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Article

Identification and Quantification of Lipopeptide Homologues Induced and Produced by Bacillus amyloliquefaciens

by
Nancy Ley-López
1,
José Basilio Heredia
2,
Cesar San Martín-Hernández
3,
Isabel Cruz-Lachica
2,
Isidro Márquez-Zequera
2,
Raymundo Medina-López
1 and
Raymundo Saúl García-Estrada
2,*
1
Facultad de Agronomía, Universidad Autónoma de Sinaloa, Carretera Culiacán-Eldorado Km 17.5, Aparatado Postal 25, Culiacán C.P. 80000, Sinaloa, Mexico
2
Centro de Investigación en Alimentación y Desarrollo, A.C. Coordinación Culiacán (CIAD), Km 5.5 Carretera a Eldorado, Campo El Diez, Culiacán C.P. 80110, Sinaloa, Mexico
3
Facultad Colegio de Postgraduados, Campus Montecillo, Carretera México-Texcoco Km 36.5, Montecillo, Texcoco C.P. 56230, Estado de México, Mexico
*
Author to whom correspondence should be addressed.
Fermentation 2023, 9(11), 944; https://doi.org/10.3390/fermentation9110944
Submission received: 17 August 2023 / Revised: 30 September 2023 / Accepted: 2 October 2023 / Published: 31 October 2023
(This article belongs to the Section Microbial Metabolism, Physiology & Genetics)
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Abstract

:
Cyclic lipopeptides (LPs) are potentially promising in the agricultural, industrial and pharmaceutical sectors. LPs have a variable hydrophilic cyclic peptide part attached to a variable fatty acid chain. One limitation of these compounds is their low availability due to their limited production by bacteria. The objective of this study was to identify and quantify homologues of LPs biosynthesized by Bacillus amyloliquefaciens using ultra-performance liquid chromatography (UPLC–MS/MS) after inducing the synthesis of these secondary metabolites using different inducers, including chemical compounds and inactive cells of Colletotrichum sp. Four homologues were identified in the iturin family (bacillomycin D), and the iturin homologue with the highest synthesis was the molecular ion m/z 1031.54, with 173.1 µg mg−1 crude extract. In addition, seven homologues were detected in the fengycin family (four of fengycin A and three of fengycin B), and the homologue with the highest content was the molecular ion m/z 1463.69 (fengycin A), with 3288 ± 528.5 ng mg−1 crude extract. Finally, five homologues were identified in the surfactin family, where the highest concentration was observed for the molecular ion m/z 1036.68, with 61.5 ± 3.01 µg mg−1 crude extract. The highest concentration of LP homologues (iturin, fengycin and surfactin) synthesized by B. amyloliquefaciens was detected in the presence of inactive cells of Coletotrichum sp., suggesting that the inducing substance is associated with the inducer’s cell envelope and could be a single protein or a structure that includes protein components.

1. Introduction

Secondary metabolites of microbial origin are not necessary for the normal development and proliferation of microorganisms that produce them. These compounds provide competitive and defensive advantages against competing microorganisms; therefore, they can have various applications in industrial, clinical and agricultural settings [1,2]. Cyclic lipopeptides (LPs) are potentially promising and contain a variable hydrophilic cyclic peptide part linked to a variable β-amino or β-hydroxy fatty acid chain, as well as present amphipathic properties that determine their mode of action; these compounds can act as antibiotics, anti-adhesives, antitumor compounds, and cleansing and foaming agents, but their action on living cells is not always clear [3,4]. These cyclic LPs are produced by various species of bacteria and fungi; however, most of the research on microbial LPs has focused on certain bacterial genera, such as Bacillus, Paenibacillus, Pseudomonas and Streptomyces [5]. Some species of the genus Bacillus are known for their wide distribution in nature, their formation of endospores with resistance to adverse environmental conditions, and their capacity to produce various secondary metabolites, mainly LPs, which are characterized by a broad antimicrobial spectrum and a strong surfactant activity on various phytopathogens, including bacteria, fungi and oomycetes [6,7]. The most studied families of LPs are surfactin, iturin and fengycin; moreover, the abovementioned bacteria are capable of producing homologues of each family of LPs [8]. The LP surfactin family is the most studied and includes potent biosurfactants that contain heptapeptides bound to a β-hydroxy fatty acid with a carbon number in the range of C12 to C16 [9,10]. The iturin LP family is composed of homologues of iturin A to E, mycosubtilin and bacillomycin D, F, L and LC; these homologues contain heptapeptides attached to a chain of β-amino fatty acids that varies in length from C14 to C17 [11,12]. The fengycin LP family includes a mixture of isoforms that vary both in the length and branching of fatty acids and in the composition of amino acids [13]. The LPs of the fengycin family contain a β-hydroxyl fatty acid chain ranging from C14 to C18 in length, which can be saturated and unsaturated and is linked to a peptide sequence of 10 amino acids (decapeptides), with 8 of which being part of a cyclic structure forming a lactone group [13,14].
These families of LPs have been considered for potential applications in industry, pharmaceuticals or agriculture [3]. However, one limitation of these applications is the low availability of LPs. In this regard, various methods have been used, such as optimization of the culture media used in bacterial growth and improvement of strains through mutagenesis [15]. In addition, inducers, such as amino acids, and supplementation with carbon and nitrogen sources have been used that significantly affect the nature and biosynthesis of LPs by bacteria [16,17]. Additionally, it has been reported that appropriate isolation of microorganisms and the conditions of the culture medium used are critical parameters for achieving good production [18]. Reports on methods for increasing the production of LPs have noted the difficulty of producing these metabolites without clarifying how inducers affect the biosynthesis of LPs.
The bacterial species Bacillus amyloliquefaciens (KX953161.1) showed significant oomyceticidal activity against Phytophthora capsici in tomato and chili plants [19]. Furthermore, it was confirmed that this species produces LPs of the fengycin and surfactin families with an oomycetic effect [7]. However, the homologues of these families of LPs produced by this bacterium that inhibit oomycete development are unknown. Therefore, it is important to identify the substances produced by this bacterium; likewise, it would be valuable to induce the synthesis of these biologically active compounds. This study used ultra-performance liquid chromatography (UPLC-MS/MS) to identify and quantify the homologues of the LP families biosynthesized by B. amyloliquefaciens (KX953161.1) exposed to stimulation from chemical compounds and inactive cells of Colletotrichum sp.

