4.5.1. Biofilm Formation by *E. coli*

Microtiter plate assay or tissue culture plate (TCP) was used for quantitative determination of *E. coli* biofilm formation in accordance with O'Toole with some modification [65]. In brief, a sterile polystyrene tissue culture plate (composed of 96 flat bottom wells) was filled with 200 µL of the diluted prepared bacterial suspension and incubated at 37 ◦C for 24 h. Then, the content of each well was gently removed by tapping the plates, and the wells were washed twice with 200 µL phosphate buffer saline (PBS) (pH 7.2) to remove free-floating 'planktonic' bacteria. Biofilms formed by adherent 'sessile' bacteria on plate were fixed by placing them in the incubator at 37 ◦C for 30 min. Then, the wells were stained with 200 µL of 1% crystal violet solution for 45 min. After that, the microtiter plates were rinsed three times with sterile distilled water to remove excess dye and left to dry for 45 min at room temperature, then de-stained by adding 200 µL ethanol. A sterile bacterial broth was used as a negative control to identify non-specific binding. A micro-ELISA reader (at 570/655 nm wavelength) was used to measure the optical densities (OD) of stained bacterial biofilms. The experiment was conducted in triplicate and the average OD values were considered. The results were graded into strong, moderate, and non or weak biofilm.

### 4.5.2. Biofilm Inhibition Assay

Catechin at MIC value was added to each well of a 96-well microplate except for the positive control well, which contained gentamicin and the negative control well, which contained bacterial broth only [66,67]. Bacterial culture (1 <sup>×</sup> <sup>10</sup><sup>8</sup> CFU/mL) in an amount of 100 µL was pipetted into each well and the well was then incubated at 37 ◦C for 18 h. After that, the content of each well was removed, and the wells were rinsed three times with distilled water and allow to dry at 60 ◦C for 45 min. Then the wells were stained with 200 µL of 1% crystal violet and incubated at room temperature for 30 min. Finally, the plates were rinsed with distilled water, de-stained with ethanol, and incubated at room temperature for 15 min. A microplate ELISA reader (model 680, Bio-Rad, Hercules, CA, USA) at 570/655 nm was used to measure the optical densities. The experiment was conducted in triplicate and the mean absorbance was considered. The percentage of biofilm inhibition was then compared with the positive control and calculated according to the following formula:

$$\% \text{ Of inhibition} = \frac{\text{OD control} - \text{OD treatment} \ast 100}{\text{OD control}} \tag{1}$$

### *4.6. In Silico Study* Molecular Docking

A docking study was conducted to predict the target protein for catechin antibiofilm action. In a previous study, the *acrA* gene that is related to antibiotic resistance of *E. coli* biofilm was identified [11]. The crystal structure of the target protein was retrieved from the Protein Data Bank database (https://www.rcsb.org/, accessed on 25 April 2022). AcrA protein (PDB-ID: 5NG5) is a component of the *E. coli* AcrAB-TolC efflux pump and consists of an AcrA subunit with six chains and 373 sequence length, an AcrB subunit with three chains and 1049 sequence length, an AcrZ subunit with three chains and 55 sequence length, and a TolC protein with three chains and 493 sequence length. The target protein was prepared for docking by removing water, adding hydrogens and kollman charges and saved as pdb format using Autodock tools 1.5.1 [68]. Then the natural ligand was extracted from the protein using Discovery Studio Visualizer software (https://discover.3ds.com/ discovery-studio-visualizer-download, accessed on 25 April 2022). The ligand and the

protein were edited in Autodock tools 1.5.1. The grid box was centered on the ligand and control docking was run using Autodock Vina 1.1.2 software [69]. The same protocol was repeated with catechin as the ligand and the 3D structure of catechin (ID: 9064) was obtained from the PubChem data base (https://pubchem.ncbi.nlm.nih.gov/, accessed on 26 April 2022). The docking simulation was repeated three times, and the average binding affinity was considered. Several parameters were evaluated, such as the binding affinity of catechin toward the target protein, the ligand interaction site, the amino acids involved in the binding pocket, and the bonds formed at the interaction site. Results were analyzed using PyMOL [70] and visualized using Discovery Studio Visualizer (Dassault Systèmes, San Diego, CA, USA).

### *4.7. Gene Expression Using Quantitative Real-Time RT-PCR*

To evaluate the effect of catechin on biofilm-related gene expression, quantitative realtime PCR (Mx3000P qPCR System, Agilent, Santa Clara, USA) was performed on *E. coli* clinical isolates with and without catechin (at the sub-inhibitory concentration = 0.5 mg/mL) [71]. *E. coli* clinical isolates (2 <sup>×</sup> <sup>10</sup><sup>5</sup> CFU) were inoculated in 250 mL of tryptic soy broth (TSB) [50]. Quick-RNA™ Fungal/Bacterial Microprep Kit was used to isolate the total RNA according to the manufacturer's instructions (Zymo, USA). The RNA was treated with DNase/RNase-Free water to remove genomic DNA. The concentration and optical absorbance of each extracted RNA were confirmed with Nanodrop (Epoch Microplate Spectrophotometer, Agilent, Santa Clara, USA) at 260 nm/280 nm and samples were kept at <sup>−</sup><sup>80</sup> ◦<sup>C</sup> to elicit DNA contamination from total isolated RNA. EasyScript® First-Strand cDNA Synthesis SuperMix kit was used to synthesize the cDNA according to the manufacturer's instructions (Transgen/China). The cDNA reaction components including Random Primer (N9) in a volume of 1 <sup>µ</sup>L, 2×ES Reaction Mix (10 <sup>µ</sup>L), EasvScript®RT/RI Enzyme Mix (1 µL), RNase-free Water (Up to 20 µL), Eluted RNA (5 µL). qRT-PCR was performed by the SYBR Green gene expression assay [27]. In each sterile PCR tube, sample components of Real MODTM Green W2 2x qPCRmix in a volume of 10 µL, Forward Primer (10 µM) in a volume of 2.0 µL, Reverse Primer (10 µM) in a volume of 2.0 µL, Template DNA (4 µL), and DNase/RNase free water (up to 20 µL) were added. The thermal program was performed as follows: initial activation step, which was optimized at 95 ◦C for 10 min, 1 cycle; denaturation step, which was optimized at 95 ◦C, for 30 s; annealing was optimized at 60 ◦C, for 30 s; extension was optimized at 72 ◦C for 30 s. Denaturation, annealing, and extension steps were performed over 40 cycles. Then there was the final extension, which was optimized at 72 ◦C, for 5 min, 1 cycle. The 16S rRNA gene was used as a housekeeping gene, while the *acrA* gene was used as a target gene for *E. coli.* The primers used in this study are listed in Table 6.

