Lysogeny broth (LB) medium, potato dextrose agar (PDA) medium and agar was obtained from Guangdong Huan Kai Biotechnology Co., Ltd. (Guangzhou, China). Trifluoroacetic acid (TFA), methanol and acetonitrile were chromatography grade, hydrochloric acid, disodium hydrogen phosphate, sodium dihydrogen phosphate, glutaraldehyde, ethanol, and sodium hydroxide were analytical pure and these were purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China).

The YA215 strain, which was isolated by our research team from the mangrove area in Beibu Gulf, Guangxi, China, was used in the current study (24, 25). Escherichia coli (CGMCC 112252), Bacillus nanotuberculosis, Bacillus subtilis CGMCC 1.14985, Bacillus subtilis CGMCC 1.15792, Staphylococcus aureus CGMCC26003, Salmonella typhimurium CGMCC11190, Shigella flexneri CGMCC110599, and Rhizopus stolonifer CGMCC3.31 were obtained from the National Center for Medical Culture Collections (CMCC, Beijing, China), and Bacillus licheniformis, Bacillus cereus, Fusarium oxysporum f.sp.cubense, and Penicillium expansum CICC 40658 were stored in a 40% glycerol solution at –80^°C in this team.

YA215 strain was grown on LB agar medium for 24 h at 37^°C, and then the colony morphology of the YA215 strain was observed, in addition a single colony was picked for Gram staining. Subsequently, strain confirmation was performed by 16S rRNA gene amplification with the following regular pair of forward and reverse primers; 27F (5′-GGTTACCTTGTTACGACTT-3′) and 1492R (5′-AGAGTTTGATTTGATCCTGGCTAG-3′). The amplification was carried out in a polymerase chain reaction (PCR) instrument (TC-512, Biometra Company, Germany) programmed using the following protocol: 94^°C for 5 min; 35 cycles of 94^°C for 30 s, 64^°C for 1 min, and 72^°C for 2 min and then a final elongation step of 72^°C for 5 min. The analyzed PCR products were sent to Sangon Biotech Co., Ltd. (Shanghai, China) for sequencing. Then, the 16S rDNA sequences were compared with the GenBank database using BLAST in NCBI data, along with homology analysis, and a phylogenetic tree was constructed using MEGA 7.0 software.

YA215 strain was activated on LB agar plates for 24 h, and then a single colony was picked and inoculated into 100 ml LB broth and incubated at 37^°C and 220 rpm for 12 h. Then, a 4% (v/v) inoculum was fermented in pill bottles at 37^°C and 220 rpm for 48 h. The cell-free supernatant was obtained after centrifugation at 4^°C and 10,000 g. The YA215LE was obtained by a combination of acid precipitation and methanol extraction method (26). Briefly, The pH of the cell-free supernatant was adjusted to 2.0 with 6 M hydrochloric acid solution, then it was left to stand overnight at 4^°C. The precipitate was then collected by centrifugation for 20 min at 4^°C at 10,000 pitate was then collects then washed twice with distilled water at pH 2.0. Afterward, the precipitate was resuspended in distilled water and the pH was adjusted to 7.0, and then it was freeze-dried. The dried sample was then extracted with methanol and the methanol soluble fraction was dried with a rotary vacuum evaporator at 45^°C. After removal of methanol, trace amounts of distilled water were added to dissolve YA215LE. The solution was then freeze-dried again and weighed for quantification.

The antibacterial activity of YA215LE was evaluated by the agar well diffusion method with minor modifications (27). Briefly, indicator strains obtained from logarithmic growth phase culture at a final concentration of 10⁶ cfu/ml were added to LB solid medium that was cooled below 50^°C. Then, the LB solid medium was poured into the Petri dishes. After the medium solidified, holes with a diameter of 6 mm were punched on the medium with a sterile punch, and then 100 μ of YA215LE solution was added to the holes. After that, the Petri dishes were left to stand overnight at 37^°C. The antibacterial activity of YA215LE was determined by measuring the diameter of the inhibition zone around the holes.

