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May  2020
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Bacteriostatic efect of lipopeptides from Bacillus subtilis N-2 on Pseudomonas putida using soybean meal by solid-state fermentation

  • Received Date: 2019-06-18
    Accepted Date: 2019-12-13
    Published online: 2020-02-25
  • Bacillus subtilis N-2 which was isolated from natto, produced lipopeptides using soybean meal as a substrate. This work aimed to purify, identify, and determine the antibacterial mechanism of lipopeptides produced by B. subtilis N-2. The fermented product obtained by solid-state fermentation was subjected to water extraction, acid precipitation, and methanol extraction. Fractions were separated and collected using a two-step ultrafiltration method and then identified by LC–MS/MS. Mass spectrometry characterization revealed the presence of four variants of iturin A that differed according to the β-amino fatty acid chain from C14 to C17 as well as the amino acid positions. A new lipopeptide (m/z 1070.3) was identified and its structure was different from the previously reported lipopeptides. The lipopeptides were shown to inhibit the growth of an isolate of Pseudomonas putida, a common pathogen in decaying fish, by changing membrane permeability. These results suggest that the lipopeptides from B. subtilis N-2 could be used as a biocontrol agent in aquaculture.
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Bacteriostatic efect of lipopeptides from Bacillus subtilis N-2 on Pseudomonas putida using soybean meal by solid-state fermentation

  • 1. School of Food Science and Engineering, Ocean University of China, Yushan Road 5, Qingdao 266003, Shandong, China

Abstract: Bacillus subtilis N-2 which was isolated from natto, produced lipopeptides using soybean meal as a substrate. This work aimed to purify, identify, and determine the antibacterial mechanism of lipopeptides produced by B. subtilis N-2. The fermented product obtained by solid-state fermentation was subjected to water extraction, acid precipitation, and methanol extraction. Fractions were separated and collected using a two-step ultrafiltration method and then identified by LC–MS/MS. Mass spectrometry characterization revealed the presence of four variants of iturin A that differed according to the β-amino fatty acid chain from C14 to C17 as well as the amino acid positions. A new lipopeptide (m/z 1070.3) was identified and its structure was different from the previously reported lipopeptides. The lipopeptides were shown to inhibit the growth of an isolate of Pseudomonas putida, a common pathogen in decaying fish, by changing membrane permeability. These results suggest that the lipopeptides from B. subtilis N-2 could be used as a biocontrol agent in aquaculture.

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Introduction
  • The lipopeptides obtained from Bacillus spp. as secondary metabolites are amphipathic molecules that contain lipids and peptides that have hydrophobic and hydrophilic moiety, respectively. The lipopeptides include structures termed as surfactin, iturin, and fengycin (Ongena and Jacques 2008). Lipopeptides have a broad spectrum of activity, kill bacteria rapidly, and show synergy with classical antibiotics (Li et al. 2017b). However, they have not yet been employed extensively in industry because lipopeptide-producing strains are not easily genetically engineered for high productivity (Chtioui et al. 2010).

    Over the past few years, solid-state fermentation (SSF) has been increasingly applied in industrial production of value-added products because of the low capital investment and low operating costs. SSF minimizes the problems of foaming and running materials due to the surfactant characteristics of antibacterial lipopeptides during liquid fermentation (Lee and Kim 2004; Zhu et al. 2013).

    Soybean meal (SBM), a co-product after oil extraction from soybean seeds, is an important and cheap protein source for food and animal feed, especially marine fish, because of its easy availability and abundant amino acids (AA) (Dai et al. 2017). Efforts have been increasingly made using SBM as a substrate in the production of lipopeptides by SSF. However, structure identification and inhibitory effects of lipopeptides are lacking when produced under SSF conditions (Biz et al. 2016; Nalini and Parthasarathi 2013; Zhu et al. 2014).

