DOI: 10.5061/dryad.c2fqz616z
title: Inhibition mechanism and antibacterial activity of natural antibacterial agent citral on bamboo mold and its anti- mildew effect on bamboo

description: Bamboo, a natural material, has been widely used in the fields of
 decoration, architecture and furniture. However, bamboo is easy to mildew
 and lose its use value. In this paper, inhibition mechanism and
 antibacterial activity of natural antibacterial agent citral on bamboo
 mold and its anti- mildew effect on bamboo were studied. The results
 showed that citral could change the shape of mycelium, destroy the
 integrity of mycelium structure, cell wall, and cell membrane structure,
 thereby causing leakage of nucleic acid, protein, and other substances in
 the cell, as well as destroy the pH balance of the inside and outside of
 the cell, to inhibit or kill mold. When the concentration of citral is
 100mg/mL, the antibacterial rates of citral against Penicillium citrinum
 (PC), Trichoderma viride (TV), Aspergillus niger (AN) and a hybrid fungi
 group comprising PC, TV, and AN (Hun) were more than 100%. However,
 compared with the direct effect of citral on mold, the antibacterial
 property of bamboo treated with citral was significantly reduced, the
 mildew proof effect can be achieved only if the concentration of citral to
 treat bamboo is increased to more than 2 times of the concentration of
 citral directly acting on mold.
description: 1.The preparation of mold suspensions The jars containing an appropriate amount of sterile water and small glass beads were placed in a high-pressure steam sterilization pot and sterilized for 30 min at 121℃ and 0.1MPa. Afterward, the mycelium and spores of the tested strains were picked with an inoculation needle under aseptic condition and placed in sterilized jars. Finally, the mold suspensions were made by shaking for 10–15 min for inoculations. 2. The preparation of citral solution A certain amount of citral was placed in a beater, then 2% (v/v) Tween-80, and a small amount of deionized water was added and mixed consistently. Next, they were moved into a volumetric flask for a constant volume. Finally, citral solutions with concentrations of 3.125, 6.25, 12.5, 25, 50,100 mg/mL were obtained. 3. Oxford Cup method to test the bacteriostatic performance of citral on bamboo mold The Oxford cup method was used to investigate the antibacterial property of citral on bamboo mold. First, the sterilized Oxford cups (8 mm in outer diameter, 6 mm in inner diameter) were placed on PDA plates with the size of 100 mm in diameter coated with 80 µL of the bacterial solution, then 80 µL of different concentrations of citral solution were injected into the cups. Second, the Petri dishes were sealed with sterile sealing film and pre-diffused for 2 h at 4 °C. Finally, the Petri dishes were incubated at 28 ±2 °C and relative humidity of 85 ±5% for two days in the incubation chamber. The cross-crossing method was used to measure the diameter of the inhibition zone. Each treatment was repeated three times, the results were averaged, and the bacteriostatic rates were calculated according to formula (1). Tween-80 treatment groups were used as controls. Bacteriostatic rate = (diameter of inhibition zone in treatment group − diameter of inhibition zone in control group)/diameter of inhibition zone in control group × 100% ----------(1) 4. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) determinations On the basis of the bacteriostatic results of citral, the concentration of citral was gradually diluted from 100mg/ mL to 0mg/ml, and the double dilution method  was used to test the MIC and MBC of citral. The PDA plates, which is 90mm in diameter, and is15 times the inner diameter of the Oxford Cup, containing different concentrations of citral were prepared by mixing different concentrations of citral uniformly, and then 80 µL of bacterial suspension was evenly coated on the surface of the PDA plates. Afterward, the culture plates were sealed with a sterile sealing film and placed in a constant temperature incubator at 28 ±2 °C and humidity of 85 ±5%. The growths of mold were observed after two days of culture, and the minimum concentration of citral for completely sterile growth was the minimum inhibitory concentration (MIC). Based on MIC determination, the culture was continued for seven days, and the minimum germicidal concentration (MBC) of completely sterile growth was taken as the minimum germicidal concentration (MBC). Tween-80 treatment groups were the control ones. 5. Effects of citral on mycelia morphology of bamboo mold A scanning electron microscope (SEM) was used to observe the effect of citral on mycelia morphology of bamboo mold. The mildew cakes with a diameter of 8.0 mm were