Purification, characterization and mode of action of enterocin, a novel bacteriocin produced by Enterococcus faecium TJUQ1

Journal Pre-proof Purification, characterization and mode of action of enterocin, a novel bacteriocin produced by Enterococcus faecium TJUQ1

Xiaoxiao Qiao, Renpeng Du, Yu Wang, Ye Han, Zhijiang Zhou PII:

S0141-8130(19)37868-7

DOI:

https://doi.org/10.1016/j.ijbiomac.2019.12.090

Reference:

BIOMAC 14117

To appear in:

International Journal of Biological Macromolecules

Received date:

28 September 2019

Revised date:

28 November 2019

Accepted date:

11 December 2019

Please cite this article as: X. Qiao, R. Du, Y. Wang, et al., Purification, characterization and mode of action of enterocin, a novel bacteriocin produced by Enterococcus faecium TJUQ1, International Journal of Biological Macromolecules(2019), https://doi.org/ 10.1016/j.ijbiomac.2019.12.090

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© 2019 Published by Elsevier.

Journal Pre-proof Purification, Characterization and Mode of Action of Enterocin, a Novel Bacteriocin produced by Enterococcus faecium TJUQ1

Xiaoxiao Qiao‡, Renpeng Du‡, Yu Wang, Ye Han, Zhijiang Zhou*

School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China ‡

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*Corresponding author: Zhijiang Zhou; [email protected]

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These authors contributed equally to this work and share the first authorship.

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Journal Pre-proof ABSTRACT: Enterococcus faecium TJUQ1 with high bacteriocin-producing ability was isolated from pickled Chinese celery. In this study, enterocin TJUQ1 was purified by ammonium sulfate precipitation, reversed-phase chromatography (Sep-Pak C8) and cation-exchange

chromatography.

The

activity

of

the

purified

bacteriocin

was

44566.41±874.69 AU/mg, which corresponds to a purification fold of 35.89±2.34. The molecular mass was 5520 Da by MALDI-TOF MS and Tris-Tricine SDS-PAGE. The result of LC-MS/MS showed that the bacteriocin shared 59.15% identity with enterocin produced by E.

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faecium GN (accession no. O34071). PCR amplification revealed that E. faecium TJUQ1 possesses a gene encoding enterocin B with 60% identity to enterocin B. Circular dichroism

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(CD) spectroscopy showed that the molecular conformation was 32.6% helix, 19.5% beta,

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12.9% turn and 35.0% random. The stability of enterocin TJUQ1 was measured. After

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exposure at 121 °C for 15 min, the residual antimicrobial activity of enterocin TJUQ1 was 85.95±1.32%. The antimicrobial activity of enterocin TJUQ1 was still active over a pH range

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of 3-11. Enterocin TJUQ1 was inactivated after exposure to proteolytic enzymes but was not

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inactivated by lipase or amylase. These results showed that enterocin TJUQ1 was a novel class II bacteriocin. Enterocin TJUQ1 showed wide antibacterial activity against food-borne

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gram-negative and gram-positive pathogens, such as Staphylococcus aureus, Listeria monocytogenes, Escherichia coli and Salmonella enterica. The MIC was 5.26±0.24 μg/mL against L. monocytogenes CMCC 1595. SEM and TEM were used to observe the changes in the morphological and intracellular organization of L. monocytogenes CMCC 1595 cells treated with enterocin TJUQ1. The results demonstrated that enterocin TJUQ1 increased extracellular electrical conductivity, facilitated pore formation, triggered the release of UV-absorbing materials, ATP and LDH, and even caused cell lysis in L. monocytogenes CMCC 1595 cells. Based on the characterization, the wide inhibitory spectrum and mode of action determined so far, enterocin TJUQ1 is a potential preservative for the food industry.

KEYWORDS: Enterocin, purification, characterization, Listeria monocytogenes, mode of action 2

Journal Pre-proof 1. Introduction Bacteriocins, antimicrobial peptides or proteins synthesized by ribosomes, are highly active against microorganisms genetically related to the microorganisms they are produced by, along with some distant bacteria, such as antibiotic-resistant isolates [1]. The bacteriocins produced by lactic acid bacteria (LAB) and their potential ability to improve the safety of food products as natural biopreservatives have drawn worldwide attention [2]. Additionally, bacteriocins from LAB are not only unharmful to the surrounding environment and human

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beings but also capable of inhibiting the growth of foodborne pathogens, such as Listeria monocytogenes, Escherichia coli, and Staphylococcus aureus [3]. Currently, nisin is an

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extensively used natural food preservative. Despite the advantages of nisin, there are some

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disadvantages, such as decreased stability and low antibacterial activity in the pH range of

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5.0-7.0; moreover, it has only a slight effect on gram-negative bacteria [4]. While nisin provides essential support, the disadvantages mentioned above lead to the restricted use of

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spectrum.

