Purication and Characterization of a Novel Bacteriocin Pediocin Z-1 Produced by Pediococcus Pentosaceus Z-1 Screened From Jinhua Ham

Background: Lactic acid bacteria (LAB) can produce bacteriostatic substances, among which bacteriocins attracted wide attention in food preservation for decades. Up to date, nisin (class I bacteriocins) has been considered to be the only bacteriocin produced by Lactococcus lactis strains for commercial use. Moreover, there are many other reports concerning the isolated bacteriocins for potential application in food industry while some exhibited a narrow bactericidal spectrum, thermal stability and low acid-base stability. Jinhua ham is a representative of traditional dry-cured meat product in China. The microbial community structure and diversity was speculated to be responsible for avor and quality formation of Jinhua ham, protecting from the spoilage microorganisms contamination. However, no studies was performed to investigate the bacteriocin from LAB in Jinhua ham. Thus, the objective of this study was to screen out high-eciency, safe and non-toxic bacteriocin-producing lactic acid bacteria (LAB) from Jinhua Ham and subsequently perform the purication, identication and characterization of its bacteriocin. Results: The bacteriocin-producing LAB was screened from Jinhua ham and then designated as Pediococcus pentosaceus Z-1 by colony morphology and 16S rDNA sequencing. The bacteriocin was then crudely extracted from the bacterial cell-free supernatant by pH adsorption, and further puried by cellulose DEAE-52 ion exchange and Sephadex G-50 chromatography columns. The tricine-SDS-PAGE electrophoresis showed a highly puried protein band with 8227.35 Da with 60 amino acids identied by MALDI-TOF-MS analysis. The bacteriocin was named as pediocin Z-1 and its antibacterial activity exhibited an acid-base stability between pH 2-10 and a thermal stability at a range of 50-110°C. The pediocin Z-1 was sensitive to proteases and showed an inhibitory effect against Gram-positive bacteria and Gram-negative bacteria, including Listeria monocytogenes, Staphylococcus aureus, Salmonella typhimurium, Salmonella potsdam and Escherichia


Background
Bacteriocins are bacteriostatic proteins or polypeptides produced by ribosomal synthesis during bacterial growth and metabolism and have attracted much attention for food preservation for decades [1]. Most lactic acid bacteria can produce a variety of bacteriostatic substances in the process of growth and metabolism, which antagonize other microorganisms. The bacteriostatic substances produced by lactic acid bacteria are mainly organic acids, hydrogen peroxide and bacteriocins [2]. Bacteriocin is a bacteriostatic protein or polypeptide produced by ribosome synthesis during the bacterial growth and metabolism and has been attracted for attention in food preservation for decades [3]. The bacteriocins are initially discovered from food isolates and can be basically divided into four categories according to their molecular mass, thermo-stability, enzymatic sensitivity and the presence of post-translational modi ed amino acid. Notably, class II bacteriocins are mostly found and own the characteristics including un-modi ed amino acids, heat-stability and generally less than 10 kDa. Class II bacteriocins has sub-groups, namely IIa (pediocin-like bacteriocins), IIb (twocomponent bacteriocins) IIc (thiol-activated bacteriocins).
Compared to the existing synthetic food preservatives, bacteriocins are considered to be the alternatives for the guarantee of food quality without health concerns [4]. However, up to date, nisin (class I bacteriocins) has been considered to be the only bacteriocin produced by Lactococcus lactis strains for commercial use. It has a molecular weight of 3354 Da with 34 amino acids and is evidenced to be nontoxic to animals and humans. As a food preservative, nisin can inhibit Gram-positive bacteria, but has no inhibitory effect on Gram-negative bacteria. Moreover, there are many other reports concerning the isolated bacteriocins for potential application in food preservation [5,6]. However, some exhibited a narrow bactericidal spectrum. For example, Guerreiro detected the Bacteriocin B231 antibacterial spectrum produced by Lactobacillus pentosus B231 and found that it only inhibited Listeria spp. [7]. A bacteriocin produced by Pediococcus pentosaceus K23-2 isolated from kimchi had good stability under acidic and neutral conditions, but its activity is sharply lost under alkaline conditions, and the bacteriocin can only inhibit Gram-positive bacteria [8]. Lactobacillus pentosus DZ35 was isolated from dry-cured meat products showing inhibitory effect on Staphylococcus aureus and Escherichia coli at the range of pH 2 -11 [9]. The pediocin GS4 is a broad spectrum bacteriocin isolated from the Pentosaceus GS4, however, it has antibacterial activity in the limited range of 30℃-50℃ and pH 5-7 [10]. The bacteriocins produced by the three enterococci isolated from Egyptian dairy products exhibited narrow-spectrum antibacterial activity, and can only inhibit Enterococcus Faecalis and Staphylococcus aureus [11]. Thus, the research for the novel bacteriocin has become one of the topical subject in the eld of food anti-corrosion.
Jinhua ham is a representative of traditional dry-cured meat product in China and possesses a variety of microorganism species. Our previous study showed that a total of 242 genera of bacteria belonging to 18 phyla was identi ed in Jinhua Ham factories [12]. The microbial community structure and diversity of Jinhua ham was speculated to be responsible for avor and quality formation of Jinhua ham, protecting from the spoilage microorganisms contamination. Therefore, the aim of this paper was to screen out high-e ciency, safe and non-toxic bacteriocin-producing LAB from Jinhua Ham. The bacteriocin was then isolated, puri ed and characterized. The puri ed bacteriocin was supposed to compare with commercialized nisin for antibacterial spectrum of activity to achieve application prospects in food preservation.

