Characterization and Antibacterial Mode of a Novel Bacteriocin with Seven Amino Acids from Lactobacillus plantarum in Guizhou Salted Radish

Traditional Chinese fermented vegetables are excellent probiotic food with probiotic lactic acid bacteria that are benefical to the health. A novel bacteriocin with molecular weight, 825 Da was found successfully from Lactobacillus plantarum 163, which was isolated from Guizhou salted radish. The complete amino acid sequence was speculated as YVCASPW based on the mass spectrometry, and was named as bacteriocin 163-1. The bacteriocin 163-1 was highly thermostable and stability over a broad pH range (pH 3-6), sensitive to protease K and pepsin, and exhibited a wide range of antimicrobial activity not only against lactic acid bacteria (LAB) but also against other foodborne pathogens including Gram-positive and Gram-negative bacteria. Bacteriocin 163-1 could disrupt the cell membrane of bacteria. The observations of the transmission electron microscopy and laser confocal microscopy on the cell membrane of Escherichia coli and Staphylococcus aureus showed that bacteriocin 163-1 could result in forming pores on the cell membrane and then cytolysis of the bacteria. The new bacteriocin with broad-spectrum antibacterial activity will be useful in preservation of vegetable, fruit and food as well agricultural bio-controlling.


Introduction
Traditional Chinese fermented vegetables are excellent probiotic food with probiotic lactic acid bacteria (LAB), which has various health benefits including anticonstipation, anticancer, antioxidative and immune-boosting.The functional ingredients from fermentation by LAB such as bacteriocins could be used as bio-preservative.LAB strains are generally recognized as safe (GRAS) microorganisms (Burdock & Carabin, 2004), and bacteriocins have also achieved GRAS status.Some bacteriocins with remarkable thermostability and pH stability show a significant inhibitory activity against spoilage and pathogenic bacteria, indicating that bacteriocin could be potentially used as an effective bio-preservative in the food industry.Some bacteriocins are already commercially available, such as nisin (produced by Lactococcus lactis), used to keep and extend the shelf life of food products in many countries in the world.However, nisin has some drawbacks when applied in the food industry.For instance, nisin does not perform the good antibacterial activity against Gram-negative bacteria, and it is only effective under an acidic environment.These limits are not positive for nisin to be applied in process and conservation for foods.Thus, an alternative novel bacteriocin with good antibacterial activities is essential for the food industry.Recently, the researcher is focusing on the mining of novel bacteriocin from LAB for such as Lb.plantarum from salted vegetable, yoghurt, fermented meat (Biscola et al., 2013;Ahmad et al., 2014;Alvarez-Sieiro et al., 2016).
Generally, most bacteriocins generated from LAB appear to share a common mode of action which form a pore in the sensitive bacterial membrane, and therefore lead to sensitive cell death (Castellano et al., 2003;Oppegård et al., 2007).In addition, other type of bacteriocins such as lantibiotic has exhibited different mode of action by binding themselves to lipid II, and therefore lead to cell death through false cell wall synthesis (Cotter et al., 2005).Furthermore, some of bacteriocins such as Pep 5 cause lysis of treated cells, which is another mode of action (Bierbaum & Sahl, 1987).The effective use of bacteriocins in food preservation requires a better understanding of their mode of action and their inhibitory action under different biochemical conditions naturally occurring in food.
In this study, the purification, identification, antimicrobial spectrum, biochemical and genetic characteristics of new bacteriocin from Lb. plantarum 163 in Guizhou salted radish were comprehensively investigated, and the action mode of the bacteriocin against to S. aureus and E. coli was discussed.

Strains, Media and Growth Conditions
Lb. plantarum 163 was screened from Chinese fermented salted radish (Hu et al., 2013) and was stored in China General Microbiological Culture Collection Center (No. 8224).The lactic acid bacteria and fungi used in this study and their respective growth conditions are listed in Table 1.

Purification of Bacteriocin 163-1
Lb. plantarum 163 was activated in MRS broth at 37 °C for 14 h without agitation.10 mL pre-culture was inoculated into 1,000 mL MRS broth, and incubated at 37 °C for 24 h without agitation.The bacterial cells then were harvested by centrifugation (6,000 g, 15 min) at 4 °C, and the cell-free supernatant collected was filtrated by 0.45 μm membrane.Then, the cell-free supernatant was separated by preparative chromatography (Waters, 600) with Waters SunFire OBD-C18 columns (19×150 mm) in a 40 min isocratic elution of 95% water-acetonitrile (5%) and containing 0.1% trifluoroacetic acid (TFA).The elution was monitored continuously at 267 nm and all individual peaks were collected.Then, all individual peaks were further purified by reversed-phase high-performance liquid chromatography (RP-HPLC, UltiMate 3000) using the following conditions: Agilent Eclipse XDB-C18 columns, (4.6 × 250 mm), followed by an isocratic elution using 95% water-acetonitrile (5%) and containing 0.1% TFA for 40 min.Each individual peak was collected and further used for antimicrobial test using Bacillus pumilus CMCC 63202 as indicator.