2. Materials and Methods

2.1. Chemical Reagents

Standards of fengycin ≥ 90% (CAS No. 102577-03-7), surfactin ≥ 98% (CAS No. 24730-31-2) and iturin A ≥ 95% (CAS No. 52229-90-0) were purchased from Sigma-Aldrich, Lipofabrik (Villeneuve d’Ascq, France). The chemical reagents and solvents used for the mobile phase (methanol, formic acid and acetonitrile) and for extraction (analytical-grade HPLC), the culture media for microbial growth, and the inducers (glutamic acid, iron, cellulose and chitin) were purchased from Sigma-Merck (KGaA, Darmstadt, Germany).

2.2. Bacterial Culture

The microorganisms evaluated in this study were provided by the Laboratory of Plant Pathology of Cen Food and Development Research Group, Culiacán Unit, Mexico. The bacterial species Bacillus amyloliquefaciens (KX953161.1) was isolated from the rhizosphere of tomato plants grown in Culiacán, Sinaloa, Mexico [19]. It was reactivated in nutrient agar (NA) at 26 ± 1 °C for 5 d. A strain of Colletotrichum sp. was reactivated in potato dextrose agar (PDA) for 10 days at 29 ± 1 °C, and then the cells were inactivated by heat at 55–65 °C for 45 min.

2.3. Biosynthesis and Extraction of Lipopeptides

For the production and extraction of LPs produced by B. amyloliquefaciens, two culture media were used. The first (bacterial inoculum) contained 200 mL of liquid Luria–Bertani (LB) broth at a pH of 7.0. The culture was incubated at 30–32 °C in an orbital shaker at 150 rpm for 20–22 h, reaching a bacterial cell concentration of 3 × 108 CFU according to the McFarland scale. The second medium, Landy, with an initial pH of 7.0 contained 20 g L−1 glucose, 5 g L−1 L-glutamic acid, 1 g L−1 K2HPO4, 1 g L−1 yeast extract, 0.5 g L−1 KCl, 0.5 g L−1 MgSO4, 1.6 mg L−1 CuSO4, 1.2 mg L−1 MnSO4 and 0.4 mg L−1 Fe2(SO4)3 [20]. A total of 750 mL of Landy medium was added to 6 Erlenmeyer flasks of 2 L each. To induce LP biosynthesis, the culture medium was supplemented with inducers (Table 1).
The samples were sterilized at 115 °C for 15 min, except in the case of the sample with inactive fungal cells, which were added to the culture medium after sterilization. Thirty milliliters of the bacterial inoculum was transferred to each flask and incubated for 6 days at 30–32 °C with shaking at 180 rpm.
Bacterial cells were extracted via centrifugation at 10,000 rpm for 12 min at 4 °C (HERMLE Z 36 HK, Labortechnik GmbH, Germany). LP extraction was performed via acid precipitation of the supernatant [21]. The supernatant without bacterial cells was acidified with 6 N HCl until reaching pH = 2.0; it was incubated for 24 h at 4 °C and centrifuged at 12,000 rpm for 20 min at 4 °C to obtain the pellet with LPs. Finally, the obtained sediment was lyophilized (FreeZone Triad Benchtop Freeze Dryer, LABCONCO, Co., Kansas, MO, EE. UU.).

