Synergistic inhibition effect of citral and eugenol against Aspergillus niger and their application in bread preservation

Food Chemistry 310 (2020) 125974

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Synergistic inhibition effect of citral and eugenol against Aspergillus niger and their application in bread preservation

T

Jian Jua,b,c, Yunfei Xiea,b,c, Hang Yua,b,c, Yahui Guoa,b,c, Yuliang Chenga,b,c, Rongrong Zhangd, ⁎ Weirong Yaoa,b,c, a

State Key Laboratory of Food Science and Technology, Jiangnan University, China School of Food Science and Technology, Jiangnan University, China c Joint International Research Laboratory of Food Safety, Jiangnan University, No. 1800 Lihu Avenue, Wuxi 214122, Jiangsu Province, China d Nantong Quanzheng Inspection and Testing Co., Ltd., No. 69, Zilang Road, Nantong City, Jiangsu Province 226000, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Essential oil Citral Eugenol Synergistic effect Antimicrobial mechanism Bread Shelf life

Consumers’ preferences for cleaner label products require the food industry to replace synthetic preservatives with natural substitutes. Therefore, the synergistic inhibitory effect of eugenol and citral (SEC) on Aspergillus niger was explored. On this basis, the antimicrobial sachet containing SEC was developed and its application potential in bread preservation was evaluated. The content of reactive oxygen species (ROS) and malondialdehyde (MDA) showed that SEC could significantly induce lipid peroxidation in cell membranes, in which citral played a leading role. The permeation experiments of SEM, TEM, propyl iodide and fluorescein diacetate showed that SEC could destroy the integrity of the cell membrane. Eugenol contributed more than citral. The OD260 and the relative conductivity of the SEC group increased by 5.2 and 4.1 times, respectively, after 8 h. Finally, the shelf life experiment of bread showed that the antimicrobial sachets containing SEC could significantly prolong the shelf life of bread without producing unpleasant odour.

1. Introduction The widespread use of chemical preservatives in food may pose a serious threat to human health (Cizeikiene, Juodeikiene, Paskevicius, & Bartkiene, 2013). Especially in the past few years, due to the emergence of food safety problems, consumers generally refuse to use chemical synthetic preservatives to control the growth of microorganisms in food (Ju, Wang, Qiao, Li, & Li, 2017). Due to the increasing market demand for safe and natural health products without chemical preservatives, relevant food departments and researchers have conducted detailed investigations to assess the feasibility of using mild preservative technology to improve product quality and safety while maintaining its good nutrition and sensation (Ju et al., 2018a). Most natural essential oils and their extracts have been listed as GRAS (Generally Recognized as Safe) by FDA (Food and Drug Administration of the United States) (Atarés & Chiralt, 2016; Ju et al., 2018b; Ruiz-Navajas, Viuda-Martos, Sendra, Perez-Alvarez, & Fernández-López, 2013) because of their high efficiency, safety and non-toxic qualities. So they can be used as substitutes for synthetic food additives. This further illustrates the good application prospects of essential oils. Eugenol is the main active component of essential oils such

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as clove, camphor and cinnamon leaves (Liu, Chen, & Chen, 2008). At present, a large number of studies have confirmed that eugenol has significant antimicrobial activity (Niu et al., 2019; Requena, Vargas, & Chiralt, 2019; Talón et al., 2019). The main antimicrobial mechanism of eugenol is that it can increase the permeability of the cell membrane and its OH group can significantly inhibit the activity of related enzymes (Woranuch & Yoksan, 2013). Because eugenol has the advantage of high antimicrobial activity, no chemical residues and no pollution to the environment, it meets the requirements of consumers on “green”, “safety” and “environmental protection” food preservatives (Wang, Chen, & Wang, 2009). Citral, the main component of lemon grass essential oil, is a natural isoprene compound and the most important representative of open-chain monoterpenes, with lemon flavour. It has been recognized by the European Commission as a condiment in food (Guo, Sun, Sun, & Sun, 2018). Previous studies have shown that citral can increase lipid oxidation of cell membranes, lead to the explosion of reactive oxygen species (ROS) and also damage the cell morphology of fungi by damaging mitochondria (Nogueira et al., 2010; Zheng et al., 2015). For some pathogenic bacteria, citral also showed good bacteriostatic activity, such as Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, etc. (Shi et al., 2016).

