Food Science and Biotechnology (2023) 32:1489–1499
https://doi.org/10.1007/s10068-023-01277-2
RESEARCH ARTICLE
Preparation and characterization of vanillin‑conjugated
chitosan‑stabilized emulsions via a Schiff‑base reaction
Jianfei Zhu1,2
· Tingting Huang1 · Xiaomei Chen1 · Dongling Tian1 · Lei Wang3,4 · Ruiping Gao1,2
Received: 24 October 2022 / Revised: 12 January 2023 / Accepted: 6 February 2023 / Published online: 27 February 2023
© The Korean Society of Food Science and Technology 2023
Abstract
In the current work, vanillin-conjugated chitosan stabilized emulsions (CSVAEs) were successfully prepared and its characterization and antibacterial properties were investigated. Under stirring condition, CSVAEs were produced by a Schiff base
reaction between the vanillin aldehyde group and the chitosan active amino group. The CSVAEs were described through
Fourier transform infrared spectroscopy, X-ray diffraction, ultraviolet spectrophotometry and thermogravimetric analysis,
which demonstrated the generation of Schiff bases between vanillin and chitosan. Furthermore, the CSVAEs displayed differences at different pH values, indicating their potential as pH-responsive materials. By studying their release behavior, pH
4 was a critical point at which the properties of the CSVAEs changed. The antibacterial tests showed that the CSVAEs had
good pH-responsive antibacterial abilities against Staphylococcus aureus and Escherichia coli.
Keywords Chitosan · Vanillin · Emulsion · Antibacterial abilities
Introduction
An emulsion is a simple and effective delivery system for
embedding and includes water-in-oil (W/O), oil-in-water
(O/W), or multiple (W/O/W or O/W/O) emulsions (McClements, 2010). Emulsions have low viscosity and uniform
size and have a protective effect on volatile, oxidative and
hydrolyzed oil components, and their delivery system can
protect, carry, and release biologically active substances
(Zhang et al., 2015a, b). In the process of preparing emulsions, some chemical synthetic surfactants are used, but
there are certain safety hazards. To avoid the potential toxicity of chemically synthesized surfactants, it is necessary to
* Jianfei Zhu
zhujf@ctbu.edu.cn
1
School of Environment and Resources, Chongqing
Technology and Business University, Chongqing 400067,
China
2
Chongqing Engineering Research Center for Processing,
Storage & Transportation of Characterized Agro–Products,
Chongqing 400067, People’s Republic of China
3
Guangxi Key Laboratory of Agricultural Resources
Chemistry and Biotechnology, Yulin 537000, China
4
College of Chemistry and Food Science, Yulin Normal
University, Yulin 537000, China
explore new natural biopolymers to prepare emulsions. To
date, a series of biopolymers, such as cellulose (Cacicedo
et al., 2016), chitin (McClements, 2010), chitosan (Azmana
et al., 2021), and polyamino acids (Kordasht et al., 2021),
have been reported as synthetic polymer replacements in a
variety of applications.
Chitosan (CS) is the only alkaline polysaccharide with
cations in nature. It is a natural biopolymer with good biological activity, safety, nontoxicity, low cost, biodegradability and antibacterial properties (Azmana et al. 2021; Wang
et al. 2020). CS is rich in amino and hydroxyl groups, allowing for the introduction of active functional substances and
construction of stimuli-responsive biomaterials, so CS has
broad application prospects in the food industry (Ahmed,
2017). Studies have confirmed that in most cases, using
acetic acid, ethanol, methanol or their mixtures as solvents,
Schiff bases can be obtained with the condensation reaction
of the chitosan active amino groups with ketone/aldehyde
compounds at room temperature or reflux temperature (Hassan et al., 2018). Tamer et al. (2016) investigated aromatic
chitosan Schiff bases through coupling with benzophenone
and 4-chlorobenzaldehyde to enhance the antimicrobial performance of chitosan. The new synthetic Schiff base derivatives can be applied as antimicrobial wound dressings to
promote wound healing. Salama et al. (2015) studied chitosan Schiff bases produced through the reaction between
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3-(4-substituted phenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde and chitosan. The special performance of chitosan
Schiff bases can be improved by modifying the molecular
structure with appropriate substituents. The Schiff base antibacterial activities are stronger than those of chitosan.