Bacterial strains
The 162 LAB strains were isolated from Jinhua ham produced by Jinnian Ham Company of Jinhua City, Zhejiang Province, and stored in deMan, Rogosa, and Sharpe (MRS) medium containing 15% glycerol at -80°C. The indicator bacteria used to determine bacteriostatic spectrum of bacteriocin were Lactobacillus plantarum, Lactobacillus cerevisiae, Lactobacillus campylobacter, Streptomyces enteri, Saprophytic staphylococci, Lactococcus lactis, Listeria monocytogenes, Staphylococcus aureus, Salmonella typhimurium, Salmonella Potsdam, Escherichia coli, provided by the key Laboratory of Zoonosis in Jiangsu Province. S. aureus was chosen as the indicator strain for antimicrobial assays. The standard strain was Lactococcus lactis (Cicc 23609) from China Industrial Culture Collection Center.

Screening of bacteriocin-producing LAB
The activated LAB at a dose of 1% (v/v) isolated from Jinhua ham were inoculated into sterile MRS medium at 30°C. The bacteria were cultured for 24 h and then precipitated by centrifugation at 8,000 g for 30 min at 4°C. The supernatant was ltered through a sterile 0.22 μm lter to obtain the cell-free fermentation solution. The bacteriostatic effect of the supernatant was tested by double-layer agar plate method and turbidity method [13] using E. coli and S. aureus as indicators. The strains with bacteriostatic effect were re-screened by successively eliminating the effect of organic acid, hydrogen peroxide and suffering the protease hydrolysis test [14,15].
Brie y, 0.1 M sterile sodium hydroxide were used to adjust the fermentation supernatant to pH of 6.0 to exclude the inhibitory effect of acetic acid and lactic acid. Catalase dissolved in 50 mM phosphate buffer was added to the acellular cell-free supernatant to make the nal concentration to 5 mg/mL. The reaction of the mixture was performed at 37°C for 2 h to eliminate the effect of hydrogen peroxide. In the protease hydrolysis test, the nal concentration of trypsin, papain and protease K (Shanghai Lanji Technology Development Co., Ltd., Shanghai, China) in bacterial supernatant was 1 mg/mL and incubation was performed at 37°C for 2 h.
Identi cation of bacteriocin-producing lactic acid bacteria The morphological and biochemical properties of the bacteriocin-producing strain were identi ed according to the Bergey's Manual of Determinative Bacteriology [16]. For assaying the 16S rDNA gene sequencing, the target strain was activated and then 1.5 mL culture medium was centrifuged at 10,000 g for 2 min at 4°C. The pellet was taken and the gene of the obtained cells were extracted according to the instructions of the bacterial genomic DNA extraction kit (Tiangen Biotech, DP302-02, Beijing, China). PCR ampli cation was carried out and procedures were as follows: denaturation at 94°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 1 min, 35 cycles, and extension at 72°C for 7 min. Then, the PCR product was sent to Shanghai Sheng gong Bioengineering Technology Service Co., Ltd.

