All growth media were obtained from Qingdao Hopebio Biotechnology Co. Ltd. (Qingdao, China). All reagents were purchased from Shanghai MacLean Biochemical Technology Co., Ltd. (Shanghai, China). The stainless steel tube, tinplate, glass, and polystyrene were supplied by Luoyang Zhenguo Packaging Material Co. Ltd. (Luoyang, China).

A total of 8 C. sakazakii strains were used in the study; among them, C. sakazakii ATCC 29544 was provided from the American Type Culture Collection (ATCC, Manassas, VA, United States), and C. sakazakii PD2, IN3, CFP6, FP1, V1, F2, and C1 were isolated from prepared dishes, instant noodles, chilled fresh pork, PIF, vegetables, fruits, and cookies, respectively, which has been reported in our previous study (Fei et al., 2018b). All strains were used to evaluate the antibacterial activity of CBCE, and C. sakazakii ATCC 29544 was used to analyze the antibacterial mechanism. Before the experiment was carried out, all strains were stored in Luria–Bertani (LB) broth with 20% glycerol (v/v) at −80°C. The above cultures were streaked on tryptone soya agar (TSA) and incubated at 37°C for 24 h. A single colony of each strain was inoculated into LB broth, followed by incubation at 37°C for 12 h for subsequent study.

The C. buds were crushed and passed through an 82-mesh sieve to obtain their powders. These powders were dissolved in sterile distilled water (SDW) with a ratio of 1:30 (m:v) and extracted at 70°C for 80 min. Then, the filtrate was collected and the filter residues were further extracted twice by the same method. All the filtrate was concentrated using a rotary evaporator (Zhejiang Nader Scientific Instrument Co. Ltd., Hangzhou, China) at 40°C for 10 h to obtain the paste of CBCE. Finally, the paste was dried into powder by a vacuum freeze-drying machine (Tokyo Riken Co., Ltd., Tokyo, Japan). Specific parameters are as follows: paste thickness is 3 mm, pre-cooling temperature is −55°C, vacuum pressure is 45 Pa, sublimation temperature is 40°C, and drying time is 16 h. The images of C. buds and CBCE powders are shown in Supplementary Figure 1, and the main chemical components of CBCE are shown in Supplementary Table 1.

The diameter of inhibition zone (DIZ) of CBCE against C. sakazakii used the Oxford cup method according to the previous report (Xu et al., 2019). Briefly, 100 μL of C. sakazakii bacterial suspensions (about 10⁷ CFU/mL) was uniformly plated on the sterilized TSA plates. Three sterile oxford cups with a diameter of 8 mm (Shanghai Precision Instrument Co., Ltd., Shanghai, China) were evenly placed on the TSA surface and gently pressed with a tweezer. Then, 200 μL of CBCE (10 mg/mL) was added in the oxford cups, followed by incubation at 37°C for 24 h. Finally, the DIZ value was determined by taking an average of the diameters of three transparent inhibition zones on the same plate.

The minimum inhibitory concentration (MIC) and minimum bactericide concentration (MBC) of CBCE against eight C. sakazakii strains were determined using the agar dilution method as previously described (Fei et al., 2018a). In brief, 500 μL of TSA media containing different concentrations of CBCE (0, 0.3125, 0.625, 1.25, 2.5, 5, 10, and 20 mg/mL) was added into sterile 24-well plates (Shanghai Precision Instrument Co., Ltd., Shanghai, China), respectively. After the TSA medium was solidified, 2 μL (10⁶ CFU/mL) of bacterial suspension was uniformly coated on the surface of agar block followed by incubation at 37°C for 24 h. MIC was defined as the lowest concentration at which CBCE completely prevented C. sakazakii from growing under visual observation. Further, 100 μL (10⁶ CFU/mL) of C. sakazakii was treated with 10, 20, 40, and 80 mg/mL of CBCE for 30 min, respectively, then was spread onto TSA plates and incubated at 37°C for 24 h. The lowest concentration at which no colonies were observed was considered as the MBC of CBCE against C. sakazakii. In addition, the TSA medium without CBCE was used as negative control, and the TSA medium containing 0.1 mg/mL ampicillin was used as positive control.

The growth curves of C. sakazakii ATCC 29544 strains with different treatments were determined according to the method described by Shi et al. (2016a), with minor modifications. One milliliter of tested bacteria cultures (OD₆₀₀ = 0.1) was added into the 96-well microtiter plates (Shanghai Precision Instrument Co., Ltd., Shanghai, China), then CBCE was added to the above solution and mixed well, and the final concentration was adjusted to 0 (control), 0.5, 1, 1.5, and 2 MIC. All samples were cultured at 37°C for 24 h, the optical density (OD)₆₀₀ values of which were measured by a multimode plate reader (Tecan, Infinite M200 PRO, Männedorf, Switzerland) every 2 h to assess bacterial growth.

