Abstract
With the increasing damage caused by foodborne pathogens to human health and the increasing attention given to healthy diets, novel food antimicrobial agents have been widely studied.
In this study, three different morphologies of citral-modified ZnO nanoparticle antimicrobial materials were prepared, and the citral-modified porous ZnO nanorod antimicrobial materials with the highest loading (60.35%) and the strongest inhibitory effect (MIC=0.2–0.1 mg/mL) were screened through a series of characterization and bacterial inhibition experiments. This novel antimicrobial material has excellent and long-lasting antimicrobial properties. It inhibited Escherichia coli by 100% when stowed at 25 °C and protected from light for 10 d and inhibited the growth of E. coli by 58.17% after being stowed under the same conditions for 60 d. Furthermore, we tested the pH change during 24 h of E. coli growth and the pH responsiveness of the materials.
The results demonstrated that under the acid-producing condition of E. coli growth, the pH-sensitive imine bond (–CH=N–) formed by the condensation of the amino of functionalized ZnO nanoparticles and citral was hydrolyzed to release the citral, which indicated that the release mechanism of citral in the antibacterial material was pH-sensitive.
The antibacterial materials in this study have broad application prospects in the field of food production and packaging in the future. Moreover, this study provides a theoretical basis for guaranteeing food quality and safety.
Introduction
The quality and safety of food are closely related to people’s lives. Microbiological, chemical, climatic, and water activities can affect the safety of food, and foodborne pathogens among them are particularly damaging, as they not only lead to reduced food quality but also cause foodborne diseases (Aladhadh, 2023). Nowadays, not only developing countries, but also developed countries are experiencing serious incidents of food-based pathogenic bacteria pollution (Mengistu and Tolera, 2020; Hailu et al., 2021). Foodborne microorganisms can cause bacterial infections and food poisoning, threatening human health and hindering economic prosperity. Therefore, the development of a fast and effective sterilization method for pathogenic bacteria in food is essential. Traditional sterilization techniques may have adverse effects on the texture and flavor of food products, but newer sterilization techniques such as plasma sterilization and pulsed electric field sterilization are challenging to implement on a large scale in industrial production due to their high economic costs and energy consumption (Guo et al., 2022; Soni and Brightwell, 2022; Wang et al., 2022). The most common method of inhibiting the growth of microorganisms in food is to use bacteriostatic agents (Zhao et al., 2021). However, when synthetic bacteriostats are used for a long time, microorganisms may develop resistance to them, which limits their large-scale application in industry and promotes research on natural antimicrobial materials as bacteriostats.
Essential oils are secondary metabolites of plants and they can be obtained by fermentation or extraction, and steam distillation is the most common method for commercial production of essential oils (Ghasemy-piranloo et al., 2020). Essential oils are known for their antibacterial, antioxidant, and anticancer activities and have been rated globally as safe, non-toxic, and effective natural antibacterial substances (Tajkarimi et al., 2010; Chen et al., 2023; Meenu et al., 2023; Mohamed Abdoul-Latif et al., 2023). The antimicrobial mechanism of essential oils has been shown to involve the disruption of bacterial cell membranes and the reduction of cell permeability (Kachur and Suntres, 2020). Previous studies have also indicated that it affects intracellular ion transport and interaction between membrane proteins and compounds (Yin et al., 2022). Alternatively, the mechanism affects the active sites of enzymatic reactions in the bacterial cell to produce an antibacterial effect (Kuttithodi et al., 2023). All of these effects disrupt the normal growth process of bacterial cell, ultimately leading to cell death. Essential oils have been recognized as excellent antibacterial ingredients due to their various advantages, but their volatility, photosensitivity, and water resistance limit their use in terms of an antibacterial agent (Al-Maqtari et al., 2021). Therefore, some researchers are dedicated to developing more scientific and reasonable methods, such as preparing relatively stable active coatings containing essential oils, emulsifying essential oils, and encapsulating essential oils in carriers for controlled release, to reduce their volatility and enhance their active utilization (Peighambardoust et al., 2022; Ren et al., 2022). Yin et al. (2023) prepared nanoparticles using the counter-solvent method with cysteine-chitosan and zein as wall materials and capsaicin as the core material. The synthesized nanomaterial was spherical in shape with a uniform size distribution, and the release rate of capsaicin was only 40.08%±4.28% after 4 h of simulated digestion in vitro, which showed excellent pH stability and ionic strength stability. In this study, essential oils were loaded by controllable chemical bonding to nanocarriers rather than by encapsulation, which would significantly decrease the impact of environment on free active substances and minimize the negative impact of essential oils on food organoleptics when applied to food packaging.
