# Lipopeptide Biosurfactants fromBacillus spp.: Types, Production, Biological Activities, and Applications in Food

Authors: Nawazish Ali; Zhengjun Pang; Fenghuan Wang; Baocai Xu; Hesham R. El-Seedi
Journal: Journal of Food Quality
 (2022)

---

## Abstract

Biosurfactants are a functionally and structurally heterogeneous group of biomolecules produced by multiple filamentous fungi, yeast, and bacteria, and characterized by their distinct surface and emulsifying ability. The genusBacillus is well studied for biosurfactant production as it produces various types of lipopeptides, for example, lichenysins, bacillomycin, fengycins, and surfactins. Bacillus lipopeptides possess a broad spectrum of biological activities such as antimicrobial, antitumor, immunosuppressant, and antidiabetic, in addition to their use in skincare. Moreover, Bacillus lipopeptides are also involved in various food products to increase the antimicrobial, surfactant, and emulsification impact. From the previously published articles, it can be concluded that biosurfactants have strong potential to be used in food, healthcare, and agriculture. In this review article, we discuss the versatile functions of lipopeptide Bacillus species with particular emphasis on the biological activities and their applications in food.


---

## Body

## 1. Introduction

Biosurfactants (BSs) could be found on the surface of microbial cell and transferred into the extracellular space by multiple filamentous fungi, and yeast (Starmerella, Candida, Ustilago, Saccharomyces, Trichosporon, and Pseudozyma) and bacteria (Nocardia, Rhodococcus, Acinetobacter, Arthrobacter, and Gordonia) [1]. They are primarily classified according to their structural characteristics, associated microorganisms, and molecular weight (MW) [2].BSs have a hydrophobic region and a hydrophilic end consisting of hydrocarbons acids, diverse fatty acids (saturated, unsaturated, linear, or branched long-chain) and carbohydrate cyclic peptide, alcohol, carboxylic acid, amino acid, and phosphate. This amphipathic framework provides an ability to reduce the surface tension at the interfaces of phases with divergent polarities, which includes emulsion (liquid-liquid) and suspension (liquid-solid), which is collectively named “dispersion” [3, 4]. BSs have also the capacity to produce molecular aggregates, for example, micelles, like the ones patented at the critical micelle concentration (CMC). The CMC of BSs is normally 1–200 mg/L, which is 10–40 times lower than that formed with chemical surfactants [5].BSs are produced through microbial fermentation, which includes yeast, fungi, and bacterial strains (Pseudomonas, Lactobacillus, Acinetobacter, Halomonas, Rhodococcus, Bacillus, Enterococcus, and Arthrobacter). Among all microbes, genus Bacillus is well studied for its biosurfactant production as it produces various types of cyclic lipopeptides/lipoproteins such as lichenysins, bacillomycin, fengycins, and surfactins [6].Lipopeptides and glycolipids are highly efficient and popular group of BSs such as surfactin and rhamnolipids, with low-MW [7–9], whereas the high-MW BSs are lipoprotein, phospholipids, and emulsion [10, 11]. Lipopeptide BSs are composed of two different regions: an acyl tail (s) and a short linear oligopeptide sequence, containing an amide bond. The hydrophobic tail contains a hydrocarbon chain, whereas the hydrophilic head contains the lipopeptide BSs peptide sequence. The peptide module includes cationic and anionic residues, as well as nonproteinaceous amino acids [12].Taking into account the unique properties ofBacillus cyclic lipopeptides, and their applications in medicine, healthcare, environment, agriculture, and food industries, their biocompatibility, bioavailability, and structural diversity attracted further attention in the last decade [13–15]. The nonribosomal peptide synthetase (NRPS) enzyme is associated with the formation of cyclic lipopeptides. Lipopeptide surfactants are classified according to their structure, with isoforms comprising a variety of D and L amino acids [16, 17]. The demand for new lipopeptides is increasing in order to broaden their application. Earlier, various studies have been conducted to establish the biotechnological production, functional qualities, and physical properties of lipopeptide surfactants. In this review article, a comprehensive study is carried out to describe the contributions of Bacillus lipopeptides in the food industry and biological activities.