2.4. Identification and Quantification of Homologues of Lipopeptides Using UPLC-MS/MS

LPs were identified and quantified using ultra-performance liquid chromatography (UPLC-MS/MS). The system consisted of a class H precision instrument (Waters) coupled to a G2–XS quadrupole and time-of-flight (Q–TOF) mass analyzer following the procedure of Lee et al. (2016) with some modifications [22]. An Acquity BEH C18 column (100 mm × 2.1 mm, 1.7 µm, Waters) was used for the separation of compounds. The binary mobile phase consisted of A: acidified water (0.1% formic acid) and B: acetonitrile. A gradient of 0–10 min, 20–90% B and 10–20 min, 100% B was applied with a flow rate of 0.4 mL min−1. The column temperature was kept at 50 °C, and the injection volume was 1 µL.
Electrospray ionization mass spectrometry (ESI-MS) was used, and mass spectra were obtained in the positive mode in a mass/charge (m/z) range of 200 to 1800. Collision energies of 10, 20, 30, 40 and 50 V, a capillary voltage of 1.5 KV, a cone voltage of 30 V, a desolvation temperature of 500 °C and a desolvation gas flow rate of 800 L h−1 were used. The compounds were identified according to the literature, their standards and fragmentation patterns. They were quantified with reference to the fengycin, surfactin and iturin standard curves. The experiments were carried out in triplicate.

2.5. Calibration Curves

The standards of surfactin (≥98%), iturin (≥95%) and fengycin (≥90%) were prepared at a concentration of 1 mg mL−1 in UPLC-grade methanol, which was selected considering the purity of each standard. Each calibration curve had seven concentrations (95, 190, 285, 475, 950, 1425 and 1900 ng mL−1). Each point on the calibration curve was measured in triplicate. The curves were constructed by plotting the peak area (signal from the MS extracted-ion chromatogram (EIC)) versus the concentration of each analyte.

2.6. Statistical Analysis

A complete randomized statistical design with six treatments was used. The data obtained were analyzed using analysis of variance (ANOVA), and comparison of means was performed with Tukey’s test (p ≤ 0.05) using the Minitab 18 software.

3. Results and Discussion

3.1. UPLC-ESI-MS Analysis

The identification of LPs produced by B. amyloliquefaciens with different inducers added to the growth medium showed that the bacterium has the capacity to produce different homologues or isoforms of LPs belonging to different families. Among the overlapping chromatographic peaks obtained using MS analysis, we focused on three groups of molecular ion peaks [M + H]+ with different m/z values. The first group of molecular ion peaks [M + H]+ was observed at the retention times (Rts) of 5.35, 5.77, 6.33 and 6.80 min, with m/z values of 1031.54, 1045.56, 1059.57 and 1073.59, respectively. In the second group of ions, the Rts were 6.14, 6.69, 6.90, 7.13, 7.15, 7.35 and 7.88 min, with m/z values of 1435.78, 1449.79, 1463.81, 1477.83, 1491.85, 1505.87 and 1519.87, respectively. In the third group of ions, the Rts were 10.49, 10.84, 11.44, 11.76 and 12.36 min, with m/z values of 994.64, 1008.65, 1022.67, 1036.68 and 1050.70, respectively (Table 2). The results of this research indicated the presence of homologues belonging to the iturin, fengycin and surfactin families of LPs reported in the literature [8,23,24].

3.2. ESI-MS/MS Analysis of the Iturin Family

According to the conducted analysis and as reported in the literature [16,23,24], the molecular masses of the detected structures correspond to four homologues of bacillomycin D, which belong to the iturin family. These four [M + H]+ ions, with molecular weights of m/z 1031.54, 1045.56, 1059.57 and 1073.59 (Table 2 and Figure 1) corresponding to the homologues detected, were used as the precursor ions and were differentiated by their fragmentation pattern. Differences in molecular weight of 14 Da intervals were observed due to the presence of fatty acid chains with variable lengths of C13, C14, C15 and C16 (Table S1) and a peptide sequence in the form of an Asn-Tyr-Asn-Pro-Glu-Ser-Thr ring, as previously reported [16,23,25].
In this study, the ionic fragmentation pattern shows an ion corresponding to each precursor ion, with a molecular weight of m/z 227.10, which could be an aliphatic fatty acid chain containing 14 carbons in the normal, iso or anteiso forms belonging to bacillomycin D (Figure 2). Bacillomycin D is characterized by its antifungal properties toward phytopathogenic fungi, acting on the structure of the cell wall, as reported by Sun et al. (2022) in a study on the antifungal effect of bacillomycin D against the fungi Fusarium graminearum and F. moniliformes [26].