Nanoparticles (NPs) are a type of nanotechnology that studies small particle materials ranging in size from 1 to 100 nm (Khan et al., 2022). Due to their small size, NPs have larger specific surface area and exhibit unique characteristics (Kavitha et al., 2023). Some nanomaterials, such as nanoliposomes (Siyadatpanah et al., 2023), and nanoemulsions (Nie et al., 2023), and biopolymeric nanoparticles (Vodyashkin et al., 2022), could be used as antibacterial carriers to encapsulate essential oil molecules, thereby enhancing the durability of their antibacterial ability. ZnO nanoparticles (ZnO-NPs) have the characteristics of small particle size, large specific surface area, and diverse micromorphological structures, as well as unique properties such as pyroelectricity, semiconductivity, and high scope for modification development (Wang et al., 2022). In addition, ZnO-NPs have been recognized as Generally Recognized As Safe (GRAS) by the US Food and Drug Administration (FDA) due to their safe, non-toxic properties (Mishra et al., 2017). ZnO-NPs possess certain antimicrobial activity by themselves, and their physicochemical properties can predict their antimicrobial and toxicological effects (Czyzowska and Barbasz, 2022). The properties of ZnO-NPs, including morphology, particle size or particle size distribution, porosity, and specific surface area, affect antimicrobial activity (Yu et al., 2020). The particle size and particle size distribution level of ZnO-NPs are key parameters to determine antimicrobial activity against pathogenic microorganisms (Sharma et al., 2019). In recent years, ZnO-NPs have been widely utilized in various fields such as optical, medical, agricultural, and horticultural industries (Du et al., 2019; Samart and Chutipaijit, 2019; Yoon and Oh, 2021; Costa et al., 2023).
The imine bond in chemical reactions is a pH-responsive covalent bond that is stable at neutral pH (~7.4) and easily hydrolyzed in acidic environments (Zhou et al., 2023), and the rate of hydrolysis is positively correlated with environmental acidity, which has been extensively practiced in biomedical hydrogels and polymeric micelles (Zhang et al., 2018; Zhang et al., 2023). Lin et al. (2023) prepared aldehyde–chitosan Schiff base compounds using chitosan and five natural aldehydes, which had better antimicrobial activity at pH 5 than at pH 7. This was attributed to the hydrolysis and breaking of the imine bond to release more aldehydes at pH 5. In addition, aldehyde–chitosan Schiff base compounds were able to extend the shelf life of broccoli (from 4 to 5–7 d) and reduce the loss of water from broccoli, which resulted in freshness preservation of the broccoli (Lin et al., 2023). In another study, smart antifungal films were fabricated by grafting aldehydes (cinnamaldehyde, citral, hydrogenated cinnamaldehyde, and citronellal) onto the surface of chitosan films by enabling the acid-catalyzed condensation of the carbonyl group with the amino group to form an imine bond. The results of antifungal activity testing in vitro of this dynamic film against two kinds of bacteria, Pseudomonas expansa and Staphylococcus griseus, showed that all the films exhibitedacterial antifungal activity, and their antifungal activity was attributed to the aldehydes released after the imine bond was broken in an acidic environment (Heras-Mozos et al., 2022).
Based on the current research status, we designed a pH-responsive intelligent release strategy for zinc oxide nanomaterials modified with essential oil. Because essential oil molecules in encapsulated form are sensitive to the environment during storage, we introduced amino groups onto the surface of ZnO-NPs to condense with aldehyde groups to form imine bonds, which controlled the release of essential oil molecules and achieved the desired antibacterial effect. In this antibacterial material, antimicrobial carriers were zinc oxide nanoparticles, imine bonds were employed as the ‘control key’ of the release process, and citral was utilized as an effective component to exert antibacterial activity. When there is a small amount of Escherichia coli in the system, only a limited quantity of acid metabolites is produced. This leads to a slight decrease in the system’s pH and the breaking of a tiny amount of imine bonds, resulting in the release of a small number of essential oil molecules to inhibit the bacteria. Conversely, when there is a high concentration of E. coli, a larger quantity of acid metabolites is produced, causing a significant decrease in the system’s pH. This condition is sufficient for the imine bond to break completely and release large amounts of essential oil. The experimental data on antimicrobial activity indicated that the synthesized materials’ adaptive pH-responsive antimicrobial system can be utilized to inhibit E. coli.
Materials and Methods
Reagents and culture medium
Zinc nitrate hexahydrate (Zn(NO3)2·6H2O, 99%), zinc acetate dihydrate ((C4H6O4)2·Zn·2H2O, 99%), hexadecyl trimethyl ammonium bromide (CTAB, 99%), β-cyclodextrin (β-CD), diethylene glycol (DEG, 98.9%), ammonium hydroxide (NH3·H2O, 99.99%), (3-aminopropyl) triethoxysilane (3-APTES, 99%), ethanol absolute (99.8%), dimethyl sulfoxide (DMSO, 99%), glutaraldehyde 25% water solution (25%), sodium chloride (NaCl, 995), tryptose soya agar (TSA), trypticase soy broth (TSB), and commercial non-fixed nano–zinc oxide were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China).
Escherichia coli strains were obtained from Shanghai Ocean University (Shanghai, China). After the initial activation, the E. coli strains were stored in a broth containing 25% glycerol and soybean trypsin at –80 °C. The strains were inoculated into a liquid medium and incubated in a constant temperature incubator at 37 °C for 24 h. The concentration of the bacterial solution was adjusted to approximately 106 CFU/mL through gradient dilution before each antibacterial experiment.