After 10⁷ CFU/mL of C. sakazakii ATCC 29544 strains were treated by 0, 1, and 2 MIC of CBCE at 37°C for 30 min, the intracellular ATP concentrations of tested bacteria were determined on the basis of the previous report (Sánchez et al., 2010). The treated strains were cracked using the ultrasound treatment, and then a treatment of 100°C for 3 min was performed to inactivate the ATP in the system. The above samples were centrifuged at 8,000 × g for 3 min at 37°C to obtain the detected supernatant. Finally, the relative luminescence value representing the intracellular ATP concentration of the tested bacteria was determined using an ATP kit (AmyJet Scientific Inc., Wuhan, China) according to the product instructions.

The method reported by Guo et al. (2019) was followed to determine the membrane potential of the tested bacteria. Briefly, 125 μL of bacterial suspension (10⁷ CFU/mL) and 0.5 μL of bis-(1,3-dibutylbarbituriis-(1,3-dibutylbarbituric acid) trimethine oxonol (Beijing Solarbio Science and Technology Co. Ltd., Beijing, China) fluorescent probe were added into a black and opaque 96-well plate and cultured at 37°C for 30 min. Then, CBCE was added to the 96-well plate, and the final concentrations were adjusted to 1 and 2 MIC, respectively. The untreated bacterial suspension was used as a control. After incubating at 37°C for 30 min, the fluorescence intensities of all samples at the excitation wavelength and emission wavelength of 492 and 515 nm, respectively were measured using a multifunctional microplate reader (Beijing Potenov Technology Co. Ltd., Beijing, China).

The effects of CBCE on pH_in of C. sakazakii ATCC 29544 strains were analyzed based on the method as described by Guo L. et al. (2020). In brief, 1.0 μM carboxyfluorescein diacetate succinimidyl ester dye as the fluorescent probe was added to 10⁸ CFU/mL of bacterial suspensions, then incubated at 37°C for 30 min. After a centrifugation at 11200 × g for 5 min, the cell pellets were obtained and suspended into sterilized normal saline (NS) followed by addition of glucose (10 mM). The mixed system was incubated at 37°C for 30 min and centrifuged at 11,200 × g for 5 min to get the cell pellets. Then, these cell pellets were washed twice and resuspended in NS. One hundred and twenty-five microliters of bacterial suspension was treated by 0, 1, and 2 MIC of CBCE in a black and opaque 96-well plate at 37°C for 20 min. The fluorescence intensities were detected using a multimode plate reader (Tecan, Infinite M200 PRO, Männedorf, Switzerland) at 25°C under the excitation wavelength of 440 nm and emission wavelength of 490 nm. The ratio of fluorescence intensities at 490/440 nm was considered as the pH_in of cells. In addition, the fluorescence intensity of the cell-free control was deducted to eliminate the background error.

About 10⁷ CFU/mL of C. sakazakii ATCC 29544 suspension was treated with 1 MIC of CBCE at 37°C for 3, 6, 9, and 12 h, respectively. The untreated C. sakazakii ATCC 29544 was used as a control. All samples were centrifuged at 11,200 × g for 5 min to obtain the cell pellets and supernatants. The pellets were resuspended in NS and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to determine the effect of CBCE on protein in bacteria according to our previous study (Fei et al., 2018a). The protein contents in supernatants were analyzed to determine the amount of protein leaking by the Coomassie bright blue method as described by He et al. (2014).

The method reported by Ma et al. (2016) was performed to determine the nucleic acid content of C. sakazakii ATCC 29544. Approximately 10⁹ CFU/mL of bacterial suspensions was treated with 0, 0.5, 1, 1.5, and 2 MIC of CBCE, respectively, then incubated at 37°C for 12 h. Every 2 h during incubation, 200 μL of cultures was absorbed and centrifuged at 11,200 × g for 10 min to obtain the supernatants. The OD₂₆₀ values of supernatants were measured by a multimode plate reader (Tecan, Infinite M200 PRO, Männedorf, Switzerland).

The effects of CBCE on the cell morphology of C. sakazakii ATCC 29544 were observed using a transmission electron microscope (TEM) according to the previous report (Fei et al., 2019). Briefly, C. sakazakii strains were treated by 0 (control), 1, and 2 MIC of CBCE for 4 h followed by centrifuging at 3,000 × g for 5 min to collect the cell pellets. The cell pellets were fixed with 0.1 M sodium phosphate buffer containing 2.5% glutaraldehyde for 12 h, then washed three times with NS. Further, the cells were stored in 1% osmium tetroxide at room temperature for 6 h followed by washing three times with NS again. After the above treatments, the cells were dehydrated with 50, 70, 90, and 100% ethanol for 10 min and embedded in Epon Lx-112 (Ladd Research, Williston, VT, United States). Finally, the samples were stained with uranyl acetate and lead citrate, then observed under TEM (Hitachi, Tokyo, Japan).

