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Journal of Functional Foods 38 (2017) 205–213
Contents lists available at ScienceDirect
Journal of Functional Foods
journal homepage: www.elsevier.com/locate/jff
Fabrication of zinc (II) functionalized L-phenylalanine in situ grafted
starch and its antibacterial activity and cytotoxicity
Chongyang Jin a, Hualin Wang a,c,⇑, Minmin Chen a, Suwei Jiang a, Qiusheng Song a, Min Pang b,c,
Shaotong Jiang b,c
a
b
c
School of Chemistry and Chemical Engineering, Hefei University of Technology, 230009 Hefei, Anhui, PR China
School of Food Science and Engineering, Hefei University of Technology, 230009 Hefei, Anhui, PR China
Anhui Institute of Agro-Products Intensive Processing Technology, 230009 Hefei, Anhui, PR China
a r t i c l e
i n f o
Article history:
Received 22 April 2017
Received in revised form 11 September
2017
Accepted 11 September 2017
Keywords:
Zinc
Amino acid
Starch
In situ grafting
Antibacterial activity
Cytotoxicity
a b s t r a c t
A facile strategy was developed to fabricate zinc (II) functionalized L-phenylalanine in situ grafted starch
(Zn(II)@L-Phe-g-St). The structure of Zn(II)@L-Phe-g-St was proposed and well characterized, and the
assays of antibacterial activity against typical bacteria (Bacillus subtilis and Escherichia coli) and cytotoxicity of Hela cell were focused on. Boc-L-phenylalanine was used to achieve L-Phe-g-St by in situ grafting
onto starch through esterification reaction after removal of Boc. It was confirmed that around 71.02% of
ANH2 of L-Phe complexed with Zn(II) by r-coordinate bonds and the coordination number of Zn(II)
approximated to 2. Zn(II)@L-Phe-g-St exhibited a good antibacterial activity against both Bacillus subtilis
and Escherichia coli with increasing its concentration, and a relatively more effective antibacterial activity
against Bacillus subtilis than Escherichia coli. Furthermore, Zn(II)@L-Phe-g-St was found to be very low toxicity towards Hela cells especially at the concentration below 40 lg ml1.
Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction
Zinc oxide nanoparticles (ZnO NPs) are well known for their
broad antibacterial properties (Akbar & Anal, 2014) in the inhibition of bacterial growth and propagation (Sirelkhatim et al.,
2015). Many research works have been reported on the investigation of the antibacterial activity of ZnO NPs (Arakha, Saleem,
Mallick, & Jha, 2015; Raghupathi, Koodali, & Manna, 2011) or their
composites such as alginate/vinyl alcohol/ZnO (Shalumon et al.,
2011), ZnO/chitosan (Petkova et al., 2014), PVP/ZnO (Selvam &
Sundrarajan, 2012) and ZnO/GO composites (Wang et al., 2014).
However, the antibacterial mechanisms of ZnO NPs have aroused
much controversy, where the two most widely accepted mechanisms are reactive oxygen species (ROS) generation (Li, Zhang,
Niu, & Chen, 2012) and membranes disturbance or disorder from
ZnO NPs precipitation on the bacterial exterior or gathering in
the cytoplasmic area or periplasm space (Zhang, Jiang, Ding,
⇑ Corresponding author at: School of Chemistry and Chemical Engineering, Hefei
University of Technology, 230009 Hefei, Anhui, PR China.
E-mail addresses: [email protected] (C. Jin), [email protected]
(H. Wang), [email protected] (M. Chen), [email protected] (S. Jiang),
[email protected] (Q. Song), [email protected] (M. Pang), [email protected]
com (S. Jiang).
http://dx.doi.org/10.1016/j.jff.2017.09.015
1756-4646/Ó 2017 Elsevier Ltd. All rights reserved.
Povey, & York, 2007). Meanwhile, the precipitation and gathering
of ZnO NPs will also damage normal human cells and exhibits cytotoxicity, especially at a relatively high NPs contents (De Berardis
et al., 2010; Lin et al., 2009).
