Synthesis, Surface and Antimicrobial Activity of Piperidine-Based Sulfobetaines

Article abstract of DOI:10.1007/s11743-016-1906-8

A new method for the preparation of new heterocyclic amine surfactants based on sulfobetaines is proposed. Interfacial activities of the surfactants obtained in aqueous solution were studied by surface tension measurements. The critical micelle concentration, surface excess concentration, minimum area per surfactant molecule, and standard Gibbs energy of adsorption were determined. The adsorption properties of these compounds depend significantly on the alkyl chain length. Alkyl chain length also affects biological properties of the new surfactants, determining the minimum inhibitory concentration and size of inhibited growth zone. The compounds have high antimicrobial activity.

Full text of DOI:10.1007/s11743-016-1906-8

J Surfact Deterg (2017) 20:151–158
DOI 10.1007/s11743-016-1906-8
ORIGINAL ARTICLE
Synthesis, Surface and Antimicrobial Activity of Piperidine-Based
Sulfobetaines
Daria Wieczorek1 Adam Dobrowolski2 Katarzyna Staszak3
•
•
•
Dobrawa Kwasniewska Patrycja Dubyk2
1
•
´
Received: 29 March 2016 / Accepted: 30 October 2016 / Published online: 15 November 2016
Ó The Author(s) 2016. This article is published with open access at Springerlink.com
Abstract A new method for the preparation of new hete-
rocyclic amine surfactants based on sulfobetaines is pro-
posed. Interfacial activities of the surfactants obtained in
aqueous solution were studied by surface tension mea-
surements. The critical micelle concentration, surface
excess concentration, minimum area per surfactant mole-
cule, and standard Gibbs energy of adsorption were
determined. The adsorption properties of these compounds
depend significantly on the alkyl chain length. Alkyl chain
length also affects biological properties of the new sur-
factants, determining the minimum inhibitory concentra-
tion and size of inhibited growth zone. The compounds
have high antimicrobial activity.
Introduction
Although amphoteric materials represent only a small
portion of total worldwide surfactant production, their
market position is increasing significantly because of their
unique properties. Their nature can make them especially
useful in applications requiring biological contact [1, 2].
The group of amphoteric surface active agents is repre-
sented by zwitterionic surfactants. Their characteristic
features are a consequence of the structure as their mole-
cules carry both negative and positive charge [1, 3]. The
molecular structure of these surfactants, in particular the
alkyl chain length, number of hydrophobic chains, nature
and number of head groups and structure of the spacer
between positively and negatively charged moieties,
strongly affects their physicochemical and biological
properties [4]. For most zwitterionic surfactants, the
cationic moiety consists of a cationic quaternary ammo-
nium group, while the anionic moiety includes a carboxylic
acid, sulfonic acid, sulfuric acid ester, or phosphoric acid
ester [5].
Keywords Sulfobetaines Á Interfacial activity Á Biological
properties
Zwitterionic molecules of the sulfobetaine type are
applied in different fields of chemistry [6]. These com-
pounds are used in production of modified polymers, e.g.,
poly(sulfobetaine methacrylate). The coexistence of posi-
tive and negative charge on the surfactant molecule gen-
erates a hydration layer as a result of strong electrostatic
interactions. Super low fouling properties of zwitterionic
materials such as poly(sulfobetaine methacrylate) arise
from this hydration layer, contributing to the reduced
protein adsorption, cell attachment, and bacterial adhesion
[7]. Polymers incorporating zwitterionic sulfobetaines have
been recognized as promising candidates for responsive
systems geared towards various potential applications such
as biosensors, catalysts, drug delivery systems, and
& Daria Wieczorek
d.wieczorek@ue.poznan.pl
1
Department of Technology and Instrumental Analysis,
Faculty of Commodity Science, Poznan University of
´
Economics and Business, al. Niepodległosci 10,
61-875 Poznan, Poland
2
3
Department of Biotechnology and Food Microbiology,
Faculty of Food Science, Wrocław University of
´
Environmental and Life Sciences, ul. Chełmonskiego 37/41,
51-630 Wroclaw, Poland
Institute of Technology and Chemical Engineering, Poznan
University of Technology, ul. Berdychowo 4, 60-965 Poznan,
Poland
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separation media [8–10]. Moreover, sulfobetaine surfac-
tants can be used as antimicrobial agents [11, 12].