Preparation and modification of ZnO nanoparticles
Preparation of ZnO nanorods
ZnO nanorods (ZNR) were prepared according to a modified literature method (Ouyang et al., 2021). First, 2.3 g CTAB, 8.0 g β-CD, and 2.0 g NH3·H2O were mixed and dissolved in 100 mL deionized water. The mixture was then ultrasonically dispersed for 30 min until it was completely dissolved. Then, 8.0 g (C4H6O4)2·Zn·2H2O were added to the mixture and stirred vigorously in a water bath maintained at a constant temperature of 45 °C for 3 h. The stirred solution was transferred to a 200-mL polytetrafluoroethylene (PTFE) high-pressure reactor and reacted in an oven at 150 °C for 12 h. The resulting precipitate was obtained by centrifugation, washed several times with deionized water and anhydrous ethanol, and then dried in a vacuum drying oven at 60 °C. The dried precipitate was placed into a crucible and heated in a muffle furnace at a rate of 3 °C/min until it reached a constant temperature of 350 °C, where it was kept for 6 h. This process resulted in the formation of ZNR.
Preparation of ZnO nanosheets
The preparation of ZnO nanosheets (ZNS) was improved using synthesis methods reported in the literature (Hezam et al., 2021). First, 0.15 g CTAB and 1.2 g NaOH were dissolved in 50 mL deionized water and the mixture was dispersed by ultrasound. The 50 mL aqueous solution of Zn(NO3)2·6H2O, with a concentration of 0.12 g/mL, was slowly added to the rapidly stirring CTAB–NaOH solution at a rate of 3 mL/min. The mixed solution was then transferred into a PTFE high-pressure reactor and subjected to a reaction in an oven at 100 °C for 24 h. After the reaction, the white precipitate was centrifuged, washed three times with deionized water and anhydrous ethanol, and finally dried in a vacuum drying oven at 60 °C for several hours to obtain ZNS.
Amino-functionalization of ZnO nanoparticles
Amino-functionalized ZnO-NPs were synthesized following the modification process described in the literature (Chelladurai et al., 2022). The amino-functionalization of ZnO-NPs was achieved using the silane coupling agent 3-APTES: ZnO-NPs (0.5 g) were sonicated and dispersed in 50 mL of the organic solvent DMSO for 45 min. Then, 400 μL 3-APTES was added to the solution and condensed and refluxed at 120 °C for 3 h. The resulting colloidal solution was centrifuged at 12 000 r/min for 20 min and then washed with anhydrous ethanol to remove any unreacted material. Finally, the precipitate was dried at 60 °C to obtain two distinct morphologies of amino-functionalized ZnO nanoparticles.
Synthesis of antibacterial materials—ZnO nanoparticles functionalized with citral
The process of functionalizing ZnO-NPs with essential oil was based on methods described in the literature, which were modified briefly (Bravo Cadena et al., 2018). First, 1 g amino-functionalized ZnO-NPs and 2 g excess citral (CIT) were dispersed ultrasonically into 50 mL anhydrous ethanol. The pH of the solution was adjusted to 8.5 with 1% NaOH and stirred at room temperature for 24 h. The mixture was then separated from the supernatant by centrifugation, and the precipitate was washed three times with deionized water. Finally, it was dried by ventilation at room temperature to obtain ZnO nanorods (ZNR@C) and ZnO nanosheets (ZNS@C) loaded with citral. At the same time, commercially available amorphous ZnO-NPs were modified with citral to synthesize SAZ@C as the control group.
Characterization of antibacterial materials and comparison and screening of antibacterial properties
The prepared samples were analyzed using standard techniques, including scanning electron microscopy (SEM), zeta potential analysis, thermogravimetric analysis (TGA), instrumental Fourier transform infrared spectroscopy (FTIR), and N2 adsorption/desorption thermocline. Some modifications were made based on previous studies.
The dried sample powder was evenly spread on a double-sided copper conductive tape, and gold particles were then sprayed onto the sample surface. Finally, SEM images of the samples were obtained by the hot-field scanning electron microscope (Hitachi Regulus 8100, Tokyo, Japan) at an accelerating voltage of 5 kV (Hou et al., 2021).
The samples were diluted to a concentration of 1 mg/mL in neutral water (pH-6.5–8.0) and sonicated at 25 °C for 20 min to create a high-dilution dispersion The particle size distribution characteristics of the samples in the sample jars were determined using a particle size meter, and the zeta potential was determined using the zeta potential analyzer (Malvern ZS90, Malvern, UK) (Sun et al., 2022).
The samples underwent thermal stability analysis using a thermogravimetric analyzer (NETZSCH STA449C, Selb, Germany) under air-filled conditions (60 mL/min) at an elevated heating rate of 10 K/min from 30 to 800 °C. The degree of functionalization of the antimicrobial carriers was ultimately evaluated based on the ratio of thermogravimetric loss in the samples (Liu et al., 2022).
A small number of solid samples was separately covered on the surface of attenuated total reflection diamonds and pressed using a constant pressure jig. The infrared spectra of the samples were measured in transmission mode at a resolution of 4 cm–1 by scanning 128 times in the wave number range of 500–4000 cm–1 using a Fourier infrared spectrometer (Nicolet Instrument, Thermo Fisher Scientific, Waltham, MA, USA) (Hou et al., 2021).
