Multifunctional alkyl ferulate esters as potential food additives: Antibacterial activity and mode of action against Listeria monocytogenes and its application on American sturgeon caviar preservation

Accepted Manuscript Multifunctional alkyl ferulate esters as potential food additives: Antibacterial activity and mode of action against Listeria monocytogenes and its application on American sturgeon caviar preservation Yu-gang Shi, Li-qing Bian, Yun-jie Zhu, Run-run Zhang, Shi-yin Shao, Yu Wu, Yuewen Chen, Ya-li Dang, Yue Ding, Hao Sun PII:

S0956-7135(18)30491-2

DOI:

10.1016/j.foodcont.2018.09.030

Reference:

JFCO 6330

To appear in:

Food Control

Received Date: 23 July 2018 Revised Date:

22 September 2018

Accepted Date: 24 September 2018

Please cite this article as: Shi Y.-g., Bian L.-q., Zhu Y.-j., Zhang R.-r., Shao S.-y., Wu Y., Chen Y.w., Dang Y.-l., Ding Y. & Sun H., Multifunctional alkyl ferulate esters as potential food additives: Antibacterial activity and mode of action against Listeria monocytogenes and its application on American sturgeon caviar preservation, Food Control (2018), doi: https://doi.org/10.1016/j.foodcont.2018.09.030. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Multifunctional alkyl ferulate esters as potential food additives:

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Antibacterial activity and mode of action against Listeria monocytogenes and its

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application on American sturgeon caviar preservation

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Yu-gang Shi a, b*, Li-qing Bian a, b, Yun-jie Zhu a, b, Run- run Zhang a, b, Shi-yin Shao a, b, Yu Wu a, b, Yue-wen Chen a

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, Ya-li Dang c, Yue Ding d, Hao Sun a, b,

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a

School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, Zhejiang 310035, China

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b

Zhejiang Provincial Collaborative Innovation Center of Food Safety and Nutrition, Zhejiang Gongshang

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University, Hangzhou, Zhejiang 310035, China

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c

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315211, China

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Key Laboratory of Animal Protein Food Processing Technology of Zhejiang Province, Ningbo University, Ningbo

Food Colloids Group, School of Food Science and Nutrition, University of Leeds, Leeds LS2 9JT, UK

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*Corresponding Author:

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Yu-gang Shi

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School of Food Science and Biotechnology, Zhejiang Gongshang University, Xiasha University

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Town, Xuezheng Str. 18, Hangzhou 310018, China

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Tel.: 86–0571–28008927

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E–mail: [email protected]

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ABSTRACT

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The alkyl ferulate esters were prepared through lipase-catalyzed reactions by using green deep

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eutectic solvent-water binary mixtures. Antibacterial effects screening of alkyl ferulate esters

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against Listeria monocytogenes demonstrated that hexyl ferulate (FAC6) exerted both bacteriostatic

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and bactericidal effects on Listeria monocytogenes (minimum inhibitory concentration: 0.1 mM,

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minimum bactericidal concentration: 0.2 mM). The antibacterial mechanism of FAC6 were further

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investigated to provide more information on practical applications as a multi-functional food

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additive. The growth curves and time-kill assay also showed the occurrence of cell lysis and

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significant inhibition of the growth of L. monocytogenes caused by FAC6. PI uptake analysis and

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massive leakage of cell constituents (K+, proteins and nucleotide) demonstrated that the membrane

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integrity and permeability were undermined by FAC6. Alterations in morphology and membrane

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hyperpolarization of L. monocytogenes cells treated with FAC6 further clearly confirmed that it

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disrupted the cell membrane. Meanwhile, FAC6 interacted with membrane proteins and affected the

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protein expression system, causing a significant change in contents, constitution and conformation

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of membrane proteins. Moreover, FAC6 could bind to L. monocytogenes DNA grooves to form

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complexes. Overall, this research highlights the effectiveness of FAC6 against L. monocytogenes,

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suggesting that FAC6 with both antioxidant and antibacterial activities can be used as an effective

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and safe multifunctional food additive for American (Amer) sturgeon caviar preservation.

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Keywords: Lipase, biocatalysis, alkyl ferulate esters, Listeria monocytogenes, antimicrobial

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activity, antibacterial mechanism, biofilm, deep eutectic solvents,

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1. Introduction

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Nowadays, concerns about healthy and natural food have asked for a prudent reconsideration in

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many fields of the food industry, thus, the development of natural antimicrobial agents, other than

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antibiotics, with broad antimicrobial spectrum has gained tremendous interest. In addition to

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essential oils, organic acids, tannins, flavonoids, etc, phenolic acids as the natural antimicrobial

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compounds are the most common non-flavonoid possessing two typical constitutive carbon

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frameworks (the hydroxycinnamic and hydroxybenzoic structure). Particular attention is given to

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hydroxycinnamic acids due to their remarkable biological properties, including the antioxidant

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activity (Phonsatta et al., 2017), amyloid β aggregation inhibition (Taguchi et al., 2017),

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antimicrobial (Cueva et al., 2010; Borges, Ferreira, Saavedra, & Simãµes, 2013) and anti-biofilm

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formation activities (Lemos et al., 2014). Among them, ferulic acid (FA) has been shown to have

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broad-spectrum antimicrobial activity against both Gram-negative and Gram-positive bacteria, as

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well as yeasts (Borges, Ferreira, Saavedra, & Simãµes, 2013). Moreover, some recent research had

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found that grafting an aliphatic chain on a carboxyl functional group of phenolic acids could

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ameliorate their hydrophilic-lipophilic balance to generate new multifunctional molecules

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(phenolipids) with combined emulsifying properties and enhanced intrinsic properties, such as

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antioxidation (Sørensen et al., 2014) and antimicrobial activity (Michiyo et al., 2002; Ou & Kwok,

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2004; Merkl, Hrádková, Filip, & Šmidrkal, 2010). Recently, the biological and antimicrobial

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activities of phenolipids have been well reviewed by Durand et al. (2017). More importantly, the

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good hydrophilic properties of phenolipids have allowed for their easy diffusion in food stuffs

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especially rich in unsaturated fatty acids.

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The physiological functions of bacteria are particularly related to subcellular structures or

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components including the cell membrane, the cytoplasm and the nucleic acids. Thus, the mode of

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bacterial inactivation caused by antibacterial agents may be confirmed by varying impacts on these

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subcellular structures of bacteria. As for cell membrane, membrane permeability and membrane

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potential (Ning et al., 2017) are mainly concerned in previous studies. In terms of bacteria proteins

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of the cell wall and cytoplasm, they are vital to maintain the physiological functions of bacteria and

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the loss of the intracellular proteins could result in the cell death. Besides the cell membrane and

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proteins, DNA damage would also cause bacterial inactivation. The antimicrobial mechanism of FA

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was attributed to their ability to disrupt the integrity of cytoplastic membrane, suppress the activity

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of enzymes involved in radical generation and also inhibit the synthesis of nucleic acids of bacteria

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(Lo & Chung, 1999, Borges, Ferreira, Saavedra, & Simãµes, 2013). Nevertheless, to the best of our

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knowledge, the intrinsic mode of action and antibacterial mechanism of alkyl ferulate esters against

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food related bacteria still remains largely unknown. In addition, few studies have been carried out

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on whether alkyl ferulate esters have the potential to be considered as novel preservatives in the

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food industry (Andrade et al., 2015). Hence, their further evaluation was undertaken to gain new

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insights into their bactericidal action on a subcellular basis.