    Gram-negative bacterial infections are one of the most significant causes of economic losses in aquaculture (Osorio et al. 2005). Pseudomonas putida is a gram-negative aerobacterium belonging to the family Pseudomonadaceae and genus Pseudomonas, which is a type of decay bacteria usually found in oxygenated soils and water environments (Altinok et al. 2006; Ladhani and Bhutta 1998). It was reported to be isolated from rotten rainbow trout, ayu, and yellowtail (Altinok et al. 2006; Kusuda and Toyoshima 1976; Wakabayashi et al. 1996). Some isolates are associated with several ulcerative and systemic diseases of marine fish, causing significant mortality over a wide geographic area (Altinok et al. 2006; López et al. 2012). Furthermore, this bacterium causes eye, ear, and wound infection in granulocyte-deficient cancer patients and in babies with a weak immune system (Korcova et al. 2005). In this study, the lipopeptides extracted from B. subtilis N-2 were used to treat the cells of P. putida to study their effects on growth and cell membrane permeability against P. putida. To our knowledge, this is the first report to control P. putida using lipopeptides.

Results

    Parameters of solid-state fermentation

  • During the fermentation process, the bacterial biomass increased as an S-shaped curve (Fig. 1). The number of bacterial cells was 3.56×108 CFU/g of wet weight at 48 h, then entered the stationary phase in which cell density decreased slightly. The crude protein content continuously decreased, very rapidly between 24 and 60 h. Lipopeptide production increased logarithmically in the early stage of fermentation, then tended to be maximized (48.79 mg/g wet weight) at 48 h. The hydrolysis degree curve of crude protein was the same as that of lipopeptides. This was similar to the growth of the bacterial cells, demonstrating the synthesis model of lipopeptides. Protease activity increased rapidly from 12 to 48 h before fermentation and reached a maximum of 31, 005 units/g at 48 h, and then continuously decreased.

    Figure 1.  Dynamic curves of product formation and substrate consumption during solid-state fermentation. Data were expressed as means±standard deviation of triplicate measurements

  • Purification and antibacterial tests

    Ultrafiltration and inhibitory effect on growth of P. putida
  • The results showed that the minimal inhibitory concentration (MIC) of the lipopeptides was 80 μg/ml. According to the results of the bacteriostatic experiment (Fig. 2), the permeate (Fig. 2a) obtained from the first step of ultrafiltration showed no bacteriostatic activity. Lipopeptides with a molecular mass greater than 10 kDa could not penetrate the filter membrane. The purified permeate (Fig. 2d) had antibacterial activity with an inhibitory zone diameter of (19.88±0.32) mm.

    Figure 2.  Schematic representation of filtration processes carried out in this study and comparison of the antibacterial effects of methanol treatment. The first step: A Retentate without MeOH, B Permeate without MeOH; the second step: C Retentate with MeOH, D Permeate with MeOH. MF, microfiltration; UF, ultrafiltration

    According to Fig. 3, when lipopeptides were added at times 0 h, 2 h, 3 h after incubation, the growth of P. putida was significantly inhibited. If lipopeptides were added at 5 h and 6 h after incubation, the bactericidal effect of lipopeptides and the bacteriostatic effect became weak.

    Figure 3.  The effect of lipopeptides on P. putida growth curve; Con: cultured without lipopeptides; 0 h, 2 h, 3 h, 5 h, 6 h: adding purified lipopeptides until the final concentration of 80 μg/ml after 0 h, 2 h, 3 h, 5 h, 6 h of culturing

    Lipopeptides also changed the cell membrane permeability. The rupture of the cell membrane caused leakage of macromolecular substances, such as nucleic acids, thereby increasing the absorbance value at 260 nm and 280 nm. As shown in Fig. 4, UV absorption of supernatant in the control group decreased with the prolongation of culture time, which was caused by the consumption of proteins, peptides, and other substances in the medium. After adding lipopeptides, the absorbance values of the experimental group increased with the prolongation of the culture time, which was different from the control group. These results indicated that lipopeptides increased the permeability of the cell membrane and caused the cell to rupture, thereby increasing the absorbance value.

