Bio-Based Antimicrobial Agents to Improve Agricultural and Food Safety E-Book
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- Herausgeber: Bentham Science Publishers
- Kategorie: Wissenschaft und neue Technologien
- Sprache: Englisch
Bio-Based Antimicrobial Agents to Improve Agricultural and Food Safety provides a comprehensive overview of the latest advancements in bio-based antimicrobial agents used to enhance food safety and agricultural production. This book highlights natural alternatives to chemical preservatives, focusing on surfactin, bacillomycin, fengycin, bacteriocins, and plant-based antimicrobials. It explores their applications in controlling microbial contamination, improving crop health, and extending food shelf life. Key topics include biopreservation techniques, biological control of pathogens, and sustainable agricultural practices. With contributions from experts worldwide, this book is an essential resource for researchers, biotechnologists, microbiologists, food scientists, and industry professionals aiming to understand modern bio-safe methods for food preservation and crop protection.
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Veröffentlichungsjahr: 2024
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FOREWORD
The use of chemical preservatives and pesticides in agriculture and food has long been a worldwide concern since it has been shown over and over again to result in food safety issues detrimental to human health and the environment. Thus, innovative modern food safety and preservation tools, especially natural bio-safe ways, are being investigated to reduce food contamination and spoilage and extend the shelf-life of food. Recently, the discovery and application of new bio-based antimicrobial agents have been the focus of many researchers, healthcare professionals, farmers, and agricultural companies to satisfy the consumers’ demand for agricultural quality and food safety. This book brings together updated, innovative technologies of antimicrobial agents originally from microorganisms and plants, as well as their application in agriculture and food. Some of the technologies presented in the book are still emerging and will be of great benefit to researchers, healthcare professionals, farmers, and agricultural companies interested in staying on the forefront of innovative bio-based antimicrobial agents to improve agriculture and food safety. It also presents some practical guided approaches that could assist in the improvement of microbial strains and food quality.
PREFACE
Food safety includes many approaches, starting from crop plantation, animal feeding, and food processing to storage, which involve microbial contamination control, quality assurance, and preservation. However, the microbiological risks in food are still one of the main sources of foodborne illnesses. In addition, the greatest losses in the food industry are attributed to microbial contamination, which dramatically affects the shelf life of food. Meanwhile, many plant pathogens influence the production of crops. Specifically, pathogenic fungi as one of the major production constraints, not only reduce crop production but also produce mycotoxins.
Nowadays, lasting exposure to chemical preservatives and pesticide residues in plants and food results in serious health impacts that are becoming global concerns. Therefore, modern safety and preservation tools, especially natural bio-safe ways, are being investigated to reduce food contamination and spoilage and to extend the shelf-life of food. This public perception has generated heightened interest in “biopreservation and biocontrol” in terms of the use of biologically producing compounds that possess a broad spectrum of antimicrobial activities as natural preservatives. Recently, the discovery and application of new bio-based antimicrobial agents have been focused on by many researchers, healthcare professionals, farmers, and agricultural companies to satisfy the consumers’ demand for agricultural quality and food safety.
The book titled “Bio-Based Antimicrobial Agents to Improve Agricultural and Food Safety” aims to bring together the most recent progress in the development and application of novel antimicrobial agents, such as lipopeptides (surfactin, fengycin, bacillomyci, and brevibacillin) from Bacillus, bacteriocins from lactic acid bacteria and others. Biopreservation and biocontrol by the new antimicrobial agent are introduced, and new biopreservation tools to improve agricultural production and food safety are discussed in this volume. This book is mainly fruitful for biotechnologists, microbiologists, food scientists, food industrial companies, and also any reader interested in recent progress in the field of new preservatives and biopreservation methods. Our volume contains eight chapters prepared by outstanding authors from China, the USA, and the UK.
List of Contributors
Surfactin Biosynthesis and its Potential Applications in Agriculture and Food System
Xiaoyu Chen1,Huawei Liu2,Zhaoxin Lu1,*Abstract
Surfactin is a biosurfactant of the lipopeptide-type that has excellent physicochemical properties and biological activity. However, surfactin’s high cost and low productivity of the wild strains restrict its large-scale manufacturing and application. Hence, numerous engineered bacteria have been utilized to boost surfactin biosynthesis. The current review includes information on the structure, physicochemical properties, and antibacterial mechanism of surfactin. This article also summarizes the regulatory network of surfactin biosynthesis, the molecular modification strategies, and the major function of surfactin, as well as its applications in agriculture, livestock, aquaculture and the food field. Finally, future prospects for surfactin research are discussed.
INTRODUCTION
Surfactin is a cyclic lipopeptide generated from Bacillus subtilis secondary metabolites, which works as a biosurfactant to lower surface tension. The ability of surfactin synthesis is broadly dispersed among B. subtilis strains, as well as B. licheniformis and B. amyloliquefaciens strains [1]. Kluge et al. (1988) hypothesized a non-ribosomal mechanism catalyzed by multi-enzymatic thiotemplates comprising the surfactin synthetase in their study on surfactin biosynthesis [2]. Surfactin has anti-bacterial, anti-fungal, anti-viral, anti-cancer, anti-mycoplasma, anti-inflammatory, thrombolytic and hemolytic action, which is of particular importance given the interest in the creation of new peptide antibiotics [1, 3-9].
Surfactin has a wide range of applications in petroleum recovery, biological pesticide production, cosmetics research and development, food processing, and pharmaceuticals, among others. According to the current notion of green sustainable development, chemically manufactured surfactants can be replaced by surfactin [10-12]. Surfactin has been examined extensively by researchers and experts since its discovery. To breed high-yielding strains and generate unique molecular structures of surfactin, researchers used physical, chemical, and genetic engineering strategies [13, 14]. Furthermore, improvement of the fermentation method for increasing surfactin yield has been extensively researched [15-21]. Surfactin synthesis, as well as its function and application, is increasingly concerned with rational genetic engineering of strains.