The samples were pretreated by degassing in a vacuum at 100 °C for 3 h under the experimental pressure to initial pressure ratio (P/P0) of 0.01–0.99, and then the N2 adsorption–desorption isotherms were obtained by conducting nitrogen adsorption–desorption experiments with an automatic adsorption analyzer at 77 K (Bayer BELSORP-mini, Tokyo, Japan) (Hou et al., 2021).
After conducting the TGA assay, ZNR@C (60.35%) and ZNS@C (54.13%), which had higher citral loadings, were selected to determine their antimicrobial activity. The two antibacterial materials were dispersed into TSB using ultrasonication. Five concentrations (0.05, 0.1, 0.2, 0.4, and 0.8 mg/mL) of suspensions containing nano-antibacterial substances were prepared and placed in test tubes. The tubes were inoculated with 100 μL of E. coli culture solution, resulting in an initial density of 106 CFU/mL of E. coli liquid. The tubes were placed in a microbial culture shaker and incubated at 37 °C for 24 h at 275 r/min. The optical density (OD) of E. coli was determined after incubation using a spectrophotometer (Thermo Fisher Multiskan FC) set at 600 nm. The number of E. coli colonies was determined using the dilution coating plate method, and the counts were transformed to log10 CFU/mL (Sysel et al., 2021). The control group was a blank control group with no samples added. All of the experimental treatments mentioned above were repeated at least three times. The formula of antibacterial rate (N) is as follows:
where A is the number of sample colonies and B is the number of colonies in the control group.
Study of antimicrobial activity and slow-release performance applications of antimicrobial materials—ZNR@C
Determination of in vitro antibacterial activity of ZNR@C
According to the growth curve of E. coli OD values, the antibacterial efficacy of antibacterial nanomaterials was determined. A concentration of 0.1 mg/mL of ZNR@C was added to a 10-mL solution of E. coli at a density of 106 CFU/mL. After repeated mixing with an oscillator, the tubes were placed in a microbiological culture shaker and incubated at 37 °C for 24 h at 275 r/min. Finally, the absorbance of the treated E. coli was measured at 600 nm using an enzymoleter. Citral and ZNR were used as control samples. All experiments were conducted three times.
Afterward, a more visual method was used to assess and compare the antimicrobial efficacy of the prepared nano-antibacterial materials by observing the number of colonies. Subsequently, the bacterial solution that was treated as described above was diluted by a factor of 100 and then inoculated onto TSA plates. The plates were then incubated at a constant temperature of 37 °C for 24 h. The resulting number of colonies on the plate was recorded.
Determination of the slow-release properties of citral in ZNR@C under various pH conditions
After determining the antibacterial activity of the three antimicrobial materials, ZNR@C was selected due to its superior antibacterial ability. Fifty milligrams of ZNR@C were added to 30 mL of phosphate buffer solution at the appropriate pH and stirred continuously at 200 r/min. Every 2 h, a 3-mL sample was taken. The absorbance was measured using a UV-visible spectrophotometer (UV1800PC, Shanghai, China), and the release of citral in the antibacterial material was calculated by substituting it into the standard curve of citral (Kumari et al., 2021).
To examine the prolonged bacterial inhibition efficacy of citral-functionalized zinc oxide porous nanorods, we stowed ZNR@C and citral at room temperature (25 °C) protected from light for 5, 10, 20, 30, and 60 d, respectively, and then determined their growth inhibitory effects on E. coli. For specific methods, refer to the above antibacterial operation procedure for ZNR@C and ZNS@C.
Study on bacteriostatic mechanism
In order to investigate the antibacterial mechanism of the antibacterial material, SEM was used to observe the morphology of E. coli before and after ZNR@C treatment. The procedure was as follows: first, ZNR@C was added to the E. coli suspension and incubated for 24 h. The suspension was then incubated at 37 °C for 24 h. After incubation, 1 mL of the suspension was centrifuged at 4000 r/min for 10 min, and the supernatant was discarded. Then, the bacterial solution was mixed with a 2.5% glutaraldehyde aqueous solution and left to fix overnight. Next, it was washed more than three times with sterile phosphate buffer solution (PBS) at pH 7.2–7.4. Finally, the solution was dehydrated using various concentrations of ethanol (30%, 50%, 70%, 90%, and 100%). Samples were prepared by adding 500 μL anhydrous ethanol, and 10 μL of the samples were air-dried on sterile slides for observation of cell morphology via SEM.
Data analysis
Plots were created using Origin 2021 software (OriginLab, Northampton, MA, USA), and experimental data were analyzed using SPSS software (version 20.0; SPSS Inc., Chicago, IL, USA) with one-way analysis of variance. Statistical significance was determined at a test level of p<0.05.
Results and Discussion
Figure 1 illustrates the preparation process of ZnO nanorods and provides the specific experimental procedure. The synthesis mechanism of ZnO nanorods can be described by the following equation:
Synthesis of ZNR@C.
Because β-cyclodextrin has a hydrophilic outer surface and hydrophobic inner surface, a large amount of Zn2+ and OH− both gather in the inner cavity of cyclodextrin due to electrostatic interaction forces under certain hydrothermal conditions, and then nucleate uniformly, growing slowly. Besides, because of the interaction between Zn(OH)42– ions and surfactant CTA+ ions, the hydrophobic CTA+ attaches to the negatively charged ionic surface, which hinders the attachment of Zn(OH)42– ions in space and charge, thus inhibiting the disordered growth of ZnO (Ouyang et al., 2021).