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On the other hand, in order to obtain such lipophilic derivatives, biocatalytic approaches are

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preferred over classical chemical synthesis (Gonzalez-Sabin, Moran-Ramallal, & Rebolledo, 2011;

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Figueroa-Espinoza & Villeneuve, 2005; Stamatis, Sereti, & Kolisis, 1999; Shi et al., 2017),

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especially for food applications, since phenolic acids are very sensitive to harsh reaction conditions

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such as high temperatures and extreme pH values (Munin & Edwards-Levy, 2011). Our research

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group has always been keen to generate potential food additives with multifunctionalities through

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biocatalytic approaches, with special focus on biocatalysis in environmentally friendly non-aqueous

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reaction media (Shi et al., 2011; 2017; 2018). Recently, we reported that lauryl ferulate was

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generated through lipase-catalyzed esterification of FA with lauryl alcohol in novel functionalized

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ionic liquid ([(EO)-3C-im][NTf2]) and the antibacterial properties of lauryl ferulate in vitro against

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three food-related bacteria (Listeria monocytogenes, Staphylococcus aureus and Escherichia coli)

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was further evaluated (Shi et al., 2017).

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In the present study, the primary aim was to evaluate the antimicrobial activity of ferulic alkyl esters

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synthesized by lipase-mediated alcoholysis in a deep eutectic solvent-water binary system (Scheme

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1). The secondary objective was to investigate the mode of action of hexyl ferulate (FAC6) against

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the food-borne pathogen, Listeria monocytogenes (L. monocytogenes). The further evaluation was

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undertaken to gain new insights into its bactericidal action on a subcellular basis. Furthermore, this

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study examined the efficacy of FAC6 on the microbial load of L. monocytogenes in Amer sturgeon

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caviar as well as its overall efficacy on the sensory quality of caviar. This is the first report to apply

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alkyl ferulate esters not only to control L. monocytogenes but also maintain the sensory quality of

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sturgeon caviar during the specific storage time.

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2. Materials and methods

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2.1. Materials

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FA (>99%) and acridine orange (AO) were purchased from Sigma Aldrich (Shanghai, China).

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Methyl ferulate (MF) and ethyl ferulate (FAC2) (>99%) were prepared in our laboratory.

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Immobilized lipase B from Candida antarctica (CalB immo Plus) with a specific activity ≥ 9000

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PLU g−1 (propyl laurate units per g) was obtained Purolite Corporation (Hangzhou, China). 3 Å and

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4 Å molecular sieves were purchased from Sinopham Chemical Reagent Co., Ltd. (Shanghai,

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China). All other chemicals of analytical or HPLC grade were obtained from commercial sources of

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China. Other organic solvents were stored over 4 Å molecular sieves, at least overnight prior to use.

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Amer sturgeon caviar was obtained from Hangzhou Qiandaohu Xunlong Sci-tech Co. Ltd

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(Hangzhou, China).

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2.2 Biocatalysis

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2.2.1 Preparation of deep eutectic solvents (DES)

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The ammonium salt (choline chloride, ChCl) and the hydrogen-bond donor (urea, U) (1:1) were

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mixed well in a flask, without any contact with air moisture. Then, the mixture was stirred at 60 °C

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for 2 hours, and a colorless liquid was obtained.

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2.2.2. General conditions for the alcoholysis

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Alkyl ferulate esters were synthesized as previously described by Durand et al., (2013) and Shi et

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al., (2017) with some modifications. The alcoholysis reactions of MF with different direct chain

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fatty alcohols (butyl, hexyl, octacyl, decyl, lauryl, hexadecyl, and octadecyl alcohols) were

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conducted in a 30 mL glass vial equipped with a tight screw-cap. 0.5 mmol of MF and 3 mmol of

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fatty alcohol were added into 10 mL of the corresponding DES with water (20%, w/w). The reaction

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was initiated by adding 100 mg of CalB immo Plus. All experiments were carried out under

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sonochemical irradiation of 150 W, at 50 oC, for an hour, and subsequently the reaction bottles were

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incubated in an orbital shaker at 200 rpm at 60 oC for 3-7 days. The supernatants were analyzed by

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TLC on Silica gel 60F254 aluminum sheets (0.2 mm thickness, Merck) and high performance liquid

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chromatography (HPLC). The conversion of MF into products was calculated as the area percentage

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of product peak against the total areas of the substrate (MF) and product peaks.

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2.2.3. Analytical procedure and purification of the product

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Reactions were followed by TLC in hexane/ethyl acetate 3:2 (v/v) with UV light at 254 nm for

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detection. HPLC analysis was performed by HPLC using a Waters pump (Waters 1525) with a UV

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detector at 325 nm and a Hypersil reversed-phase C18 column (25 cm×4.6 mm, 5 µm). Elution was

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conducted with acetonitrile/water (95:5, v/v) at a flow rate of 1 mL min-1 and a column temperature

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at 35 oC. The 1H-NMR spectra were recorded on a Bruker AVANCE 500MHz spectrometer using

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CDCl3 as solvent and TMS as internal standard. The ESI-MS were obtained on Agilent

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1946A-MSD. After the completion of reaction, any solid including lipase was removed by

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centrifugation and lipase was washed thoroughly with hexane for reuse. The supernatants were

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concentrated by vacuum drying in a rotary evaporator, and the target product was purified by a

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silica gel column chromatography using hexane/ethyl acetate (96:4, v/v) as the eluent and/or

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crystallization.

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2.3. Bacterial strains and culture conditions

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Listeria monocytogenes ATCC 19115 (L. monocytogenes) and Escherichia coli ATCC 25922 (E.

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coli) were obtained from National Center For Medical Culture Collections (Beijing, China), and

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were grown and maintained in Tryptone Soya broth (TSB) and on Trypticase Soy agar (TSA)

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(Hangzhou Microbial Reagent Co. Ltd, China). Strains were maintained on TSA slants at 4 oC.

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Cells were prepared by 16-h culture in TSB at 37 oC. A 16 h culture was diluted with TSB to

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achieve an inoculum of 106 CFU mL-1 approximately. The number of cells in the suspensions was

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determined by duplicate plating from ten-fold serial dilution on TSA and counting the colonies after

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incubation at 37 oC for 24 h.

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2.4. Antimicrobial activity and antibacterial mechanism

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2.4.1. Minimum inhibitory concentration (MIC) and minimum bactericide concentration (MBC)

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Broth macrodilution assay (Wilson et al., 2005) with some modification was used to determine MIC

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and MBC values. Serial two-fold dilutions of the alkyl ferulate esters (0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4,

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12.8 and 25.6 mM) were prepared with TSB. One tube without the alkyl ferulate ester was used as

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the negative control. Inocula were transferred into all the tubes to achieve the initial bacterial

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inoculum of approximate 106 CFU mL-1. All tubes were incubated at 37 °C for 24 h, a 1 mL portion

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was removed from each tube for colony counting by decimal dilution in 0.85% (w/v) sodium

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chloride solution, and plated out onto TSA. Each concentration was assayed in triplicate. The MIC

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and MBC were defined as the lowest concentrations resulting in maintenance or reduction of

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inoculums viability compared to the negative control after 24 h, and the lowest concentration where

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no less than 99% of the initial inoculums are killed after 24 h, respectively.

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2.4.2. Growth curve and time-kill kinetics analysis

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The indicator was cultured to exponential phase (OD600 nm= 0.5-0.6) at 37 oC and 125 µL of the

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culture was added into each well on 96-well microtiter plates. The alkyl ferulate ester was dissolved

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in the cultures at the final concentrations of 1×MIC, 2×MIC and 4×MIC, and samples without the

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alkyl ferulate ester were set as the negative control. Bacteria were further cultured at 37 oC, and cell

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growth was monitored by enzyme micro-plate reader at 600 nm at 0.5 h intervals, and lysis was

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observed. The killing kinetics of the alkyl ferulate ester against tested bacteria was performed

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according to the method by D’Arrigo et al., (2010). Cultures of bacteria with a density of 106 CFU

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mL-1 were exposed to the alkyl ferulate ester broth dilutions with various concentrations. All the

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solutions were incubated at 37 oC under 180 rpm agitation. To count colony forming units (CFU

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mL-1) after 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8 and 12 h of incubation, aliquots (1 mL) were removed

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from each tube and decimally diluted in sterile saline(0.85%, w/v, sodium chloride), and spotted

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onto TSA at 37 oC for 12-18 h. The theoretical detection limit was 10 CFU mL-1, corresponding to 1

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Log CFU mL-1.