## 2. Classes of Lipopeptides Produced byBacillus spp

Lipopeptides are a subgroup of microbial surfactants, for example, surfactin, fengycin, iturin, lichenysin, and kurstakin [18]. The types or classifications of lipopeptides surfactants are mainly based on the amino acid sequences and various strains of Bacillus spp. producing lipopeptides such as B. subtilis, B. cereus, B. thuringiensis, B. globigii, B. amyloliquefaciens, B. megaterium, B. pumilus, and B. licheniformis. [19–22] (Table 1 and Figure 1).Table 1
Lipopeptide-producing strains and their applications.
LipopeptidesLP-producing bacterial strainBiological applicationRef.SurfactinB. methylotrophicus DCS1Antioxidant, antimicrobial, and antiadhesive activities[23]B. mojavensis A21Diesel biodegradation[24]B. mojavensis PRC101Antagonism againstFusarium verticillioides (fungal species infecting maize)[25]B. subtilisInhibitory activity against phytopathogenicFusarium sp.[26]B. atrophaeus L193Aphicidal activity against the aphid Rhopalosiphum padi (pest in cereal crops)[27]B. subtilis SAS-1 B. amyloliquefaciens BR-15Engine oil degradation efficiently augmented (75–94%)[28]B. subtilis SPB1Antioxidant activity, chelating activity, histological study proved effective treatment of complicated wound healing and skin diseases[29]B. subtilis strain ATCC6633Microbial enhanced oil recovery[30]B. subtilis 573Decreased viability of breast cancer cell lines, T47D and MDA-MB-231 and nontumor fibroblast cell line (MC-3 T3-E1)[31]B. natto TK-1Therapeutic agent, anti-inflammation[32]B. subtilis ANR 88Effective in the synthesis of silver as well as gold nanoparticles[33]B. pumilusSilver nanoparticles produced as antimicrobial and nematicide[34]B. subtilis LSFM-05Nontoxic dispersion in biotechnology and nanotoxicology.[35]Surfactin AB. (SPB) NH-100 and NH-217Biocontrol agent against bakanae diseases in rice[36]B. stratosphericus sp. A15Antioxidant and antibacterial activity, wound healing activity by connective tissue regeneration, thickened epidermal layer, and keratinocyte formation[37]PumilacidinB. stratosphericus FLU5Hydrocarbon removal from contaminated soil, negligible cytotoxic effect against the mammalian cells HEK293[38]LipopeptideB. subtilis SPB1Anti-obesity effect through the inhibition of lipid digestive and liver dysfunction enzymes[39]Iturin A, fengycinB. amyloliquefaciens DSM 23117Biocontrol agent against a common fungal phytopathogen botrytis cinerea[40]Bacillomycin DB. amyloliquefaciens fiply 3AKilling human cancer cell line viz. A549 (alveolar adenocarcinoma), A498 (renal carcinoma) and HCT-15 (colon adenocarcinoma) while not effecting the normal cell line L-132 (pulmonary epithelial cells)[41]Iturin AB. subtilisInhibiting chronic myelogenous leukemia in vitro via paraptosis, apoptosis, and inhibition of autophagy[42]LipopeptideB. mojavensis ifo 15718Pharmaceutical applications as it possesses antibacterial activity against pathogenS. aureus and lack of toxicity to PC12 and PBMC cells[43]Iturin AB. amyloliquefaciens PPCB004Activity against postharvest fungal pathogens on stored fruits[44]FengycinB. amyloliquefaciens FZB42Antifungal activity against fusarium moniliforme (rice bakanae disease), fusarium oxysporum (root rot) and trichoderma atroviride (ear rot and root rot)[45]Surfactin homologsB. amyloliquefaciens MD4-12Synergistic Antimicrobial effects against various gram-positive and Gram-negative bacteria[46]B. subtilis NH 217, B. amyloliquefaciens FZB42Excellent biofilm formation, antifungal activity against various phytopathogen and their associated diseases[47]Bacillomycin, FengycinB. methylotrophicus XT1 CECT 8661Effective biocontrol agent againstB. cinerea infection, antioxidant triggerer in different fruits[48]Iturin A, Fengycin CB. Subtilis EA-CBOO15Iturin A inhibitedM. fijiensis mycelial growth, and fengycin C displayed strong inhibitory activity on ascospore germination[49]Iturin A, surfactinB. SubtilisBroad hypocholesterolemic activities, immune-modulators, toxins, and enzyme inhibitors[50]Surfactin, FengycinB. subtilis fmbj (CGMCC no. 0934)Effective against Newcastle disease virus (NDV) and infectious bursal disease virus (IBDV)[51]LichenysinB. licheniformis VS16Biofilm inhibition, removal of heavy metals[52]B. licheniformis Ali5Enhanced oil recovery and motor oil removal from contaminated sand[53]B. licheniformis W16Excellent emulsification and microbial enhanced oil recovery[54]SurfactinB. subtilis C9Effective biocontrol agent controlling cladoceran grazers in algal cultivation system[55]Lipopeptide biosurfactantB. pseudomycoides OR 1Biocontrol agent against food-borne pathogensE. coli (MTTC 43), Klebsiella pneumoniae (MTVV 530) and staphylococcus aureus (MTCC 96)[56]Novel cyclic lipopeptide C18B. pseudomycoides BS6Bioremediation and recycling waste cooking oil[57]PumilacidinB. safensis CCMA-560Thermal stable and microbial enhanced oil recovery[58]LipopeptideB. sp. H20-1Antagonistic effect against sulfate-reducing bacteria[59]LipopeptideB. cereus UCP1615Bioremediation of petroleum derivative in soil and water with above 90% removal[60]PaenibacterinPaenibacillus elgii HOA73Effective bio-pesticide against diamondback moth Plutella xylostella (destructive insect pest)[61]PaenibacterinPaenibacillus thiaminolyticusOSY-SEMinimized endotoxemia showed low toxicity against human kidney sell line (ATCC CRL-2190)[62]LipopeptideB. altitudinis MS16Promising emulsification and antifungal activity[63]LipopeptideB. brevisExcellent emulsifier and antibacterial effects[64]LipopeptideB. subtilis SPB1Toothpaste formulation[65]Figure 1
Chemical structures of some lipopeptides: (a) surfactin, (b) lichenysin, (c) iturin, (d) fengycin.
(a)(b)(c)(d)


### 2.1. Surfactin

Surfactin belongs to the lipopeptides family, which was firstly isolated by Arima et al. in 1968 and produced by manyBacillus with surfactant activities [66]. Surfactin (1036 Da) is an amphipathic cyclic lipopeptide biosurfactant produced by many strains of the bacterial genus Bacillus. The surfactin molecule was firstly screened from the culture media of B. subtilis strains and applied as a clotting inhibitor [67, 68].Surfactin is composed of a heptapeptide (ELLVDLL) along with chiral sequence LLDLLDL linked withβ-hydroxy (fatty acid chain) of carbon chain (C12–C16) and forms a close cyclic lactone ring structure. The structure of surfactin consists of both hydrophobic (located at 2–4, 6, and 7) and hydrophilic (located at 1 and 5) part [69]. Surfactin displays a stable and conserved folding in aqueous solutions, and negatively charged amino acids, Glu and Asp, exhibit polar domain. Moreover, it is also soluble in organic solvents, for example, dichloromethane, ethanol, chloroform, butanol, and methanol [70].The peptide part represents topology like “horse-saddle” and is called theβ-sheet structure in the backbone folding, which believe that these structural traits contribute to the broad spectrum of biological properties of surfactin [71, 72].Naturally, many isoforms of surfactin present, which only differ with their physicochemical properties such as (1) type of amino acid of peptide ring at 2nd, 4th, and 7th positions, and (2) branching of hydroxyl fatty acid moiety and chain length. What’s more, isoforms also depend upon theBacillus strain and other factors such as media, environmental, and nutritional conditions of substrate [73, 74]. Previously, studies reported that surfactin shows potent antitumoral, antiviral, anticoagulant, inhibitors of enzymes, and antimicoplasma activities [75].


### 2.2. Lichenysin

Lichenysin a lipopeptide produced most ofB. licheniformis strains, and it has excellent surfactant and chelating agent for Ca2+ and Mg2+ [76–79]. Lichenysin was also reported to exert antimicrobial, anti-inflammatory, antitumor, and immunosuppressive properties. Besides good biological activities, it also has hemolytic activity [79]. These traits of lichenysin are caused by the amphiphilic nature of the lipopeptide. Structurally, lichenysin consists of amino acids (7) and a β-hydroxy fatty acid along with C12–C17 carbon atoms. Many isoforms of lichenysin are present in nature, for example, lichenysin A [80–82]. The structure of lichenysin is very similar to surfactin and differs with the substitution of glutamine with glutamic acid in the first amino acid position [82]. However, this small difference markedly increases the surfactant properties of lichenysin [79].


### 2.3. Kurstakin

Kurstakin is a low-molecular-weight lipopeptide mainly produced and isolated fromBacillus thuringiensis kurstakin HD-1. The amino acid sequence of kurstakin was reported as follows: Thr-Gly-Ala-Ser-His-Gln-Gln. The fatty acyl chain of kurstakin is linked with N-terminal amino acid residue by amide bond, and every lipopeptide consists of lactone linkage among carboxyl terminal amino acid and hydroxyl group in the side chain of the serine residue [83, 84].


