Chemical composition and anti-biofilm activity of essential oil from Citrus medica L. var. sarcodactylis Swingle against Listeria monocytogenes

Industrial Crops & Products 144 (2020) 112036

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Chemical composition and anti-biofilm activity of essential oil from Citrus medica L. var. sarcodactylis Swingle against Listeria monocytogenes

T

Zhipeng Gaoa,*, Weiming Zhonga, Kangyong Chena, Puyu Tanga, Jiajing Guob,* a

Hunan Engineering Technology Research Center of Featured Aquatic Resources Utilization, College of Animal Science and Technology, Hunan Agricultural University, Changsha, 410128, Hunan Province, China b Hunan Agriculture Product Processing Institute, Hunan Academy of Agricultural Sciences, Changsha, 410125, Hunan Province, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Citrus medica L. var. sarcodactylis swingle Fingered citron Essential oil Anti-Biofilm Listeria monocytogenes

Listeria monocytogenes is an important foodborne pathogen that could cause listeriosis and poses potential threats to human health. Citrus medica L. var. sarcodactylis Swingle, also known as Fingered Citron, is a citrus species widely cultivated in South China. The aim of this work was to investigate the anti-biofilm efficacy of essential oil from Fingered Citron (FCEO) against L. monocytogenes. The crystal violet assay showed that FCEO exhibited strong anti-biofilm activity on both inhibiting biofilm formation and eradicating preformed biofilm. XTT assay indicated that FCEO significantly increased the metabolic active cells in the biofilm. Confocal scanning laser microscopy images and COMSTAT assay revealed that FCEO destroyed the intact architecture of biofilm, reduced biofilm biomass, thickness and substratum coverage. The morphological changes of cells in the biofilm were observed by scanning electron microscopy, which showed that FCEO could cause the wrinkle, collapse, lysis and cytoplasmic content leakage of cells. Our results also indicated that FCEO displayed its anti-biofilm activity through both biofilm dispersal and cell killing. Our findings provide evidence that FCEO exhibits strong anti-biofilm activity and might be used as a potential agent to control L. monocytogenes biofilm.

1. Introduction Listeria monocytogenes is a Gram-positive bacterium widely existing in different environments, it’s also able to survive in many harsh environments such as high salty, low temperature and low pH (Gandhi and Chikindas, 2007). L. monocytogenes can cause listeriosis, which is a kind of severe foodborne disease with high hospitalization and mortality rate. In the USA, 1651 cases of listeriosis were reported during 2009–2011 and the mortality rate was 21% (Silk et al., 2013). In the European Union, 2480 cases were reported in 2017 and the mortality rate was 13.8% (Authority et al., 2018). Meanwhile, pregnant women, newborns, the aged and people with low immunity are more sensitive to be infected by L. monocytogenes. Although L. monocytogenes widely exist in different environments, they are more preferable to contaminate and live in food products. According to the data supplied by the European Union summary report, fish and fishery products is the food category with the highest detection rate of L. monocytogenes infection in Poland, Germany, Bulgaria, the Czech Republic and the Netherlands (Authority et al., 2018). L. monocytogenes is able to form biofilm by adhering to a variety of abiotic and biotic surfaces (Mizan et al., 2015). Biofilm is organized bacterial

⁎

communities surrounded by the extracellular polymeric substance. Once established, biofilms are more resistant to antimicrobials and more difficult to eradicate than planktonic cells (Davies, 2003; Møretrø and Langsrud, 2004; Mah and Toole (2001); Stewart and Costerton, 2001). Furthermore, L. monocytogenes cells in biofilms could cause recurrent contamination in food products (Leonard et al., 2010). Thus, L. monocytogenes biofilm existing in food and food processing environment has become a serious problem for food safety and human health. Hence, finding efficacy antimicrobial agents against L. monocytogenes biofilm has become a crucial and urgent task all over the world. Recently, many studies focused on “natural and green” antimicrobials (such as herbs, antimicrobial peptides or essential oils) in order to find alternative agents of chemical antimicrobial drugs. Essential oils (EOs) extracted from plants were generally recognized as safe (GRAS) by the US Food and Drug Administration, several studies have shown that EOs have strong antimicrobial and anti-biofilm activity against variety of microorganisms (Bączek et al., 2017; Cui et al., 2019; Elhidar et al., 2019; Geraci et al., 2017; Hu et al., 2019; Manoharan et al., 2017). Citrus medica L. var. sarcodactylis Swingle, also known as Fingered Citron, is a citrus species widely cultivated in south China and usually used as a kind of Chinese traditional medicine

Corresponding authors. E-mail addresses: [email protected] (Z. Gao), [email protected] (J. Guo).

https://doi.org/10.1016/j.indcrop.2019.112036 Received 15 August 2019; Received in revised form 5 December 2019; Accepted 6 December 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.