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2.4.3. Propidium iodide uptake test

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The propidium iodide (PI) uptake test was conducted according to the method described by Park

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and Kang (2013) with some modifications to evaluate the cell membrane integrity. After inoculation,

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all the solutions were incubated at 37 oC under 180 rpm agitation for 24 h. A 5 mL portion from

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each tube was removed and then centrifuged at 6000 g at 4 °C for 15 min. Cells pellets were

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washed ×3 with PBS (0.01 M, pH 7.2, 0.14 M NaCl) and then resuspended in the same buffer (10

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mL) with the final cells concentration of 106 CFU mL-1. The alkyl ferulate ester was added at the

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final concentration of 1×MIC and then incubated at 37 oC under shaking conditions at 180 rpm for 4

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h. A PI stock solution of 1 mg mL-1 was prepared. After the alkyl ferulate ester treatment, cells were

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incubated with PI in the dark at 37 oC for 10 min. For evaluation of PI uptake, fluorescence was

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monitored in a fluorimeter (RF-5301PC, Shimadzu, Japan) using an excitation wavelength of 495

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nm and an emission wavelength of 500-700 nm. Both slit widths were kept at 5-nm. The parallel

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sample without the alkyl ferulate ester was used as the negative control.

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2.4.4. Cell constituents’ release

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The release of cell constituents into supernatant was measured according to the method described

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by Lv et al., (2011) and Diao et al., (2014) with some modifications. Cells were collected by

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centrifugation at 5000 g for 10 min, washed ×3 with PBS (0.01 M, pH 7.2, 0.14 M NaCl), and

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resuspended in the same buffer. 5 mL of cell suspension were incubated at 37 oC under agitation in

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the presence of the alkyl ferulate ester. Then, 2 mL of each sample was centrifuged at 10,000 g for 5

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min. Control groups containing bacterial supernatant without the alkyl ferulate ester treatments

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were tested similarly. The concentrations of proteins in supernatants were determined by Bradford

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assay. The amounts of DNA and RNA released from the cytoplasm into the supernatant were

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estimated by the detection of absorbance at 260 nm. Potassium ion concentrations in TSB in

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supernatants were determined by the method by Shao et al., (2018). Cells in in TSB with a density

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of 106 CFU mL-1 were exposed to FAC6 and were incubated at 37 oC under 180 rpm. After 0, 0.5, 2,

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3, and 6 h of incubation, aliquots of 1 mL was removed from each tube and centrifuged at 8000 g

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for 15 min, then the suspension was harvested through filtration using a 0.45-µm nylon membrane.

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0.1 mL filtrate was diluted with 4.9 mL HNO3 solution (2%) for measuring the amount of

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extracellular potassium ions by using inductively coupled plasma mass spectrometry (ICP-MS;

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Agilent 7700x, Agilent Technologies Japan, Tokyo, Japan).

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2.4.5. Evaluation of zeta potential

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The zeta potential of the bacterial suspensions was determined as reported by Borges et al., (2013)

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with some modifications. Various bacteria were incubated overnight in TSB at 37 oC 180 rpm. The

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cells were harvested with centrifugation at 5000 g for 10 min and washed with sterile distilled water.

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The cell suspensions were adjusted to OD640 nm = 0.2± 0.02. A volume of 1.8 mL of this culture was

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added to 200 uL of test compound (to a final concentration of 1.6 mM) and incubated for 1 h at 30

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the bacterial suspensions in zeta potential cells (DTS1060, Malvern) was measured using a Nano

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C and 120 rpm. A negative control was prepared with sterile distilled water. The zeta potential of

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Zetasizer (Malvern Instruments) equipment at the room temperature.

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2.4.6. Analysis of membrane proteins

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SDS-PAGE analysis was carried out to determine bacterial membrane proteins before and after the

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alkyl ferulate ester pretreatment using the method by Laemmli (1970) with some modifications. L.

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monocytogenes was incubated in TSB at 37 oC for 14 h. The alkyl ferulate ester was added to the

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suspension of activated bacterial cells to obtain a final concentration of 0.1 mM. A control sample

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without the alkyl ferulate ester was prepared. All tubes were incubated at 37 oC for 4 h. Bacterial

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cells were collected by centrifugation (6000 g, 4 oC) for 10 min and were washed ×3 with PBS

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(0.01 M, pH 7.2, 0.14 M NaCl), then bacterial membrane proteins were extracted by using kits

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(BestBio, Shanghai, China). Then, the Marker (Takaba, Dalian, China) and membrane proteins

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samples were run through the separating gel at 100 V for 90 min. The gel was dyed with Coomassie

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Brilliant Blue R250 for 4 h. Afterwards, the gel was decolorized with a common decoloring agent.

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After one night, protein bands were visualized on the gels.

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The fluorescence spectra of membrane proteins in the absence and presence of KI or the alkyl

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ferulate ester were determined using a spectrofluorometer (Model F-7000, Hitachi, Japan)

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according to a slightly modified method (Wang, Wang, Zeng, Xu, & Brennan, 2017). A 3.0 mL

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bacteria suspension (109–1010 CFU mL-1) in 0.85% saline solution with different concentrations of

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potassium iodide (KI) or the alkyl ferulate ester was incubated at 25 oC for 1 h. The fluorescence

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emission spectra of the bacterial samples were recorded under an excitation wavelength of 258, 280,

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or 296 nm (slits = 5.0 nm).

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2.4.7. The effect of FAC on bacterial genomic DNA

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TIANamp Bacteria DNA Kit (Tiangen Biotech, co., LTD) was used to extract L. monocytogenes

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genomic DNA according to the operation instruction. The purity of the extracted DNA was

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determined by the value of OD260 nm/OD280 nm (1.978), and the DNA concentration was determined

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by measuring the absorbance at 260 nm using a spectrophotometer (Shimadzu UV-2600

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spectrometer, Tokyo, Japan) at room temperature. Interaction of the alkyl ferulate ester with bacteria

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genomic DNA was studied with a method (Ebrahimipour et al., 2015) with minor modifications.

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Absorption spectral titrations were performed at a constant concentration of the alkyl ferulate ester

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(0.4 mM) in PBS (0.01 M, pH 7.2, 0.14 M NaCl) while gradually increasing the DNA concentration.

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After incubation at 25 oC for 10 min, the absorbance spectra of FAC6 were measured across a

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wavelength range from 230 nm to 400 nm (Shimadzu UV-2600 spectrometer, Tokyo, Japan).

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The competitive displacement assay was performed as reported earlier (Sarwar, et al., 2017). AO

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displacement assay was carried out by adding 5 µM of AO to 50 µM of genomic DNA. Excitation

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of the AO–genomic–DNA complex was done at 490 nm and emission spectra was recorded between

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500 and 700 nm and then titrated with varying concentration of FAC6 (0–0.5 mM).

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The bacteria genomic DNA and FAC6 were dissolved in 10 mM Tris–HCl buffer (pH 7.2) and then

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mixed to obtain various samples with constant DNA concentration (400 µg mL-1) and increasing

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FAC6 concentrations (0, 1 and 2×MIC). After incubation at 37 oC for 10 min, electrophoresis was

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performed to separate and visualize DNA fragment in a 0.8% agarose gel at 130 V for 25 min.

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Genomic DNA was stained with the Gelred dye.

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2.4.8. Biofilm formation assay

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The biofilm formation was quantified in 96-well microtiter plates as previously described by

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Narisawa et al. (2005) with slight modifications. The overnight-grown cultures were diluted 1:100

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in TSB containing fixed concentrations of the alkyl ferulate ester, removed to 96-well microplates at

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200 µL per well and incubated at 37 °C without shaking for 48 h. The microplates were then

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washed ×5 with 450 µL sterile distilled water. The biofilm was stained with 250 µL of 2% crystal

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violet per well and incubated at 25 oC for 45 min. The wells were washed five times with 450 µL

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water and allowed to air dry. 200 µL of 70% ethanol was then added to each well to dissolve the

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crystal violet dye. Then, the OD600nm was recorded for each well using a microplate reader

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(Multiskan Spectrum 1500, Thermo Electron Corporation, USA). The biofilm content was obtained

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by subtracting the average absorbance of the control wells from each sample well. The averages and

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standard deviations were calculated from the results of five replicate wells.