Corresponding author at: State Key Laboratory of Food Science and Technology, Jiangnan University, China. E-mail address: [email protected] (W. Yao).

https://doi.org/10.1016/j.foodchem.2019.125974 Received 17 September 2019; Received in revised form 27 November 2019; Accepted 27 November 2019 Available online 04 December 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.

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2.3. Determination of malondialdehyde content

At present, the research on antimicrobial activity of eugenol and citral has been widely reported. Their application potential has also been further confirmed in the preservation of fruits, vegetables, and meat products (Huang et al., 2019; Ju et al., 2017; López-Romero et al., 2018; Wan et al., 2018). However, the main problem is that the actual dosage of essential oil used in food preservation is generally higher than the theoretical value obtained under laboratory conditions, which leads to the fact that the amount of essential oil needs to be increased in specific food preservation applications in order to achieve better antimicrobial effect. Therefore, the high concentration of essential oil may affect the quality of food itself, especially the flavour of food. The synergistic effect of essential oils may be an effective way to solve this problem. According to the existing research, the main microorganisms causing the spoilage of baked food are A. niger and P. roqueforti (Axel et al., 2016; Clemente, Aznar, & Nerín, 2019; Ju et al., 2017; Lavermicocca, Valerio, & Visconti, 2003). However, the research on synergistic inhibition effect of essential oil on fungi in baked food is very limited. Therefore, the purpose of this study was to evaluate the synergistic inhibition effect of eugenol and citral (SEC) on A. niger and then to explore the effect of antimicrobial sachets containing SEC on bread shelf life. From the point of view of synergistic antimicrobials, it can provide reference for reducing the dosage of plant essential oil in food preservation.

Malondialdehyde (MDA) was determined as described previously (Yang, Sun, Deng, Wang, & Sun, 2017). A 1 g sample of treated mycelium was ground with liquid nitrogen and nine volumes of PBS buffer was added to prepare a slurry that was centrifuged at 12000 rpm for 15 min at 4 °C. The supernatant was obtained and the MDA content was measured according to the kit instructions. Each treatment was repeated three times. 2.4. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) Spore suspension of A. niger at a cell density of 1 × 106 colonyforming units (cfu)/ml was suspended in potato dextrose water medium and incubated for 48 h at 28 °C with shaking at 120 rpm. After centrifugation at 6000×g for 5 min, the cell pellet was resuspended in phosphate-buffered saline (PBS, pH 7.2), and eugenol and citral were added to the same final concentration to 1 MIC culture as SEC and incubated for 8 h at 28 °C. Anhydrous ethanol was used as a negative control. Sample preparation for SEM was performed as described previously. The samples were incubated under vacuum for 10 min in 4% glutaraldehyde diluted in 0.1 M phosphate buffer (pH 7.0), rinsed with the same buffer for 8 h at 4 °C. Samples were further dehydrated in a graded ethanol series (50%, 70%, 90%), critical-point-dried using CO2 and were finally mounted on specimen stubs, coated with gold and examined with a scanning electron microscope (Hitachi S-4800 FESEM, Japan). The preparation of TEM samples was slightly modified according to the method described by Kong et al. (2019). After treatment, the samples were harvested, and pre-fixed in 3% glutaraldehyde and kept at 4 °C for 8 h. Afterwards, the samples were dehydrated in a graded series of ethanol solutions (50%, 70% and 90%) for a period of 15 min in each alcohol dilution, acetone:embedding solution (3:1) for 2–3 h, and acetone:embedding solution (1:1) for 2–3 h. They were then soaked in 100% embedding solution overnight and finally immobilized in a graded oven (37 °C overnight, 45 °C for 12 h and 60 °C for 24 h). Ultrathin sections (approximately 50–60 nm in thickness) were stained with 3% uranyl acetate and lead citrate and then examined by TEM (Tecnai G2 20 S-Twin, FEI Company, Hillsboro, USA).

2. Materials and methods 2.1. Materials and antifungal agents A. niger (ATCC 16404) was obtained from the China Center of Industrial Culture Collection. Potato dextrose agar medium was purchased from Hope Bio-Technology Co., Ltd. (Qingdao, China). Eugenol (CAS-No. 93-53-0; 98%) and citral (CAS-No. 5392-40-5; 97%) were purchased from J&K Scientific Co., Ltd. (Shanghai, China). The MIC concentration of eugenol and citral synergistic action on A. niger was determined to be 0.23 mg/ml, respectively. The 1 MIC of SEC solution was obtained by mixing eugenol and citral at 1:1 (v/v).