### 2.4. Iturin

Iturin are an important class of lipopeptides with a molecular mass of ∼1.1 kDa. Iturin A consists of two parts: (a) C14–C17 (amino fatty acids) and (b) seven amino acid residues (heptapeptides; Asn-Tyr-Asn-Gln-Pro-Asn-Ser). Iturin (D and E) varies from iturin A due to the presence of a free carboxyl group in iturin D and carboxymethyl group in iturin E. The structure of iturin shows that it has an amphiphilic character [85, 86]. Iturin molecule is of great interest because of their biological activities and physicochemical traits and used in oil, pharmaceutical, and food industries. Almost all strains of Bacillus subtilis produce iturin lipopeptide, and its operon ranges from 38 to 40 kb in size and contains four open reading frames such as ItuA, ItuB, ItuC, and ItuD [87]. Iturin lipopeptide also contains mojavensin, mycosubtilin, bacillomycin D, bacillomycin F, and bacillomycin L, which differ in amino acid sequences of the heptapeptides [88]. Iturin was reported to exert potent antifungal activity against, Botrytis cinerea, Alternaria alternate, and Penicillium expansum. Moreover, it also has strong surface activity and destabilizing effect [89].


### 2.5. Fengycins

Fengycins are lipopeptides mainly produced by the genera ofBacillus and Paenibacillus. Fengycins have strong antifungal activity and markedly affect filamentous fungi [90]. Fengycins are decapeptides and C14–C19 to β-hydroxy fatty acid chain, which showed potent antifungal activity [91, 92]. There are two subclasses of Fengycins, namely, Fengycin A and Fengycin B, that only differ from each by the amino acid attached at position 6. Fengycin B contains Val at position 6, whereas Fengycin A contains Ala. Fengycins (A and B) were firstly reported in B. subtilis strain by Vanittanakom et al. [93]. The closely related fengycin type was reported and named plipastatin due to the position of amino acids L- Tyr and D- Tyr [94].


## 2.1. Surfactin

Surfactin belongs to the lipopeptides family, which was firstly isolated by Arima et al. in 1968 and produced by manyBacillus with surfactant activities [66]. Surfactin (1036 Da) is an amphipathic cyclic lipopeptide biosurfactant produced by many strains of the bacterial genus Bacillus. The surfactin molecule was firstly screened from the culture media of B. subtilis strains and applied as a clotting inhibitor [67, 68].Surfactin is composed of a heptapeptide (ELLVDLL) along with chiral sequence LLDLLDL linked withβ-hydroxy (fatty acid chain) of carbon chain (C12–C16) and forms a close cyclic lactone ring structure. The structure of surfactin consists of both hydrophobic (located at 2–4, 6, and 7) and hydrophilic (located at 1 and 5) part [69]. Surfactin displays a stable and conserved folding in aqueous solutions, and negatively charged amino acids, Glu and Asp, exhibit polar domain. Moreover, it is also soluble in organic solvents, for example, dichloromethane, ethanol, chloroform, butanol, and methanol [70].The peptide part represents topology like “horse-saddle” and is called theβ-sheet structure in the backbone folding, which believe that these structural traits contribute to the broad spectrum of biological properties of surfactin [71, 72].Naturally, many isoforms of surfactin present, which only differ with their physicochemical properties such as (1) type of amino acid of peptide ring at 2nd, 4th, and 7th positions, and (2) branching of hydroxyl fatty acid moiety and chain length. What’s more, isoforms also depend upon theBacillus strain and other factors such as media, environmental, and nutritional conditions of substrate [73, 74]. Previously, studies reported that surfactin shows potent antitumoral, antiviral, anticoagulant, inhibitors of enzymes, and antimicoplasma activities [75].


## 2.2. Lichenysin

Lichenysin a lipopeptide produced most ofB. licheniformis strains, and it has excellent surfactant and chelating agent for Ca2+ and Mg2+ [76–79]. Lichenysin was also reported to exert antimicrobial, anti-inflammatory, antitumor, and immunosuppressive properties. Besides good biological activities, it also has hemolytic activity [79]. These traits of lichenysin are caused by the amphiphilic nature of the lipopeptide. Structurally, lichenysin consists of amino acids (7) and a β-hydroxy fatty acid along with C12–C17 carbon atoms. Many isoforms of lichenysin are present in nature, for example, lichenysin A [80–82]. The structure of lichenysin is very similar to surfactin and differs with the substitution of glutamine with glutamic acid in the first amino acid position [82]. However, this small difference markedly increases the surfactant properties of lichenysin [79].


## 2.3. Kurstakin

Kurstakin is a low-molecular-weight lipopeptide mainly produced and isolated fromBacillus thuringiensis kurstakin HD-1. The amino acid sequence of kurstakin was reported as follows: Thr-Gly-Ala-Ser-His-Gln-Gln. The fatty acyl chain of kurstakin is linked with N-terminal amino acid residue by amide bond, and every lipopeptide consists of lactone linkage among carboxyl terminal amino acid and hydroxyl group in the side chain of the serine residue [83, 84].


## 2.4. Iturin

Iturin are an important class of lipopeptides with a molecular mass of ∼1.1 kDa. Iturin A consists of two parts: (a) C14–C17 (amino fatty acids) and (b) seven amino acid residues (heptapeptides; Asn-Tyr-Asn-Gln-Pro-Asn-Ser). Iturin (D and E) varies from iturin A due to the presence of a free carboxyl group in iturin D and carboxymethyl group in iturin E. The structure of iturin shows that it has an amphiphilic character [85, 86]. Iturin molecule is of great interest because of their biological activities and physicochemical traits and used in oil, pharmaceutical, and food industries. Almost all strains of Bacillus subtilis produce iturin lipopeptide, and its operon ranges from 38 to 40 kb in size and contains four open reading frames such as ItuA, ItuB, ItuC, and ItuD [87]. Iturin lipopeptide also contains mojavensin, mycosubtilin, bacillomycin D, bacillomycin F, and bacillomycin L, which differ in amino acid sequences of the heptapeptides [88]. Iturin was reported to exert potent antifungal activity against, Botrytis cinerea, Alternaria alternate, and Penicillium expansum. Moreover, it also has strong surface activity and destabilizing effect [89].


## 2.5. Fengycins

Fengycins are lipopeptides mainly produced by the genera ofBacillus and Paenibacillus. Fengycins have strong antifungal activity and markedly affect filamentous fungi [90]. Fengycins are decapeptides and C14–C19 to β-hydroxy fatty acid chain, which showed potent antifungal activity [91, 92]. There are two subclasses of Fengycins, namely, Fengycin A and Fengycin B, that only differ from each by the amino acid attached at position 6. Fengycin B contains Val at position 6, whereas Fengycin A contains Ala. Fengycins (A and B) were firstly reported in B. subtilis strain by Vanittanakom et al. [93]. The closely related fengycin type was reported and named plipastatin due to the position of amino acids L- Tyr and D- Tyr [94].