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2.4.9. Scanning electron microscopy (SEM) analysis

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To determine the efficacy of ferulic acid and the alkyl ferulate ester and the morphological changes

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of bacteria, SEM studies were carried out as previously reported by Shao et al. (2018) with some

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modifications. L. monocytogenes were cultured to logarithmic stage at 37 oC. After incubation,

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bacterial suspension were harvested by centrifugation (6000 g, 4 oC) for 15 min, washed ×3 with

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PBS (0.1 M, pH 7.0, 0.14 M NaCl), then resuspended in 4 mL PBS. The above washed bacteria

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cells (1 mL) were taken into 9 mL PBS and L. monocytogenes were treated with FAC6. The control

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and samples were incubated at 37 oC for 0.5 h and 4 h. After incubation, cells were harvested by

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centrifugation (4000 g, 4 oC) for 10 min and washed ×3 with PBS, and then fixed with 0.5-1 mL 2.5%

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(v/v) glutaraldehyde in PBS for 15 min at each step; then postfixed with 1% OsO4 in PBS for 15

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min at each step. Samples were further dehydrated through a gradient of ethanol solutions, subject

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to critical-point drying, coated with gold-palladium in Hitachi Model E-1010 ion sputter for 4-5 min,

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and observed in Hitachi Model TM-1000 SEM (Hitachi, Tokyo, Japan).

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2.4.10. Transmission electron microscopy (TEM) analysis

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TEM was used to observe changes in the intracellular organization of L. monocytogenes treated

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with ferulic acid or the alkyl ferulate ester. Bacteria were cultured as described in the section 2.4.9

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and then collected by centrifugation after 0.1 and 4 h. Samples were soaked in a 1:1 mixture of

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absolute acetone/embedding solution for 2 h and then changed to pure embedding solution

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overnight. Ultrathin sections were stained and observed in Hitachi Model H-7650 TEM (Hitachi,

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Tokyo, Japan).

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2.5. Storage test on Amer Sturgeon Caviar

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2.5.1. In situ antibacterial activity

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The Amer sturgeon caviar samples were sent frozen in a metal-canning jars from the manufacturer.

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They were confirmed to be L. monocytogenes-free prior to the start of experiments. For

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enumeration of L. monocytogenes, a final L. monocytogenes concentration of 102 CFU g-1 was

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obtained through the inoculation of an aliquot (100 µL) of L. monocytogenes culture at 104 CFU

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mL-1 into 10 g sample. The alkyl ferulate ester (0.1 mM) was transferred into the L.

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monocytogenes-inoculated samples, followed by incubation at 10 oC. Bacteria for the samples at 10

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o

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samples, 90 mL of sterile saline water was added to each 10 g-sample, homogenized and serially

300

diluted before plated onto TSA. Following the incubation of inoculated plates at 35 oC for 24 h,

301

viable counting was performed and number of CFU g-1 calculated. Each experiment was performed

302

in triplicate.

303

2.5.2. Sensory evaluation

304

Sensory analysis was performed on Amer sturgeon caviar samples from batches Control and FAC6

305

after 7 days of storage at 10 °C. A panel comprised of 15 judges was employed to define the

306

descriptive sensory profile. The judges were trained in 3 preliminary sessions according to

307

Sorrentino et al., (2018). Random samples were evaluated by assigning a score between 1 (poor)

308

and 9 (excellent) for positive descriptors, and between 9 (unacceptable) and 1 (excellent) for the

309

negative one (off-flavour).

310

2.6. Statistical analysis

311

All of the experiments were performed in triplicate and the mean ± standard deviation values were

312

reported. The significant differences between the two groups were examined using t-test. A P value

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< 0.05 denoted the presence of a statistically significant difference.

314

3. Results and discussion

315

3.1. Lipophilization of FA through Lipase-catalyzed alcoholysis

316

DES exhibit outstanding solvation properties that are strongly influenced by hydrogen bonding and

317

result in a high affinity to all compounds capable of offering electrons or protons. Thus, DES could

318

be an alternative to ionic liquids as efficient green media for lipase-catalyzed reactions. Phenolic

319

compounds can undoubtedly be dissolved in DES because several intermolecular hydrogen bonds

320

of them had been established. Previous study showed that faster reaction and higher conversion

321

rates were obtained in ChCl:U-water mixtures as compared to in ChCl:Gly-water mixtures and the

322

pH can favor lipase activity could explain its superior performance in ChCl:U (Durand, Lecomte,

323

Baréa, Dubreucq, Lortie, & Villeneuve, 2013). faster reaction and higher conversion rates for this

324

reaction were found in ChCl:U-water mixtures than in ChCl:U. Moreover, an apparent equilibrium

325

could be reached after 7 day in ChCl:U-water mixtures. The effect of the chain length of fatty

326

alcohol on alcoholysis of MF was studied by using different natural fatty alcohols with varying

327

carbon n-alkyl chain lengths from C4 to C16 (Scheme 1). The tendency of conversion of MF with

328

various fatty alcohol chains was relatively different. The reaction became the most effective when

329

MF reacted with n-hexanol to generate FAC6, equilibrium achieved within 3 days with almost 94%

330

conversion of MF. These results are in agreement with a previous report (Durand, Lecomte, Baréa,

331

Dubreucq, Lortie, & Villeneuve, 2013), indicating that the length of fatty alcohol chains plays an

332

important role.

333

3.2. MICs and MBCs of alkyl ferulate esters

334

The antimicrobial efficiency of FA and its alkyl esters (from FAC2 to FAC12) against L.

335

monocytogenes and E. coli strains were screened. The MIC and MBC values of FA and its alkyl

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esters, FAC2–FAC12, against L. monocytogenes are shown in Table 1. The alkyl chain length was

337

found to be associated with the antibacterial activity to a large extent. The theoretical partition

338

coefficients (clog P) of them were used here since lipophilicity is an important property for the

339

antibacterial activity of alkyl ferulate esters. In general, a clear tendency for the antibacterial

340

activity of alkyl ferulate esters again L. monocytogenes was observed: MIC or MBC values first

341

decreased and then increased with increasing length of the alkyl ester chain, except FAC12,

342

maximizing at alkyl chain length between C4 and C6. The similar phenomenon was described by

343

Kubo et al. (2004) for alkyl gallates against Bacillus subtilis. Moreover, a simple modification of

344

the lipophilicity of FA (see FA vs. FAC2) led to a noticeable decline in MIC and MBC values,

345

which is in accordance with the results obtained by Merkl et al. (2010) within similar systems.

346

Furthermore, in order to assess the spectrum of the antimicrobial activity of alkyl ferulates, MIC

347

analysis was carried on an Gram-negative bacterium, E. coli. Interestingly, we found that

348

Gram-negative bacteria, E. coli (MIC= 6.4), was more susceptible to FA than Gram-positive

349

bacteria, L. monocytogenes (MIC= 10), and similar result was also reported by Borges et al. (2013).