    Figure 4.  Changes of ultraviolet absorption of supernatant liquid as a function of time at 260 nm (a) and 280 nm (b). Con: no lipopeptide; 0, 2, 3, 5, 6 h: lipopeptides added at 0, 2, 3, 5, and 6 h after culturing

  • Effect of lipopeptides on microscopic characteristics of P. putida
  • The morphological changes of P. putida before and after lipopeptide treatment were observed by scanning electron microscopy. The cells of the control (Fig. 5a, b) were full and uniform, of which the edges and outline were neat and clear, while after lipopeptide treatment, bacterial cells (Fig. 5c, d) showed irregular edges, rough surface, folds, and cells became slender and irregular, indicating that lipopeptides caused abnormalities in the morphology of P. putida.

    Figure 5.  SEM of P. putida treated by lipopeptides. a, b: P. putida without lipopeptide treatment; c, d: P. putida with lipopeptide treatment. Magnification: 20.0 k (a, c) and 50.0 k (b, d)

  • Identification of lipopeptides

  • The positive ion current chromatogram of HPLC–MS is shown in Fig. 6a. The lipopeptide fraction (Fig. 6b) referred to at retention time 4.68 min appeared in a series of [M+H]+ m/z 1043.6, 1057.5, 1070.3, and 1084.4. Their mass charge was exactly one methylene group (-CH2).

    Figure 6.  Positive ion current chromatogram of HPLC–MS (a) and LC–ESI–MS spectrum (b); retention time 4.68 min for antimicrobial lipopeptides

    Figure 7a showed the LC–ESI–MS/MS spectrum of the precursor ion at m/z 1043.6. A series y+ fragment at m/z 243 (Asn5-Gln4), 406 (Tyr6-Asn5-Gln4), 745.3 (β-NH fatty acid-Asn7-Tyr6-Asn5-Gln4), 832 (Ser1-β-NH fatty acid-Asn7-Tyr6-Asn5-Gln4), and the b+ fragment ion 212 (Pro3-Asn2), 299 (Pro3-Asn2-Ser1), 524 (Pro3-Asn2-Ser1-β-NH fatty acid), 638 (Pro3-Asn2-Ser1-β-NH fatty acid-Asn7), 801 (Pro3-Asn2-Ser1-β-NH fatty acid-Asn7-Tyr6), 915 (Pro3-Asn2-Ser1-β-NH fatty acid-Asn7-Tyr6-Asn5) were found. The sequence was Pro3-Asn2-Ser1-C14β-NH fatty acid-Asn7-Tyr6-Asn5-Gln4 (Fig. 7a), which was assigned as iturin A. Similarly, m/z 1057.5 was the same homolog as the m/z 1043.6 amino acid sequence with a difference in carbon chain length, C14 and C15, respectively.

    Figure 7.  LC–ESI–MS/MS spectrum of the iturin precursors. a Iturin precursor ion [M+H]+ at m/z 1043.6 at retention time 4.68 min; b Iturin precursor ion [M+H]+ at m/z 1070.3 at retention time 5.85 min

    Figure 7b shows the LC–ESI–MS/MS spectrum of the precursor ion at m/z 1070.3. A series y+fragment at m/z 426 (Ser4-Asn3-Gln2-Pro1), 541 (Asn5-Ser4-Asn3-Gln2-Pro1), 703 (Tyr6-Asn5-Ser4-Asn3-Gln2-Pro1), 817 (Asn7-Tyr6-Asn5-Ser4-Asn3-Gln2-Pro1), and the b+ fragment ion 252 (β-NH fatty acid), 366 (β-NH fatty acid-Asn7), 643 (β-NH fatty acid-Asn7-Tyr6-Asn5), 730 (β-NH fatty acid-Asn7-Tyr6-Asn5-Ser4), 844 (β-NH fatty acid-Asn7-Tyr6-Asn5-Ser4-Asn3), 972 (β-NH fatty acid-Asn7-Tyr6-Asn5-Ser4-Asn3-Gln2) were found in the MS/MS spectrum of m/z 1070.3. The structural formula of m/z 1070.3 is shown in Fig. 7b, the fatty acid chain length was C16. The corresponding m/z 1084.4 was different from m/z 1070.3, as the chain length was C17.