## 3. Production, Isolation, and Characterization ofBacillus Lipopeptides

Lipopeptide surfactants are produced by many microbes including bacteria, fungi, and yeast. However, herein, we mainly focus on the production ofBacillus lipopeptides. The biosurfactants are synthesized from the extracellular or intracellular part of microbes. Notably, biosurfactants are produced during the stationary and exponential phase, whereas the biosurfactant production is predominate in the death phase. Reduction in surface tension to 8 mJm−2 is the minimum value to be considered when producing the biosurfactant. The various strains of Bacillus spp. produced novel lipopeptides such as B. licheniformis, and B. circulans. Furthermore, details of lipopeptide production from various Bacillus along with fermentation conditions are presented in Tables 1 and 2.Table 2
Strategies and mechanisms used to enhance lipopeptide production byBacillus sp.
Strategy usedFactors evaluatedStrainResultsBS natureRef.RSM, CCDBrewery waste and (carbon, nitrogen, agitation speed, temperature, pH)B. subtilis N3–1P657 g.l−1Surfactin[95]RSM, BBD(d-glucose, sucrose, xylose), hydrocarbons (hexadecane, diesel, benzene, heptane), nitrogen source (NaNO3, NH4NO3, (NH4)2SO4, NaHCO3, urea)Bacillus sp. SS1052.65 g.l−1Surfactin[96]CCD, PBDSucrose, glucose, starch, peanut oil, potato), nitrogen source (peptone, beef extract, trypsin, yeast extract), other variables (MgSO4, KCl, KH2PO4, FeSO4.6H2O, NH4Cl, MnSO4, CuSO4, sodium glutamate)B. subtilis N70.706 g.l−1Surfactin[97]RSM, PBD, (TFAT) two factors at timeNitrogen source ((NH4)2SO4, KNO3, NaNO3, NH4Cl, beef extract, yeast extract)B. subtilis KLP20150.98 g.l−1Surfactin[98]RSM, OFAT one factor at a timeCarbon source (glucose, fructose, sucrose, xylose, rhamnose, soluble starch), nitrogen source (NH4Cl, C6H17N3O7, urea, peptone, soybean meal), metal ions (ZN2+, Fe3+, Mg2+, Mn2+, Ca2+, K+)Bacillus sp. BH0720.027 g.l−1IturinA A[99]OFATSingle and multidose Fe2+B. megaterium4.2 g.l−1Surfactin[100]22 factorial design, RSMGlucose and yeast extractB. subtilis EA-CB00150.78, 0.355 g.l−1Fengycin, Iturin A[101]PBD, CCDCarbon source (glucose, maltose, dextrose, mannitol, sorbitol, galactose, xylose, starch), nitrogen source (KNO3, (NH4)2SO4, NaNO3, soy flour, peptone, casein hydrolysate, yeast extract urea)B. amyloliquefaciens MD4-121.25 g.l−1Surfactin[102]CCD, Fed-batch strategyFed-batch strategy (pH-stat, DO-stat, constant rate feeding, combined feeding), Sunflower oil, NaNO3, MgSO4.7H2O, yeast extractAneurinibacillus thermoaerophilus HAKO111.2 g.l−1Surfactin[103]ANN-GAGlucose, urea, SrCl2, and MgSo4B. circulans MTCC 82814.38 g.l−1Unidentified[104]ANN-GALp concentration, Ca2+, pHB. licheniformis, B. megaterium45% oil recoveryLipopeptide[105]BBDGlucose, glutamic acid, temperature, NaClB. mojavensis 144.12 g.l−1Lipopeptide[106]BBDOptimization of non-nutritional factors (inoculum age and size, pH, agitation, aeration, temperature)B. subtilis SPB13.3 g.l−1Lipopeptide[107]Media composition and characteristicsNitrogen source ((NH4)2SO4, KNO3, NH4NO3, NH4Cl, peptone, beef extract, yeast extract), carbon source (glucose, sucrose, fructose, maltose, sorbitol, starch)B. velezensis KLP20162.5 g.l−1Lipopeptide[108]n-paraffin, n-dodecane, n-hexadecane, sunflower oil, canola oil, sucrose, glycerol, diesel fuel, n-tetradecane, nitrogen source ((NH4)2SO4, NaNO3, KNO3, urea, peptone, yeast extract, beef extract), metal and sulfur source (FeSO4, MnSO4, MgSO4), and C/N ratio, pHPaenibacillus sp. D94.11 g.l−1New lipopeptide[109]Different culture media, shaking speed of shaker, liquid and solid fermentation, attapulgite powderB. natto NT-61.94-fold increasedIturin A, surfactin[110]Different culture media, vine-trimming shoots, glucose, hemicellulosic hydrolysate, and cellulosic hydrolysateB. tequilensis ZSB101.52 g.l−1Lipopeptide[111]Taguchi methodCarbon source (sucrose, whey, crude oil)), NaCl, Na2HPO4, NaH2PO4, and (NH4)2SO4B. cereus1.8 g.l−1Lipopeptide[112]Use of cheap substrate/raw materialCarbon source (orange peel, citrus medica peels, banana peels, and potato peels), inoculum size, incubation time, temperature, substrate concentrationB. licheniformis KC7109731.796 g.l−1Lichenysin[113](Butter milk, poultry-transforming waste flour, inoculum size), submerged fermentationB. subtilis SPB112.61 g.l−1Lipopeptide[114]Corn steep liquor (CSL), iron, manganese, and magnesiumB. subtilis #5734.8 g.l−1Surfactin[115]Potato peels, temperature, pH, saline conditionsB. pumilus DSVP183.2 g.l−1Iturin A[116]Grape seed flourB. amyloliquefaciens C50.80 g.l−1Bacillomycin D[117]Distiller grains (DGS, coculture fermentation with B. B. amyloliquefaciens X82B. amyloliquefaciens MT453.4 g.l−1Surfactin[118]Palm oil, waste glycerol, immobilized on chitosanBacillus Sp. GY 199.8 g.l−1Lipopeptide[119]Solid state fermentation (SSF)Soybean flour, rice straw, starch, yeast extract, kinetic parameters (iso- and nonisothermal process, isothermal and nonisothermal process in fermenter)B. amyloliquefaciens XZ-173(55.83 mg/gds)Lipopeptide[120]Soybean flour, rice straw, glycerol, maltose, pH, water content, inoculum size, fermentation time, temperatureB. amyloliquefaciens XZ-17315.03 mg/gdsSurfactin[121]Wheat bran, rice straw, soybean flour, temperature, pH, water content, inoculum sizeB. subtilis CCTCCM20720970.90 mg/gdsLipopeptide[122]Rice bran husk, sunflower oil, coconut oil cake, cotton oil cake, corn cob, orange peel, jackfruit peel, sugarcane leaf, pineapple peel, banana leaf, cheese whey, dry yeast cells, pongamia seed cake, jatropha seed cake ground oil cake, glucose with MSMB. amyloliquefaciens3-fold increasedIturin A[123]Olive leaf residue flour, olive cake flourB. subtilis SPB10.3067 g.l−1Lipopeptide[124]Mutagenesis induced enhanced yieldUV and gamma ray-induced mutagenesisB. subtilis HS01212-fold increasedSurfactin[125]Random mutagenesis using gamma irradiationB. subtilis UTB11.8-fold increasedIturin A[126]UV irradiation, nitrosoguanidine, and ion beam mutagenesisB. amyloliquefaciens ES-2-410.3-fold increasedLipopeptide[127]Combination of UV irradiation and nitrous acid treatmentB. subtilis SPB12-fold increasedLipopeptide[51]Genome shufflingGenome shufflingB. amyloliquefaciens FMB382-fold increasedSurfactin[128]Genome shuffling and gene (fenA) expressionB. amyloliquefaciens ES-2-48.30-fold increasedFengycin[129]Mutagenesis (UV, nitrosoguanidine, atmospheric, and room temperature plasma)B. amyloliquefaciens LZ-52.03-fold increasedIturin A[130]Protoplast fusionB. mojavensis JF-20.382 g.l−1Lipopeptide[131]Recombinant strains for higher yieldSurfactin promoter THY-15 was replaced to THY-15/pg3-srfA. Then introduced a Vitreoscilla hemoglobin (VHb) gene into engineered strain to obtain a novel THY-15/pg3-srfA (VHb)B. subtilis THY-1510.02 g.l−1Surfactin C15[132]Loc gene expressed, the fosmid N13 with whole Loc gene screened from B. velezensis 916 genomic library, the cassette fused with IPTG inducible promoter Pspac induced in the fosmid N13 resulted N13+spec and N13+PSSpec transformed to obtained derivative strains FZBNPLOC and FZBPSLOCB. velezensis FZB4215-fold increasedLocillomycins[133]Enhanced transcription of iturin A biosynthetic genes was implemented by inserting a strong promoter C2up into upstream of theitu operon, fermentation optimization using RSM and furthermore, overexpression of pleiotropic regulator DegQB. amyloliquefaciens LL38-fold increasedIturin A[134]Cloning of the biosurfactant genes sfp, sfp0, sfpA into BioS a, BioS b, BioS c, recombinant strains after cloning of biosurfactant genes in toE. coli. (E. coli DH5ά)B. subtilis SK3202-fold increasedLipopeptide[135]Wild type, overexpression of THY-7-Pgrac-ycxA, overexpression of THY-7-Pgrac-krsE, overexpression of THY-7-Pgrac-yerPB. subtilis 1680.55, 1.15, 0.93, 1.67 g.l−1Surfactin[136]Using CRISPRi 20 genes were repressed,yrpC, racE, murC genes were inhibited individually. Furthermore, combination inhibition of bkdAA and bkdAB genesB. subtilis4.69-fold increasedSurfactin[137]Replacement of PsrfA with Pg3B. subtilis0.55–9.74 g l−1Surfactin[138]Insertion ofsfp gene from Paenibacillus sp. D9 into E. coliPaenibacillus sp. D93-fold increasedPaenibacterin[139]