The inhibitory abilities of CBCE against C. sakazakii ATCC 29544 in biofilm on stainless steel tube, tinplate, glass, and polystyrene were assessed according to the report of Fei et al. (2020). In brief, all the materials were sterilized and cut into 50 mm × 20 mm coupons then placed into 50-mL tubes containing 30 mL of bacterial suspensions (10⁷ CFU/mL) followed by incubation at 4°C for 24 h. The coupons were washed twice with SDW for 10 s and cultured in 30 mL of LB broth at 25°C for 24 h then washed twice with SDW for 10 s. The samples were treated with 0, 1, and 2 MIC of CBCE and collected at 0, 10, 20, and 30 min, respectively, then washed twice with 300 mL of SDW for 10 s. Then, 30 mL of sterile phosphate-buffered saline and 3 g glass bead (300–600 μm) were added into the tubes containing the coupon, followed by swirling for 5 min. Finally, the plate count method was used to determine the viable counts of C. sakazakii in phosphate-buffered saline (PBS, pH 7.2). The PBS suspension was serially diluted and plated onto TSA plates, followed by incubation at 37°C for 24 h to enumerate the viable count of C. sakazakii.

All experiments except SDS-PAGE and TEM analysis were repeated three times. The data were expressed as mean values ± standard deviation (SD). The significance test was performed by analysis of variance (ANOVA) in the SPSS 20.0 software (SPSS Inc., Chicago, IL, United States). P < 0.05 was considered as significant difference.

The DIZs, MICs, and MBCs of CBCE against eight C. sakazakii strains are given in Table 1. The results showed that the DIZs ranged from 14.55 ± 0.44 to 14.84 ± 0.38 mm, and no significant difference (P > 0.05) was found between these strains. The MICs and MBCs of CBCE against all C. sakazakii strains were 10 and 20 mg/mL, respectively.

As shown in Figure 1, compared with the control group, the growth rates of C. sakazakii ATCC 29544 treated by CBCE were obviously reduced and slowed down with the increase of CBCE concentration. When C. sakazakii was treated by 1 MIC of CBCE, the logarithmic phase of C. sakazakii disappeared. When treating by 2 MIC, C. sakazakii stagnated in the delayed stage and stopped growing.

The effect of CBCE on the intracellular ATP concentration of C. sakazakii ATCC 29544 is shown in Figure 2. Compared to the control group, the intracellular ATP concentration of C. sakazakii ATCC after treatments with 1 and 2 MIC of CBCE decreased significantly (P < 0.05) with the increase of concentration.

Figure 3 illustrates the changes in membrane potential of C. sakazakii ATCC 29544 after treatments with 0, 1, and 2 MIC of CBCE. It can be seen that the cell membrane potential of experimental groups was significantly increased in comparison with the control (P < 0.05), indicating that the membrane potential depolarization occurred in cells treated with CBCE.

As shown in Figure 4A, the pH_in value had a good linear relationship with the fluorescence ratio of 490 nm and 440 nm (y = −1.1701x + 12.917, R² = 0.9929). Figure 4B shows that with the addition of CBCE, the pH_in value of C. sakazakii ATCC 29544 decreased significantly, and there were significant differences among the three groups (P < 0.05). Specifically, after treatments with 1 and 2 MIC of CBCE, the pH_in value dropped from 7.68 ± 0.52 to 4.21 ± 0.19 and 3.01 ± 0.10, respectively.

The changes in protein levels of C. sakazakii ATCC 29544 after treatment with 1 MIC of CBCE are presented in Figure 5. The SDS-PAGE image (Figure 5A) showed the changes in the bacterial protein where the protein bands were obviously weaker and almost disappeared when 1 MIC of CBCE was applied for 12 h. The Coomassie bright blue test result (Figure 5B) showed that the amount of protein leaking from the bacteria significantly increased at different treatment times.

The amounts of nucleic acid leakage from C. sakazakii ATCC 29544 cells were expressed as the absorbance value at 260 nm and given in Figure 6. After treatments with CBCE, the OD_{260 nm} values of supernatants increased significantly with the increase of concentrations, suggesting that CBCE could cause a large amount of nucleic acid leakage from the tested cells.

After treatments with 1 and 2 MIC of CBCE for 4 h, TEM was used to observe the changes in C. sakazakii ATCC 29544 cell morphology and is shown in Figure 7. The untreated cell was used as the control (Figure 7A), which showed regular morphology, smooth surface, and uniform cytoplasm. After treatments with CBCE, the deformation, collapse, and leakage of cell fluid occurred in the tested cells, and the damage degree of cell morphology was more serious with the increase of CBCE concentration (Figures 7B,C).

The inactivation activities of CBCE against C. sakazakii ATCC 29544 in biofilm on stainless steel tube (Figure 8A), glass (Figure 8B), tinplate (Figure 8C), and polystyrene (Figure 8D) were evaluated. After treatments with CBCE, the number of viable bacteria in biofilm on the surfaces of the four materials was significantly reduced as compared to the control group (P < 0.05), and about 6.5 log CFU/mL of strains could be completely inactivated after a 1 MIC-30 min treatment or 2 MIC-20 min treatment. In addition, among the four materials, CBCE has the best antibacterial ability against C. sakazakii in biofilm on the polyetherone resin, followed by the stainless steel tube, tinplate, and glass.