Sulfobetaines differ from each other in the length of and
the presence of hydroxyl groups in the spacer separating
the quaternary ammonium center from the sulfonate group.
Besides different spacers, the amines used for the synthesis
of these surfactants can also be different [13].
CH2), 2.91 (s, 2H, CH2SO3-), 3.27 (m, 2H, CH2), 3.46 (m,
2H, CH2N?), 3.55 (m, 2H, CH2N?), 3.70 (m, 2H, CH2N?),
4.00 (m, 2H, CH2N?). 13C NMR (CDCl3) d = 14.16, 18.1,
19.6, 20.8, 21.4, 22.6, 23.7, 26.4, 29.3, 31.8, 47.4, 53.1,
57.8, 59.1. IR = 1034, 1196, 2853, 2920 cm-1. Anal.
Calcd: C, 62.25; H, 10.66; N, 4.03; S, 9.22. Found: C, 61.08;
H, 10.53; N, 3.73; S, 8.63. mp 172–173 °C, yield 62%.
The significant interest in zwitterionic surfactants
prompted us to prepare a series of sulfobetaine surfactants
using a piperidine moiety with N-alkyl substituents of
variable chain length (C10–C16) with an N-alkyl C3 or C4
spacer with a terminal sulfonate group. We aimed to
determine surface activities by study of surface tension.
Additionally, microbial activity against both Gram positive
and Gram negative bacteria and one yeast species was
examined. The effect of the chemical structure (alkyl
length chain or length of spacer between quaternary
ammonium center and sulfonate group) of these surfactants
on their properties is discussed.
N-Dodecyl-N-(propylpiperidinium-3-sulfate) (P12S3)
1H NMR (CDCl3) d = 0.88 (m, 3H, CH3), 1.26 (m, 14H,
CH2), 1.35 (s, 2H, CH2) 1.70–1.84 (m, 6H, 3CH2), 1.98 (s,
2H, CH2), 2.17 (s, 2H, CH2), 2.91 (s, 2H, CH2SO3-), 3.26
(m, 2H, CH2), 3.46 (m, 2H, CH2N?), 3.53 (m, 2H,
CH2N?), 3.69 (m, 2H, CH2N?), 4.49 (m, 2H, CH2N?). 13
C
NMR (CDCl3) d = 13.9, 18.0, 19.6, 20.7, 21.3, 22.5, 23.5,
26.4, 29.4, 31.7, 47.4, 52.9, 58.0, 59.0. IR = 1033, 1177,
2851, 2919 cm-1. Anal. Calcd: C, 64.00; H, 10.93; N,
3.73; S, 5.53. Found: C, 63.37; H, 11.48; N, 3.43; S, 7.91.
mp 174–175 °C, yield 56%.
Materials and Methods
Synthesis Procedures
N-Tetradecyl-N-(propylpiperidinium-3-sulfate) (P14S3)
1H NMR (CDCl3) d = 0.88 (3H, CH3), 1.26 (m, 18H,
CH2), 1.35 (s, 4H, 2CH2), 1.70–1.81 (m, 6H, 3CH2), 2.02
(s, 2H, CH2), 2.18 (s, 2H, CH2SO3-), 2.91 (m, 2H, CH2),
3.26 (m, 2H, CH2N?), 3.41 (m, 2H, CH2N?), 3.58 (m, 2H,
CH2N?), 3.72 (m, 2H, CH2N?). 13C NMR (CDCl3)
d = 14.0, 18.3, 19.7, 20.9, 21.5, 22.6, 23.5, 26.5, 29.3,
31.8, 47.5, 49.9, 53.1, 57.8, 59.2. IR = 1035, 1197, 2853,
2920 cm-1. Anal. Calcd: C, 65.51; H, 11.17; N, 3.47; S,
7.94. Found: C, 64.58; H, 11.67; N, 3.22; S, 7.41. mp
173–174 °C, yield 48%.