Figure 2 illustrates the process for preparing oxidative nanosheets and the specific experimental procedure. The synthesis mechanism of ZnO flakes follows the following equation:
Synthesis of ZNS@C.
The equation illustrates the importance of OH− in initiating the reaction and controlling the reaction rate. Reactions (4) and (5) occur faster when there is more OH– in the solution and reaction rate (6) increases. First, CTAB is ionized into CTA+, and Zn(OH)42– ions are bond with CTA+. The negatively charged ions in the reaction system were well condensed on the positively charged ion surface through electrostatic interaction. Hydrophobic CTA+ adheres to the surface of positively charged ions, hindering the adhesion of Zn(OH)42– ions in space and charge, thus inhibiting the disordered growth of zinc oxide and forming a sheet form (Guo et al., 2014).
Figure 3 shows the morphology of ZnO nanorods, nanosheets, and commercially available disordered nanoparticles before and after functionalization as observed via SEM. The ZnO nanorods, before and after functionalization (Figures 3A and 3B), exhibited a uniformly shaped rod-like structure with a length ranging from 5 to 10 μm and a bottom diameter of approximately 3–5 μm. The ZnO nanosheets, both before and after functionalization (Figures 3C and 3D), exhibited a uniformly shaped flake morphology, with a length ranging from 3 to 8 μm and a width of 500 nm. This size and morphology are consistent with those previously reported (Hezam et al., 2021). The commercially available disordered ZnO nanoparticles, both before and after functionalization (Figures 3E and 3F), exhibited variable morphology with non-uniform particle sizes, with granular, blocky, and needle-like morphologies. Comparison before and after functionalization revealed that amino functionalization did not affect the structure of the materials, which confirms that subsequent modification treatments do not alter the integrity or morphology of the nanomaterial matrix.
SEM images of ZnO nanorods (A, B), nanosheets (C, D), and commercial nanoparticles (E, F) before and after functionalization.
The zeta potentials of ZnO nanomaterials are presented in Figure 4, both before and after functionalization with the amino group and essential oil molecules. The zeta potential is a crucial parameter for predicting the surface charge of nanomaterials. As shown in the figure, ZnO nanomaterials functionalized with positively charged 3-APTES amino significantly increased from smaller positive charge numbers to large positive charge numbers (Rokicka-Konieczna et al., 2020; Mahalanobish et al., 2022). After citral was chemically bonded to the ZnO nanomaterials, the surface charge of the material was slightly reduced. The reason for this may be that certain active ingredients, such as essential oils, produce negative charges after modifying the nanocarriers, thereby decreasing the zeta potential of the ZnO nanomaterials (Chen et al., 2020).
Zeta potentials of self-synthesized ZnO nanorods, self-synthesized ZnO nanosheets, and commercial ZnO nanoparticles before and after modification.
Figure 5 displays the thermogravimetric analysis curves of ZNR, ZNS, and SAZ, as well as those of the ZnO-NPs after amino functionalization and citral modification. Because of the greater volatility of citral under high thermal conditions, thermogravimetric curves can be used to determine the total amount of citral bonded to the nanocarriers. As depicted in the figure, the weight loss curve of unmodified ZnO nanocarriers exhibited only one stage, namely, a slight decrease in the weight of ZnO nanocarriers with a minor weight loss within the experimental temperature range. This can be ascribed to the small amount of water molecules absorbed and residual reaction solvent during the synthesis of the nanocarriers (Rotjanasuworapong et al., 2021). Similar to the unmodified material, the antimicrobial carrier functionalized with citral showed a weight reduction in the preliminary stage before reaching 150 °C. It is conjectured that this phenomenon is likely due to the thermal evaporation of adsorbed water or solvent on its surface. During the second stage (200–450 °C), the weight loss of the materials was mainly caused by the decomposition of the citral bonded to the carriers, which simultaneously proved that citral was successfully bonded onto the surface of ZnO-NPs (Sun et al., 2022). From the thermogravimetric curves, it is evident that ZNR exhibited the highest degree of citral functionalization (60.35%), followed by ZNS (54.13%). SAZ had the lowest degree of essential oil functionalization (15.63%) due to its variable morphology, fewer internal pores, and uneven pore size. Therefore, subsequent characterization and experiments were conducted on ZNR and ZNS.
Thermogravimetric assay curves of ZNRs, ZNSs, and SAZs before and after functionalization.