3.3. ESI-MS/MS Analysis of Fengycin A and B

The second group of ions [M + H]+ detected, with molecular weights of m/z 1435.78, 1449.79, 1463.81, 1477.83, 1491.85, 1505.87 and 1519.87 (Figure 3), correspond to seven homologues of the fengycin family; according to the molecular masses reported in the literature [8,27,28], they are considered precursor ions in the MS/MS analysis. These ions differ in molecular weight in multiples of 14 Da (corresponding to a CH2 group), and this difference is attributed to the variability in the length of the fatty acid chain of each homologue [8,9].
The fragmentation profiles of the precursor ions with m/z 1435.78, 1449.79, 1463.81 and 1477.83 share a pair of ions with molecular masses of m/z 966.46 and 1080.54 (Figure 4A–D). These ions originate from fengycin A and have been reported with the amino acid sequence Glu-Orn-Tyr-Thr-Glu-Ala-Pro-Gln-Tyr-Ile, Orn-Tyr-Thr-Glu-Ala-Pro-Gln-Tyr-Ile; they are attributed to the neutral losses of fatty acid-Glu and fatty acid-Glu-Orn from the N-terminal segment with C14 to C17 [8,27,29]. On the other hand, in the fragmentation of the precursor ions of m/z 1491.85, 1505.87 and 1519.87, a pair of ions with molecular masses of m/z 994.49 and 1108.57 was observed (Figure 4E–G). These results are attributed to the losses of neutral fatty acid-Glu and fatty acid-Glu-Orn from the N-terminal segment of fengycin B with the amino acid sequence Glu-Orn-Tyr-Thr-Glu-Val-Pro-Gln-Tyr-Ile, Orn-Tyr-Thr-Glu-Val-Pro-Gln-Tyr-Ile with C18 to C20 [9,27,29]. The fengycin B ions identified have a molecular weight that is 28 Da more than that of the fengycin A ions, which indicates the presence of Ala in the lactone ring (or amino acid chain) in fengycin A and the presence of Val in fengycin B [27,30]. The ion fragmentation products with molecular masses of m/z 996.46, 1080.54, 994.49 and 1108.57 of the precursor ions have been used as fingerprints to quickly detect fengycin A and B [27]. Therefore, the ions detected in this study correspond to homologues of fengycin A and B (Table S2). These homologues have been reported to have antimicrobial activity against Botryospharia dothidea and Candida albicans [8,31]. Liu et al. (2019) reported that fengycin A exhibited powerful antifungal activity against F. graminearum and observed that fengycin A altered the integrity of the fungal membrane by reducing the ergosterol content, affecting the structure and stability of the membrane, and interacting with and binding to genomic DNA [32].

3.4. ESI-MS/MS Analysis of Surfactin

The third group of ions [M + H]+, with molecular weights of m/z 994.64, 1008.65, 1022.67, 1036.68 and 1050.70 (Table 2 and Figure 5), correspond to five homologues of the surfactin family, according to the reported molecular masses [33,34,35]. This family consists of a mixture of isoforms with the cyclic peptide chain Glu-Leu-Leu-Val-Asp-Leu-Leu, where one amino acid is replaced by another amino acid [36,37]. Similar to the previous groups of ions detected, this group of ions of the surfactin family differs in molecular weight in multiples of 14 Da (corresponding to a CH2 group) due to the presence of fatty acid chains with variable length [35,36,38].
In the fragmentation profile of the precursor ions, a common fragment with a weight of m/z 685.44 was observed (Figure 6 and Table S3), corresponding to the previously reported Val-Leu-Asp-Val-Leu-Leu amino acid sequence [36]. This fragment is considered a base ion confirming the structure of surfactin [38]. Similarly, the ion with m/z 441.26 (Figure 6) was observed for all the precursor ions with the amino acid sequence Leu-Leu-Val-Asp [35]. These surfactin homologues have shown bioactive properties toward biological membrane structures. For example, surfactin caused a significant reduction in the intracellular content of iron, manganese and zinc in Candida albicans compared to the cells of the control treatment. In addition, the expression of genes involved in the synthesis of ergosterol decreased, thus affecting the growth of the fungus [39].

