Antimicrobial compounds
Cinnamon oil was extracted from Cinnamomum zeylanicum leaves by steam distillation method. It was purchased from Erin Limited Company, Australia. Cinnamon oil stock solution was prepared by emulsifying cinnamon oil in deionized water with 1% Tween-80 by stirring 30 min to get a colloidal suspension for use within 24 h with final cinnamon oil concentrations of 207 and 414 mg/mL, respectively.
Chemicals and reagents
Cinnamaldehyde [99.5%, chromatographic pure (GCP)] and cinnamyl alcohol (99%, GCP) were purchased from Aladdin, Shanghai, China. HPLC-grade methylene dichloride was purchased from Tianjin Shield Specialty Chemical Co., Ltd., Tianjin, China. HPLC-grade acetonitrile and methanol were purchased from Tedia Company, Inc., Ohio, USA. Other solvents and chemicals were purchased from Dingguo biological technology Co., Ltd., Shanghai, China. Ultrapure water was purified on a Milli-Q system (Millipore, Bedford, USA). Millipore syringe filters (Millex-GP, 0.22 mm pore size) were purchased from Nihon Millipore, Tokyo, Japan.
Shewanella putrefaciens preparation
Shewanella putrefaciens was isolated from spoiled fish and identified by China Center of Industrial Culture Collection. When shipped to our laboratory, the strain was cultured twice in nutrient broth (NB) at 30 °C for 24 h, then streaked on nutrient agar (NA) slants and cultured under the same conditions. The slants were stored at 4 °C and sub-cultured monthly until use. Before each experiment, stock cultures were propagated through two consecutive 24-h growth cycles in NB at 30 °C and then cultivated to the exponential phase (5 h). The working cultures contained approximately 108 CFU/mL S. putrefaciens were obtained by diluting the exponential phase cells in nutrient broth.
Treatments of S. putrefaciens
Each 50 mL working culture containing approximately 108 colony-forming unit (CFU)/mL S. putrefaciens was transferred into 100-mL test tube. These test tubes were treated as follows: group one without adding cinnamon oil was used as control (CK); group two was added cinnamon oil with the final concentration of 207 μg/mL (C1); group three contained 207 μg/mL cinnamon oil and irradiated by GI (C1+G); group four only was irradiated (G); group five was added cinnamon oil with the final concentration of 414 μg/mL (C2). G and C1+G were irradiated 0.080 kGy GI as soon as possible after cinnamon oil treatment at the Institute of Crops and Nuclear Technology Utilization, Zhejiang Academy of Agricultural Sciences, China. The data of 207 and 414 μg/mL were the ½ MIC (minimal inhibitory concentration) and MIC values of cinnamon oil for 108 CFU/mL S. putrefaciens. The doses were conducted by using 55-cm distance to the irradiation source with 30 min. The self-contained GI source was 137Cs with an approximate dose rate of 0.10 kGy/min. The dose rate was established by using National Physical Laboratory (Middlesex, United Kingdom) dosimeters. After irradiation, all samples were transported to our laboratory within 180 min. Moreover, the nutrient broths containing 207 and 414 μg/mL cinnamon oil without S. putrefaciens were also used as controls marked as CC1 and CC2, respectively. Each treatment group contained 5 test tubes. The assays were tested in triplicate, and values are presented as mean ± standard deviation of replicated measurements.
Cinnamon oil analysis
The cinnamon oil was analyzed using a GC–MS system (Agilent 7890A/5975C) on a HP-5MS capillary column (30.0 m × 250 μm × 0.25 μm) using helium as the carrier gas with a split flow was 1.5 mL/min and a 100:1 split ratio. The initial oven temperature of GC was 50 °C, and programmed to 250 °C at a rate of 30 °C/min and then kept constant at 250 °C for 10 min. The inject volume was 0.5 μL and the source temperature was 230 °C. MS was taken at 70 eV and a mass range of 29–450 amu. A library search was carried out using NIST98.L database. Relative percentage amount was calculated from total ions chromatograms (TIC) by the computer (Ooi et al. 2006).