In the first step of N-alkylpiperidine synthesis, piperidine
(0.04 mol) was reacted with alkyl bromide (0.02 mol). The
reaction was carried out for several hours at room tem-
perature using diethyl ether as solvent. The resulting pre-
cipitate was filtered off, while excess solvent was
evaporated from the solution. Liquid piperidine derivatives
with 10-, 12-, 14-, and 16- carbon chains were obtained.
In the next step, to get N-alkyl-N-(propylpiperidinium-3-
sulfate) or N-alkyl-N-(butylpiperidinium-4-sulfate), the
1,3-propane or 1,4-butane sultone (0.1 mol), respectively,
was dissolved in ethyl acetate and N-alkylpiperidine
(0.1 mol) was then added. The mixture was left for several
days with protection against ambient moisture. The product
was filtered off and the crude product was recrystallized
from methanol/ethyl acetate. The reaction scheme is shown
in Fig. 1.
N-Hexadecyl-N-(propylpiperidinium-3-sulfate) (P16S3)
1H NMR (CDCl3) d = 0.88 (3H, CH3), 1.20 (m, 26H,
CH2), 1.71–1.81 (m, 6H, 3CH2), 1.99 (s, 2H, CH2), 2.17 (s,
2H, CH2SO3-), 2.89 (m, 2H, CH2), 3.27 (m, 2H, CH2N?),
3.43 (m, 2H, CH2N?), 3.57 (m, 2H, CH2N?), 3.75 (m, 2H,
CH2N?). 13C NMR (CDCl3) d = 14.0, 18.3, 19.7, 20.8,
21.4, 22.6, 26.6, 29.4, 31.8, 47.4, 53.0, 57.8, 59.1.
IR = 1035, 1165, 2853, 2920 cm-1. Anal. Calcd: C,
66.98; H, 11.16; N, 3.26; S, 7.44. Found: C, 65.42; H,
11.79; N, 2.90; S, 6.66. mp 165–166 °C, yield 35%.
Synthesis Results
All synthesized surfactants and their abbreviations are
presented in Table 1. The structures of the obtained com-
pounds were confirmed by spectroscopic methods and
elemental analysis and are presented below.
N-Decyl-N-(butylpiperidinium-4-sulfate) (P10S4)
1H NMR (CDCl3) d = 0.90 (m, 3H, CH3), 1.26 (m, 12H,
CH2), 1.61 (4H, CH2), 1.92 (m, 8H, CH2), 2.28 (m, 2H,
CH2SO3-), 2.87 (m, 2H, CH2), 3.19 (m, 2H, CH2N?), 3.30
(m, 2H, CH2N?), 3.46 (m, 2H, CH2N?), 3.65 (m, 2H,
CH2N?). 13C NMR (CDCl3) d = 13.9, 19.7, 20.2, 22.4,
N-Decyl-N-(propylpiperidinium-3-sulfate) (P10S3)
1H NMR (CDCl3) d = 0.88 (m, 3H, CH3), 1.26 (m, 12H,
CH2), 1.79 (m, 6H, 3CH2), 2.02 (s, 2H, CH2), 2.19 (s, 2H,
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153
Fig. 1 Synthesis route
23.5, 24.2, 25.7, 26.3, 27.5, 29.2, 31.6, 48.1, 50.0, 54.4,
58.8, 59.6. IR = 1034, 1184, 2855, 2926 cm-1. Anal.
Calcd: C, 63.16; H, 10.80; N, 3.88; S, 8.86. Found: C,
61.47; H, 11.13; N, 3.50; S, 7.21. mp 194–195 °C, yield
48%.