Imine (C=N) bonds are formed through Schiff base condensation or the condensation of carbonyl compounds and
amino compounds (Sharma et al., 2020). C=N bonds are
generally resistant to neutral and alkaline conditions but are
susceptible to hydrolysis in acidic media (Yu et al., 2021).
C=N bonds can endow dynamic reversible polymer networks with excellent stimulus-responsive and self-healing
properties, and their reactions are reversible and responsive
to pH and have been extensively investigated in biomedical fields (Zhang et al., 2021). Because of this behavior,
chitosan-based Schiff bases (CSBs) are employed as superior pH sensors. Hsu et al. (2020) found that pH-responsive
indocyanine green (ICG)-carrying chitosan-based micelles
display significant potential in cancer therapy. Xu et al.
(2018) investigated chitosan self-healing hydrogels based
on dynamic imine bonding, which has been widely studied
because of its advantages, such as simple fabrication, excellent biocompatibility and automatic repair under physiological conditions.
Vanillin (VA), a phenolic aldehyde, is one of the most
preferred flavoring materials worldwide and has a wide
range of applications in the beverage, food, pharmaceutical
and perfume industries (Martău et al., 2021). It has -CHO
and phenol-OH functional groups, which have strong reactivity, can be oxidized and cross-linked by themselves,
Fig. 1 Schiff base reaction between VA and CS
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J. Zhu et al.
and can also combine with food macromolecules. Moreover, vanillin has antibacterial and antioxidant activities
against yeast, mold and bacteria and can also be employed
as a food preservative (Bezerra et al., 2017). The water
alcohol extract of vanillin possesses antioxidant properties, so it can be utilized as a food preservative. The vanillin aldehyde group has been proven to play a critical
role in antifungal activity (Romero-Cortes et al., 2019).
The position of the side groups on the benzene ring may
be a significant structural characteristic leading to such
effects (Sinha et al., 2008). There is a reaction between the
vanillin aldehyde group and the amino group of chitosan,
which produces the Schiff base of chitosan. In contrast to
bare vanillin or chitosan, the Schiff base of chitosan has
superior antibacterial performance (Stroescu et al., 2015).
Figure 1 shows the condensation reaction between vanillin
and chitosan active amino groups that yields Schiff bases.
In this work, the possibility of the generation of a
Schiff base from VA and CS at low pH was investigated.
CS solution and VA were chosen as the continuous and
dispersed phases, respectively, to prepare CSVAEs at different pH values. The CSVAEs were characterized with
FT-IR, XRD, UV‒Vis, and TGA. The emulsion stability
(ESI), centrifugation stability (Ke), particle size, rheological properties, and antibacterial activity of the CSVAEs at
different pH values were analyzed, with the aim of providing a theoretical foundation for further research on VA
and CS in emulsions at different pH values. The results
indicate that the CSVAEs obtained in our study may be
suitable for use as antibacterial agents for food applications with pH-responsive release characteristics.
Preparation and characterization of vanillin‑conjugated chitosan‑stabilized emulsions…
Materials and methods
Materials
CS (with a deacetylation degree and molecular weight of
approximately 95% and 300 kDa, respectively) was provided by Zhejiang Golden-Shell Pharmaceutical Co., Ltd.
(Zhejiang, China). Medium-chain triglycerides (MCTs) was
obtained from Shanghai Yuanye Bio-Technology Co., Ltd.
(Shanghai, China). VA was obtained from Shanghai Macklin
Biochemical Co., Ltd. (Shanghai, China). Other reagents
were provided by Sinopharm Chemical Reagent Co., Ltd.
(Shanghai, China). Unless otherwise specified, all of the
reagents were of analytical grade.