350

However, in this study, E. coli was less susceptible than L. monocytogenes to some of alkyl ferulate

351

esters (Table 1), such as FAC2, FAC4 and FAC6, which is in accordance with the data reported by

352

Kubo et al. (2002). Some explanations could be proposed: 1) The activity of FA was strongly

353

dependent on pH, salt type, and concentration, whereas its corresponding phenolipids become

354

independent of these influence factors. 2) The antimicrobial activity of alkyl ferulates is associated

355

with a fine balance between affinity for the lipid bilayers of cell membranes and the ability to cause

356

disruption of the membrane, which is strikingly dependent on both variety of bacteria and the length

357

of the alkyl ester side chain (Kubo, Fujita, Nihei, & Nihei, 2004). In our case, lipophilization of FA

358

to the optimized alkyl ferulate esters caused an alteration in the mechanism of action with improved

359

the antibacterial properties of FA, exhibiting a cut-off effect at four or six carbons chain length

360

against L. monocytogenes. In addition, The MIC values obtained in this study are in the range of

361

those described in other works attained with alkyl ferulates and related compounds which reviewed

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by Guzman et al., (2014). Merkl et al., (2010) also reported that phenolic acids alkyl esters showed

363

a broad spectrum of antimicrobial activity, with MIC values between 5–10 mM and 2.5–5 mM

364

against E. coli and L. monocytogenes, respectively. In this study, FAC6 presented the lowest MIC

365

(0.1 mM for L. monocytogenes; 0.4 mM for E. coli) and MBC (0.2 mM for L. monocytogenes)

366

values. Overall, FAC6 had an excellent antimicrobial activity and promising potential as

367

antimicrobial. Therefore, it was chosen to be further investigated its potential antibacterial

368

mechanism against L. monocytogenes and the practical application for Amer sturgeon caviar

369

preservation.

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370

3.3. Growth curve and time-kill analysis

372

The Gram-positive bacteria L. monocytogenes were used for the study of potential antibacterial

373

mechanism of FAC6. Growth curves of it with and without treatment by FAC6 were measured (Fig.

374

1(A)). For the control, the indicator kept exponential-phase growth. In contrast, the OD600nm values

375

of L. monocytogenes decreased when exposed to FAC6, which ascertain clearly the lytic action of

376

FAC6 to L. monocytogenes (Tsuchido, Ahn, & Takano, 1987; Sitohy, Mahgoub, Osman, El-Masry,

377

& Al-Gaby, 2013). The concentration-dependent and time-dependent bactericidal activities of FAC6

378

can be observed. Time-kill assays was used to examine the rate at which different concentrations of

379

FAC6 kill bacteria. FAC6 at 1× or 2×MIC led to a rapid decrease in bacterial number for L.

380

monocytogenes. Furthermore, the viability of the cells decreased significantly to 0 Log CFU mL−1

381

after 4 h of incubation with FAC6 at 2×MIC compared to nearly 8.7 Log CFU mL−1 of the control

382

(Fig. 1(B)). Therefore, FAC6 had bactericidal action mode with concomitant cell lysis against L.

383

monocytogenes at the tested concentrations. Furthermore, the rapidly bactericidal activity stemmed

384

from FAC6 at high concentrations pointed towards a mechanism of action, infiltration of bacterial

385

membrane.

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3.4. Integrity and permeability of cell membrane

388

To verify the effect of FAC6 on the membrane integrity of L. monocytogenes, the PI uptake assay

389

was performed since PI can invade cytoplasm and bind to DNA once the cell membrane integrity is

390

destroyed (Park & Kang, 2013). Fig 2(A) showed that PI uptake values for L. monocytogenes

391

treated with FAC6 at 1×MIC for 4 h were significantly higher than those treated without FAC6.

392

Moreover, the percentage of cells suffering from membrane damage was increased with increasing

393

the concentration of FAC6 from 0.1 mM to 1.6 mM. The PI uptake results indicated that FAC6

394

compromised the integrity of the cytoplasmic membrane and pores can form in the cell membrane.

395

Moreover, L. monocytogenes exhibited significant morphological damage with pores when exposed

396

to 1×MIC FAC6, which was observed in SEM and TEM analysis (Fig. 3). Therefore, it can be

397

proposed that FAC6 may act as the antimicrobial agent that weakens the cell membrane, inducing

398

alteration on its integrity and, consequently, the bacterial physicochemical characteristics.

399

The permeability of the membranes to intracellular K+ was also investigated to assess the effect of

400

FAC6 on cytoplasmic membrane. Fig. 2 (B) showed that K+ efflux from L. monocytogenes treated

401

with FAC6 at 1×MIC and 2×MIC significantly accelerated for the first 2 h as compared to the

402

control group, indicating an alternation in the permeability of cytoplasmic membranes. As the

403

degree of bacterial cell membrane damage is more conspicuous, large molecules leach out from the

404

cells including protein and genetic nucleotide materials. Fig. 2(C) and (D) showed the release of

405

cell constituents when bacteria were treated with 1×MIC FAC6. After 1 h, the protein leakage from

406

L. monocytogenes in the control was 2.52 µg mL-1, while the leakage amount of protein from cells

407

treated with 1×MIC FAC6 were 5.57 µg mL-1. In addition, the absorbance at 260 nm of supernatant

408

of L. monocytogenes treated with FAC6 at 1×MIC were 2.4 times as high as that of the control.

409

Noteworthily, the leakage amount of proteins in L. monocytogenes cells treated with FAC6 at

410

1×MIC for the first 2 h accounted for 83.3% of the overall leakage amount for 6 h (6.62 µg mL-1).

411

Similarly, the leakage of 260-nm absorbing substances at 2 h accounted for 94.1% of the leakage

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amount in L. monocytogenes treated with FAC6 for 6 h. Such a tendency suggested that the

413

treatment of FAC6 at 1×MIC for 2 h could greatly destroy bacterial membrane integrity and

414

permeability, causing proteins and nucleotide leaking, which is in accordance with the data acquired

415

by Chen et al. (2017) who reported that bacterial membrane integrity could be greatly disrupted,

416

causing proteins leaking, when cells were exposed to 10 mg mL-1 sugar beet molasses polyphenols

417

for 2 h. These results inferred that FAC6 probably acted on the cytoplasmic membrane by inducing

418

membrane damage, resulting in the leakage of cell constituents due to a loss of membrane integrity.

419

3.5. Effects of FAC6 on the apparent zeta potential of cells

420

The surface charge of cells could be measured based on their zeta potential in the presence of an

421

electric field under defined pH and ionic concentrations. The time course of zeta potential

422

measurements of L. monocytogenes treated or untreated with FAC6 were presented in Fig. 2 (E).

423

The change in zeta potential of treated L. monocytogenes showed different patterns compared to the

424

control. Normally, the surface charge of bacterial cells is negative due to the presence of anionic

425

groups (e.g., carboxylate and phosphate groups) in their membranes. The surface charge of L.

426

monocytogenes was altered to more negative values after exposure to FAC6 (p <0.05), indicating

427

cells treated with FAC6 leading to cell membrane hyperpolarization. Hyperpolarization caused by

428

natural products is being increasingly documented as a significant type of membrane damage (Li,

429

Wang, Xu, Zhang, & Xia, 2014). Consistent with our results, Bennik et al. (1998) reported that

430

bactericidal effect of mundticin against L. monocytogenes was associated with a rapid alteration of

431

the membrane potential (hyperpolarization of cell membrane), which indicates the dissipation of

432

ionic gradients as a result of pore-formation. Therefore, this redistribution of ions induced by FAC6

433

might affect the overall bacterial metabolic activity of L. monocytogenes, leading to some

434

biosynthetic pathway inhibition. Noteworthily, in terms of effects of alkyl ferulates in the surface

435

charge of cells, we noted these findings are inconsistent with the results reported by Andrade et al.

436

(2015) who reported that the surface charge of S. aureus and E.coli to less negative values were

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obtained after exposure to caffeic acid esters.

438

3.6. Effect of FAC6 on bacterial morphology

440

The morphological and intracellular alternations of L. monocytogenes were studied following

441

treatment with FAC6 using SEM and TEM analysis to gain a better understanding of the mode of

442

antibacterial action of FAC6. Untreated cells (Fig. 3 (A0)) retained regular and typical morphology,

443

showing a plump and smooth surface. In contrast, L. monocytogenes treated with FAC6 at 1×MIC

444

for 0.5 h were damaged with big holes in the cell poles of some bacteria as a result of cell lysis (Fig.

445

3(A1)). As the treatment time was prolonged to 4 h, cells (Fig. 3(A2)) were damaged severely, with

446

debris as well as extensive lysis of the cells. More partial cell wall and membrane of L.