**Table 6.** The primer sequences for the real time qPCR analysis.


### *4.8. Statistical Analysis*

All experiments were performed in triplicate and data were expressed as mean ± standard deviation using GraphPad Prism 9 software for Mac, www.graphpad.com (GraphPad, San Diego, CA, USA). The data were analyzed using one-way ANOVA followed by Tukey test. For the gene expression, unpaired T-test was performed. A significant difference was considered at the level of *p* < 0.01. Data for the percentage of biofilm inhibiting activity of catechin were presented using Microsoft Excel.

### **5. Conclusions**

Biofilm-forming *E. coli* places a significant burden on the health of the population. With a high rate of recurrence, *E. coli* becomes increasingly resistant to antimicrobial treatments and more difficult to eradicate. Catechin isolated from *Canarium patentinervium* Miq. exhibited strong biofilm inhibiting activity by reducing the expression of the biofilmrelated *acrA* gene. This study highlights how important natural products are for treating infectious diseases that are resistant to the currently available antimicrobials.

**Author Contributions:** Conceptualization, M.R.; methodology, N.J. and Y.K.M.; software, A.F and N.J.; validation, M.R. and N.H.A.; formal analysis, A.F. and N.J. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Data Availability Statement:** The data used and/or analyzed in this study are available from the corresponding author on reasonable request.

**Conflicts of Interest:** The authors declare that they have no conflict of interest.

### **References**


*Article*

**Jiaman Xu 1,2, Quan Lin 1,2, Maokun Sheng 1,2, Ting Ding 1,2, Bing Li <sup>3</sup> , Yan Gao <sup>4</sup> and Yulong Tan 1,2,\***


**\*** Correspondence: tanyulong@qau.edu.cn; Tel.: +86-532-5895-7918

**Abstract:** Food contamination caused by food-spoilage bacteria and pathogenic bacteria seriously affects public health. *Staphylococcus aureus* is a typical foodborne pathogen which easily forms biofilm. Once biofilm is formed, it is difficult to remove. The use of nanotechnology for antibiofilm purposes is becoming more widespread because of its ability to increase the bioavailability and biosorption of many drugs. In this work, chitosan nanoparticles (CSNPs) were prepared by the ion–gel method with polyanionic sodium triphosphate (TPP). Cinnamaldehyde (CA) was loaded onto the CSNPs. The particle size, potential, morphology, encapsulation efficiency and in vitro release behavior of cinnamaldehyde–chitosan nanoparticles (CSNP-CAs) were studied, and the activity of CA against *S. aureus* biofilms was evaluated. The biofilm structure on the silicone surface was investigated by scanning electron microscopy (SEM). Confocal laser scanning microscopy (CLSM) was used to detect live/dead organisms within biofilms. The results showed that CSNP-CAs were dispersed in a circle with an average diameter of 298.1 nm and a zeta potential of +38.73 mV. The encapsulation efficiency of cinnamaldehyde (CA) reached 39.7%. In vitro release studies have shown that CA can be continuously released from the CSNPs. Compared with free drugs, CSNP-CAs have a higher efficacy in removing *S. aureus* biofilm, and the eradication rate of biofilm can reach 61%. The antibiofilm effects of CSNP-CAs are determined by their antibacterial properties. The minimum inhibitory concentration (MIC) of CA is 1.25 mg/mL; at this concentration the bacterial cell wall ruptures and the permeability of the cell membrane increases, which leads to leakage of the contents. At the same time, we verified that the MIC of CSNP-CAs is 2.5 mg/mL (drug concentration). The synergy between CA and CSNPs demonstrates the combinatorial application of a composite as an efficient novel therapeutic agent against antibiofilm. We can apply it in food preservation and other contexts, providing new ideas for food preservation.

**Keywords:** chitosan; nanoparticle; antibiofilm; cinnamaldehyde; *Staphylococcus aureus*

### **1. Introduction**

Foodborne diseases are prominent health problems. Due to the long food chain and complex pathogen sources, food is easily contaminated. Meat, poultry, eggs, aquatic products, dairy products and other foods are very vulnerable to *Staphylococcus aureus* [1]. *S. aureus* often contaminates food in the following ways: via food-processing personnel, when food-processing staff or cooks' hands or clothes are contaminated with germs and are not cleaned; when the food itself is contaminated before processing or is contaminated during processing, resulting in enterotoxin and food poisoning; when the packaging of cooked food products is not sealed and the contents are contaminated during transportation; and when, e.g., dairy cows suffer from suppurative mastitis or there is local purulence in livestock, contamination can occur in other parts of the body, etc. Biofilm can also form on

**Citation:** Xu, J.; Lin, Q.; Sheng, M.; Ding, T.; Li, B.; Gao, Y.; Tan, Y. Antibiofilm Effect of Cinnamaldehyde-Chitosan Nanoparticles against the Biofilm of *Staphylococcus aureus*. *Antibiotics* **2022**, *11*, 1403. https://doi.org/10.3390/ antibiotics11101403

Academic Editor: Wolf-Rainer Abraham

Received: 6 September 2022 Accepted: 2 October 2022 Published: 13 October 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Qingdao 266071, China

the surfaces of food and food-processing equipment [2]. Bacterial biofilm refers to a large number of aggregated, membrane-like substances formed by bacteria adhering to a contact surface and secreting polysaccharide matrix, fibrin, lipid protein, etc. [3,4]. The extracellular polymers on the surface of the biofilm are intricately aggregated, forming a complex and orderly overall structure, which effectively protects the stability of the biofilm on the carrier surface. The microorganisms in the biofilm occur together in the form of clusters and are firmly adsorbed on the surface of the carrier. Using common physicochemical methods is often difficult to completely remove biofilm. The physical methods include ultrasonic [5,6], low-current and other methods, such as applications of electromagnetic and shock waves. Although physical methods can meet the requirements for removing biofilms, it is difficult to achieve the desired effect for some large-scale processing equipment. The chemical methods include disinfectants and antibiotics, etc. [7]. These common chemical disinfectants, such as sodium hypochlorite and chlorine dioxide, and antibiotics, such as penicillin and tetracycline, cannot achieve ideal removal effects, and their use will also lead to the development of bacterial drug resistance [8]. Therefore, new drugs to overcome biofilm resistance and eliminate biofilm-protected bacteria need to be developed.