### 3.1. Substrates

Many substrates mainly consist of hydrophobic mixtures, vegetable oils, waste products, dairy products, etc. and are used for the production of lipopeptide-based surfactants. To minimize the production cost of lipopeptide-based surfactants, renewable and low-cost substrates were applied as presented in Tables1 and 2. Moreover, it is also necessary to select substrates with a high nutritional value for the growth of microbes. One of the best methods used is to apply organic matter such as industrial waste, oil substrate, and agro-based materials. Interestingly, these waste materials provide distinct energy source for microbes with effective surfactants production.


### 3.2. Production of Biosurfactants by Using Agro-Industrial Waste

Agro-industrial waste is an ideal choice for the production of lipopeptide and helps in the industrial waste management. Agro-industrial wastes contain both carbon and lipids along with other necessary nutrients, which are the major requirement for the growth of biosurfactant-producing microbes. Previously, many researchers successfully utilized various agro-industrial wastes such as sugarcane molasses, date molasses, cassava flour, rice straw, corn, fruits and vegetable wastes, bran, and others for the production of biosurfactant [115, 140–146].Molasses is the key waste product of sugar and date industries, and it has gained a lot of attention for the production of biosurfactant. This popularity to use as a substrate for biosurfactant production is mainly due to its low cost and rich source of dry matter (75%), protein (2.5%), nonsugar organic matter (9–12%), minerals (potassium, calcium, phosphorus, and magnesium), and other components (thiamine, biotin, inositol, and pantothenic acid). The sugar content in the molasses ranges from 48 to 56%, making it ideal for the growth of various microorganisms [147–149].Makkar and Cameotra [150], Saimmai et al. [142], and Joshi et al. [151] reported the biosurfactant production from Bacillus subtilis strains (MTTCC 2423, MTCC1427, and SA9) and Bacillus licheniformis TR7) by using molasses as carbon source. In another study, Joshi et al. [54] documented that cane molasses and date molasses used as a carbon source enhance the production of lichenysin-A-like lipopeptide by using Bacillus licheniformis W16. Rane et al. [33] conducted a study to utilized agro-industrial wastes (molasses, banana peels, orange peels, whey, potato peels, and bagasse) as a substrate for the production of biosurfactant by Bacillus subtilis ANR 88. Their study results revealed that biosurfactant production in the molasses substrate as a carbon source was higher (0.24 g/L) compared to other agro-industrial wastes. Moreover, they also found that by optimizing the conditions (ammonium ferric citrate 0.25%, molasses 4%, and pH 7), the yield of biosurfactant significantly increases to 2-fold (0.513 g/L).Recently, Al-Dhabi et al. [141] used date molasses as a carbon source for the production of biosurfactant from Bacillus subtilis strain Al-Dhabi-130. They found that using date molasses as a carbon source yields the biosurfactant to 74 mg/g substrate and can be used for the large-scale production of biosurfactant .Peanut oil cake is a novel agro-waste, which can be used for the production of lipopeptide. Nalini et al. [152] reported that maximum lipopeptide production was obtained (8.18 g) from peanut oil cake as a substrate by using B. cereus strain SNAU01. Paraszkiewicz et al. [153] also observed that lipopeptide surfactants such as surfactin and iturin can be produced by Bacillus strains using carrot peel as substrate.


### 3.3. Production of Lipopeptides by Using Oil Waste

Wastes from oil processing industries represent one of the best and readily available renewable substrates for microbial biosurfactant production. The hydrophobic substrate containing media such as oil helps microbes to produce lipopeptide surfactant. Sunflower, olive oil, coconut oil, and canola are the main oil made from oil industries and considered the best carbon source for biosurfactant production [22, 38, 143, 154].Ostendorf et al. [143] reported the excellent production of lipopeptide biosurfactants by Bacillus stratosphericus strain FLU5 using waste vegetable oils (olive oil, corn oil, and residual frying oil). In another study, Md Badrul Hisham et al. [155] observed the excellent yield of surfactin by Bacillus sp. HIP3 when using used cook oil as a substrate (2%).