Antimicrobial Spectrum of Bacteriocin 163-1
The bacteriocin 163-1 from preparative chromatography were used to evaluate the antimicrobial activity against indicator organisms (seven of positive bacteria, three of gram-negative bacteria and three of fungi).Antibacterial activity of bacteriocin 163-1 was measured using agar well diffusion assay method (Ruixiang et al., 2015).

Effects of Enzyme, Temperature and pH on the Activity of Bacteriocin 163-1
The effect of Protease K (30 U/mg), Trypsin (2.5 KU/mg), α-Chymotrypsin (1 KU/mg) and Pepsin (3 KU/mg) on the activity of bacteriocin 163-1 was determined.The purified bacteriocin 163-1 from preparative chromatography was incubated at the optimum pH at 37 °C for 3 h) with the enzymes of the final concentration of 5 mg/mL.The influence of temperature on the activity of bacteriocin 163-1 was examined by treating at 60 °C, 80 °C and 100 °C for 10, 20 and 30 min, respectively in a thermostatic water bath and at 121 °C for 20 min in an autoclave.Lastly, the pH value of the purified bacteriocin 163-1 was adjusted to 2-10 and then kept at 37 °C for 3 h in a thermostatic water bath.The residual antimicrobial activities were measured after enzymatic, temperature and pH treatments using agar diffusion assay method using Bacillus pumilus CMCC 63202 as the indicator (Gao et al., 2010;Zhang et al., 2013).

Effect of Bacteriocin 163-1 on the Sensitive Cell Growth and Time-Kill Kinetics
The activity unit of bacteriocin 163-1 was defined as the reciprocal of the highest dilution with antimicrobial activity and was expressed in activity units (AU) per milliliter (Barefoot & Klaenhammer, 1983;Pucci et al. 1988).
In order to determine the effect of bacteriocin 163-1 on the sensitive cell growth, S. aureus (ATCC 25923) or E. coli (ATCC 25922) were activated in LB medium, then bacteriocin 163-1 were added at a concentration of 6.4 Au/mL and 12.8 Au/mL.The sterile distilled water treatment was used as a control while the sterile LB medium was used as a baseline.The OD 600 of all treatments were measured at 2, 3, 4, 5, 6, 7, 8, 10, 12 and 16 h.In order to determine the Time-kill kinetics, S. aureus (ATCC 25923) or E. coli (ATCC 25922) was activated in LB medium and incubated at 37 °C for 6 hours with agitation.Then bacteriocin 163-1 was added at a concentration of 12.8 AU/mL and the bacteria were counted at 0.5, 1, 1.5, 2, 2.5 and 3 h.

Effect of Bacteriocin 163-1 on Release of Proteins and Nuclear Acid in Cells
To determine the effect of bacteriocin 163-1 on release of proteins and nuclear acid in bacteria cells, S. aureus (ATCC 25923) or E. coli (ATCC 25922) were activated in LB medium, and incubated at 37 °C for 6 hours with agitation, then bacteriocin 163-1 were added at a concentration of 6.4 AU/mL.The bacterial cells then were harvested by centrifugation (6,000 g, 15 min) at 4 °C.The OD 280 and OD 260 of the cell-free supernatants were measured by 0.5, 1, 1.5, 2, 2.5 and 3 h.The sterile distilled water was used as a control while the sterile LB medium was used as a baseline.
S. aureus (ATCC 25923) or E. coli (ATCC 25922) were activated and incubated at 37 °C for 5 hours with agitation, then bacteriocin 163-1 were added at a concentration of 6.4 Au/mL for the determination of the lactic dehydrogenase (LDH) release.After bacterial cells were harvested by centrifugation at 6000 g for 15 min at 4 °C, the LDH contents of the cell-free supernatants were measured at 0.5, 1, 1.5, 2, 2.5 and 3 h using LDH kit (Nanjing Jiancheng Bioengineering Institute, China).The sterile distilled water treatment was used as a control while the sterile LB medium was used as a baseline.

Transmission Electron Microscopy (TEM) Observations on the Sensitive Cell Membrane Treated with
Bacteriocin 163-1 S. aureus (ATCC 25923) or E. coli (ATCC 25922) were activated in LB medium, and incubated at 37 °C for 8 hours (E. coli 4 h) with agitation, then bacteriocin 163-1 were added at a concentration of 6.4 AU/mL, incubated at 37 °C for 2 hours, bacterial cells were harvested by centrifugation at 6,000 g for 15 min at 4 °C.The bacterial cells were washed by 2.5% Gluteraldehyde twice, finally bacterial cells were fixed by 2.5% Gluteraldehyde and the cell membrane observation was done by using Transmission Electron Microscopy (HITACHI, H-600, Japan).The sterile distilled water treatment was used as a control.