2.2. Fluorescence microscopy 2.5. Release of cell components Fluorescence microscopy was performed as described previously (Ji et al., 2018). A spore suspension of A. niger (1 × 106 cfu/ml) was suspended in potato dextrose water medium (Hope Bio-Technology Co., Ltd. Qingdao, China), and incubated for 36 h at 28 °C with constant shaking (120 rpm). The precipitate was obtained by centrifugation at 8000×g for 5 min at 28 °C, and suspended in phosphate buffer (PB, pH 7.2). Meanwhile, SEC was added to the cultures at a final concentration of 1 MIC, and cultures were incubated at 28 °C. Absolute ethyl alcohol was used as a negative control. After incubation for 8 h, cells were harvested and centrifuged at 8000×g for 2 min, washed three times with PB. Then the corresponding dye was added into and incubated with the cell. After incubation in the dark at 37 °C for 30 min, cells were harvested and centrifuged at 12,000×g for 2 min, and then washed three times with PB. Cell viability was examined using 50 mg/l fluorescein diacetate (FDA; Cat. no. F7378; Sigma) and 20 mg/l propidium iodide (PI; Cat. no. P3566; ThermoFisher); Intracellular reactive oxygen species (ROS) levels were measured using 10 μM 2,7-dichlorodihydrofluorescein diacetate (DCHF-DA) and an oxidation-sensitive probe (Molecular Probes, Eugene, OR, USA). After staining, spores were examined under a Zeiss Axioskop 40 fluorescence microscope (Zeiss, Germany) using the following parameters: for FDA and DCHF-DA detection (EX BP 450–490, FT 510, LT515); for PI (BP 546/12, FT 580, LP 590). Three independent experiments were performed.

Referring to Huang, Qian, Jiang, and Zheng (2019) method, with slight modifications, spore suspension of A. niger at a cell density of 1 × 106 (cfu)/ml was suspended in potato dextrose water medium and incubated for 48 h at 28 °C with shaking at 120 rpm. After centrifugation for 15 min at 4000×g, the mycelia were collected and suspended in phosphate buffer (pH 7.0) after washing 3 times with sterile water. Eugenol, citral and SEC with MIC concentration were added into the above sample for 8 h. Samples were centrifuged for 2 min at 12,000 rpm to collect supernatant. Absorption was measured at 260 nm by ultraviolet spectrophotometer. PBS (pH 7.0) was used in control group for correction. 2.6. Relative conductivity The effects of eugenol, citral and SEC treatments on the extracellular conductivity of A. niger were measured by a micro conductometer. The method of culture and treatment was described in Section 2.5. The extracellular conductivity was measured and compared with absolute ethanol. 2.7. Sachet elaboration According to the method of Passarinho et al. (2014), 1 g corn porous 2

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starch (particle size is 10–50 μm) was precisely weighed and put into a grinding triangular bottle. Eugenol and citral were then added in a ratio of 1:1 in 5 ml. After capping, they were put into a constant temperature water bath oscillator (150 rpm) for 60 min at 35 °C. After a certain period of oscillation, the essential oil can enter the porous starch through osmotic diffusion and form microcapsules. The residual essential oil on the porous starch surface was washed with absolute ethanol by vacuum filtration. The microencapsule was collected after drying at 28 °C for 30 min. A sample of 1 g microcapsule was placed in sachets made of nonwoven tissue (thickness: 60 μm; tensile strength: 12.02 MPa; resistance to puncture: 2.16 N), which was a porous, polypropylene-based material that allowed the diffusion of the volatile compounds from essential oil. Sachets were then heat-sealed and exposed to ultraviolet radiation (110 V, 254 nm) for 2 min/side before being used for testing. EO-free sachets were used as a control.