Cinnamaldehyde (CA) is an active component extracted from cinnamon bark, which has a variety of effects, such as anticancer, antifungal and antibacterial activities [9–11]. It has been reported that CA can repress bacteria, yeasts and filamentous molds by inhibiting ATPases, cell-wall biosynthesis and by changing membrane structure and integrity [11]. Albano et al. confirmed that CA had the ability to reduce the growth of *Staphylococcus epidermidis* in planktonic state, inhibit biofilm formation and eradicate formed biofilm [12]. Yu et al. found that *Campylobacter* strains treated with 15.63 µg/mL CA exhibited significantly decreased bacterial auto-aggregation, motility, exopolysaccharide production and soluble protein levels, which proved that it had the ability to remove biofilm [13]. CA also had an inhibitory effect on *Candida albicans* by inhibiting the release of its virulence factors [14]. Kot et al. found that trans-cinnamaldehyde is a promising antibiofilm agent for use in MRSA-biofilm-related infections [15].

Since the antibacterial properties of essential oils (EOs) are limited by their high volatility and water-insolubility, the stability of EOs has become a key issue in recent years [16]. Nanoparticles (NPs) have good biocompatibility and can be more stable for the release of EOs and have been widely used in encapsulating EOs [17]. NPs loaded with an EO can protect the EO from the external environment to prolong its inhibitory effect on microorganisms. Chitosan (CS) is an alkaline polysaccharide containing more free amino groups. It is used as a biological carrier, having good safety and biocompatibility, and has been identified as having an effective antibacterial membrane effect [18–22]. When the pH is lower than 10, the amino groups will be protonated and positively charged. The lipid layer on the surface of the biofilm is negatively charged and easy to electrostatically adsorb with positively charged chitosan particles [23]. As a pharmaceutical carrier, chitosan nanoparticles (CSNPs), with the advantages of slow or controlled drug release, can improve drug solubility and stability, thereby enhancing drug effects and reducing the side effects of drugs [24]. This paper mainly studied the antibiotic film effect of chitosan nanoparticles with cinnamaldehyde.

### **2. Results**

### *2.1. Characterization of Nanoparticles*

The surface morphologies and morphologies of CSNPs were shown by TEM. CSNP-CAs had smooth surfaces and nearly spherical shapes (Figure 1). The average diameters of the CSNPs and CSNP-CAs were estimated to be 167.3 nm and 298.1 nm, respectively. The surface zeta potentials of the CSNPs and CSNP-CAs were (+34.60) mV and (+38.73) mV, respectively. The average amount of CA on the carriers was 39.7%.

The surface zeta potentials of the CSNPs and CSNP-CAs were (+34.60) mV and (+38.73)

The surface zeta potentials of the CSNPs and CSNP-CAs were (+34.60) mV and (+38.73)

mV, respectively. The average amount of CA on the carriers was 39.7%.

mV, respectively. The average amount of CA on the carriers was 39.7%.

*Antibiotics* **2022**, *11*, 1403 3 of 13

**Figure 1.** TEM image of CSNP-CAs. **Figure 1.** TEM image of CSNP-CAs. **Figure 1.** TEM image of CSNP-CAs.

#### *2.2. In Vitro Release Studies 2.2. In Vitro Release Studies 2.2. In Vitro Release Studies*

It can be seen from the figure (Figure 2) that the release effect of CSNP-CAs can be significantly divided into two stages. The drug release was rapid in the early stage, including the release of unencapsulated CA adsorbed on the surface of the nanoparticles. The drug release was slow in the later stage, which was due to the gradual release of the drug inside the NPs. The cumulative release amount was about 77.01% in 7 h, and the release tended to be gentle after 10 h. The cumulative release amount reached 86.00% in 48 h. It can be seen from the figure (Figure 2) that the release effect of CSNP-CAs can be significantly divided into two stages. The drug release was rapid in the early stage, including the release of unencapsulated CA adsorbed on the surface of the nanoparticles. The drug release was slow in the later stage, which was due to the gradual release of the drug inside the NPs. The cumulative release amount was about 77.01% in 7 h, and the release tended to be gentle after 10 h. The cumulative release amount reached 86.00% in 48 h. It can be seen from the figure (Figure 2) that the release effect of CSNP-CAs can be significantly divided into two stages. The drug release was rapid in the early stage, including the release of unencapsulated CA adsorbed on the surface of the nanoparticles. The drug release was slow in the later stage, which was due to the gradual release of the drug inside the NPs. The cumulative release amount was about 77.01% in 7 h, and the release tended to be gentle after 10 h. The cumulative release amount reached 86.00% in 48 h.

**Figure 2.** In vitro release profiles for CA from CA-loaded chitosan nanoparticles, using an initial Time (h) **Figure 2.** In vitro release profiles for CA from CA-loaded chitosan nanoparticles, using an initial weight ratio of chitosan to CA of 4:1. Data are presented as means ± SDs, *n* = 3. **Figure 2.** In vitro release profiles for CA from CA-loaded chitosan nanoparticles, using an initial weight ratio of chitosan to CA of 4:1. Data are presented as means ± SDs,*n* = 3.

weight ratio of chitosan to CA of 4:1. Data are presented as means ± SDs, *n* = 3.

