Introduction

The bacterial species Bacillus amyloliquefaciens can secrete secondary metabolites with biological activity as a response to the stimuli of the external microbiota (^{Gimenez et al., 2021}; ^{Bai et al., 2023}; ^{Ley-López et al., 2023}). These compounds are an important source of novel chemical structures for biotechnology, and a large number of secondary microbial metabolites have been identified (^{Yan et al., 2020}; ^{Posa et al., 2023}). Likewise, they are characterized by having surfactant properties, including the non-ribosomal cyclic lipopeptides of the iturin, surfactin and fengycin families (^{Carolin et al., 2021}; Bai et al., 2023). These metabolites have different biological abilities, such as antitumor, hemolytic and antimicrobial properties; the latter is associated with activity against a variety of phytopathogenic microorganisms (bacteria, fungi and oomycetes), causing cell lysis and leakage through binding to the plasma membrane (^{Zhou et al., 2020}), a main mechanism which helps the biological activities of the lipopeptides to take place. Such is the case of the inhibition of the mycelial growth of Sclerotium rolfsii by the lipopeptides synthesized by B. subtilis (^{Abdel-Gayed et al., 2019}; ^{Zhao et al., 2017}). The antifungal activity of the fungicidal lipopeptid on Fusarium oxysporum inhibits the mycelial growth of the phytopathogen (^{Nam et al., 2015}) and the fungicidal lipopeptide is said to affect the structural and morphological characteristics of the cell membrane (Zhao et al., 2017). It also induces apoptosis at low concentrations and necrosis at high concentrations when the fungal cells are treated with fengycin (Gimenez et al., 2021).

The structural composition of the lipopeptides consists of a variable hydrophilic peptide macrocycle, joined to a lateral chain of fatty acid residue (^{Zhao et al., 2017}). In addition, these lipopeptide families display counterparts, similar structures or identical molecular weights that differ in the length in the fatty acid chains and in the composition of amino acids of the peptide ring (^{Biniarz et al., 2017}; ^{Ley-López et al., 2023}). The biosynthesis of lipopeptides by Bacillus spp. begins with the assembly by successive addition of both proteinogenic and non-proteinogenic amino acids with the synthesis of non-ribosomal peptide synthetases (NRPS), as well as by the attachment of fatty acids to the NRPS in the form of acetyl-CoA for straight chain fatty acids and isobutyryl-CoA, isovaleryl-CoA and methyl butyryl-CoA for branched fatty acid (^{Chooi et al., 2010}; ^{Zhu et al., 2021}). The members of the family of the surfactin lipopeptide have been reported to present a length in the fatty acid chain from C12 to C16 (^{de Faria et al., 2011}; ^{Jajor et al., 2016}), whereas the iturin family presents an acid chain length of C14 a C17 (^{Jin et al., 2020}) and the fengicin family has chain lengths from C14 to C18, which may be saturated or unsaturated (Sa et al., 2018). There is a basic structural diversity of bacterial cyclic lipopeptides, in which the carboxyl group of the fatty acid can form a peptide bond with an amino acid residue located in the side chain of the lipopeptide (^{Urajová et al., 2016}; Biniarz et al., 2017). Likewise, the fatty acid can be incorporated into the peptide structure through cyclization, frequently at carbon C2 or C3 via β-amino or β-hydroxy groups (^{Duitman et al., 1999}; ^{Mareš et al., 2014}). This diversity in the structure of the peptide macrocycle and the length of the fraction of the fatty acids of the lipopeptides present amphipatic properties that play a decisive role in their mode of action and can act as antibiotics, anti-adhesives, cleaning and foaming agents, but their action on living cells is not always clear (^{Tomek et al., 2015}; Biniarz et al., 2017). ^{Youssef et al. (2005}) observed that the fatty acid composition is fundamental to the biosurfactant activity of the lipopeptides, displaying a significantly positive correlation between the mass percentage of iso-3-OH-C14 fatty acid and the specific activity of the lipopeptide. Therefore, the composition of the fatty acids is important for the biological properties of the lipopeptides such a the surfactin, iturin and fengycin familis (Chooi et al., 2010; ^{Yan et al., 2020}). ^{Bai et al. (2023}) analyzed lipopeptides with GC-MS and described the main differences between the lipopeptide counterparts, which arise from the fatty acid chain length and the amino acids in the peptide chain. Likewise, ^{Qian et al. (2020}) reported that the bacillomycin D counterparts with C14 and C15 fatty acids presented antifungal activity; in both studies, the highest fatty acid mass content corresponded to carbon chain lengths of C14 and C15. However, new structural types of lipopeptide composition are still being reported, highlighting their rich chemical diversity and variability in fatty acid chain length.