Bazzaz, 2016; Zhang, 2018). These published research results could play a better supporting role to the above conclusions. 3.2. Changes in MDA content MDA is one of the most important products of lipid peroxidation and is elevated following exposure to ROS, which subsequently damages phospholipids, enzymes, nucleic acids and biofilms and can reflect the degree of lipid peroxidation (Chen, Xu, Cheng, Gao, & Zhang, 2018; Jayashree & Subramanyam, 2000; Zaccaria et al., 2015). It can be seen from Fig. 2 that the amount of MDA produced increased gradually in the three different treatment groups. In particular, SEC caused a substantial increase in MDA in A. niger cells that was significantly (p < 0.05) higher than that in the other treatment groups. The MDA contents in the control, eugenol, citral and SEC groups were 3.77, 6.25, 9.03 and 13.15, respectively. However, it can also be clearly seen from the figure that the content of MDA in the citral treatment group was significantly (p < 0.05) higher than that in the eugenol treatment group. We found that the contribution of citral to peroxidation damage of cell membrane lipids was greater than that of eugenol. This further confirmed the conclusion in 3.1 that citral could induce ROS outbreak and then result in a rapid increase in MDA content.

2.8. Determination of shelf life of bread Wheat flour was provided by Zhongyu Co., Ltd. (Shandong Province, China). Yeast was provided by Angel Yeast Co., Ltd. (Hubei Province, China). A Dongling bread machine (DL-JD08) was provided by Guangdong Dongling Electric Co., Ltd. (Guangdong, China). The bread material included: water 90 ml, high-gluten flour 200 g, white granulated sugar 30 g, milk powder 7 g, salt 2 g, egg 50 g, butter 20 g, and yeast 3 g. The following breads were prepared: Control samples: without antimicrobial sachet; Experimental samples: with antimicrobial sachets. The detailed packaging process is shown in Schematic diagram S1. The packaged bread was stored in incubators at 25 °C and 35 °C, respectively. Samples were checked daily to detect visible mold colonies. The day before the first mildew spots appeared on bread was recorded as shelf life.

3.3. Changes of spore morphology and cell ultrastructure The results of SEM analyses of A. niger spores are shown in Fig. 3A. Spores of control groups were smooth and full, with no cracks, wrinkles or dryness, and they were in a normal growth state. By contrast, cracking and dissolution were evident on the surface of spores from eugenol treated A. niger, and more of the citral treated spores exhibited wrinkling and dryness. Previous studies have shown that eugenol could (p < 0.05) increase the permeability of the phospholipid bilayer significantly (Wang et al., 2009; Woranuch & Yoksan, 2013; Zhang et al., 2019). Similarly, some researchers have found that citral treatment could distort mycelia (Tao, Duan, Fan, & Huang, 2015). In the SEC group, the spores of A. niger showed more obvious wizened and lysis change than those treated with eugenol or citral alone, and even appeared with holes. Thus it can be seen that the main function of eugenol may be to destroy the permeability of cell membranes, leading to cell decomposition or dissolution, while citral mainly affects cell morphology, leading to cell shrinkage or atrophy. TEM analyses showed that the mycelial cell walls of A. niger blank controls were clear with clear boundaries, of uniform thickness, oval in shape, mitochondria were clearly visible, vacuoles and other organelles were well ordered, and cells were in a normal growth state (Fig. 3B). By contrast, mycelial cell walls of eugenol treated A. niger was dissolved, blurred and along with cell membranes, the internal structure of the cell was chaotic and disordered, and numerous vacuoles were fused in the cytoplasm, forming larger vacuoles. The mycelium cell wall of citraltreated A. niger appeared to be weaker than that of the eugenol-treated group, and the internal structure of cells was relatively intact compared with eugenol treated cells. However, in the SEC combined treatment group, the mycelium cell walls of A. niger exhibited severe dissolution and deformation. The internal structure of cells was disordered, and some organelles were absent. Thus, eugenol played a leading role in destroying the integrity of cell membranes. At the same time, it further proved the conclusion in 3.4 that eugenol could better destroy the permeability or integrity of cell membrane. Previous results showed that thymol could increase cell membrane potential and alter membrane permeability in Fusarium graminearum, and similar phenomena were observed in Vibrio parahaemolyticus treated with tea polyphenols (Kong et al., 2019; Liu et al., 2017).