To avoid the cytotoxicity from the precipitation and gathering
of ZnO NPs, zinc (II) complexes have shown a great potential. Some
representative researches are focused on chitosan–Zn(II) (Wang,
Du, & Liu, 2004), catechins–Zn(II) (Zhang, Jung, & Zhao, 2016)
and Schiff base ligands–Zn(II) complexes (Chohan, Arif, & Sarfraz,
2007; Montazerozohori, Yadegari, Naghiha, & Veyseh, 2014). As
indicated from these achievements, the choice of appropriate
ligands plays an important role in antibacterial activity and cytotoxicity of the targeting Zn(II) complexes. Amino acids or their
derivatives can be served as good ligands for Zn(II) complexes
due to the characteristic in structure and nutrition. Several typical
amino acid Schiff bases have been used as ligands for the fabrication of Zn(II) complexes with good antibacterial activity (Boghaei
& Gharagozlou, 2007; Chohan et al., 2007). We also noticed the
investigations of Zn(II) complexes of proline-glycine and glycine
reported by these research groups (Arjmand, Parveen, &
Mohapatra, 2012; Hoppilliard, Rogalewicz, & Ohanessian, 2001;
Wang, Tang, Ma, & Feng, 2010). However, no attention was focused
on the antibacterial activity of amino acid–Zn(II) complexes.
206
C. Jin et al. / Journal of Functional Foods 38 (2017) 205–213
Starch, one of the most common ingredients in food products, is
an inexpensive, versatile and available natural polymer composed
entirely of glucose units linked together by glycosidic bonds (Miao
et al., 2013). As the hydroxyl groups on the starch molecules can
provide active sites for esterification, some functionalized starch
esters can be easily obtained by the esterification with organic or
inorganic acids, such as fatty acid starch esters (Winkler,
Vorwerg, & Wetzel, 2013), starch phosphate (Passauer, Bender, &
Fischer, 2010) and starch sulfate (Cui, Liu, Wu, & Bi, 2009). It is well
known that amino acids have been widely used in food products as
nutrient supplements, which play a crucial role in human nutrition
and health maintenance (Cui, Fang, Zhou, & Yang, 2014). Our interesting is the characteristic of amino acid molecular structure, in
which the ANH2 groups can be served as ligands in metal ion complexes, meanwhile, the ACOOH groups can work as precursors in
esterification. In order to remain the integrity of the ANH2 group
during esterification, the Boc-amino acid was employed as starting
material in the present work.
Taking advantages of starch, amino acid and zinc, we proposed
a facile strategy to fabricate zinc (II) functionalized L-phenylalanine
in situ grafted starch (Zn(II)@L-Phe-g-St). Representative bacteria
Bacillus subtilis (B. subtilis, Gram-positive) and Escherichia coli
(E. coli, Gram-negative) were used as test bacteria to evaluate
antimicrobial activity, and HeLa cells for cytotoxicity investigation.
The structure of Zn(II)@L-Phe-g-St was proposed and well characterized, and more attentions were focused on the assays of antibacterial activity and cytotoxicity of Zn(II)@L-Phe-g-St. Our data
suggest that Zn(II)@L-Phe-g-St has strong ability to kill bacteria at
low concentrations and does not affect the viability of HeLa cells.
2. Materials and methods
2.1. Materials
Corn starch (moisture 8.42%, total amylose 25.60%, all on dry
basis), Boc-L-phenylalanine (Boc-L-Phe, 98.5%; Boc, tertbutoxycarbonyl), zinc oxide (ZnO, 99%), dimethyl sulfoxide (DMSO,
99.8%), trifluoroacetic acid (TFA, 99.5%) and yeast extract powder
were purchased from Aladdin Chemical Reagent Co., Ltd. (Shanghai, China). Sodium chloride, sodium hydroxide, hydrochloric acid,
ethyl acetate and ethanol were obtained from Sinopharm Chemical
Reagent Co., Ltd. (Shanghai, China). Peptone and beef extract were
provided by Aoboxing Product (Shanghai, China). Hank’s balance
salt solution (HBSS) was purchased from Beijing Solarbio Science
& Technology Co. Ltd. (Beijing, China). Dulbecco’s modified Eagle’s
medium (DMEM)/ high glucose, 0.25% trypsin 1X and fetal bovine
serum (FBS) were provided by HyClone Laboratories Inc. (Utah,
USA). All the chemicals and reagents were of analytical grade and
all solutions were prepared with Milli-Q water from a purification
system (Millipore, Bedford, MA, USA).
Bacillus subtilis (B. subtilis, ATCC 6633) was preserved in BeNa
Culture Collection (Beijing, China). Escherichia coli (E. coli, 8099)
was provided by the China Center of Industrial Culture Collection
(Beijing, China). HeLa cells were obtained as a gift from the
Research Institute of Toxicology at School of Food Science and
Engineering in Hefei University of Technology (Hefei, Anhui).