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nisin; therefore, it is necessary to explore novel bacteriocins with a broad antimicrobial

Food spoilage caused by the growth and metabolism of pathogens is a worldwide and

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serious problem that causes enormous economic losses. Moreover, food-borne pathogens can produce toxic substances that can cause great harm to human health [5]. L. monocytogenes, which can cause listeriosis disease and subsequent foodborne epidemics, is one of the most pernicious foodborne pathogens [6]. It is found in a wide variety of food, such as vegetables, meat, dairy and seafood [7]. L. monocytogenes has strong survival ability in various conditions, including a wide pH range, high salt concentration and low temperature, and can form biofilms in food processing facilities, resulting in the contamination of food products [8]. Therefore, agents that could prevent or control L. monocytogenes contamination are imminently needed in the food industry [9]. However, a crucial requirement is that the agents must be safe and natural food preservatives as opposed to chemical agents since potential problems from chemical preservatives in foods are attracting the attention of consumers. Information on biochemical characteristics, antibacterial activity under various 3

Journal Pre-proof conditions, antibacterial spectrum, and mode of action is required to promote practical application of bacteriocins [10]. More specifically, bacteriocins primarily affect the cytoplasmic membrane through electrostatic attracting, causing the release of ions, intracellular nucleic acid and proteins through pores formed in the phospholipid bilayer. Eventually, because of the formation of pores and the insertion of bacteriocins, the cells will lyse [9]. However, in some antimicrobial systems, there are some discovered differences [11]. Our previous study isolated a strain, Enterococcus faecium TJUQ1, and the bacteriocin

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activity of this strain was enhanced 1.78-fold by the response surface method (RSM) [12]. This present study aimed to describe the purification and characterization of the bacteriocin

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produced by E. faecium TJUQ1, as well as the mode of action against L. monocytogenes, to

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develop a highly efficient and natural preservative for application in the food industry.

2. Materials and methods

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2.1. Bacterial strains and growth conditions

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E. faecium TJUQ1 isolated from pickled Chinese celery (Kunming, Yunnan Province, China) [12] was used for the production of bacteriocin. The strain was cultured in modified

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MRS medium (30 g/L sucrose, 15 g/L tryptone, 6 g/L yeast extract, 15.20 g/L beef extract, 1.93 g/L K2HPO4, 4 g/L anhydrous sodium acetate, 1.5 g/L ammonium citrate, 2 mmol/L ZnCl2, and 1.0 mL/L Tween-80, and the initial pH was adjusted to 7.19) at 30 °C for 18 h under static conditions [12]. All indicator organisms and culture conditions are listed in Table 1. L. monocytogenes CMCC 1595 was selected as the indicator strain for antimicrobial assays at various purification steps and for determining the mode of action of the bacteriocin. 2.2. Purification of enterocin TJUQ1 The enterocin TJUQ1 was purified through ammonium sulfate precipitation, reversed-phase chromatography (Sep-Pak C8) and cation-exchange chromatography. Production of the bacteriocin required 50 mL of E. faecium TJUQ1 cultured in 1 L of modified MRS at 30 °C for 18 h overnight. The cells were removed by centrifugation at 4 °C and 8000 ×g for 10 min. Next, ammonium sulfate was added to the collected supernatant 4

Journal Pre-proof gently to reach 65% saturation and then stirred for 12 h at 4 °C. After centrifugation (8000 ×g, 20 min, 4 °C), the sediment was resuspended in 20 mM (pH 7) potassium phosphate buffer and dialyzed for 12 h at 4 °C using a 2 kDa cut-off membrane [13]. Using a rotary evaporator, the crude bacteriocin sample was condensed to one-third of the original liquid volume (RE-52, Shanghaizhixin, China). The active sample was loaded onto a Sep-Pak C8 6 cc Vac Cartridge (Waters Millipore, USA) balanced with acetonitrile. Then, various concentrations of acetonitrile (0, 30, 50 and

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100% (v/v)) to elute the protein by adding 0.05% (v/v) trifluoroacetic acid. The active fraction obtained was freeze-dried and resuspended in 10 mM (pH 7) potassium phosphate buffer.

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The concentrated antibacterial fraction was further purified by cation-exchange

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chromatography (Sartobind® S15, Sartorius Stedim Biotech GmbH, Germany). Different

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concentrations of NaCl (0, 0.1 M, 0.33 M, 1 M) in 10 mM (pH 7) potassium phosphate buffer were used to elute the bacteriocin. An agar well diffusion assay was used to assay

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antibacterial activity against L. monocytogenes CMCC 1595 after each purification process

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step [14]. Titers were defined as the highest dilution reciprocal that restrained the growth of the indicator bacteria [15]. A Bradford assay was performed to determine the protein

standard [16].

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concentration after each step of purification using bovine serum albumin (BSA) as the

2.3. Molecular mass determination and LC-MS/MS analysis of enterocin TJUQ1 Tris-Tricine SDS-PAGE (Solarbio P1325, Beijing, China) with a 16.5% (m/v) separating gel was primarily used to determine the molecular size of purified enterocin TJUQ1 [17]. The purified bacteriocin preparation and molecular mass marker (Solarbio PR1900, Beijing, China) were run on the gel at 30 V for 60 min and then at 100 V for 4 h. Half of the gel was stained using the Coomassie brilliant blue staining method to determine the molecular mass. To assay the antibacterial activity of the protein band, the other half of the gel was washed with sterile water, and nutrient soft agar containing L. monocytogenes CMCC 1595 was placed on top of the gel. Furthermore, MALDI-TOF mass spectrometry (MS) (Shimadzu, Japan) was used to measure the molecular mass of enterocin TJUQ1. The band was cut and 5

Journal Pre-proof sent to Gene Create Company (Wuhan, China) to determine the protein sequence. The amino acid sequence was determined by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) (ekspertTM nanoLC; AB Sciex Triple TOF 500-PLUS). 2.4. Detection of the enterocin gene Total DNA of strain E. faecium TJUQ1 was used as a template in polymerase chain reaction (PCR) to detect the enterocin genes. PCR amplification of the target genes (ent A, ent B, ent L50A, and ent L50B, ent P) and specific primers are listed in Table S1 [18]. Agarose

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gel electrophoresis was used to analyze the PCR products, which were then purified and sequenced by GENEWIZ company (Tianjin, China). Sequences were aligned against

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sequences in GenBank of NCBI and translated to amino acid sequences using the

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Genetyx-Win program.