The infrared spectra of citral, ZNS, and ZNR materials before and after functionalization with citral are shown in Figure 6. FTIR analysis revealed that the peaks at wavelengths 581 cm−1 and 602 cm−1 for unmodified and citral-functionalized ZNS and ZNR corresponded to the stretching of the Zn–O bond. The values of these peaks may vary between 430 cm−1 and 610 cm−1 due to the different synthesis methods of the ZnO-NPs (Sharma and Garg, 2022). ZNR and ZNS exhibit a wide absorption peak at 3356 cm−1, which is likely associated with the characteristic absorption of the hydroxyl group in ZnO-NPs (Hong et al., 2009). In the spectra of citral, the FTIR spectral peaks near 2918 cm−1 and 2858 cm−1 are attributed to the stretching vibrations of CH3 and CH2, respectively; the absorption bands at 1671 cm−1 and 1717 cm−1 are attributed to the stretching vibrations of C=C and C=O, respectively; and the absorption bands at 1438 cm−1 and 1192 cm−1 are attributed to the coupling of bending vibrations and C–O stretching vibrations in the OH plane (Tian et al., 2018). In ZNS@C and ZNR@C, the characteristic bands of ZnO-NPs were observed at 581 cm−1 and 602 cm−1, respectively, and the relevant characteristic bands of citral were also detected. These characteristic peaks indicated the successful synthesis of antimicrobial materials ZNS@C and ZNR@C.
Fourier transform infrared spectroscopy of ZNRs and ZNSs before and after functionalization.
To gain a deeper understanding of the structure and porosity of ZnO nanoparticles, we analyzed ZNR and ZNS using N2 adsorption–desorption isotherms (Figure 7). According to the International Union of Pure and Applied Chemistry (IUPAC) classification, ZnO-NPs with various morphologies at high relative pressures exhibit V-shaped isotherms and H3-type hysteresis lines (P/P0 ranging from 0.1 to 1). The reason is that these materials are non-porous or macroporous, while ZnO-NPs are characterized by a homogeneous structure (Liu et al., 2009; Ouyang et al., 2021). The synthesized ZNR is a macroporous material, as determined by Figures 3A and 3B, while ZNS is a non-porous material, as shown in Figures 3C and 3D. Furthermore, the pore size distribution of ZNR is wider than that of ZNS. The pore size distribution of ZNS ranges mainly between 0 nm and 10 nm, while that of ZNR is mainly distributed between 0–20 nm and 200–400 nm. The number of pores in ZNR is higher, indicating that it has a larger pore volume to accommodate citral. ZnO-NPs binded with essential oil molecules, as shown in Figure 7C, exhibit a significant reduction in pore volume, which laterally confirms the thermogravimetric curve results, and that ZNR has the highest binding rate of citral.
N2 adsorption–desorption and pore size comparison of ZNR (A), ZNS (B), ZNR@C (C), and ZNS@C (D).
It can be seen from Table 1 that the specific surface area, average pore size, and pore volume of ZnO nanoparticles with different morphologies exhibited a decreasing trend before and after modification. This phenomenon could be attributed to the binding of citral, which occupies the pores and reduces their parameters, in agreement with the previous trend of pore parameter changes in essential oil molecules functionalized nanomaterials in the literature (Lu et al., 2023).
Pore structural parameters of ZNSs and ZNRs
| Materials | Surface area (m2/g) | Average pore diameter (nm) | Pore volume (cm3/g) |
|---|---|---|---|
| ZNR | 38.79 | 62.25 | 0.9364 |
| ZNS | 25.14 | 33.95 | 0.2134 |
| ZNR@C | 17.67 | 37. 53 | 0.3075 |
| ZNS@C | 11.56 | 26.17 | 0.0843 |
| Materials | Surface area (m2/g) | Average pore diameter (nm) | Pore volume (cm3/g) |
|---|---|---|---|
| ZNR | 38.79 | 62.25 | 0.9364 |
| ZNS | 25.14 | 33.95 | 0.2134 |
| ZNR@C | 17.67 | 37. 53 | 0.3075 |
| ZNS@C | 11.56 | 26.17 | 0.0843 |
Pore structural parameters of ZNSs and ZNRs
| Materials | Surface area (m2/g) | Average pore diameter (nm) | Pore volume (cm3/g) |
|---|---|---|---|
| ZNR | 38.79 | 62.25 | 0.9364 |
| ZNS | 25.14 | 33.95 | 0.2134 |
| ZNR@C | 17.67 | 37. 53 | 0.3075 |
| ZNS@C | 11.56 | 26.17 | 0.0843 |
| Materials | Surface area (m2/g) | Average pore diameter (nm) | Pore volume (cm3/g) |
|---|---|---|---|
| ZNR | 38.79 | 62.25 | 0.9364 |
| ZNS | 25.14 | 33.95 | 0.2134 |
| ZNR@C | 17.67 | 37. 53 | 0.3075 |
| ZNS@C | 11.56 | 26.17 | 0.0843 |
The antimicrobial activity of the materials was evaluated by determining the MIC (minimum inhibitory concentration) against representative foodborne microorganisms such as E. coli. Figure 8 displays the growth inhibition rates of E. coli following 24 h of treatment with varying concentrations of ZNR, ZNS, ZNR@C, and ZNS@C in comparison to untreated E. coli. In Figure 8A, we compared the effects of two unmodified antimicrobial carriers on the growth of E. coli. Both carriers showed low inhibition in the range of 0.05–0.8 mg/mL. The antibacterial property is attributed to the fact that zinc oxide nanomaterial itself is an inorganic metal antimicrobial agent, which can disrupt cell membranes (Gharpure and Ankamwar, 2020), bind to protein DNA, generate reactive oxygen species (Saha et al., 2020), interfere with the amplification process of bacterial DNA to alter gene expression, and have a direct bactericidal effect on Gram-negative bacteria such as E. coli. However, there was no significant difference in the inhibition ability of ZNR and ZNS at the same concentrations, indicating that the two initial morphologies had little effect on the antimicrobial capacity. ZNR@C exhibits stronger antibacterial properties than ZNR, as shown in Figure 