2.2. Sample preparation
2.2.1. L-phenylalanine in situ grafted starch preparation
A weight of 15.0 g corn starch was added to 35.0 ml of Milli-Q
water and vigorously stirred at 70 °C for 10 min before adjusting
the pH value of suspension to 4.0 with 0.5 M hydrochloric acid.
Subsequently, the esterification of Boc-L-Phe with starch was performed at 70 °C for 30 min by slowly adding 3.0 g of Boc-L-Phe to
above suspension at pH 4.0. At the end of esterification, the suspension was filtered. In order to assure the purity of Boc-L-Phe
in situ grafted starch (Boc-L-Phe-g-St), the obtained precipitate
was thoroughly washed by Milli-Q water three times before being
dried at 40 °C for 24 h in a vacuum oven. Subsequently, the Boc-LPhe-g-St was resuspended in 20 ml of Milli-Q water and 20 ml trifluoroacetic acid (TFA) was added to for the removal of Boc group
at pH 4.0 (adjusting by 2.0 M hydrochloric acid) at room temperature. After 2.5 h of reaction, the obtained precipitate was thoroughly washed by Milli-Q water three times for removing the
impurities to obtain L-phenylalanine in situ grafted starch (L-Pheg-St).
2.2.2. Zinc (II) functionalized L-phenylalanine in situ grafted starch
preparation
Assisted with stirring, the as-prepared L-Phe-g-St was thoroughly suspended in a three-necked flask containing 30.0 ml of
Milli-Q water, and then 0.46 g of ZnO was added. After 2.0 h of
functionalization reaction at 45 °C, 5.00 ml of ethyl alcohol was
added to and the mixture was kept stirring for another 15 min
before filtration. In order to remove the residues, obtained precipitate was thoroughly washed Milli-Q water for three times. After
being dried at 40 °C for 24 h in a vacuum oven, zinc (II) functionalized L-phenylalanine in situ grafted starch (Zn(II)@L-Phe-g-St) was
obtained.
2.3. Morphology and structure analysis
Scanning electron microscopy (SEM, SU8020, Hitachi, Japan)
equipped with energy dispersive X-ray spectroscopy (EDS) was
used to observe the morphologies of samples and analyze the surface of Zn(II)@L-Phe-g-St. The sample surface was sputter-coated
with a layer of gold and the applied accelerating voltages were of
5 kV for both starches and bacteria. X-ray Diffraction (XRD) was
performed on a D/MAX2500 V diffractometer (Rigaku, Japan) using
Cu Ka radiation (k = 0.15418 nm) to determine the crystal phase of
Zn(II)@L-Phe-g-St. Fourier transform infrared spectroscopy (FTIR)
spectra were used to investigate the interaction of the components
in Zn(II)@L-Phe-g-St and conducted on a Nicolet 6700 spectrometer
(Thermo Nicolet, Madison, WI, USA) in the range of 4000–400 cm1
using KBr pellets. X-ray photoelectron spectroscopy (XPS) performed by Mg Ka radiation with an ESCALAB 250 (Thermo-VG Scientific, USA) was used to examine the chemical structure and
composition of samples.
2.4. Antibacterial activity assay
In the experiments, the concentrations of bacteria were determined by measuring optical density at 600 nm (OD600) using a
spectrophotometer (UV-754PC, Shanghai Jinghua Technology
Instruments Co., Ltd., Shanghai, China). According to manufacturers’ culturing guidelines, B. subtilis and E. coli strains were cultured
overnight in Luria-Bertani (LB) culture medium on a shaker platform (37 °C, 200 rpm), respectively, and then diluted until the concentration of the culture medium reached 104 CFU/ml of bacteria
for further experiments.
A volume of 1.0 ml as-prepared culture medium was again
diluted to 100.0 ml with a fresh LB medium containing 1.0, 2.0,
3.0, and 4.0 mg of Zn(II)@L-Phe-g-St (i.e. 10.0, 20.0, 30.0 and
40.0 lg ml1, respectively) before being incubated in 250.0 ml
Erlenmeyer flasks for 24 h on the shaker platform (37 °C,
200 rpm). The growth curves of B. subtilis and E. coli after exposure
to Zn(II)@L-Phe-g-St were plotted with OD600 versus time by measuring the value of OD600 at an interval of 4 h within 24 h (specimen with starch concentration at 10.0 lg ml1 was used as a
C. Jin et al. / Journal of Functional Foods 38 (2017) 205–213
control). For an intuitive comparison, the inhibition efficiency (IE)
of Zn(II)@L-Phe-g-St against bacteria was determined as follows:
IE ¼
Spec
ODCtrl
600 OD600
ODCtrl
600
100%
ð1Þ
Spec
where ODCtrl
600 and OD600 represent the OD600 of control and culture
medium at time t, respectively. Note that, the antibacterial activity
evaluation of Zn(II)@L-Phe-g-St at higher contents was provided in
supporting information and the results were shown in Supplementary Fig. S1 due to the safety dosge.
