Bacteriocin-producing strains were inoculated into MRS broth and cultured at 37°C for 24 h. After incubation, the colony color and morphology of the strain were observed. A light microscope (CX31, 10 × 100/Oil, Olympus, Tokyo, Japan) was used to observe the colony morphology characteristics of the strain.

Genomic DNA was extracted using bacterial genomic DNA extraction kit (Solarbio, Beijing Solarbio Technology Co., Ltd., Beijing, China). 16S rDNA gene sequence was amplified using the forward primer 5′-AGAGTTTGATCCTGGCTCAG-3′ and the reverse primer 5′-CTACGGCTACCTTGTTACGA-3′. The PCR amplification products were recovered by AxyPrep DNA Gel Recovery Kit (Solarbio, Beijing Solarbio Technology Co., Ltd., Beijing, China) and sequenced by Shanghai Paisano Co., Ltd. (Shanghai, China). 16S rRNA gene sequences were submitted to the NCBI database for homology analysis using the BLAST tool. A phylogenetic tree was constructed by MEGA6 software.

The CFS was concentrated twice using a rotary evaporator at 55°C, mixed with ethyl acetate in a ratio of 1:2, stirred at 100 rpm at 25°C for 2 h, and placed overnight for stratification. The upper organic phase was pooled and evaporated under a vacuum at 55°C using a rotary evaporator (Yarong, SY-5000, Shanghai, China) to obtain the crude bacteriocin extract (18). Samples were stored at 4°C for further analysis.

In all, 2 ml of crude bacteriocin extract was initially filtered with 0.22 μm filter membrane, then samples were loaded in Sephadex G-25 gel chromatography column (Henghuibio, 1.5 cm × 80 cm, Beijing, China) to eliminate impurities, The samples were eluted with sterile water (pH5.5) with a linear gradient at a flow rate of 0.5 ml/min and measured by UV detector at 280 nm. The fractions were collected according to an automatic collection device (19). Using S. aureus as an indicator strain, the antimicrobial activity of each fraction was detected by agar well diffusion method.

The fractions with antimicrobial effect were pooled, concentrated, dried, and dissolved in PBS (pH 5.5) as well as loaded onto the Sephadex LH-20 gel chromatography column (Henghuibio, 1.5 cm × 80 cm, Beijing, China) and eluted by 80% methanol at a flow rate of 0.25 ml/min with a UV detector at 280 nm. Each of fractions was collected, and antimicrobial activity was detected (17). The active fractions with antimicrobial activity were pooled for full-wavelength scanning, with the maximum absorption peak as the detection wavelength for the next purification.

Fractions with antimicrobial effect collected from the Sephadex LH-20 gel chromatography column (Henghuibio, 1.5 cm × 80 cm, Beijing, China) were filtered with 0.22-μm filter membrane and further purified by RP-HPLC (SCG100-V2, Suzhou Sepure Instruments Co., Ltd., Suzhou, China) (20). The purification conditions were as follows: C18 column (Phenomenex Jupiter C18, 4.6 mm × 250 mm, Phenomenex, USA), mobile phase A comprised 85% water and 15% acetonitrile (containing 0.07% trifluoroacetic acid), gradient: 100% A within 35 min, flow rate at 1 ml/min, the volume of samples loaded into the system was 500 μl. UV detector was set at 220 nm (21). A component of each peak was collected, concentrated by freezing, and then, dissolved in PBS (pH 5.5). Antimicrobial activity was detected by agar well diffusion method using S. aureus as an indicator strain.