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(Commission, 2010). Few studies have shown that FCEO exhibited potential antibacterial activity (Abdollahzadeh et al., 2014; Jadhav et al., 2013; Lou et al., 2017), but little is known for its anti-biofilm activity. To our best knowledge, this is the first study demonstrated the anti-biofilm activity of FCEO against L. monocytogenes. The aim of this work was to evaluate the anti-biofilm activity of FCEO against L. monocytogenes and provide further theoretical basis for its application.

1 × 106 CFU/ml. Wells with no FCEO and bacterial suspension added were used as positive control and negative control respectively. After that, the plate was incubated at 37 °C for 24 h. The sample with the lowest concentration of FCEO showing no visible growth of L. monocytogenes was recorded as the MIC.

2. Materials and methods

The inhibition effect of FCEO against L. monocytogenes biofilm was determined by crystal violet staining assay. Overnight cultures of L. monocytogenes were diluted 200-fold by the fresh broth in 24 well microtiter plates (Corning, USA), the final volume of bacterial suspension in each well was 2 mL, a sterilized coverslip (6 mm in diameter) was embedded in each well before dilution. After that, FCEO with different concentrations (0.5 × MIC and 1 × MIC) were added to each well, wells without FCEO added were used as positive control and wells without bacterial cultures added were used as negative control. The MIC value was 4% (v/v) which has been tested in our previous study. For both control and experimental groups, Tween 20 was added to each well at a final concentration of 1% to increase the solubility of FCEO. Then the microtiter plate was incubated in static culture for 24 h at 37 °C, after that the wells were gently washed thrice with phosphate buffered saline (PBS) to remove planktonic cells, then the biofilm attached to the coverslip was stained with 1% crystal violet solution for 30 min at room temperature, washed thrice with PBS and air-dried, then images were taken by using a Nikon camera. After photographing the crystal violet stains combined with biofilm were dissolved by 95% ethanol. Finally, the absorbance was measured with a microplate reader (Thermo, USA) at 630 nm. The percentage of inhibition was calculated according to the formula:

2.5. The biofilm inhibition assay

2.1. Bacterial strains and culture L. monocytogenes (ATCC19115) was purchased from Guangdong Microbiology Culture Center (GMCC, Guangdong, China) and stored at −80 °C, the strain was incubated by using Brain Heart Infusion broth (BHI, Guangdong Huankai Microbial Sci. and Tech. Co., Ltd, China) at 37 °C. 2.2. Essential oil extraction Fingered Citron samples were collected from Zhejiang Province (China) in November 2017. The extraction of FCEO was carried out by using steam distillation. Briefly, 500 g peel of Fingered Citron was subjected to steam distillation in 2000 mL distilled water for 3 h, and then the essential oil was collected and dried by anhydrous sodium sulfate. FCEO was stored at 4 °C in brown sealed glass vials. 2.3. Chemical composition of FCEO The chemical composition of FCEO was analyzed using gas chromatography-mass spectrometry (GC-MS). Gas chromatography analysis was performed as described in our previous study (J. Guo et al., 2019). The GC-MS analysis was carried out by using an Agilent 7890A GC equipped with a Gerstel MPS autosampler, coupled with an Agilent 5975C MSD detector. The chromatographic separation was performed on an HP-5MS capillary column (30 m × 0.25 mm i.d., film thickness 0.25 μm). The GC oven temperature program was as follows: initial temperature was 40 °C for 1 min, increased to 220 °C by 3 °C/min and held for 25 min, then increased to 250 °C by 5 °C/min and finally held for 10 min. Helium (1 mL/min) was used as the carrier gas. The injector temperature was 250 °C and the detector temperature was 280 °C. The mass spectrometer was carried out with 70 eV EI and the mass scan range was from 35 to 350 amu/s, the temperature of ion source was 230 °C. The data of FCEO components was analyzed by matching with the National Institute of Standards and Technology (NIST 08).