Determination of bacteriocin activity
The antimicrobial activity of the bacteriocin was determined by using the agar-well diffusion method [17]. The wells were added with 100 µL of cell-free supernatant and active fractions during the puri cation process which was subjected to a serial two-fold dilutions. The activity of bacteriocin was de ned as the reciprocal of the highest dilution [18] with a clear zone of inhibition in the indicator lawn and expressed as arbitrary unit per milliliter (AU/mL). Protein concentration was measured by using BCA protein kit (Thermo Scienti c, 23227, USA). The speci c activity of bacteriocin (AU/mg) was calculated by the ratio of the bacteriocin activity (AU/mL) over the protein concentration (mg/mL).

Extraction of crude bacteriocin
The crude bacteriocin was extracted by pH adsorption method according to Daba et al [19] with a minor modi cation. The activated strain of 1% (v/v) was inoculated into MRS medium and cultured for 24 h at 30°C.
The fermentation broth was then incubated at 70°C for 30 min. After the fermentation broth was cooled to room temperature, the pH of the broth was adjusted to 6.0. The bacteriocins were allowed to adsorb onto the cells by gentle stirring for 12 h and then the bacterial cells were collected by centrifugation at 8,000 g for 30 min. The bacterial cells were washed twice with 50 mM sodium phosphate buffer (PBS, pH 6.0). Desorption of bacteriocin from the cell was carried out by adjusting the pH to 2.0 with magnetic stirring in 100 mM NaCl. The supernatant was ltered with 0.22 μm lter and dialyzed with 50 mM PBS (pH 6.0). The crude bacteriocin supernatant was freeze-dried and stored at -80 °C for further puri cation.

Puri cation of bacteriocin
The bacteriocin was puri ed by the cellulose DEAE-52 ion exchange chromatography and Sephadex G-50 chromatography. Brie y, The 5 mL of crude bacteriocin dissolved in 50 mM PBS (pH 7.4) at concentration of 1 mg/ml was loaded onto a 100 mL DEAE-52 ion exchange chromatography column. The bacteriocin was stepwise eluted with PBS buffer (pH=7.4) containing 0, 0.2, 0.4 and 0.6 M NaCl. The e uent was collected with 5 mL per tube, setting the ow rate as 1 mL/min. The eluted peaks were collected and detected at 280 nm. The fractions with antibacterial activity was mixed and dialyzed with PBS buffer for 48 h and then freeze-dried. The bacteriocin was then solubilized in 20 mM PBS (pH 7.4) and loaded onto the Sephadex G-50 column. The active peak with bacteriostatic activity was combined and freeze-dried for further analysis.
The gel samples were prepared by protein solution with equal volume of 2×Tricine loading buffer (30% (v/v) glycerol, 8% (w/v) SDS, 3.1% (w/v) DTT, 0.02% (w/v) Coomassie Brilliant Blue G-250, and 250 mM Tris-HCl, pH 6.8). Samples were boiled at 95°C for 5 min. Equal protein amount (10 µg) of each sample was loaded onto a gel system containing 18% polyacrylamide separating gel and 4% polyacrylamide stacking gel. The electrophoresis was run at a constant voltage of 90 V for 120 min. The gel was stained and destained by Silver staining kit (Sangon Biotech, C500029, Shanghai, China) and the gel was imaged by Gel DOC XR gel imaging system (ChemiDoc XRS+, Bio-Rad Laboratories, Inc., USA).
The molecular mass and sequence of the puri ed bacteriocin was determined by Matrix-assisted laser desorption ionization-time of ight mass spectrometry (MALDI-TOF-MS). The puri ed bacteriocin was mixed with equal volume of the matrix solution containing a-cyano-4-hydroxy cinnamic acid dissolved in 0.1% TFA and 50% acetonitrile. Then 1 μL of the solution was dried on the sample target plate for the MALDI analysis (Test Center of Yangzhou University, Jiangsu, China).