8B. ZNR@C completely inhibited the growth of E. coli in the concentration range 0.2–0.8 mg/mL. Whereas ZNR partially inhibited the growth of E. coli within the concentration range of 0.05–0.1 mg/mL, the inhibition decreased from 88% to 3% as the concentration decreased. These findings are consistent with previous studies (Lu et al., 2023). Figure 8D shows that the antibacterial effect of ZNS@C, which did not achieve 100% inhibition of E. coli growth at concentrations of 0.2 mg/mL and below, is slightly inferior to that of ZNR@C. The main reasons for this are as follows: on the one hand, the binding rates of citral to ZNR and ZNS are different, as illustrated in Figure 5; on the other hand, the surface of the ZnO-NPs carriers after citral functionalization was positively charged, and the positive value was larger than that of ZNS@C, while the surface of E. coli cells was negatively charged, so the electrostatic interaction force between ZNR@C and E. coli was stronger than that of ZNS@C. The antimicrobial activity of ZnO nanocarriers functionalized with citral is primarily sourced from the interaction between citral with cell membranes (Kachur and Suntres, 2020), leading to the leakage of contents and disruption of cell integrity. Ultimately, this results in cell lysis and death.
Effects of different concentrations of antibacterial carriers—ZNR, ZNS (A), and citral-functionalized antibacterial materials—ZNR@C and ZNS@C (B, C, D), on the growth rate of E. coli (p<0.05).
The antimicrobial activity of carriers is influenced by various factors such as morphology, particle size, pore size, and essential oil loading. These factors predominantly affect the surface potential of newly synthesized nanomaterials and the loading of antimicrobial compounds to affect the antibacterial capacity (Velumani et al., 2022). Thus, ZNS@C could more easily attach to the cell surface and exert its bacteriostatic effect and ZNR@C was selected for subsequent in vitro antimicrobial experiments, as well as long-lasting bacterial inhibition and slow-release experiments.
The results of the in vitro antimicrobial activity of ZNR@C are presented in Figure 9. The inhibitory effect of the three samples on the growth of E. coli was demonstrated in Figure 9A by measuring the OD values of E. coli suspensions treated with ZNR, ZNR@C, and CIT. The bacterial inhibition of ZNR@C was not significant during the 0–4 h interval, probably due to the pH dependence of the controlled release of citral from this material. During the 4-h duration of action, E. coli produced acidic by-products to rapidly decrease the pH, at which the imine bonds in ZNR@C were only marginally broken, releasing a small number of citral. During the 4–24 h interval, ZNR exhibited some inhibitory ability, but it was weaker than that of the ZNR@C antimicrobial material. The trend and OD values of the bacterial solutions treated with ZNR@C and citral were approximately the same, indicating that the inhibitory ability of the synthesized antimicrobial material, ZNR@C, was mainly due to the presence of citral.
Comparison of antibacterial properties of antibacterial materials at 0.01 mg/mL concentration (A) and results of antibacterial coating plates at different concentrations of ZNR@C (B).
It can also be directly observed from Figure 9B that the antibacterial effect of the sample material on E. coli is as follows: within the concentration range of 0.2–0.8 mg/mL, ZNR@C had the strongest antibacterial ability and achieved complete inhibition. Compared to the high volatility and potent antimicrobial properties of pure citral, ZNR@C demonstrated superior stability and antimicrobial properties at different concentrations (0.1 mg/mL and 0.05 mg/mL).
Figure 10A represents the pH change of E. coli in TSB culture medium over 24 h. It can be observed that the pH of E. coli in TSB initially decreased sharply, then leveled off, and finally showed a slow increase as the incubation time increased. As shown in Figure 9, the antimicrobial effect of ZNR@C was not evident within the first 0–4 h. The reason for this may be the rapid proliferation of E. coli cells due to nutrient enrichment, resulting in the production of metabolites, which in turn caused a rapid drop in system pH. Based on the fit, the standard curve of citral was calculated in Figure 10B as y=0.1070+1.8905x, R2=0.9991. Figure 10C shows the release rate of citral from ZNR@C at different pH values (7.4, 6.5, and 5.0). As the system becomes more acidic, the material releases a greater amount of essential oil molecules and the release rate is faster. At pH 5.0, the release of essential oil molecules from the material increased rapidly to 80% within 12 h, and the maximum release exceeded 90%. In contrast, at a pH of 7.4, the release of essential oil molecules from the material remained at no more than 10% after 24 h. As the pH decreases and acidification increases, the imine bond connecting the amino group to citral is broken, releasing a significant amount of essential oil molecules to produce an antimicrobial effect (Zeng et al., 2017; Ren et al., 2021). Starting from the fifth hour, the pH of the bacterial growth environment reached between 5 and 6; as shown in Figure 10C, this pH is favorable for the release of citral. The growth rate of the E. coli colony slowed down between hours 15 and 24 due to the depletion of nutrients in the medium and the accumulation of harmful metabolites. E. coli continued to a later stage, which may result in apoptosis due to the discharge of contents, and the pH value gradually increased to 5.86.