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2.5. Circular Dichroism (CD) spectra of enterocin TJUQ1

A J-810 spectrometer (JASCO, Japan) was purged with N2 gas, and the CD spectra of the

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samples were recorded from 240 to 190 nm [19]. Stock solutions of enterocin TJUQ1 were

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prepared in deionized water, and the CD spectra of an enterocin TJUQ1 solution (50 μg/mL final concentration) were recorded. Each measurement was repeated three times, and the path

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length of the sample cell was 0.1 cm. The instrument parameters were as follows: bandwidth, 2.5 nm; step resolution, 0.1 nm; response time, 0.5 s; and scan speed, 50 nm/min. To avoid interference caused by solvent, optical equipment and impurities, the CD spectrum of the deionized water was subtracted from the CD spectrum of the enterocin TJUQ1 solution. 2.6. Stability of purified enterocin TJUQ1 after treatment with enzymes, pH, heat, organic solvents, and detergents The purified enterocin TJUQ1 was used to measure the stability of bacteriocin. The enzyme sensitivity of enterocin TJUQ1 was assessed by incubating with various enzymes including trypsin, papain, protease K, pepsin, amylase, and lipase (1 mg/mL). After incubation at 37 °C for 3 h, the reaction was terminated, and the enzymes were inactivated by heating for 5 min at 100 °C [20]. To analyze the pH sensitivity of enterocin TJUQ1, the pH of enterocin TJUQ1 was adjusted in the range from 3 to 11 with 1 M NaOH and 1 M HCl [21]. 6

Journal Pre-proof The bacteriocin was incubated at 37, 60, 80 or 100 °C for 30 min and at 121 °C for 15 min to detect its thermostability. A total of 0.2 mL of solvent (chloroform, ethanol, acetone, acetonitrile or benzene) was added to 0.2 mL of enterocin TJUQ1 and mixed thoroughly. Then, mixtures were placed in tubes, which were incubated for 3 h at 37 °C, and the antibacterial activity was assessed [22]. Different detergents, including urea, Tween-80, Tween-20, and SDS, at a concentration of 1% (v/v) were added to an Eppendorf tube containing 0.5 mL of bacteriocin sample. The mixtures were incubated at 37 °C for 3 h, then

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proceeded to antibacterial activity assay. All treatments were performed using purified enterocin TJUQ1 (50 μg/mL). Residual antibacterial activity was analyzed against L.

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bacteriocin sample was used as the positive control.

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monocytogenes CMCC 1595 by the agar well diffusion method, and the group with untreated

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2.7. Antibacterial spectrum and minimum inhibitory concentration (MIC) values of enterocin TJUQ1

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The antibacterial spectrum of purified enterocin TJUQ1 was evaluated on the indicator strains listed in Table 1. The double dilution method was used to investigate the MIC of

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enterocin TJUQ1 against various indictor strains [23]. Briefly, 90 μL of bacterial suspension

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(106 CFU/mL) was added to 10 μL of 2-fold serial dilutions of enterocin TJUQ1 in microtiter plates, and the microplates were incubated for 16 h at 30 °C. While, the bacterial suspension added with nisin as the control group. To determine the bacterial growth in each tube, the absorbance of the supernatants was measured at 600 nm using a Multiskan FC microplate reader (Thermo, Shanghai, China). The MIC was confirmed as the lowest bacteriocin concentration that completely inhibited the indicator strain. 2.8. Time-killing kinetics The time-killing kinetics of L. monocytogenes CMCC 1595 were performed as previously described [24]. The OD600 value of the strain was 0.6, then exposed to 1/4 MIC, 1/2 MIC, MIC, 2 MIC enterocin TJUQ1 for 5 h. The OD600 values were measured at proper time intervals. Indicator bacteria grown without the enterocin TJUQ1 were the control group. 2.9. Determination of electrical conductivity 7

Journal Pre-proof The electric conductivity value was adopted to explain the cell membrane permeability of L. monocytogenes CMCC 1595 and illustrate the mode of action of enterocin TJUQ1 to some degree [25]. The bacteria were centrifuged and resuspended in deionized water. Different concentrations (0, MIC, 2 MIC) of enterocin TJUQ1 were added to the cell suspensions and incubated at 30 °C. The same treatment with no bacteriocin was the control group. At fixed time points, 3 mL of each sample was centrifuged to remove cells, and the supernatant was filtered through a 0.22 μm nitrocellulose membrane. Then, the electric

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conductivity was measured with a conductivity meter (Leici, Shanghai, China). 2.10. Measurement of UV-absorbing materials

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The integrity of the cell membrane was examined to survey the release of proteins and

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nucleic acids. Enterocin TJUQ1-treated and control cell suspensions were sampled every 30