Preparation of CSVAEs
CSVAEs were generated in accordance with the application
of Chen et al. (2017) with slight changes. The aqueous phase
was produced by dissolving 1055 mg of CS in 100 mL of
acetic acid aqueous solution (1%, v/v) with gentle stirring
at a speed of 300 r/min and 25 °C for 12 h and then removing air bubbles by centrifugation at 2000 r/min for 20 min.
The oil phase was obtained by dissolving VA (200 mg) in
MCTs (5 mL) under gentle stirring at 300 r/min and 25 °C
for 1 h. Subsequently, 95 mL of the water phase was added
to 5 mL of the oil phase with a burette over half an hour to
form a O/W emulsion, which was stirred at 900 r/min and
25 °C for five hours. Afterward, the ultimate CSVAE was
acquired through shearing at 20,000 rpm and 25 °C for ten
minutes. Finally, the CSVAEs were collected by adjusting
the pH to 4.0 or were processed into powder in a vacuum
freeze-drying instrument for 48 h. The CSVAE-dried samples, called CS-VA, were obtained and kept in a desiccator
until further analysis.
X‑ray diffraction
XRD analysis was implemented in accordance with El
Knidri et al. (2016) by utilizing an XRD-6100 diffractometer (Shimadzu, Japan). The samples were then analyzed in
the 2θ range between 0° and 40° with a 5°/min step size, and
Cu-Kα radiation at 20 mA and 40 kV was used.
Fourier transform infrared spectroscopy
An FTIR spectrometer (IRPrestige-21, Shimadzu, Japan)
was applied to analyze the FT-IR spectra based on the protocol of Agarwal et al. (2018). Potassium bromide powder
and the CS-VA complex (3 mg) were mixed at a ratio of
100:1 (w/w) and compressed with a tablet press. Next, a
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TGS detector was utilized to scan 32 times at frequencies
between 4000 and 500 ­cm−1. Potassium bromide was used
as the background, and it was subtracted before each scan.
All the readings were taken at RT (25 ± 1 °C).
Thermal gravimetry analysis
By applying a thermogravimetric analyzer (TGA-60, Shimadzu, Japan), the TGA curves of films were examined
based on the procedure presented by Moussout et al. (2016).
Ten milligrams of sample was studied by derivative thermogravimetry (DTG) and thermogravimetry (TG) by heating
from 30 to 600 °C at a heating rate of 10 ˚C/min.
Determination of CS‑VA by ultraviolet
spectrophotometry
The dried CS, VA, and CSVAE samples were dissolved in
acetic acid-sodium acetate buffer solution at pH 1 (0.1 mg/
mL). The absorption curves of the CS, VA and CSVAE solutions from 200 to 800 nm were obtained through a UV‒visible spectrophotometer (UV1100 II, Tianmei, China) using
the blank solvent as a reference.
The dried samples of CSVAEs were solubilized in acetate-sodium acetate buffer solution (0.5%, v/v) at pH values of 1–7. After one day, the UV absorption curves of the
CSVAEs from 200 to 800 nm were detected using the blank
solvent as a reference.
Determination of emulsifying properties
ESI and ­Ke are important indicators of the functional properties of emulsions, which can reflect the ability of the complexes to form emulsions at the oil–water interface. Emulsion stability refers to the ability of the formed emulsion to
remain dispersed and without oil flocculation or flocculation
for a certain period of time. The ESI and K
­ e of CSVAEs were
identified based on the approach of Zou et al. (2020) with
a slight variation. CSVAEs (100 μL) were diluted 50 times
with SDS (0.10%) solution. Utilizing a spectrophotometer
(UV1100 II, Shanghai Tianmei, China), the absorbance of
the diluted solution was detected at 500 nm. The absorbance
calculated immediately (­ A0) and 30 min (­ A30) after preparation of the emulsion was applied to determine the ESI. Fifty
microliters of CSVAEs was diluted 100 times using 0.10%
SDS, and the absorbance of the emulsion was determined
at 500 nm as A
­ 1, followed by centrifugation at 4000 r/min
for ten minutes in a 10-mL centrifuge tube with a diameter of 1 cm. The absorbance of the supernatant was measured at 500 nm as ­A2.