Antifungal activity was evaluated by the agar well diffusion method with minor modifications (28). Mycelial blocks (6 mm in diameter) were obtained from the central part of the 7 days-old fungal indicator strain and then placed onto the center of a culture dish loaded with fresh sterile PDA medium. After 4 days of incubation at 28^°C, the medium was perforated (5 mm diameter) with a sterile punch at a distance of 2 cm from the mycelium, and 100 μl of YA215LE solution was added to the holes. The culture dish was continued to be incubated at 28^°C for 4 days, and then the diameter of the inhibition zone was measured.

The YA215LE obtained from the fermentation broth of the YA215 strain was first was purified on a Sephadex G-25 (Shanghai Xiamei Biochemical Technology Development Co., Ltd., China) gel filtration column (1.6 × 100 cm). The samples were eluted with phosphate buffered saline (PBS) (20 mM, pH 7.0) at a flow rate of 0.3 ml/min, and the absorbance was monitored at 280 nm. An automatic collection device was used to collect the eluent for 10 min per tube. The antibacterial activity of each canal was determined by using the agar well diffusion method as described before. The active eluent collected was further purified by semipreparative RP-HPLC using the method previously described (29) with modifications. The 100 μl sample solution was injected into the RP-HPLC (Waters Company, MA, USA) through a semipreparative column (XBridge™BEH130 C18, 10 × 250 mm). The mobile phase consisted of eluent A (0.1% v/v TFA) in ultrapure water and eluent B (methanol with 0.1% v/v TFA). The sample was separated using the following gradient: 0–50 min: 5–100% B; 50–51 min: 100–5% B; 51–60 min with 5% B. The flow rate of the mobile phase was set to 2 ml/min. The UV detector detected the elution peak at 280 nm and the same elution peak fractions were collected together and they were freeze-dried to detect their antimicrobial activity.

The purity of the elution peak with the highest antibacterial activity obtained by semipreparative RP-HPLC was measured by an analytical RP-HPLC system equipped with a C18 analytical column (4.6 × 250 mm, 100 Å, 5 μm, Waters, USA) with the same elution procedure. The injection volume was 20 μl, and the flow rate of the mobile phase was set to 0.5 ml/min.

The identification of the eluted fraction with antibacterial activity obtained by RP-HPLC was performed by LC-MS/MS analysis as described previously (30, 31). Mass spectrometry detection equipment with C18 analytical column (Acclaim PepMapTM RSLC, C18, 100 Å, 2, 50 μm analytical columnivity obtained by R) was used to identify antimicrobial active compounds. The mobile phases consisted of eluent A (2% acetonitrile, 0.1% formic acid, 97.9% water) and eluent B (98% acetonitrile, 0.1% formic acid, 1.9% water). The column was eluted by the following gradient: 0–5 min with 2–12% B; 5–30 min with 12–20% B; 30–43 min with 20–32% B; 43–48 min with 32–98% B; 48–58 min with 98% B, and the flow rate of the mobile phase was set to 300 nl/min. The mass spectrometry measurement conditions were: positive ion scan, voltage 1.9 kV, temperature 270°C, energy 29% higher energy collision induced dissociation (HCD), and scan range m/z 300–1,800.

The MIC of BVYA1 was determined by the 2-fold dilution method using a sterile 96-well plate (32). 100 μl of 30 μg/ml BVYA1 was added to wells containing 100 μl of fresh LB broth medium and serially diluted. Then 100 μl of the indicator bacteria (10⁸ cfu/ml) was added to each well. The MIC value of BVYA1 was determined as the lowest concentration at which growth did not occur. The MIC was investigated in duplicate wells and triplicate experiments.