Surfactin transcriptional structural characterization and bacteriostatic mechanism are discussed in this review. In addition, the genetically modified method and novel molecular structure of surfactin are summarized briefly, as well as its function and application in agricultural production, livestock, aquaculture, and food field. This provides a better reference for further exploring and industrialization of surfactin.
STRUCTURE AND PHYSICO-CHEMICAL PROPERTIES OF SURFACTIN
Surfactin, a 1036 Da amphipathic cyclic lipopeptide, is made up of a heptapeptide with the chiral sequence LLDLLDL connected with β-hydroxy fatty acid with a chain length of 12 to 16 carbon atoms to form a cyclic lactone ring structure. In addition, the carboxyl group of β-hydroxy fatty acid is linked to the N-terminus of the 7th amino acid of the peptide backbone by an amide bond, and a lactone bond connects the hydroxy group to the C-terminal carboxyl group of the heptapeptide, generating a closed ring structure [22-25]. L-Glu1-L-Leu2-D-Leu3-L-Val4- L-Asp5-D-Leu6-L-Leu7 is a typical chiral sequence of surfactin’s heptapeptide-ring. The hydrophobic group of surfactin is attached to hydrophobic amino acid residues at positions 2, 3, 4, 6 and 7, as well as a length chain of β-hydroxy fatty acid. The heptapeptide’s hydrophilic group is formed by the cyclic backbone and the amino acids at positions 1 and 5 (which introduce two negative charges to the molecule) [26, 27]. In addition, the amino acids at positions 2, 4, and 7 are highly variable, allowing them to undergo a variety of changes [28-31]. Natural surfactin is a combination of isoforms A, B, C and D obtained from the production strain with varied physiological properties [32]. Many surfactin variations and homologs with different fatty acid chain lengths, types, and positions of amino acids have been identified [30, 33-36].
Bonmatin et al. (1994) analyzed the three-dimensional configuration of surfactin using high-resolution two-dimensional nuclear magnetic resonance (1H NMR) and molecular imaging techniques [37]. As a result, surfactin exists in the aqueous phase and at the water/air interface as a β-sheet structure with a characteristic horse-saddle conformation [37]. Residues 2 and 6 face each other on one side of the molecule, between the acidic Glu-1 and Asp-5 side chains, which define a minor polar domain [38]. On the opposite side, residue 4 faces the lipidic chain, which forms a large hydrophobic domain and incorporates the side-chains of residues 3 and 7 to a lesser extent, explaining its amphiphilic character and strong surfactant properties [39]. This conformation causes negatively charged amino acid residues on the ring to form potential divalent cation-binding holes. The fatty acid chain is fully extended on the other side of the ring, allowing it to participate in micelle production or penetrate the phospholipid bilayer [40]. Tsan et al. (2007) exploited NMR technology to characterize the structure of surfactin in polar and non-polar environments, obtaining a low-energy, stable 3D horse-saddle conformation, which differs from Bonmatin’s [39]. In this conformation, the molecule residues 1 and 5 are located on the same side of the molecule, and the remainder of non-polar amino acids are on the other side except for the molecule residue 4 [39].
Surfactants with different chemical structures have different critical micelle concentration (CMC) values and self-aggregation forms, including micellar, hexagonal, cubic, and lamellar [31]. The self-aggregation structure of surfactants is also associated with intermolecular electrostatic interactions, ionic strength, polarity, and temperature of the solvent [41]. Surfactin is a powerful surfactant with a large molecular weight and complex conformation that can drop water surface tension from 72 mN.m-1 to 27 mN.m-1 at a concentration as low as 10 µM, far below the critical micelle concentration in water and about two orders of magnitude lower than most other detergents [11, 17]. Surfactin has the ability to form rod-like micelles with an aggregation number of ~170 [42]. Surfactin can also reduce the interfacial tension of water/dodecane from 52 mN m-1 to 2.45 mN m-1 at concentrations comparable to CMC [43].
ANTIBACTERIAL MECHANISM
Surfactin-membrane Interaction
Surfactin possesses considerable antibacterial effects on both gram-positive and gram-negative bacteria, and it is difficult to develop drug resistance due to its molecular structure. Surfactin’s antibacterial mechanism is intimately linked to its physio-chemical properties, however, it has yet to be clarified. The antimicrobial peptide surfactin must first be attracted to the surfaces of the target bacterial cell membrane [44, 45]. Surfactin subsequently penetrates into the membrane through hydrophobic interactions to impact both the hydrocarbon chain order and the membrane thickness, and this interaction process can be further facilitated as conformational changes of peptide cycle [46]. Surfactin, an amphiphilic molecule, destabilizes the membrane and disturbs its integrity [47]. Surfactin dimerization in the bilayer is important for membrane instability and content leakage [48]. The hypothetical mechanism that surfactin would interact with the membrane exhibits a complex pattern, comprising inserting into lipid bilayers, chelating mono- and divalent cations, and modifying membrane permeability by forming “barrel-stave” and “carpet” or lysis of membrane by a detergent-like mechanism (the toroidal model) [44, 45, 49, 50]. However, there is no evidence of the mechanisms between surfactin and membrane. Many investigations have identified the detergent-like model as a typical mechanism of amphipathic molecules [4, 51]. This detergent-like model is supported by a report by Deleu et al. [52].