The ESI and K
­ e were calculated according to the following equations:
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J. Zhu et al.
ESI(min) =
Ke (%) =
A0
× 30
A30 − A0
A1 − A2
× 100
A1
(1)
(2)
where ­A0 is the absorbance measured immediately, ­A30 is
the absorbance at 30 min, ­A1 is the absorbance before centrifugation, and ­A2 is the absorbance after centrifugation.
Particle size measurement for CSVAEs
The size distribution (D50) of volume-average droplets of
freshly produced emulsions at pH 1 to 7 was calculated by
the approach of Xiong et al. (2018) with a few changes using
a BT-2001 Baite laser particle size analyzer (Dandong Baite
Instrument Co., Ltd., Liaoning, China). All emulsions were
diluted 100 times with distilled water prior to testing. All
determinations were performed at least three times.
Rheological measurement for CSVAEs
The viscosity of the CSVAEs at pH 3–7 was determined
using a Thermo HAAKE rotational rheometer (HAAKE
MARS 60, Shanghai, China) at 25 °C using a plate with an
angle of 1° and 60.00 mm radius. When the shear rate was
constant (100 ­s−1) for 180 s, 100 data points were recorded
for each logarithmic cycle. When the shear rate was dynamic
(0.001 to 1 000 ­s−1) for 180 s, 200 data points were recorded
for each logarithmic cycle. The viscosity of rotational shear
was reported as a function of the shear rate in logarithmic
coordinates. The rheology experiments were conducted at
least three times.
Antimicrobial effect of CSVAEs
The antibacterial effect of the CSVAEs at pH 3–7 was
determined in accordance with the approach of Nguyen
et al. (2019) with a few changes, and S. aureus (IID 980)
and E. coli (IID 5208) were utilized in the current work.
S. aureus and E. coli were incubated on nutrient agar and
cultivated, and suspensions at approximately ­106 to ­107 cfu/
mL were prepared. The suspension (100 μL) was aspirated
into a nutrient agar dish and spread evenly with a smear
stick. Three holes were punched in the medium using sterile pipette tips (100–1000 µL), and the medium in the agar
wells was then removed with a sterile needle. Each well was
spaced more than 25 mm apart in the center and more than
15 mm from the periphery of the Petri dish. The CSVAEs
(50 µL) were aspirated into each agar well (three wells to
a set), and the Petri dish was covered. Petri dishes without
inhibitor and inhibitor solvent were used as positive controls,
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and dishes without bacteria were applied as the negative
controls. The dishes were inoculated at 37 °C in a biochemical incubator. The results were observed and recorded after
16 to 18 h.
Statistical analysis
The significance of the acquired findings was determined
through one-way analysis of variance (ANOVA) with 95%
confidence intervals. SPSS software version 24.0 was
employed for statistical analysis. Figures were made by
Origin 8.6.
Results and discussion
XRD analysis
Figure 2a displays the XRD patterns of CS-VA and CS. Both
CS and CS-VA had peak values at 20–22° (2θ). CS had two
sharp diffraction peaks near 11° and 22° (2θ), attributed to
the type I and type II crystals of CS, which can be used to
characterize the degree of crystallization of CS (Anirudhan
et al., 2016). After modification by the Schiff base reaction,
the small diffraction peak at 19° (2θ) disappeared, indicating
that the molecular chain stacking structure originally formed
by hydrogen bonding in CS was destroyed. Compared to that
of bare CS, the intensity of the CS-VA diffraction peak from
10 to 15° (2θ) became broader and flatter, which implies
excellent compatibility between vanillin and chitosan. After
modification by the Schiff base reaction, the crystallization
peak of CS at 12° (2θ) was weakened, indicating that the
microcrystalline structure of chitosan was destroyed and that
the crystallinity of the polymer was reduced (Zhang et al.,
2015a, b). These results confirm the chemical reaction of
vanillin with chitosan, which destroyed the original hydrogen bonding force, changed the crystallization properties,
and resulted in the broadening and weakening of the peak.