The indicator strain E. coli in the log phase of growth was incubated with BVYA1 (2 × MIC) at 37^°C for 2 h. The bacterial cells treated with PBS (pH 7.4) served as a control. Bacterial cells were collected by centrifugation at 5,000 rpm for 15 min. Bacterial cells were washed three times with 20 mM PBS (pH 7.4). The bacterial cells were then fixed with 2.5% glutaraldehyde at 4^°C overnight. After that, the bacterial cells were washed three times with 20 mM PBS. The bacterial cells were then dehydrated in a series of graded ethanol (30–100%) for 10 min at each concentration. Afterward, the bacterial cells were freeze-dried and sputtered for gold plating prior to the assay (33). The prepared samples were examined using SEM (FEI Quattro S, America) at an accelerating voltage of 8.0 kV.

The effect of BVYA1 on bacterial membrane integrity was measured using a calcein-AM/propidium iodide (PI) double staining kit (Beijing Solarbio Science and Technology Co., Ltd) performed according to a previously described method (34) with some modifications. Log phase E. coli cells were suspended in 100 μl of 10 mM PBS (pH 7.4) at a concentration of 2 × 10⁶ cells/ml and then treated with 50 μl BVYA1 (2 × MIC) for 30 min at 37^°C. Cells were then collected by centrifugation at 8,000 × g for 10 min and resuspended in 100 μl of PBS containing 0.1 μl of calcein-AM stock solution. After incubation at 37^°C for 25 min, 5 μl of PI stock solution was added to the reaction system. After continued incubation at 37^°C for 5 min, the cells were washed three times with PBS and then fixed on microscope glass slides by air-drying. Imaging was performed using a fluorescence microscope (DM4B; Leica, Germany). The control sample was treated with PBS, and observed under the same conditions.

Each assay was performed in triplicate. The experimental data are presented as the mean ± standard deviation.

To classify the YA215 strain more differently at the species level, observation of its cellular morphology and homologous analysis of its 16S rDNA sequences were conducted. YA215 strain was found to be yellowish and round with irregular colony edges after 24 h incubation at 37^°C (Figure 1A). Through Gram staining and SEM, the cells of strain YA215 were observed to be typically rod-shaped with blunt ends under Gram staining and SEM (Figures 1B,C). At the molecular level, PCR amplification of the 16S rRNA gene of the YA215 strain was sequenced and submitted to GenBank. BLAST results indicated that the constructed phylogenetic tree revealed 96% similarity between the YA215 strain and the Bacillus velezensis CBMB205 strain (Figures 1D,E). The strain YA215 was thus identified as B. velezensis and was named as B. velezensis YA215.

The antimicrobial activity of the YA215LE was evaluated by the agar well diffusion method. The YA215LE exhibited a wide spectrum of antibacterial and antifungal activities. The YA215LE exhibited a good inhibitory effect on gram-positive bacteria, such as Bacillus licheniformis, Bacillus cereus, Staphylococcus aureus CGMCC26003, Bacillus nanotuberculosis, Bacillus subtilis CGMCC1.14985, and Bacillus subtilis CGMCC1.15792, and on gram-negative bacteria, such as Escherichia coli CGMCC44102, Salmonella typhimurium CGMCC11190, and Shigella flexneri CGMCC110599. In the antifungal activity assay, YA215LE also exhibited strong inhibitory effects on Fusarium oxysporum f.sp.cubense, Rhizopus stolonifer CGMCC3.31, and Penicillium expansum CICC 40658 (Figure 2). These results suggest that YA215LE exhibits broad-spectrum antimicrobial effects.