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min. The supernatants were prepared as described before, but phosphate-buffered saline (50 mM) was used instead of deionized water. The concentration of ultraviolet-absorbing

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materials was measured using a UV–visible spectrophotometer (1800PC, Shanghai, China) at

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260 and 280 nm for nucleic acids and protein, respectively. 2.11. Release of adenosine triphosphate (ATP) and lactate dehydrogenase (LDH)

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Intracellular ATP and LDH leak into the medium when the cell membrane was destroyed. Therefore, the leakage of ATP and LDH was also examined to elucidate the disruption of cell membrane integrity [26]. To measure the ATP released from the cells, an ATP Test Kit (A095, Nanjingjiancheng, China) was used based on the manufacturer's instructions. The LDH concentration was determined with an LDH Kit (BC0680, Solarbio, Beijing, China) and a UV–visible spectrophotometer (UV-1800PC, Shanghai, China). 2.12. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) The ultrastructural alterations of L. monocytogenes CMCC 1595 exposed to enterocin TJUQ1 were investigated by SEM and TEM. Exponential-phase indicators with an OD600 of 0.6 were treated with enterocin TJUQ1 at the MIC and incubated for 2 h at 30 °C. The control group was grown without the bacteriocin. The enterocin TJUQ1-treated and control samples were harvested, washed with potassium phosphate buffer, and fixed with 2.5% (V/V) 8

Journal Pre-proof glutaraldehyde at 4 °C for 4 h. Then, the samples were dehydrated with gradient alcohol solutions (30%, 50%, 70%, 85%, 90% and 100%). The specimens were examined by scanning electron microscopy (S4800, Hitachi, Japan). The pretreatment for TEM was the same as that for SEM. After being fixed and dehydrated, the specimens were embedded in hard resin. The resin samples were cut into thin slices (70 nm) by an ultramicrotome (PowerTome-PC, RMC, USA) and stained with alkaline lead citrate and alcoholic uranyl acetate. Finally, the intracellular organization was examined with a transmission electron

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microscope (JEM-2000FX, Hitachi, Japan) at an operating voltage of 120 kV. 2.13. Statistical Analysis

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Data were expressed as the means ± standard error from three independent variables.

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JMP software version 9.0.2 (SAS Institute 171 Inc.) was applied for the statistical analysis.

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This study used SigmaPlot version 10.0 software (Systat Software Inc. San Jose, CA) for the

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3. Results and discussion

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graph and statistical analysis.

3.1. Purification of enterocin TJUQ1

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Enterocin TJUQ1 was purified in three steps. The purification process results are shown in Table 2. The culture supernatant from E. faecium TJUQ1 was extracted by ammonium sulfate precipitation, and a crude extract was obtained with a special activity of 5538.21±156.37 AU/mg and a purification fold of 4.46±0.41. Then, the extraction was loaded on a reversed-phase cartridge (Sep-Pak C8) and eluted. At this stage of purification, the purification fold was 16.73±1.22, and the activity reached 20774.48±558.95 AU/mg. After cation-exchange chromatography, a fairly pure bacteriocin preparation (35.89±2.34-fold) was obtained. We obtained a higher purification fold for enterocin TJUQ1 than enterocin LR/6, for which specific activity was increased 6.37-fold [27]. Du et al. [28] reported that the specific activity of plantaricin GZ1-27 increased 10.55-fold after three-step purification. 3.2. Molecular mass determination of enterocin TJUQ1

9

Journal Pre-proof Tris-Tricine SDS-PAGE showed a peptide band between approximately 3.3 and 6.5 kDa (Figure 1A). The band of enterocin TJUQ1 had inhibitory ability against L. monocytogenes CMCC 1595. MALDI-TOF MS showed a sharp peak, corresponding to 5520 Da (Figure 1B). Similar results have been reported for bacteriocins produced by other E. faecium, such as enterocin LR/6 (6 KDa) [27], enterocin LD3 (4.1 KDa) [29] and enterocin LM-2 (between 3.5 and 6.4 kDa) [30]. LC-MS/MS analysis results of a fragment of enterocin TJUQ1 are shown in Figure S1. The fragment was analyzed with Proteome Discoverer 2.1 software. Enterocin

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TJUQ1 was searched in the NCBI database; it had a low homology with other studied bacteriocins and only 59.15% identity with enterocin B (accession no. O34071). Thus,

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3.3. Detection of bacteriocin gene

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enterocin TJUQ1 represents a novel enterocin.

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The genomic DNA extracted from E. faecium TJUQ1 was PCR-amplified with primers targeting five known enterocin genes generating positive results only for enterocin B. The

The

deduced

amino

acid

sequence

was

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CP028727.1).