2.9. Sensory evaluation The acceptability of bread containing antimicrobial sachets was assessed throughout storage. The sensory evaluation team of bread consisted of 16 members (8 males and 8 females). The sensory attribute overall impression was rated using the 9-point hedonic scale to assess liking and disliking, with terms varying from dislike extremely to like extremely (Table S1). In addition, members of the sensory evaluation team are allowed to provide additional comments. Sensory tests were carried out in an appropriate laboratory. 2.10. Statistical analysis All experiments were performed in triplicate, and data was analysed using OriginLab-9s (OriginLab Corporation, Hampton, MA, USA). Mean values were compared by Duncan’s new multiple range tests, and statistically significant differences were set at p < 0.05. 3. Results and discussion 3.1. SEC induced accumulation of reactive oxygen species (ROS) Accumulation of ROS could induce apoptosis or even necrosis through cellular oxidative stress. SEC strongly induced the accumulation of ROS in A. niger cells according to DCHF-DA fluorescence signal measurements. There were no spores associated with green fluorescence in control groups. However, the proportion of cells stained in the citral treatment group was significantly (p < 0.05) higher than that of the eugenol treatment group but lower than the SEC group (Fig. 1), indicating that citral may cause oxidation-induced ROS outbreaks in A. niger cell membrane lipids. Previous studies have shown that terpenoids can significantly (p < 0.05) induce lipid peroxidation in eukaryotic cell membranes (Huang et al. (2019); Khameneh, Diab, Ghazvini, &

3.4. SEC treatment leaded to loss of cell membrane integrity and viability To further explore the underlying mechanism by which SEC exerted 3

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Fig. 1. SEC induces the accumulation of ROS. A is the results of DCHF-DA staining of A. niger (bar = 20 μm); B is the percentage of DCHF-DA staining. Different superscript letters indicates significant differences (p < 0.05).

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It can be seen from Fig. 4 that A. niger control cells were readily stained with FDA, yielding high fluorescence intensity. However, cells of A. niger incubated with SEC were not stained (Fig. 4A and C), indicating that SEC caused a loss of activity. Similarly, more cells in the SEC groups were stained by PI than in the control groups (Fig. 4B and D), indicating that a significant percentage of cells lost membrane integrity. However, the percentage of cells stained in eugenol-treated groups was significantly (p < 0.05) higher than that in citral treated groups (Fig. 4D), indicating that eugenol was significantly (p < 0.05) more destructive to the cell membrane of A. niger than citral. Because PI is impermeable and usually excluded from living cells, these results indicated that cells exhibiting red fluorescence (PI) were dead. These results were consistent with those of FDA staining, which accumulated in living cells as fluorescein after uptake. Based on these results, as in previous studies, it was confirmed that eugenol could better break the integrity of the cell membrane (Woranuch & Yoksan, 2013; Zhang, Zhang, Ma, Wang, & Ding, 2018).

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Fig. 2. Changes in MDA content. Different superscript letters indicates significant differences (p < 0.05).

As shown in Fig. 5A, SEC could significantly (p < 0.05) increase the component release of A. niger cells at OD260nm. After 8 h incubation, the OD260 nm value of SEC was 0.29 which was significantly (p < 0.05) higher than that of the eugenol group (0.21) and citral group (0.18). The relative conductivity data in Fig. 5B also showed a similar trend,

antifungal action, FDA and PI were employed to investigate cell viability and membrane integrity of A. niger cells using a fluorescence microscope.

Fig. 3. Observation of spore morphology (bar = 5 μm) and cell ultrastructure (bar = 500 nm) by SEM and TEM. 4

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Fig. 4. SEC treatment leads to loss of cell membrane integrity and viability. A is the results of PI staining of A. niger (bar = 20 μm); C is the percentage of PI staining. B is the results of FDA staining of A. niger (bar = 20 μm); D is the percentage of FDA staining. Different superscript letters indicates significant differences (p < 0.05).

colonial spots on the 6 d during storage at 25 °C. However, the bread in the experimental group had obvious colonial spots on the 15 d. Similar trends were also observed at 35 °C. The bread in the control group had obvious fungal colonies on the 4 d, while the bread in the experimental group had slight colonial spots on the 10 d. It can be seen that the antimicrobial sachet has a good protective effect on bread.

that was, the relative conductivity of the SEC group was the highest, followed by the eugenol group and citral group. The increase of OD260 and relative conductivity meant that the cell membrane was destroyed, leading to the leakage or extravasation of the cell contents. Previous studies have also demonstrated the effects of citronellal or terpineol on component release and conductivity of Penicillium cell. The results showed that these two substances could increase the permeability of the cell membrane thus leading to the leakage of intracellular contents and increase of conductivity (Ouyang, Jia, Tao, & He, 2014; Wu, OuYang, & Tao, 2016).