laid on the flourishing culture medium for seven days and placed on the surface of the medium plates with citral at various concentrations (0, MIC, and MBC). Then, the samples were cultured at 28 ±2 °C and 85±5% humidity. After four days, the cakes were cut as the test samples under an electron microscope. The samples were fixed with 2.5% glutaraldehyde at 4°C for 12 h. After that, the samples were washed thrice with phosphate buffer solution (PBS, pH 7.0) for 15 min each. Subsequently, the samples were fixed with 1% osmium solution for 2 h. After fixing, the samples were rinsed thrice with PBS solution for 15 min each. The samples were then dehydrated in ethanol series gradients (30, 50, 70, 80, 90, and 95%, v/v) for 15 min at a time. Finally, they were dehydrated with anhydrous ethanol for 20 min. The final samples were freeze-dried, gold-sprayed, and observed in a SU8010 SEM. 6. Effects of citral on the microstructure of bamboo mold cells Transmission electron microscopy (TEM) was used to observe the microstructures of mold cells. The preliminary treatment method of mold and electron microscope samples treated with citral was the same as that of 5., except that the samples were treated with pure acetone solution for 20 min after gradient dehydration with ethanol series. Then the samples were permeated, embedded, sliced, and dyed. Finally, the microstructures of mycotic cells were observed in a JEM-1200 TEM. 7. Effects of citral on the release of cellular materials of bamboo mold By simplifying Paul's method, the effects of citral on the release of cellular materials were investigated. Mold spores were cultured for seven days, washed thrice with PBS solution (pH 7.0), and suspended in buffer solution. Then appropriate spore suspensions were taken and treated with citral with concentrations of 0, MIC, MBC for 0, 30, 60, and 120 min, respectively. Afterward, 5 mL samples were collected and centrifuged at 12000 r/min for 5 min. Then, the supernatant was taken and measure the absorbance at 260 nm with the UV-1800 spectrophotometer. The control group was calibrated with PBS (pH 7.0). 8. Effect of citral on the extracellular pH of bamboo mold cells The extracellular pH of mold cells treated with citral was measured with a micro pH/mV meter. The treatment method of spore suspensions was identical to that in 7. After treatment, 5 mL spore suspensions were taken to measure the extracellular pH, and each group was repeated three times. Tween-80 treatment groups were the control ones. 9. Anti - mildew test of bamboo treated with citral solution Bamboo strips were placed in a pressurized tank with citral solution, and then removed to dry for later use. Then, anti-mold properties of citral were examined according to the “Test method for anti-mildew agents in controlling wood mould and stain fungi” (GB/T 18261-2013) [20]: Spore suspensions of<i>Penicillium citrinum</i> (PC), <i>Trichoderma viride</i> (TV), <i>Aspergillus niger </i>(AN), and a hybrid fungi group comprising PC, TV, and AN (Hun) were smeared in Petri dishes containing the plate medium. After 2 minutes, every Petri dish put a sterilized U-shaped solid glass rod. Next, the Petri dishes were placed in an incubator at a temperature of 28±2℃ and a relative humidity of 85±5% for mildew cultivation. After the molds were successfully cultivated, the bamboo strips were placed on the U-shaped glasses, and the edge of the petri dishes were sealed with parafilm, repeat 3 times for each group. Finally, the Petri dishes with bamboo strips were placed into the incubator for a mildew resistance test. Every other day, the bamboo strips in the incubator infected by PC, TV, AN, and Hun were observed and recorded and the infection values took the average of the results (see Table3). On the 28th day, photographs of the bamboo samples were taken, and the area of the bamboo strips infected by the fungus were observed and analyzed to determine the infection levels of the bamboo strips (Table 1) and the prevention and control effectiveness were calculated according to formula (2), and the anti-mold properties of the citral were analyzed. E=(1-<i>D</i><sub>1</sub><i>D</i><sub>0</sub>)×100%---------------（2） where E is the anti-mould efficiency (%), D<sub>1</sub> is the average infection ratio of extracted specimens, and D<sub>0</sub> is the average infection ratio of control specimens. The anti-mould efficiency of a specific group of specimens is defined as the mean value of their E values against the three individual mildews and the mildew mix.
description: 1. Diameters of the inhibition zone  2.
 Inhibition rates 3.  Minimal inhibitory concentration
 (MIC) and minimal bactericidal concentration (MBC) 4.
 Absorbance 5. pH 6. Mean infection
 value 7.Anti-mold efficiency