Effects of branched-chain amino acids on surfactin structure and antibacterial activity in Bacillus velezensis YA215

Effects of branched-chain amino acids on surfactin structure and antibacterial activity in Bacillus velezensis YA215 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effects of branched-chain amino acids on surfactin structure and antibacterial activity in Bacillus velezensis YA215 Futian Yu, Yuanyuan Shen, Yiyang Pang, Heliang Fan, Mingyuan Liu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4522872/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Jul, 2024 Read the published version in World Journal of Microbiology and Biotechnology → Version 1 posted 10 You are reading this latest preprint version Abstract Antibiotics are essential for combating pathogens; however, their misuse has led to increased resistance, necessitating the search for effective, low-toxicity alternatives. Surfactin, due to its unique structure, exhibits significant antibacterial activity without easily inducing resistance, making it a focus of current research. Nonetheless, the effects of branched-chain amino acids (BCAAs) on surfactin's structure and activity are not well understood. This study examines the influence of BCAAs (L-valine, L-leucine, and L-isoleucine) on the lipopeptide (surfactin) produced by B. velezensis YA215. Process optimization revealed that adding 1 g/L of L-Leu and L-Ile, and 0.5 g/L of L-Val, maximizes surfactin production. Surfactin levels peaked with L-Val and L-Ile at 36 h, while L-Leu reached its maximum at 24 h. Notably, L-Val supplementation resulted in the highest relative surfactin content. Antimicrobial testing demonstrated that BCAAs significantly enhance the antibacterial effects of lipopeptides against Escherichia col i and Staphylococcus aureus , with Val showing the most pronounced effect. The addition of BCAAs notably altered the composition of surfactin fatty acid chains. Specifically, Val increased the proportions of iso C14 and iso C16 β-hydroxy fatty acids from 13.3% and 4.216–23.803% and 8.31%, respectively. Additionally, the amino acid composition at the 7th position of the peptide chain changed significantly, especially with Val addition, which increased the proportion of C14 [Val 7] surfactin by 3.29 times. These structural changes are likely associated with the enhanced antibacterial activity of surfactin. These findings provide valuable insights into the roles of BCAAs in microbial fermentation, underscoring their importance in metabolic engineering to enhance the production of bioactive compounds. Branched-chain amino acids. Bacillus velezensis. Lipopeptide. Surfactin. Antibacterial activity. Fatty acid composition Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Foodborne pathogens not only present substantial economic risks to the food industry but also give rise to severe illnesses and, in some instances, human fatalities. This is an issue of public concern that demands global attention (Du et al. 2021 ). As per the World Health Organization's data, more than 600 million people suffer from foodborne illnesses annually, resulting in 420,000 deaths attributed to the consumption of contaminated food (Kirk et al. 2015 ). E. coli and S. aureus stand out as the most perilous pathogens responsible for foodborne diseases, hospitalizations, and fatalities on a global scale (Bajpai et al. 2022 ). Given the potential health risks associated with chemical preservatives and the emergence of multidrug-resistant bacteria, the food industry is compelled to explore natural compounds, such as bio-preservatives and antimicrobials, to ensure food safety. Surfactin exhibits remarkable antibacterial activity due to its unique molecular structure. Huang et al. demonstrated that surfactin and iturin significantly inhibit Salmonella enteritidis in meat (Huang et al. 2009 ). Further research by Huang et al. established the minimum inhibitory concentration (MIC) of surfactin at 31.25 µg/mL for Bacillus cereus and 15.625 µg/mL for Escherichia coli (Huang et al. 2012 ). Li et al. reported that surfactin’s MIC and minimum bactericidal concentration (MBC) against methicillin-resistant Staphylococcus aureus range from 512–1024 µg/mL and 1024–2048 µg/mL, respectively (Li et al. 2023 ). Ali et al. found that surfactin has an MIC of 50 µg/mL against clinical pathogens including Pseudomonas aeruginosa MTCC424, Escherichia coli MTCC43, Klebsiella pneumoniae MTCC9751, and methicillin-resistant Staphylococcus aureus (Ali et al. 2022 ). Shan et al. showed that Propionibacterium acnes is killed by surfactin in a time- and dose-dependent manner (Shan et al. 2021 ). These findings highlight surfactin's broad-spectrum antibacterial efficacy against various pathogens. Additionally, surfactin disrupts phospholipid bilayers by inserting itself into cell membranes, causing perforations that lead to cell death. This mode of action is less likely to induce resistance, providing a robust foundation for surfactin's potential as a next-generation antibiotic. Its unique bactericidal mechanism not only offers a novel approach to combat bacterial and fungal infections but also presents a promising solution to the issue of antibiotic resistance associated with conventional treatments. Surfactin is a cyclic lipopeptide composed of a heptapeptide ring linked to a β-hydroxy fatty acid chain ranging from C12 to C17 carbon atoms(Hamley et al. 2013 ). Industrial applications of surfactin have been constrained by production limitations, but these have been ameliorated as engineered strains now yield 10–20 g/L of surfactin(Hu et al. 2019 ). The isomeric nature of surfactin significantly impacts its functional activity, yet current research on surfactin isomers remains insufficient. Thus, it is imperative to investigate how the structural isomerism of surfactin influences its bioactivity, thereby providing a theoretical foundation for its application in various fields. The functional activity of surfactin isomers, particularly variations in their fatty acid structures and amino acid sequences, shows significant differences. For example, when the fourth position in the peptide ring component of surfactin changes from L-Val to L-Ile, the critical micelle concentration value is halved, but boundary stability markedly increases(Bonmatin. et al. 1995). Additionally, the chain length of surfactin's fatty acids can affect both its surface activity and bioactivity. Li et al. found that the longer the fatty acid chain of surfactin, the stronger its surface activity(Li et al. 2011 ). Dhali et al. discovered that surfactin with a C14 fatty chain has superior foaming ability compared to those with C13 and C15(Dhali et al. 2017 ). Barale et al. observed that C15 surfactin in a lipopeptide mixture exhibits good antibacterial activity (1600 AU/ml) against antibiotic-resistant foodborne Bacillus cereus and human pathogen Staphylococcus aureus(Barale et al. 2022 ). Deleu et al. concluded that the longer the fatty acid chain, the higher the hemolytic activity, and the lower the critical micelle concentration(Deleu et al. 2003 ). The configuration of surfactin's fatty acid chain also directly influences its antibacterial and surface activities, with the configuration intensity ranked as linear (n), iso, and anteiso. Research by Dufour et al. revealed that linear synthetic C14 surfactin displays minimal or no hemolytic behavior compared to cyclic natural C14 surfactin(Dufour et al. 2005 ). Youssef et al. found that iso-odd fatty acid isomers have higher oil displacement activity than n-even fatty acid isomers, highlighting their potential advantages in specific applications(Youssef et al. 2005 ). Moreover, the proportion of lipopeptide configurations significantly affects their activity. An increase in the proportion of branched configurations results in a notable reduction in the surface activity of lichenysin, whereas a decrease enhances surface activity(Yakimov et al. 1996 ). Liu et al. demonstrated that the higher the content of C15 fatty chain-containing surfactin components at the same working concentration, the greater its efficiency in oil sand cleaning and crude oil displacement(Liu et al. 2015 ). These findings underscore the substantial impact of surfactin isomerism on its functional activity. Therefore, a deeper understanding of the mechanisms underlying surfactin isomerism is crucial for its applications. Surfactin is synthesized through a complex mechanism mediated by nonribosomal peptide synthetases (NRPS) encoded by the srfA operon, which comprises srfAA, srfAB, srfAC, and srfAD(Nakano et al. 1991 ). These enzymes preferentially utilize amino acids and fatty acid residues present in the cytoplasm as substrates(Yang et al. 2015 ). Therefore, amino acids and fatty acids are critical precursors for surfactin biosynthesis. Although fatty acid precursors are essential for the structural formation of surfactin, the direct addition of lipids or free fatty acids to the culture medium does not effectively enhance surfactin production and may even inhibit its synthesis(Yao et al. 2014 ). Conversely, the addition of amino acids directly influences surfactin isomerism by altering the composition of amino acid residues in the peptide chain and the variations in β-hydroxy fatty acid residues, thereby also affecting surfactin yield. Peypoux et al. reported that adding L-valine or L-isoleucine as nitrogen sources selectively enhanced the production of Val7-surfactin(Peypoux et al. 1992). Liu et al. found that adding Arg and Gln to the culture medium of the B. subtilis T8942 strain increased the even β-hydroxy fatty acid fractions (C14, C16), while adding Cys, His, Met, Ser, or Thr increased the odd β-hydroxy fatty acid fractions (C13, C15). Furthermore, the addition of branched-chain amino acids (L-valine, L-leucine, and L-isoleucine) resulted in more significant changes in the β-hydroxy fatty acid residues: L-leucine increased the proportion of iso odd β-hydroxy fatty acid residues, L-isoleucine increased the proportion of anteiso odd β-hydroxy fatty acid residues, and L-valine increased the proportion of iso even β-hydroxy fatty acid residues in the product(Liu et al. 2012 ). In summary, the addition of amino acids significantly impacts the structure of surfactin, notably altering its isomeric forms. However, there is limited literature on how branched-chain amino acids (L-Leu, L-Ile, L-Val) influence the structure and antimicrobial activity of surfactin, necessitating further investigation. This study aims to optimize the fermentation conditions and amino acid supplementation for the B. velezensis YA215 strain, exploring the effects of branched-chain amino acids on the growth, lipopeptide production (mainly surfactin), and antimicrobial activity of B. velezensis YA215. Special attention is given to the regulatory role of branched-chain amino acids (L-Leu, L-Ile, L-Val) in the composition of surfactin during fermentation. By exogenously adding branched-chain amino acids to the culture medium, the optimal conditions for their addition were first determined. Subsequently, the effects of branched-chain amino acids on the growth of B. velezensis YA215, lipopeptide production (primarily surfactin), and antimicrobial activity were analyzed. Finally, the influence of branched-chain amino acids on the structure of surfactin was examined using liquid chromatograph mass spectrometer /mass spectrometer (LC-MS/MS) and gas chromatography-mass spectrometry (GC/MS). Materials and methods Experimental reagents Lysogeny broth (LB), LB nutrient agar, and soluble starch medium (SS) were procured from