Inhibitory spectrum of puri ed bacteriocin
The inhibitory spectrum of the puri ed bacteriocin was assayed by using the method of [13]. The indicator bacteria were Lactobacillus plantarum, Lactobacillus cerevisiae, Lactobacillus campylobacter, Streptomyces enteri, Saprophytic staphylococci, Lactococcus lactis, Listeria monocytogenes, Staphylococcus aureus, Salmonella typhimurium, Salmonella Potsdam, Escherichia coli. Nisin (Yuanye Shanghai China) was considered as a control, using the Oxford Cup method to determine the antibacterial activity. The diameter of the inhibition zone was recorded to obtain inhibition spectrum of bacteriocin.
Physico-chemical properties of the puri ed bacteriocin The effects of protease, pH, and temperature on activity of bacteriocin and nisin were detected. The proteases including proteinase K, pepsin and trypsin at 1 mg/mL was incubated with equal amount of bacteriocin and nisin for 2 h at 37°C. The pH of the puri ed bacteriocin and nisin solution was adjusted to 2-10, respectively and incubated at 37°C water bath for 30 min. The effect of temperatures at 45, 70, 90, 100, and 110°C on bacteriocin activity was evaluated by incubation at respective water bath for 30 min. The antibacterial activity of bacteriocin against S. aureus among different treatments was determined using agar well diffusion method and the diameters of inhibition zone were recorded [20].

Statistical analysis
Each assay was performed in triplicate. The data are presented as means ± standard deviation. Signi cant differences between groups were evaluated by analysis of variance (FACTORIAL ANOVA) using the software package SAS (Version 9.13) and the plot was made by Sigmaplot 10.0 (Sigmaplot Lab Corporation; USA). P<0.05 was considered as statistically signi cant.

Results And Discussion
Screening of bacteriocin-producing LAB The 159 strains of LAB in 162 strains isolated from Jinhua ham were observed to inhibit the growth of S. aureus and/or E. coli (Supplementary Material, Table S1). This was consistent with the view that most of the LAB reported by Birri et al [21] can produce antibacterial substances to inhibit the growth of other microorganisms. Besides bacteriocins, it was known that the metabolites produced by LAB including organic acids and hydrogen peroxide also exhibited anti-bacterial activity. Thus, it is necessary to exclude the effect of organic acids and hydrogen peroxide on inhibition of bacterial growth. As shown in Table 1, after the elimination of the organic acid, there was 8 strains showed inhibitory activity on indicator bacteria, among which strain L1, L3 and L7 only inhibited S. aureus. Strain L2, L4, L5, L6 and L8 had inhibitory effects on both indicator strains. The inhibition zone diameters among the detected strains were signi cantly different (P<0.05).
In order to screen the bacteriocin-producing LAB that can both inhibit the growth of representative Gramnegative and Gram-positive microbes, strains of L2, L4, L5, L6 and L8 were selected to perform the hydrogen peroxide exclusion test. Catalase was supposed to catalyze the hydrogen peroxide to eliminate its effect. After the elimination of hydrogen peroxide, the antibacterial effect of strain L8 disappeared, suggesting the bacteriostatic substance produced by strain L8 was presumed to be the hydrogen peroxide. The strain L2 still had antibacterial activity against S. aureus, and the diameter of the inhibition zone was 10.02±0.53 mm, but showed no antibacterial ability on E. coli. Strains L4, L5 and L6 had inhibitory effects on both two indicator bacteria. The bacteriostatic diameter of cell-free supernatant in strain L5 against E. coli was 11.36±0.54 mm, which was signi cantly larger than the inhibition zone diameter of strains of L4 and L6 (P<0.05). The results indicated that in addition to organic acids and hydrogen peroxide, the bacteriostatic component in fermentation supernatant of strain L4, L5 and L6 was possibly to be bacteriocin. Generally, the bacteriocin was small molecular protein and polypeptide, thus the protease detection test was done to evidence the supernatant of strains L4, L5 and L6 with anti-bacterial activity contained proteinaceous substances (Supplementary Material, Table S2). The antibacterial activity of L4 and L6 still has a weak antibacterial activity after trypsin and proteinase K digestion when E. coli was used as indicator strain. Notably, the antibacterial activity of the supernatant of strain L5 showed no inhibitory effect on both indicator strains after the enzymes digestion.
Therefore, strain L5 is selected for further bacteriocin puri cation and characterization.