Analysis of membrane fatty acids in S. putrefaciens
The working cultures of S. putrefaciens with different treatments at 180 min were centrifuged for 10 min at 5000g, and the cell pellet was harvested and re-suspended in phosphate-buffered saline (0.1 M, pH 7.0), afterwards frozen at −80 °C and freeze-dried using a freeze-dryer (FD-1-50, Bo Yikang Co. Ltd., Beijing, China). The samples were submitted for membrane fatty acid extraction. Extraction of fatty acid from cellular materials was carried out as described by Evans et al. (1998).
Lipid samples were trans-methylated for analysis of their acyl groups as fatty acid methyl esters (FAME). The samples of total lipid extract were evaporated to dryness in a round-bottom flask using a boiling water bath. To the dried samples, heated under a reflux condenser (20–30 cm), 10 mL of KOH in methanol (0.2 M) and 1 mL of heptane were added. After 10 min, 5 mL of boron trifluoride (BF3) was added and followed, after 2 min, by 4 mL of hexane. After waiting for 1 min, the samples were cooled. A saturated solution of Na2SO4 was added to the samples, and after settling into a two-phase system, the upper layer was taken and transferred into a vial. The samples were stored at −30 °C until further analysis (Dussault et al. 2009).
GC-MS analysis was performed using a Thermo Trace 1300 Gas Chromatograph equipped with a Thermo TG-WAXMS capillary column (dimensions: 30 m × 0.25 mm × 0.25 μm; Thermo Scientific, USA), coupled to a Thermo ISQ LT Single Quadrupole Mass Spectrometer (MS) through a heated transfer line (220 °C). Helium (99.999%) was used as the carrier gas with a constant flow rate of 1 mL/min and a 1:50 split ratio. The GC inlet temperature is 220 °C. 1 mL aliquots were injected using an AI 1310 autosampler, and the GC oven was programmed to hold 120 °C for 5 min, then raise the temperature by 6 °C/min to 210 °C, which was held for 5 min, then raise the temperature by 1 °C/min to 230 °C, which was held for 20 min. The MS was operated with the ion source at 220 °C. The solvent delay is 1.46 min. FAME peaks were identified by comparison of their retention times with those of a standard solution (GLC NESTLE 37 Component FAME MIX) and quantified with the internal standard. In all cases, the mass spectrometer was operated in the electron ionization mode (EI) at 70 eV. The retention times and the characteristic fragments of the EI mass spectra were obtained from m/z 20–400 with the scan rate of 500 amu/s. The most abundant ions and/or ions with-out apparent cross-contribution and interferences were chosen as target ions for the quantification (SIM mode) (Schummer et al. 2009).
Electrophoretic analysis
All the implements must be precooled at −20 °C before the tests. The membrane protein (MP) was prepared with Bacterial Membrane Extraction Kit (BestBio, Shanghai, China) according to the instructions with minor modification. The working cultures of S. putrefaciens with different treatments at 180 min were centrifuged at 10,000g for 5 min at 0 °C, then washed with PBS buffer (0.1 M, pH 7.0) for two times. The extraction buffer A (500 μL, combined with 2 μL Protease inhibitor) was added to the sediments (20 mg), then put on ice for 2–3 h with shaking for 30 s with every 30 min. The mixture was then centrifuged at 12,000g for 5 min at 0 °C, the supernatant was collected, and then 10 μL. Extraction buffer B was added to the supernatant, then kept at 37 °C for 10 min. The mixture was centrifuged at 1000g for 5 min at 37 °C. The under layer samples were the membrane protein (MP). MP concentration was measured with enhanced bicinchoninic acid (BCA) Protein Assay Kit (Beyotime Biotechnology, Jiangsu, China) according to the manufacturer’s instructions. The samples may need to be diluted further to same concentration with appropriate volume of membrane protein dissolution buffer in the kit. The loaded concentration of different treatments’ MP was adjusted to 1 mg/mL. 20 μL of protein lysates was then mixed with 5 μL 5× SDS gel-loading buffer (60 mM Tris–HCl, pH 6.8, 14.4 mM dithiothreitol, 2% SDS, 25% glycerol, and 0.1% bromphenol blue), boiled at 100 °C for 5–10 min before use or stored at −20 °C until use. The loaded volume was 15 μL. Sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to measure the molecular weight changes of MP, using 12% separating gel and a 5% stacking gel. The running buffer was 25 mM Tris-192 mM glycin-0.1% SDS, pH 8.3. The power supply was settled at 80 V during the stacking of the proteins, then at 120 V. Gels were stained with Coomassie Brilliant Blue R250 and destained with a solution of methanol and acetic acid to visualize proteins (Carraro and Catani 1983).