2.5. Cell culture and fluorescence image
HeLa cells were grown in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% FBS. All cells were supplemented
with an antibiotic antimycotic solution (0.1 mg ml1 penicillin and
0.1 mg ml1 streptomycin) and incubated in a CO2 incubator
(37 °C, 5% CO2).
Fluorescent images were taken to observe the live and attached
Hela cells exposure to Zn(II)@L-Phe-g-St using fluorescent microscopy (Olympus BX43, Japan) by a 10X ocular lens and 20X objective lens, and excitation at 488 nm provided by a Multi Ar laser on
BWA detection. A detailed description of the process was provided
in Supplementary Section 2.
2.6. Cytotoxicity assay
After an incubation of Hela cells in the 96-well plates in a CO2
incubator for 24 h, Zn(II)@L-Phe-g-St with fresh culture medium
at different concentrations was injected to the plate and also incubated for 24 h. CCK-8 assay and a microplate reader (Bio-Rad, USA)
were used to determine cell viability (CV) by measuring optical
density at 450 nm (OD450) of culture medium (specimen with
starch concentration at 10.0 lg ml1 was used as a control), the
value of CV was calculated as follows:
CV ¼
Blank
ODTest
450 OD450
Blank
ODCtrl
450 OD450
ð2Þ
Ctrl
Blank
where ODTest
450 , OD450 and OD450 are the OD450 of Zn(II)@L-Phe-g-St,
control and blank (without Hela cells and Zn(II)@L-Phe-g-St) culture
mediums, respectively. A detailed description of the process was
provided in Supplementary Section 3.
2.7. Statistical analysis
Unpaired Student’s t-test was used in the evaluation of statistical analysis. Data were means ± standard deviation (SD) and a p
value < 0.05 was considered statistically significant. Each experiment is representative of three independent experiments.
3. Results and discussion
3.1. Synthesis routine and mechanism of Zn(II)@L-Phe-g-St
Starch is a multi-hydroxyl polymer with three hydroxyl groups
per monomer, which can work as active sites for the esterification
of the carboxyl groups to achieve the in situ grafting
L-phenylalanine (L-Phe) onto starch. On the other hand, the
nitrogen atoms of the amino groups on L-Phe can also
provide unshared lone-pair electrons to form complex with
Zn(II) ion. Therefore, Boc-L-phenylalanine (Boc-L-Phe, Boc, tertbutoxycarbonyl) was employed in the present work. The synthesis
routine and mechanism of Zn(II)@L-Phe-g-St was proposed as
depicted in Scheme 1. In brief, Boc-L-Phe was first in situ grafted
207
onto starch to form Boc-L-Phe-g-St through esterification reaction.
After removing Boc, Zn(II) was complexed with L-Phe-g-St to form
Zn(II)@L-Phe-g-St by r-coordinate bonds. The relative information
was confirmed in details afterwards.