Inhibition percentage ODcontrol]×100%

(%)

=

[(ODcontrol

−

ODsample)

/

2.6. The biofilm eradication assay 2.6.1. The culture of preformed biofilm and the treatment procedures Overnight cultures of L. monocytogenes were diluted 200-fold by the fresh broth in 24 well microtiter plates, the final volume of bacterial suspension in each well was 2 mL, a sterilized coverslip was embedded in each well before dilution. Then the microtiter plates were incubated for 24 h at 37 °C, after that, the suspension (planktonic cells) was gently removed and the fresh broth was added into each well, repeated this procedure three times (in total, 72 h incubation) to make biofilms mature. Then the suspension was gently removed and 2 mL of fresh broth containing FCEO (1 × MIC concentration) was added into each well, wells without FCEO added were used as positive control. For both control and experimental groups, Tween 20 was added to each well at a final concentration of 1% to increase the solubility of FCEO. Then the microtiter plates were incubated at 37 °C for 8, 16 and 24 h respectively. After that, the following assays (biofilm biomass assay, biofilm metabolic activity assay, SEM and CLSM assay) were carried out.

2.4. Evaluation of the antibacterial activity 2.4.1. Agar diffusion assay The bacterial suspensions were grown up overnight at 37 °C with shaking. Then 100 μl of bacterial suspension (1 × 106 CFU/ml) was spread onto the solidified plate. After that, a sterile filter paper disc (6 mm) containing 6 μl of FCEO was placed on the center of the plate. After standing for 10 min, the plate was incubated at 37 °C for 18 h. Finally, the diameter of the inhibition zone (DIZ) was measured and described as mean ± SD.

2.6.2. Biofilm biomass assay The biofilm biomass was also quantified by crystal violet staining assay. Briefly, after the treatment procedures mentioned in 2.5.1, the same procedures were carried out as mentioned in 2.4 (the biofilm inhibition assay). The percentage of eradication was calculated according to the formula:

2.4.2. Determination of minimum inhibitory concentration (MIC) The MIC was determined using the microdilution broth method. The bacterial suspensions were grown up overnight at 37 °C with shaking, then diluted 50-fold by fresh broth and sub-cultured until the logarithmic phase. FCEO was dissolved in fresh broth at a serial two-fold (from 8% to 0.125% v/v) in sterile 96-well tissue culture plate at a final volume of 200 μl each well, Tween 20 was added to increase the solubility of EO at a final concentration of 1% v/v. Then the bacterial suspension was added to each well to reach a final concentration of

Eradication percentage ODcontrol]×100%

(%)

=

[(ODcontrol

−

ODsample)

/

2.6.3. Biofilm metabolic activity assay The biofilm metabolic activity was measured by XTT assay (Cell 2

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Proliferation Kit II, Sigma-Aldrich) (Adukwu et al., 2012; Laird et al., 2012; Sivaranjani et al., 2016). After the treatment procedures mentioned in 2.5.1, the suspension was gently removed and the wells were washed thrice with PBS, then 50 μL XTT working solution and 100 μL PBS were added to each well, after that the microtiter plates were covered and incubated for 24 h at 37 °C, finally the absorbance was measured with a microplate reader at 450 nm.

Table 1 Chemical composition of FCEO.