#### *2.3. Antibacterial Activity of CA and CSNP-CAs* The antibacterial activity of CA against *S. aureus* strains (NCTC 8325, RN6390, 15981,

*2.3. Antibacterial Activity of CA and CSNP-CAs*

*Antibiotics* **2022**, *11*, 1403 4 of 13

The antibacterial activity of CA against *S. aureus* strains (NCTC 8325, RN6390, 15981, Col) were evaluated in vitro by measuring MIC and MBC values. The MIC and MBC values for this strain were 1.25 mg/mL and 2.5 mg/mL when the MIC of CSNP-CAs was 2.5 mg/mL. Col) were evaluated in vitro by measuring MIC and MBC values. The MIC and MBC values for this strain were 1.25 mg/mL and 2.5 mg/mL when the MIC of CSNP-CAs was 2.5 mg/mL. *2.4. Inhibition Activity on Biofilm Formation of CA and CSNP-CAs*

#### *2.4. Inhibition Activity on Biofilm Formation of CA and CSNP-CAs* As can be seen in Figure 3, biofilm reduction was achieved with all the concentrations

As can be seen in Figure 3, biofilm reduction was achieved with all the concentrations of CA tested in this work. Both free CA and CSNP-CAs showed good biofilm inhibition. The activities of free CA and loaded CA both increased with the increase in CA concentration, indicating that the inhibition activities of free CA and loaded CA with respect to biofilm were concentration-dependent. For free CA, there was almost no biofilm formation when the concentration was greater than 1.25 mg/mL. In the same concentration range, the inhibitory activity of loaded CA (CSNP-CAs) was slightly lower than that of free CA. of CA tested in this work. Both free CA and CSNP-CAs showed good biofilm inhibition. The activities of free CA and loaded CA both increased with the increase in CA concentration, indicating that the inhibition activities of free CA and loaded CA with respect to biofilm were concentration-dependent. For free CA, there was almost no biofilm formation when the concentration was greater than 1.25 mg/mL. In the same concentration range, the inhibitory activity of loaded CA (CSNP-CAs) was slightly lower than that of free CA.

**Figure 3.** Inhibition effects of NPs on biofilm in 96-well microplates. The results represent the means and standard deviations (error bars) of three independent experiments. *\* p* < 0.05 for comparison between the untreated and treated groups. **Figure 3.** Inhibition effects of NPs on biofilm in 96-well microplates. The results represent the means and standard deviations (error bars) of three independent experiments. \* *p* < 0.05 for comparison between the untreated and treated groups.

#### *2.5. Antibiofilm Activity on Mature Biofilm of CA and CSNP-CAs 2.5. Antibiofilm Activity on Mature Biofilm of CA and CSNP-CAs*

In order to evaluate the effect of CSNP-CAs on the established biofilm, we assessed the antibiofilm effect of CA and CSNP-CAs by repeated treatment for two days. Figure 4 shows the antibiofilm activity of CSNP-CAs and CA on mature biofilm at various CA concentrations after 48 h of treatment. As can be seen from the figure, the biofilm was removed at each concentration, but the effect was different. The removal effect of the CSNP-CAs was better than that of free CA. When the drug concentration reached 4 × MIC, the clearance rate of CSNP-CAs for the biofilm was 48.10% and that of free CA was 38.66%. In order to evaluate the effect of CSNP-CAs on the established biofilm, we assessed the antibiofilm effect of CA and CSNP-CAs by repeated treatment for two days. Figure 4 shows the antibiofilm activity of CSNP-CAs and CA on mature biofilm at various CA concentrations after 48 h of treatment. As can be seen from the figure, the biofilm was removed at each concentration, but the effect was different. The removal effect of the CSNP-CAs was better than that of free CA. When the drug concentration reached 4 × MIC, the clearance rate of CSNP-CAs for the biofilm was 48.10% and that of free CA was 38.66%.

**Figure 4.** Disruption effects of NPs on mature biofilm in 96-well microplates. The results represent the means and standard deviations (error bars) of three independent experiments. *\* p* < 0.05 for comparison between the untreated and treated groups. **Figure 4.** Disruption effects of NPs on mature biofilm in 96-well microplates. The results represent the means and standard deviations (error bars) of three independent experiments. \* *p* < 0.05 for comparison between the untreated and treated groups. **Figure 4.** Disruption effects of NPs on mature biofilm in 96-well microplates. The results represent the means and standard deviations (error bars) of three independent experiments. *\* p* < 0.05 for comparison between the untreated and treated groups.

#### *2.6. Antibiofilm Effect of CSNP-CAs on Food-Grade Silicone 2.6. Antibiofilm Effect of CSNP-CAs on Food-Grade Silicone*

*2.6. Antibiofilm Effect of CSNP-CAs on Food-Grade Silicone* The biofilm grown on food-grade silicone platelets was observed by SEM (Figure 5). *S. aureus* biofilm grown without CA treatment exhibited the typical 3D morphology with dense structures and water channels (Figure 5A). Under the same conditions but treated with free CA and CSNP-CAs, the biofilm showed structural changes (Figure 5B–C). CA can be removed by bactericidal action to form biofilms (Figure 5B). The loading of CA and NPs enhanced the removal of biofilms (Figure 5C). The silicone surface showed more The biofilm grown on food-grade silicone platelets was observed by SEM (Figure 5). *S. aureus* biofilm grown without CA treatment exhibited the typical 3D morphology with dense structures and water channels (Figure 5A). Under the same conditions but treated with free CA and CSNP-CAs, the biofilm showed structural changes (Figure 5B–C). CA can be removed by bactericidal action to form biofilms (Figure 5B). The loading of CA and NPs enhanced the removal of biofilms (Figure 5C). The silicone surface showed more blank areas, with only single or no cells sticking to the surface. The biofilm grown on food-grade silicone platelets was observed by SEM (Figure 5). *S. aureus* biofilm grown without CA treatment exhibited the typical 3D morphology with dense structures and water channels (Figure 5A). Under the same conditions but treated with free CA and CSNP-CAs, the biofilm showed structural changes (Figure 5B–C). CA can be removed by bactericidal action to form biofilms (Figure 5B). The loading of CA and NPs enhanced the removal of biofilms (Figure 5C). The silicone surface showed more blank areas, with only single or no cells sticking to the surface.