Nano liquid chromatography coupled with tandem mass spectrometry analysis was performed to determine the molecular mass and amino acid sequence of the purified bacteriocin. The bacteriocin purified by RP-HPLC (SCG100-V2, Suzhou Sepure Instruments Co., Ltd., Suzhou, China) was reduced by 10 mM DL-dithiothreitol (Sigma-Aldrich, St. Louis, MO, USA) at 56°C for 1 h and alkylated by 50 mM iodoacetamide (Sigma-Aldrich, St. Louis, MO, USA) at room temperature in dark for 40 min. Lyophilize the extracted bacteriocin to near dryness. Resuspend peptides in 20 μl of 0.1% formic acid (Sigma-Aldrich, St. Louis, MO, USA) before LC-MS/MS analysis. The analysis of bacteriocin was conducted by nano LC-MS/MS in an Easy-nLC 1200 system (Thermo Fisher Scientific, Waltham, MA, USA) coupled with a Q Exactive™ Hybrid Quadrupole-Orbitrap™ Mass Spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) with an electrospray nanospray source. An in-house built reverse-phase nanocolumn (150 μm × 15 cm, 1.9 μm, 100 Å, Dr. Maisch GmbH, Germany) packed with Acclaim PepMap was used. Mobile phase A consisted of 0.1% formic acid in water, mobile phase B comprised 20% of 0.1% formic acid in water- 80% acetonitrile. Liquid chromatography linear gradient: from 4 to 8% B for 2 min, from 8 to 28% B for 43 min, from 28 to 40% B for 10 min, from 40 to 95% B for 1 min, and from 95 to 95% B for 10 min. Total flow rate is at 600 nL/min. A first-order mass spectrometry spectrum was obtained in a full-scan positive ion mode of m/z 300–1800. The second-order mass spectrometry data were collected using collision-induced dissociation to capture the b and y ions (22). The data acquisition and analysis were conducted by PEAKS Studio 8.5 software.

Lactic acid bacteria can produce organic acids and hydrogen peroxide, which can inhibit the growth of other strains (26, 27). Both of them have been reported to act on the cytoplasmic membrane by neutralizing its electrochemical potential and increasing its permeability, resulting in bacteriostasis and ultimately death of susceptible bacteria (28). In this study, isolating and screening bacteriocin-producing strains from 2,000 plant-derived strains by agar well diffusion method with E. coli and S. aureus as indicator strains were conducted. Results indicated that 25 strains were initially isolated after eliminating the contribution of organic acid (Table 1). Further elimination of the contribution of hydrogen peroxide, only strain W3-2 exhibited the strongest antimicrobial activity with the diameters of the inhibition zone against E. coli and S. aureus exceeded 16 mm (Figure 1). Therefore, strain W3-2 was selected for subsequent tests.

In order to confirm the antimicrobial active substance is bacteriocin, the CFS of strain W3-2 was treated with trypsin, pepsin, and proteinase K, respectively. As shown in Table 2, results displayed that the diameters of the inhibition zone against S. aureus decreased from 16.68 ± 0.04 to 10.25 ± 0.18 mm, 10.55 ± 0.24 mm, and 13.22 ± 0.15 mm after being treated with trypsin, pepsin, and proteinase K, respectively. These results indicated that bacteriocin was the main antimicrobial substance in CFS of W3-2. Bacteriocin can damage the structure of cell membrane, making the cell membrane form pores, leading to cell rupture and the outflow of small molecules, such as potassium ions, magnesium ions, phosphorus ions, amino acids, and ATP (29). The low level of ATP and ion deficiency in cells result in the inhibition of the synthesis of DNA, RNA, proteins, and polysaccharides, ultimately leading to cell death (30). Bacteriocin BM1122 led to the separation of the plasma wall of S. aureus and the formation of pores in E. coli and could inhibit the formation of biofilm, disrupt the normal cell cycle, thus damage the integrity of cell membrane (31). In addition, bacteriocin can also induce cell death by changing the permeability of cell membrane or the activity of intracellular enzymes, inhibiting the germination of spores and the respiration of sensitive bacteria interfering with the normal metabolism of nucleic acids, proteins, and other substances in cells (32).

Strain W3-2 was further identified by colony morphology and 16S rRNA gene sequence analysis. As shown in Figure 2A, the colonies of strain W3-2 were medium-sized, raised, creamy white, rounded, and with neat edges. It is a gram-positive strain with single or arranged in chains, rod-shaped, and spore-free (Figure 2B). The strain W3-2 was amplified by PCR, and the gel electrophoresis results of the PCR are shown in Figure 2C. Results showed that the 16S rRNA gene sequence length of strain W3-2 was 1,466 bp, and the homology with Lactobacillus plantarum was more than 99.86% (Figure 2D). Combined morphological characteristics with 16S rRNA gene sequence analysis, strain W3-2 was identified as L. plantarum. Swapnil et al. isolated a bacteriocin-producing L. plantarum from the colon part of honey bee and the rectum region of stomach (33). Mills et al. isolated L. plantarum from handmade cheese that can inhibit the growth of Listeria (34).