FT‑IR analysis
Figure 2b shows the FT-IR patterns of CS, VA, CS-VA,
and the mixture of CS and VA (CS + VA). The FT-IR spectrum of the pure vanillin powder exhibited characteristic
peaks at 2868 and 3290 ­cm−1, which are associated with
the methyl group of VA and –OH, respectively (Kamaraj
et al., 2017). The VA characteristic peak at 1660 ­cm−1 was
caused by the C=O stretching vibration of the aldehyde
group (García-Castañeda et al., 2021). Furthermore, the
peak at 1080 ­cm−1 reveals the existence of ether groups
in pure VA (Gnanasekar et al., 2020). The bare chitosan
powder spectrum shows bands at 3200–3600 ­cm−1, which
are attributed to N–H and O–H stretching (Ma et al.,
Preparation and characterization of vanillin‑conjugated chitosan‑stabilized emulsions…
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Fig. 2 Characterization of the samples. a XRD patterns of CS and CS-VA, b FTIR spectra of VA, CS, CS+VA, and CS–VA, c TG curves of CS
and CS–VA, and d DTG curves of CS and CS–VA
2017), and the peaks at 1598 and 1074 ­cm−1 are attributed to primary amide and glycosidic bond stretching. In
the FT-IR spectrum of CS + VA, the characteristic peaks
of CS and VA alone still exist, indicating that no new
chemical groups formed. The spectrum of CS-VA displays a peak attributed to C = N stretching vibrations at
1670 ­cm−1, confirming the formation of an imine group
(Li et al., 2018). The Schiff base reaction consumed the
amino group, so the stretching vibration peak at 1598 ­cm−1
disappeared, which is proof of the modification of chitosan
(Huang et al., 2021). The monosubstituted characteristic
peak of the aromatic ring in VA appeared at 736 ­c m −1,
indicating that the derivative contained the benzene ring
structure of VA (Kamaraj et al., 2017).
TGA analysis
The DTG and TGA curves of the CS-VA and CS powders are shown in Fig. 2c and d. The differences between
CS-VA and CS can be seen from these curves. For CS,
weight loss occurs in three steps. The first CS mass loss
stage, from approximately 30 to 160 °C, is primarily
attributed to water loss, with a mass loss of approximately
10.0%. The second stage, from approximately 200–300 °C,
corresponds to chitosan decomposition (heat and oxidation), and the mass loss was approximately 80.0%. The
third stage occurs above 350 °C, caused by disruption of
the pyranose ring structure and decomposition of residual
carbon (Cestari et al., 2005).
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J. Zhu et al.
The trends of the thermogravimetric curves of CS and
CS-VA were roughly the same, indicating that the thermal
stability of CS-VA modified by a Schiff base was not significantly different from that of CS. The mass loss in the first
stage was approximately 10% due to evaporation of surface
water (Neto et al., 2005). The mass loss in the second stage
was reduced compared to that of CS, probably due to the
introduction of benzene rings into the CS segment by VA,
which increases intramolecular and intermolecular hydrogen bonds. The mass loss of CS-VA increased in the third
stage, by approximately 20.0%, because the decomposition
of the aromatic structure of VA needed to be carried out at a
higher temperature, thus increasing the mass loss of CS-VA
at high temperature. This also appears to reflect the existence of C=N bonds (Schiff base), which are thermostable
and require high temperatures to decompose. The thermogravimetric curves show that the CS thermal stability was
evidently enhanced, indicating that a new phase formed after
the reaction between CS and VA (Cestari et al., 2005).
acidic medium, the absorption peaks of CS-VA were similar
to those of VA, and the wavelength of the maximum absorption peak did not shift significantly. The UV‒Vis spectra
indicate that in acidic medium, the absorption of CS-VA was
the cause of the release of VA.