3.2. Structure and morphology investigation
Fig. 1A shows the FT-IR spectra of starch, L-Phe-g-St and Zn(II)
@L-Phe-g-St. In spectrum of starch (Fig. 1A(a)), characteristic peaks
were presented at: 3353 cm1 (OAH stretching, broad), 2930 and
2870 cm1 (asymmetric and symmetric ACH2 stretching, respectively), 1648 cm1 (OAH bending), 1462, 1421 and 1370 cm1
(ACH2 bending), 1024 cm1 (CAO stretching of CAOAC in the
polysaccharide) 1160 and 1082 cm1 (CAO stretching in anhydroglucose ring) (Simsek, Ovando-Martinez, Marefati, Sjӧӧ, &
Rayner, 2015). As expected, the characteristic peaks of starch were
presented in the spectrum of L-Phe-g-St (Fig. 1A(b)). In comparison
with the spectrum of starch, we also noted in Fig. 1A(b): peaks at
3343 and 3266 cm1 associated with stretching vibration of
ANH2
in
L-Phe
(Rutnakornpituk,
Puangsin,
Theamdee,
Rutnakornpituk, & Wichai, 2011), a new peak at 1725 cm1 attributed to stretching vibration of [email protected] in ester carbonyl groups from
the esterification between the AOH in starch and ([email protected])OH in
1
L-Phe (Fan, Luo, Sun, Qiu, & Li, 2013), a peak at 1627 cm
assigned
to bending vibration of NH, strengthened peaks at 1082 and
1024 cm1 (CAO stretching) due to the coverage of ([email protected])AO
stretching. Additionally, a peak at 3025 cm1 was due to unsaturated CAH stretching vibration in aromatic ring, peaks at 1497
and around 1410 cm1 were assigned to the substituted benzene
ring-modes, and the peaks at 697 and 752 cm1 were ascribed to
the out-of-plane deformations of CAH in aromatic ring. The above
information suggested the successful esterification of starch with
L-Phe and the formation of L-Phe-g-St. Here, the removal of Boc
could be confirmed by the comparison of L-Phe-g-St with
Boc-L-Phe in FT-IR spectra (Supplementary Fig. S2). When Zn(II)
was coordinated with L-Phe-g-St to form Zn(II)@L-Phe-g-St,
r-coordinate bonds formed between Zn(II) and N atom in ANH2
as indicated in Fig. 1A(c): lower shifts of ANH2 stretching (from
3343 and 3266 cm1 to 3332 and 3266 cm1, respectively) and
NH bending (from 1627 cm1 to 1624 cm1).
Fig. 1(B–D) illustrate XPS full-scan of Zn(II)@L-Phe-g-St and survey spectrum of Zn 2 p, and N 1 s. As indicated in Fig. 1B, all the
peaks on the curve were ascribed to Zn, O, C and N elements.
Due to the spin-orbit coupling, the spectrum of Zn 2 p split into
Zn 2p3/2 and 2p1/2 which separately presented at 1022.18 and
1045.48 eV (Fig. 1C), and the peak separation between 2p3/2
and 2p1/2 (23.30 eV) indicated the presence of Zn (II) ions in
Zn(II)@L-Phe-g-St (Hwang et al., 2011). Fig. 1D showed the signal
deconvolution of N 1 s after Gaussian curve fitting, indicating
two chemically different N species in Zn(II)@L-Phe-g-St. One lower
peak at 399.6 eV was assigned to the coordinate N (N?Zn(II))
(Dianzhong & Bo, 1993). The other peak at 400.3 eV was attributed
to amino functional groups having a hydrogen atom (CANH2)
(Nelson, Balasundaram, & Webster, 2006). On the basis of the ratio
of the relative areas, it could be determined that around 71.02% of
ANH2 had coordinated with Zn(II) to form Zn(II)@L-Phe-g-St. Furthermore, according to the atomic% of N and Zn (Supplementary
Table S1), the coordination number of Zn(II) was determined and
approximated to 2 as indicated in Scheme 1.
The native starch granule showed a shape of flat spheroid with a
diameter ranging from 20 to 30 lm and a smooth surface with no
evidence of cracks or holes (Fig. 2A). As it could be seen from
Fig. 2B, Zn(II)@L-Phe-g-St still remained the shape of native starch,
suggesting the successful in situ esterification reaction of L-Phe on
native starch. The corresponding EDS spectra of Zn(II)@L-Phe-g-St
208
C. Jin et al. / Journal of Functional Foods 38 (2017) 205–213
Scheme 1. Synthesis routine, mechanism and chemical structue of Zn(II)@L-Phe-g-St.
(Fig. 2C) revealed the presence of Zn element. Additionally, no ZnO
particles were observed on the surface of Zn(II)@L-Phe-g-St, implying the completely formation of coordinated Zn(II). Direct evidence
could be achieved from the information presented in XRD patterns
as shown in Fig. 2D. The ZnO used in our experimental showed the
typical diffraction peaks of hexagonal wurtzite ZnO (space group
P63/mc) (Tang, Wang, Yao, & Li, 2014), which disappeared in pattern of Zn(II)@L-Phe-g-St.