**Figure 5.** SEM images of *S. aureus* biofilm formations on medical-grade silicone surfaces with media **Figure 5.** SEM images of *S. aureus* biofilm formations on medical-grade silicone surfaces with media without treatment (**A**) and with CA (**B**) and CSNP-CA treatments (**C**). **Figure 5.** SEM images of *S. aureus* biofilm formations on medical-grade silicone surfaces with media without treatment (**A**) and with CA (**B**) and CSNP-CA treatments (**C**).

without treatment (**A**) and with CA (**B**) and CSNP-CA treatments (**C**). The images obtained with CLSM (Live/Dead staining) confirmed the results. In the control group (Figure 6A), a thick green structure (live cells) could be seen, indicating a mature biofilm structure with active cells. Free CA treatment (Figure 6B) resulted in a reduction in biofilm thickness. Biofilms had less biomass and reduced biofilm thickness, and more biofilms were stained red (dead cells). After the treatment with CSNP-CAs (Figure 6C), biofilms had less biomass and reduced biofilm thickness, the biofilm was almost completely red (dead cells) and the structure of the biofilm was destroyed. In conclusion, The images obtained with CLSM (Live/Dead staining) confirmed the results. In the control group (Figure 6A), a thick green structure (live cells) could be seen, indicating a mature biofilm structure with active cells. Free CA treatment (Figure 6B) resulted in a reduction in biofilm thickness. Biofilms had less biomass and reduced biofilm thickness, and more biofilms were stained red (dead cells). After the treatment with CSNP-CAs (Figure 6C), biofilms had less biomass and reduced biofilm thickness, the biofilm was almost completely red (dead cells) and the structure of the biofilm was destroyed. In conclusion, the CSNP-CAs could better destroy the structure of biofilm and kill the cells. The images obtained with CLSM (Live/Dead staining) confirmed the results. In the control group (Figure 6A), a thick green structure (live cells) could be seen, indicating a mature biofilm structure with active cells. Free CA treatment (Figure 6B) resulted in a reduction in biofilm thickness. Biofilms had less biomass and reduced biofilm thickness, and more biofilms were stained red (dead cells). After the treatment with CSNP-CAs (Figure 6C), biofilms had less biomass and reduced biofilm thickness, the biofilm was almost completely red (dead cells) and the structure of the biofilm was destroyed. In conclusion, the CSNP-CAs could better destroy the structure of biofilm and kill the cells.

the CSNP-CAs could better destroy the structure of biofilm and kill the cells.

**Figure 6.** CLSM images of *S. aureus* biofilm formations on medical-grade silicone surfaces without treatment (**A**) and with CA (**B**) and CSNP-CA treatments (**C**). Biofilms were stained with the Live/Dead® BacLight™ Bacterial Viability and Counting Kit. CLSM reconstructions show the threedimensional staining patterns for live cells (SYTO-9, green) and dead cells (propidium iodide, red). Magnification, ×10. **Figure 6.** CLSM images of *S. aureus* biofilm formations on medical-grade silicone surfaces without treatment (**A**) and with CA (**B**) and CSNP-CA treatments (**C**). Biofilms were stained with the Live/Dead® BacLight™ Bacterial Viability and Counting Kit. CLSM reconstructions show the threedimensional staining patterns for live cells (SYTO-9, green) and dead cells (propidium iodide, red). Magnification, ×10. **Figure 6.** CLSM images of *S. aureus* biofilm formations on medical-grade silicone surfaces without treatment (**A**) and with CA (**B**) and CSNP-CA treatments (**C**). Biofilms were stained with the Live/Dead® BacLight™ Bacterial Viability and Counting Kit. CLSM reconstructions show the threedimensional staining patterns for live cells (SYTO-9, green) and dead cells (propidium iodide, red). Magnification, ×10.

*Antibiotics* **2022**, *11*, 1403 6 of 13

#### *2.7. Effect of CA on Cell-Wall Integrity of S. aureus 2.7. Effect of CA on Cell-Wall Integrity of S. aureus 2.7. Effect of CA on Cell-Wall Integrity of S. aureus*

As shown in Figure 7A, the content of extracellular alkaline phosphatase in the bacterial solution treated with CA was increased by more than threefold. The content of alkaline phosphatase reached the maximum at about 8 h, and it tended to be stable after 8 h. It could be inferred that CA could destroy the cell wall of *S. aureus* and increase the permeability of the cell wall. As shown in Figure 7A, the content of extracellular alkaline phosphatase in the bacterial solution treated with CA was increased by more than threefold. The content of alkaline phosphatase reached the maximum at about 8 h, and it tended to be stable after 8 h. It could be inferred that CA could destroy the cell wall of *S. aureus* and increase the permeability of the cell wall. As shown in Figure 7A, the content of extracellular alkaline phosphatase in the bacterial solution treated with CA was increased by more than threefold. The content of alkaline phosphatase reached the maximum at about 8 h, and it tended to be stable after 8 h. It could be inferred that CA could destroy the cell wall of *S. aureus* and increase the permeability of the cell wall.

for *S. aureus* treated with CA. **Figure 7.** The changes in AKP concentration (**A**), K<sup>+</sup> concentration (**B**) and protein concentration (**C**) for *S. aureus* treated with CA. **Figure 7.** The changes in AKP concentration (**A**), K<sup>+</sup> concentration (**B**) and protein concentration (**C**) for *S. aureus* treated with CA.

**Figure 7.** The changes in AKP concentration (**A**), K<sup>+</sup> concentration (**B**) and protein concentration (**C**)

#### *2.8. Effect of CA on Membrane Permeability of S. aureus 2.8. Effect of CA on Membrane Permeability of S. aureus* Figure 8 clearly shows the effect of CA on the morphology of *S. aureus.* In the control group without CA, the bacterial cells were spherical and relatively intact, and the bacterial

*2.8. Effect of CA on Membrane Permeability of S. aureus*

*Antibiotics* **2022**, *11*, 1403 7 of 13

Figure 8 clearly shows the effect of CA on the morphology of *S. aureus.* In the control group without CA, the bacterial cells were spherical and relatively intact, and the bacterial cell membrane was smooth and not broken (Figure 8A). When the concentration of CA was 1 × MIC, *S. aureus* lost its original spherical state, many cells collapsed and cell membranes even ruptured (Figure 8B). After adding CA, the permeability of cell membranes increased, leading to the outflow of potassium ions and proteins in cells. In Figure 7B, when CA was added to the bacterial solution, the concentration of extracellular potassium ions increased gradually and reached a peak at 5 h. Figure 8 clearly shows the effect of CA on the morphology of *S. aureus.* In the control group without CA, the bacterial cells were spherical and relatively intact, and the bacterial cell membrane was smooth and not broken (Figure 8A). When the concentration of CA was 1 × MIC, *S. aureus* lost its original spherical state, many cells collapsed and cell membranes even ruptured (Figure 8B). After adding CA, the permeability of cell membranes increased, leading to the outflow of potassium ions and proteins in cells. In Figure 7B, when CA was added to the bacterial solution, the concentration of extracellular potassium ions increased gradually and reached a peak at 5 h. cell membrane was smooth and not broken (Figure 8A). When the concentration of CA was 1 × MIC, *S. aureus* lost its original spherical state, many cells collapsed and cell membranes even ruptured (Figure 8B). After adding CA, the permeability of cell membranes increased, leading to the outflow of potassium ions and proteins in cells. In Figure 7B, when CA was added to the bacterial solution, the concentration of extracellular potassium ions increased gradually and reached a peak at 5 h.