3.7. Sensory analysis The sensory acceptability of bread during storage was evaluated in the experimental group (Table S2). The sensory score of bread stored at 25 °C was 7.0 on the 12 d of storage, which was in a state of moderate liking. The sensory score of bread stored at 35 °C was 6.2 on the 9 d, which was between moderate liking and slight liking. In addition, the addition of antimicrobial sachets did not significantly affect the sensory quality of bread by analysing the additional comments from team members. Based on the above results, it can be reasonably assumed that the shelf life of bread should be extended at commercial storage temperature. This method has great practical application potential. However, the actual shelf life of bread products needs to be verified by

3.6. Bread challenge test As shown in Fig. 6A and B, the shelf life of bread can be extended to 13 d by using antimicrobial sachets at 25 °C compared with the control group. And the shelf life of bread was prolonged to 9.5 d by using antimicrobial sachets compared with the control group at 35 °C. It can be seen from Fig. 6C and D that physical changes occur in bread during storage at 25 °C and 35 °C, respectively. It can be clearly seen from the picture that the bread in the control group had obvious

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Fig. 5. Effects of SEC on cellular component release and relative conductivity. A is the OD260nm; B is the relative conductivity. Different superscript letters indicates significant differences (p < 0.05). 5

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Fig. 6. Shelf life of bread. A and C represent the shelf life and physical changes of bread stored at 25 °C, respectively. B and D represent shelf life and physical changes of bread stored at 35 °C, respectively.

increasing the number of experimental samples in the coming days.

Acknowledgements

4. Conclusion

The work described in this article was supported by the National Key Technology R&D Program in the 13th Five Year Plan of China (2018YFC1602300), Natural Science Foundation of Jiangsu Province (BK20171139), Yangtze River Delta Project of Shanghai (18395810200), Forestry science and technology innovation and extension project of Jiangsu Province (No. LYKJ [2017] 26), National first-class discipline program of Food Science and Technology (JUFSTR20180509), Science and technology project of Jiangsu Bureau of Quality and Technical Supervision (KJ175923 and KJ185646), China Postdoctoral Science Foundation funded project (2018M642165), and Science and Technology Plan of Changzhou City (CE20172002).

Against A. niger, eugenol combined with citral (SEC) appear to be a potent bacteriostatic combination. Analysis of the antimicrobial mechanism indicated that direct damage to the cell membranes of A. niger may explain the combined antimicrobial activity of SEC. Among the two components, eugenol is mainly responsible for the permeability of damaged cell membrane, whereas citral mainly causes membrane lipid peroxidation, which leads a burst in ROS. In addition, the shelf life test of bread showed that the antimicrobial sachets containing SEC had great potential for practical application as antifungal active packaging. Antimicrobial sachets do not produce unpleasant sensory effects.

Appendix A. Supplementary data

CRediT authorship contribution statement

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodchem.2019.125974.

Jian Ju: Data curation, Writing - original draft, Writing - review & editing. Yunfei Xie: Visualization, Investigation. Hang Yu: Visualization, Investigation. Yahui Guo: Software, Validation. Yuliang Cheng: Software, Validation. Rongrong Zhang: Supervision. Weirong Yao: Conceptualization, Methodology, Software.

References Atarés, L., & Chiralt, A. (2016). Essential oils as additives in biodegradable films and coatings for active food packaging. Trends in Food Science & Technology. 48, 51–62. Axel, C., Brosnan, B., Zannini, E., Peyer, L. C., Furey, A., Coffey, A., & Arendt, E. K. (2016). Antifungal activities of three different Lactobacillus species and their production of antifungal carboxylic acids in wheat sourdough. Applied Microbiology and Biotechnology, 100(4), 1701–1711. Chen, H. Y., Xu, R. T., Cheng, Z. X., Gao, Q., & Zhang, J. (2018). Mechanism of H2O2 on oxidative stress of Aspergillus niger. Biotechnology Bulletin, 34(4), 201–207. Cizeikiene, D., Juodeikiene, G., Paskevicius, A., & Bartkiene, E. (2013). Antimicrobial activity of lactic acid bacteria against pathogenic and spoilage microorganism