3.3. Antibacterial activity assay
The standard shake flask method was performed to evaluate
antibacterial activity of Zn(II)@L-Phe-g-St towards a representative
Gram-positive bacterium, B. subtilis and a representative Gramnegative bacterium, E. coli and the corresponding growth curves
and inhibition efficiencies were shown in Fig. 3(A1, B1) and Fig. 3
(A2, B2), respectively. The value of OD600 reflected the amount of
bacterial cell growth. As expected, the growth curve of B. subtilis
exposure to the control gave a representative dynamic cycle
including three phases (Fig. 3A1): lag phase, exponential phase,
and stabilization phase. As compared with the control, the values
of OD600 decreased with the increase of the concentration of Zn
(II)@L-Phe-g-St at given culture time. As bacteria needed to adapt
themselves to external environment at lag phase, they matured
but not yet able to divide themselves, which limited their reproduce and caused a slow increase in bacteria number. After exposure to Zn(II)@L-Phe-g-St, partial bacteria were killed due to the
touching onto Zn(II). Therefore, the presence of Zn(II)@L-Phe-g-St
caused a decrease of OD600 with increasing its concentration. At
exponential phase, bacteria reproduced at their maximum rates
and the bacteria number increased as an exponential function of
time because there was no restriction by nutrient or metabolic
products (Thiel et al., 2007). In comparison with the control, a good
antibacterial activity against B. subtilis was well indicated from the
significant decline, which appeared at the growth curve with
increasing the concentration of Zn(II)@L-Phe-g-St. As time prolonged, the depletion of nutrients limited the bacterial growth.
As a result, the growth curve of control showed a slight increase
before reaching a plateau at stabilization phase. In sharp contrast,
a significant decline appeared at each curve of Zn(II)@L-Phe-g-St at
this phase, and the decline became more serious at higher Zn(II)@LPhe-g-St concentration due to antibacterial activity of Zn(II). As
could be seen from Fig. 3A2, each inhibition efficiency curve
showed a similar variation trend corresponding to three phases.
At lag phase, the increase of bacteria number was in a lower level.
Thereby, the inhibition growth of bacterial cells by Zn (II) played
the principal role at this phase and resulted in relatively high inhibition efficiency at incubation of 4 h. As the bacteria reproduction
rate increased fast and reached its maximum value at the medexponential phase for the control, the inhibition of Zn(II) could
not effectively inhibit bacteria reproduction and leaded to a decline
in inhibition efficiency. After then, the rate gradually slowed down
and the amount of bacteria tended to be stable, hence, the inhibition of Zn(II) was becoming effectively again and resulted in an
increase in inhibition efficiency curve.
As for E. coli, similar variation trends were present at the growth
curves (Fig. 3B1) and inhibition efficiencies (Fig. 3B2). We also
observed Zn(II)@L-Phe-g-St showed slightly poor antibacterial
209
C. Jin et al. / Journal of Functional Foods 38 (2017) 205–213
(A)
(B)
164 8
b
3353 2930
1462
2870
1421
1 725
c
3025
1370
1160
1082 1024
840
1 497
1627
752
697
1340
1389
14 10
334 3 3266
1082 1024
1500
1000
1200
500
1000
Wavenumber (cm )
Zn2p
Zn 2p3/2
Zn 2p1/2
1035
1040
600
400
200
0
1045
1050
Binding energy (eV)
(D)
N 1s
Intensity (counts/s)
(C)
1030
800
Binding energy (eV)
-1
Intensity (counts/s)
C 1s
1624
4000 3500 3000 2000
1025
N 1s
Zn LMM
1088
3332 3257
1020
O 1s
N KLLZ n 2p 1/2
Z n 2p 3/2
Intensity (counts/s)
Transmittance (%)
a
N Zn(II)
C- NH
394
396
398
400
402
2
404
406
408
410
Binding energy (eV)
Fig. 1. (A) FT-IR spectra of starch (a), L-Phe-g-St (b) and Zn(II)@L-Phe-g-St (c); XPS full-scan of Zn(II)@L-Phe-g-St (B): survey spectrum of (C) Zn 2p, and (D) N 1s.
Fig. 2. SEM images of starch (A) and Zn(II)@L-Phe-g-St (B); EDS spectrum (C) from (B), showing the presence of Zn and XRD pattern of ZnO and Zn(II)@L-Phe-g-St(D).
activity towards E. coli than B. subtilis at given Zn(II)@L-Phe-g-St
concentration, which may be due to the different outer membrane
structure. Generally, the cell wall of Gram-positive bacteria (e.g. B.