3.5. Quantification of Lipopeptide Homologues

Among the identified homologues of LPs, the same compounds were observed in all treatments; however, the total content of the compounds varied significantly between treatments. In this study, the presence of competing microorganisms in the culture medium with the producer bacteria increased the production of these secondary metabolites. Consequently, a higher production of homologues in the three families of LPs identified (iturin, fengycin and surfactin) was obtained in the presence of inactivated fungal cells (Table 3, Table 4 and Table 5). The bacillomycin D homologue with the highest concentration was the molecular ion of m/z 1031.54, with 173.1 ± 7.14 µg mg−1 crude extract (Table 3).
In the fengycin family, the representative homologue was the molecular ion of m/z 1463.69 corresponding to fengycin A, with 3288 ± 528.5 ng mg−1 crude extract. This ion has been reported as being predominant or present with high abundance among the compounds produced by B. subtilis [9]. The ions with molecular weights of m/z 1477.79 and 1435.79 of fengycin A were produced at lower concentrations than in the first group, with 385 ± 50.0 and 350 ± 47.0 ng mg−1 crude extract, respectively (Table 4).
Regarding the homologues of the surfactin family, the molecular ion with m/z 1036.68 had the highest concentration, with 61.5 ± 3.01 µg mg−1 crude extract, and was the predominant ion with a Leu-Leu-Asp-Val-Leu-Leu-GluOMe amino acid sequence [35] (Table 5). Moreover, a lower concentration of homologues in the fengycin and surfactin families was observed in the extracts induced with cellulose (Table 4 and Table 5). Unlike the homologues of surfactin and fengycin, bacillomycin D showed lower concentrations in the treatments with glutamic acid, iron and cellulose, which might be because the concentrations used were not optimal for the production of these LPs by the bacteria. The use of inducers at appropriate concentrations significantly promotes the synthesis of bioactive metabolites such as LPs [7,40,41].
One of the strategies to increase the production of microbial LPs is to add different carbon sources based on sugar and nitrogen to the culture media with the producing bacteria [41,42,43]. However, it is important to consider the C/N ratio due to its positive or negative influence on the synthesis of LPs [18,42]. Some carbohydrates, such as glycerol, glucose and arabinose, promote the synthesis of secondary metabolites and enzymatic activity involving signaling proteins in the form of LPs [15,44,45].
As reported, some genes involved in protein production are less expressed when there are large amounts of carbon and nitrogen sources, such as glucose, which may impact the activity of some Bacillus genes since these compounds can be toxic in high concentrations [42]. Likewise, bacterial cells in stressful environments carry out a predominant synthesis of proteins that are vital for their adaptive stress response, which can be caused by the environment or by the presence of natural enemies [44]. Thus, it is likely that the bacteria under study were threatened by the presence of inactive fungal cells.
Therefore, a suitable nutrient or inducer can stimulate bacteria to increase the production of LPs. Qian et al. [16] added the amino acid L-glutamine (L-gln) to the growth culture medium of the bacterium B. subtilis and observed significant production of the LP bacillomycin D, in addition to an increase in the expression of genes involved in the biosynthesis of this LP. However, they found that not all amino acid residues in the cyclic peptide chains of this LP were beneficial for its production, such as L-aspartic (L-asp) and L-cysteine (L-cys), which completely inhibited the synthesis of bacillomycin D [16]. Therefore, the production of compounds may be associated with amino acids that provide sources of organic nitrogen for cell development, and cell growth is important for the biosynthesis of target products [46]. However, Wang et al. [46] and Qian et al. [16] reported that cellular growth is linked not only to the production of LPs but also to the synthetases and regulatory proteins related to these compounds. Similarly, they mentioned that amino acids that regulate LP biosynthesis through synthetase and regulatory proteins should be studied further.
In this study, B. amyloliquefaciens produced a greater amount of LP homologues in the presence of heat-attenuated cells of Colletotrichum sp. A previous study mentioned that the use of inactivated or even partially denatured cells as inducers affected the biosynthesis of antimicrobials, and this effect was associated with an inducer cell wall protein of approximately 58 kDa [18,47]. It is worth mentioning that these studies focused on the production of bacteriocins, while for LP biosynthesis, there is almost no information. Likewise, it has been reported that the mycelial structure of C. gloeosporioides is destroyed due to disintegration of the cell wall and deformation of hyphae by the activity of the bacteria B. amyloliquefaciens and B. subtilis. These results suggest that the membrane and cell wall of C. gloeosporioides are the main targets of antimicrobial substances produced by Bacillus species [5,17].
According to previous literature and the results of our study, it can be considered that the inducing substance is associated with the cell envelope of inactivated fungal cells and could be a single protein or a structure that includes protein components. There is great variability in the components constructing the fungal cell wall, including β-1,3-glucan, mixed β-1,3/1,4-glucan, β-1,6-glucan, chitin, α-1,3-glucan, mannan, galactomannan and melanin [48]. This last fungal component has diverse applications as a result of its multiple biological properties and presents structural diversity [49], allowing it to act as an inducer for the synthesis of LPs. Furthermore, fungal cell tissue can provide available nutrients to antagonistic bacteria that produce chitinases in the presence of fungi, a common trait in bacteria that exhibit antifungal activity. However, chitinolytic activity alone is insufficient to explain lysis in fungal hyphae [48,50,51]. This may indicate that these bacteria could produce antimicrobial compounds such as LPs. This demonstrates the complexity of the fungal cell wall and makes it a prime target for bacterial attack, as antagonistic bacteria need to rapidly produce a wide variety of antimicrobial compounds to degrade cell wall components and compromise fungal structural integrity [48]. Therefore, the presence of specific fungal compounds can help bacteria achieve their goal [52]. Therefore, these results provide information and the basis for further studies regarding the interaction mechanisms between fungi and antagonistic bacteria to enhance the production of LPs with antimicrobial activity.