Others
Increasing evidence indicates that antimicrobial peptides have other intracellular targets. Previous reports illustrated that lipopeptides could suppress chromosomal DNA replication, mRNA synthesis, protein synthesis, and cell wall formation [44, 53-56]. Surfactin, iturin, and fengycin are mixed lipopeptides from B. subtilis that have the ability to bind DNA. Huang et al. (2010) confirmed that lipopeptides (surfactin and iturin) isolated from B. subtilis fmbJ could inhibit certain enzymes’ activity [57].
THE REGULATORY NETWORK OF SURFACTIN BIOSYNTHESIS
A global surfactin biosynthesis pathway in B. subtilis was explored and constructed based on the reported surfactin production process. Six modules were divided into functional groups, i.e., glycolysis and tricarboxylic acid (TCA) cycle, fatty acid biosynthesis, amino acids biosynthesis, nicotinamide adenine dinucleotide phosphate (NADPH) generation, modularly enzymatic surfactin synthesis, and surfactin secretion and resistance [58, 59].
The upstream precursor supply unit is divided into 4 modules, providing basic precursors for cell development and surfactin synthesis, including amino acids, carbon skeleton, NADPH, ATP, etc. The module for NADPH production contains part of the pentose phosphate pathway, which mainly produces NADPH and pentose for cellular metabolism, which belong to module 4. This module is not discussed in this chapter.
Upstream Precursor Supply Unit
Module 1 represents the utilization of carbon or glycogen, as well as the glycolytic pathway and TCA cycle, which provide the carbon skeleton and energy for cellular metabolism. Zhi et al. (2017) reported that the genes encoding sugar transporter, permease and sucrose 6-phosphate hydrolase were sacP, murP and sacA, respectively, and they were considerably expressed in B. amyloliquefaciens MT45, with a high-producing surfactin [59]. The genes involved in key enzymes in the glycolytic pathway can be regulated through CcpA. In addition, CcpA can directly or indirectly regulate citZ gene expression associated with the TCA cycle [60]. CcpA is associated with Streptococcus gordonii adhesin gene expression, development, and biofilm formation [61]. However, the network between CcpA and surfactin production has not been established [62]. This should be examined further in the future.
Their biosynthesis pathway is rarely noticed, although the fatty acids attached to the N-terminal are key elements of surfactin, comprising the hydrophobic tail [14, 63, 64]. The branched-chain fatty acids are the main components among produced surfactin variants, accounting for around 78% of the total variants [65]. Most genes involved in the fatty acid synthesis were up-regulated in B. amyloliquefaciens MT45, which produced highly surfactin [59]. Hence, the fatty acid biosynthesis system, especially branched-chain fatty acids, is also critical for the synthesis of surfactin in addition to non-ribosomal peptide synthetase (NRPS). It is initiated from the precursor acetyl-CoA. In addition, pyruvate dehydrogenase (phdABCD) catalyzes the conversion of pyruvate to acetyl-CoA (Fig. 1). Hu et al. (2019) showed that there are positive regulators of fatty acid synthesis, including acetyl-CoA carboxylase (accABCD, which catalyzes acetyl-CoA to form malonyl-CoA), malonyl-CoA (fabD, which catalyzes malonyl-CoA to form malonyl-ACP), and β-ketoacyl-ACP synthases (FabH, which catalyzes malonyl-ACP and branched chain α-ketoacyl CoAs to form β-keto acyl ACP) [13]. β-ketoacyl-acyl carrier protein synthase III (FabH) catalyzes the condensation of malonyl-acyl carrier protein (ACP) with acetyl-CoA to form β-ketobutyryl-ACP, which is the initial step in straight-chain saturated fatty acid biosynthesis.
FabH catalyzes the condensation of malonyl-acyl carrier protein (ACP) with acetyl-CoA to form β-ketobutyryl-ACP, the first step in the biosynthesis of linear saturated fatty acids. Isobutyryl-CoA, isovaleryl-CoA, and α-methylbutyryl-CoA are branched-chain fatty acids synthesis precursors that can be produced from the branched-chain amino acid L-val, L-leu, and L-isoleu, respectively [13]. Kraas et al. (2010) proved that the activation of the activity of the acyl-CoA ligases LcfA and LcfB in B. subtilis caused the subsequent activation of 3-hydroxy long-chain fatty acids, and the long-chain fatty acids finally activated by CoA were considered substrate for B. subtilis, the initiation of surfactin synthesis (Fig. 1) [66]. This represents module 2 of surfactin biosynthesis.
Fig. (1)) The general regulatory network of surfactin biosynthesis [58].The amino acids synthesis module is involved in the biosynthesis of the surfactin components Glu, Asp, Val, and Leu. The branched-chain amino acids (L-isoleucine, L-valine and L-leucine) play three roles in surfactin biosynthesis (Fig. 1): branched-chain amino acids biosynthesis, branched-chain fatty acid and CoA-activated 3-hydroxy fatty acids precursor biosynthesis, and NRPS-catalyzed synthesis. The biosynthesis of the branched-chain amino acids shares the enzyme system encoded by ilvBN, ilvGM, ilvIH, ilvC, ilvD, and ilvE (acetohydroxy acid synthase I, acetohydroxy acid synthase II, acetohydroxy acid synthase III, acetohydroxy acid isomeroreductase, dihydroxy acid dehydratase, branched-chain amino acid aminotransferase) [67, 68]. Besides, an enzyme complex encoded by leuACDB is involved in the production of L-leucine from α-keto-isovalerate. The branched-chain α-keto acid dehydrogenase complex converts these intermediates into the corresponding branched-chain acyl-CoA precursors: α-methylbutyryl-CoA, isobutyryl-CoA, and isovaleryl-CoA [69]. Subsequently, the action of FabH condenses these branched-chain acyl-CoAs and malonyl-ACP into 3-keto-4-methylhexanoyl-ACP, 3-keto-4-methylvaleryl-ACP, and 3-keto-5-methylhexanoyl-ACP, which then participate in fatty acid synthesis [70]. In addition, some previous reports suggested that CodY inhibits the transcription of three gene operons (ilvD, ybgE, ilvBHC-leuABCD) involved in branched-chain amino acid biosynthesis [71], as well as a gene operon (bkd) involved in the biosynthesis of branched-chain keto acids, which is the precursor of branched-chain fatty acids [69, 71]. The majority of newly discovered leucine network predictions were related to genes involved in leucine degradation, such as bcd and bkL, which code for branched-chain amino acid dehydrogenase and E3 lipoamide dehydrogenase (lpdV), respectively [69].