2.6.4. Scanning electron microscopy (SEM) assay The morphological observation of cells in the biofilm was carried out by using SEM as described in our previous study (Gao et al., 2015). After the treatment procedures mentioned in 2.5.1, the coverslip attached with mature biofilm was washed thrice with PBS to remove planktonic cells, and then fixed with 2.5% glutaraldehyde for 4 h at 4 °C, subsequently washed thrice with PBS and gradually dehydrated with gradient ethanol (10%, 30%, 50%, 70%, 90% and 100%) for 15 min respectively. Finally, the samples were freeze-dried, coated with gold film and observed by SEM (Hitachi SU8010). 2.6.5. Confocal laser scanning microscopy (CLSM) and COMSTAT analysis CLSM and COMSTAT analysis were used to investigate the architecture and characteristics of biofilm. After the treatment procedures mentioned in 2.5.1, the coverslip attached with mature biofilm was washed thrice with PBS and fixed with 4% paraformaldehyde (PFA) for 4 h at 4 °C, then cells in biofilm were stained by using Filmtracer™ LIVE/DEAD™ Biofilm Viability Kit (FilmTracer™, Molecular Probes®, Thermo Fisher) for 30 min in the dark at room temperature, then gently washed thrice with PBS. After that, the samples were observed by CLSM (Zeiss LSM880) by using a 63×/1.40 oil objective at 488 nm excitation. COMSTAT 2 software was used to evaluate the biomass, average thickness and surface volume ratio of biofilm (Heydorn et al., 2000). 2.7. Statistical analysis All experiments were carried out in triplicates. Statistical analysis was analyzed by using the Student's t-test with GraphPad Prism 6 software, P < 0.05 was considered to be significant and the significant changes were indicated by asterisks in all figures. 3. Results 3.1. Chemical composition of FCEO FCEO was yellowish with a pleasant smell, and the yield was 1.45 % (v/w). The chemical composition of FCEO was shown in Table 1. A total of 113 components were identified which represented 93.4% of the total composition. The most abundant compound was terpenes. D-limonene (44.87 %) was the major compound, followed by α-pinene (3.36 %), β-myrcene (2.73 %), β-ocimene (2.61 %), carveol (2.32 %), camphene (1.88 %), α-vetivone (1.84 %), β-pinene (1.83 %), 2,4,6octatriene (1.78 %), limonene oxide (1.73 %), α-bisabolene epoxide (1.42 %), caryophyllene oxide (1.33 %), α-cadinol (1.17 %) and alloaromadendrene oxide (1.15 %), these major compounds comprised 70.02% of the total composition. Except for these 14 major compounds mentioned above, the other 99 components were also found with a lower percentage (0.01 %-0.98 %). 3.2. Inhibition effect of FCEO against L. Monocytogenes biofilm The inhibition effect of FCEO against L. monocytogenes biofilm was tested by crystal violet staining assay. As shown in Fig. 1A, FCEO significantly inhibited the biofilm formation of L. monocytogenes (P < 0.05). The inhibitory rate at 0.5 × MIC and 1 × MIC were 52 % and 100 % respectively. The FCEO at 1 × MIC completely inhibited the biofilm formation, which meant it might be a promising anti-biofilm agent against L. monocytogenes.

Number

Compound

Percentage (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74

D-Limonene α-Pinene β-Myrcene β-Ocimene Carveol Camphene α-Vetivone β-Pinene 2,4,6-Octatriene Limonene oxide α-Bisabolene epoxide Caryophyllene oxide α-Cadinol Alloaromadendrene oxide Santolina triene Cubenene trans-p-Mentha-2,8-dienol Mandelamide Allylcyclopentene Limonene glycol Decanal α-Terpilenol Ledene oxide-(II) Isoaromadendrene epoxide trans-Isolimonene γ-Terpinene Nerol Sabinene Spathulenol δ-Elemene cis-Linaloloxide Nerol acetate Caryophyllene Elemene (E)-β-Famesene n-Hexadecanoic acid Terpenol Cyclopentadiene α-Farnesene β-Humulene α-Dihydroionone Copaene Cryptone Globulol 2-Methoxytoluene Linalool α-helmiscapene Epianastrephin Perillaldehyde Prenal Cycloheptene oxide Benzyl Alcohol Dicumene Humulene epoxide I dihydro-(-)-Neoclovene-(II) α-himachalene α-Ionol 3,8-p-Menthadiene Cyclooctane β-Sinensal Bioallethrin Cyclodecane Aciphyllene Aciphyllene Nonanal α-Isophorone cis-p-Mentha-1(7),8-dien-2-ol γ-cadinene Eremophylene Cyclooctanone β-Terpilenol 2-ethyl-4-Pentenal Ocimene α-Muurolene

44.87 3.36 2.73 2.61 2.32 1.88 1.84 1.83 1.78 1.73 1.42 1.33 1.17 1.15 0.98 0.92 0.78 0.77 0.73 0.69 0.65 0.57 0.55 0.53 0.51 0.5 0.49 0.48 0.47 0.46 0.45 0.44 0.43 0.43 0.41 0.37 0.36 0.33 0.32 0.32 0.32 0.31 0.27 0.26 0.25 0.24 0.24 0.24 0.22 0.22 0.22 0.21 0.21 0.21 0.21 0.2 0.2 0.19 0.18 0.18 0.18 0.17 0.17 0.17 0.16 0.16 0.16 0.16 0.15 0.14 0.14 0.13 0.13 0.13

(continued on next page) 3

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Table 1 (continued) Number

Compound

Percentage (%)

75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113

α-Myrcene Octopamine Limona ketone Squalene β-Cubebene Octanal citronellal Isobornyl acetate Decyl oxirane 5-Dodecyne n-Decanoic acid β-helmiscapene Mecytosine α-cedrene epoxide 1-Nonanol 4-Decyne cis-Dihydrocarvone Allethrolons Neodihydrocarveol Ledene Dodecanal Thujone α-Calacorene Leaf alcohol Nonane 3-Methylacetophenone δ-Guaiene β-Citral Acorenone Longipinene epoxide Hexanal 7-methyl-3-Octyne Verbenol 2-Hexenal 1-Hexanol Nonanoic acid Norbornadieone Cosmene 2-Hexenol