(m, 2H, CH2N?), 3.46 (m, 2H, CH2N?), 3.61 (m, 2H,
CH2N?). 13C NMR (CDCl3) d = 13.8, 19.6, 20.2, 22.4,
23.4, 24.3, 25.6, 26.2, 27.2, 29.3, 31.6, 48.0, 50.1, 53.8,
57.2, 58.7. IR = 1035, 1186, 2855, 2921 cm-1. Anal.
Calcd: C, 64.78; H, 11.05; N, 3.60; S, 8.23. Found: C, 60.56;
H, 10.78; N, 3.35; S, 7.53. mp 175–176 °C, yield 31%.
N-Dodecyl-N-(butylpiperidinium-4-sulfate) (P12S4)
N-Tetradecyl-N-(butylpiperidinium-4-sulfate) (P14S4)
1H NMR (CDCl3) d = 0.881 (m, 3H, CH3), 1.26 (m, 16H,
CH2), 1.65 (m, 4H, CH2), 1.91 (m, 8H, CH2), 2.28 (m, 2H,
CH2SO3-), 2.86 (m, 2H, CH2), 3.20 (m, 2H, CH2N?), 3.30
1H NMR (CDCl3) d = 0.88 (m, 3H, CH3), 1.26 (m, 20H,
CH2), 1.35 (m, 2H, CH2), 1.94 (m, 8H, CH2), 2.26 (m, 2H,
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Table 1 Surface properties and adsorption parameters of surfactants
Surfactant CMC
cCMC
pC20
PCMC
DG0m
ASz 9 108 BSz 9 102 C? 9 106 Corr
(–)
DGabs Amin 9
(kJ/mol) 1019 (m2)
(mmol/dm3) (mN/m) (mol/dm) (mN/m) (kJ/mol) (mol/dm3)
(mol/m2)
P10S3
P12S3
P14S3
P16S3
P10S4
P12S4
P14S4
P16S4
8.70
3.97
0.18
0.005
9.78
1.79
0.11
0.002
29.01
39.81
38.23
36.66
23.95
31.59
34.08
31.93
2.95
3.97
4.22
5.66
2.73
3.30
4.47
6.19
42.59
31.79
33.37
34.93
47.65
40.01
37.52
39.67
–21.43
–23.35
–30.91
–39.68
–21.14
–25.29
–32.12
–41.92
2511
21.35
9.44
6.44
2.75
2.59
0.99
4.31
3.34
2.30
1.28
0.976 –20.3
0.992 –27.4
0.995 –34.2
0.994 –61.4
0.992 –21.0
0.987 –25.7
0.994 –36.0
0.977 –53.2
2.58
6.03
1350
84.78
0.0012
18,710
2454
8.58
6.41
3.28
16.76
3.85
14.81
11.57
7.91
4.98
39.89
0.036
7.23
4.42
12.95
CH2SO3-), 2.90 (m, 2H, CH2), 3.19 (m, 2H, CH2N?), 3.26
(m, 2H, CH2N?), 3.43 (m, 2H, CH2N?), 3.66 (m, 2H,
CH2N?). 13C NMR (CDCl3) d = 14.1, 19.8, 20.4, 22.6,
23.5, 24.1, 25.4, 26.5, 27.8, 29.5, 31.8, 48.3, 50.1, 54.4,
56.9, 59.9. IR = 1035, 1193, 2850, 2919 cm-1. Anal.
Calcd: C, 66.19; H, 11.27; N, 3.36; S, 7.67. Found: C,
62.14; H, 10.95; N, 2.80; S, 8.39. mp 182–183 °C, yield
25%.
(ATCC 10536) and Pseudomonas aeruginosa (ATCC
15442); (3) and yeast Candida albicans (ATCC 10231).
All the microorganisms were obtained from culture col-
lection of the Department of Biotechnology and Food
Microbiology (UELS, Wrocław). The bacteria were grown
in nutrient broth medium at 37 °C, except B. subtilis
(ATCC 6633) which was grown at 30 °C. The yeast was
grown in YPD medium at 30 °C. Agar was added to the
medium at a concentration of 2% when necessary. The
investigation of all three classes of microorganism allows a
robust assessment of antimicrobial activity of these piper-
idine-based sulfobetaines.