Intermediary Transcriptional Driving Unit
Surfactin production requires a modular enzymatic manufacturing process known as the intermediary transcriptional drive unit. This was known as module 5. Surfactin biosynthesis and regulation is a complicated process that involves multiple genes and quorum sensing [58, 72, 73]. Surfactin synthesis is linked to competent pathways, spore formation, cell mobility, and biofilm formation in B. subtilis [74, 75] (Fig. 1). ComX, a pheromone of B. subtilis, binds to the membrane protein histidine kinase ComP and induces its autophosphorylation, and this phosphate group is conveyed to the regulatory protein ComA. Phosphorylated ComA combines with a specific region of srfA, which is the promoter of surfactin synthetase and activates RNA polymerase to trigger transcription of srfA [76, 77]. Hamoen et al. (2003) suggested that nutritional stress during the late exponential phase could stimulate the global regulatory mechanism, including ComP-ComA and Spo0A-AbrB, to further induce the expression of srfA [78]. The transcription and expression of the comS gene embedded in srfAB determines the initiation of competence development of B. subtilis [89]. Except for ComX, the competence stimulating factor (CSF) encoded by the phr gene also participates in the synthesis and regulation of surfactin [78].
The extracellular precursor peptide Phr enters into the cell through the oligopeptide transport system Opp (the transmembrane output way of Phr has not been verified) and exerts the phosphatase inhibitory action by combining with the Rap protein, according to Pottathil and Lazazzera [79]. Auchtung et al. (2006) demonstrated that once the Phr peptide binds to the Rap protein, ComA retains an active phosphorylation state, boosting the transcription of the srfA gene and indirectly regulating the surfactin synthesis [80]. The Opp is a significant and conserved transporter utilized by the Bacillus species and other microorganisms to import peptides, and the Opp operon consists of five subunits (OppA, OppB, OppC, OppD, and OppF) in B. subtilis [62]. It was discovered that oppA is involved in surfactin production and regulation mechanisms. Wang et al. (2019) and Bongiorni et al. (2005) studied B. subtilis intracellular Rap proteins and found that short-peptides Phr may specifically decrease Rap proteins such as RapA, RapC, RapE, RapF, RapG, RapI, RapK [62, 81]. It has also been proven that ComX and PhrC have a favorable effect on surfactin production [61]. RghR inhibits DegU binding to DNA, suppressing srfA expression [82, 83]. RghR and srfA have similar expression patterns [59].
ComA and SigA bind directly to the srfA promoter to initiate transcription, while AbrB, as a transition state regulator, may help surfactin synthesis [59]. B. subtilis also produces Abh, a protein with N-terminal DNA-binding domains that are quite similar to those of AbrB [84]. Abh disruption had a stronger derepressed effect on the srfA operon than abrB deletion, demonstrating Abh’s negative regulation of srfA expression [85]. However, in the abh deletion strain, AbrB, which binds to the srfA promoter, was preserved, but the Abh binding ability is poor without abrB [85]. The global regulator CodY could repress transcription of CitB (aconitase) involved in the citric acid cycle and GltAB (glutamate synthase) involved in glutamate formation [86]. This indicated that deleting codY might be beneficial for supplying energy and precursor for surfactin biosynthesis. Another potential repressor Spx suppresses srfA expression by blocking the interaction between ComA and RNAP in the promoter region via competition for an overlapping site in the α-CTD [87]. SinI inhibits the constitutive synthesis of surfactin by positively regulating matrix formation and indirectly suppressing srfA expression [88]. SrfA was recently reported to be positively regulated by PhoP in B. subtilis under phosphate-limiting conditions [89]. Up-regulated expression of SrfAA, SrfAB, and SrfAC was observed, suggesting that the global regulatory protein CcpA may positively regulate srfA operon expression [14]. In addition, the synthetase genes SrfAA, SrfAC, and SrfAD, which are cis-acting elements located at the 5’UTR of ribonuclease E, could be simultaneously regulated by sRNA rne5. Previous reports indicate that the key regulators of srfA transcription and the efficient surfactin synthesis mechanism are not well understood [13].