subtilis) is composed of a thick peptidoglycan layer containing
lipoteichoic acid, whereas the cell wall of Gram-negative bacteria
(e.g. E. coli) consists of a thin peptidoglycan layer between the
inner and outer lipid membranes (Brown, Wolf, Prados-Rosales, &
Casadevall, 2015). Bacteria contain genes that responsible for the
transport of Zn ions (Lindsay & Foster, 2001). The presence of
lipoteichoic acid had a more tendency for positive ions to be
C. Jin et al. / Journal of Functional Foods 38 (2017) 205–213
(A1)
1.0
Control
10.0 µg/ml
20.0 µg/ml
30.0 µg/ml
40.0 µg/ml
OD600 nm
0.8
0.6
0.4
0.2
(A2)
100
Inhibition efficiency (%)
210
80
0.0
60
40
20
10.0 µg/ml
20.0 µg/ml
30.0 µg/ml
40.0 µg/ml
0
0
4
8
12
16
20
24
0
4
8
(B1)
1.4
Control
10.0 µ/ml
20.0 µg/ml
30.0 µg/ml
40.0 µg/ml
1.2
OD600 nm
1.0
12
16
0.8
0.6
0.4
(B2)
100
80
40
20
10.0 µg/ml
20.0 µg/ml
30.0 µg/ml
40.0 mg/ml
0
0.0
4
8
12
24
60
0.2
0
20
Time (h)
Inhibition efficiency (%)
Time (h)
16
20
24
Time (h)
0
4
8
12
16
20
24
Time (h)
Fig. 3. Growth curves of B. subtilis (A1) and E. coli (B1) exposure to control and Zn(II)@L-Phe-g-St; Inhibition efficiencies of of B. subtilis (A2) and E. coli (B2) exposure to Zn(II)
@L-Phe-g-St. The data were representative of results from three independent experiments and expressed as the means standard deviations (SD). The Zn(II)@L-Phe-g-St
showed significant inhibition efficiency with comparison to the control (p < 0.05).
attached on Gram-positive bacteria (Esmailzadeh, Sangpour,
Shahraz, Hejazi, & Khaksar, 2016), which would break the equilibrium of Zn ions and lead to bacterial death. Therefore, the antibacterial activity of Zn(II)@L-Phe-g-St against B. subtilis was relatively
more effective with comparison to E. coli. Similar results were also
observed by other researchers (Kim & An, 2012; Li, Deng, Deng, Liu,
& Li, 2010).
Fig. 4 shows SEM images of E. coli cells after exposure to the
Zn(II)@L-Phe-g-St. The E. coli cells showed a normal rod-shaped
morphology with smooth surface (Fig. 4A). After exposure to
Fig. 4. SEM images of E. coli exposure to Zn(II)@L-Phe-g-St at concentration of (a) 0.0, (b) 20.0, (c) 30.0 and (d) 40.0 lg ml1.
C. Jin et al. / Journal of Functional Foods 38 (2017) 205–213
100
Cell viability (% of control)
*
*
80
*
60
40
a diameter of 12.0 lm (Fig. 4D). It could be deduced that the
antibacterial process was composed of two stages: Zn(II) ions
anchored to the cell membrane and destroyed it, which could kill
partial bacteria directly (Stoimenov, Klinger, Marchin, &
Klabunde, 2002). After then, the reactive oxygen species (ROS)
arisen from the oxidative stress by the Zn(II) ions penetrated inside
the cell disrupt the zinc homeostasis in the bacteria (Applerot et al.,
2009), which damaged lysosomal and mitochondria and leaded to
the death of bacteria cells eventually (Ma, Williams, & Diamond,
2013).
3.4. Cytotoxicity investigation
20
0
211
Control
10
20
30
40
50
60
70
-1
Concentration ( µg ml )
Fig. 5. Viability of Hela cells after exposure to Zn(II)@L-Phe-g-St (37 °C, 24 h). (*)
p < 0.05 for Zn(II)@L-Phe-g-St with comparison to control. The data were representative of the results from repeated experiments (n = 3) and expressed as the
mean ± SD.
Zn(II)@L-Phe-g-St, the disequilibrium of Zn(II) ions destroyed the
cell membrane. When the cytoplasm flew away from the cell, the
cell shrank with some wrinkles or even a few cracks on surface
(Fig. 4B). At low dose of Zn(II)@L-Phe-g-St, only partial bacteria
had been killed, and then the living bacteria spread out again after
digested all the starch. Therefore, no obvious aggregation phenomenon was observed. With increasing Zn(II)@L-Phe-g-St concentrations, the attached cell membranes were seriously destroyed
and more cytoplasm had flowed away from them. As a result, the
membranes collapsed and aggregated to a similar spherical cluster
sized about 7.0 lm in diameters at a concentration of 30.0 lg ml1
(Fig. 4C). When the concentration was high at 40.0 lg ml1, the
cluster was enlarged and turned into a spherical bacteria ball with
Hela cells were selected to test the cytotoxicity of
Zn(II)@L-Phe-g-St. The cell viability of Hela cells were assayed after
exposure to Zn(II)@L-Phe-g-St for 24 h by CCK-8 assay (Fig. 5),
herein, the viability was determined by the change of formazan
dye associated with the activity of mitochondrial dehydrogenase.