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. 6

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Nogueira, J. H., Gonçalez, E., Galleti, S. R., Facanali, R., Marques, M. O., & Felício, J. D. (2010). Ageratum conyzoides essential oil as aflatoxin suppressor of Aspergillus flavus. International Journal of Food Microbiology, 137(1), 55–60. Ouyang, Q. L., Jia, L., Tao, N. G., & He, X. L. (2014). Inhibitory effect of alpha-terpineol on Penicillium italicum. Journal of Food Science, 11, 7–15. Passarinho, A. T. P., Dias, N. F., Camilloto, G. P., Cruz, R. S., Otoni, C. G., Moraes, A. R. F., & Soares, N. D. F. F. (2014). Sliced bread preservation through oregano essential oilcontaining sachet. Journal of Food Process Engineering, 37(1), 53–62. Requena, R., Vargas, M., & Chiralt, A. (2019). Eugenol and carvacrol migration from PHBV films and antibacterial action in different food matrices. Food Chemistry, 277, 38–45. Ruiz-Navajas, Y., Viuda-Martos, M., Sendra, E., Perez-Alvarez, J. A., & Fernández-López, J. (2013). In vitro antibacterial and antioxidant properties of chitosan edible films incorporated with Thymus moroderi or Thymus piperella essential oils. Food Control, 30(2), 386–392. Shi, C., Song, K., Zhang, X., Sun, Y., Sui, Y., Chen, Y., & Xia, X. (2016). Antimicrobial activity and possible mechanism of action of citral against Cronobacter sakazakii. PLoS One, 11(7), e0159006. Talón, E., Lampi, A. M., Vargas, M., Chiralt, A., Jouppila, K., & González-Martínez, C. (2019). Encapsulation of eugenol by spray-drying using whey protein isolate or lecithin: Release kinetics, antioxidant and antimicrobial properties. Food Chemistry, 295, 588–598. Tao, N. G., Duan, X. F., Fan, F., & Huang, S. R. (2015). Inhibitory effects of citral and octanal mixture on Penicillium digitatum. Modern Food Science and Technology, 31(6), 73–77. Wan, J., Hu, Y., Ai, T., Ye, S., Huang, Q., Yang, Q., & Li, B. (2018). Preparation of thermoreversible eugenol-loaded emulgel for refrigerated meat preservation. Food Hydrocolloids, 79, 235–242. Wang, C. Y., Chen, C. T., & Wang, S. Y. (2009). Changes of flavonoid content and antioxidant capacity in blueberries after illumination with UV-C. Food Chemistry, 117(3), 426–431. Woranuch, S., & Yoksan, R. R. (2013). Eugenol-loaded chitosan nanoparticles: Thermal stability improvement of eugenol through encapsulation. Carbohydrate Polymers, 96, 578–585. Wu, Y., OuYang, Q., & Tao, N. (2016). Plasma membrane damage contributes to antifungal activity of citronellal against Penicillium digitatum. Journal of Food Science and Technology, 53(10), 3853–3858. Yang, S., Sun, L. L., Deng, S. L., Wang, Z. Y., & Sun, H. (2017). Effects of Trichoderma oxysporum fermentation broth on antioxidant system and lipid peroxidation of tomato early blight. Chinese Agricultural Science Bulletin, 33(5), 90–94. Zaccaria, M., Ludovici, M., Sanzani, S., Ippolito, A., Cigliano, R., Sanseverino, W., & Reverberi, M. (2015). Menadione-induced oxidative stress re-shapes the oxylipin profile of Aspergillus flavus and its lifestyle. Toxins, 7(10), 4315–4329. Zhang, M. (2018). Prevention and treatment of postharvest black spot disease of sweet potato by perillaldehyde and its mechanism of pathogenic fungi (Master's thesis)Jiangsu Normal University). Zhang, L. H., Yu, Q., Yang, Y., Chen, X. L., Wang, C., & Wang, J. (2019). Comparison of effects of diatomite loaded eugenol sustained release on quality of different strawberry varieties. Modern Food Science and Technology, 35(2), 94–101. Zhang, L., Zhang, J., Ma, L. Y., Wang, Q., & Ding, W. (2018). Preparation of eugenol nanocapsules by self-assembly method and its performance characterization. Journal of Food Science, 39(8), 198–204. Zheng, S., Jing, G., Wang, X., Ouyang, Q., Jia, L., & Tao, N. (2015). Citral exerts its antifungal activity against Penicillium digitatum by affecting the mitochondrial morphology and function. Food Chemistry, 178, 76–81.