Identi cation of Pediococcus pentosaceus
The target strain L5 was primarily identi ed by traditional method of colony morphology and Gram staining.
The strain L5 showed tetrad and bigeminal forming cocci with a smooth, circular, convex and ivory-white in color of colony ( Fig. 1A and Fig 1B). The Gram staining of strain L5 was purple, indicating L5 as a Grampositive bacteria. In the sugar fermentation test, strain L5 can utilize amygdalin, L-arabinose, D-cellobiose, Dfructose, D-galactose, glucose, sucrose, D-maltose, D-mannose, and melibiose while it cannot be able to metabolize lactose, inulin, D-trehalose, melezitose and D-mannitol, ra nose, D-ribose, D-xylose, D-Sorbitol and L-rhamnose (Supplementary Material, Table S3). Based on the above results, it can be primarily speculated that the assignment of the strain L5 is Pediococcus pentosaceus. The 16S rDNA was sequenced and identi ed for further demonstration of the target strain. A PCR ampli cation product of approximately 1200 bp was obtained, and a single band were shown to be bright in the agarose gel (Fig. 1C). A comparison based on the alignment results from GenBank using the BLAST software on the NCBI website prove a 98% homology between the target strain and Pediococcus pentosaceus M58834.1. Accordingly, phylogenetic tree was drawn based on the sequence alignment (Fig. 1D). This was consistent with the physiological and biochemical results, con rming that the target strain L5 was a Pediococcus pentosaceus.

Recently, bacteriocins produced by Pediococcus pentosaceus have been isolated from other resources. Carolina
Gutiérrez-Cortés et al. [22] obtained some Pediococcus pentosaceus including P. pentosaceus 63, 145, 146 and 147 from minas cheese, showing bacteriocin-producing ability. Bacteriocinogenic strains classi ed as P. pentosaceus with anti-Listeria activity were identi ed from Brazilian artisanal cheese [20], intestine of Mimachlamys nobilis [23], ermented Appam batter [24]. According to Ramanjeet Kaur et al [25], Pediococcus pentosaceus LB44 could grow and produce bacteriocin under acidic and alkaline conditions. The strain L5 in current study which was designated as P. pentosaceus Z-1 isolated from Jinhua ham expand the bacteriocin production scope from food resources of fermented meat product.