Qingdao Hope Bio-Technology Co., Ltd.; NaOH, HCl, Na2HPO4, and NaH2PO4 were of analytical grade, purchased from Dongguan Sparta Chemical Co., Ltd.; L-isoleucine (L-Ile), L-leucine (L-Leu), and L-valine (L-Val) were of analytical grade, obtained from Solarbio; trichloroacetic acid was of chromatographic grade, sourced from Macklin; methanol and acetonitrile were of chromatographic grade, acquired from Fisher Chemical. Microbial strains The microbial strains used in this study were B. velezensis YA215 (NCBI number: CP121465.1), Escherichia coli (CGMCC 112252), and Staphylococcus aureus (CGMCC 26003). Effect of fermentation time on surfactin production by B. velezensis YA215 A glycerol stock of B. velezensis YA215 was inoculated onto LB agar plates and incubated at 37°C for 24 h. A single colony was then transferred to LB broth and cultured at 37°C with shaking at 220 r/min for 12 h until reaching the logarithmic phase. Subsequently, 4% (v/v) of this seed culture was transferred into four 1 L flasks containing 600 mL of SS medium. Three of the flasks were supplemented with 1 g/L of L-Leu, L-Ile, and L-Val, respectively, while the fourth flask served as the control without any branched-chain amino acids. Samples were taken at 12, 24, 36, 48, and 60 h, centrifuged at 10,000 g for 10 minutes at 4°C to obtain cell-free supernatants. The relative content of surfactin in the fermentation supernatants was measured using high-performance liquid chromatography (HPLC), with peak areas integrated and the relative surfactin content calculated using the peak area normalization method. Effect of amino acid concentration on surfactin production by B. velezensis YA215 Following the optimization of fermentation time for the addition of branched-chain amino acids to the SS medium, we further optimized the concentration of these amino acids. SS medium was divided into three groups, each supplemented with L-Leu, L-Ile, or L-Val at concentrations of 0.05 g/L, 0.1 g/L, 0.25 g/L, 0.5 g/L, 1 g/L, and 1.5 g/L. The fermentation was conducted using the previously optimized time, followed by centrifugation at 10,000 g for 10 minutes at 4°C to obtain cell-free supernatants. The relative content of surfactin was determined using HPLC, with peak areas integrated and the relative surfactin content calculated using the peak area normalization method. Effect of branched-chain amino acid addition on B. velezensis YA215 growth and lipopeptide production Under the optimized fermentation conditions, the impact of branched-chain amino acids on the growth and lipopeptide production of B. velezensis YA215 was assessed. A needle was used to transfer a glycerol stock of B. velezensis YA215 to LB agar plates, which were incubated at 37°C for 24 h. A single colony was then inoculated into LB broth and cultured at 37°C with shaking at 220 r/min until reaching the logarithmic phase. Subsequently, 4% (v/v) of this seed culture was transferred into four 1 L flasks containing 600 mL of SS medium. Three of the flasks were supplemented with 1 g/L of L-Leu, L-Ile, and L-Val, respectively, while the fourth flask served as the control without any amino acids. The flasks were incubated at 37°C with shaking at 220 r/min for 36 h. After incubation, the supernatants and cells were separated by centrifugation at 10,000 g for 10 minutes at 4°C. The pH of the fermentation supernatants was measured; the biomass was determined after freeze-drying the cells. The lipopeptide yield was assessed following the extraction method described in section lipopeptide extraction . Lipopeptide extraction The fermentation supernatant obtained above was used as the starting material for lipopeptide extraction. The pH of the supernatant was adjusted to 2.0 by adding 6 M HCl and left to precipitate overnight at 4°C. The precipitate was collected by centrifugation at 10,000 g for 10 minutes at 4°C, washed twice with distilled water at pH 2.0, and the pH was then adjusted to 7.0. The precipitate was resuspended in distilled water, freeze-dried, and extracted with methanol. The methanol-soluble fraction was dried using a rotary vacuum evaporator at 45°C. The residue was redissolved in a small amount of methanol, freeze-dried again, and weighed to quantify the lipopeptides. Impact of branched-chain amino acids on the antibacterial activity of lipopeptides The antibacterial activity of crude lipopeptide extracts from B. velezensis YA215, with added branched-chain amino acids, was evaluated using the agar diffusion method against E. coli and S. aureus as indicator organisms. Indicator bacteria from logarithmic phase cultures were incorporated into cooled LB solid medium to a final concentration of 10 6 CFU/mL. After pouring the LB medium into sterile petri dishes and allowing it to solidify, 6 mm wells were punched, and 100 µL of crude lipopeptide extract was added to each well. The plates were incubated overnight at 37°C, and the antibacterial activity was assessed by measuring the diameter of inhibition zones around the wells (Kost et al. 2022 , Alahyaribeik S et al. 2024). Effect of branched-chain amino acids on surfactin fatty acid composition The fatty acid composition of surfactin was analyzed using GC-MS (Singh et al. 2021 ). Lipopeptide samples (2 mg) obtained after the addition of branched-chain amino acids were hydrolyzed in 5 mL tubes with 1.0 mL of 6 mol/L HCl at 110°C for 24 h. Post-hydrolysis, the mixture was transferred to test tubes and extracted thrice with 3 mL dichloromethane. The organic phases were combined and evaporated to dryness at 60°C. The dried samples were esterified with 1.0 mL of 10% H2SO4-methanol solution at 55°C for 6 h. The reaction was terminated by adding 3 mL distilled water, and the esterified fatty acids were extracted thrice with 3 mL n-hexane. The combined organic phases were evaporated to dryness at 40°C and redissolved in 1.0 mL methanol for GC-MS analysis. The GC-MS was equipped with a 7890B gas chromatograph and a 5977A mass spectrometer, using an HP-5MS capillary column (30 m × 0.25 mm × 0.25 µm). The ion source temperature was set to 230°C with ionization energy at 70 eV. Helium (99.999%) was used as the carrier gas at a flow rate of 1.0 mL/min, with the injector temperature at 250°C and an injection volume of 1.0 µL at a split ratio of 10:1. The temperature program started at 100°C (held for 4 min), increased to 250°C at 4°C/min (held for 5 min), and finally to 280°C at 3°C/min (held for 1 min)(Isa et al. 2020 ). Effect of branched-chain amino acids on the amino acid composition of surfactin The changes in the amino acid composition of surfactin after the addition of branched-chain amino acids were analyzed using LC-MS/MS. The LC-MS/MS analysis was performed on an Acclaim PepMapTM RSLC nano-LC system coupled with a Q Exactive™ hybrid quadrupole-Orbitrap mass spectrometer, following previously described procedures. Samples were loaded onto a C18 trapping column (Acclaim PepMap® 100, C18, 100 Å, 3 µm, 75 µm × 2 cm; Thermo Fisher Scientific Inc.) and eluted and separated on an Acclaim PepMap RSLC C18 analytical column (Acclaim PepMapTM RSLC, C18, 100 Å, 2 µm, 50 µm × 15 cm). The mobile phases comprised solvent A (2% acetonitrile, 0.1% formic acid, 97.9% water) and solvent B (98% acetonitrile, 0.1% formic acid, 1.9% water). The gradient for column elution was as follows: 0–5 min, 2–12% B; 5–30 min, 12–20% B; 30–43 min, 20–32% B; 43–48 min, 32–98% B; 48–58 min, 98% B. The flow rate was set to 300 nL/min. The mass spectrometer operated in positive ion mode with a spray voltage of 1.9 kV, capillary temperature of 270°C, and collision energy of 29% HCD. The full scan resolution was set to 70,000 for m/z 445.12003, with a scan range of m/z 300-1,800 . Statistical analysis All experiments were conducted with a minimum of three repetitions, and the mean values ± standard deviations (SD) were calculated. Statistical distinctions between groups were assessed through one-way analysis of variance (ANOVA) using SPSS software (version 20.0; SPSS, Inc., Chicago, IL) (p < 0.05). Results and discussion Influence of fermentation time on surfactin production by B. velezensis YA215 To enhance surfactin yield, we optimized the fermentation duration in SS medium supplemented with branched-chain amino acids. Specifically, 1 g/L of L-Leu, L-Ile, and L-Val were added, and the effect of different fermentation times (12, 24, 36, 48, and 60 h) on surfactin content was analyzed using high-performance liquid chromatography (HPLC). As illustrated in Fig. 1 , surfactin content peaked at 36 h without any branched-chain amino acid supplementation. When L-Val and L-Ile were added, the surfactin levels also reached their maximum at 36 h, mirroring the trend seen in the control group. However, with L-Leu addition, the peak surfactin content was achieved earlier, at 24 h. Among the four groups, the highest surfactin yield was observed in the L-Val group, significantly surpassing the other groups, while the yields in the L-Leu and L-Ile groups were lower than the control SS group. These findings indicate that branched-chain amino acids not only alter the timing of maximum surfactin production by B. velezensis YA215 but also affect the overall surfactin yield, providing essential theoretical insights for optimizing fermentation conditions. Impact of branched-chain amino acids concentration on surfactin production by B. velezensis YA215 Following the optimization of fermentation time with branched-chain amino acid supplementation in SS medium, the concentration of these amino acids was further optimized. Different concentrations of L-Leu, L-Ile, and L-Val were added to the fermentation medium, and the surfactin content in the fermentation supernatant was measured using HPLC at the previously optimized fermentation times. As shown in Fig. 2 , surfactin content increased with the concentration of L-Leu and L-Ile, peaking at 1 g/L. For L-Val, surfactin content also increased with concentration but reached its maximum at 0.5 g/L. Among the three amino acids, L-Val addition resulted in the highest surfactin production, while L-Ile addition resulted in the lowest. These experimental results provide critical data for optimizing surfactin production, particularly regarding the timing and concentration of branched-chain amino acid supplementation. The data offer a solid experimental foundation for increasing surfactin yield and have significant implications for future research and applications in this field(Zhou et al. 2019 ). Previous studies have demonstrated that the addition of branched-chain amino acids can significantly impact surfactin synthesis during microbial fermentation(Balakrishnan et al. 1996). Researchers have observed that adding these amino acids at specific fermentation time points enhances the yield of the target product, aligning with our finding that surfactin content maximized at 36 h with L-Val addition. Furthermore, our results highlight potential optimization opportunities concerning branched-chain amino acid concentrations. Specifically, surfactin production peaked at 1 g/L for L-Leu and L-Ile, corroborating findings by Yao et al., which indicated that moderate amino acid concentrations promote surfactin production during biological fermentation(Yao et al. 2014 ). Impact of branched-chain amino acids supplementation on biomass and lipopeptide production by B. velezensis YA215 Under the optimized fermentation conditions, the influence of branched-chain amino acids on the growth and lipopeptide production of B. velezensis YA215 was thoroughly examined. Supplementing the SS medium with branched-chain amino acids affected both the