Discussion
  • During solid-state fermentation, the high concentration of lipopeptides produced by microorganisms lysed the membranes and caused the decrease of apparent biomass after a period of fermentation (Heerklotz and Seelig 2007). Moreover, from 24 to 60 h there was a drastic decrease in crude protein concentration when the production of lipopeptides rose rapidly. A good correlation was shown between crude protein consumption and lipopeptides production, which requires protein. The highest production yield at 48 h was approximately 48.79 mg of crude lipopeptides per gram of solid material, which was similar to the results of Zouari et al. (2014) and Zhu et al. (2012). After 60 h of solid-state fermentation, the yield of lipopeptides began to decrease. The dynamic change of yield was consistent with the results of Zhu et al. (2012). This was mainly attributed to bacterial self-resistance to the accumulation of lipopeptides, as lipopeptides can be used as a nutrient (Tsuge et al. 2001).

    Ultrafiltration is a common method used to purify lipopeptides (Hentati et al. 2019). If it is higher than critical micelle concentration (CMC), the monomers of lipopeptides gather to form micelles. However, if methanol is added to the micelle solution, lipopeptides can be recovered as the monomer (Isa et al. 2007). Membrane ultrafiltration separation processes are highly suitable for large-scale processing as they do not need large quantities of organic solvents (Coutte et al. 2013).

    In addition to the extraction and purification of lipopeptides, the aim of the current work was to investigate their effects on P. putida. Song et al. (2016) demonstrated that the whole cells and protoplasts of Pseudomonas aeruginosa PAO1 and B. cereus were disrupted by lipopeptides produced by B. amyloliquefaciens. Listeria monocytogenes was also inhibited by lipopeptides (Li et al. 2017a). To the best of our knowledge, this is the first report showing that lipopeptides inhibited the growth of P. putida.

    Grau-Campistany et al. (2015) provided strong evidence that lipopeptides altered the bacterial membrane permeability barrier. Furthermore, Greber et al. (2019) reported that lipopeptides showed a significant effect on cell membrane permeability. The difference in inhibition activity is probably due to the differences in the lipid bilayer composition between gram-positive bacteria and gram-negative bacteria. We investigated the effect of lipopeptides on cell membrane permeability by UV absorption at 260 nm and 280 nm and scanning electron microscope (SEM). Macromolecular substances (nucleic acid and proteins) were shown to leak after the addition of lipopeptides. The cell membrane of a large number of cells also shrank (Fig. 5). According to these results and Greber et al. (2019), the action of lipopeptides was on the cell membrane of P. putida.

    Reverse-phase (RP) high-performance liquid chromatography (HPLC) is highly suitable for the purification of molecules with different hydrophilicity and has been used to discriminate between the homologs of lipopeptides (Yang et al. 2015). Iturins, fengycins, and surfactins can be eluted by 40%–50% (Yuan et al. 2011), 50%–70% (Villegas-Escobar et al. 2013), and 85%–100% (Liu et al. 2009) acetonitrile in water, respectively, on a C18 column in an RP-HPLC system. According to LC–MS, the precursor ions at m/z 1043.6, 1057.5, 1070.3, and 1084.4 were hypothesized to be a series of homolog molecules of iturin with 14 or multiples of 14 Da difference in their molecular ion species. Two major fractions (m/z 1043.6, 1070.3) were considered as precursor ions for MS/MS analysis and are described hereafter.

    LC–ESI–MS/MS showed that the amino sequence at m/z 1043.6 was Pro3-Asn2-Ser1-β-NH fatty acid-Asn7-Tyr6-Asn5-Gln4, a typical structure of iturin A, so [M+H]+ at m/z 1057.5 was hypothesized to be the homolog of iturin A. However, the amino sequence at m/z 1070.3 was β-NH fatty acid-Asn7-Tyr6-Asn5-Ser4-Asn3-Gln2-Pro1, which was different from the typical structure of iturin A, and different from previously reported lipopeptides. This result indicated that B. subtilis N-2 produced at least two different lipopeptides. In summary, the different structures of lipopeptides were good references for future research.