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gene sequence showed 91% homology with E. faecium FSIS1608820 (GenBank number

RIKQHFIQRWSKCGAAIAGGLFGIPKGPLHGLLGLQMYTLNAT. No identical sequence

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was acquired from the database, while only 60% identity with enterocin B was observed (accession No. ADR70740.1). These results indicate that enterocin TJUQ1 (accession No. MN736844) is a novel bacteriocin. Similar results have been reported for the enterocin B gene detected in E. faecium FL31 [18]. Enterocins of E. faecium and E. faecalis such as enterocin A, enterocin P, enterocin Q, enterocin L50 and enterocin B are class II bacteriocins [18]. 3.4. Molecular conformation of enterocin TJUQ1 The CD spectrum revealed minima at 206 and 220 nm, and the ellipticity at 220 nm versus the ellipticity at 206 nm (R value) was 1.79 (Figure S2). The results were 32.6±0.8% helix, 19.5±1.0% beta, 12.9±0.4% turn and 35.0±1.2%, which totaled 100%, with restriction. The secondary conformation obtained by CD spectroscopy revealed that bacteriocin TSU4i contains 23.7±0.2% α-helical 17.1±0.1% β-sheet and 59.2±1% random coil conformations 10

Journal Pre-proof [31]. 3.5. Stability of enterocin TJUQ1 Table 3 shows the effects of enzymes, pH, temperature, organic solvents and detergents on the antibacterial ability of enterocin TJUQ1. When treated with proteolytic enzymes (protease K, trypsin, pepsin and papain), the enterocin TJUQ1 was absolutely inactivated, but it was not influenced by lipase and amylase. The above results demonstrated that enterocin TJUQ1 was a peptide that did not contain lipid carbohydrate groups. These results are in

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agreement with the bacteriocins from E. faecium JCM 5804T [32] and E. faecium LM-2 [30]. Enterocin TJUQ1 remained relatively stable at pH 3-11. However, plantaricin GZ1-27 lost

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most of its activity at pH 7.0 and was fully inactive at pH values > 8.0 [28]. This result was

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similar to previous findings; neutral and alkaline conditions readily inactivate most

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bacteriocins, including nisin, plantaricin JLA-9 [20], and bacteriocin R1333 [24]. Moreover, after treatment at 60, 80, and 100 °C for 30 min, the antibacterial activity remained stable.

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The residual activity was maintained at 85.95±1.32% after autoclaving for 15 min at 121 °C.

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Enterocin TJUQ1 was stable after heat treatment, which was in accordance with other bacteriocins, such as lactocin MXJ32A [33] and bacteriocin MM4 [21]. Compared to nisin,

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enterocin TJUQ1 had better thermal stability. Organic solvents and detergents did not affect the antibacterial activity of enterocin TJUQ, which is in accordance with enterocin MMRA [34]. The same report indicated thatsome solvents did not change the activity of bacteriocin CV7, but Tween-20, Tween-80 and urea decreased the activity [35]. Liu [22] reported that Tween-80, Tween-20, EDTA and urea did not reduce the activity of bifidocin A; however, with SDS-36%, the activity of bifidocin A was lost. This means that solvents and detergents could cause structural changes in bacteriocin. Investigation of the newly detected enterocin TJUQ showed that it has the merits of being degradable by various proteases, including a molecular weight of approximately 5.5 kDa, remarkable pH and thermal stability, bactericidal in nature and potent antilisterial activity, which are characteristic features of class II bacteriocins [36, 37]. Therefore, enterocin TJUQ was a novel class II bacteriocin. 3.6. Antibacterial spectrum and MIC values of enterocin TJUQ1 11

Journal Pre-proof Table 1 shows the antibacterial spectrum and MIC values of enterocin TJUQ1. Enterocin TJUQ1 revealed observably antibacterial activity against gram-positive bacteria, for instance S. aureus and Bacillus. Moreover, enterocin TJUQ1 was able to prevent the growth of L. monocytogenes. This finding was consistent with the characteristics of class II bacteriocins [38]. The MIC of enterocin TJUQ1 against L. monocytogenes CMCC 1595 was 5.26±0.24 μg/mL. The antibacterial ability of enterocin TJUQ1 was dramatically enhanced, achieving a 10-fold increase, compared with enterocin CRL35 against L. monocytogenes LS01 (MIC

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value of 57 μg/mL) [39] and enterocin AS-48 against L. monocytogenes (MIC value of 50 μg/mL) [40]. Based on this result, the mode of action of enterocin TJUQ1 was analyzed at the

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MIC. Like most LAB bacteriocins, enterocin TJUQ1 could also inhibit genetically related

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LAB strains. Moreover, the inhibitory ability of enterocin TJUQ1 against gram-negative

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bacteria, for instance E. coli and Salmonella enterica give it greater potential as a biological preservative candidate. The enterocin TJUQ1 did not have inhibitory ability against fungi

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such as Moniliella pollinis BH010, Saccharomyces cerevisiae, Botrytis cinereal, Fusarium

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oxysporum and Fusarium graminearum, while it could inhibit the growth of Zygosaccharomyces rouxii. However, nisin did not show inhibitory ability against E. coli, S.

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enterica and Z. rouxii as the control group. Some researchers have reported that E. faecium bacteriocins can effectively inhibit foodborne pathogens. For example, enterocin LM-2 produced by E. faecium LM-2 showed a broad inhibitory spectrum, especially against L. monocytogenes, S. aureus, E. coli, Salmonella and Bacillus [30]. The inhibition spectrum of enterocin MMRA was relatively broad, preventing the growth of Listeria spp. and many other gram-negative and gram-positive bacteria [34]. 3.7. Time-killing kinetics A time-kill curve assay was used to reveal the concentration and time of the activity of enterocin TJUQ1. As observed in Figure 2A, the ability of different concentrations (1/4 MIC, 1/2 MIC, MIC, 2 MIC) of enterocin TJUQ1 to prevent or kill L. monocytogenes CMCC 1595 within 5 h was determined. On the basis of the value of OD600, at 1/4 MIC and 1/2 MIC, the L. monocytogenes CMCC 1595 was restrained. At concentration higher than the MIC, enterocin 12