4. Conclusions

In this study, homologues of the fengycin, surfactin and iturin families produced by the bacterial species Bacillus amyloliquefaciens exposed to different inducers, such as chitin, cellulose, iron, glutamic acid and heat-inactivated cells of Colletotrichum sp., were identified and quantified. However, the production of LP homologues was not successful when this bacterium were under the stress of cellulose, chitin, iron and glutamic acid as inducers. In contrast, the antagonistic bacterium produced a higher concentration of LP homologues in the presence of inactivated fungal cells, suggesting that the inducing substance is associated with fungal cell envelope and could be a single protein or a structure that includes protein components. According to the results of this study, B. amyloliquefaciens is considered an effective candidate for the synthesis of different analytes, such as homologues of bacillomycin D, fengycin A and B, and surfactin, which demonstrate antifungal effects on different phytopathogenic species. This information provides the basis for future studies on the induction mechanisms involving inactive fungal cells with the growth of antagonistic bacteria and their effect on the production of LPs with antimicrobial activity.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/fermentation9110944/s1, Table S1. Composition of homologs of the iturin lipopeptide family synthesized by Bacillus amyloliquefaciens and analyzed using ultra-performance liquid chromatography (UPLC-MS/MS); Table S2. Composition of homologs of the fengycin lipopeptide family synthesized by Bacillus amyloliquefaciens and analyzed using ultra-performance liquid chromatography (UPLC-MS/MS); Table S3. Composition of homologs of the surfactin lipopeptide family synthesized by Bacillus amyloliquefaciens and analyzed using ultra-performance liquid chromatography (UPLC-MS/MS).

Author Contributions

Conceptualization, N.L.-L.; Validation, J.B.H.; Software, C.S.M.-H.; Resources, I.M.-Z.; Data curation, R.M.-L.; Supervision, I.C.-L.; Investigation, N.L.-L.; Methodology, N.L.-L.; Project administration, R.S.G.-E.; Writing—Original draft, N.L.-L.; Writing—Review and editing, R.S.G.-E.; Visualization, R.S.G.-E. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by National Council of Humanities, Sciences and Technologies (CONAHCYT) and Food and Development Research Center (CIAD).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data used to support the findings of this study are included within the article.

Conflicts of Interest

The authors declare no conflict of interest.