0.13 0.12 0.11 0.11 0.11 0.1 0.1 0.1 0.1 0.09 0.09 0.09 0.09 0.09 0.08 0.08 0.08 0.08 0.08 0.08 0.07 0.07 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.04 0.04 0.04 0.03 0.03 0.03 0.02 0.02 0.01

Total

Fig. 2. Effect of FCEO on the metabolic activity of cells in preformed L. monocytogenes biofilm at different treated times determined by the XTT assay. Bars represented the mean values (Mean ± SD; n = 3).

get a complete disruption. 3.4. The effect of FCEO on the metabolic activity of preformed L. Monocytogenes biofilm Since the CV assay stained both viable and non-viable cells in biofilm (Bauer et al., 2013), here XTT assay was carried out to quantify the metabolic active cells in the biofilm. As shown in Fig. 2, the number of metabolic active cells in biofilm increased significantly (P < 0.05) after exposed to FCEO at different time points. But there was no timedependent tendency since it showed no difference among these three treated time points. 3.5. The effect of FCEO on the architecture and characteristics of L. Monocytogenes biofilm Furthermore, CLSM and COMSTAT analysis were used to investigate the architecture and characteristics of biofilms. The images acquired by CLSM were represented in Fig. 3A, a thick and mature biofilm with an intact three-dimensional structure was formed in the control group. But after exposed to FCEO, the biomass decreased dramatically with timedependent and the architecture of biofilm was disturbed remarkably. COMSTAT analysis was used to transform the information from CLSM images to numerable data as shown in Fig. 3B. When taken the COMSTAT analysis and CLSM observations together, some obvious characteristics were found out. Firstly, the biofilm biomass reduced significantly which was agreed with the crystal violet staining assay. Secondly, the mean thickness of biofilm decreased dramatically, but there was no significant difference between 16 and 24 h treatment. Thirdly, the substratum coverage of biofilm declined significantly, but it showed no difference between 8 and 16 h. Fourthly, after exposed to FCEO, the intact architecture of biofilm was destroyed, many gaps

93.4

a: match with reference GC retention index. b: Peak area obtained by GC/FID.

3.3. Eradication effect of FCEO against preformed L. Monocytogenes biofilm The eradication effect of FCEO against preformed biofilm was also assessed. As shown in Fig. 1B, FCEO exhibited remarkable eradicative ability against preformed biofilm with time-dependent (P < 0.05). The eradicative rates were 56.6%, 61.7%, and 67.3% respectively after 8, 16 and 24 h treatment. However, the preformed biofilm was not eradicated completely even after 24 h treatment, which implied that a higher concentration of FCEO or a longer treated time should be used to

Fig. 1. Anti-biofilm activity of FCEO against L. monocytogenes determined by crystal violet staining assay. (A) The inhibition effect against L. monocytogenes biofilm formation at different concentrations. (B) The eradication effect against preformed L. monocytogenes biofilm at different treated times. The purple coverslips on the top indicated the biofilm stained by crystal violet. PC and NC represented positive control and negative control respectively.

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Fig. 3. CLSM images and COMSTAT analysis of L. monocytogenes biofilm untreated (Control) and treated by FCEO at different treated times (8, 16, and 24 h). (A) CLSM images. (B) COMSTAT analysis. Bars represented the mean values (Mean ± SD; n = 3).

4. Discussion

(structures like hollows and holes) were observed in CLSM images. Only micro-colonies but not mature biofilm existed after 8 and 16 h treatment, most of the cells turned to be scattered and spread on a thin monolayer after 24 h treatment.