Downstream Efflux and Resistance Unit
Surfactin resistance gene expression is covered in Module 6 (Fig. 1). In terms of its antibiotic activity, self-resistance is essential for the high productivity of surfactin [59]. Wu et al. (2019) illustrated that SwrC and AcrB may collaborate to efficiently secrete surfactin [14]. Besides, SwrC is also required for surfactin efflux and self-resistance in B. subtilis [63]. The liaRSFGHI operon, annotated as genes associated with the resistance of daptomycin (a structural analog of surfactin), was highly expressed in high-yielding surfactin strains, particularly for liaH and liaI, and its over-expression during the late stage of fermentation might facilitate surfactin production [59]. The mechanism of action of Lia protein is unclear; however, it is thought to be important in preventing envelope stress and maintaining membrane integrity [14]. Hamon et al. (2001) found that the transcription factors SpoOA, sigma-H and AbrB can regulate biofilm development in B. subtilis [90]. SpoOA is related to the formation of spores and the synthesis of antibacterial peptides. Chen et al. (2012) reported that the histidine kinase (KinC) on the cell membrane could sense the K+ infiltration caused by surfactin attack on the cell membrane, phosphorylate Spo0A, and stimulate extracellular biofilm membrane matrix formation. Surfactin produced in response to stress not only initiates biofilm formation but also inhibits surfactin synthesis. This is an organism’s self-protection strategy to promote optimum survival [91]. Hamon et al. (2004) showed that Rap-Phr interacts with regulatory proteins (SpoOA, ComA, DegU) and is involved in the regulation of spores and competence, whereas Rap and Phr regulate the intracellular concentration of ComA-P [92-94]. Tsuge et al. (2001) proposed that yerP was a key factor in surfactin resistance [95]. Resistance, nodulation, and cell division (RND) family proton motive force-dependent efflux pumps only characterized in gram-negative strains have similarities with YerP. In addition, yerP is the first RND-like gene characterized in gram-positive bacteria, and it is supposed to be involved in surfactin secretion [95].
STRATEGIES OF MOLECULAR MODIFICATION FOR ENHANCING SURFACTIN PRODUCTION
A global surfactin biosynthesis pathway in B. subtilis was explored and constructed based on the reported surfactin production process. However, we mainly focus on surfactin's molecular modification aspects. Surfactin has attracted considerable attention in scientific research and industrial applications because of its biological and physicochemical properties. According to previous reports, the yield of surfactin of most wild strains is extremely low (50-100 mg/L) [24]. This is a hindrance in its large-scale production and application. Surfactin synthesis is too expensive to meet the demands of commercial applications. The strategy is mainly carried out from the following two aspects: (1) Reduce production costs by focusing on fermentation processes, as well as the optimization or development of efficient biosynthetic processes, such as medium formula and fermentation conditions. However, it is difficult to achieve breakthroughs in surfactin yield by utilizing traditional mutation breeding or fermentation optimization. In addition, it is a cost-effective strategy to investigate cheaper biomass as raw materials, such as agricultural or industrial waste, which caters to the theme of green sustainable development of the era and is supposed to be developed further. (2) Strain screening and molecular modification by genetic engineering are the most straightforward and effective ways to develop high-producing surfactin strains, and this can fundamentally solve the problem of low yield. In addition, for downstream surfactin production, several low-cost and efficient separation and purification technologies or means could aid production and save costs. The vast genetic sequence of the srfA operon, which encodes surfactin synthase (over 25 kb), and the complex biosynthetic regulation of a quorum sensing system are major concerns [59, 96]. B. subtilis has been modified to boost surfactin production primarily by exchanging promoters and editing the srfA operon, overexpressing the potent surfactin exporter to enhance its secretion and efflux, and modifying transcription to regulate srfA genes [97-101]. With the development of genetic engineering and synthetic biology, the development of high-transformation and high-productivity strains will be realized in the near future. Surfactin yield in genetic engineering is summarized in Table 1. In addition, novel surfactin variants are obtained through genetic engineering are being increasingly observed and explored. Table 2 shows novel surfactin variants.
APPLICATIONS IN AGRICULTURE AND FOOD SYSTEM
Surfactin Application in Agricultural Production
Lipopeptides’ antibacterial or antifungal properties boost the effect of B. subtilis’s competitiveness in the natural environment [99]. Currently, B. subtilis is regarded as a safe bacterium. Due to their capacity to suppress plant pathogenic bacteria, their secondary metabolites could be effectively exploited for biological control in agriculture [114]. Additionally, a prior study demonstrated that B. subtilis might be employed in agricultural production to produce its secondary metabolite surfactin in addition to promoting plant development and cleaning soil [115, 116].
Due to their potent antagonistic effects on microbial plant pathogens, a number of Bacillus strains are utilized as biocontrol agents. The pathogenic fungus Botrytis cinerea and Cladosporium fulvum were effectively inhibited by chitinase-producing biocontrol agent B. subtilis strain G3. The solid culture filtrate was used to create antifungal compounds; further investigating revealed that these antifungal agents were iturin, surfactin, and chitinase, which were isolated from crude proteins by acid precipitate. Surfactin and chitinase also prevented the growth of pores and germ tubes [117]. The endophytic isolate B. subtilis SCB-1’s antifungal capability was demonstrated by the results of Hazarika et al. While other metabolites were not seen, lipopeptide surfactin was found in the bacterial extract after mass spectrometric analysis. It was discovered that this isolate had antagonistic action toward a variety of fungal pathogens, including those belonging to the genera Saccharicola, Cochliobolus, Alternaria, and Fusarium. This bacterial strain produces volatiles and antifungal chemicals like surfactin that can be utilized to treat fungal infections. Additionally, this bacterial isolate's biocontrol capacity against several Fusarium strains has created opportunities for its use as a promising biocontrol agent in the near future [118]. It has previously been demonstrated that the tomato rhizosphere-isolated Bacillus strain SZMC 6179J has excellent in vitro antagonistic properties against the most important fungal pathogens of tomatoes, including Alternaria solani, Botrytis cinerea, Phytophthora infestans and Sclerotinia sclerotiorum, as well as a number of Fusarium species. It belongs to the B. subtilis subsp. group, according to taxonomic analyses. The critical gene can be proposed as the genetic determinant of biocontrol properties in B. subtilis, and it is found in the sequenced genome of strain SZMC 6179J [119]. Surfactin is an extracellular antibiotic that is synthesized by this gene. Fengycin (F), surfactin (S), and mycosubtilin (M) are three naturally occurring lipopeptides produced by B. subtilis that have been investigated within the context of biocontrol development. Their antifungal properties were assessed on two strains of Venturia inaequalis, an ascomycete fungus that causes apple scab, in vitro in a liquid medium. Tebuconazole, an active ingredient from the triazole family, was responsive to these two strains in a sensitive and less sensitive manner, respectively. These three compounds were examined alone, in binary (FS, FM, SM) and ternary mixes (FSM). All lipopeptide delivery methods demonstrated antifungal efficacy in experiments using the delicate strain. Overall, this study highlights the variety of ways that lipopeptides affect apple scab strains [120].