biomass and lipopeptide yield of the YA215 strain. As depicted in Fig. 3 A, a slight decrease in biomass was observed at 36 h of fermentation with the addition of branched-chain amino acids, although the reduction was not pronounced. Figure 3 B shows that the pH of the fermentation supernatant decreased after 36 h, especially with the addition of Ile and Val, potentially contributing to the biomass reduction. However, this decline in biomass did not exhibit a clear trend. In contrast, lipopeptide yield varied with branched-chain amino acid supplementation, with a significant increase in lipopeptide production observed in the Val-supplemented group compared to the control, whereas Leu and Ile supplementation resulted in decreased lipopeptide yields (Fig. 3 C). This variation in lipopeptide yield differs from the previously discussed surfactin production trends, as lipopeptides comprise surfactin, iturin, and fengycin families(Ongena et al. 2008), and changes in surfactin alone do not fully represent lipopeptide yield trends. These results underscore the multifaceted roles of branched-chain amino acids in microbial fermentation systems, affecting not only specific metabolite yields but also biomass and environmental parameters such as pH. This understanding provides valuable insights into the regulatory mechanisms of microbial fermentation processes. Moreover, in microbial metabolic engineering applications, it is crucial to consider the comprehensive effects of branched-chain amino acid supplementation on the overall production process to design and optimize fermentation conditions more accurately. This study not only offers a solid experimental foundation for surfactin production but also provides valuable references for engineering improvements in related microbial fermentation systems. Additionally, the observed increase in lipopeptide yield with branched-chain amino acid supplementation, particularly with Val, aligns with findings by Zhou et al., who reported that appropriate supplementation of branched-chain amino acids stimulates target product production in similar microbial fermentation experiments, likely due to their role in metabolic pathways(Zhou et al. 2019 ). Effect of branched-chain amino acids on the antibacterial activity of lipopeptides from B. velezensis YA215 Following the optimization of fermentation conditions, the antibacterial activity of lipopeptides extracted from fermentation supernatants with branched-chain amino acid supplementation was evaluated using the acid precipitation and ethanol extraction method. The antibacterial effect against E. coli and S. aureus was assessed using an inhibition zone assay with equal lipopeptide concentrations across four sample groups. As illustrated in Fig. 4 and detailed in Table 1 , lipopeptides from fermentation media supplemented with the three amino acids exhibited superior antibacterial effects against S. aureus and E. coli compared to the control group. Notably, the antibacterial effect was significantly higher in the Val-supplemented samples than in those supplemented with Leu and Ile. Furthermore, the lipopeptides demonstrated a stronger inhibitory effect against E. coli than S. aureus . As bioactive molecules, lipopeptides are categorized into surfactin, iturin, and fengycin families(Ongena et al. 2008), each exhibiting different antibacterial properties. Surfactin, for instance, shows notable antibacterial activity but limited antifungal activity(Buchoux et al. 2008 ), whereas iturin possesses strong antifungal activity(Hua et al. 2023 , Yaraguppi et al. 2023 ), and fengycin, with lower hemolytic activity, displays potent antifungal properties(Fu et al. 2022 , He et al. 2023 ). Previous studies have also indicated that surfactin is the primary compound responsible for antibacterial activity. Our prior research corroborated these findings, identifying surfactin as the principal antibacterial component produced by the YA215 strain(Yu et al. 2022 ). The consistent trend in surfactin yield changes with amino acid supplementation further supports the correlation between surfactin content and lipopeptide antibacterial efficacy. These findings provide valuable clues for understanding the bioactivity of lipopeptides produced by the YA215 strain and their mechanisms of antibacterial action, offering useful references for microbial metabolic engineering in the production of bioactive substances. Table 1 Lipopeptide inhibition results of B. velezensis YA215 strain. Sample S.aureus E.coli Diameter of inhibition zone(mm) SS 10.03 ± 0.21 a 15.17 ± 0.15 a SS + Leu 11.73 ± 0.21 b 17.3 ± 0.29 b SS + Ile 12.83 ± 0.35 c 18.7 ± 0.32 c SS + Val 15 ± 0.56 d 24 ± 0.26 d Note: different letters indicate significant differences between groups (p < 0.05). Effect of branched-chain amino acids on the hydrolyzed fatty acids of lipopeptides from B. velezensis YA215 The increased antibacterial activity of surfactin is not solely linked to its production quantity but also closely associated with structural and conformational changes. In this section, the relationship between the enhanced antibacterial activity of surfactin and its structural modifications is explored. By hydrolyzing lipopeptides extracted from four sample groups, changes in fatty acids were analyzed to infer structural information about surfactin. Initially, the extracted lipopeptides were subjected to high-temperature hydrolysis with hydrochloric acid under identical concentration conditions, resulting in free β-hydroxy fatty acids. These acids were then reacted with methanol under acidic conditions to form β-hydroxy fatty acid methyl esters, which were subsequently detected using GC-MS. Comparative analysis of the total ion chromatograms (Fig. 5 ) and the extracted ion chromatograms with an m/z of 103 (Fig. 6 ) revealed that the total types of β-hydroxy fatty acids did not change with branched-chain amino acid supplementation; all four sample groups exhibited 14 chromatographic peaks. In the mass spectrometry analysis, β-hydroxy fatty acid methyl esters generated M-1, M-18, and M-50 fragment ion peaks due to the loss of H atom, H 2 O, and (CH 3 OH + H 2 O) respectively. Through comparison with the NIST database, it was determined that these 14 fatty acid methyl esters had molecular weights ranging from 230 to 300, differing by 14 units, corresponding to β-hydroxy fatty acid methyl esters with varying carbon chain lengths. β-Hydroxy fatty acid methyl esters with the same carbon chain length exhibited n, iso, or anteiso structures due to branching differences(Cheng et al. 2013 ). It was found that the retention time of β-hydroxy fatty acid methyl esters had a linear relationship with their carbon number, and the retention time followed the order iso < anteiso < n for esters with the same carbon number(Hosono et al. 1983). Based on this analysis, the lipopeptide samples contained β-hydroxy fatty acids with various chain lengths and branching structures, such as iso C12, iso C13, anteiso C13, n C14, iso C14, iso C15, anteiso C15, n C15, iso C16, n C16, iso C17, anteiso C17, and n C18, highlighting the diversity and structural complexity of the fatty acid chains. Further analysis of the β-hydroxy fatty acid content variation with different carbon chain lengths (Fig. 7 ) showed that, compared to the control group, the proportions of C12 and C13 β-hydroxy fatty acids decreased with branched-chain amino acid addition. With Val supplementation, the content of C14 β-hydroxy fatty acids increased compared to the control, while other chain lengths decreased. With Leu supplementation, the contents of C15 and C17 β-hydroxy fatty acids increased, whereas other chain lengths decreased. With Ile supplementation, the contents of C16, C17, and C18 β-hydroxy fatty acids increased, while other chain lengths decreased. Further exploration of the conformational changes in β-hydroxy fatty acids with different chain lengths (Fig. 8 ) indicated that, compared to the control group, the contents of n C12 β-hydroxy fatty acids, iso C13 β-hydroxy fatty acids, and anteiso C13 β-hydroxy fatty acids decreased with branched-chain amino acid addition. With Val supplementation, the proportion of iso C14 β-hydroxy fatty acids increased from 13.30–23.80%, and iso C16 from 4.22–8.31%, while other conformations decreased. With Leu supplementation, the proportions of iso C15, anteiso C15, iso C17, and anteiso C17 β-hydroxy fatty acids increased, especially iso C15, which rose from 11.42–26.81%, while other conformations decreased. With Ile supplementation, the proportions of n C14, anteiso C15, n C16, anteiso C17, n C17, and n C18 β-hydroxy fatty acids increased, particularly n C16, which significantly rose from 10.37–20.54%, while other carbon chain lengths decreased. The analysis of the experimental data revealed that the addition of branched-chain amino acids altered the content and configuration of β-hydroxy fatty acids of various chain lengths. This phenomenon might be due to the specific structural effects of these branched chains on the formation and antibacterial activity of surfactin. Liu et al. discovered that adding Arg and Gln to the culture medium of B. subtilis T8942 increased the even-numbered β-hydroxy fatty acids (C14, C16), while adding Cys, His, Met, Ser, or Thr increased the odd-numbered β-hydroxy fatty acids (C13, C15) (Liu et al. 2012 ). Furthermore, they noted that the addition of branched-chain amino acids (L-Val, L-Leu, and L-Ile) led to significant changes in hydroxy fatty acid residues: L-Leu markedly increased the proportion of iso odd-numbered hydroxy fatty acid residues; L-Ile enhanced the proportion of anteiso odd-numbered hydroxy fatty acid residues; and L-Val elevated the proportion of iso even-numbered hydroxy fatty acid residues. Our experimental results also indicated that Val addition significantly increased the content of C14 β-hydroxy fatty acids, while Leu and Ile additions notably increased the contents of C15, C16, C17, and C18 β-hydroxy fatty acids, highlighting the impact of branched-chain amino acids on surfactin's structure and configuration. Impact of branched-chain amino acids on the peptide chains of surfactin in B. velezensis YA215 The changes in the peptide chain composition of surfactin under the same concentration conditions in four sample groups were analyzed using LC-MS/MS, providing critical insights into its molecular structure. Table 2 shows that the SS fermentation produced surfactin with peptide chains Glu-Leu-Leu-Val-Asp-Leu-Leu and Glu-Leu-Leu-Val-Asp-Leu-Val, connected to C13-C16 fatty acid chains, known as [Leu 7]surfactin and [Val 7]surfactin, respectively, as well as [Leu 7]surfactin linked to C12 fatty acid chains. Upon Leu addition, [Val 7]surfactin was converted to [Leu 7]surfactin, resulting in a single peptide chain structure of [Leu 7]surfactin. With Ile addition, the Val at the 7th position in the peptide chains of C12, C14, and C16 surfactin was replaced by Leu, unifying the peptide chain structure as [Leu 7]surfactin. The C13 surfactin peptide chain structure remained consistent with the control SS. However, the C15 surfactin peptide chains exhibited two structures: [Leu 7]surfactin and [Leu/Ile 4]surfactin, where Val at the 4th position was replaced by Leu or Ile. Upon Val addition, the C13 surfactin peptide chain displayed two structures: one with Leu at the 7th position replaced by Val, forming [Val 7]surfactin, and another consistent with the control SS, [Leu 7]surfactin. The peptide chain structures of C12, C14, C15, and C16 surfactin were consistent with the control SS, each containing two structures, but the