Conclusion
  • In this study, lipopeptides were isolated from B. subtilis N-2 by SSF, and the maximum production of the lipopeptide was 48.79 mg/g. Two different structures of lipopeptides (iturin A and β-NH fatty acid-Asn7-Tyr6-Asn5-Ser4-Asn3-Gln2-Pro1) were elucidated through tandem mass spectrometry. Furthermore, we investigated the identified lipopeptides (MIC 80 μg/ml) and showed that it mainly inhibited the growth of P. putida by disrupting cell membrane permeability. This is an interesting feature that gives importance to B. subtilis N-2 and the lipopeptides for future uses in aquaculture.

Materials and methods

    Bacterial strain and culture conditions

  • Bacillus subtilis N-2 was isolated from natto (Wang et al. 2014) and P. putida was isolated from fish preserved in laboratory of Applied Microbiology, Ocean University of China. Both bacteria were cultured on nutrient agar medium (NA: 3 g/L beef extract, 10 g/L peptone, 5 g/L sodium chloride, 18 g/L agar and pH 7.0) and kept at 4 ℃. For activation, B. subtilis N-2 was inoculated into 50 ml of nutrient broth medium (NB: 3 g/L beef extract, 10 g/L peptone, 5 g/L sodium chloride, pH 7.0) in 250-ml Erlenmeyer flask and incubated for 16 h at 37 ℃ and 160 r/min.

    For solid-state fermentation, B. subtilis N-2 was inoculated into sterile substrates (containing 50 g of soybean meal, 1.28 g of K2HPO4, glucose 0.85 g, KCl 0.17 g, MgSO4 0.21 g, sodium glutamate 0.85 g, FeSO4 0.13 g, MnSO4 0.26 g, CuSO4 0.13 g, distilled water 35 ml, pH 7.0) with a level of 10% (v/w) inoculum, mixed under sterile conditions, then aerobically cultured at 37 ℃ for 48 h.

  • Parameters of solid-state fermentation

  • The fermented substrates were sampled every 12 h until 96 h. Bacterial biomass was determined using the method described by Sella et al. (2008); the crude protein content was determined by Kjeldahl method (Wang et al. 2016); the degree of hydrolysis (DH) by ninhydrin method (Sun et al. 2006); the protease activity by spectrophotometric methods (Chinnadurai et al. 2018); and the yield of lipopeptides by dry weight (Zhu et al. 2012).

  • Extraction and purification of lipopeptides

  • The fermented substrates were mixed (1:10, w/v) with Milli-Q water for 1 h and centrifuged at 10, 000 g for 10 min to remove insoluble matter. 6 mol/L HCl was added to the supernatant fluid to acheive a final pH of 2.0. The crude lipopeptides were precipitated overnight at 4 ℃. After centrifugation at 10, 000 g for 20 min at 4 ℃, the crude lipopeptides were extracted with a 20% (v/w) solution of methanol in water for 5 h and the filtrate was dried using a rotary vacuum evaporator and freeze-drier after centrifugation. The lyophilized powder (also crude lipopeptides) was dissolved in Milli-Q water.

    Millipore tubular ultrafiltration separation, of which the membrane cut-off molecules were smaller than 10, 000 Da, was used to purify the lipopeptides. First, an ultrafiltration membrane separation unit was used with the lipopeptide aqueous solutions and the retentate was collected. The pH of the retentate was then adjusted to pH 7.0 with 1 mol/L NaOH. Next, a 20% (v/w) solution of methanol (dispersed micelles) was added and then stirred for 2 h. The ultrafiltration membrane was again used with the lipopeptide aqueous solutions. The resulting permeate (purified lipopeptides) was dried using a vacuum freeze-drier. The purified lipopeptides were subsequently dissolved in Milli-Q water for further analysis (Ma et al. 2016).