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Fermentation 09 00944 g001
Figure 1. Chromatograms of homologues belonging to the LP iturin family as determined in the crude extract obtained from B. amyloliquefaciens exposed to the inducer of inactivated fungal cells.
Figure 1. Chromatograms of homologues belonging to the LP iturin family as determined in the crude extract obtained from B. amyloliquefaciens exposed to the inducer of inactivated fungal cells.
Fermentation 09 00944 g001
Fermentation 09 00944 g002
Figure 2. Mass spectra with collision energy ranging from 30 to 50 V for the identification of bacillomycin homologues produced by B. amyloliquefaciens.
Figure 2. Mass spectra with collision energy ranging from 30 to 50 V for the identification of bacillomycin homologues produced by B. amyloliquefaciens.
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Fermentation 09 00944 g003
Figure 3. Chromatograms of homologues belonging to the LP fengycin A (A–D) and B (E–G) family as determined in the crude extracts obtained from B. amyloliquefaciens exposed to the inducer of inactivated fungal cells.
Figure 3. Chromatograms of homologues belonging to the LP fengycin A (A–D) and B (E–G) family as determined in the crude extracts obtained from B. amyloliquefaciens exposed to the inducer of inactivated fungal cells.
Fermentation 09 00944 g003
Fermentation 09 00944 g004
Figure 4. Mass spectra with collision energy ranging from 30 to 50 V for the identification of fengycin A (A–D) and B (E–G) homologues produced by B. amyloliquefaciens.
Figure 4. Mass spectra with collision energy ranging from 30 to 50 V for the identification of fengycin A (A–D) and B (E–G) homologues produced by B. amyloliquefaciens.
Fermentation 09 00944 g004
Fermentation 09 00944 g005
Figure 5. Chromatograms of homologues belonging to the LP surfactin family as determined in the crude extracts obtained from B. amyloliquefaciens exposed to the inducer of inactivated fungal cells.
Figure 5. Chromatograms of homologues belonging to the LP surfactin family as determined in the crude extracts obtained from B. amyloliquefaciens exposed to the inducer of inactivated fungal cells.
Fermentation 09 00944 g005
Fermentation 09 00944 g006
Figure 6. Mass spectra with collision energy ranging from 30 to 50 V for the identification of surfactin homologues produced by B. amyloliquefaciens.
Figure 6. Mass spectra with collision energy ranging from 30 to 50 V for the identification of surfactin homologues produced by B. amyloliquefaciens.
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Table 1. Inducers added to the cultures for lipopeptide synthesis by Bacillus amyloliquefaciens.
Table 1. Inducers added to the cultures for lipopeptide synthesis by Bacillus amyloliquefaciens.
TreatmentsInductorsConcentration
T1Glutamic acid4 g L−1
T2Without inducer-
T3Iron0.3 mg L−1
T4Inactive cells of Colletotrichum sp.mL−1 (1 × 106)
T5Cellulose20 g L−1
T6Chitin4 g L−1
Table 2. Parent ion fragments obtained from the crude extract synthesized by Bacillus amyloliquefaciens for the identification of homologues of LPs belonging to the surfactin, fengycin and iturin families.
Table 2. Parent ion fragments obtained from the crude extract synthesized by Bacillus amyloliquefaciens for the identification of homologues of LPs belonging to the surfactin, fengycin and iturin families.
LPs (Family)HomologuesRetention Times (min)Exact Mass [M + H]+Fragments
IturinBacillomycin5.351031.54227.1028, 754.4310, 978.4970, 1014.5230, 1031.5444
Bacillomycin5.771045.56227.1028, 768.4553, 992.5057, 1011.5144, 1028.5370, 1045.5626
Bacillomycin6.331059.57227.1028, 782.4656, 1007.5164, 1025.5273, 1042.5546, 1059.5754
Bacillomycin6.801073.59227.0997, 796.4755, 1020.5471, 1039.5442, 1073.5944
FengycinFengycin A6.141435.78966.4698, 1080.5443, 1435.7811
Fengycin A6.691449.79966.4698, 1080.5442, 1449.7935
Fengycin A6.901463.81351.1653, 966.4698, 1080.5443, 1463.8191
Fengycin A7.131477.83966.4507, 1080.5443, 1477.8335
Fengycin B7.151491.85994.4919, 1108.57, 1491.8516
Fengycin B7.351505.87994.4919, 1108.57, 1505.8722
Fengycin B7.881519.87349.1362, 994.4919, 1108.57, 1519.8787
SurfactinSurfactin10.49994.64373.1229, 441.2964, 653.4440, 685.4539, 976.6290, 994.6469
Surfactin 10.841008.65441.2964, 667.4569, 685.4431, 990.6501, 1008.6597
Surfactin 11.441022.67441.2681, 685.4485, 1004.6674, 1022.6791
Surfactin 11.761036.68441.2645, 685.4485, 1018.6786, 1036.6897
Surfactin 12.361050.70441.2650, 685.4539, 1032.6882, 1050.7029