Identification of the composition of YA215LE was assessed using liquid chromatography-mass spectrometry (LC-MS) analysis. The strain produced molecules with m/z corresponding to cyclic lipopeptides from different families that were previously described in the Bacillus genera, namely, surfactins, iturins, and fengycins (35). Iturin homologs with molecular ion peaks [M + H]⁺ of 993.41, 1007.59, 1021.57, 1035.46, 1049.48, and 1063.44 were detected, corresponding to C12 to C17 hydroxy fatty acid chains (Figures 3A,B). Mycosubtilin homologs belonging to the iturin family with molecular ion peaks [M + H]⁺ 1043.55, 1057.45, 1071.46, 1085.59, and 1099.60 were also detected, corresponding to C14 to C18 hydroxy fatty acid chains (Figure 3B). For the molecules biosynthesized from the YA215 strain, the m/z values were attributed to analogs of surfactin. These analogs contain various fatty acid tails, i.e., C12 (m/z 994.64 [M + H]⁺, m/z 1016.62 [M + Na]⁺), C13 (m/z 1008.66 [M + H]⁺, m/z 1030.64 [M + Na]⁺), C14 (m/z 1022.67 [M + H]⁺, m/z 1044.65 [M + Na]⁺), C15 (m/z 1036.69 [M + H]⁺, m/z 1058.67 [M + Na]⁺), and C16 (m/z 1050.70 [M + H]⁺, 1072.68 [M + Na]⁺) (Figures 3C,D). These m/z values could also correspond to many other surfactin analogs that contain amino acid modifications at different locations on the peptide chain. Finally, fengycin A analogs with different possible fatty acid chains were detected, i.e., C15 (m/z 1449.81 [M + H]⁺), C16 (m/z 1463.82 [M + H]⁺), C17 (m/z 1477.82 [M + H]⁺), C18 (m/z 1491.83 [M + H]⁺), C19 (m/z 1505.85 [M + H]⁺), and C20 (m/z 1519.86 [M + H]⁺) (Figures 3E,F). Fengycin B analogs with different possible fatty acid chains were also detected, i.e., C14 (m/z 1433.78 [M + H]⁺), C15 (m/z 1447.80 [M + H]⁺), C16 (m/z 1461.82 [M + H]⁺), C17 (m/z 1475.84 [M + H]⁺), and C18 (m/z 1489.85 [M + H]⁺) (Figures 3E,F).

First, three separation fractions were obtained from the separation of YA215LE by gel column chromatography (Figure 4A). After testing the antibacterial activity of the three separation fractions, the results showed that P3 exhibited good antibacterial activity (Figure 4B). The P3 fraction was further separated by semiprepared column liquid phase separation, and the results showed that the P3 component was more a complex with 10 components (Figure 4C). Among the separation fractions, the P3-4 fraction exhibited the highest antibacterial activity against E. coli (Figure 4D). This fraction was then was further checked by an analytical RP-HPLC system. The results showed that the P3-4 fraction was considered a pure product (Figure 4E) and was named BVYA1. BVYA1 was collected and concentrated, and further identified and analyzed by LC-MS/MS.

LC-MS/MS analysis in the positive ionization mode was carried out to identify the BVYA1 lipopeptide. First, based on the results of the primary mass spectrometry, it can be concluded that the most abundant fragment ions in the positive ion mode were observed at m/z 994.66, while ions at m/z 980.62 and 1008.66 with a lower intensity were also observed (Figure 5A). The molecular ions in these peaks differed by 14 days from each other, which illustrated that the three components might be homologs. According to the mass numbers reported in the literatures (36–39), the parent ions at m/z 980.62, 994.66 and 1008.66 could be attributed to surfactins of the lipopeptides class. Second, MS/MS analysis was used to further analyze the parent ions at m/z 994.66, 980.62, and 1008.66. De novo peptide sequencing was performed by evaluating the b- and y-type consecutive ions. The parent ions at m/z 980.62, 994.66, and 1008.66 were found to contain the same peptide sequence, and their peptide sequences were identified as Glu–Leu/Ile–Leu–Val–Asp–Leu–Leu/Ile. In addition, the fatty acid moieties of the parent ions at m/z 980.62, 994.66, and 1008.66 comprised chains of different lengths from C11 to C13 (Figures 5B–D).

The MIC of the purified BVYA1 was determined by a broth microdilution assay in a sterile 96-well microtiter plate. It can be seen from Figure 6, Table 1 show that the MICs of BVYA1 to Escherichia coli, Salmonella, and Shigella were 7.5l, 15, and 15 μg/ml, respectively. The results show that BVYA1 exhibits significant antibacterial activity.