  • Antibacterial tests

    Inhibitory effect of purified lipopeptides on the growth of P. putida
  • Oxford Cup method: the minimum inhibitory concentration (MIC) was determined according to the procedure recommended by the Clinical Laboratory Standards Institute (Gök et al. 2016). Briefly, 100 μl of P. putida with 107 CFU/ml was coated onto nutrient agar plates and Oxford cups put on the plates. Then, 200 μl gradient dilutions of purified lipopeptides (10 μg/ml~200 μg/ml were added to the Oxford cups, respectively, and cultured at 37 ℃ for 24 h. The MIC was determined as the lowest concentration of lipopeptides that inhibited bacterial growth.

    Effect on the growth curve of P. putida. 103 CFU/ml of P. putida was cultured in 100 ml liquid NB medium at 37 ℃ shaking at 160 r/min for 24 h. Lipopeptides were added at the final concentration of 1×MIC at times 0 h, 2 h, 3 h, 5 h, 6 h after inoculation of P. putida, respectively. OD600 was determined at different times.

    The effect of lipopeptides on cell membrane permeability was measured at 260 nm and 280 nm by UV spectrophotometer (Yang et al. 2017). The control contained no lipopeptide. 103 CFU/ml of P. putida was cultured in 100 ml liquid NB medium at 37 ℃ shaking at 160 r/min for 24 h. Lipopeptides were added at the final concentration of 1×MIC at times 0 h, 2 h, 3 h, 5 h, 6 h after inoculation of P. putida, respectively. OD260 and OD280 were determined at different times.

  • Effect of lipopeptides on microscopic characteristics of P. putida
  • A total of 103 CFU/ml of P. putida was inoculated into 100 ml liquid NB medium at 37 ℃ shaking at 160 r/min. After 5 h of incubation, 1 ml of sterile water was added to the control group, while lipopeptides were added to the experimental group until the final concentration of 1×MIC at 160 r/min at 37 ℃.

    P. putida was collected and fixed to an electron microscope with 2.5% glutaraldehyde (Heerklotz and Seelig 2007). The image was observed by A JSM 7800f scanning electron microscope (SEM) (JEOL, Japan). The acceleration voltage was fixed to 5 kV.

  • Identification of lipopeptides

  • The structure of the lipopeptides was identified by LC–MS/MS with a Zorbax SB-Aq-C18 column (Agilent, Santa Clara, USA) (4.6×150 mm, 5 μm particle size). Standard iturin A was bought from Sigma (I1774, St. Louis, MO, USA). The column was eluted with solvent A (acetonitrile containing 0.1% formic acid) and solvent B (water containing 0.1% formic acid), which were used with a flow rate of 200 μl/min at 35 ℃. 100 μl aliquot was injected into the system and gradient strategy for 0~8.5 min, 45% solvent A~55% solvent A; 8.5~20 min, 55% solvent A.

    Mass spectra from 200 to 2200 m/z were recorded in the positive ionization mode. The electrospray source was operated at a spray voltage of 4.5 kV, a capillary voltage of 35 V and a capillary temperature of 320 ℃.

  • Statistical analysis

  • All experiments were performed at least three times. Data were expressed as means±standard deviation of triplicate measurements and analyzed by the SPSS statistics program (Version 22, USA). One-way analysis of variance (ANOVA) and Duncan's multiple range test were carried out to test the significant differences among various treatments. In all cases, P < 0.05 was used to determine statistical significance.

Acknowledgements
  • This study was supported by the National Key R & D Program of China (2017YFC1600703) and the National Natural Science Foundation of China (31471657).

Authors contributions
  • ML, HM, and QK designed the experiment. ML, TZ, and XF performed the research. ML, HM, and QK analyzed the data and wrote the manuscript.

Compliance with ethical standards
  • Conflict of interest The authors declare that the research was conducted in the absence of any commercial or fnancial relationships that could be construed as a potential confict of interest.

    Animal and human rights statement This paper followed the ethical guidelines for animal and human rights established by Ocean University of China (Permit Number, SD2007695).

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