole is not inferior than caspofungin, micafungin or amphotericin B in the presence
of 50% human serum against Candida albicans and Candida parapsilosis biofilms. Med.
Mycol. 57, 573-581. https://doi.org/10.1093/mmy/myy108
Narasimhan, B., Judge, V., Narang, R., Ohlan, R., Ohlan, S., 2007. Quantitative structure–activity
relationship studies for prediction of antimicrobial activity of synthesized 2,4-hexadienoic
acid
derivatives.
Bioorg.
Med.
Chem.
Lett.
17,
5836-5845.
https://doi.org/10.1016/j.bmcl.2007.08.037
Nishihama, Y., Yoshinaga, J., Iida, A., Konishi, S., Imai, H., Yoneyama, M., Nakajima, D.,
Shiraishi, H., 2016. Association between paraben exposure and menstrual cycle in female
university
students
in
Japan.
Reprod.
Toxicol.
63,
107-113.
https://doi.org/10.1016/j.reprotox.2016.05.010
Piper, P., W., 2011. Resistance of yeasts to weak organic acid food preservatives. Adv. Appl.
Microbiol. 77, 97-113. https://doi.org/10.1016/B978-0-12-387044-5.00004-2
Plumridge, A., Hesse, S., J. A., Watson, A., J., Lowe, K., C., Stratford, M., Archer, D., B., 2004.
The weak acid preservative sorbic acid inhibits conidial germination and mycelial growth of
Aspergillus niger through intracellular acidification. Appl. Environ. Microbiol. 70, 3506-
3511. https://doi.org/10.1128/AEM.70.6.3506-3511.2004
Plumridge, A., Melin, P., Stratford, M., Novodvorska, M., Shunburne, L., Dyer, P. S., Roubus, J.,
A., Menke, H., Stark, J., Stam, H., Archer, D., B., 2010. The decarboxylation of the weak-
acid preservative, sorbic acid, is encoded by linked genes in Aspergillus spp. Fungal. Genet.
Biol. 47, 683-692. https://doi.org/10.1016/j.fgb.2010.04.011
Plumridge, A., Stratford, M., Lowe, K., C., Archer, D., B., 2008. The weak-acid preservative
sorbic acid is decarboxylated and detoxified by a phenylacrylic acid decarboxylase, PadA1,
in the spoilage mold Aspergillus niger. Appl. Environ. Microbiol. 74, 550-552.
https://doi.org/10.1128/AEM.02105-07

Qu, D., Jiang, M., Huang, D., Zhang, H., Feng, L., Chen, Y., Zhu, X., Wang, S., Han, J., 2019.
Synergistic effects of the enhancements to mitochondrial ROS, p53 activation and
apoptosis generated by aspartame and potassium sorbate in HepG2 cells. Molecules 24,
457 https://doi.org/10.3390/molecules24030457.
Raposa, B., Pónusz, R., Gerencsér, G., Budán, F., Gyöngyi, Z., Tibold, A., Hegyi, D., Kiss, I.,
Koller, Á., Varjas, T., 2016. Food additives: Sodium benzoate, potassium sorbate,
azorubine, and tartrazine modify the expression of NFκB, GADD45α, and MAPK8 genes.
Physiol. Int. 103, 334-343. http://dx.doi.org/10.1556/2060.103.2016.3.6
Roszak, J., Smok-Pniążek, A., Domeradzka-Gajda, K., Grobelny, J., Tomaszewska, E., Ranoszek-
Soliwoda, K., Celichowski, G., Stępnik, M., 2017. M. Inhibitory effect of silver
nanoparticles on proliferation of estrogen-dependent MCF-7/BUS human breast cancer
cells induced by butyl paraben or di-n-butyl phthalate. Toxicol. Appl. Pharmacol. 337, 12-
21. https://doi.org/10.1016/j.taap.2017.10.014
Rowe, R., C., Sheskey, P., J., Quinn, M., E., 2009. Handbook of pharmaceutical excipients sixth
ed. Pharmaceutical Press and American Pharmacists Association, London, Chicago.
Shabir, G., Anwar, F., Sultana, B., Khalid, Z., M., Afzal, M., Khan, Q., M., Ashrafuzzaman, M.,
2011. Antioxidant and antimicrobial attributes and phenolics of different solvent extracts
from leaves, flowers and bark of gold hohar [Delonix regia (Bojer ex Hook.) Raf.].
Molecules 16, 7302-7319. https://doi.org/10.3390/molecules16097302
Smith, C., N., Alexander, B., R., 2005. The relative cytotoxicity of personal care preservative
systems in Balb/C 3T3 clone A31 embryonic mouse cells and the effect of selected
preservative systems upon the toxicity of a standard rinse-off formulation. Toxicol. In
Vitro 19, 963-969. https://doi.org/10.1016/j.tiv.2005.06.014
Stratford, M., Nebe-von-Caron, G., Steels, H., Novodvorska, M., Ueckert, J., Archer, D., B.,
2013. Weak-acid preservatives: pH and proton movements in the yeast Saccharomyces
cerevisiae.
Int.
J.
Food
Microbiol.
161,
164-171.
https://doi.org/10.1016/j.ijfoodmicro.2012.12.013
Stratford, M., Steels, H., Nebe-von-Caron, G., Avery, S., V., Novodvorska, M., Archer, D., B.,
2014. Population heterogeneity and dynamics in starter culture and lag phase adaptation of
the spoilage yeast Zygosaccharomyces bailii to weak acid preservatives. Int. J. Food
Microbiol. 181, 40-47. https://doi.org/10.1016/j.ijfoodmicro.2014.04.017
Tzatzarakis, M., N., Tzatsakis, A., M., Lotter, M., M., Shtilman, M., I., Vakalounakis, D., J., 2000.
Effect of novel water-soluble polymeric forms of sorbic acid against Fusarium oxysporum
f.sp.
radicis-cucumerinum.
Food
Addit.
Contam.
17,
965-971.
https://doi.org/10.1080/02652030010002289
Ullah, A., Orij, R., Brul, S., Smits, G., J., 2012. Quantitative analysis of the modes of growth
inhibition by weak organic acids in saccharomyces cerevisiae. Appl. Environ. Microbiol. 78,
8377-8387. https://doi.org/10.1128/AEM.02126-12
Valkova, N., Lépine, F., Labrie, L., Dupont, M., Beaudet, R., 2003. Purification and
characterization of PrbA, a new esterase from enterobacter cloacae hydrolyzing the esters
of 4-hydroxybenzoic acid (parabens) J. Biol. Chem. 278, 12779-12785.
https://doi.org/10.1074/jbc.M213281200
Wang, C., Deng, Q., Han, D., Zhang, L., 2012. Effects of benzalkonium chloride and potassium
sorbate on airway ciliary activity. ORL. 74, 149-153. https://doi.org/10.1159/000337830
Wang, J., Ma, M., Yang, J., Chen, L., Yu, P., Wang, J., Gong, D., Deng, S., Wen, X., Zeng, Z.,
2018. In vitro antibacterial activity and mechanism of monocaprylin against escherichia coli
and staphylococcus aureus. J. Food Prot. 81, 1988-1996. https://doi.org/10.4315/0362-
028X.JFP-18-248.

Graphical abstract