He et al. (2021) explored the effect of surfactin on the colonization of B. subtilis XF-1 in the phyllosphere of Chinese cabbage [121]. Static culture and biofilm assays on swimming and swarming agars and in 24-well plates, respectively, were used to examine the growth curve, motility, and biofilm formation of strain XF-1. Then, using analyses of leaf-microbe interactions and colonization assay, the bacterial adhesion to plant leaf surfaces and colonization in plant tissues of XF-1 were evaluated. In accordance with the findings, XF-1’s swimming, swarming, and ability to build biofilms were all dramatically reduced after 24 hours by 36.8%, 43.9%, and 53.9%, respectively. According to their findings, surfactin promoted XF-1 colonization in the phyllosphere of Chinese cabbage. Additionally, research offered theoretical guidance for enhancing strain XF-1's agricultural application. The cooperative effect of arbuscular mycorrhiza fungi (AMF) Glomus versiforme and B. vallismortis HJ-5 were tested against cotton Verticillium wilt using a greenhouse pot experiment, and higher performance liquid chromatography (HPLC) and tablet testing were used to identify any potential underlying mechanism [122]. AMF and HJ-5 demonstrated synergistic suppression against Verticillium wilt disease when compared to the control group. The disease index was reduced by 50.82%, root weight rose by 125.00%, and HJ-5 colonization in the rhizosphere increased by 23.80%. HPLC analysis showed that HJ-5 was capable of producing antifungal substances such as iturin A and surfactin. Dual inoculation enhanced the concentration of iturin A and surfactin by 13.38% and 11. 27%, respectively, compared to single inoculation with HJ-5. These findings indicated that the antifungal lipopeptides surfactin and iturin A may have played critical roles in the development of resistance to Verticillium wilt disease in cotton [122].
In addition, the storage and processing of fruits, vegetables and agricultural products were evaluated in relation to the use of lipopeptides surfactin. According to Tao et al. (2011), the mixed lipopeptides produced by B. subtilis fmbJ, which included surfactin and fengycin, had antifungal action, and the minimal inhibitory concentration (MIC) of the mixture was 2.0 mg/mL [123]. Furthermore, it was discovered that fengycin with surfactin could delay the development of peach soft rot and inhibit the pathogen (Rhizopus stolonifer). Additionally, mycelium surface morphological changes, membrane penetration, internal structure disintegration, and content leaking all contribute to the bacteria’s quick death. Everywhere potato is cultivated year after year, black scurf is a major cause for worry and is primarily controlled through seed treatment prior to sowing and other cultural techniques. A novel biosurfactant strain of B. subtilis HussainT-AMU and its culture filtrate were evaluated against Rhizoctonia solani in vitro and in vivo based on 16sRNA molecular and biochemical tests. Under the dual culture approach, a significant suppression of pathogen mycelium growth was seen in comparison to control. Fourier transform–infrared spectroscopy was used to identify the lipopeptide group-surfactin as the largely purified biosurfactant. The results of the bioagent were also assessed under laboratory and field circumstances. Under pot and field circumstances, the incidence of disease was reduced by up to 71% and 50%, respectively, and there was an improvement in tuber yield and plant growth. In addition, this novel biosurfactant was lipopeptide surfactin in structure [124]. The results of the current investigation, therefore, showed that the R. solani pathogen can be controlled by B. subtilis HussainT-AMU in an eco-friendly way, and its potential can be further explored with an extensive research study on various applications. This novel biosurfactant producer will aid in the creation and adoption of new biocontrol tactics in the future [124]. The biotrophic oomycete Plasmopara viticola, which is the root cause of grapevine downy mildew, is successfully controlled by B. subtilis GLB191. Researchers found that GLB191 supernatant is highly effective against downy mildew in this investigation, and the activity is due to both direct action against the pathogen and promotion of plant defense (induction expression of defense gene and callose production). The cyclic lipopeptides fengycin and surfactin were found in the supernatant by high-performance thin-layer chromatography analysis. As a result of their direct anti-oomycete activity against P. viticola to promote plant defense, surfactin and fengycin in the supernatant can shield a natural strain of GLB191 from downy mildew, as shown in this work. Overall, this study indicates that GLB191 supernatant may be a potential biocontrol agent for grapevine downy mildew [125]. The Fusarium oxysporum f. Cubense (FOC)-caused banana fusarium wilt is an important disease that affects bananas and has a significant negative impact on the global and Chinese banana industries. With its wide range of hosts, illness, and other characteristics, Fusarium oxysporum f. Cubense race 4 (Foc4) has a significant emphasis on prevention and control of the object. Some species of Bacillus already have enormous potential as commercial bacteri a compared to the current chemical control and other approaches. Ye et al. (2017) explored the mechanism of lipopeptide antibiotic produced by strain ZJ6-6 and the mechanism of action to Foc4 [126]. In a pot experiment, strain ZJ6-6 considerably decreased the prevalence of powdery bananas infested with Foc4; the disease index was 57.2 and 85.6,. The average plants were 21.7 cm in height and weighed 62.5 g when fresh. The control group’s average plant height and fresh weight were 15.8 cm and 43.6 g, respectively. The large increase in plant height and fresh weight suggests that ZJ6-6 not only effectively controls Foc4 but also promotes plant growth. The liposin antibiotic surfactin was found in the crude extract of strain ZJ6-6 after LC-MS analysis, and the discovered mass-to-charge ratio (m/z) was close to 1008, 1022, 1036, and 1050. It is hypothesized that C13–15 surfactin A, C14–16 surfactin B, or C16 surfactin C may be present in the crude extract of lipopeptide. These findings offered a theoretical foundation for the creation of a new commercial biocontrol because they showed that surfactin produced by B. amyloliquefaciens ZJ6-6 demonstrated effective disease control on bananas.