The UV‒Vis spectra of CS-VA in the range of pH 1–7 are
shown in Fig. 3b. To explore the pH-responsive behavior of
CS-VA, it was placed in different pH environments, and the
change in absorbance was examined at 330 nm after 24 h.
Under different pH conditions, the maximum absorption
wavelength of CS-VA was located at approximately 330 nm,
which showed no obvious shift. As the pH decreased, the
absorption intensity of CS-VA at 300–360 nm gradually
increased, which indicates that CS-VA could release VA at
different degrees in response to pH changes. When the pH
was further reduced (pH < 4), the difference between the
UV absorbances decreased, which may be due to the acidsensitive C=N bonds in CS-VA being hydrolyzed to a large
extent under strong acidic conditions (Zhai et al., 2018).
Analysis of the UV‒Vis spectra
Analysis of emulsifying properties
The UV‒Vis spectra of CS, VA, and CS-VA are shown in
Fig. 3a. There are no chromophores in the chemical structure of CS, so there was no obvious absorption peak in the
ultraviolet spectrum. Because the chemical structure of
VA contains a benzene ring and a carbonyl group, it has a
strong absorption peak in the UV‒Vis spectrum, and vanillin absorbs ultraviolet light in the 278–308 nm wavelength
range (Krasaekoopt & Jongyin, 2017). Due to the effect of
solvent polarity, the maximum absorption wavelength of
the solution redshifted (Bilokin et al., 2009). The maximum
absorption wavelength of VA was approximately 330 nm. In
The ESI and ­Ke curves of CSVAEs at pH 1–7 are shown
in Fig. 4a. The emulsifying stability of the CSVAEs at pH
1–7 was studied by ESI. From the results, the ESI increased
from 238.46 to 500.62 min and gradually increased with
increasing pH. As CS and CS-VA were both dissolved in
the solution at pH < 4 (Ono et al., 2000), the ESI values
of CSVAEs at pH 1–3 seem to be the same. The ESI was
strongly dependent on the interaction in CS molecules (Li
& Xia, 2011), which influences the CSVAEs particle size
distribution. When pH < 4, CS-VA decomposed into uniform
and small molecules, and the coagulation phenomenon did
Fig. 3 UV‒Vis spectra of the samples. a UV‒Vis spectra of CS, VA and CSVAEs. b UV‒Vis spectra of CSVAEs at pH 1–7
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Preparation and characterization of vanillin‑conjugated chitosan‑stabilized emulsions…
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Fig. 4 Emulsifying properties of the samples. a ESI and ­Ke curves of CSVAEs at pH 1–7 and b particle size distributions of CSVAEs at pH 1–7
not easily occur; therefore, the stability of the emulsion was
improved. At pH values from 4 to 7, the ESI decreased, perhaps because CS and VA continuously underwent dynamic
reversible cross-linking reactions in neutral solutions,
decreasing the stability of the emulsion.
During the centrifugation process, the centrifugal force
aggravated the stratification of the emulsion, and the stability of the emulsion could be judged more quickly. Centrifugal stability was expressed by the centrifugal stability coefficient (Ke). The lower the centrifugal stability factor is, the
better the emulsion stability (Zhang et al., 2020). From the
results, with increasing pH, Ke showed an increasing trend,
from 15.28 to 62.10%. The reason may be that at lower pH,
the network structure of CS chains reduced the diffusion of
droplets in the emulsion, stabilized contact between droplets,
and improved the centrifugal stability of the emulsion (Li
& Xia, 2011). At higher pH values, the network structure of
CS was destroyed, since dynamic cross-linking of VA and
CS also decreased the solubility of CS.