As it could be seen from Fig. 5, the viability loss of cell resulted
from Zn(II)@L-Phe-g-St was dose-depended manner. The
Zn(II)@L-Phe-g-St showed no obvious viability loss below the concentration of 40 lg ml1. The cell viability still approximated to
80% even at a high concentration of 60 lg ml1, indicating low
toxicity of Zn(II)@L-Phe-g-St to Hela cells. As ROS was produced
by superoxide, which was strongly dependent on intracellular
Zn (II) (Shen et al., 2013). When the extracellular Zn (II) dissociated
from and the coordinated on Zn(II)@L-Phe-g-St were insufficient to
produce enough intracellular Zn(II), no cytotoxicity would produced (Buerki-Thurnherr et al., 2013; Shen et al., 2013; Wang
et al., 2014). Therefore, Zn(II)@L-Phe-g-St showed very low toxicity
towards Hela cells especially at the concentration below
40 lg ml1. Similar results were observed in ZnO nanoparticles
(Li et al., 2008) or its composites (Liu, Ai, Yuan, & Lu, 2011;
Zhang, Xiong, Ren, Xia, & Kong, 2012) at low concentrations.
To visualize the viability loss in dose-depended manner as
shown in Fig. 5, rhodamine 123 (Rh-123), a cell-permeable and
Fig. 6. Fluorescence microscope images of HeLa cells exposure to Zn(II)@L-Phe-g-St at concentration of (A) 0.0, (B) 20.0, (C) 40.0 and (D) 60.0 lg ml1; White arrows: dead
cells.
212
C. Jin et al. / Journal of Functional Foods 38 (2017) 205–213
cationic dye specific for mitochondria, was used for the dye of HeLa
cells before being subjected to fluorescence microscope analysis.
The living HeLa cell showed a small round ball with clear boundary
in bright green fluorescence without exposure to Zn(II)@L-Phe-g-St
(Fig. 6A). After exposure to Zn(II)@L-Phe-g-St at relatively low concentration of 20.0 lg ml1, no obvious change in morphology was
observed (Fig. 6B), suggesting no cytotoxicity towards HeLa cells.
With increasing the concentration to 40.0 lg ml1, we observed
the appearance of dead cell as indicated from Fig. 6C. When the
dyed cytoplasm flowed out from the broken cell membrane, the
clear boundary of the round ball disappeared, meanwhile, the fluorescence ball was enlarged and became dimming. As expected,
more dead Hela cells were found in Fig. 6D with Zn(II)@L-Pheg-St concentration at 60.0 lg ml1, however, the living cells displayed an absolute advantage in total number over dead cells.
Thereby, the results from fluorescence microscope analysis further
confirmed the conclusions drawn from CCK-8 assay.
4. Conclusion
In summary, a facile strategy was developed to fabricate
Zn(II)@L-Phe-g-St. The in situ grafting of Boc-L-Phe onto starch
was achieved through esterification reaction, after removing Boc,
Zn(II) was complexed with L-Phe-g-St by r-coordinate bonds.
The structure of Zn(II)@L-Phe-g-St was proposed and well
characterized, in which around 71.02% of ANH2 had coordinated
with Zn(II) and the coordination number of Zn(II) was confirmed
to be approximated to 2. Zn(II)@L-Phe-g-St showed a good
antibacterial activity against both B. subtilis and E. coli, and the
corresponding inhibition efficiency enhanced with the increase of
Zn(II)@L-Phe-g-St concentrations. Because of difference structure
in outer membrane of bacteria, Zn(II)@L-Phe-g-St exhibited a relatively more effective antibacterial activity against B. subtilis than
E. coli. In additional, cytotoxicity results of Zn(II)@L-Phe-g-St
displayed very low toxicity towards Hela cells especially at the
concentration below 40 lg ml1, and the cell viability loss
was in a dose-depended manner. The proposed strategy may be
favorable in the design and fabrication of similar metal ion
functionalized amino acids in situ grafted starch, and the resulting
Zn(II)@L-Phe-g-St may have potential as an ideal food additives due
to its better antimicrobial activity and less toxicity.
Acknowledgement
The research was supported by National Natural Science
Foundation of China (31371859).
Conflict of interest
The authors declare no competing financial interest.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.jff.2017.09.015.
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