Jia et al. (2013) investigated that the difference in strains affected the cucumber wilt disease [127]. According to the research, mutant B1020 could effectively suppress cucumber wilt disease at 2 weeks, with a control efficacy of over 70%, and at 3 weeks, with a reduced efficacy of 36.2%. Only 29.1% of controls were effective at suppressing mutant B841 after three weeks. According to the findings, the levels of surfactin produced in nutrient broth by Bacillus strains did not improve their ability to suppress cucumber Fusarium wilt in nursery substrate. Further research must be carried out in the future. The mycotoxigenic phytopathogenic fungus contamination, such as Fusarium moniliforme contamination, in maize kernels may not only affect seed germination but also cause mycotoxicosis in animals and humans. F. moniliforme cannot be effectively controlled by fungicides on maize kernels. Hence, efficient bioactive compounds are required to prevent plant and animal diseases associated with F. moniliforme contamination in cereals. In a previous report, the effects of surfactin on the growth of pakchoi and other crops as well as the use of surfactin in fertilizer, were investigated [128]. The germination rate and biomass were used as evaluating indexes when surfactin was applied at various concentrations to treat Arabidopsis, pakchoi and wheat seeds. The crop’s germination and biomass peaked at less than 0.4 g/L of surfactin. Although more surfactin led to inhibition, lesser surfactin was advantageous for seed germination and growth. The activities of the enzymes amylase, peroxidase (POD), and catalase (CAT) in the surfactin group (0.6 g/L) were enhanced by 103.02%, 71.74% and 73.95%, respectively, in comparison to that in the control group, according to discovered enzyme activities. The effects of surfactin on stomatal opening, soaking area, waxy layer of leaves, surface tension of foliar fertilizer solution, plant development, and nutrient absorption in pakchoi were also investigated by this research. The results showed that 0.4 g/L surfactin promoted seeding growth and biomass and enhanced absorption of Fe and Mn by 26.01% and 231.48%, respectively. It was also found that the absorption of spraying liquid and leaf fertilizer could be promoted by the surface, which reduces the tension of foliar fertilizer solution, dissolves the leaf waxy layer, opens the stomatal opening, improves the wetting area and increases the permeability of nutrients. These provided the basis for improving the utilization rate and development of related additives.
Surfactin is a well-known antimicrobial lipopeptide with excellent antifungal activities against a number of phytopathogenic fungi and may have implications for agriculture. Mildew is the main cause of losses in grains during storage, both in quality and quantity. In high temperature and humidity, paddy easily gets mildew and accumulates mycotoxins. In order to sustainably grow agriculture, new types of biological anti-mildew agents must be investigated due to the effects of conventional pesticides on the environment and the disease's rising drug resistance. Reversed-phase high-performance liquid chromatography (RP-HPLC) was used to isolate and identify the metabolites from B. subtilis subsp. and B. subtilis natto Bna05, which included two kinds surfactin variants: V7-surfactin variants and I/L7-surfactin variants. Wang et al. (2017) evaluated their antifungal activities subsequently [129]. Utilizing response surface design and a single factor test, the liquid fermentation process was optimized. The following conditions allowed Bna05 to produce antifungal compounds: 30 °C temperature, 37 h of culture, and an initial pH of 6.6 for medium. Soy peptone (or tryptone or peptone) 1.0 g, glucose 2.0 g, beef extract 0.3 g, Na2HPO4 0.15 g, KH2PO4 0.1 g, yeast extract 0.3 g, glutamate 0.06 g, aspartic acid 0.06 g, and H2O 100 mL were the ingredients in the appropriate medium formula. The fungal inhibition rate (FIR%) against Aspergillus niger of the supernatant after fermentation optimization was 87.02%, which was increased by 65.69% compared with the FIR% of the supernatant before optimization. Additionally, a light microscope and scanning electron microscope (SEM) were used to investigate the antifungal mechanisms. The observation of the light microscope showed that A. niger spore germination was delayed and hyphal growth inhibited as a result of the treatment with either of the three RP-HPLC isolates. The spores and mycelium assembled together in the presence of another active compound F2, and some drop-like materials were observed around the mycelium in the presence of either V7-surfactin variants or I/L7-surfactin variants. These drop-like objects might be formed by the fungus’s cell content leaking out; I/L7-surfactin variants caused this drop phenomenon more obviously than V7-surfactin variants. Additionally, SEM showed that the hypha treated by either of the three isolates exhibited shriveling and distortion, and the spore head showed signs of severs collapse. In contrast, the actively growing hyphae were plump and exuberant in untreated cultures. The preliminary antifungal synergy study indicated that the combination of either V7-surfactin or I/L7-surfactin with F2 showed a synergistic antifungal effect; there was no synergy effect between V7-surfactin and I/L7-surfactin. Subsequently, the antifungal spectrum and stability under different conditions of sample supernatant solid phase extraction liquid (SPEL) were analyzed, most of the tested fungi, including A. niger, A. flavus, Rhizopus sp, Penicillium expansum and A. fumigatus, were sensitive to SPEL. Although SPEL was tolerable to high temperature and a wide range of pH values, metal ions such Mg2+, Zn2+, Fe 2+ and Ca2+ should be avoided at temperatures higher than 80 °C and in a strong acidic and alkaline medium. SPEL’s in vitro antifungal impact on paddy was then investigated. The results showed that SPEL with an additive content of 200 pg SPEL/g paddy could reduce the number of molds in the control from a serious health hazard level to a critical level, and fatty acid values were reduced