**Figure 8.** SEM images of *S. aureus* untreated (**A**) and treated (**B**) with cinnamaldehyde for 4 h. **Figure 8.** SEM images of *S. aureus* untreated (**A**) and treated (**B**) with cinnamaldehyde for 4 h.

Meanwhile, the concentration of protein in the bacterial suspension also increased gradually (Figure 7C). Compared with the control, the protein content increased six times. This result showed that the addition of CA could rupture the cells of *S. aureus* and cause cytoplasmic leakage. Meanwhile, the concentration of protein in the bacterial suspension also increased gradually (Figure 7C). Compared with the control, the protein content increased six times. This result showed that the addition of CA could rupture the cells of *S. aureus* and cause cytoplasmic leakage. gradually (Figure 7C). Compared with the control, the protein content increased six times. This result showed that the addition of CA could rupture the cells of *S. aureus* and cause cytoplasmic leakage. The damaged membranes of *S. aureus* cells were observed by flow cytometry, as

Meanwhile, the concentration of protein in the bacterial suspension also increased

The damaged membranes of *S. aureus* cells were observed by flow cytometry, as shown in Figure 9. The figure shows three different types of cells, namely, FITC-/PI- living cells (Q3), FITC+/PI+ dead cells (Q2) and FITC-/PI+ necrotic cells and debris (Q1). It can be seen from the figure (Figure 9A) that almost all control cells were clustered in the Q3 type, which proves that *S. aureus* has a complete cell-membrane structure in this state. However, after CA treatment (Figure 9B), the majority of cells changed from Q3 to Q1, which revealed that the integrity of *S. aureus* membranes was disrupted after CA treatment. This phenomenon is consistent with the results observed in SEM experiments*.* All experiments proved that cell membranes were damaged after CA treatment. The damaged membranes of *S. aureus* cells were observed by flow cytometry, as shown in Figure 9. The figure shows three different types of cells, namely, FITC-/PI- living cells (Q3), FITC+/PI+ dead cells (Q2) and FITC-/PI+ necrotic cells and debris (Q1). It can be seen from the figure (Figure 9A) that almost all control cells were clustered in the Q3 type, which proves that *S. aureus* has a complete cell-membrane structure in this state. However, after CA treatment (Figure 9B), the majority of cells changed from Q3 to Q1, which revealed that the integrity of *S. aureus* membranes was disrupted after CA treatment. This phenomenon is consistent with the results observed in SEM experiments. All experiments proved that cell membranes were damaged after CA treatment. shown in Figure 9. The figure shows three different types of cells, namely, FITC-/PI- living cells (Q3), FITC+/PI+ dead cells (Q2) and FITC-/PI+ necrotic cells and debris (Q1). It can be seen from the figure (Figure 9A) that almost all control cells were clustered in the Q3 type, which proves that *S. aureus* has a complete cell-membrane structure in this state. However, after CA treatment (Figure 9B), the majority of cells changed from Q3 to Q1, which revealed that the integrity of *S. aureus* membranes was disrupted after CA treatment. This phenomenon is consistent with the results observed in SEM experiments*.* All experiments proved that cell membranes were damaged after CA treatment.

**Figure 9.** The permeability of *S. aureus* cell membranes before (**A**) and after CA treatment (**B**). **Figure 9.** The permeability of *S. aureus* cell membranes before (**A**) and after CA treatment (**B**). **Figure 9.** The permeability of *S. aureus* cell membranes before (**A**) and after CA treatment (**B**).

### **3. Discussion**

Nowadays, there is an urgent need to develop a method that can efficiently solve the biofilm problem of foodborne pathogens without causing drug resistance in pathogens. NPs are defined as particles or materials at the nanometer scale [25]. The antimicrobial agent loaded on NPs can be protected from sequestering drugs by the biofilm matrix [26]. The antibacterial agent CA selected in this experiment is a safety additive recognized by the Food and Drug Administration (FDA) [27]. The maximum experimental amount used in this paper (5 mg/mL) was much lower than the maximum addition amount (286 mg/mL) specified by the FDA. CA can inhibit the synthesis of bacterial cell walls and affect membrane permeability and enzyme systems. In the experiment, the drug concentration of 1 × MIC led to a significant increase in the content of alkaline phosphatase, which proved that CA can destroy the integrity of bacterial cell walls. The destruction of the bacterial cell walls led to the death of the bacteria, which was consistent with the conclusion of Shreaz et al. [11]. Therefore, this article supports the method of loading CA on CSNPs to clear biofilm and kill bacteria.

Regarding drug delivery, in vitro release profiles for CA from NPs were evaluated. CSNP-CAs showed a typical release profile, with rapid-burst release at the beginning, followed by sustained release. The initial burst may have been due to the drug loading on the surface of the material, after which a persistent pattern was established due to the release of the drug from the NPs. This phenomenon is more effective for biofilm clearance, as higher initial drug doses reduce the antibiotic resistance of surviving bacteria in biofilms [28].

CSNP-CAs were not as effective in inhibiting biofilm formation as free CA, precisely because the CSNPs had a sustained-release effect, the drug concentration released in the time period considered here was small and the antibiofilm effect was weak. As shown in this work, CSNP-CAs were more effective in removing biofilms than free CA. The unique antibiofilm properties of chitosan were mainly attributed to its polycationic nature, conferred by the amino functionality (NH2) of the N-acetylglucosamine unit [29–31]. The positive charge of chitosan reacts electrostatically with negatively charged biofilm components, such as EPS, proteins and DNA, resulting in an inhibitory effect on bacterial biofilms [32,33]. At the same time, nanoparticles can also carry drugs into biofilm and release drugs to act on the bacteria themselves and kill bacteria. Therefore, CSNP-CAs are more effective in scavenging biofilms than free CA.