The chemical composition of FCEO was also investigated by other studies. Here, four relevant studies were selected, including C. medica L. var. Sarcodactylis collected from China (Chongqing Province) (Wu et al., 2013), C. medica L. var. sarcodactylis collected from China (Zhejiang Province) (Lou et al., 2017), C. medica collected from Greece (Mitropoulou et al., 2017) and C. medica L. collected from France (Lota et al., 1999). As shown in Table 2, the first six major components of these studies were listed and the results were quite different from each other, but we could also find some common characteristics. Firstly, Dlimonene was the component found in all studies with the highest percentage although their percentage was different. Secondly, α-pinene (4 times), terpinene (3 times) and myrcene (3 times) were found with high frequency. These tendencies indicated that limonene, α-pinene, terpinene, and myrcene might be the crucial components for the antibiofilm activity of FCEO. The different chemical composition of FCEO in different studies might be caused by their different geographical regions, genetic variations, harvesting seasons, extract methods or environmental factors (Diao et al., 2013; J.-j. Guo et al., 2018) In the current study, our data showed that FCEO was efficient against L. monocytogenes biofilm. There were also few studies focusing on the anti-biofilm activity of FCEO against other bacteria species, such as Staphylococcus aureus and Pseudomonas aeruginosa. Zhang H et al. investigated the anti-biofilm activity of FCEO extracted by different methods against S. aureus biofilm and showed that FCEO extracted by hydrodistillation extraction (the same method used in our study) completely inhibited the biofilm formation at a concentration of 0.75 mg/mL (Zhang et al., 2019). Lou Z et al. also found that FCEO exhibited an inhibitive effect on S. aureus biofilm formation with a 55.6 % inhibiting rate at a concentration of 0.24 mg/mL (Lou et al., 2017). In contrast, Wang E et al. found that FCEO exhibited only little inhibitive ability against both S. aureus (inhibition rate: 11.6%) and P. aeruginosa (inhibition rate: 10%) biofilm at a concentration of 4.0 mg/mL (Wang et al., 2019). Taken together, some tendencies were concluded. Firstly,

3.6. The effect of FCEO on the morphological changes of cells in L. Monocytogenes biofilm SEM was used to demonstrate the changes on both the community and cellular level. The SEM results were presented in Fig. 4. Firstly, the changes of biofilm structure were observed by low magnification images, cells in the control group were tightly aggregated and clustered together to form intact mature biofilm structure. Meanwhile, some channels were found in the biofilm which might be helpful for exchanging nutrient and discharging metabolic waste of cells. In contrast, after exposed to FCEO, the integrity of the biofilm architecture was obviously disrupted and the biomass decreased significantly. After 8 h treatment, both macro-colonies and small cell clusters were observed, and some macro-colonies still exhibited three-dimensional architectures. While after 16 h treatment, only small clusters but no macrocolonies were found. When treated for 24 h, only small cell aggregates were scattered on the coverslip, and they were almost located in one layer. These results were highly coincident with CLSM and COMSTAT analysis. Secondly, the morphological changes of cells in the biofilm were observed by high magnification images. Cells in the control group were intact with smooth surface, but obvious morphological changes were observed after exposure to FCEO. Wrinkled and collapsed cell surface were presented after 8 h treatment. The lysis of cells was observed after 16 and 24 h treatment, and the percentage of lytic cells at 24 h was much higher than 16 h, but the whole lysis was often found after 24 h treatment. Some fragments were also found out of cells which might be the cytoplasmic content leaked from lytic cells.

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Fig. 4. SEM micrographs of L. monocytogenes biofilm untreated (Control) and treated by FCEO at different treated times (8, 16, and 24 h). The scale bar represented 500 nm.

Table 2 Chemical composition of FCEO from different studies. Num.

Collected from China (Zhejiang) -Our study

Collected from China (Chongqing)

Collected from Chian (Zhejiang)

Collected from Greece

Collected from France

1

D-limonene (44.87 %) α-pinene (3.36 %) β-myrcene (2.73 %) β-ocimene (2.61 %) carveol (2.32 %) camphene (1.88 %)

limonene (33.84 %)

D-limonene (50.04%) c-terpinene (32.11%) a-pinene (2.75) 1,3,6-Octatriene3,7- dimethyl-, (Z) (1.64%)

limonene (64.35 %)

limonene (51.9 %)

geranio (2.65 %)

γ-terpinene (31.3 %)

nerol (2.57 %)

geranial (2.6 %)

myrcene (2.31 %)

α-pinene (1.9 %)

neral (cis-citral) (0.71%) linalool (0.71 %)

β-pinene (1.8%)

2 3 4 5 6

γ-terpinene (22.88 %) 3-carene (9.01 %) α-pinene (7.73 %) α-thujone (5.09 %) β-pinene (3.18 %)

(1R)-2,6,6-Trimethylbicyclo[3.1.1]hept-2-ene (1.63%) Cyclohexene, 1-methyl-4-(1-methylethylidene) (1.30%)

6

myrcene (1.6 %)

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promising anti-biofilm agent against L. monocytogenes. Further investigation will be carried out to explore whether FCEO could eradicate L. monocytogenes biofilm formed on food processing surfaces. Our future studies will also focus on the anti-biofilm mechanism of FCEO.