## 3.1. Substrates

Many substrates mainly consist of hydrophobic mixtures, vegetable oils, waste products, dairy products, etc. and are used for the production of lipopeptide-based surfactants. To minimize the production cost of lipopeptide-based surfactants, renewable and low-cost substrates were applied as presented in Tables1 and 2. Moreover, it is also necessary to select substrates with a high nutritional value for the growth of microbes. One of the best methods used is to apply organic matter such as industrial waste, oil substrate, and agro-based materials. Interestingly, these waste materials provide distinct energy source for microbes with effective surfactants production.


## 3.2. Production of Biosurfactants by Using Agro-Industrial Waste

Agro-industrial waste is an ideal choice for the production of lipopeptide and helps in the industrial waste management. Agro-industrial wastes contain both carbon and lipids along with other necessary nutrients, which are the major requirement for the growth of biosurfactant-producing microbes. Previously, many researchers successfully utilized various agro-industrial wastes such as sugarcane molasses, date molasses, cassava flour, rice straw, corn, fruits and vegetable wastes, bran, and others for the production of biosurfactant [115, 140–146].Molasses is the key waste product of sugar and date industries, and it has gained a lot of attention for the production of biosurfactant. This popularity to use as a substrate for biosurfactant production is mainly due to its low cost and rich source of dry matter (75%), protein (2.5%), nonsugar organic matter (9–12%), minerals (potassium, calcium, phosphorus, and magnesium), and other components (thiamine, biotin, inositol, and pantothenic acid). The sugar content in the molasses ranges from 48 to 56%, making it ideal for the growth of various microorganisms [147–149].Makkar and Cameotra [150], Saimmai et al. [142], and Joshi et al. [151] reported the biosurfactant production from Bacillus subtilis strains (MTTCC 2423, MTCC1427, and SA9) and Bacillus licheniformis TR7) by using molasses as carbon source. In another study, Joshi et al. [54] documented that cane molasses and date molasses used as a carbon source enhance the production of lichenysin-A-like lipopeptide by using Bacillus licheniformis W16. Rane et al. [33] conducted a study to utilized agro-industrial wastes (molasses, banana peels, orange peels, whey, potato peels, and bagasse) as a substrate for the production of biosurfactant by Bacillus subtilis ANR 88. Their study results revealed that biosurfactant production in the molasses substrate as a carbon source was higher (0.24 g/L) compared to other agro-industrial wastes. Moreover, they also found that by optimizing the conditions (ammonium ferric citrate 0.25%, molasses 4%, and pH 7), the yield of biosurfactant significantly increases to 2-fold (0.513 g/L).Recently, Al-Dhabi et al. [141] used date molasses as a carbon source for the production of biosurfactant from Bacillus subtilis strain Al-Dhabi-130. They found that using date molasses as a carbon source yields the biosurfactant to 74 mg/g substrate and can be used for the large-scale production of biosurfactant .Peanut oil cake is a novel agro-waste, which can be used for the production of lipopeptide. Nalini et al. [152] reported that maximum lipopeptide production was obtained (8.18 g) from peanut oil cake as a substrate by using B. cereus strain SNAU01. Paraszkiewicz et al. [153] also observed that lipopeptide surfactants such as surfactin and iturin can be produced by Bacillus strains using carrot peel as substrate.


## 3.3. Production of Lipopeptides by Using Oil Waste

Wastes from oil processing industries represent one of the best and readily available renewable substrates for microbial biosurfactant production. The hydrophobic substrate containing media such as oil helps microbes to produce lipopeptide surfactant. Sunflower, olive oil, coconut oil, and canola are the main oil made from oil industries and considered the best carbon source for biosurfactant production [22, 38, 143, 154].Ostendorf et al. [143] reported the excellent production of lipopeptide biosurfactants by Bacillus stratosphericus strain FLU5 using waste vegetable oils (olive oil, corn oil, and residual frying oil). In another study, Md Badrul Hisham et al. [155] observed the excellent yield of surfactin by Bacillus sp. HIP3 when using used cook oil as a substrate (2%).


## 4. Isolation, Purification, and Characterization of Lipopeptides

Lipopeptides are mostly synthesized by bacterial genusBacillus. The bacterial cells are grown in their respective media with specific conditions (varied from strains to strains) to produce lipopeptides prior to their separation by centrifugation. Malfanova et al. [156] grew bacterial cells (60 h at 28°C) subjected to centrifugation (13,000 rpm for 10 min) to obtain crude lipopeptides. The obtained supernatant was acidified by using HCl acid, while the precipitate was extracted with methanol and further concentrated by vacuum evaporation [156, 157]. The crude extract was purified by many methods such as gel filtration in Sephadex column and high-performance liquid chromatography, and the collected eluent was further subjected to MALDI-TOF-MS/LC-MS/MS-MS/NMR/FTIR [53].


## 5. Pharmacological Activities

### 5.1. Anticancer

Bacillus lipopeptides are considered versatile bioactive compounds with potent antitumor activity. For example, surfactin has been documented to exert antitumor activity towards human colon carcinoma cell lines (HCT15 and HT29), Ehrlich’s ascites carcinoma cells, and breast cancer cell lines (T47D and MDA-MB-231) [31, 104, 158]. Surfactin inhibits the growth of transformed cells via cell cycle arrest and induction of apoptosis and suppresses ERK (extracellular-signal-regulated kinase) and PI3 K/Akt pathway [158]. In another study, Liu et al. [159] reported that surfactin-like lipopeptides purified from B. subtilis Hs0121 exert cytotoxicity towards Bcap-37 breast cancer cells (IC50: 29 ± 2.4 μM). Surfactin was also documented to inhibit the LoVo colon cancer cells (IC50: 26 μM) [158] (Figure 2). What’s more, Wang et al. [160] observed that B. subtilis natto T-2 with crude cyclic lipopeptides (CLPs) showed a cytotoxic effect against human K562 leukemia cells. Surfactin was also reported to exhibit the cytotoxic effect on hepatocellular carcinoma [159, 160]. Recently, Hong et al. [161] have reported that five Surfactin isomers produced by B. pumilus HY1 during Cheonggukjang fermentation markedly inhibited the growth of two cancer cell lines (MCF-7 and Caco-2).Figure 2
Pharmacological activities of lipopeptides.Fengycin, a lipopeptide produced by various strains ofB. subtilis, was reported to exert strong anticancer activity on colon cancer cell line HT29 and human lung cancer cell line 95D [162, 163]. Similarly, Bacillus lipopeptide (iturin) was also reported to possess a broad spectrum of anticancer activity on several cell lines (e.g., HepG2, Caco-2, BT474, MDA-MB-231, MCF-7, HUVEC, BIU-87, BRL-3A, A549, and K562 cells [42, 164–171].