The slow-release properties of ZNR@C antimicrobial materials. (A) pH change of E. coli grown in TSB; (B) standard curve of citral; and (C) release curve of citral from ZNR@C material.
It has been experimentally confirmed that ZNR@C, an antimicrobial material, functions as a pH-responsive essential oil controlled-release system. In this system, a small number of metabolites is produced when there is a low concentration of E. coli in the system, which lowers the pH slightly and opens a small number of imine bonds to release essential oil molecules for inhibiting the bacteria; whereas a large number of metabolites produced by E. coli can significantly lower the pH of the system, and the imine bonds are sufficiently destroyed to release a large number of essential oil molecules for inhibiting the bacteria (Li et al., 2018; Lu et al., 2023).
Figure 11 displays the growth inhibition rates of ZNR@C and citral against E. coli on different days of placement (5, 10, 20, 30, and 60 d). The results indicate that ZNR@C exhibits excellent long-lasting bacterial inhibition ability. It inhibited 100% of E. coli growth on day 10 and maintained 58.17% inhibition even on day 60. This performance surpasses that of synthetic antimicrobial materials previously reported in the literature (Si et al., 2021). This suggests that citral does not simply diffuse freely onto the ZNR, but rather forms strong covalent bonds with the nanomaterials (Peña-Gómez et al., 2019). The decrease in bacterial inhibitory ability observed between day 20 and day 60 can be attributed to the collapse of the microstructure of the ZnO-NP nanocarriers and the dilution of the essential oil concentration due to its hygroscopicity. In contrast, pure citral completely evaporated on day 20 and therefore could not exert its excellent bacteriostatic effect against E. coli.
The growth reduction of antibacterial materials with different storage days on E. coli: ZNR@C (A) and CIT (B).
SEM analysis of E. coli before and after ZNR@C treatment was performed to further investigate the antimicrobial mechanism of the antimicrobial material. As shown in Figure 12, the surface of E. coli before antimicrobial treatment appeared relatively smooth, and the cell membrane and cell wall were intact, with the complete cell morphology of E. coli. This is consistent with the pictures shown in the referenced literature. After antimicrobial treatment, the cell surface appeared concave and exhibited partial contraction, which may be due to the interaction between citral and the lipids present on the bacterial cell membrane, increasing the permeability of the cell membrane, leading to the leakage of the cell and subsequently affecting bacterial activity (Firmanda et al., 2023).
Scanning electron microscopy of E. coli before and after being treated with ZNR@C.
Conclusions
After screening by experiments, this study synthesized a novel zinc oxide porous nanorod (ZNR) using the synthetic template (β-cyclodextrin) to allow aggregation of ZNRs to grow and the surfactant (CTAB) to restrict its growth direction. Then, ZNR was modified with 3-APTES amino functionalization and covalently bound to citral via pH-sensitive imine bonds to obtain a high-quality, pH-responsive antimicrobial material (ZNR@C). The results revealed that this loading mode of chemical bonding could effectively decrease the volatilization of citral and enhance its antimicrobial activity. The positive charge on the surface of the synthesized ZNR@C can attract the negatively charged Escherichia coli, so as to achieve a better antibacterial effect. The weakly acidic metabolites produced during the growth of E. coli in the system of the novel antimicrobial material ZNR@C can lower the pH of the system, break down the imine bond, and release the essential oil to inhibit bacteria. The more E. coli there are, comparatively the pH of the system becomes more acidic, the higher the rate of imine bond disruption of ZNR@C is, the more essential oil molecules are released, and the better the antibacterial effect, thus confirming that this novel antimicrobial material, ZNR@C, is a pH-responsive essential oil-controlled-release system that effectively inhibits E. coli.
In conclusion, this study presents a feasible method for immobilizing beneficial natural antimicrobial compounds onto nanocarriers through covalent bonding, provides new insights for the research and development of food packaging materials utilizing natural essential oils, increases the application prospects of natural antimicrobial essential oils in the food antibacterial field (for example, bacterial filtration of liquid foods), and provides a theoretical foundation for the application of this innovative antimicrobial material in reducing the risk of foodborne illness.
Author Contributions
Yanan Fan: Conceptualization, data curation, formal analysis, writing original draft, review & editing, visualization, and software. Qixiang Xu: Conceptualization, data curation, formal analysis, writing original draft, and visualization. Keyu Ren: Software, review & editing, and visualization. Mengge Zhai: Conceptualization, methodology, and resources. Guozheng Xing: Conceptualization, methodology, and data curation. Yishan Song: Supervision, review & editing, project administration, and funding acquisition. Yongheng Zhu: Visualization, supervision, review & editing, project administration, and funding acquisition.
Funding
This research was funded by the National Natural Science Foundation of China (No.32272399).
Conflict of Interest
The authors declare that they have no known competing financial interests or persona relationships that could have appeared to influence the work reported in this paper.
References
Author notes
Yanan Fan and Qixiang Xu are co-first authors of the paper.