Journal Pre-proof TJUQ1 remarkably decreased the number of bacteria. In particular, at 2 MIC, the OD600 was reduced to 0.22±0.03 in 5 h. This indicated that the lower levels of the bacteriocin could inhibit bacterial growth but could not thoroughly kill the bacteria. In conclusion, exposure time and concentration can affect the antibacterial activity of enterocin TJUQ1. To gain further insight into the mechanism of enterocin TJUQ1 against L. monocytogenes CMCC 1595, the molecular and cellular changes of samples with enterocin TJUQ1 were explored. Others reported that bacteriocin CH4 can inhibit the growth of L. monocytogenes [41], and a

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time-kill curve assay was conducted. The growth of L. monocytogenes was prevented, but there was no dramatic reduction at MIC (31 μg/mL) and 2 MIC. The cell number was

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decreased below 2 log CFU/mL at 16 MIC.

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3.8. Effect of enterocin TJUQ1 on cell membrane permeability

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Extracellular electrical conductivity can reflect the cytoplasmic membrane of L. monocytogenes CMCC 1595. Compared with control cells, enterocin TJUQ1-treated cells

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revealed a dramatic improvement in the electrical conductivity, which was observed after 0.5

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h. The electrical conductivity of samples (MIC, 2 MIC) was promptly enhanced in 2 h, after which the conductivity leveled off (Figure 2B). The electric conductivity of the samples was

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112.54±3.93 and 161.36±4.25 μS/cm with MIC and 2 MIC, respectively, after 5 h. Compared with the lower concentration of enterocin TJUQ1, the permeability of the bacterial membranes was significantly destroyed at higher concentrations. The above results indicated that enterocin TJUQ1 disrupted the cell membrane permeability of L. monocytogenes CMCC 1595 in a dose-dependent matter, inducing an increase in the extracellular electrical conductivity value. Similar increases have been recorded in L. monocytogenes LS01 cell suspensions treated with the antimicrobial peptide enterocin CRL35 [39]. 3.9. Effect of enterocin TJUQ1 on cell membrane integrity The results revealed that enterocin TJUQ1 caused the leakage of nucleic acids (Figure 3A) and proteins (Figure 3B) from treated L. monocytogenes CMCC 1595, and the increase in protein was very similar to the increase in nucleic acids. After the treatment of enterocin TJUQ1 for 5 h, the OD260 values at the MIC rose gradually from 0.08±0.01 to 0.78±0.03, and 13

Journal Pre-proof those at the 2 MIC rose greatly from 0.11±0.01 to 1.03±0.02. The OD280 values at various enterocin TJUQ1 concentrations also increased to 0.62±0.03 (MIC) and 0.86±0.03 (2 MIC). The optical density at 2 MIC ascended much more rapidly than those at MIC. Additionally, no noticeable changes in the OD260 and OD280 of the control cells were observed. Nucleic acids and proteins are the significant components of cells, and their losses are determined as irreversible damage to the cytoplasmic membrane [42]. Other bacteriocins, such as plantaricin LPL-1 against L. monocytogenes 54002 [10] and CH4 against L. monocytogenes [41], have

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been found to exhibit similar results. The effect of enterocin TJUQ1 on the extracellular ATP levels of L. monocytogenes

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CMCC 1595 cells are presented in Figure 4A. After treatment with different concentrations of

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enterocin TJUQ1 for 5 h, the concentration of extracellular ATP reached 147.57±4.51

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μmol/gprot and 233.96±5.63 μmol/gprot at MIC and 2 MIC, respectively. The untreated sample was found to be 22.14±0.38 μmol/gprot. In comparison to pentocin 31-1 against L.

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monocytogenes NICPBP 54002 [43] or lactocin 705 against Lactobacillus plantarum CRL691

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[44], enterocin TJUQ1 can cause the release of an abundance of ATP. The rapid leakage of ATP indicated that enterocin TJUQ1 had an adverse influence on the integrity of the cell

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membrane.

LDH is an enzyme involved in energy metabolism. It is present in the cytoplasm, and increases in its extracellular concentration indicate cell damage [45]. Therefore, LDH is commonly used as a cell lysis signal in biochemical studies [46]. As shown in Figure 4B, the concentration of released LDH gradually increased in 2 h, while it rapidly increased to 19.63±0.34 U/mL (MIC) and 26.86±0.53 U/mL (2 MIC) at 4 h. However, the concentration of the control was approximately 5.52±0.19 U/mL at all times. Yi [47] reported that with the extension of time, the LDH content of L. monocytogenes affected by bacteriocin BM1157 increased from 0.11 to 0.18 nM. Therefore, compared with enterocin TJUQ1, which caused a rapid increase in the extracellular LDH, the disruption of the cell membrane integrity caused by BM1157 was subtle. 3.10. SEM and TEM analyses 14

Journal Pre-proof The impact of enterocin TJUQ1 on the morphology of L. monocytogenes CMCC 1595 cells was investigated with SEM. As shown in Figure 5A, untreated L. monocytogenes CMCC 1595 cells exhibited intact cell structures, as observed by plump spheres, smooth membrane surfaces and striated cell walls. When exposed to enterocin TJUQ1 for 1 h, the cell morphology was altered, with slight gaps and some small holes on the cell surface (Figure 5B). When the experiment time was extended to 3 h, the cell walls exhibited noticeable holes and some were even completely dissolved (Figure 5C). These results demonstrated that

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enterocin TJUQ1 affected changes in the morphology of L. monocytogenes CMCC 1595 cells in a time-dependent manner.