N-Hexadecyl-N-(butylpiperidinium-4-sulfate) (P16S4)
1H NMR (CDCl3) d = 0.88 (m, 3H, CH3), 1.26 (m, 22H,
CH2), 1.61 (m, 4H, CH2), 1.93 (m, 8H, CH2), 2.28 (m, 2H,
CH2SO3-), 2.89 (m, 2H, CH2), 3.17 (m, 2H, CH2N?), 3.26
(m, 2H, CH2N?), 3.52 (m, 2H, CH2N?), 3.64 (m, 2H,
CH2N?). 13C NMR (CDCl3) d = 14.1, 19.8, 20.4, 22.6,
23.6, 24.2, 25.6, 26.5, 27.6, 29.6, 31.9, 48.3, 50.2, 54.4,
59.0, 59.6. IR = 1033, 1183, 2851, 2921 cm-1. Anal.
Calcd: C, 67.57; H, 11.26; N, 3.15; S, 7.21. Found: C,
64.21; H, 11.40; N, 2.64; S, 7.19. mp 143 °C, yield 22%.
Determination of Antimicrobial Properties by Agar
Diffusion Assay
Agar diffusion assay (well diffusion assay) was used for
testing the antimicrobial activity of the newly synthesized
surfactants. Wells were made in seeded agar and the test
samples were introduced directly into these wells. After
incubation, the distances between the edge of the well and
the end of the clear zones around the wells were mea-
sured. Briefly, on a sterile Petri dish containing appro-
priate agar medium, 200 lL of bacterial or yeast
overnight cultures washed in saline solution and adjusted
to OD600 = 1 was applied (which corresponds to
6 9 106 cells of yeast and 2 9 108 cells of bacteria).
Next, four wells per plate were made with a sterile Pas-
teur pipette (8.4 mm diameter). Subsequently, 100 lL of
aqueous solution of surfactants (5 mg/mL) was poured
into each well and incubated 6 h in 4 °C to achieve full
diffusion of the solution tested in the agar medium. Next,
prepared Petri plates were incubated at 30 °C or 37 °C for
48 h and zones of inhibited growth around each well were
measured. On each plate, the three wells contained,
respectively, a solution of surfactant, a control, and sterile
saline solution. All assays were carried out three times.
The standard deviation of all analyzed zones of the
antimicrobial activity did not exceed 3%.
Determination of Surface Activity
The surface tension of the aqueous solutions of surfactants
was measured by the Du Nou¨y ring method with a K12
¨
KRUSS tensiometer, with resolution 0.01 mN/m, at a
constant temperature of 21 °C. The deviation between
three replicate measurements was in the range of
0.05–0.22 mN/m. Measurements were made for the aque-
ous solution at the initial concentration of 50 mM; other
solutions were obtained by the serial dilution method.
Microorganism and Culture Conditions
The antimicrobial activity of the surfactants was tested on
the following strains of microorganisms: (1) Gram positive
bacteria Staphylococcus aureus (ATCC 9538), Bacillus
subtilis (ATCC 6633), and Enterococcus hirae (ATCC
10542); (2) Gram negative bacteria Escherichia coli
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155
Determination of Minimum Inhibitory
Concentration (MIC)
The microdilution method was used to determine the
minimum inhibitory concentrations (MICs) of the new
synthesized surfactants. The experiments were performed
in 100-well microplates (honeycomb) with use of a Bio-
screen C microbial growth monitoring system (Oy Growth
Curves Ab Ltd., Finland). Portions of 200 lL of an
appropriate medium (nutrient broth for bacteria and YPD
for yeast) containing different concentrations of one sur-
factant (0.01–5 mg/mL) were dispensed into the wells of a
microplate. Growth control wells did not contain any sur-
factant. All wells (except blank controls for each concen-
tration of each surfactant) were inoculated with 5 lL of
overnight bacterial or yeast washed cultures (diluted to
reach final OD600 = 0.1). The plates were incubated for
48 h at 30 or 37 °C under constant agitation. The growth of
microorganisms was monitored by measuring optical den-
sity at 420-560 nm every 20 min, and the data were col-
lected using Bioscreen C software. Assays were carried out
three times in five replicates (five wells for each concen-
tration of surfactant).