Recently, in the Bacillus amyloliquefaciens KX953161.1 bacterial strain with oomycidal activity on Phytophthora capsici (^{Ley-López et al., 2018}), several counterparts were identified in the families of (fengycin, surfactin and bacillomycin) with differences in molecular weight, due to the presence of fatty acid chains with varying lengths. However, the lipid profile of the lipopeptide extract synthesized by this bacterium is unknown, as is the quantity of the fatty acids detected (Ley-López et al., 2023). Due to this, interest has been placed on the composition of the fatty acids in the structure of the lipopeptides produced by this bacterium. The aim of this study was to determine the composition and quantification of the fatty acids in the structures of the lipopeptides synthesized by the bacterium B. amyloliquefaciens KX953161.1, using gas chromatography with flame detection (GC-FID) and determine the effect of the lipopeptides produced by the bacteria in this study on the microscopic morphology of the

F. oxysporum MG557870 and S. rolfsii OM510466 mycelia.

Materials and Methods

Biological material. The strains evaluated (bacteria and fungi) for this study were provided by the strain collection of the Phytopathology Laboratory at the Research Center for Food and Development, Culiacán Unit, Mexico. B. amyloliquefaciens B17 (KX953161.1) isolated from the tomato plant rhizosphere, was reactivated on nutrient agar (NA) at 27 °C. F. oxysporum MG557870 (^{Isaac et al., 2018}) and S. rolfsii OM510466 (^{García-León et al., 2022}) were isolated from roots of vegetable crops (tomato and chili). Moth microorganisms were identified based on morphological characteristics, using specific taxonomic keys, and pathogenicity tests had previously been performed on them. They were reactivated on potato dextrose agar (PDA) for 10 d at 28-30 °C. The culture media for microbial growth were purchased from Sigma- Aldrich^®, USA.

Production of lipopeptides. For the synthesis of lipopeptides by the bacterium B. amyloliquefaciens KX953161.1, a bacterial inoculum was used in 50 mL of Luria-Bertani (LB) liquid culture medium (casein peptone 10.0 g L^-1, yeast extract 5.0 g L^-1, and sodium chloride10.0 g L^-1 with a pH of 7.0). It was incubated at 30±1 °C in an orbital shaker at 150 rpm for 18-20 h, reaching a bacterial cell concentration of 3x10⁸ CFU, according to the McFarland scale. The second culture medium consisted of 500 mL of Landy medium (glucose 20 g L^-1, L-glutamic acid 5 g L^-1, yeast extract 1 g L^-1, K2HPO4 1 g L^-1, MgSO4

0.5 g L^-1, KCl 0.5 g L^-1, CuSO4 1.6 mg L^-1, Fe2 (SO4)3 0.4 mg L^-1, MnSO4 1.2 mg L^-1)

with an initial pH of 7.0 (^{Landy et al., 1948}). Both culture media were sterilized at 121

°C for 15 min. Later, 30 mL of the bacterial inoculum were transferred to the Landy medium and incubated for 6 d at 30±1 °C, stirred at 180 rpm.

The extraction of bacterial cells was performed by centrifuging (HERMLE^® Z 36 HK, Germany) at 10,000 rpm for 12 min at 4 °C. The extraction of the lipopeptides was performed with the method of acid precipitation of the supernatant (^{Unás et al., 2018}). The supernatant without bacterial cells was acidified by adding HCl 6N, until a pH of 2.0 was obtained pH=2.0; it was incubated for 24 h at 4 °C, and to obtain the pellet with the lipopeptides, it was centrifuged at 12,000 rpm for 20 min a 4 °C. Finally, it was lyophilized and stored until use (FreeZone Triad Benchtop Freeze Dryer, LABCONCO^®, USA).

Analysis of fatty acids with CG-FID. The process involves the hydrolytic fragmentation of the bond between the peptide/protein part and the lipid portions. Therefore, the resulting fatty acid chains were derivatized to fatty acid methyl esters (FAME) and their subsequent analysis with GC-FID. For the derivatization, the MAK174 extraction kit (Sigma^®), was used, following the protocols by ^{Smyth et al. (2014}). In the analysis of the lipid part of the material, 25 mg of the purified and lyophilized material was used, which was washed with hexane to remove the free fatty acids. The product was collected in an amber glass vial for subsequent gas chromatography analysis.