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TEM was performed to observe in much details the ultrastructural and intracellular

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changes of L. monocytogenes CMCC 1595 cells. Without enterocin TJUQ1, the membrane

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integrity of the bacterial cells was preserved and exhibited a dense and even distribution of the cytoplasm (Figure 5D). In contrast, samples treated with enterocin TJUQ1 for 1 h showed

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pores shapes in the cell wall, the cytoplasmic contents flowed out, and the cytoplasm became

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loose (Figure 5E). At 3 h, the cellular structure was obviously destroyed, and the cell membrane became damaged. Therefore, the cells no longer had the basic structure of the

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membrane (Figure 5F). These micrographs clearly demonstrated that enterocin TJUQ1 induced noticeable destruction of the intracellular organization and structure of L. monocytogenes CMCC 1595 cells.

The TEM and SEM analyses indicated that the mode of action of enterocin TJUQ1 against L. monocytogenes CMCC 1595 includes forming pores on the membrane and even cellular lysis, which implied that it destroys the cell membrane in a progressive process. This, combined with the results described above, indicated that enterocin TJUQ1 increases the cell membrane permeability, pore formation, and destruction of the cell structure, leading to the leakage of intracellular macromolecules. Similar results were reported for enterocin LD3 [48], bacteriocin BMP11 [49] and lactocin MXJ 32A [33].

4. Conclusion 15

Journal Pre-proof In this study, enterocin TJUQ1 was purified by ammonium sulfate precipitation, reversed-phase chromatography and cation-exchange chromatography. Purified enterocin TJUQ1 exhibited a specific activity of 44566.41±874.69 AU/mg, which corresponds to a purification fold of 35.89±2.34, and the molecular mass of enterocin TJUQ1 was found to be 5520 Da by MALDI-TOF MS. LC-MS/MS and PCR amplification indicated that enterocin TJUQ1 is a novel bacteriocin. Its secondary structure conformation was 23.6% helix, 17.5% beta, 23.9% turn and 35.0% random. Enterocin TJUQ1 showed a wide-spectrum antibacterial

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ability against both gram-negative and gram-positive food-borne pathogens and excellent stability after different treatments. The mode of action of enterocin TJUQ1 against L.

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monocytogenes CMCC 1595 showed that enterocin TJUQ1 increased membrane permeability,

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destroyed membrane integrity, and eventually led to cell death. In conclusion, enterocin

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TJUQ1 produced by E. faecium TJUQ1 is a novel class II bacteriocin, which was first reported in the current study. These results revealed that enterocin TJUQ1 is a promising and

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Acknowledgements

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attractive natural biologic preservative in the food preservation industry.

This work was financially supported by the Key Technology R&D Program of Tianjin, China

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Journal Pre-proof Tables Table 1 Antimicrobial Spectrum of Enterocin TJUQ1. medium and temperature MIC indicator strains

source (℃)

(μg/mL)

Gram-positive bacteria ATCC 6538

LB, 30

46.50 ± 1.81

Micrococcus luteus

ATCC 4698

LB, 30

23.77 ± 1.12

Bacillus subtilis BH072

FS, TJU

LB, 37

11.54 ± 0.81

Bacillus licheniformis

ATCC 10716

LB, 30

Listeria innocua

DSMZ 20649T

BHI, 30

5.74 ± 0.12

Listeria monocytogenes

CMCC 1595

BHI, 30

5.26 ± 0.24

Lactobacillus plantarum

FS, TJU

MRS, 30

23.75 ± 0.95

Lactobacillus sakei

DSMZ 20017T

MRS, 30

11.46 ± 0.63

Pediococcus acidilactici

PA003

MRS, 30

24.17 ± 1.22

Lactococcus lactis

NZ9000

MRS, 30

23.65 ± 0.95

MRS, 30

11.68 ± 0.57

faecalis

FS, TJU

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TG2

22.16 ± 0.92

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Enterococcus

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Staphylococcus aureus

Gram-negative bacteria Escherichia coli DH5α

ATCC 68233

LB, 30

49.20 ± 1.52

Escherichia coli BL21

ATCC 68004

LB, 30

45.50 ± 1.84

Salmonella enterica

ATCC 7378

LB, 30

47.30 ± 1.43

Salmonella paratyphi A

CGMCC 1.18

LB, 30

–

FS, TJU

PDA, 28

–

FS, TJU

PDA, 28

–

Fungi Moniliella

pollinis

BH010 Saccharomyces cerevisiae 22

Journal Pre-proof Zygosaccharomyces FS, TJU

PDA, 30

25.42 ± 1.32

Botrytis cinerea

CGMCC 3.4584

PDA, 30

–

Fusarium oxysporum

CGMCC 3.2830

PDA, 28

–

Fusarium graminearum

CGMCC 3.6862

PDA, 28

–

rouxii

ATCC, American Type Culture Collection; CMCC, China Microbiological Collection Center;

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DSMZ, Deutsche Sammlungvon Mikroorganismenund Zellkulturen; FS, TJU, Food Science Laboratory of Tianjin University.