FCEO exhibited different anti-biofilm ability against different bacterial species, P. aeruginosa biofilm was more difficult to inhibit than L. monocytogenes and S. aureus. This might be caused by the bacterial type or cell structure, P. aeruginosa is a Gram-negative bacteria with an outer membrane which could be a barrier to prevent the penetration of FCEO, but both L. monocytogenes and S. aureus are Gram-positive bacteria without outer membrane (J.-j. Guo et al., 2018; Trombetta et al., 2005). Moreover, FCEO showed different anti-biofilm ability against the same bacterial species. The inhibition rate of S. aureus biofilm in the above studies was 100%, 55.6%, and 11.6% respectively. This might be caused by the Fingered Citron samples, which might be collected from different geographical regions or at different harvesting seasons. These factors might influence the chemical composition and finally resulted in their different anti-biofilm abilities. Besides FCEO, the anti-biofilm ability of other EOs extracted from different plants was also explored. Jadhav S et al. showed that EO from Achillea millefolium was efficient to eradicate preformed Listeria biofilm with an eradication rate of 52.2% at 1 × MIC (Jadhav et al., 2013). Leonard C M et al. found that EOs from Syzygium aromaticum and Mentha spicata exhibited great potential to eradicate L. monocytogenes biofilm, in contrast, EOs from Cymbopogon citratus and Lippia rehmannii caused biofilm enhancement rather than inhibition (Leonard et al., 2010). Our previous work also showed that EO from Citrus Changshan huyou Y. B. Chang exhibited strong anti-biofilm activity against L. monocytogenes (J. Guo et al., 2019). In our study, the metabolic activity of cells in biofilm increased significantly after exposure to FCEO. There are also other studies showed the increased or unchangeable metabolic activity of cells after the treatment of EOs. Adukwu et al. found that, after treated with grapefruit EO, four S. aureus strains showed no reduction in metabolic activity and one S. aureus strain showed increased metabolic activity (Adukwu et al., 2012). Kwiecinski et al. reported an increase in metabolic activity in S. aureus biofilm after treated with tea tree EO (Kwieciński et al., 2009). The reasons for such a phenomenon were unknown. In my opinion, a probable explanation might be that the cells in biofilms increased their metabolic activities as a response to the rapid change of environment and stress (FCEO treatment), but whether this is the case for the phenomenon remains to be determined in the future. According to the results and observations in our study, we speculated two potential ways to explain how FCEO eradicated L. monocytogenes biofilm. Firstly, after 8 h treatment, the biomass decreased significantly but the lysis of cells was not observed. These results indicated that dispersal rather than killing of cells might be the main function of FCEO during this period. Secondly, after 16 and 24 h treatment, except for the decrease of biomass, the lysis of cells was also observed. These findings implied that both killing and dispersal might concurrence during this period. Polystyrene microtiter plates were often used as the adhesive medium for biofilm research in the lab. Except for polystyrene microtiter plates, stainless steels which were often used in the food processing industry were also used in other studies. Sadekuzzaman M et al. demonstrated the anti-biofilm ability of thyme and tea tree EOs against L. monocytogenes formed on stainless steels, the results showed that thyme and tea tree EOs significantly reduced the biofilm cells to 1.5 log CFU/ cm2 and 2.0 CFU/cm2 at 1 × MIC respectively (Sadekuzzaman et al., 2018). Desai M A et al. also found that both thyme and oregano EOs could completely eradicate 4-day-old preformed biofilm on stainless steels at a 0.5% concentration (Desai et al., 2012). These findings indicated that EOs have the potential to be used to eradicate L. monocytogenes biofilm in the food processing industry.