Table 3. Quantification of homologues of LPs belonging to the iturin family synthesized by B. amyloliquefaciens (µg mg−1 of crude extract).
Table 3. Quantification of homologues of LPs belonging to the iturin family synthesized by B. amyloliquefaciens (µg mg−1 of crude extract).
Inducer TreatmentBacillomycin (1031.54)Bacillomycin (1045.55)Bacillomycin (1059.57)Bacillomycin (1073.58)
Glutamic acid39.4 ± 4.09 c,d0.1 ± 0.01 c8.5 ± 1.09 b,c4.6 ± 0.64 b
Mly B17 *54.3 ± 3.97 b0.1 ± 0.02 c16.6 ± 1.81 b3.7 ± 0.69 b
Iron32.5 ± 3.13 d0.2 ± 0.06 b6.7 ± 0.87 c3.6 ± 0.56 b
Inactive cells173.1 ± 7.14 a0.4 ± 0.05 a108.5 ± 3.80 a10.7 ± 1.97 a
Cellulose37.5 ± 1.99 c,d0.1 ± 0.02 c8.4 ± 1.86 b,c2.8 ± 0.20 b
Chitin43.4 ± 3.56 c0.1 ± 0.01 c14.1 ± 0.13 b,c2.7 ± 0.49 b
By column, the means that do not share the same letters indicate significant differences between the samples, Tukey (p ≤ 0.05). * Control treatment.
Table 4. Quantification of homologues of LPs belonging to the fengycin family synthesized by B. amyloliquefaciens (ng mg−1 of crude extract).
Table 4. Quantification of homologues of LPs belonging to the fengycin family synthesized by B. amyloliquefaciens (ng mg−1 of crude extract).
Inducer TreatmentFen A (1435.79)Fen A (1449.79)Fen A (1463.79)Fen A (1477.79)Fen B (1491.79)Fen B (1505.79)Fen B (1519.9)
Glutamic acid94 ± 12.8 b53 ± 8.2 c,d656 ± 111.7 b143 ± 30.0 b54 ± 10.9 c13 ± 1.5 b33 ± 2.6 c,d
Mly B17 *74 ± 10.9 b146 ± 40.2 b749 ± 191.3 b134 ± 30.2 b128 ± 21.1 ab18 ± 4.0 b28 ± 6.1 c,d
Iron74 ± 12.0 b35 ± 8.8 d753 ± 99.5 b115 ± 20.2 b46 ± 10.7 c17 ± 2.8 b35 ± 6.6 c
Inactive cells350 ± 47.0 a216 ± 32.2 a3288 ± 528.5 a385 ± 50.0 a168 ± 34.5 a45 ± 5.5 a182 ± 3.8 a
Cellulose45 ± 7.0 b96 ± 7.5 b,c427 ± 105.3 b84 ± 19.6 b100 ± 6.0 b,c11 ± 3.7 b22 ± 2.1 d
Chitin92 ± 17.5 b112 ± 9.1 b,c756 ± 112.6 b146 ± 37.7 b97 ± 20.5 b,c13 ± 2.8 b59 ± 3.7 b
By column, the means that do not share the same letters indicate significant differences between the samples, Tukey (p ≤ 0.05). * Control treatment.
Table 5. Quantification of homologues of LPs belonging to the surfactin family synthesized by B. amyloliquefaciens (µg mg−1 of crude extract).
Table 5. Quantification of homologues of LPs belonging to the surfactin family synthesized by B. amyloliquefaciens (µg mg−1 of crude extract).
Inducer TreatmentSurfactin (994.65)Surfactin (1008.65)Surfactin (1022.66)Surfactin (1036.68)Surfactin (1050.69)
Glutamic acid0.8 ± 0.09 b1.5 ± 0.16 b15.8 ± 3.02 b27.8 ± 1.94 b0.3 ± 0.03 b
Mly B17 *0.3 ± 0.02 b0.9 ± 0.06 b,c6.6 ± 0.92 c,d21.6 ± 3.01 b,c0.4 ± 0.02 b
Iron0.6 ± 0.08 b1.2 ± 0.15 b,c12.0 ± 2.67 b,c18.6 ± 2.66 c,d0.4 ± 0.24 b
Inactive cells4.2 ± 0.80 a7.2 ± 0.82 a42.5 ± 3.85 a61.5 ± 3.01 a1.5 ± 0.28 a
Cellulose0.2 ± 0.01 b0.5 ± 0.09 c4.2 ± 0.79 d13.8 ± 2.01 d0.2 ± 0.03 b
Chitin0.3 ± 0.09 b0.6 ± 0.04 b,c6.0 ± 1.06 c,d15.0 ± 1.99 d0.2 ± 0.02 b
By column, the means that do not share the same letters indicate significant differences between the samples, Tukey (p ≤ 0.05). * Control treatment.
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Ley-López, N.; Heredia, J.B.; Martín-Hernández, C.S.; Cruz-Lachica, I.; Márquez-Zequera, I.; Medina-López, R.; García-Estrada, R.S. Identification and Quantification of Lipopeptide Homologues Induced and Produced by Bacillus amyloliquefaciens. Fermentation 2023, 9, 944. https://doi.org/10.3390/fermentation9110944

AMA Style

Ley-López N, Heredia JB, Martín-Hernández CS, Cruz-Lachica I, Márquez-Zequera I, Medina-López R, García-Estrada RS. Identification and Quantification of Lipopeptide Homologues Induced and Produced by Bacillus amyloliquefaciens. Fermentation. 2023; 9(11):944. https://doi.org/10.3390/fermentation9110944

Chicago/Turabian Style

Ley-López, Nancy, José Basilio Heredia, Cesar San Martín-Hernández, Isabel Cruz-Lachica, Isidro Márquez-Zequera, Raymundo Medina-López, and Raymundo Saúl García-Estrada. 2023. "Identification and Quantification of Lipopeptide Homologues Induced and Produced by Bacillus amyloliquefaciens" Fermentation 9, no. 11: 944. https://doi.org/10.3390/fermentation9110944

APA Style

Ley-López, N., Heredia, J. B., Martín-Hernández, C. S., Cruz-Lachica, I., Márquez-Zequera, I., Medina-López, R., & García-Estrada, R. S. (2023). Identification and Quantification of Lipopeptide Homologues Induced and Produced by Bacillus amyloliquefaciens. Fermentation, 9(11), 944. https://doi.org/10.3390/fermentation9110944

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