An Agilent 7890B gas chromatograph was mounted onto a flame ionization detector (FID). The samples were injected using an Agilent GC Automatic Sampler 80 with split/splitless injection ports with a VF-5ms capillary column, 30 m x 0.25 mm x 0.25 µm, under the conditions shown in Table 1. The temperature of the injector was 250 °C, the carrying gas was helium with a flow of 1.0 µL min^-1, Pulsed Splitless mode. To determine the possible compound detected, a spectral comparison was performed in the NIST Library (NIST 2011b Mass Spectral Library using NIST MS Search Rev. 2011b or the Probability Based Matching (PBM) Search Format as a part of Agilent Technologies MS WorkStation Software Version 7.0).

Quantification of fatty acids. Fatty acids detected were quantified using the area standardization method; this was done directly from the chromatographic run of the sample, adding the areas of all the peaks eluted in the sample run. The total represents 100% of the area of each peak identified as fatty acids. The fat determination was carried out using the AOAC method 920.39. This parameter was obtained with 3 g of lyophilized lipopeptides in a Goldfish extraction thimble (LABCONCO^®, USA). The extraction was performed using anhydrous petroleum ether for 4 h. The extract obtained with the solvents was evaporated until dry in an oven at 105 °C. Finally, the calculation of fat was carried out using the following equation.

%EE= Percentage of ethereal extract.

Pe= Weight of extract (weight of the dry cup at the end of the extraction and its constant weight before extraction).

Pm= Weight of sample.

Inhibiting activity by extraction with lipopeptides. The minimum inhibiting concentration (MIC) of the extract with the lipopeptides synthetized by B. amyloliquefaciens KX953161.1 on F. oxysporum MG557870 and S. rolfsii OM510466 was determined using the diffusion method on dishes. This consisted in placing discs, 6 in diameter, containing fungal mycelia, in the middle of a Petri dish with PDA and placing Whatman paper discs, 6 mm in diameter, impregnated with the concentrations of 5, 10, 15, 20, 25, 30 and 40 µg mL^-1 of lipopeptides, incubated at 26±1 °C for 6 d. The MIC was determined visually, based on the minimum concentration of sample solution required to guarantee that no fungal growth was observed. All analyses were performed in triplicate. The MIC was considered for the ultrastructural observation samples of the fungi through scanning electron microscopy (SEM).

Ultrastructural analysis via MEB. For the structural observation of the fungi with SEM analysis, F. oxysporum MG557870 spores and hyphae were taken, along with S. rolfsii OM510466 mycelia, aged 6 and 3 days, respectively. They were placed in 2 mL Eppendorf tubes and the lipopeptide suspension, with the concentration obtained from the MIC test for each fungus was added; they were then let to stand for 10 min. Subsequently, they were fixated in 2.5% glutaraldehyde (prepared in 0.1 M sodium phosphate buffer) at 4 °C for 24 h, then rinsed three times with phosphate buffer (0.02 M) and later fixated with 2% osmium tetroxide for 2 h at 20 ºC. Dehydration was carried out with a gradually ascending series of ethanol (30, 50, 75 and 95%) for10 minutes each, dried with CO2 and covered using Nanotech (ES-2030 HITACHI^®, Japan) cathodic pulverization. The samples were kept in a drier until the test with a scanning electron microscope (Philips, SEM-505, Netherlands) run at 30 kV.

Statistical analysis. The data obtained from the determination of fatty acids and MIC were analyzed using an analysis of variance (ANOVA), and the means comparison was carried out using Tukey's test (p≤0.05) using the Minitab 19 software.

Results

GC-FID analysis. The fatty acids found in the lipopeptides synthetized by the bacterium

B. amyloliquefaciens KX953161.1 were analyzed after derivatization with GC-FID. The chromatogram obtained shows 10 main peaks with a retention time of 12.82, 13.30, 14.58, 15.04, 16.43, 17.13, 18.25, 19.49, 19.97 and 21.33 min, each of which corresponds to the fatty acids detected (Figure 1). β-hydroxylated C13 to C18 fatty acids were detected, and myristic (C14) and palmitic (C16) fatty acids were identified (Table 2). In the analysis of the total content of each fatty acid identified, β-hydroxylated C14 (tetradecanoic) fatty acid (Figure 2) was observed to have the highest content, with 8.254±0.031 ng mg^-1 of sample, followed by the β-hydroxylated C13 (tridecanoic) and C16 (palmitic) fatty acids, with 4.304±0.064 and 4.100±0.120 ng mg^-1, respectively (Table 2).

Figure 1 Chromatogram of the lipid profile of the lipopeptides synthesized by Bacillus amyloliquefaciens KX953161.1, analyzed by CG-MS. 