Analysis of CSVAE particle size
The distribution of particle size and the mean particle size
reflect the stability of emulsion systems. From Fig. 4b, the
mean particle size of the emulsion progressively increased
as the pH increased, but the change was not significant at
pH 1–3. The smallest average particle size was 3.04 μm,
and the largest was 76.42 μm. The reason may be that when
pH < 4, the CSVAEs were fully acidly hydrolyzed (Zhai
et al., 2018); thus, the difference in particle size was not significant (P > 0.05). The particle size of the emulsion changed
abruptly when the pH was changed from 6 to 7. The transition from acid to neutral conditions for the CSVAEs affected
not only affected the Schiff base reaction between CS and
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VA, but also the acidolysis degree of CS. The solubility of
CS decreases in a neutral medium, and CS combines into
larger particles (Hu et al., 2005); thus, the formed emulsion
has a larger particle size. The particle size also reflected the
acidolysis degree of CS. The particle size was associated
with the emulsion stability. The smaller the emulsion particle size was, the more uniform the distribution and the more
stable the emulsion (Yang et al., 2019).
The particle size distribution of the CSVAEs is shown
in Fig. 4c. The emulsions were all monomodal and monodisperse systems, indicating that the oil droplets were uniform in size and that the dispersed phase distribution in the
emulsion was concentrated. At pH 1–3, CS and CS-VA were
decomposed into uniform and small molecules. Their curves
all show a single peak for a monodisperse system, indicating that the oil droplets were uniform in size and that the
dispersed phase distribution in the emulsion was concentrated. At pH 4–7, the particle size distribution range of the
CSVAEs was obviously widened, and the peak value was
reduced, indicating that the emulsion stability was poor. The
CSVAEs exhibited a bimodal distribution, which is indicative of a polydisperse system. The reasons may be associated
with the reduction in the extent of the Schiff base reaction
and CS solubility. In accordance with the particle size distribution and the mean particle size, pH 4 was the critical point
of CSVAE stability change.
Rheological analysis of CSVAEs
The above results showed that the properties of the
CSVAEs at pH 1–3 were similar, so the pH range of 3–7
was tested in the following research. The most direct
method used to test the viscosity of samples is to fix
the shear rate and test the viscosity of CSVAEs within a
J. Zhu et al.
certain period of time. The steady-state shear viscosities
of the CSVAEs at pH 3 to 7 are shown in Fig. 5a. From
the results, the deviation of the sample data points within
180 s was very small, and the average viscosity within
180 s was taken. The lowest viscosity of the five CS-VA
emulsions was 3.92 mPas, and the highest was 94.76
mPas. The viscosities of the CS-VA emulsions increased
gradually with increasing pH, and at pH 3, 4, and 5, they
were not significant. When the pH rose to 6, the viscosity
increased significantly, and from pH 6 to 7, the viscosity
of the CS-VA emulsion changed abruptly, increasing from
12.92 to 94.76 mPa s. The reason may be that CS decomposed in the acidic solvent (Zhai et al., 2018); when the pH
increased, chitosan aggregated and cross-linked into large
particles again, thereby increasing the viscosity.
The rotational shear viscosities of the CSVAEs in the
shear rate range 1­ 0–3 ≤ γ̇̇ ≤ ­103 s at pH 3 to 7 are shown in
Fig. 5b. The viscosities of the CSVAEs at pH 3 to 7 show
the significant shear thinning features and high viscosity of
pseudoplastic fluid. The CSVAEs displayed similar behavior
at all shear times; the viscosity of the CSVAEs was shear
rate-dependent at low shear rates ­(10–3 ≤ γ ̇ ≤ ­10–1 s), but
at higher and moderate shear rates ­(10–1 ≤ γ̇̇ ≤ ­103 s), it
remained almost constant (El-Hefian et al., 2010). When the
impact of pH on the CSVAEs was investigated, at low shear
rates ­(10–3 ≤ γ̇̇ ≤ ­10–1 s), the shear thinning behavior was
remarkable. At low shear rates, the viscosity of the CSVAEs
increased gradually with increasing pH, but at higher shear
rates, it remained the same value.