447

monocytogenes cells disappeared and more pores on the surface were observed. Besides, numerous

448

blebs were evident on the surface of the FAC6 treated bacteria (Fig 3(A1) and (A2) indicated by

449

white arrows). Overall, the results indicated that 1×MIC FAC6 damaged the external structure of L.

450

monocytogenes and the FAC6-induced impairment of treated cells occurred in a time-dependent

451

manner.

452

The TEM images of the control showed a similar morphology as SEM, exhibiting a homogeneous

453

electron density in the cytoplasm (Fig. 3(B0)), while the cells treated with FAC6 at 1×MIC for 0.5 h

454

appeared aberrant properties, including the lysis of the cell walls and the leakage of cytoplasmic

455

contents (Fig. 3(B1)), which was consistent with PI uptake assay and supported the results of the

456

cell constituents’ leakage assay. In addition, a damaged intracellular organization with a

457

condensation of nuclear material and vacuolization could be noticeably observed. As the treatment

458

time was prolonged to 4 h, cells completely disintegrated as revealed in Fig. 3(B2). The integrity of

459

the cell membrane layer structure was destroyed seriously, showing discontinuous and ruptured

460

surface of cells. Complete loss of cytoplasm in the treated cells and the leaked cytoplasm around

461

cell surrounding could be easily found. Apparently, the results showed that FAC6 simultaneously

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caused not only the cell membrane dysfunction but also the significant internal damage of target

463

cells.

464

Many previous electron microscopy observations for L. monocytogenes have reported that the

465

effects of various antimicrobial agents on bacterial cell morphology were different, such as cell

466

distortion, completely lysed bacteria or ghost-like appearances, separation of cytoplasmic

467

membrane from cell wall, and leakage of cytoplasmic contents. In our study, FAC6 led to

468

considerable lysis of the cell envelope and leakage of cytoplasmic materials of L. monocytogenes

469

with increased cell wall perforation, whereas cell distortions or adhesion could not be observed. In

470

contrast, L. monocytogenes exposed to FA (0.1 mM) had a more distorted or shrinkage surface (Fig

471

3 (A3)) compared than the cells treated with FAC6 at the same concentration. Moreover, the

472

percentage of distorted cells and degree of distortion increased with the increase of FA treatment

473

(Fig 3 (A4)). Distortions or adhesion was commonly found in the cells treated with bioactive

474

phenolic compounds, such as safflower seed meal extract (Son, Kang, & Song, 2017) and sugar

475

beet molasses polyphenols (Chen, Zhao, Meng, & Yu, 2017). On the other hand, the intracellular

476

organization and morphology of L. monocytogenes cells were both susceptible to FAC6. The mode

477

of action from FAC6 is also different from that induced by bacteriocins, such as bifidocin A (Liu,

478

Ren, Zhao, Cheng, Wang, & Sun, 2017) and BMP 11 (Yi et al., 2018). Compared to the morphology,

479

the intracellular organization of cells was prone to being damaged by these bacteriocins, with

480

alterations in intracellular organization occurring earlier and more severely than the morphological

481

changes. According to the above observation, we inferred that FAC6 have an impact on the cell

482

envelope of L. monocytogenes in a different manner from that of above antimicrobial agents. Thus,

483

cell envelope-associated mechanisms were the main action for FAC6 to kill microorganisms.

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484

485

3.7. Membrane protein

486

The abundant perturbations and severe disruption in bacterial morphology caused by FAC6 20

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treatment point to the bacterial membrane as the main target. Therefore, the capacity of direct

488

interaction with membrane proteins was evaluated to shed light on their mode of action. SDS–

489

PAGE profiles of membrane proteins from L. monocytogenes treated with FAC6 are shown in Fig. 4.

490

The protein bands of the control bacteria showed strong intensities, whereas the protein bands faded

491

or even disappeared after treatment with FAC6 at 1×MIC for 6 h. Moreover, as for untreated

492

bacteria, there were two thick bands (R3 for 63.6 kDa and R6 for 58.1 kDa) among 14 major bands

493

in Line 1. After treated with FAC6, the amount of R3, R9 and R10, especially R3, significantly

494

reduced, while one band (R6) even disappeared in Line 2. Two new bands (R4 and R5) appeared in

495

lane 2 and the amount of R11 increased. The disappearance of protein bands probably involved in

496

interfering with gene expression and protein synthesis. This result was consistent with a previous

497

report of Zeng et al. (2010), who suggested that polyphenols may have an effect on cellular proteins

498

by disrupting cellular proteins or inhibiting their synthesis.

499

Tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe) are the main fluorophores in membrane

500

proteins. The fluorescence of residues on the surface of membrane proteins can only be quenched

501

by KI, whereas it cannot affect the fluorescence spectra of the residues situated inside or in crannies

502

of the membrane protein. Therefore, the location of Trp, Tyr, and Phe can be qualitatively

503

determined. Fig.5 (A0), (B0) and (C0) show a remarkable fluorescence decrease of Phe and no

504

significant quenching effects of Trp and Tyr residues with increasing KI concentration, indicating

505

that Phe residues are mainly located outside while Trp and Tyr residues are mainly situated inside

506

the membrane. The fluorescence spectra of Phe, Trp, and Tyr residues in the presence of FAC6 with

507

various concentrations are shown in Fig.5 (A1), (B1) and (C1). The maximum emission intensity of

508

residues gradually decreased accompanied by the red shift, indicating that the binding of FAC6 to

509

membrane proteins occurred, and addition of FAC6 render the microenvironment of Phe, Trp, and

510

Tyr residues more hydrophilic. Therefore, it can be inferred that interaction of FAC6 with L.

511

monocytogenes cell membrane proteins changed the conformation of these biomacromolecules

512

(Wang, Wang, Zeng, Xu, & Brennan, 2017).

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513

3.8. Effect of FAC6 on DNA of L. monocytogenes

515

Besides the cell wall and membrane, the bacterial genome might be another antibacterial target of

516

FAC6. The disruption of DNA could inhibit gene expression, subsequently block the enzyme and

517

receptor synthesis, resulting in the death of bacteria. There are two major modes related to the

518

non-covalent interactions of DNA with small molecules (DNA-binding): intercalation, involving the

519

insertion of a planar molecule between DNA base pairs, and groove binding (Liu, Ren, Zhao, Cheng,

520

Wang, & Sun, 2017), which could be distinguished through the method of ultraviolet absorption

521

spectroscopy. For comparative purpose we have included UV–vis absorption spectra of FAC6 alone

522

(5×10-5 M) and genomic DNA (10.2×10-5 M) alone showing distinct peaks without overlap (Fig. 6

523

(A)). The absorption spectra of FAC6 with different concentrations of DNA are shown in Fig. 6 (B)

524

having maximum absorbance at 325 nm. When the FAC6 was titrated with DNA, a hyperchromism

525

effect centered at 325 nm with a minor shift was observed, indicating the formation of complex

526

between FAC6 and DNA. Thus, intercalative mode of binding can be excluded and groove binding

527

along with electrostatic interaction appears to be more acceptable mode of binding (Ebrahimipour et

528

al., 2015; Wu et al., 2017).

529

Based upon the variation in absorbance (Fig. 6 (B)), the intrinsic binding constant (K) of the FAC6

530

with DNA was determined according to modified Benesi–Hildebrand equation (Sarwar et al., 2017):

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=

+

×

(1)

532

where Ao and A are the absorbance of FAC6 in the absence and presence of DNA,

533

are the molar extinction coefficients of FAC6 alone and the bound complex respectively, K is the

534

binding constant and C is the DNA concentration. The double reciprocal plot of Ao/(A-Ao) vs. 1/C

535

is linear (Fig. 6 (C)). The value of binding constant was estimated from the ratio of the intercept to

536

that of slope of the above mentioned plot. The value of K for the interaction of FAC6 with DNA

537

was 2.18×104 M-1, which is similar to those reported for well-established groove biding agents 22

and

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538

(Strekowski ea al., 1989). The high value of the binding constant for a minor groove binding might

539

be a consequence of the helicoidal geometry of the complex. We also calculated the change in

540

Gibbs free energy (∆G) using following equation:

541

∆ =−

(2)

where T is the absolute temperature in Kelvin and R is gas constant(R = 1.987 cal mol−1K−1).Value

543

of ∆G was found to be –5.9 kcal mol-1 suggesting the binding process to be spontaneous.