Changes of cinnamon oil in nutrient broth with S. putrefaciens
GC–MS was used to analyze chemical compositions of cinnamon oil in nutrient broth (NB) of C1 at 0 and 180 min. GC–MS analysis was performed using a Thermo Trace GC Ultra system equipped with a Thermo TR-5MS capillary column (dimensions: 30 m × 250 mm × 0.25 mm; Thermo Scientific, Runcorn, UK) operating with helium as a carrier gas, coupled to a Thermo ITQ 1100 mass spectrometer (MS) through a heated transfer line (230 °C). The GC injector (230 °C) was operated in a pulsed split mode (50:1); 1 μL aliquots were injected using an autosampler. The initial oven temperature of GC was 100 °C, and programmed to 130 °C at a rate of 20 °C/min, then programmed to 170 °C at a rate of 5 °C/min, after then, programmed to 230 °C at a rate of 25 °C/min and then kept constant at 230 °C for 5 min. The MS was operated with the ion source at 230 °C and a damping flow of 1 mL/min. MS was taken at 70 eV and a mass range of 35–425 amu. The solvent delay time was 3 min. A library search was carried out using NIST98.L database. Relative percentage amount was calculated from TIC by the computer (Ooi et al. 2006).
Distribution of cinnamaldehyde and cinnamyl alcohol
After the analysis of cinnamon oil changes in broth in C1, we found that in the nutrient broth, cinnamaldehyde decreased and cinnamyl alcohol emerged (data are shown in results section). Thus, the distribution of cinnamaldehyde and cinnamyl alcohol in nutrient broth and S. putrefaciens was analyzed using HPLC system (Waters 2695, Milford, USA) consisting of quaternary gradient pump, autosampler, column oven, and photodiode array detector (PDA, Waters 2996). Chromatographic data were acquired using Empower software. The HPLC column consisted of a Waters symmetry C18 column (200 mm × 4.6 mm × 5 mm) connected to Nova-Pak C18 Guard-PakTM guard column (2 mm × 4 mm × 5 mm). The gradient elution was employed using deionized water and acetonitrile at 30 °C for 20 min. The flow rate was set at 1 mL/min. A volume of 10 μL of sample was injected into HPLC system for analysis. The detection wavelengths were set at 290 and 250 nm for cinnamaldehyde and cinnamyl alcohol, respectively. To calculate their concentration, the standard curves ranging from 0.2 to 200 μg/mL were used to obtain a linear relationship between concentrations of drugs versus peak area response, which resulted in a R
2 (coefficient of determination) value of 0.9999. A re-equilibration period of 5 min was used between individual runs. Because only treatments of C1, C1+G, and C2 were treated with cinnamon oil, so here the changes of cinnamaldehyde and cinnamyl alcohol of C1, C1+G, and C2 at 0 and 180 min were observed. In order to observe the effect of S. putrefaciens, the changes of cinnamaldehyde and cinnamyl alcohol of CC1 and CC2 at 0 and 180 min were also detected.
Statistical analysis
One-way analysis of variance and Duncan’s multiple ranges tests were employed to determine the effect of the combination of cinnamon oil and GI treatments on fatty acids, cinnamaldehyde, and cinnamyl alcohol. Calculations were performed using SPSS software Base 19.0 (SPSS, Inc., Chicago, IL, USA). Differences between means were considered significant at p ≤ 0.05.