This experiment has confirmed that CSNP-CAs have the effect of removing biofilm and that the antibacterial effect of cinnamaldehyde plays an important role in it. CA destroyed the cell wall of *S. aureus* and changed the permeability of the cell membrane, resulting in the outflow of potassium ions, alkaline phosphatase, protein and other substances. Alkaline phosphatase is an enzyme between the cell wall and cell membrane. Under normal circumstances, its activity cannot be detected outside the cell. When the cell wall or cell membrane is damaged, alkaline phosphatase will penetrate the outside of the cell and threaten the integrity of the cell wall. Inoue et al. found that potassium-ion leakage provided direct evidence of membrane damage [34]. The content of extracellular potassium ions in the bacterial solution treated with 1 × MIC of CA increased continuously, which clearly showed the damage to the bacteria caused by CA. Since most of the proteins in the cell are present in the cytoplasm, leakage of the cytoplasm leads to increased levels of extracellular proteins. Propidium iodide (PI) is a nucleic-acid dye that cannot penetrate an intact cell membrane. Since the membranes of injured cells and necrotic cells are destroyed, PI can enter the cell membranes of injured cells and necrotic cells [35], so that the cells can be stained with red fluorescence [36]. Phospholipid serine (PS) exists only on the inner side of the cell membrane in normal cells. When a specific injury occurs, the PS acid on the inner side of the plasma membrane is redistributed, flipped from the inside of the cell membrane to the outside of the cell membrane, and exposed on the outer surface of the cell. PS eversion can be detected by Annexin V. Double staining with FITC-labeled Annexin V and PI enables sensitive detection of live, injured and dead cells via flow cytometry. Therefore, the intracellular PI content can verify the permeability of the cell membrane. The changes in the permeability of the cell membrane and plasma membrane of *S. aureus* lead to cell death.

In summary, we have verified that CA can destroy the cell membrane: on the one hand, it can change the cell-membrane potential and cause the leakage and death of potassium ions, proteins and other contents; on the other hand, CA acts on the cell membrane to redistribute PS inside the plasma membrane, which damages the cell membrane. SEM images can also clearly show changes in cell morphology and even cell rupture. In addition, the combination of CSNPs and CA makes it easier for CA to enter the mature biofilm, to achieve the effect of clearing biofilm.

### **4. Materials and Methods**

### *4.1. Bacterial Strains and Growth Media*

At present, most *S. aureus* strains have drug resistance. We used four different strains (NCTC 8325, RN6390, 15981, Col) for the determination of MIC. *S. aureus* RN6390 can easily form biofilm and has been wildly used for bovine mastitis infection assays in experiments [37,38], so we used RN6390 to conduct the following detailed experiments. The strain was cultured overnight in Tryptic Soy Broth (TSB) medium from Solarbio (Beijing, China) at 37 ◦C, 220 rpm.

CS (low-molecular-weight; degree of deacetylation: 75–85%) and CA (≥95%) were purchased from Macklin (Shanghai, China). Other reagents were purchased from Solarbio (Beijing, China).

### *4.2. Preparation of Nanoparticles*

CSNPs were prepared by the ion crosslinking method with polyanionic sodium triphosphate (TPP). In brief, CS was dissolved in acetic acid (1%) and stirred overnight to bring the concentration of chitosan to 1 mg/mL. Then, TPP was dissolved in deionized water for 2 h. TPP solution was mixed dropwise with the CS solution under stirring conditions (volume ratio CS: TPP = 3:1). CSNPs were separated by ultracentrifugation (12,000 rpm, 30 min) and then resuspended in PBS.

### *4.3. Preparation of CSNP-CAs*

For the preparation of the CSNP-CAs, CA (1%, *v*/*v*) was dissolved in ethanol (25%, *v*/*v*) and slowly dropped into the prepared chitosan solution under mechanical agitation to ensure that the volume ratio of CA to CS solution was 1:4. Then, the TPP solution was added to the mixed solution to ensure that the volume ratio of CS solution to TPP solution was 3:1. The mixed solution continued to be stirred at 600 rpm for 4 h. The prepared liquid was centrifuged at 12,000 rpm, and the precipitate was washed with distilled water. The CSNP-CA suspensions were freeze-dried and stored at −20 ◦C before use.

### *4.4. Particle Size and Zeta Potential of NPs*

The size and surface charge of the NPs were measured with a ZetaSizer Nano ZS90 (Malvern Instruments, Malvern, UK). CSNP suspensions were analyzed by dynamic light scattering (DLS) for size and potential determinations. The morphological characteristics were confirmed with transmission electron microscopy (TEM, JEM-1200EX, 80kv, Tokyo, Japan).

### *4.5. Drug Encapsulation*

The standard curve of CA was determined by UV spectroscopy (Persee, TU-1810, Beijing, China) at 291 nm. The amounts of CA encapsulated in the NPs were determined by centrifugation. The CSNP-CA solution was centrifuged at 12,000 rpm for 30 min. Then, the

absorbance of the supernatant was detected by UV spectroscopy. The CA loading capacity (LC) was calculated according to:

$$\mathbf{L}\mathbf{C} = (\mathbf{A} - \mathbf{B})/\mathbf{C} \tag{1}$$

where A = total amount of CA, B = total amount of non-loaded CA and C = weight of the NPs.

### *4.6. In Vitro Release Test*

The in vitro drug-release effect of the CSNP-CAs was passed by dynamic dialysis. The CSNP-CAs were transferred into a dialysis bag (with a retained relative molecular weight of 3500) and 50 mL of PBS buffer was used as the release medium. The dialysis bag was kept at a constant temperature of 37 ◦C and agitated at 70 rpm. At predetermined time intervals, 5 mL of dialysate was taken and supplemented with the same amount of release medium. The samples were measured at 291 nm with a UV spectrophotometer, 3 times for each time point, and an in vitro drug-release curve for the CSNP-CAs was drawn.

### *4.7. Determinations of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)*

The determination of the MIC was carried out according to the method described by Tan et al [3]. Stock solutions of CA were kept in 25% (*v*/*v*) ethanol to enhance their solubility in suspension. Antimicrobial effects of CA were evaluated using a serial twofold dilution method. CA was initially diluted in 25% (*v*/*v*) ethanol (1:100 *v*/*v*) and then in TSB medium. Serial twofold dilutions for CA were prepared to obtain concentrations ranging from 10 to 0.15 mg/mL. A quantity of 100 <sup>µ</sup>L of bacteria at a final concentration of <sup>1</sup> <sup>×</sup> <sup>10</sup><sup>5</sup> CFU/mL in TSB was added into the wells of 96-well microplates. Free CA (10, 5, 2.5, 1.25, 0.62, 0.31, 0.15 or 0 mg/mL) was added to each well. The microplate was incubated at 37 ◦C for 24 h at 150 rpm. MIC refers to the lowest drug concentration that inhibits the growth of bacteria in culture medium after 18 to 24 hours of culture in vitro. MBC refers to the minimum drug concentration capable of killing cultured bacteria after 18 to 24 hours in vitro.