544

AO displacement assay was carried out to further confirm the mode of binding of FAC6 with DNA,

545

since AO is a sensitive fluorescence dye that binds to DNA through intercalation. The fluorescence

546

intensity of AO–DNA system decreases as small molecules replace AO after intercalating into DNA

547

base pairs. However, as shown in Fig. 6 (D), the fluorescence intensity of OA-DNA is not decreased

548

as subsequent addition of FAC6, indicating that FAC6 is not able to bind to DNA by intercalative

549

mode and it interacts with DNA via groove binding.

550

Agarose gel electrophoresis of genomic DNA and DNA with FAC6 was used to evaluate whether it

551

could induce the DNA damage. As shown in Fig. 6 (E), the bands of DNA treated with different

552

concentrations of FAC6 were similar to that of control, which indicated FAC6 had no ability to

553

cause significant DNA damage.

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3.9. Effect of FAC6 at sub-MICs on the inhibition of biofilm formation

556

FAC6 is safe (Nakauchi et al.,2011) and if it had biofilm inhibitory activity, it would be suitable to

557

be used in controlling biofilms in the food applications. And microbial biofilms represent a

558

distinguished bacterial physiology featured by a multicellular phenotype that is intrinsically

559

different from planktonic bacteria. Fig. 6(F) showed that FAC6 exhibits the specific ability for

560

impairing the formation of biofilm by L. monocytogenes. Compared to the control, the biofilm

561

formation from L. monocytogenes with 1/2 ×MIC FAC6 reduced by 2.2 times. And the extent of

562

inhibition of biofilm formation caused by FAC6 was relatively concentration-dependent. The

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563

physical factors such as nature of electric charge of the cell surface or physical interaction between

564

cell and solid surface might be of more importance for this inhibition (Furukawa, Akiyoshi, O'Toole,

565

Ogihara, & Morinaga, 2010). On the other hand, the growth of bacteria was nearly unaffected by

566

FAC6 at sub-MICs.

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3.10. Application of FAC6 in Amer Sturgeon Caviar Samples

569

Since some sea food products, such as fish roe, are prevalently vulnerable to the growth of L.

570

monocytogenes, especially at abusive storage temperatures (Takahashi et al., 2012). Moreover,

571

caviar, rich in protein and high unsaturated fatty acids, is more susceptible to food pathogens and

572

oxidation, which spoiled flavour, aroma, taste, nutritional value and overall quality of foods. Thus,

573

in this study FAC6 as a multifunctional additive with antioxidant and antimicrobial activities was

574

used to improve the shelf-life and the safety of food. Firstly, experiments were conducted to

575

evaluate efficiency of the treatments with FAC6 to the L. monocytogenes growth in artificially

576

inoculated Amer sturgeon caviar at 10 oC for one week. The results of the effect of FAC6 treatments

577

on the reduction of L. monocytogenes growth in the treated sturgeon caviar and untreated control

578

are presented in Fig. 7 (A). As the negative control, approximately 102 CFU g-1 was inoculated into

579

caviar samples, and L. monocytogenes cell numbers rose to 8.4 Log CFU g-1 in caviar for 7 days of

580

incubation at 10 oC. In contrast, FAC6 was effective in inhibiting L. monocytogenes growth. This

581

reduction continued throughout the total storage period of 7 days with final L. monocytogenes

582

numbers reduced to 1.2 Log CFU g-1 for FAC6 treatments. Because of a relatively short shelf life

583

for most of the seafood products retailed (less than 2-3 days), preventing L. monocytogenes growth

584

during these 3 days is absolutely satisfactory. Fig. 7 (B) shows the results of the sensory analysis of

585

Amer sturgeon caviar stored at 10 °C for 7 d. Amer sturgeon caviar treated with FAC6 were highly

586

appreciated by panellists, as demonstrated by scores ranging from 7.2 to 8.8 for texture, general

587

appearance, flavor and colour, whereas a lower score was attributed to the presence of off-flavours.

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588

4. Conclusion

590

This work investigated the antibacterial properties of enzymatically synthesized FAC6 against L.

591

monocytogenes. FAC6 exhibited significant antimicrobial activity against tested bacterial and

592

anti-biofilm formation. The potential antibacterial subcellular mechanisms of FAC6 were related to

593

permeability and integrity of cell envelops, resulting in leakage of some cellular components

594

(proteins, 260 nm absorbing materials and K+). FAC6 might bind with membrane protein to disrupt

595

proteins activity or inhibit their synthesis. It could also bind to L. monocytogenes DNA grooves to

596

form complexes. More broadly, we expect this study to find use of alkyl ferulate esters generated by

597

biotransformation on their own or as adjuvants, which may highlight a potential way forward to

598

tackle foodborne infections and biofilms in food processing environment in the future.

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599

Acknowledgements

601

The work was supported by National Natural Science Foundation of China (21106131 and 31771945), Academic

602

Exchanges and Talent Training Program (2017SICR109), Zhejiang Provincial Program for Overseas High-Level

603

Experts Introduction (Z20170407), as well as Food Science and Engineering the Most Important Discipline of

604

Zhejiang Province (JYTsp20142101).

605

Abbreviation

606

Ferulic acid, FA

607

Methyl ferulate, MF

608

Ethyl ferulate, FAC2

609

Butanol ferulate, FAC4

610

Hexyl ferulate, FAC6

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25

Decanol ferulate, FAC10

613

Dodecanol ferulate, FAC12

614

Deep eutectic solvents, DES

615

Acridine orange, AO

616

Propidium iodide, PI

617

Tryptophan, Trp

618

Tyrosine, Tyr

619

Phenylalanine, Phe

620

Listeria monocytogenes, L. monocytogenes

621

Escherichia coli, E. coli

622

Staphylococcus aureus, S. aureus

623

American sturgeon caviar, Amer sturgeon caviar

624

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612

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Octanol ferulate, FAC8

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611

References

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TABLES and FIGURE Legends

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Table 1 Minimum inhibitory concentration (MIC) and minimum bactericide concentration (MBC)

754

of FA and its alkyl ester derivatives (FAC2–C12) against L. monocytogenes and E. coli.

755

Fig. 1. (A) Growth curve and (B) Time-kill curves of FAC6 at the different concentration against L.

757

monocytogenes. The error bars represent the standard deviations.

758

Fig. 2. (A) The fluorescence spectra of propidium iodide (PI) in L. monocytogenes cells treated with

759

FAC6 for 4 h. (B) Potassium ion efflux of L. monocytogenes cell suspensions in TSB. Effect of

760

1×MIC FAC6 on cell constituents’ release, (C) protein and (D) UV-absorbing substances OD260 nm

761

of L. monocytogenes. Different letters in the same group of bacteria means significant differences (p

762

< 0.05). (E) The effect to FAC6 on zeta potential of L. monocytogenes. All results are the means ±

763

SD (n=3).

764

Fig. 3. Scanning electron microscopy (SEM) of L. monocytogenes. (A0), (A1) and (A2) were SEM

765

images of control, treatment for 0.5 h and 4 h by FAC6 at 1×MIC, respectively. (A3) and (A4) were

766

SEM images of treatment for 0.5 h and 4 h by FA at 0.1 mM, respectively. (B0), (B1) and (B2) were

767

TEM images of control, treatment for 0.5 h and 4 h by FAC6 at 1×MIC, respectively. Bleb

768

formation (white arrows), condensation of nuclear material (red arrows 1), vacuolization (red

769

arrows 2), loss of cytoplasm (red arrows 3), discontinuity and ruptured cell surface (red arrows 4),

770

as well as leaked cytoplasm material around cell surrounding (red arrows 5) are visible.