Fig. 2 Surface tension isotherms for j P10S3, d P12S3, m P14S3,
. P16S3
Results and Discussion
Surface Activity
The most important property of surfactants is the ability to
lower surface and interfacial tension. Another interesting
property of aqueous surfactant solution is the formation of
micelles above the critical concentration (CMC), which
can be determined from the experimental results of the
surface tension for a series of aqueous solutions of different
concentrations. Figures 2 and 3 presents adsorption iso-
therms of sulfobetaines studied. Their CMC values were
determined from bilateral extrapolation of the straight
sections of the isotherms, with results are presented in
Table 1.
Fig. 3 Surface tension isotherms for j P10S4, d P12S4, m P14S4,
. P16S4
the surfactants studied seems to be similar to that in past
studies of zwitterionic surfactants.
Moreover, from the results presented in Table 1 it can
be concluded that the nominal values of CMC are higher
for propane derivatives than for butane ones. This can be
explained by an increase in their hydrophobicity. The same
observations were made by Staszak et al. for sulfobetaines
with morpholinium moiety [13].
As shown in Table 1, the CMC values decrease with
increasing alkyl chain length. The relationship between the
number of carbon atoms in the alkyl chain (n) and CMC
can be expressed by the following equation:
It is hard to find a similar correlation between CMC and
the length of the spacer. For instance, Cheng et al. [16]
studied amido-amine-based cationic gemini surfactants
with propyl and hexyl spacer groups and showed that
derivatives of the compounds with longer alkyl chain in the
spacer group had higher CMC values; however, the authors
did not comment on the reasons for this phenomenon.
The reduction in surface tension depends on the
replacement of solvent molecules with the surfactant ones
logðCMCÞ ¼ AÀB Â n;
ð1Þ
where A is a constant characteristic of a particular ionic
head at a given temperature and B depends on the type of
surfactant. For cationic and anionic surfactants B is close to
0.3 [14], for nonionic [15] and zwitterionic ones it is 0.5
[5]. The values of B for N-alkyl-N-(propylpiperidinium-4-
sulfate) and N-alkyl-N-(butylpiperidinium-4-sulfate) are
equal to 0.55 and 0.61, respectively. Thus, the behavior of
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at the interface. The efficiency of a surfactant in reducing
surface tension should reflect its concentration at the
interface relative to that in the bulk liquid phase and can be
described by the parameters cCMC, PCMC, and pC20. The
value of surface tension at CMC (cCMC) is in the range of
29.0–39.8 and 25.4–34.1 mN/m for propane and butane
derivatives, respectively. Thus the ability to reduce the
value of surface tension at CMC, corresponding to the
surface activity, is higher for butane derivatives than for
the corresponding propane ones. There was no observation
of alkyl chain length dependence of this ability.
(C?), the minimum molecular area in the adsorption layer
at the saturated interface (Amin), and the Gibbs free energy
of adsorption (DGads), according to the equations [19]
BSzc0
RT
C
1
¼
;
ð3Þ
1
Amin
¼
;
ð4Þ
ð5Þ
C NA
1
Sz
DG ¼ RT lnðAminÞ;
ads
where c0, R, T, and NA stand for interfacial tension of
solvent (here water), gas constant, temperature, and Avo-
gadro’s constant, respectively. The values of correlation
coefficients (cor) for the Szyszkowski isotherms, presented
in Table 1, indicated a good fit of the experimental data to
the proposed model.