### 5.2. Hemolytic Activity

The lipopeptide surfactants induce the hemolysis of human erythrocytes due to their detergent effect and membrane forming ability. Therefore, lipopeptide surfactants are used as potent inhibitors of fibrin clot formation. Arima et al. [172] reported for the first time that the surfactin potently inhibits the fibrin clot formation via abrogating the conversion of fibrin monomer into fibrin polymer. Bernheimer and Avigad studied the inhibition of fibrin clot formation and hemolysis of erythrocyte by subtilysin derived from B. subtilis since 1970 [173] (Figure 2).The hemolytic activity of lipopeptide iturin A was studied by Aranda et al. They documented that iturin dependently exerts hemolytic activity on human erythrocytes. The underlying mechanism of action was that iturin A induced hemolysis via colloid-osmotic mechanism and K+ leakage followed by hemoglobin release [85]. In another study, Dehghan-Noudeh et al. [174] documented that B. subtilis ATCC 6633-derived lipopeptide surfactant attenuated potent hemolytic effect in comparison with chemical surfactants such as hexadecyl trimethyl ammonium bromide, sodium dodecyl sulfate, tetradecyl trimethyl ammonium bromide, and benzalkonium chloride.


### 5.3. Anti-Inflammatory

Previously, it was reported that lipopeptides exert anti-inflammatory activities via several pathways such as modulation of the TLR4 (Toll-like receptor 4), inhibition of lipoteichoic acid (LTA)-induced NF-κB, activator of transcription-1 (STAT-1), interaction with cytosolic phospholipase A2 (PLA2), and increase in the phosphorylation of STAT-3 [88] (Figure 3).Figure 3
The proposed anti-inflammatory mechanism ofBacillus subtilis lipopeptides.Moreover, it also impairs the antigen-presenting function of macrophages, suppresses the LPS-induced expression of cluster of differentiations (CD40, CD54, CD80), and inhibits the activation of CD4+ T-cells [32, 88].It was also documented that surfactin markedly inhibits the overproduction pro-inflammatory mediators (IL-6, tumor necrosis factor alpha or TNF-alpha, and interleukin beta or IL-1β), prostaglandin E2, monocyte chemoattractant protein-1, NO, and reactive oxygen species (ROS), and suppresses the expression of MMP-9 (matrix metallopeptidase 9), COX-2 (cyclooxygenase-2), and iNOS (inducible nitric oxide synthase) [175].


### 5.4. Antibacterial Activity

The demand for new antimicrobial agents significantly increases due to the resistance of pathogenic microorganisms towards already present antimicrobial drugs. Surfactin, a lipopeptide, was reported to exert antibacterial activity against various pathogenic bacteria. Beside surfactin, otherBacillus-related lipopeptides were also reported to possess well-known inhibitory activity towards the growth of pathogenic bacteria [176–179].Huang et al. [178] reported that surfactin and fengycin produced by the strain B. subtilis fmbj effectively inactivate endospores of B. cereus. The lipopeptide mainly damages the surface structure of the spores. B. velezensis strain H3-isolated surfactin isoforms were reported to active against P. aeruginosa, St. aureus, Klebsiella pneumoniae, and Mycobacterium [179].In another study, fengycin isoforms isolated from marineBacillus strain markedly inhibited the growth of various bacteria such as K. aerogenes, Citrobacter fruendii, Micrococcus flavus, Proteus vulgaris, Alcaligenes faecalis, E. coli, and Serratia marcescens [180].Lipopeptide antibiotic subtulene A isolated from the culture filtrate ofB. subtilis SSE4 was reported to inhibit the growth of Gram-positive and Gram-negative bacterial strains such as Stenotrophomonas maltophilia, Enterobacter cloacae, and Xanthomonas campestris [181]. Fengycin and surfactin lipopeptides containing culture filtrate of the endophytic B. amyloliquefaciens was reported to potently inhibited the growth of all tested Gram-negative ones except Ochrobactrum anthropi and all Gram-positive bacteria tested except B. [157].Recently, a study conducted by Lv et al. [177] has also reported that B. amyloliquefaciens C-1 fermentation supernatant contains a mixture containing surfactin and fengycin, which inactivate the growth of Clostridium difficile (bacteria that can infect the bowel and cause diarrhoea). Iturin analog isolated from Bacillus strain was reported to inactivate the growth of Xanthomonas arboricola and Pseudomonas syringae [176].


### 5.5. Antifungal and Biocontrol

It has been documented thatBacillus lipopeptides exert a wide array of antifungal activities. Briefly, iturin markedly inhibits the growth of nematophagous fungi, wood-staining fungi, Aspergillus ﬂavus, Penicillium roqueforti, and Colletotrichum demiatium [19, 182–186], whereas fengycin was reported to inhibit the Fusarium graminearum, Botrytis cinerea, and Podosphaera fusca [187, 188].More detailed investigations conducted by various researchers reported that lipopeptides exert morphological changes such as hyphal swellings, changed organization of mitochondria, decreased intracellular pH, esterases, and mitochondria activities, and decreased hydrophobicity of the hyphae [48, 189].Desmyttere et al. [190] conducted a study to explore the antifungal activities of lipopeptides isolated from B. subtilis against apple scab disease causing Venturia inaequalis strains. Their study results revealed that Bacillus subtilis lipopeptide mixtures containing (fengycin, surfactin, and mycosubtilin) markedly inhibited the growth of V. inaequalis S755 and V. inaequalis rs552.Han et al. [191] documented that B. amyloliquefaciens L-H15-derived peptides (iturin A with C15β-amino fatty acid and cyclic peptide with a molecular weight of 852.4 Da) exhibited strong antagonism against Fusarium oxysporum, Rhizoctonia solani, and Phytophthora capsici.Dimkić et al. [176] studied five different lipopeptide-producing strains of Bacillus (SS-10.7, SS-12.6, SS-13.1, SS-27.2, and SS-38.4), and their extracts were further tested against Pseudomonas syringae pv. aptata (P16) and Xanthomonas arboricola pv. juglandis (301, 311, and 320). The results revealed that Bacillus strains mostly produced kurstakins, iturins, surfactins, and fengycins lipopeptides. Moreover, they reported that ethyl acetate extracts exert more favorable effect on phytopathogens.Botrytis cinerea is a necrotrophic fungi, which infects more than 200 plant species including fruits and vegetables. Toral et al. [48] conducted a study to determine anti-B. cinerea activity of lipopeptides isolated from Bacillus XT1 CECT 8661. They observed that lipopeptide-rich extract mainly containing surfactin, bacillomycin, and fengycin potently inhibits the growth of B. cinerea. What’s more, SEM (scanning electron microscope) and TEM (transmission electron microscope) analysis revealed that lipopeptides alter the morphology of the phytopathogen.