Inhibiting activity and ultrastructural observation of the fungi. In the MIC analysis fir the antifungal effect with the lipopeptides synthesized by B. amyloliquefaciens KX953161.1, with the minimum concentration of 20 µg mL^-1 of lipopeptides, an inhibiting halo of 0.95 mm was observed in the mycelial growth of F. oxysporum MG557870 and for S. rolfsii OM510466 with 15 µg mL^-1 of lipopeptides, an inhibiting halo of 1.5 mm was observed (Table 3).

Figure 2 Mass spectrum of fatty acid C14 (Tetradecanoic) of the extract with lipopeptides synthesized by B. amyloliquefaciens KX953161.1. 

The minimal inhibition concentrations with lipopeptides of 15 and 20 µg mL^-1 on S. rolfsii OM510466 and F. oxysporum MG557870 respectively were used for the SEM observation analysis of spores and the mycelia of fungi related to diseases, in order to try to understand the action mechanism of lipopeptides. The F. oxysporum MG557870 hyphae, not treated with lipopeptides, displayed smooth surfaces, without any damage or cracks (Figure 3B). Normal growth and germination were observed in the spores, with a relatively smooth surface (Figure 3D), whereas in the hyphae treated with lipopeptides, deformities, cracks and wrinkles were found on the surface (Figure 3A). In the spores, it was found that most were unable to germinate successfully; some of the ones that did germinate were abnormal and deformed, with twists, distortions and shrinkage (Figure 3C). In the untreated S. rolfsii OM510466 mycelium, a similar pattern to the one in F. oxysporum MG557870 was observed, with no disturbances on the surface, and it displayed normal development (Figure 4B and D), whereas in pore formation was observed on the fungal cell surface, leading to rupture, deformation and a rough texture (Figure 4A and C).

Figure 3 Micrographs (MEB) of spores and hyphae from Fusarium oxysporum MG557870 after the treatment with lipopeptides synthesized by Bacillus amyloliquefaciens KX953161.1. A and C: spores and hyphae treated with lipopeptides (1,500x, bar= 10 µm and 10,000x, bar= 1 µm, respectively); B and D: spores and hyphae without lipopeptides (1,500x, bar= 10 µm y 10,000x, bar= 1 µm, respectively). 

Figure 4 Micrographs (MEB) of mycelia from Sclerotium rolfsii OM510466 after the treatment with lipopeptides synthesized by Bacillus amyloliquefaciens KX953161.1. A and C: mycelia treated with lipopeptides (1,500x, bar= 10 µm and 5,000x, bar= 5 µm, respectively); B y D: micelium without lipopeptides (1,500x, bar= 10 µm y 5,000x, bar= 5 µm, respectively). 

Discussion

Some species of the Bacillus genus produce a variety of antimicrobial compounds, such as B. subtilis and B. amyloliquefaciens, which produce various lipopeptide families (bacillomycin, fengycin and surfactin), as well as diverse counterparts of these families (^{Carolin et al., 2021}; ^{Qian et al., 2020}; ^{Ley-López et al., 2023}).

Microbial lipopeptides are a type of molecules with a structural composition that consists of a variable hydrophillic peptide macrocycle, joined to a lateral fatty acid residue chain (^{Zhao et al., 2017}). These lipopeptides can reduce surface and interfacial tension of the biofilms, and can eventually alter the membrane structure, reported as the main mechanism of lipopeptides, which helps biological activities take place (Zhao et al., 2017). Many of these molecules with active membrane properties play an important part in the formation of pores on the membrane, causing leakage of the cytoplasm to the outside of the cell (^{Liu et al., 2010}), the formation of ionic channels, acting as cation transporters (^{Sheppard et al., 1991}) and presenting detergent-like effects (^{Heerklotz and Seelig, 2007}), which can lead to cytotoxic effects on the vegetative and reproductive structures of competing fungi. The lipopeptide surfactin presents surface activity effects; it inserts itself into lipid bilayers, solubilizes the fluid phase of the phospholipids, chelates monovalent and divalent cations, and alters membrane permeability through the formation of channels or membrane solubilization via a detergent-like mechanism (^{Deleu et al., 2013}). This lipopeptide can form channels, independent to voltage in biofilms and disrupt membrane integrity and the permeability of ions, including Ca²⁺ and K⁺, which can lead to membrane rupture (^{Inès and Dhouha 2015}; ^{Ostroumova et al., 2010}). The lipopeptide bacillomyxin L presents a strong antifungal activity and it is believed to cause membrane rupture; this antifungal activity is associates with its interaction with intracellular targets (^{Zhang et al., 2013}). Likewise, fengycin has been reported to affect the structural and morphological characteristics of the biological membranes, and in high concentrations, it can completely disintegrate the lipid layers (Deleu et al., 2005).