Extraction, puri cation and identi cation of bacteriocin
The bacteriocin in cell-free supernatant produced by P. pentosaceus Z-1 was stepwise puri ed by cell adsorption, DEAE-52 exchange chromatography and Sephadex G50 gel ltration (Fig. 2). There were four protein peaks after gradient salt elution from DEAE-52 exchange chromatography. The inhibition zone of fraction c against S. aureus was seen to be obviously larger than fraction a while fraction b and c showed no inhibitory zone on S. aureus (Fig. 2A). The fraction c were pooled and processed to Sephadex G50 gel ltration. As shown in Fig. 2B, two protein peaks were observed and fraction b with a big inhibition zone were preferred to be the puri ed bacteriocin. The anti-bacterial activity of the active fraction during the puri cation process was summarized in Table 2. The bacteriocin extracted from cell-free supernatant by cell adsorption method had led to an improvement of the speci c activity by 6.59 fold with a 61.93% recovery. The puri ed bacteriocin from the active fraction of Sephadex G-50 column showed the speci c activity as high as 8712.05 AU/mg, resulting in 40.10 fold increase of speci c activity compared to the cell-free supernatant. In addition, the yield of the total activity was gradually decreased during the puri cation process as the speci c activity increased. This was consistent with the basic principle of puri cation procedures and similar results were also reported, such as the puri cation of plantaricin ZJ008 and plantaricin K25 [26,27]. It is suggested that the bacteriocin in cell-free supernatant in present study is effectively puri ed.
The purity of the bacteriocin was originally identi ed by Tricine-SDS-PAGE. Abundant protein bands were present in cell-free supernatant with a broad protein molecular range of 1.2-27 kDa (Fig. 2C). As the processing of puri cation, miscellaneous proteins were excluded and a single protein band at the range of 4.6-10 kDa was emerged in the last active fraction, indicating that the bacteriocin was highly puri ed. The bacteriocin in the current study was named as pediocin Z-1. The molecular weight of pediocin Z-1 was further detected as 8227.35 Da by MALDI-TOF-MS (Fig. 2D). The purity of the pediocin Z-1 was 96.62% based on the peak area ratio calculation in the total ion chromatogram. As shown in Fig. 2E, the amino acid sequence of pediocin Z-1 was predicted to be MAITLKTELL DQKMTEVFDC SNDQTPLRDA MCNHVMDDNG HDTMKTIAEA KKWENMNDAE by the MASCOT searching. The sequence of Pediocin Z-1 showed no homology with other known bacteriocins using protein BLAST against the GenBank database (https://blast.ncbi.nlm.nih.gov/Blast) and antimicrobial peptide database (http://aps.un mc.edu/AP/main.php). Thus, pediocin Z-1 was considered as a novel bacteriocin produced by P. pentosaceus.
The pediocin Z-1 was preliminarily categorized into class II bacteriocin according to its molecular weight [28], but possessing no conserved motif (YGMGVxC). Similarly, plantaricin GZ1-27 reported by Du et al. [29] belonged to the class II of bacteriocins. However, there was also no the characteristic sequence of Class IIa bacteriocins. Among the 60 amino acid residues of pediocin Z-1, it was composed of 35.8% hydrophobic residues (Ala, Ile, Leu, Met, Phe, Pro, Val) and 64.2% polar residues with 20.0% (Glu, Asp) acidic and 15.7% basic (Arg, Lys, His) amino acids, indicating the pediocin Z-1 with a amphiphilic structure of class II peptide. The Nterminus of the pediocin Z-1 was consisted of the hydrophobic residues and the C-terminus contained a high percentage of hydropholic residues. The structure of the bacteriocin was regularly linked with the mode of action and basic anti-bacterial principle of the bacteriocin [30]. The most-elucidated mechanism of bacteriostatic effect was pore formation, referring to that membrane channel of cells was destroyed by binding of the bacteriocin peptides with membrane components, such as phosphatidylglycerol. Likewise, the pediocin Z-1 is putative to interrupt the integrity membrane of sensitive microorganism by using its amphiphilic structure to cause cell wall depolarization while additional investigations are needed to elucidate the underlying mechanism.

Antibacterial spectrum
The antibacterial spectrum of Pediocin Z-1 was shown in Table 3. Result showed that besides Gram-positive bacterias, the growth of Gram-negative bacteria including S. typhimurium, S. Potsdam and E. coli were suppressed by pediocin Z-1, indicating that pediocin Z-1 was supposed to exhibit a broad antibacterial activity.
However, nisin can only inhibit the Gram-positive bacteria which was agreement with earlier reports [31]. Moreover, the inhibition zone of Lactobacillus, Streptomyces enteri and Lactococcus lactis by nisin was slightly higher than that of pediocin Z-1 (P<0.05). In the test of S. staphylococci and L. monocytogenes, there was no signi cant difference between pediocin Z-1 and nisin (P>0.05). Notably, pediocin Z-1 showed a stronger antibacterial activity than nisin when using S. aureus as an indicator strain (P<0.05). It is indicated that pediocin Z-1 and nisin can both achieve the inhibitory effect of the growth of Gram-positive bacteria, however, the bacteriostaticability was dependent on the species of the detected bacteria.