proportion of [Val 7]surfactin notably increased (Fig. 9 ). Quantitative analysis indicated that the proportion of C14 [Val 7]surfactin increased by 3.29 times compared to the control SS, aligning with the previously observed fatty acid changes. This could be attributed to the addition of Val in the SS medium, enhancing the antibacterial activity of lipopeptides. Additionally, it was observed that adding L-Val and L-Ile to the medium selectively increased the yield of [Val 7]surfactin, whereas the addition of L-Leu resulted in a lower yield of [Val 7]surfactin. This selective effect may be associated with the cross-relationships between amino acid biosynthesis and metabolic pathways. This finding is consistent with previous studies on the regulatory role of amino acids in lipopeptide biosynthesis. Peypoux et al. found that supplementing the medium with L-Val or L-Ile as nitrogen sources selectively enhanced the production of Val7-surfactin (Peypoux et al. 1992). Table 2 Results of changes in the structure of the peptide chain of surfactin. Retention Time (min) Main LC-MS Peak(m/z) β-OH fatty acids Peptide sequence SS 12.99–14.10 994.57 C12 N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C 13.57 1008 C13 N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C 14.29 1008 C14 N-Glu-Leu-Leu-Val-Asp-Leu-Val-C 14.09 1022 C14 N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C 14.64 1022 C15 N-Glu-Leu-Leu-Val-Asp-Leu-Val-C 14.37 1036 C15 N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C 15.27 1036 C16 N-Glu-Leu-Leu-Val-Asp-Leu-Val-C 15.01 1050 C16 N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C SS + Leu 12.71–14.10 994 C12 N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C 13.54 1008 C13 N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C 14.26 1008 C14 N-Glu-Leu-Leu-Val-Asp-Leu-Val-C 14.06,14.60 1022 C14 N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C 14.51,15.37 1036 C15 N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C 14.92 1050 C16 N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C SS + Ile 12.71–14.10 994 C12 N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C 13.53 1008 C13 N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C 1008 C14 N-Glu-Leu-Leu-Val-Asp-Leu-Val-C 14.07,14.60 1022 C14 N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C 14.40,14.94,15.30 1036 C15 N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C 15.15,16.18 1050 C16 N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C 15.66 1050 C15 N-Glu-Leu-Leu-Leu/Ile-Asp-Leu-Leu-C SS + Val 12.71–13.20 994 C12 N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C 13.20–14.10 994 C13 N-Glu-Leu-Leu-Val-Asp-Leu-Val-C 13.57 1008 C13 N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C 14.29 1008 C14 N-Glu-Leu-Leu-Val-Asp-Leu-Val-C 14.04 1022 C14 N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C 14.59 1022 C15 N-Glu-Leu-Leu-Val-Asp-Leu-Val-C 14.49 1036 C15 N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C 15.20 1036 C16 N-Glu-Leu-Leu-Val-Asp-Leu-Val-C 15.01 1050 C16 N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C Conclusion This study underscores the significant impact of branched-chain amino acids (BCAAs) on the structure and antibacterial activity of surfactin produced by Bacillus velezensis YA215. Process optimization revealed that adding 1 g/L of L-Leu and L-Ile, and 0.5 g/L of L-Val, maximizes surfactin production. Surfactin levels peaked with L-Val and L-Ile at 36 h, while L-Leu reached its maximum at 24 h. Notably, L-Val supplementation yielded the highest relative surfactin content. Antimicrobial assays revealed that BCAAs markedly improved the antibacterial efficacy of lipopeptide against Escherichia col i and Staphylococcus aureus , with L-Val demonstrating the most significant effect. Additionally, BCAA incorporation significantly modified the composition of surfactin fatty acid chains. In particular, L-Val addition increased the proportions of iso-C14 and iso-C16 β-hydroxy fatty acids and altered the amino acid composition at the 7th position of the peptide chain. These structural modifications are likely linked to the enhanced antibacterial activity of surfactin. 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15:40:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4522872/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4522872/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11274-024-04088-7","type":"published","date":"2024-07-27T16:16:24+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":58564280,"identity":"db821d17-672e-4026-8426-31b80247b6f6","added_by":"auto","created_at":"2024-06-18 09:38:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":41965,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of fermentation time on surfactin production by \u003cem\u003eB. velezensis\u003c/em\u003e YA215 strain.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4522872/v1/b0c803ecf6d8ce81ca4a6986.png"},{"id":58564274,"identity":"51f6e51a-0a3f-4e5c-a446-9d8e7310b036","added_by":"auto","created_at":"2024-06-18 09:38:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":58577,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of amino acid addition on surfactin production by \u003cem\u003eB. velezensis\u003c/em\u003eYA215 strain.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4522872/v1/b90c942fe12edfaf97de5b72.png"},{"id":58564275,"identity":"916dc34a-4268-4aa1-bf39-e9ee9da810a2","added_by":"auto","created_at":"2024-06-18 09:38:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":519496,"visible":true,"origin":"","legend":"\u003cp\u003eResults of the effect of branched-chain amino acids on the growth and lipopeptide production of \u003cem\u003eB. velezensis\u003c/em\u003eYA215 strain (A: Changes in biomass of \u003cem\u003eB. velezensis\u003c/em\u003e YA215 strain, B: Changes in pH of fermentation supernatant, C: Changes in lipopeptide production).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4522872/v1/8c3838a57097c73b90a31ce8.png"},{"id":58564285,"identity":"5b028712-1843-4e3b-b3af-719e055b6b04","added_by":"auto","created_at":"2024-06-18 09:38:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":12857313,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of branched-chain amino acids on the inhibition of lipopeptides in \u003cem\u003eB. velezensis\u003c/em\u003e YA215 strain (Inhibitory effect of equal concentrations of lipopeptides (2 mg/mL) on \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus, \u003c/em\u003eSS samples were fermentation medium blank group samples, SS+Leu, SS+Ile and SS+Val were Leu, Ile and Val addition treatment groups, respectively, and MeOH was a methanol solvent negative control group sample).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4522872/v1/0b4eb61c7fd19a74ac9321f3.png"},{"id":58564978,"identity":"ea936be3-2d5b-4a7e-8f73-f887e62586de","added_by":"auto","created_at":"2024-06-18 09:46:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":39030,"visible":true,"origin":"","legend":"\u003cp\u003eTotal ion chromatogram of the esterified fatty acid molecules.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4522872/v1/ae4314d12d2d7a059420edba.png"},{"id":58564279,"identity":"0913247e-7987-431b-97c5-4f47bcfccea6","added_by":"auto","created_at":"2024-06-18 09:38:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":834553,"visible":true,"origin":"","legend":"\u003cp\u003eExtracted ion chromatogram of esterified fatty acid molecules (m/z=103).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4522872/v1/c675f3c610dd56693772efe1.png"},{"id":58564286,"identity":"a31d44c5-ffaf-41a8-8ba6-966b8e7835c7","added_by":"auto","created_at":"2024-06-18 09:38:15","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":300191,"visible":true,"origin":"","legend":"\u003cp\u003eLength distribution of esterified fatty acid chains.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4522872/v1/d0613091449411af26a51b4b.png"},{"id":58564284,"identity":"55a5ce01-71b3-46d2-a00b-9cea1fdbf4b8","added_by":"auto","created_at":"2024-06-18 09:38:15","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":300874,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of chain conformations of esterified fatty acids.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-4522872/v1/0059189dfb59cba434594704.png"},{"id":58564975,"identity":"d1986dd2-e9ad-46bc-a35b-6a1ecff88650","added_by":"auto","created_at":"2024-06-18 09:46:14","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":396076,"visible":true,"origin":"","legend":"\u003cp\u003eResults of the effect of Val addition on the structure of surfactin peptide chains.\u003c/p\u003e","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4522872/v1/7cb0d798242ca4a0442ded19.jpeg"},{"id":61596515,"identity":"02261c8d-b47a-4736-a6f9-4ecbba3396ed","added_by":"auto","created_at":"2024-08-01 17:28:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":25740593,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4522872/v1/7ff9e643-da8a-4b09-8661-c4dbca813410.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of branched-chain amino acids on surfactin structure and antibacterial activity in Bacillus velezensis YA215","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFoodborne pathogens not only present substantial economic risks to the food industry but also give rise to severe illnesses and, in some instances, human fatalities. This is an issue of public concern that demands global attention (Du et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). As per the World Health Organization's data, more than 600\u0026nbsp;million people suffer from foodborne illnesses annually, resulting in 420,000 deaths attributed to the consumption of contaminated food (Kirk et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e stand out as the most perilous pathogens responsible for foodborne diseases, hospitalizations, and fatalities on a global scale (Bajpai et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Given the potential health risks associated with chemical preservatives and the emergence of multidrug-resistant bacteria, the food industry is compelled to explore natural compounds, such as bio-preservatives and antimicrobials, to ensure food safety.\u003c/p\u003e \u003cp\u003eSurfactin exhibits remarkable antibacterial activity due to its unique molecular structure. Huang et al. demonstrated that surfactin and iturin significantly inhibit \u003cem\u003eSalmonella enteritidis\u003c/em\u003e in meat (Huang et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Further research by Huang et al. established the minimum inhibitory concentration (MIC) of surfactin at 31.25 \u0026micro;g/mL for \u003cem\u003eBacillus cereus\u003c/em\u003e and 15.625 \u0026micro;g/mL for \u003cem\u003eEscherichia coli\u003c/em\u003e (Huang et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Li et al. reported that surfactin\u0026rsquo;s MIC and minimum bactericidal concentration (MBC) against methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e range from 512\u0026ndash;1024 \u0026micro;g/mL and 1024\u0026ndash;2048 \u0026micro;g/mL, respectively (Li et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Ali et al. found that surfactin has an MIC of 50 \u0026micro;g/mL against clinical pathogens including \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e MTCC424, \u003cem\u003eEscherichia coli\u003c/em\u003e MTCC43, \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e MTCC9751, and methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (Ali et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Shan et al. showed that \u003cem\u003ePropionibacterium acnes\u003c/em\u003e is killed by surfactin in a time- and dose-dependent manner (Shan et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These findings highlight surfactin's broad-spectrum antibacterial efficacy against various pathogens. Additionally, surfactin disrupts phospholipid bilayers by inserting itself into cell membranes, causing perforations that lead to cell death. This mode of action is less likely to induce resistance, providing a robust foundation for surfactin's potential as a next-generation antibiotic. Its unique bactericidal mechanism not only offers a novel approach to combat bacterial and fungal infections but also presents a promising solution to the issue of antibiotic resistance associated with conventional treatments.