Bacteriocin 163-1 Localization on Celluar
S. aureus (ATCC 25923) or E. coli (ATCC 25922) were activated in LB medium, and incubated at 37 °C for 8 hours (E. coli 6 h) with agitation, then fluorescein isothiocyanate (FITC)-Bacteriocin 163-1 were added at a concentration of 1 mg/L, and incubated at 37 °C for 0.5 hours with agitation (in dark), bacterial cells were harvested by centrifugation at 6,000 g for 15 min at 4 °C, then the bacterial cells were washed by 2.5% Gluteraldehyde twice.Then, the bacterial cells were suspended using 2.5% Gluteraldehyde (in dark) and finally Bacteriocin is an active peptide produced by bacteria.From previous studies, many bacteriocins produced by Lb. plantarum have been found and identified, such as plantaricin Y produced by Lb. plantarum 510 (Chen et al., 2014), plantaricin LD1 Produced by Lb. plantarum LD1 (Gupta & Tiwari, 2014).Some of Lb. plantarum could produce more than one type of plantaricin, for instance Lb. plantarum C11 could produce plantaricin A, plantaricin F, plantaricin E (Hauge et al., 1999).In our previous work, Lb. plantarum 163 could produce plantaricin 163 which consist of 32 amino acid with the molcecular weight of 3553 Da (Hu et al., 2013).

Antimicrobial Spectrum of Bacteriocin 163-1
The antimicrobial activities of bacteriocin 163-1 was shown in Table 1.The bacteriocin 163-1 significantly inhibited the growth of Gram-positive bacteria (S. aureus, Listeria monocytogenes, Bacillus pumilus, Bacillus cereus, Micrococcus luteus, Lactobacillus thermophilus, Lactobacillus rhamnosus) and the Gram-negative bacteria (E.coli, Pseudomonas aeruginosa, and Pseudomonas fluorescens), but had no antimicrobial activity against fungi such as Penicillium notatum, Aspergillus niger, and Rhizopus nigricans, indicated that bacteriocin 163-1 had a broad antimicrobial spectrum.As shown in previous literature, most of bacteriocin, such as plantaricin F, plantaricin E, plantaricin J could only showed antibacterial activity against homologous species (Anderssen et al., 1998), whereas only a few plantaricin appeared to be a broad spectrum of antimicrobial activity.A bacteriocin with broad antibacterial activity may be more useful and valuable in agro-food industry.

Effects of Enzyme, Temperature and pH on the Activity Bacteriocin 163-1
Effects of enzyme, temperature and pH on the activity bacteriocin 163-1 were investigated and the results were shown in Table 2.The antimicrobial activity of bacteriocin 163-1 was found to be fully lost after enzymatic treatments with protease K, pepsin, and partially lost after enzymatic treatments with trypsin, α-Chymotrypsin, thus highlighting the typical property of a peptide.
Further, the stability of bacteriocin 163-1 was tested at 60 °C, 80 °C, 100 °C and 121 °C and under a different pH (2-10).Surprisingly, it was observed that the activity recovery of bacteriocin 163-1was more than 90% by treatments of 60-121 °C in pH 4, appeared to be good thermostability.On other hand, its recovery was more than 70% when tested at pH 3-10, of which showed remarkable pH stability in pH 3-8.The result was consistent with previous findings about bacteriocin peptides such as plantaricin MG (Gong et al., 2010), plantaricin 163 (Hu et al., 2013).The good physicochemical properties of bacteriocin 163-1 could offer an essential promise for its application in the processing and preservation of foods as well bio-controlling of plant diseases.

Effect of Bacteriocin 163-1 on Cell Growth and Time-Kill Kinetics S. aureus and E. coli
The effect of bacteriocin 163-1 on cells growth and Time-kill kinetics of S. aureus and E. coli was shown in Figure 2. The growths of S. aureus and E. coli were partly inhibited when they were treated with bacteriocin 163-1 at a concentration of 6.4 AU/mL (Figures 2a and 2b).However, S. aureus growth was fully inhibited, but E. coli growth was done before 8 h of culture when bacteriocin 163-1 was added at a concentration of 12.8 AU/mL (Figures 2a and 2b), suggested S. aureus was more sensitive to bacteriocin 163-1 than E. coli and it needed higher concentration of the bacteriocin to fully inhibit growth of E. coli.The growth of S. aureus was highly inhibited after 1 h with the treatment of 12.8 AU/mL (Figure 2c).The growth of E. coli was significantly reduced from 0.5 h to 3 h, and a 5-log reduction was observed after 3 h when treated with bacteriocin 163-1 at a concentration of 12.8 AU/mL (Figure 2d).ectron Micros Figure 4a), wh and the holes istorted.In the cavity cells du he results of T the bacteria.T n cell death.shown in Fig Figures 5a and  nent Figure 1 MALDI-[4*C Figure 2 growth by Figure 3. a the cell-fr LDH o Bacterioci Figure 4 slitting, ro

Table 1 .
Antimicrobial activity profile of bacteriocin 163-1 produced from the isolated Lactobacillus plantarum 163