### *4.8. Growth of Biofilm*

Biofilm formation in 96-well microplates was supported and assayed as described previously [39]. The microplates were incubated at 37 ◦C for 24 h without shaking. After treatment with different concentrations of CA and CSNP-CAs, the wells were washed 3 times with PBS to remove impurities and plankton. Then, each well was stained with 100 µL of 0.1% (*w*/*v*) crystal violet (CV) solution for 15 min. A quantity of 100 µL of 30% (*v*/*v*) acetic acid was used for extraction. After shaking, the absorbance was measured at 570 nm.

### *4.9. Removal of Biofilm*

Biofilm was grown in a 96-well microplate without CSNP-CAs for 48 h, as described above. Then, CSNP-CAs and free CA solution with different concentrations were added to each well to remove the biofilm. After 24 h, the microplates were washed with PBS, and the removal ability was detected by CV staining.

### *4.10. Antibiofilm Effect on Food-Grade Silicone Surfaces*

Scanning electron microscopy (SEM) and biofilm-formation analyses were conducted according to the method of Tan et al. [4]. Biofilms were formed and treated with CA and CSNP-Cas, as described above, on silicone (7 mm in diameter; Hongxiang, Jiaxing, China) as the surface substrate. Afterwards, the biofilm was fixed with 3% glutaraldehyde at 4 ◦C overnight and then dehydrated in a series of ethanol solutions for 20 min each (70%, 80%, 96% and 100%). After chemical dehydration with tert-butanol (Macklin, Shanghai, China), the samples were coated with gold and analyzed via SEM (VEGA3, TESCAN, Brno, Czech Republic), using a 10 kV accelerating voltage.

### *4.11. Live/Dead Assay*

Biofilm cells were stained with a Live/Dead® BacLight™ Bacterial Staining Kit (40274, Yeasen, Shanghai, China), according to the manufacturer's protocol, for 30 min at 37 ◦C in the dark. Images of biofilm were analyzed by confocal laser scanning microscopy (CLSM; ZEISS, LSM900, Oberkohen, Germany).

### *4.12. Effect of CA on Membrane Permeability of S. aureus*

To the bacterial suspension, 1 × MIC CA was added at 37 ◦C, and PBS buffer was used as the control. Samples were taken hourly to measure potassium-ion concentration. The determination steps for potassium-ion contents were performed according to the instructions provided with the Nanjing Jiancheng potassium ion kit, and the absorbance was measured at 440 nm (Persee, TU-1810, Beijing, China). All the experiments were carried out in triplicate.

### *4.13. Effect of CA on Cell Wall Integrity of S. aureus*

To the bacterial suspension, 1 × MIC CA was added at 37 ◦C, and PBS buffer was used as the control. Samples were periodically sampled by hourly sampling and centrifuged at 1000 rpm for 10 min, and the supernatant was saved for assay. The instructions provided with the Nanjing Jiancheng alkaline phosphatase determination kit (AKP) were referred to for the determination steps, and absorbance was measured at 520 nm.

### *4.14. The Morphologies of the Bacteria Observed by SEM*

The bacterial suspension was cultured to the logarithmic phase, centrifuged at 4000 rpm for 10 min and the supernatant was discarded. Then, the precipitation was fixed with 2.5% (*v*/*v*) glutaraldehyde at 4 ◦C overnight, followed by dehydration in a series of ethanol solutions (10%, 30%, 50%, 70%, 80%, 90%, 95%, 100%) for 20 min. Then, the samples were placed in 50% and 100% tert-butanol solutions (Macklin, China) for chemically dehydration for 15 min and vacuum dried for 48 h. Then, the samples were sprayed with gold and analyzed via SEM (VEGA3, TESCAN, Brno, Czech Republic).

### *4.15. Flow Cytometry Analysis*

*S. aureus* was collected by centrifugation (10,000 rpm, 10 min, 4 ◦C). The bacterial suspension was washed 3 times with sterile PBS buffer and diluted to bacterial suspension OD<sup>600</sup> = 0.01. Then, 2 × MIC CA was added to the bacterial suspension prior to incubation for 4 h. Flow cytometry analysis was conducted according to the method of Deng et al., with minor modifications [40]. A quantity of 1 mL *S. aureus* bacterial suspension was added to 5 µL of FITC and 5 µL of PI staining solution (Meilunbio, Dalian, China), placed in the dark at 37 ◦C for 30 min and then detected by flow cytometry (Facs AriaIII, BD, New York, NY, USA).

### *4.16. Statistical Analysis*

All the experiments were carried out in triplicate. Statistical analyses were performed with SPSS 19.0 (SPSS Inc., Chicago, IL, USA). Means ± standard deviations (SDs) were calculated for each experiment. Statistical significance was determined by *t*-test analysis, with significant differences determined at *p* < 0.05.

### **5. Conclusions**

The results showed that the antibiofilm activity of CA against *S. aureus* was enhanced when loaded on CSNPs. The antibiofilm activity of CSNP-CAs was determined by biofilm inhibition, biofilm disruption and biofilm live/dead bacterial changes on food-grade silicone surfaces. Synthetic CSNP-CAs can be used as potential therapeutics to control *S. aureus* biofilm formation in the future. Furthermore, CSNPs can be used as platforms to design more chemically modified agents or be loaded with other antibiofilm agents for more functional drug-delivery systems.

**Author Contributions:** Conceptualization, Y.T. and J.X.; methodology, J.X.; software, J.X.; validation, J.X., Q.L. and M.S.; formal analysis, B.L.; investigation, Y.G.; resources, Y.T.; data curation, J.X.; writing—original draft preparation, J.X.; writing—review and editing, Y.T. and T.D.; visualization, M.S.; supervision, Y.T.; project administration, Y.T.; funding acquisition, Y.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors gratefully acknowledge the Young Taishan Scholars Program of Shandong Province (tsqn202103094) and the Talent Research Foundation of Qingdao Agricultural University (665/1121020).

**Conflicts of Interest:** The authors declare no conflict of interest.

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