The results show that changes in the rheological properties of CSVAEs at different pH values were mainly related to
the physicochemical properties of chitosan rather than those
of vanillin. The reason may be that the cross-links of CS-VA
may be destroyed in the shearing process, and the relative
Fig. 5 Rheological properties of the samples. a Constant viscosity of CSVAEs at pH 3–7 and b dynamic viscosity of CSVAEs at pH 3–7
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Preparation and characterization of vanillin‑conjugated chitosan‑stabilized emulsions…
molecular mass of CS was much larger than that of VA. This
mainly reflects the rheological properties of CS.
Analysis of the antibacterial activity of CSVAEs
At pH 3–7, the activities of the CSVAEs and CS against
the chosen microorganisms, S. aureus (gram-positive), and
E. coli (gram-negative) bacteria, are reflected in Fig. 6a–c,
respectively. The results show that both CS and the CSVAEs
exhibited inherent antimicrobial properties, and their antibacterial activity decreased with increasing pH. CS and
CSVAEs were more effective against E. coli than against S.
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aureus. Moreira et al. (2011) reported that CS antibacterial
activity may be attributed to the polycationic character of
molecules, which allows interaction with polymers generated at the surface of bacterial cells and the formation of
polyelectrolyte complexes. At the same pH, the antibacterial ability of the CSVAEs towards S. aureus and E. coli
was superior to that of the CS emulsions, especially in neutral solutions. At pH 7, the inhibition zone diameters of the
CSVAEs towards S. aureus and E. coli were 2.0 mm and
4.0 mm, respectively, but the inhibition zone diameters of
the CS towards both S. aureus and E. coli was 0 mm. Many
studies have reported that the degree of polymerization of
Fig. 6 Antibacterial properties of the samples. a Inhibition zones of CS and CS-VA, b Inhibition zone diameter of CSVAEs and CS for E. coli at
pH values from 3 to 7 and c inhibition zone diameter of CSVAEs and CS for S. aureus at pH values from 3 to 7
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CS is reduced and that its antibacterial activity is enhanced.
CS hardly dissolved in neutral solution, indicating that it
had almost no antibacterial ability in neutral solution, but
the CSVAEs still retained antibacterial activity. Xu et al.
(2011) reported that CS-based Schiff bases display superior
antimicrobial activity compared to bare CS. A report by Xu
et al. (2011) suggested that the phenyl substitution of CSBs
can have a considerable impact on the antimicrobial efficiency of CSBs if chitosan Schiff bases are produced from
aryl aldehydes. CSVAEs are normally produced by rapid
condensation of the carbonyl group of the VA of aldehydes
with the amino group of CS through eliminating water molecules. They retain their antibacterial ability in acidic or
neutral solutions. When the pH is gradually decreased, its
antibacterial ability against S. aureus and E. coli increases.
In conclusion, CS-VA had good pH-responsive controlled release properties in different pH buffer solution
systems. The UV release curves show that the prepared
derivatives had good acid-responsive properties, showing
obvious release differences under strong acid (pH 1), moderately strong acid (pH 4–5) and neutral (pH 7) conditions.
The emulsion stability of CSVAEs was explored at different pH values, emulsion centrifugation stabilities, particle
sizes, particle size distributions and rheological properties
and found that pH 4 is a critical point for the change in the
properties of CSVAEs. Moreover, the CSVAEs had evident
suppressive effects on S. aureus and E. coli and antibacterial
effects under slightly acidic conditions at pH 6 (the inhibition zone diameters for S. aureus and E. coli were 5.8 mm
and 8.5 mm, respectively). The suppressive effects under
slightly acidic conditions are stronger than those under the
neutral condition at pH 7.0 (the inhibition zone diameters for
S. aureus and E. coli were 2 mm and 4 mm, respectively).
Compared with bare CS, CSVAEs have better antibacterial
properties. Therefore, acid-sensitive imine bonds can act as
pH-responsive "switches" for antimicrobial release.
Acknowledgements The project was supported by the Open Fund of
Guangxi Key Laboratory of Agricultural Resources Chemistry and
Biotechnology (Grant No. 2022KF06).
Declarations
Conflict of interest The authors declare no conflict of interest.
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