Physicochemical properties of Pediocin Z-1
The effect of temperature on Pediocin Z-1 activity was presented in Fig. 3A. When heat treatments of bacteriocin gradually increased from 50℃ to 110℃, the antibacterial activity of both nisin and pediocin Z-1 signi cantly decreased (P<0.05). The antibacterial activity of nisin was signi cantly higher than that of pediocin Z-1 at room temperature (shown in control treatment) and the range of 50-80℃ (P<0.05, Fig. 3A).
There was no signi cant difference in antibacterial activity between the two groups at 90℃. Nevertheless, pediocin Z-1 showed a higher diameter of bacteriostatic zone than nisin when the treatment temperature was 100℃. Moreover, as the temperature processed to 110℃, the inhibition zone of pediocin Z-1 group was 11.52±0.36 mm while no bacteriostatic of nisin was observed. It is suggested that pediocin Z-1 had a more heat stability than nisin, as it was a typical characteristic of class IIa bacteriocin.
As it was shown in Fig. 3B, the pediocin Z-1 presented the antibacterial activity between pH 2-10, having a wider pH tolerance range than that of nisin. The antibacterial activity of pediocin Z-1 was signi cantly lower than that of nisin between pH 2.0 and 4.3 (P<0.05). The bacteriostatic ability of nisin decreased between pH 4.3 and 7.0 while the antibacterial activity of pediocin Z-1 was signi cantly increased at that range. The antibacterial activity of pediocin Z-1 was strongest at pH 6.0 and the diameter of bacteriostatic zone was 24.74±0.33 mm.
Compared to the report of Zhu et al. [26], a bacteriocin of Plantaricin ZJ008 produced by Lactobacillus Platarum ZJ008 isolated from fresh milk exhibited narrow pH stability at the range of 4.0-5.0. The antimicrobial activity of two bacteriocins isolated from Portuguese fermented sausage decreased obviously when pH values below 5.0 and above 7.0 [32]. Thus, the results in current study enabled pediocin Z-1 possessing antibacterial activity have a wide pH range when applied in food preservation.
Both nisin and pediocin Z-1 can be degraded by pepsin, proteinase K, trypsin and papain (Table 4). This was consistent with the view that the class IIa bacteriocin was sensitive to proteases and was not to α-amylase [33].
Similar effect was observed for acidocin LCHV, its anti-bacterial activity was lost after the treatment of proteolytic enzymes [34]. Nisin and pediocin Z-1 are not sensitive to lipase amylase and amylase, demonstrating that the essence of pediocin Z-1 was a protein, not a glycoprotein. It was indicated that the bacteriocin can be degraded by proteases in the human digestive system and possessed no adverse effects to the human health. Above all, the physicochemical properties of pediocin Z-1 was preliminarily determined, suggesting that the discovered bacteriocin in the current study, pediocin Z-1, can be applied as a potent food additive.

Conclusions
The primary outcome in this paper was to screen a bacteriocin-producing lactic acid bacteria from Jinhua ham. Pediococcus pentosaceus Z-1 was identi ed and evidenced to produce bacteriocin by eliminating the effect of hydrogen peroxide and organic acid and the protease test. Pediocin Z-1 was stepwise puri ed by the pH adsorption, cellulose DEAE-52 ion exchange and Sephadex G-50 chromatography columns. A purity of 96.62% of pediocin Z-1 was obtained from and determined as molucular weight of 8227.35 Da by MALDI-TOF-MS.
Pediocin Z-1 showed an inhibitory effect on Gram-positive and Gram-negative bacteria and was stable at acidic and alkaline pHs as well as at high temperature. Our results suggest that pediocin-Z-1 had a great potential in applying as a novel food preservative.   Note: "-" indicated no bacteriostatic activity referring to the diameter zone was not signi cantly different with the blank well; "*" indicated there were signi cant differences in inhibition zone between pediocin Z-1 and nisin groups (P<0.05). "NS" showed that there were no signi cant differences between two bacteriocin groups (P>0.05). Table 4 The inhibition zone diameter of pediocin Z-1 and nisin by enzymatic digestion.  Puri cation and identi cation of bacteriocin. Puri cation of bacteriocin was performed using cellulose DEAE-52 exchange chromatography (A) and Sephadex G50 gel chromatography (B) and active fraction of each column was identi ed, respectively. Tricine-SDS-PAGE gel showed the initial purity of bacteriocin during the puri cation process (C, 1 referred to cell-free fermentation supernatant, 2 for crude protein extracted by cell adsorption, 3 for active faction c from DEAE-52 column, and 4 for active faction b from Sephadex G50 column). Matrix-assisted laser desorption/ionization time-of-ight mass spectrometry was used to identify the puri ed bacteriocin (D) and the fragment ions map of m/z indicated the molecular weight of the puri ed bacteriocin was 8.23 kDa (E).