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–, no inhibition.

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Journal Pre-proof Table 2 Purification Results of Enterocin TJUQ1. protein

activity

specific

purification

(mg/mL)

(AU/mL)

activity

fold

volume purification stage (mL) (AU/mg) culture

807.14 ± 1000.00

1241.75 ±

0.65 ± 0.21

1.00 ± 0.00

supernatant

35.47

45.36

10583.52 ±

5538.21 ±

sulfate

50.00

1.91 ± 0.32 283.76

10.00

275.64

558.95

16935.23

44566.41 ±

±337.59

874.69

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cation-exchange

16.73 ± 1.22

0.38 ± 0.12

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chromatography

na

Specific activity is the ratio of activity to that of protein.

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a

20774.48 ±

0.53 ± 0.11

chromatography

5.00

11010.47 ±

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reversed-phase

24

4.46 ± 0.41

156.37

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precipitation

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ammonium

35.89 ± 2.34

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Table 3. Stability of purified enterocin TJUQ1 after treating with enzymes, pH, heat, organic solvents, and detergents. factors

residual antibacterial activity (%)

protease K

0.00 ± 0.00

papain

0.00 ± 0.00

pepsin

0.00 ± 0.00

amylase

100.00 ± 0.96

lipase

100.00 ± 0.83

pH 98.19 ± 0.65

5

98.84 ± 1.23

7

99.65 ± 1.64

9

93.36 ± 0.95

37 ℃, 30 min 60 ℃, 30 min 80 ℃, 30 min

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temperature

89.82 ± 1.44

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11

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3

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0.00 ± 0.00

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trypsin

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enzymes

99.42 ± 0.83 97.58 ± 1.57 96.89 ± 1.13

100 ℃, 30 min

91.71 ± 0.78

121 ℃, 15 min

85.95 ± 1.32

solvents ethanol

98.65 ± 0.94

chloroform

97.98 ± 1.17

acetone

97.83 ± 0.42

25

Journal Pre-proof acetonitrile

98.36 ± 0.77

benzene

98.97 ± 1.36

detergents 98.57 ± 1.26

Tween-80

98.23 ± 0.95

Tween-20

97.65 ± 0.67

SDS

97.52 ± 0.78

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urea

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Figure captions

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Figure 1. (A) Tris-Tricine SDS-PAGE of enterocin TJUQ1. Lane M: low molecular marker; lane 1: culture supernatant of enterocin TJUQ1; lane 2: Stained half of the gel showing

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proteins present in partially purified enterocin TJUQ1. (B) MALDI-TOF MS mass spectrum

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of enterocin TJUQ1.

Figure 2. (A) Time-killing curves of enterocin TJUQ1 on L. monocytogenes CMCC 1595. (B) Changes of electric conductivity value from L. monocytogenes CMCC 1595 after treatment with different concentrations of enterocin TJUQ1.

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Figure 3. Extracellular UV-absorbing materials from L. monocytogenes CMCC 1595 cells

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were detected at 260 nm (A) and 280 nm (B).

Figure 4. Extracellular ATP (A) and LDH levels (B) from L. monocytogenes CMCC 1595 after treatment with different concentrations of enterocin TJUQ1.

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Figure 5. SEM and TEM images of L. monocytogenes CMCC 1595 cells: (A and D) control;

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(B and E) treated with MIC enterocin TJUQ1 for 1 h; (C and F) treated with MIC enterocin

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TJUQ1 for 3 h.

29

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Xiaoxiao Qiao: Conceptualization, Methodology, Investigation, Writing - Original Draft. Renpeng Du: Data Curation, Investigation, Formal analysis, Writing - Review & Editing. Yu Wang: Software, Validation, Supervision. Ye Han: Resources, Supervision, Project administration.

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Zhijiang Zhou: Writing - Review & Editing, Funding acquisition.

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The present study described the purification, characterization and mode of action of enterocin TJUQ1, which is generated by Enterococcus faecium TJUQ1. The activity of the purified bacteriocin was 44566.41±874.69 AU/mg, which corresponds to a purification fold of 35.89±2.34. The molecular mass was 5520 Da by MALDI-TOF MS and Tris-Tricine SDS-PAGE. LC-MS/MS and PCR amplification indicated that enterocin TJUQ1 is a novel bacteriocin. The residual antimicrobial activity of enterocin TJUQ1 was 85.95±1.32% after exposure at 121 °C for 15 min. The antimicrobial activity of enterocin TJUQ1 was still active over a pH range of 3-11. These results showed that enterocin TJUQ1 was a novel class II bacteriocin. Enterocin TJUQ1 showed wide antibacterial activity against food-borne gram-negative and gram-positive pathogens. The mode of action of enterocin TJUQ1 against L. monocytogenes CMCC 1595 showed that enterocin TJUQ1 increased membrane permeability, destroyed membrane integrity, and eventually led to cell death.

31

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An enterocin TJUQ1 was purified from Enterococcus faecium TJUQ1 through three steps. Tris-Tricine SDS-PAGE, MALDI-TOF MS and LC-MS/MS indicated that the enterocin was a novel enterocin. Enterocin TJUQ1 showed a broad-spectrum antibacterial activity against both Gram-positive and Gram-negative food-borne pathogens.

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The mode of action of enterocin TJUQ1 against L. monocytogenes CMCC 1595 was studied.

32