The effectiveness (PCMC) of a surfactant to reduce sur-
face tension can be measured by the surface pressure,
PCMC = c0 – cCMC, attained at the critical micelle con-
centration, since the reduction of the tension below CMC is
relatively insignificant [3]. The higher the value of PCMC is,
the more effectively the surface tension value is reduced.
This effectiveness of butane derivatives was higher.
The values of both free energies of adsorption and
micellization decreased with increasing number of carbon
atoms in the alkyl chain. However, the values for the C10
surfactants were more negative for DG0m, indicating that the
compounds obtained exhibited greater tendency to form
micelles than to adsorb at the water–air interface. With
increasing length of the alkyl group the surfactant tendency
to get adsorbed at the water–air interface is greater than
that of micelle formation.
A convenient measure of the efficiency of adsorption is
the negative logarithm of the concentration of surfactant in
the bulk phase needed to produce 20 mN/m reductions in
the surface tension of the solvent, pC20. High values indi-
cate that surfactants more efficiently adsorb at the interface
and reduce surface tension. As follows from data in
Table 2, the pC20 values increase with increasing alkyl
chain length for both homologous series. This fact indicates
that greater reduction in surface tension is achieved for
compounds with longer alkyl chain used at a smaller
concentration. The same tendency was observed for sul-
Estimated values of C? decrease and Amin increase with
increasing aliphatic chain length in the surfactant mole-
cules. Thus, the adsorption efficiency is higher for the
surfactant molecules with shorter aliphatic chain. The
results obtained indicate that the structure of adsorption
monolayer depends on the hydrophobicity of the com-
pounds studied. Molecules with short aliphatic chains are
much more densely populated at the saturated air–water
interface. These results are in contrast to those obtained for
other betaine-type surfactants [13, 17, 20]. However, in a
few papers no relationship between Amin and alkyl length
chain has been observed [21] or like here Amin increased
with increasing aliphatic chain length in the surfactant
molecules [22–24].
fopropane betaines H-(CH2)m N?(CH3)2(CH2)3SO3 with
-
m = 12, 14, 16, 18 and sulfobutane betaines H-(CH2)n
-
N?(CH3)2(CH2)4SO3 with n = 12, 14, 16, 18 [17].
The standard free energy of micellization (DG0m) was
calculated from the equation
DG0m ¼ RT ln XCMC
:
ð2Þ
Moreover, the surface tension data were fitted to
Szyszkowski’s equation [18] and the following parameters
were estimated: the surface excess at the saturated interface
Table 2 Zones of inhibited
growth and MICs of the
surfactants on the panel of
tested microorganisms
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157
Antimicrobial Properties of Surfactants
The results show that QACs are generally more effective
against Gram positive bacteria in comparison to Gram
negative ones. QACs are positively charged compounds
that are naturally attracted to negatively charged substances
such as bacterial proteins essential for the structure and
enzymatic activities of the cell. For the studied piperidine-
based sulfobetaines there are no significant differences
between results of MIC, as well as zones of inhibited
growth of microorganism, for Gram positive and Gram
negative bacteria. The similar effect on both the Gram
positive and Gram negative microorganisms illustrates the
preferred broad-spectrum activity of the compounds stud-
ied. The same effect was observed for series a of N-alkyl
betaines (C8, C12, C16, C18) [30]. The MICs of the
betaine series decreased with increasing chain length. The
best microbiological activity was obtained for compounds
with 16 carbon atoms, with MICs of 61 and 120 lM for
S. aureus and E. coli, respectively. The MIC values
obtained for the surfactants studied are very small. Ward
et al. [12] evaluated this parameter for some sulfopropyl-
betaine copolymers. The lowest observed MIC value was
1.1 mg/mL for E. coli and 1.3 mg/mL for S. aureus [12].