from unsuitable for storage standard to a suitable for storage standard. The content of aflatoxin B1 was decreased by 74.0%. Besides, SPEL could inhibit paddy germination effectively. These data revealed that SPEL is valuable for the exploration of new antifungal agents for paddy storage [129]. A previous report investigated the mechanism and effect of surfactin in controlling rice sheath blight and rice bacterial leaf blight [130]. The laboratory virulence test results showed that surfactin possessed certain virulence to rice sheath blight fungus and Xanthomonas oryzae pv. oryzae; the concentration for 50% of maximal effect (EC50) values are 19.76 μg/mL and 16.49 μg/mL, respectively. In the pot plant efficacy test, the surfactin solution with an effective concentration of 100 μg/mL had a 58.29% control effect on rice sheath blight disease; the average control effect on rice sheath blight disease spots was 55.61%, and the inhibitory effect on rice bacterial blight disease spot was 64.21%. In the field efficacy test, surfactin solution had an overall control effect of 44.64% against rice sheath blight and an overall control effect of 49.71% against rice bacterial blight. It had an effective concentration of 100 μg/mL. In addition, the effects of mycotoxicosis and the antifungal surfactin derived from Brevibacillus brevis KN8(2) on seed germination and F. moniliforme were examined. The results revealed that surfactin inhibited and damaged the F. moniliforme hyphae in vitro. Surfactin damaged the DNA and protein and decreased the glutathione concentration in F. moniliforme, according to agarose gel electrophoresis, biochemical, and sodiumdodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Surfactin's antifungal properties also inhibited F. moniliforme from growing in maize kernels, preventing the spread of diseases that affect plants and animals associated with F. moniliforme contamination of maize kernels. These findings revealed that surfactin might be an effective bio-fungicide for controlling plant disease. On the basis of these findings, surfactin from B. brevis KN8(2) serves as an effective bio-fungicide to control F. moniliforme, which is associated with plant and animal diseases [131]. Jiang et al. (2016) investigated the antibacterial mechanism of the antibacterial substance [ΔLeu6] surfactin secreted by B. subtilis LS-9 against F. moniliforme and F. graminearum [132]. The [ΔLeu6] surfactin inhibited F. moniliforme, with an inhibition rate of 95.88%. Using electron microscopy, F. moniliforme and F. graminearum mycelia were observed. The [ΔLeu6] surfactin caused the formation of F. moniliforme and F. graminearum mycelis cell membranes on the surface of many of the sags. Internal organelles formed cannot be distinguished, and the cell interior color becomes shallow, and hyphae are decreased. Furthermore, using isobaric tags for relative and absolute quantitation technology (iTRAQ), the number of up-regulated proteins for F. graminearum was found to be 103 and the down-regulated proteins was 225. There were 328 total proteins; 10.9% of the identified total protein ratio was taken into account. Hydrolases and lyases are increased, functional proteins on the membrane are up-regulated, and cell growth and protein metabolism are markedly inhibited according to an analysis of up-regulated protein function clustering. Further, the effects of [ΔLeu6] surfactin on F. moniloforme growth and Fiimonisin vitamin B1 synthesis were investigated. The results show that the inhibitory effect of higher [ΔLeu6] surfactin concentration is more obvious. The data also had similar impacts on the synthesis of deoxynivalenol and the growth of F. graminearum. In addition, B. subtilis LS-9-produced surfactin has been successfully used for corn storage.
On the other hand, it was claimed that the addition of surfactin might considerably increase the rate of organic matter decomposition in fertilizer and the effectiveness of composting [133]. Surfactin also has a cosolvent effect on pesticides [134]. This means that it can help pesticides operate better and break down organic pesticides, hence decreasing pollution and harm to the soil and the ecosystem.
According to its performance, surfactin exhibits strong resistance to plant pathogens, infections, and insect pests, as well as the solubility of pesticides, and this is very important for agricultural planting and production. The production and safety of agricultural products are strongly tied to the safety of the food industry, which serves as a source of food raw materials and is the first barrier to food safety. In the near future, surfactin has higher prospects for use in the storage and processing of agricultural products.
Surfactin Application in Livestock and Aquaculture
The easily absorbed and digested amino acids are antibacterial lipopeptide components. They can be used to fully or partially replace antibiotics in the feed for livestock and poultry to reduce damage to animals. Peptides can strongly suppress the growth of gram-positive bacteria when added to feed, support the growth of livestock and poultry, and improve feed utilization. In addition, some reports show that peptides primarily function in the digestive system and are hardly ever absorbed in the intestine. Humans or animals are typically unaffected by residual problems in bodies following peptide surfactin consumption. The intestinal flora of livestock and poultry can be regulated by peptides, and no bacterial resistance was observed. The peptide is worthy of being recognized as an environmentally friendly feed addition owing to its stable, non-toxic, no side effects, no residues, and no bacterial resistance properties. Furthermore, the thermal stability of Bacillus lipopeptide ensures lower loss of activity and its stable antibacterial activity during the process of granulation. The addition of surfactin might considerably increase the digestion and utilization of the oil nutrients in the feed, preventing indigestion of fat feed by animal pups.
It was reported that surfactin’s effects in inhibiting the Avian influenza virus (H9N2) and Newcastle Virus (NDV) were explored [135