CRediT authorship contribution statement Zhipeng Gao: Funding acquisition, Conceptualization, Project administration, Writing - original draft. Weiming Zhong: Investigation, Formal analysis. Kangyong Chen: Investigation, Visualization. Puyu Tang: Investigation, Formal analysis. Jiajing Guo: Project administration, Validation, Supervision. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This study was supported by the Science and Technology Innovation Project of Hunan Province (S2017NCTPSQ0064), the Key Research and Development Project of Hunan Province (2016NK2109), Hundred Talents Plan of Hunan Province and Shennong Scholar Program of Hunan Agricultural University (540499818005). References Abdollahzadeh, E., Rezaei, M., Hosseini, H., 2014. Antibacterial activity of plant essential oils and extracts: The role of thyme essential oil, nisin, and their combination to control Listeria monocytogenes inoculated in minced fish meat. Food Control 35, 177–183. https://doi.org/10.1016/j.foodcont.2013.07.004. Adukwu, E., Allen, S.C., Phillips, C.A., 2012. The anti-biofilm activity of lemongrass (Cymbopogon flexuosus) and grapefruit (Citrus paradisi) essential oils against five strains of Staphylococcus aureus. J. Appl. Microbiol. 113, 1217–1227. https://doi.org/ 10.1111/j.1365-2672.2012.05418.x. Authority, E.F.S., Prevention, E. C. f. D.Control, 2018. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2017. Efsa J. 16, e05500. https://doi.org/10.2903/j.efsa.2018.5500. Bączek, K.B., Kosakowska, O., Przybył, J.L., Pióro-Jabrucka, E., Costa, R., Mondello, L., Gniewosz, M., Synowiec, A., Węglarz, Z., 2017. Antibacterial and antioxidant activity of essential oils and extracts from costmary (Tanacetum balsamita L.) and tansy (Tanacetum vulgare L.). Ind. Crops Prod. 102, 154–163. https://doi.org/10.1016/j. indcrop.2017.03.009. Bauer, J., Siala, W., Tulkens, P.M., Van Bambeke, F., 2013. A combined pharmacodynamic quantitative and qualitative model reveals the potent activity of daptomycin and delafloxacin against Staphylococcus aureus biofilms. Antimicrob. Agents Chemother. 57, 2726–2737. https://doi.org/10.1128/AAC.00181-13. Commission, C.P., 2010. Pharmacopoeia of the People’s Republic of China. 1 (2010). China Medical Science Press. Cui, H., Zhang, C., Li, C., Lin, L., 2019. Antibacterial mechanism of oregano essential oil. Ind. Crops Prod. 139, 111498. https://doi.org/10.1016/j.indcrop.2019.111498. Davies, D., 2003. Understanding biofilm resistance to antibacterial agents. Nat. Rev. Drug Discov. 2, 114. https://doi.org/10.1038/nrd1008. Desai, M.A., Soni, K.A., Nannapaneni, R., Schilling, M.W., Silva, J.L., 2012. Reduction of Listeria monocytogenes biofilms on stainless steel and polystyrene surfaces by essential oils. J. Food Prot. 75, 1332–1337 https://doi.org/10.4315/0362-028X.JFP-11-517. Diao, W.-R., Hu, Q.-P., Feng, S.-S., Li, W.-Q., Xu, J.-G., 2013. Chemical composition and antibacterial activity of the essential oil from green huajiao (Zanthoxylum schinifolium) against selected foodborne pathogens. J. Agric. Food Chem. 61, 6044–6049. https://doi.org/10.1021/jf4007856. Elhidar, N., Nafis, A., Kasrati, A., Goehler, A., Bohnert, J.A., Abbad, A., Hassani, L., Mezrioui, N.-E., 2019. Chemical composition, antimicrobial activities and synergistic effects of essential oil from Senecio anteuphorbium, a Moroccan endemic plant. Ind. Crops Prod. 130, 310–315. https://doi.org/10.1016/j.indcrop.2018.12.097. Gandhi, M., Chikindas, M.L., 2007. Listeria: a foodborne pathogen that knows how to survive. Int. J. Food Microbiol. 113, 1–15. https://doi.org/10.1016/j.ijfoodmicro. 2006.07.008. Gao, Z.P., Nie, P., Lu, J.F., Liu, L.Y., Xiao, T.Y., Liu, W., Liu, J.S., Xie, H.X., 2015. Type III secretion system translocon component EseB forms filaments on and mediates autoaggregation of and biofilm formation by Edwardsiella tarda. Appl. Environ. Microbiol. 81, 6078–6087. https://doi.org/10.1128/AEM.01254-15. Geraci, A., Di Stefano, V., Di Martino, E., Schillaci, D., Schicchi, R., 2017. Essential oil components of orange peels and antimicrobial activity. Nat. Prod. Res. 31, 653–659. https://doi.org/10.1080/14786419.2016.1219860. Guo, J.-j., Gao, Z.-p., Xia, J.-l., Ritenour, M.A., Li, G.-y., Shan, Y., 2018. Comparative

5. Conclusion In conclusion, the results presented in this work suggested that Fingered Citron essential oil was effective against L. monocytogenes biofilm. These results strongly supported that FCEO may be used as a 7

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