\u003c/p\u003e \u003cp\u003eSurfactin is a cyclic lipopeptide composed of a heptapeptide ring linked to a β-hydroxy fatty acid chain ranging from C12 to C17 carbon atoms(Hamley et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Industrial applications of surfactin have been constrained by production limitations, but these have been ameliorated as engineered strains now yield 10\u0026ndash;20 g/L of surfactin(Hu et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The isomeric nature of surfactin significantly impacts its functional activity, yet current research on surfactin isomers remains insufficient. Thus, it is imperative to investigate how the structural isomerism of surfactin influences its bioactivity, thereby providing a theoretical foundation for its application in various fields.\u003c/p\u003e \u003cp\u003eThe functional activity of surfactin isomers, particularly variations in their fatty acid structures and amino acid sequences, shows significant differences. For example, when the fourth position in the peptide ring component of surfactin changes from L-Val to L-Ile, the critical micelle concentration value is halved, but boundary stability markedly increases(Bonmatin. et al. 1995). Additionally, the chain length of surfactin's fatty acids can affect both its surface activity and bioactivity. Li et al. found that the longer the fatty acid chain of surfactin, the stronger its surface activity(Li et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Dhali et al. discovered that surfactin with a C14 fatty chain has superior foaming ability compared to those with C13 and C15(Dhali et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Barale et al. observed that C15 surfactin in a lipopeptide mixture exhibits good antibacterial activity (1600 AU/ml) against antibiotic-resistant foodborne Bacillus cereus and human pathogen Staphylococcus aureus(Barale et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Deleu et al. concluded that the longer the fatty acid chain, the higher the hemolytic activity, and the lower the critical micelle concentration(Deleu et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The configuration of surfactin's fatty acid chain also directly influences its antibacterial and surface activities, with the configuration intensity ranked as linear (n), iso, and anteiso. Research by Dufour et al. revealed that linear synthetic C14 surfactin displays minimal or no hemolytic behavior compared to cyclic natural C14 surfactin(Dufour et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Youssef et al. found that iso-odd fatty acid isomers have higher oil displacement activity than n-even fatty acid isomers, highlighting their potential advantages in specific applications(Youssef et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Moreover, the proportion of lipopeptide configurations significantly affects their activity. An increase in the proportion of branched configurations results in a notable reduction in the surface activity of lichenysin, whereas a decrease enhances surface activity(Yakimov et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). Liu et al. demonstrated that the higher the content of C15 fatty chain-containing surfactin components at the same working concentration, the greater its efficiency in oil sand cleaning and crude oil displacement(Liu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). These findings underscore the substantial impact of surfactin isomerism on its functional activity. Therefore, a deeper understanding of the mechanisms underlying surfactin isomerism is crucial for its applications.\u003c/p\u003e \u003cp\u003eSurfactin is synthesized through a complex mechanism mediated by nonribosomal peptide synthetases (NRPS) encoded by the srfA operon, which comprises srfAA, srfAB, srfAC, and srfAD(Nakano et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). These enzymes preferentially utilize amino acids and fatty acid residues present in the cytoplasm as substrates(Yang et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Therefore, amino acids and fatty acids are critical precursors for surfactin biosynthesis. Although fatty acid precursors are essential for the structural formation of surfactin, the direct addition of lipids or free fatty acids to the culture medium does not effectively enhance surfactin production and may even inhibit its synthesis(Yao et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Conversely, the addition of amino acids directly influences surfactin isomerism by altering the composition of amino acid residues in the peptide chain and the variations in β-hydroxy fatty acid residues, thereby also affecting surfactin yield. Peypoux et al. reported that adding L-valine or L-isoleucine as nitrogen sources selectively enhanced the production of Val7-surfactin(Peypoux et al. 1992). Liu et al. found that adding Arg and Gln to the culture medium of the B. subtilis T8942 strain increased the even β-hydroxy fatty acid fractions (C14, C16), while adding Cys, His, Met, Ser, or Thr increased the odd β-hydroxy fatty acid fractions (C13, C15). Furthermore, the addition of branched-chain amino acids (L-valine, L-leucine, and L-isoleucine) resulted in more significant changes in the β-hydroxy fatty acid residues: L-leucine increased the proportion of iso odd β-hydroxy fatty acid residues, L-isoleucine increased the proportion of anteiso odd β-hydroxy fatty acid residues, and L-valine increased the proportion of iso even β-hydroxy fatty acid residues in the product(Liu et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In summary, the addition of amino acids significantly impacts the structure of surfactin, notably altering its isomeric forms. However, there is limited literature on how branched-chain amino acids (L-Leu, L-Ile, L-Val) influence the structure and antimicrobial activity of surfactin, necessitating further investigation.\u003c/p\u003e \u003cp\u003eThis study aims to optimize the fermentation conditions and amino acid supplementation for the \u003cem\u003eB. velezensis\u003c/em\u003e YA215 strain, exploring the effects of branched-chain amino acids on the growth, lipopeptide production (mainly surfactin), and antimicrobial activity of \u003cem\u003eB. velezensis\u003c/em\u003e YA215. Special attention is given to the regulatory role of branched-chain amino acids (L-Leu, L-Ile, L-Val) in the composition of surfactin during fermentation. By exogenously adding branched-chain amino acids to the culture medium, the optimal conditions for their addition were first determined. Subsequently, the effects of branched-chain amino acids on the growth of \u003cem\u003eB. velezensis\u003c/em\u003e YA215, lipopeptide production (primarily surfactin), and antimicrobial activity were analyzed. Finally, the influence of branched-chain amino acids on the structure of surfactin was examined using liquid chromatograph mass spectrometer /mass spectrometer (LC-MS/MS) and gas chromatography-mass spectrometry (GC/MS).\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperimental reagents\u003c/h2\u003e \u003cp\u003eLysogeny broth (LB), LB nutrient agar, and soluble starch medium (SS) were procured from Qingdao Hope Bio-Technology Co., Ltd.; NaOH, HCl, Na2HPO4, and NaH2PO4 were of analytical grade, purchased from Dongguan Sparta Chemical Co., Ltd.; L-isoleucine (L-Ile), L-leucine (L-Leu), and L-valine (L-Val) were of analytical grade, obtained from Solarbio; trichloroacetic acid was of chromatographic grade, sourced from Macklin; methanol and acetonitrile were of chromatographic grade, acquired from Fisher Chemical.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMicrobial strains\u003c/h2\u003e \u003cp\u003eThe microbial strains used in this study were \u003cem\u003eB. velezensis\u003c/em\u003e YA215 (NCBI number: CP121465.1), \u003cem\u003eEscherichia coli\u003c/em\u003e (CGMCC 112252), and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (CGMCC 26003).\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of fermentation time on surfactin production by\u003c/b\u003e \u003cb\u003eB. velezensis\u003c/b\u003e \u003cb\u003eYA215\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA glycerol stock of \u003cem\u003eB. velezensis\u003c/em\u003e YA215 was inoculated onto LB agar plates and incubated at 37\u0026deg;C for 24 h. A single colony was then transferred to LB broth and cultured at 37\u0026deg;C with shaking at 220 r/min for 12 h until reaching the logarithmic phase. Subsequently, 4% (v/v) of this seed culture was transferred into four 1 L flasks containing 600 mL of SS medium. Three of the flasks were supplemented with 1 g/L of L-Leu, L-Ile, and L-Val, respectively, while the fourth flask served as the control without any branched-chain amino acids. Samples were taken at 12, 24, 36, 48, and 60 h, centrifuged at 10,000 g for 10 minutes at 4\u0026deg;C to obtain cell-free supernatants. The relative content of surfactin in the fermentation supernatants was measured using high-performance liquid chromatography (HPLC), with peak areas integrated and the relative surfactin content calculated using the peak area normalization method.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of amino acid concentration on surfactin production by\u003c/b\u003e \u003cb\u003eB. velezensis\u003c/b\u003e \u003cb\u003eYA215\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFollowing the optimization of fermentation time for the addition of branched-chain amino acids to the SS medium, we further optimized the concentration of these amino acids. SS medium was divided into three groups, each supplemented with L-Leu, L-Ile, or L-Val at concentrations of 0.05 g/L, 0.1 g/L, 0.25 g/L, 0.5 g/L, 1 g/L, and 1.5 g/L. The fermentation was conducted using the previously optimized time, followed by centrifugation at 10,000 g for 10 minutes at 4\u0026deg;C to obtain cell-free supernatants. The relative content of surfactin was determined using HPLC, with peak areas integrated and the relative surfactin content calculated using the peak area normalization method.