isolated from food and their control in wheat bread. Food Control, 31(2), 539–545. Clemente, I., Aznar, M., & Nerín, C. (2019). Synergistic properties of mustard and cinnamon essential oils for the inactivation of foodborne moulds in vitro and on Spanish bread. International Journal of Food Microbiology, 298, 44–50. Guo, D., Sun, H. H., Sun, Z., & Sun, S. C. (2018). Antimicrobial activity of citral against Vibrio parahaemolyticus. Journal of Food Science, 33(3), 122–130. Huang, F., Kong, J., Ju, J., Zhang, Y., Guo, Y., Cheng, Y., & Yao, W. (2019). Membrane damage mechanism contributes to inhibition of trans-cinnamaldehyde on Penicillium italicum using Surface-Enhanced Raman Spectroscopy (SERS). Scientific Reports, 9(1), 490. Huang, Q., Qian, X., Jiang, T., & Zheng, X. (2019). Effect of eugenol fumigation treatment on chilling injury and CBF gene expression in eggplant fruit during cold storage. Food Chemistry. Jayashree, T., & Subramanyam, C. (2000). Oxidative stress as a prerequisite for aflatoxin production by Aspergillus parasiticus. Free Radical Biology and Medicine, 29(10), 981–985. Ji, D., Chen, T., Ma, D., Liu, J., Xu, Y., & Tian, S. (2018). Inhibitory effects of methyl thujate on mycelial growth of Botrytis cinerea and possible mechanisms. Postharvest Biology and Technology, 142, 46–54. Ju, J., Wang, C., Qiao, Y., Li, D., & Li, W. (2017). Effects of tea polyphenol combined with nisin on the quality of weever (Lateolabrax japonicus) in the initial stage of freshfrozen or chilled storage state. Journal of Aquatic Food Product Technology, 26(5), 543–552. Ju, J., Xie, Y., Guo, Y., Cheng, Y., Qian, H., & Yao, W. (2018a). Application of edible coating with essential oil in food preservation. Critical Reviews in Food Science and Nutrition, 1–14. Ju, J., Xie, Y., Guo, Y., Cheng, Y., Qian, H., & Yao, W. (2018b). The inhibitory effect of plant essential oils on foodborne pathogenic bacteria in food. Critical Reviews in Food Science and Nutrition, 1–12. Ju, J., Xu, X., Xie, Y., Guo, Y., Cheng, Y., Qian, H., & Yao, W. (2017). Inhibitory effects of cinnamon and clove essential oils on mold growth on baked foods. Food Chemistry, 240, 850–855. Khameneh, B., Diab, R., Ghazvini, K., & Bazzaz, B. S. F. (2016). Breakthroughs in bacterial resistance mechanisms and the potential ways to combat them. Microbial Pathogenesis, 95, 32–42. Kong, J., Zhang, Y., Ju, J., Xie, Y., Guo, Y., Cheng, Y., & Yao, W. (2019). Antifungal effects of thymol and salicylic acid on cell membrane and mitochondria of Rhizopus stolonifer and their application in postharvest preservation of tomatoes. Food Chemistry, 285, 380–388. Lavermicocca, P., Valerio, F., & Visconti, A. (2003). Antifungal activity of phenyllactic acid against molds isolated from bakery products. Applied and Environmental Microbiology, 69(1), 634–640. Liu, S. M., Chen, Z. L., & Chen, J. X. (2008). Study on the antibacterial effect of eugenol in vitro. Journal of Food Science, 29(9), 122–124. Liu, H. M., Zhang, W. T., Wu, Y. Y., Sun, L. J., Wang, Y. L., Liu, Y., & Ji, H. W. (2017). Synergistic antimicrobial effect and mechanism of lipopeptides and tea polyphenols on Vibrio parahaemolyticus. Journal of Food Science, 38(13), 14–19. López-Romero, J. C., Valenzuela-Melendres, M., Juneja, V. K., García-Dávila, J., Camou, J. P., Peña-Ramos, A., & González-Ríos, H. (2018). Effects and interactions of gallic acid, eugenol and temperature on thermal inactivation of Salmonella spp. in ground chicken. Food Research International, 103, 289–294. Niu, D., Wang, Q. Y., Ren, E. F., Zeng, X. A., Wang, L. H., He, T. F., & Brennan, C. S. (2019). Multi-target antibacterial mechanism of eugenol and its combined inactivation with pulsed electric fields in a hurdle strategy on Escherichia coli. Food Control, 106, 106742.

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