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. 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