Additionally, the antimicrobial activity of piperidine-based
compounds was shown for 4-amidopiperidine-C12
(4AP12), the base form of 4-dodecaneamidopiperidine HCl
[31]. Similar to surfactants obtained in this study, the
4AP12 has broad-spectrum activity against both Gram
negative and Gram positive bacteria and fungi. Moreover,
the authors of the cited work compared 4AP12 with its
analogue with 16 carbon atoms in its acyl chain. The
results showed comparable activity of both compounds
against the tested microorganisms. For some bacterial and
fungal strains the values of MIC were higher for 4AP16
(A. baumannii, C. glabrata ATCC CBS138), for some
lower (S. aureus ATCC 25923). The possible antimicrobial
mechanism of piperidine sulfobetaines is connected to their
disruption of the cell membrane of microorganisms and
subsequent cell lysis. The study suggests that all newly
synthesized surfactants show antimicrobial activities, but
the highest activity was determined for surfactants P16S4
and P16S3, i.e., those with the longest alkyl chain.
Antimicrobial activities of the surfactants studied were first
evaluated by the well diffusion method. The solutions of
each surfactant added to the wells were found to inhibit the
growth of almost all microorganisms tested, except the
strain of P. aeruginosa, which was resistant to all surfac-
tants (Table 2). The weakest effect was observed for sur-
factants P10S3 and P10S4 for which no inhibition was
noted for four and three microorganisms, respectively, and
very small zones were measured for the remaining
microorganisms. The strongest effect was observed for
surfactant P12S3 against C. albicans (7.58 mm) and for
Gram positive bacteria with the largest zone for P14S4
surfactant when tested on E. hirae (7.3 mm), S. aureus
(6.88 mm), and B. subtilis (6.23 mm). Gram negative
bacteria E. coli was sensitive to all surfactants, except
P10S3 and P10S4, but with much smaller zones of inhib-
ited growth compared to those obtained for Gram positive
bacteria (max zone for P12S3 = 3.85 mm). Also, we [12]
reported a higher antibacterial activity of studied surfac-
tants against Gram positive (B. subtilis and S. aureus) than
Gram negative bacteria (E. coli and P. aeruginosa), which
corresponds to the results obtained in this work [25].
Comparison of the sizes of inhibited growth zones deter-
mined for the surfactants studied with those evaluated for
the surfactants used as disinfectants shows that the newly
obtained sulfobetaines are much more active. The effec-
tiveness of cetylpyridinium chloride (CPC) against S. au-
reus and B. subtilis measured as the size of the inhibition
growth zone was 2.0 mm, while the zone measured for
E. coli was 1.7 mm [26]. For more accurate study of
antimicrobial activity of the newly synthesized surfactants
the values of MICs for selected strains were determined by
the microdilution method. The lowest MIC was observed
for the surfactants P16S4, P16S3 (0.01 mg/mL) and P14S4,
P14S3 (0.05 mg/mL) which indicates the highest antimi-
crobial activity. The highest MIC values were observed for
P10S3 and P10S4, which indicates the weakest antimi-
crobial properties, which is in the agreement with previ-
ously described plate diffusion experiments. Increasing the
alkyl chain length improved the antimicrobial properties.
Such a phenomenon is unique and is related to the structure
of the polar head, which has been confirmed by literature
reports [27, 28]. The effect of alkyl chain length was briefly
discussed for quaternary ammonium compounds (QACs)
[29]. They are the most prevalent forms of cationic sur-
factants used today because they have a broad spectrum of
microbiological activities over wide pH ranges and are
used in industry, agriculture, hospitals, and housekeeping.
The highest antimicrobial activity of a homologous series
(C12–C16) of alkyldimethylbenzylammonium chlorides is
for compounds with 14 carbon atoms in the alkyl chain.
Conclusions
The new sulfobetaine surfactants not only reduce the sur-
face tension and form aggregates but also inhibit the
growth of microorganisms even when applied in low
concentrations. The surface properties, as well as antimi-
crobial activity of surfactants, dependent on their structure,
the length of carbon chain and spacers between positively
and negatively charged head groups. It was found that
surfactants, both propane and butane derivatives, with the
123

158
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Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://crea
tivecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
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