This study identified different β-hydroxylated fatty acids, with different C13 to C18 chain lengths (Table 2), which can be attached to the cyclic lipopeptides, which were identified as counterparts of the lipopeptides fengycin, surfactin and bacillomycin, synthetized by this bacterium, in other studies (^{Ley-López et al., 2023}). This diversity in the chain length of fatty acids provide the shape and composition of lipopeptide isomers. The variation of the position of the functional groups in small organic molecules is responsible for the biological activity (^{Routhu et al., 2021}). The lipid effect of the lipopeptides synthetized by B. subtilis and B. mojavensis reported by Yousset et al. (2005) displayed variability in the C13 to C16 fatty acid chain, which were present as mixtures of isomers and ^{Bai et al. (2023}) reported that the synthesis of fatty acids by B. amyloliquefaciens covered the chain of fatty acids from C14 to C18. They also mention that, in some cases, fatty acids 3-OH-C14 (tetradecanoic) and 3-OH-C15 (pentadecanoic) together make the majority of the lipopeptide fatty acids. Thus, for some Bacillus species, tetradecanoic fatty acid has been the main isomer (^{Qian et al., 2020}: ^{Besson et al., 1992}; ^{Youssef et al., 2005}). In addition, a positive relation was proven to exist between the percentage content of tetradecanoic fatty acid and specific surfactant activity. It was proposed that the branched-chain fatty acid (in this case, iso-C14) could provide the optimal hydrophilic-lipophilic balance required for an optimum surface activity (Youssef et al., 2005). Besson et al. (1992) were the first to report β-hydroxylated tetradecanoic fatty acid as a constituent found in a surfactant lipopeptide synthesized by Bacillus subtilis and that the percentages of β- hydroxylated fatty acids vary, depending on the antibiotic- producing strain and can be influenced by the addition of amino acids to the growth medium of the producing bacterium. It has also been reported that under pressure (presence of competing organisms) the B. subtilis bacterium increases the production of lipopeptides, with a powerful antimicrobial activity (both antifungal and antibacterial); furthermore, they confirmed a substantial accumulation of fatty acids in response to the high pressure (^{Kumar et al., 2020}; Ley-López et al., 2023). Due to this, an appropriate induction with amino acids should be carried out to increase the production of the β- hydroxylated tetradecanoic fatty acid in the lipopeptide and purify this compound to carry out specific biological activity studies.

According to ^{Youssef et al. (2005}) and the results from this study, in which a higher content of the β- hydroxylated tetradecanoic was obtained, along with an inhibiting effect on the fungi F. oxysporum MG557870 and S. rolfsii OM510466 (Figure 3 and 4), it can be inferred that this fatty acid, a component of lipopeptides, is related to the compound's biological activity. Antifungal compounds synthesized by bacteria of the Bacillus genus have been reported to cause damage to the structure and morphology of F. oxysporum, S. rolfsii and Aspergillus cells (^{Xu et al., 2020}; ^{Abdel-Gayed et al., 2019}; ^{Gong et al., 2014}). Additionally, the number of carbon atoms in the fatty acid chain is another important factor that determines the biological capacity of some lipopeptides-greater hydrophobicity of the fatty acids enhances their biological capacity (^{de Faria et al., 2011}). The results of this study indicate that the composition and variation in the chain length β- hydroxylated fatty acids in biosurfactant lipopeptides are important for their antifungal activity.

For future studies related to biological activity, it is recommended to purify the C14 fatty acid together with the lipopeptide, as well as to induce the bacterium to produce specific fatty acids and enhance the biological effect of the lipopeptides.

Conclusions

In the lipid profile of the sample with lipopeptides synthesized by the bacterium Bacillus amyloliquefaciens KX953161.1, there is a variation in the length of the fatty acid chain from C13 to C18 β-hydroxylated. These fatty acids found in the lipopeptides are composed mainly of the fatty acid β-hydroxylate C14 (tetradecanoic) with 27.5% of the total of fatty acids, which increases its probability with the biological activity of the lipopeptide. These compounds synthesized by B. amyloliquefaciens KX953161.1 displayed an antifungal effect on F. oxysporum MG557870 and S. rolfsii OM510466. The effect of the lipopeptides was observed through ultrastructural analysis of the fungal cell wall, which revealed damages to the structure and morphology of the fungal cells.