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of branched-chain amino acid addition on\u003c/b\u003e \u003cb\u003eB. velezensis\u003c/b\u003e \u003cb\u003eYA215 growth and lipopeptide production\u003c/b\u003e\u003c/p\u003e \u003cp\u003eUnder the optimized fermentation conditions, the impact of branched-chain amino acids on the growth and lipopeptide production of \u003cem\u003eB. velezensis\u003c/em\u003e YA215 was assessed. A needle was used to transfer a glycerol stock of \u003cem\u003eB. velezensis\u003c/em\u003e YA215 to LB agar plates, which were incubated at 37\u0026deg;C for 24 h. A single colony was then inoculated into LB broth and cultured at 37\u0026deg;C with shaking at 220 r/min until reaching the logarithmic phase. Subsequently, 4% (v/v) of this seed culture was transferred into four 1 L flasks containing 600 mL of SS medium. Three of the flasks were supplemented with 1 g/L of L-Leu, L-Ile, and L-Val, respectively, while the fourth flask served as the control without any amino acids. The flasks were incubated at 37\u0026deg;C with shaking at 220 r/min for 36 h. After incubation, the supernatants and cells were separated by centrifugation at 10,000 g for 10 minutes at 4\u0026deg;C. The pH of the fermentation supernatants was measured; the biomass was determined after freeze-drying the cells. The lipopeptide yield was assessed following the extraction method described in section \u003cspan refid=\"Sec5\" class=\"InternalRef\"\u003elipopeptide extraction\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eLipopeptide extraction\u003c/h2\u003e \u003cp\u003eThe fermentation supernatant obtained above was used as the starting material for lipopeptide extraction. The pH of the supernatant was adjusted to 2.0 by adding 6 M HCl and left to precipitate overnight at 4\u0026deg;C. The precipitate was collected by centrifugation at 10,000 g for 10 minutes at 4\u0026deg;C, washed twice with distilled water at pH 2.0, and the pH was then adjusted to 7.0. The precipitate was resuspended in distilled water, freeze-dried, and extracted with methanol. The methanol-soluble fraction was dried using a rotary vacuum evaporator at 45\u0026deg;C. The residue was redissolved in a small amount of methanol, freeze-dried again, and weighed to quantify the lipopeptides.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eImpact of branched-chain amino acids on the antibacterial activity of lipopeptides\u003c/h2\u003e \u003cp\u003eThe antibacterial activity of crude lipopeptide extracts from \u003cem\u003eB. velezensis\u003c/em\u003e YA215, with added branched-chain amino acids, was evaluated using the agar diffusion method against \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e as indicator organisms. Indicator bacteria from logarithmic phase cultures were incorporated into cooled LB solid medium to a final concentration of 10\u003csup\u003e6\u003c/sup\u003e CFU/mL. After pouring the LB medium into sterile petri dishes and allowing it to solidify, 6 mm wells were punched, and 100 \u0026micro;L of crude lipopeptide extract was added to each well. The plates were incubated overnight at 37\u0026deg;C, and the antibacterial activity was assessed by measuring the diameter of inhibition zones around the wells (Kost et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Alahyaribeik S et al. 2024).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eEffect of branched-chain amino acids on surfactin fatty acid composition\u003c/h2\u003e \u003cp\u003eThe fatty acid composition of surfactin was analyzed using GC-MS (Singh et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Lipopeptide samples (2 mg) obtained after the addition of branched-chain amino acids were hydrolyzed in 5 mL tubes with 1.0 mL of 6 mol/L HCl at 110\u0026deg;C for 24 h. Post-hydrolysis, the mixture was transferred to test tubes and extracted thrice with 3 mL dichloromethane. The organic phases were combined and evaporated to dryness at 60\u0026deg;C. The dried samples were esterified with 1.0 mL of 10% H2SO4-methanol solution at 55\u0026deg;C for 6 h. The reaction was terminated by adding 3 mL distilled water, and the esterified fatty acids were extracted thrice with 3 mL n-hexane. The combined organic phases were evaporated to dryness at 40\u0026deg;C and redissolved in 1.0 mL methanol for GC-MS analysis. The GC-MS was equipped with a 7890B gas chromatograph and a 5977A mass spectrometer, using an HP-5MS capillary column (30 m \u0026times; 0.25 mm \u0026times; 0.25 \u0026micro;m). The ion source temperature was set to 230\u0026deg;C with ionization energy at 70 eV. Helium (99.999%) was used as the carrier gas at a flow rate of 1.0 mL/min, with the injector temperature at 250\u0026deg;C and an injection volume of 1.0 \u0026micro;L at a split ratio of 10:1. The temperature program started at 100\u0026deg;C (held for 4 min), increased to 250\u0026deg;C at 4\u0026deg;C/min (held for 5 min), and finally to 280\u0026deg;C at 3\u0026deg;C/min (held for 1 min)(Isa et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEffect of branched-chain amino acids on the amino acid composition of surfactin\u003c/h2\u003e \u003cp\u003eThe changes in the amino acid composition of surfactin after the addition of branched-chain amino acids were analyzed using LC-MS/MS. The LC-MS/MS analysis was performed on an Acclaim PepMapTM RSLC nano-LC system coupled with a Q Exactive\u0026trade; hybrid quadrupole-Orbitrap mass spectrometer, following previously described procedures. Samples were loaded onto a C18 trapping column (Acclaim PepMap\u0026reg; 100, C18, 100 \u0026Aring;, 3 \u0026micro;m, 75 \u0026micro;m \u0026times; 2 cm; Thermo Fisher Scientific Inc.) and eluted and separated on an Acclaim PepMap RSLC C18 analytical column (Acclaim PepMapTM RSLC, C18, 100 \u0026Aring;, 2 \u0026micro;m, 50 \u0026micro;m \u0026times; 15 cm). The mobile phases comprised solvent A (2% acetonitrile, 0.1% formic acid, 97.9% water) and solvent B (98% acetonitrile, 0.1% formic acid, 1.9% water). The gradient for column elution was as follows: 0\u0026ndash;5 min, 2\u0026ndash;12% B; 5\u0026ndash;30 min, 12\u0026ndash;20% B; 30\u0026ndash;43 min, 20\u0026ndash;32% B; 43\u0026ndash;48 min, 32\u0026ndash;98% B; 48\u0026ndash;58 min, 98% B. The flow rate was set to 300 nL/min. The mass spectrometer operated in positive ion mode with a spray voltage of 1.9 kV, capillary temperature of 270\u0026deg;C, and collision energy of 29% HCD. The full scan resolution was set to 70,000 for m/z 445.12003, with a scan range of m/z 300-1,800 .\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were conducted with a minimum of three repetitions, and the mean values\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations (SD) were calculated. Statistical distinctions between groups were assessed through one-way analysis of variance (ANOVA) using SPSS software (version 20.0; SPSS, Inc., Chicago, IL) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e \u003cb\u003eInfluence of fermentation time on surfactin production by\u003c/b\u003e \u003cb\u003eB. velezensis\u003c/b\u003e \u003cb\u003eYA215\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo enhance surfactin yield, we optimized the fermentation duration in SS medium supplemented with branched-chain amino acids. Specifically, 1 g/L of L-Leu, L-Ile, and L-Val were added, and the effect of different fermentation times (12, 24, 36, 48, and 60 h) on surfactin content was analyzed using high-performance liquid chromatography (HPLC). As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, surfactin content peaked at 36 h without any branched-chain amino acid supplementation. When L-Val and L-Ile were added, the surfactin levels also reached their maximum at 36 h, mirroring the trend seen in the control group. However, with L-Leu addition, the peak surfactin content was achieved earlier, at 24 h. Among the four groups, the highest surfactin yield was observed in the L-Val group, significantly surpassing the other groups, while the yields in the L-Leu and L-Ile groups were lower than the control SS group. These findings indicate that branched-chain amino acids not only alter the timing of maximum surfactin production by \u003cem\u003eB. velezensis\u003c/em\u003e YA215 but also affect the overall surfactin yield, providing essential theoretical insights for optimizing fermentation conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eImpact of branched-chain amino acids concentration on surfactin production by\u003c/b\u003e \u003cb\u003eB. velezensis\u003c/b\u003e \u003cb\u003eYA215\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFollowing the optimization of fermentation time with branched-chain amino acid supplementation in SS medium, the concentration of these amino acids was further optimized. Different concentrations of L-Leu, L-Ile, and L-Val were added to the fermentation medium, and the surfactin content in the fermentation supernatant was measured using HPLC at the previously optimized fermentation times. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, surfactin content increased with the concentration of L-Leu and L-Ile, peaking at 1 g/L. For L-Val, surfactin content also increased with concentration but reached its maximum at 0.5 g/L. Among the three amino acids, L-Val addition resulted in the highest surfactin production, while L-Ile addition resulted in the lowest.\u003c/p\u003e \u003cp\u003eThese experimental results provide critical data for optimizing surfactin production, particularly regarding the timing and concentration of branched-chain amino acid supplementation. The data offer a solid experimental foundation for increasing surfactin yield and have significant implications for future research and applications in this field(Zhou et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Previous studies have demonstrated that the addition of branched-chain amino acids can significantly impact surfactin synthesis during microbial fermentation(Balakrishnan et al. 1996). Researchers have observed that adding these amino acids at specific fermentation time points enhances the yield of the target product, aligning with our finding that surfactin content maximized at 36 h with L-Val addition. Furthermore, our results highlight potential optimization opportunities concerning branched-chain amino acid concentrations. Specifically, surfactin production peaked at 1 g/L for L-Leu and L-Ile, corroborating findings by Yao et al., which indicated that moderate amino acid concentrations promote surfactin production during biological fermentation(Yao et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eImpact of branched-chain amino acids supplementation on biomass and lipopeptide production by\u003c/b\u003e \u003cb\u003eB. velezensis\u003c/b\u003e \u003cb\u003eYA215\u003c/b\u003e\u003c/p\u003e \u003cp\u003eUnder the optimized fermentation conditions, the influence of branched-chain amino acids on the growth and lipopeptide production of \u003cem\u003eB. velezensis\u003c/em\u003e YA215 was thoroughly examined. Supplementing the SS medium with branched-chain amino acids affected both the biomass and lipopeptide yield of the YA215 strain. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, a slight decrease in biomass was observed at 36 h of fermentation with the addition of branched-chain amino acids, although the reduction was not pronounced. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB shows that the pH of the fermentation supernatant decreased after 36 h, especially with the addition of Ile and Val, potentially contributing to the biomass reduction. However, this decline in biomass did not exhibit a clear trend. In contrast, lipopeptide yield varied with branched-chain amino acid supplementation, with a significant increase in lipopeptide production observed in the Val-supplemented group compared to the control, whereas Leu and Ile supplementation resulted in decreased lipopeptide yields (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). This variation in lipopeptide yield differs from the previously discussed surfactin production trends, as lipopeptides comprise surfactin, iturin, and fengycin families(Ongena et al. 2008), and changes in surfactin alone do not fully represent lipopeptide yield trends. These results underscore the multifaceted roles of branched-chain amino acids in microbial fermentation systems, affecting not only specific metabolite yields but also biomass and environmental parameters such as pH. This understanding provides valuable insights into the regulatory mechanisms of microbial fermentation processes. Moreover, in microbial metabolic engineering applications, it is crucial to consider the comprehensive effects of branched-chain amino acid supplementation on the overall production process to design and optimize fermentation conditions more accurately. This study not only offers a solid experimental foundation for surfactin production but also provides valuable references for engineering improvements in related microbial fermentation systems. Additionally, the observed increase in lipopeptide yield with branched-chain amino acid supplementation, particularly with Val, aligns with findings by Zhou et al., who reported that appropriate supplementation of branched-chain amino acids stimulates target product production in similar microbial fermentation experiments, likely due to their role in metabolic pathways(Zhou et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of branched-chain amino acids on the antibacterial activity of lipopeptides from\u003c/b\u003e \u003cb\u003eB. velezensis\u003c/b\u003e \u003cb\u003eYA215\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFollowing the optimization of fermentation conditions, the antibacterial activity of lipopeptides extracted from fermentation supernatants with branched-chain amino acid supplementation was evaluated using the acid precipitation and ethanol extraction method. The antibacterial effect against \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e was assessed using an inhibition zone assay with equal lipopeptide concentrations across four sample groups. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and detailed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, lipopeptides from fermentation media supplemented with the three amino acids exhibited superior antibacterial effects against \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e compared to the control group. Notably, the antibacterial effect was significantly higher in the Val-supplemented samples than in those supplemented with Leu and Ile. Furthermore, the lipopeptides demonstrated a stronger inhibitory effect against \u003cem\u003eE. coli\u003c/em\u003e than \u003cem\u003eS. aureus\u003c/em\u003e. As bioactive molecules, lipopeptides are categorized into surfactin, iturin, and fengycin families(Ongena et al. 2008), each exhibiting different antibacterial properties. Surfactin, for instance, shows notable antibacterial activity but limited antifungal activity(Buchoux et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), whereas iturin possesses strong antifungal activity(Hua et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, Yaraguppi et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and fengycin, with lower hemolytic activity, displays potent antifungal properties(Fu et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, He et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Previous studies have also indicated that surfactin is the primary compound responsible for antibacterial activity. Our prior research corroborated these findings, identifying surfactin as the principal antibacterial component produced by the YA215 strain(Yu et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The consistent trend in surfactin yield changes with amino acid supplementation further supports the correlation between surfactin content and lipopeptide antibacterial efficacy. These findings provide valuable clues for understanding the bioactivity of lipopeptides produced by the YA215 strain and their mechanisms of antibacterial action, offering useful references for microbial metabolic engineering in the production of bioactive substances.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLipopeptide inhibition results of \u003cem\u003eB. velezensis\u003c/em\u003e YA215 strain.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS.aureus\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eE.coli\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eDiameter of inhibition zone(mm)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSS\u0026thinsp;+\u0026thinsp;Leu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSS\u0026thinsp;+\u0026thinsp;Ile\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSS\u0026thinsp;+\u0026thinsp;Val\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eNote: different letters indicate significant differences between groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of branched-chain amino acids on the hydrolyzed fatty acids of lipopeptides from\u003c/b\u003e \u003cb\u003eB. velezensis\u003c/b\u003e \u003cb\u003eYA215\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe increased antibacterial activity of surfactin is not solely linked to its production quantity but also closely associated with structural and conformational changes. In this section, the relationship between the enhanced antibacterial activity of surfactin and its structural modifications is explored. By hydrolyzing lipopeptides extracted from four sample groups, changes in fatty acids were analyzed to infer structural information about surfactin. Initially, the extracted lipopeptides were subjected to high-temperature hydrolysis with hydrochloric acid under identical concentration conditions, resulting in free β-hydroxy fatty acids. These acids were then reacted with methanol under acidic conditions to form β-hydroxy fatty acid methyl esters, which were subsequently detected using GC-MS. Comparative analysis of the total ion chromatograms (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) and the extracted ion chromatograms with an m/z of 103 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) revealed that the total types of β-hydroxy fatty acids did not change with branched-chain amino acid supplementation; all four sample groups exhibited 14 chromatographic peaks. In the mass spectrometry analysis, β-hydroxy fatty acid methyl esters generated M-1, M-18, and M-50 fragment ion peaks due to the loss of H atom, H\u003csub\u003e2\u003c/sub\u003eO, and (CH\u003csub\u003e3\u003c/sub\u003eOH\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO) respectively. Through comparison with the NIST database, it was determined that these 14 fatty acid methyl esters had molecular weights ranging from 230 to 300, differing by 14 units, corresponding to β-hydroxy fatty acid methyl esters with varying carbon chain lengths. β-Hydroxy fatty acid methyl esters with the same carbon chain length exhibited n, iso, or anteiso structures due to branching differences(Cheng et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). It was found that the retention time of β-hydroxy fatty acid methyl esters had a linear relationship with their carbon number, and the retention time followed the order iso\u0026thinsp;\u0026lt;\u0026thinsp;anteiso\u0026thinsp;\u0026lt;\u0026thinsp;n for esters with the same carbon number(Hosono et al. 1983). Based on this analysis, the lipopeptide samples contained β-hydroxy fatty acids with various chain lengths and branching structures, such as iso C12, iso C13, anteiso C13, n C14, iso C14, iso C15, anteiso C15, n C15, iso C16, n C16, iso C17, anteiso C17, and n C18, highlighting the diversity and structural complexity of the fatty acid chains.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurther analysis of the β-hydroxy fatty acid content variation with different carbon chain lengths (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) showed that, compared to the control group, the proportions of C12 and C13 β-hydroxy fatty acids decreased with branched-chain amino acid addition. With Val supplementation, the content of C14 β-hydroxy fatty acids increased compared to the control, while other chain lengths decreased. With Leu supplementation, the contents of C15 and C17 β-hydroxy fatty acids increased, whereas other chain lengths decreased. With Ile supplementation, the contents of C16, C17, and C18 β-hydroxy fatty acids increased, while other chain lengths decreased.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurther exploration of the conformational changes in β-hydroxy fatty acids with different chain lengths (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e) indicated that, compared to the control group, the contents of n C12 β-hydroxy fatty acids, iso C13 β-hydroxy fatty acids, and anteiso C13 β-hydroxy fatty acids decreased with branched-chain amino acid addition. With Val supplementation, the proportion of iso C14 β-hydroxy fatty acids increased from 13.30\u0026ndash;23.80%, and iso C16 from 4.22\u0026ndash;8.31%, while other conformations decreased. With Leu supplementation, the proportions of iso C15, anteiso C15, iso C17, and anteiso C17 β-hydroxy fatty acids increased, especially iso C15, which rose from 11.42\u0026ndash;26.81%, while other conformations decreased. With Ile supplementation, the proportions of n C14, anteiso C15, n C16, anteiso C17, n C17, and n C18 β-hydroxy fatty acids increased, particularly n C16, which significantly rose from 10.37\u0026ndash;20.54%, while other carbon chain lengths decreased.\u003c/p\u003e \u003cp\u003eThe analysis of the experimental data revealed that the addition of branched-chain amino acids altered the content and configuration of β-hydroxy fatty acids of various chain lengths. This phenomenon might be due to the specific structural effects of these branched chains on the formation and antibacterial activity of surfactin. Liu et al. discovered that adding Arg and Gln to the culture medium of B. subtilis T8942 increased the even-numbered β-hydroxy fatty acids (C14, C16), while adding Cys, His, Met, Ser, or Thr increased the odd-numbered β-hydroxy fatty acids (C13, C15) (Liu et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Furthermore, they noted that the addition of branched-chain amino acids (L-Val, L-Leu, and L-Ile) led to significant changes in hydroxy fatty acid residues: L-Leu markedly increased the proportion of iso odd-numbered hydroxy fatty acid residues; L-Ile enhanced the proportion of anteiso odd-numbered hydroxy fatty acid residues; and L-Val elevated the proportion of iso even-numbered hydroxy fatty acid residues. Our experimental results also indicated that Val addition significantly increased the content of C14 β-hydroxy fatty acids, while Leu and Ile additions notably increased the contents of C15, C16, C17, and C18 β-hydroxy fatty acids, highlighting the impact of branched-chain amino acids on surfactin's structure and configuration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eImpact of branched-chain amino acids on the peptide chains of surfactin in\u003c/b\u003e \u003cb\u003eB. velezensis\u003c/b\u003e \u003cb\u003eYA215\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe changes in the peptide chain composition of surfactin under the same concentration conditions in four sample groups were analyzed using LC-MS/MS, providing critical insights into its molecular structure. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows that the SS fermentation produced surfactin with peptide chains Glu-Leu-Leu-Val-Asp-Leu-Leu and Glu-Leu-Leu-Val-Asp-Leu-Val, connected to C13-C16 fatty acid chains, known as [Leu 7]surfactin and [Val 7]surfactin, respectively, as well as [Leu 7]surfactin linked to C12 fatty acid chains. Upon Leu addition, [Val 7]surfactin was converted to [Leu 7]surfactin, resulting in a single peptide chain structure of [Leu 7]surfactin. With Ile addition, the Val at the 7th position in the peptide chains of C12, C14, and C16 surfactin was replaced by Leu, unifying the peptide chain structure as [Leu 7]surfactin. The C13 surfactin peptide chain structure remained consistent with the control SS. However, the C15 surfactin peptide chains exhibited two structures: [Leu 7]surfactin and [Leu/Ile 4]surfactin, where Val at the 4th position was replaced by Leu or Ile. Upon Val addition, the C13 surfactin peptide chain displayed two structures: one with Leu at the 7th position replaced by Val, forming [Val 7]surfactin, and another consistent with the control SS, [Leu 7]surfactin. The peptide chain structures of C12, C14, C15, and C16 surfactin were consistent with the control SS, each containing two structures, but the proportion of [Val 7]surfactin notably increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Quantitative analysis indicated that the proportion of C14 [Val 7]surfactin increased by 3.29 times compared to the control SS, aligning with the previously observed fatty acid changes. This could be attributed to the addition of Val in the SS medium, enhancing the antibacterial activity of lipopeptides.\u003c/p\u003e \u003cp\u003eAdditionally, it was observed that adding L-Val and L-Ile to the medium selectively increased the yield of [Val 7]surfactin, whereas the addition of L-Leu resulted in a lower yield of [Val 7]surfactin. This selective effect may be associated with the cross-relationships between amino acid biosynthesis and metabolic pathways. This finding is consistent with previous studies on the regulatory role of amino acids in lipopeptide biosynthesis. Peypoux et al. found that supplementing the medium with L-Val or L-Ile as nitrogen sources selectively enhanced the production of Val7-surfactin (Peypoux et al. 1992).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResults of changes in the structure of the peptide chain of surfactin.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRetention Time (min)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMain LC-MS Peak(m/z)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eβ-OH fatty acids\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePeptide sequence\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12.99\u0026ndash;14.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e994.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN-Glu-Leu-Leu-Val-Asp-Leu-Leu-C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1008\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN-Glu-Leu-Leu-Val-Asp-Leu-Leu-C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1008\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN-Glu-Leu-Leu-Val-Asp-Leu-Val-C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN-Glu-Leu-Leu-Val-Asp-Leu-Leu-C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN-Glu-Leu-Leu-Val-Asp-Leu-Val-C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e 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align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN-Glu-Leu-Leu-Val-Asp-Leu-Leu-C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1008\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN-Glu-Leu-Leu-Val-Asp-Leu-Val-C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14.06,14.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e 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colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSS\u0026thinsp;+\u0026thinsp;Ile\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12.71\u0026ndash;14.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e994\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN-Glu-Leu-Leu-Val-Asp-Leu-Leu-C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1008\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e 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\u003cp\u003e1008\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN-Glu-Leu-Leu-Val-Asp-Leu-Val-C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN-Glu-Leu-Leu-Val-Asp-Leu-Leu-C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN-Glu-Leu-Leu-Val-Asp-Leu-Val-C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1036\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN-Glu-Leu-Leu-Val-Asp-Leu-Leu-C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1036\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN-Glu-Leu-Leu-Val-Asp-Leu-Val-C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1050\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN-Glu-Leu-Leu-Val-Asp-Leu-Leu-C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study underscores the significant impact of branched-chain amino acids (BCAAs) on the structure and antibacterial activity of surfactin produced by \u003cem\u003eBacillus velezensis\u003c/em\u003e YA215. Process optimization revealed that adding 1 g/L of L-Leu and L-Ile, and 0.5 g/L of L-Val, maximizes surfactin production. Surfactin levels peaked with L-Val and L-Ile at 36 h, while L-Leu reached its maximum at 24 h. Notably, L-Val supplementation yielded the highest relative surfactin content. Antimicrobial assays revealed that BCAAs markedly improved the antibacterial efficacy of lipopeptide against \u003cem\u003eEscherichia col\u003c/em\u003ei and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, with L-Val demonstrating the most significant effect. Additionally, BCAA incorporation significantly modified the composition of surfactin fatty acid chains. In particular, L-Val addition increased the proportions of iso-C14 and iso-C16 β-hydroxy fatty acids and altered the amino acid composition at the 7th position of the peptide chain. These structural modifications are likely linked to the enhanced antibacterial activity of surfactin. Our findings provide valuable insights into the role of BCAAs in microbial fermentation, highlighting their importance in metabolic engineering to boost the production of bioactive compounds. This research underscores the potential of BCAAs to optimize surfactin yield and efficacy, offering a promising approach to developing effective, low-toxicity antimicrobial agents as alternatives to conventional antibiotics.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlahyaribeik S, Nazarpour M (2024) Peptide recovery from chicken feather keratin and their anti-biofilm properties against methicillin-resistant Staphylococcus aureus (MRSA). 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Microbiologyopen 8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://doi.org/10.1002/mbo3.794\u003c/span\u003e\u003cspan address=\"10.1002/mbo3.794\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"world-journal-of-microbiology-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wibi","sideBox":"Learn more about [World Journal of Microbiology and Biotechnology](https://www.springer.com/journal/11274)","snPcode":"11274","submissionUrl":"https://submission.nature.com/new-submission/11274/3","title":"World Journal of Microbiology and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Branched-chain amino acids. Bacillus velezensis. Lipopeptide. Surfactin. Antibacterial activity. Fatty acid composition","lastPublishedDoi":"10.21203/rs.3.rs-4522872/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4522872/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAntibiotics are essential for combating pathogens; however, their misuse has led to increased resistance, necessitating the search for effective, low-toxicity alternatives. Surfactin, due to its unique structure, exhibits significant antibacterial activity without easily inducing resistance, making it a focus of current research. Nonetheless, the effects of branched-chain amino acids (BCAAs) on surfactin's structure and activity are not well understood. This study examines the influence of BCAAs (L-valine, L-leucine, and L-isoleucine) on the lipopeptide (surfactin) produced by \u003cem\u003eB. velezensis\u003c/em\u003e YA215. Process optimization revealed that adding 1 g/L of L-Leu and L-Ile, and 0.5 g/L of L-Val, maximizes surfactin production. Surfactin levels peaked with L-Val and L-Ile at 36 h, while L-Leu reached its maximum at 24 h. Notably, L-Val supplementation resulted in the highest relative surfactin content. Antimicrobial testing demonstrated that BCAAs significantly enhance the antibacterial effects of lipopeptides against \u003cem\u003eEscherichia col\u003c/em\u003ei and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, with Val showing the most pronounced effect. The addition of BCAAs notably altered the composition of surfactin fatty acid chains. Specifically, Val increased the proportions of iso C14 and iso C16 β-hydroxy fatty acids from 13.3% and 4.216\u0026ndash;23.803% and 8.31%, respectively. Additionally, the amino acid composition at the 7th position of the peptide chain changed significantly, especially with Val addition, which increased the proportion of C14 [Val 7] surfactin by 3.29 times. These structural changes are likely associated with the enhanced antibacterial activity of surfactin. These findings provide valuable insights into the roles of BCAAs in microbial fermentation, underscoring their importance in metabolic engineering to enhance the production of bioactive compounds.\u003c/p\u003e","manuscriptTitle":"Effects of branched-chain amino acids on surfactin structure and antibacterial activity in Bacillus velezensis YA215","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-18 09:38:09","doi":"10.21203/rs.3.rs-4522872/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-17T17:38:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-17T03:23:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-14T19:38:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"218706098787814095552450799785078335381","date":"2024-06-12T01:21:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"57931115260546170778809066943176213556","date":"2024-06-06T20:22:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"129193074401375543705865237707000135026","date":"2024-06-06T15:03:27+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-06T12:16:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-05T06:24:59+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-04T08:46:29+00:00","index":"","fulltext":""},{"type":"submitted","content":"World Journal of Microbiology and Biotechnology","date":"2024-06-03T15:38:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"world-journal-of-microbiology-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wibi","sideBox":"Learn more about [World Journal of Microbiology and Biotechnology](https://www.springer.com/journal/11274)","snPcode":"11274","submissionUrl":"https://submission.nature.com/new-submission/11274/3","title":"World Journal of Microbiology and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c70d93ed-e897-482f-9a90-693564b0ad30","owner":[],"postedDate":"June 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-01T17:10:59+00:00","versionOfRecord":{"articleIdentity":"rs-4522872","link":"https://doi.org/10.1007/s11274-024-04088-7","journal":{"identity":"world-journal-of-microbiology-and-biotechnology","isVorOnly":false,"title":"World Journal of Microbiology and Biotechnology"},"publishedOn":"2024-07-27 16:16:24","publishedOnDateReadable":"July 27th, 2024"},"versionCreatedAt":"2024-06-18 09:38:09","video":"","vorDoi":"10.1007/s11274-024-04088-7","vorDoiUrl":"https://doi.org/10.1007/s11274-024-04088-7","workflowStages":[]},"version":"v1","identity":"rs-4522872","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4522872","identity":"rs-4522872","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}