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Fig. 4. Electrophoresis map of membrane protein of L. monocytogenes treated with 1×MIC FAC6

772

for 4 h. Line 1: untreated bacteria; Line 2: FAC6-treated bacteria at 1×MIC for 4 h.

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Fig. 5. Fluorescence spectra of amino acid residues Phe (λex = 258 nm), Trp (λex = 280 nm), and

774

Tyr (λex = 296 nm) of L. monocytogenes membrane proteins in the various concentrations of KI

775

(A0, B0 and C0) and FAC6 (A1, B1 and C1) at different concentrations, respectively.

776

Concentrations of KI were 0, 5, 10, 15, 20, 25, 30 mM for curves 1 to 7; concentrations of FAC6

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were 0, 1/48, 2/48, 3/48, 4/48, 5/48, 6/48, 7/48, 8/48 MIC for curves 1 to 9. The downward arrows

778

indicate raising the concentrations of KI or FAC6.

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Fig. 6. (A) UV–vis absorption spectra of FAC6 alone (5×10-5 M) and genomic DNA of L.

780

monocytogenes (10.2×10-5 M) alone showing distinct peaks without any overlap. (B) UV-vis

781

absorption spectra of FAC6 (4×10-4 M) in the presence of increasing amounts of genomic DNA

782

(0-4.5×10-5 M). (C) Modified Benesi–Hildebrand plot for the corresponding UV spectra of FAC6 in

783

presence of genomic DNA. Changes in absorbance of FAC6 upon addition of DNA at 325 nm was

784

taken into consideration for calculation of binding constant. (D) Fluorescence titration of AO–DNA

785

complex with FAC6. AO–DNA complex was excited at 480 nm and emission spectra were recorded

786

from 490 nm to 600 nm. (E) Electrophoresis map of DNA extracted from L. monocytogenes treated

787

with different concentrations of FAC6. (F) Effect of different concentrations of FAC6 on the biofilm

788

formation of L. monocytogenes. The error bars represent the standard deviations, and the asterisk

789

indicates a statistically significant difference between each other.

790

Fig. 7. (A) L. monocytogenes cell number in artificially inoculated Amer sturgeon caviar treated

791

with FAC6 versus untreated control stored for 7 days at 10 oC. (B) Sensory profiles of fresh Amer

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sturgeon caviar treated with FAC6 or control and stored for 7 days at 4 °C.

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SCHEME

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Shi et al.

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Scheme 1. Lipase-catalyzed synthesis of alkyl ferulate esters and their antibacterial activity.

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Table 1 Minimum inhibitory concentration (MIC) and minimum bactericide concentration (MBC)

805

of FA and its alkyl ester derivatives (FAC2–FAC12) against L. monocytogenes and E. coli. L. monocytogenes MIC

MBC

MIC

MBC

(mM)

(mM)

(mM)

(mM)

Ferulic acid

FA

H

10

20

Ferulic alkyl

FAC2

C2H5

1.6

6.4

FAC4

C4H9

0.1

0.2

FAC6

C6H13

0.1

0.2

FAC8

C8H17

25.6

FAC10 C10H21 FAC12 C12H25

RI PT

R

25.6

1.421

1.6

>25.6

2.176

0.8

>25.6

3.234

0.4

25.6

4.292

>25.6

1.6

>25.6

5.350

25.6

>25.6

>25.6

>25.6

6.408

3.2

>25.6

3.2

>25.6

7.466

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Theoretical estimated using ChemBioDraw Ultra 13.0 program.

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clogPa

6.4

esters

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E. coli

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Figure captions

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(A)

(B)

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Fig. 1. (A) Growth curve and (B) Time-kill curves of FAC6 at the different concentration against L.

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monocytogenes. The error bars represent the standard deviations.

818

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(B)

(D)

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(E) Fig. 2. (A) The fluorescence spectra of propidium iodide (PI) in L. monocytogenes cells treated with

822

FAC6 for 4 h. (B) Potassium ion efflux of L. monocytogenes cell suspensions in TSB. Effect of

823

1×MIC FAC6 on cell constituents’ release, (C) protein and (D) UV-absorbing substances OD260 nm 36

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824

of L. monocytogenes. Different letters in the same group of bacteria means significant differences (p

825

< 0.05). (E) The effect to FAC6 on zeta potential of L. monocytogenes. All results are the means ±

826

SD (n=3).

827

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(B0)

(B1)

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(A3)

(A4)

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Fig. 3. Scanning electron microscopy (SEM) of L. monocytogenes. (A0), (A1) and (A2) were SEM

832

images of control, treatment for 0.5 h and 4 h by FAC6 at 1×MIC, respectively. (A3) and (A4) were

833

SEM images of treatment for 0.5 h and 4 h by FA at 0.1 mM, respectively. (B0), (B1) and (B2) were

834

TEM images of control, treatment for 0.5 h and 4 h by FAC6 at 1×MIC, respectively. Bleb

835

formation (white arrows), condensation of nuclear material (red arrows 1), vacuolization (red

836

arrows 2), loss of cytoplasm (red arrows 3), discontinuity and ruptured cell surface (red arrows 4),

837

as well as leaked cytoplasm material around cell surrounding (red arrows 5) are visible.

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Fig. 4. Electrophoresis map of membrane protein of L. monocytogenes treated with 1×MIC FAC6

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for 4 h. Line 1: untreated bacteria; Line 2: FAC6-treated bacteria at 1×MIC for 4 h.

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(A1)

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(C1)

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Fig. 5. Fluorescence spectra of amino acid residues Phe (λex = 258 nm), Trp (λex = 280 nm), and

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Tyr (λex = 296 nm) of L. monocytogenes membrane proteins in the various concentrations of KI 41

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((A0), (B0) and (C0)) and FAC6 ((A1), (B1) and (C1)) at different concentrations, respectively.

853

Concentrations of KI were 0, 5, 10, 15, 20, 25, 30 mM for curves 1 to 7; concentrations of FAC6

854

were 0, 1/48, 2/48, 3/48, 4/48, 5/48, 6/48, 7/48, 8/48 MIC for curves 1 to 9. The downward arrows

855

indicate raising the concentrations of KI or FAC6.

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(B)

(D)

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(E)

(F)

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Fig. 6. (A) UV–vis absorption spectra of FAC6 alone (5×10-5 M) and genomic DNA of L.

860

monocytogenes (10.2×10-5 M) alone showing distinct peaks without any overlap. (B) UV-vis 43

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absorption spectra of FAC6 (4×10-4 M) in the presence of increasing amounts of genomic DNA

862

(0-4.5×10-5 M). (C) Modified Benesi–Hildebrand plot for the corresponding UV spectra of FAC6 in

863

presence of genomic DNA. Changes in absorbance of FAC6 upon addition of DNA at 292 nm was

864

taken into consideration for calculation of binding constant. (D) Fluorescence titration of AO–DNA

865

complex with FAC6. AO-DNA complex was excited at 480 nm and emission spectra were recorded

866

from 490 nm to 600 nm. (E) Electrophoresis map of DNA extracted from L. monocytogenes treated

867

with different concentrations of FAC6. (F) Effect of different concentrations of FAC6 on the biofilm

868

formation of L. monocytogenes. The error bars represent the standard deviations, and the asterisk

869

indicates a statistically significant difference between each other.

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(A)

(B)

Fig. 7. (A) L. monocytogenes cell number in artificially inoculated Amer sturgeon caviar treated

876

with FAC6 versus untreated control stored for 7 days at 10 oC. (B) Sensory profiles of fresh Amer

877

sturgeon caviar treated with FAC6 or control and stored for 7 days at 10 °C.

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Highlights:

Alkyl ferulate esters were synthesized by lipase-mediated alcoholysis in DES

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Antibacterial activities of ferulate esters against L. monocytogenes were evaluated The mode of action of FAC6 against L. monocytogenes was studied

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FAC6 effectively maintain the sensory quality of sturgeon caviar