The ethyl acetate extracted bacteriocin produced by L. plantarum W3-2 was purified by gel filtration chromatography. As shown in Figure 3A, there were four elution peaks in the purification process of Sephadex G-25. It was proved that only the second elution peak had antimicrobial activity against S. aureus, while the other three elution peaks had no inhibitory effect. The eluent of tubes 59–70 was collected and concentrated (Figure 3B). After further purification by Sephadex LH-20 (Figure 3C), a single elution peak with similar antimicrobial activity was appeared. The eluent corresponding to the 44th–51th tubes was collected for subsequent purification (Figure 3D). There were five absorption peaks after being purified by RP-HPLC (Figure 3E). Except for the one at 5.578 min, the other absorption peaks had no antimicrobial effect. Therefore, eluent at peak time of 5.578 min was collected for further analysis.

Eluent at a retention time of 5.578 min was collected and identified by LC-MS/MS. As shown in Figure 4A, the primary mass spectrometry displayed that the molecular weight of the bacteriocin was 618.26 Da and named as plantaricin W3-2. The primary mass spectrum peak was further analyzed by De novo, and the secondary mass spectrum was finally obtained. As shown in Figure 4B, the entire amino acid sequence was detected as AVEEE. Plantaricin W3-2 is a polypeptide composed of five amino acids with antimicrobial activity. Bacteriocins, with the same molecular weight and amino acid sequence, have not been reported in the present study.

Most of the molecular weights of bacteriocins reported at present are between 1 and 10 KDa. For example, plantaricin SLG1 produced by L. plantarum SLG1 isolated from yak cheese has a molecular weight of 1083.25 Da (35); plantaricin JY22 produced by L. plantarum JY22 isolated from the intestine of golden carp has a molecular weight of 4.1 KDa (36); and plantaricin H5 produced by L. plantarum H5 isolated from the intestinal flora of sturgeon has a molecular weight of 3.0 KDa (37). Plantaricin W3-2 has a molecular weight of 618.26 Da, which belongs to the small molecular weight bacteriocin. Similarly, the molecular weight of the plantarum C010 is 260.1161 Da produced by L. plantarum C010 isolated from cow manure (17).

Thermal stability of plantaricin W3-2 was envaulted. As depicted in Figure 5A, compared to control group, no significant difference in antimicrobial effect on S. aureus by plantaricin W3-2 was observed after exposure to 60, 70, and 80°C for 10 min. After being treated at 100°C, 82.2% antimicrobial activity was retained. Further, the increase in temperature to 121°C, and 73.4% antimicrobial activity was kept. Those results elucidated that plantaricin W3-2 had good thermal stability. The reason may be ascribed to small hydrophobic proteins, almost without tertiary structure, with strong hydrophobic region and stable cross-linking structure. The good thermal stability of the plantaricin W3-2 is similar to the bacteriocin produced by L. plantarum SLG1 isolated from yak cheese, which exhibits good antimicrobial activity even after being heated at 100°C for 20 min (35).

Results of pH stability of plantaricin W3-2 are shown in Figure 5B, and the antimicrobial activity of plantaricin W3-2 was significantly affected by the change in pH. It was enhanced by acidic environment, while inhabited by basic environment. The antimicrobial activity on S. aureus was the largest at pH 2, with diameter of 24.6 mm. The inhibition zone was gradually decreased with the increase in pH and retained 64.3% antimicrobial activity at pH 11. These results proved that plantaricin W3-2 not only had a good antimicrobial effect in acidic environment but also retained its function in a basic environment.

Ten kinds of common pathogenic bacteria in food were used as indicator strains to test the antimicrobial effect of plantaricin W3-2. Results showed that the diameters of the inhibition zone of plantaricin W3-2 against S. aureus, Listeria monocytogenes, and B. cereus reached 28 mm. For B. subtilis, M. luteus, E. coli, P. aeruginosa, Salmonella, Shigella, Proteus, and other indicator strains, the diameter of the inhibition zone of plantaricin W3-2 was around 21 mm (Table 3). These results demonstrated that plantaricin W3-2 had broad-spectrum antimicrobial activity.