### 5.6. Antiviral Activity

It has been well documented that lipopeptides such as surfactin possess a broad spectrum of antiviral activity against SARS-CoV-2, herpes simplex virus (HSV-1 and HSV-2), Newcastle disease virus, Semliki Forest virus, murine encephalomyocarditis virus, Simian immunodeficiency virus, vesicular stomatitis virus, transmissible gastroenteritis virus, porcine parvovirus feline calicivirus, pseudorabies virus, and bursal disease virus, porcine epidemic diarrhoea virus, and viral hemorrhagic septicemia virus. The chemical structure of surfactin lipopeptide, for example, length of the carbon chain, makes it fit for the inactivation of various viruses [51, 192–194]. Moreover, it was also observed that surfactin more significantly inactivates the enveloped viruses such as herpes viruses and retroviruses compared with nonenveloped viruses [195]. This may be due to the physicochemical interaction among membrane active property of surfactin and the virus lipid membrane [196]. Surfactin permeates into the lipid bilayer and results in the complete disintegration of the envelope containing the viral proteins involved in virus adsorption and penetration to the target cells [195].


### 5.7. Antiadhesion and Antibiofilm

Surface adhesion and biofilm formation are the mechanisms by which most of bacteria are used for their survival. Lipopeptides have the potential to decrease the interfacial tension and surface of biofilms. In numerous ways, lipopeptides disrupt the membrane structure. For example, surfactin gets inserted into the lipid bilayers, chelates monovalent and divalent cations, solubilizes the fluid phospholipid phase, and modifies the membrane permeability [197]. Surfactin may form voltage-independent channels in biofilms, and these channels disturb the membrane integrity and permeability, leading to membrane disruption [13, 198]. Iturin isoform (mycosubtilin) produced by B. subtilis interacts with membranes via its sterol alcohol group and exhibits resistance to fungi [199]. B. circulans strain showed antiadhesive property towards many bacteria species [200]. Similarly, various Bacillus strains inhibit biofilm formation [6, 78–80].


### 5.8. Others

Bacillus lipopeptides were also reported in wound healing and oral care products [65]. Zouari et al. [29] documented how the B. subtilis SPB1 biosurfactant supplementation improves the liver function, hyperlipidemia, and hypertriglyceridemia in high-fat-high-fructose (HFHF) diet-fed rats. In another study, the same research group also observed that B. subtilis SPB1 biosurfactant treatment improves the renal functions and inhibits angiotensin I-converting enzyme (ACE) in HFHF diet-fed rats [39]. Moreover, B. subtilis strain containing surfactin was shown to effectively kill the larval and pupal stages of mosquito species, for example, Aedes aegypti, Culex quinquefasciatus, and Anopheles stephensi [195].


## 5.1. Anticancer

Bacillus lipopeptides are considered versatile bioactive compounds with potent antitumor activity. For example, surfactin has been documented to exert antitumor activity towards human colon carcinoma cell lines (HCT15 and HT29), Ehrlich’s ascites carcinoma cells, and breast cancer cell lines (T47D and MDA-MB-231) [31, 104, 158]. Surfactin inhibits the growth of transformed cells via cell cycle arrest and induction of apoptosis and suppresses ERK (extracellular-signal-regulated kinase) and PI3 K/Akt pathway [158]. In another study, Liu et al. [159] reported that surfactin-like lipopeptides purified from B. subtilis Hs0121 exert cytotoxicity towards Bcap-37 breast cancer cells (IC50: 29 ± 2.4 μM). Surfactin was also documented to inhibit the LoVo colon cancer cells (IC50: 26 μM) [158] (Figure 2). What’s more, Wang et al. [160] observed that B. subtilis natto T-2 with crude cyclic lipopeptides (CLPs) showed a cytotoxic effect against human K562 leukemia cells. Surfactin was also reported to exhibit the cytotoxic effect on hepatocellular carcinoma [159, 160]. Recently, Hong et al. [161] have reported that five Surfactin isomers produced by B. pumilus HY1 during Cheonggukjang fermentation markedly inhibited the growth of two cancer cell lines (MCF-7 and Caco-2).Figure 2
Pharmacological activities of lipopeptides.Fengycin, a lipopeptide produced by various strains ofB. subtilis, was reported to exert strong anticancer activity on colon cancer cell line HT29 and human lung cancer cell line 95D [162, 163]. Similarly, Bacillus lipopeptide (iturin) was also reported to possess a broad spectrum of anticancer activity on several cell lines (e.g., HepG2, Caco-2, BT474, MDA-MB-231, MCF-7, HUVEC, BIU-87, BRL-3A, A549, and K562 cells [42, 164–171].


## 5.2. Hemolytic Activity

The lipopeptide surfactants induce the hemolysis of human erythrocytes due to their detergent effect and membrane forming ability. Therefore, lipopeptide surfactants are used as potent inhibitors of fibrin clot formation. Arima et al. [172] reported for the first time that the surfactin potently inhibits the fibrin clot formation via abrogating the conversion of fibrin monomer into fibrin polymer. Bernheimer and Avigad studied the inhibition of fibrin clot formation and hemolysis of erythrocyte by subtilysin derived from B. subtilis since 1970 [173] (Figure 2).The hemolytic activity of lipopeptide iturin A was studied by Aranda et al. They documented that iturin dependently exerts hemolytic activity on human erythrocytes. The underlying mechanism of action was that iturin A induced hemolysis via colloid-osmotic mechanism and K+ leakage followed by hemoglobin release [85]. In another study, Dehghan-Noudeh et al. [174] documented that B. subtilis ATCC 6633-derived lipopeptide surfactant attenuated potent hemolytic effect in comparison with chemical surfactants such as hexadecyl trimethyl ammonium bromide, sodium dodecyl sulfate, tetradecyl trimethyl ammonium bromide, and benzalkonium chloride.


## 5.3. Anti-Inflammatory

Previously, it was reported that lipopeptides exert anti-inflammatory activities via several pathways such as modulation of the TLR4 (Toll-like receptor 4), inhibition of lipoteichoic acid (LTA)-induced NF-κB, activator of transcription-1 (STAT-1), interaction with cytosolic phospholipase A2 (PLA2), and increase in the phosphorylation of STAT-3 [88] (Figure 3).Figure 3
The proposed anti-inflammatory mechanism ofBacillus subtilis lipopeptides.Moreover, it also impairs the antigen-presenting function of macrophages, suppresses the LPS-induced expression of cluster of differentiations (CD40, CD54, CD80), and inhibits the activation of CD4+ T-cells [32, 88].It was also documented that surfactin markedly inhibits the overproduction pro-inflammatory mediators (IL-6, tumor necrosis factor alpha or TNF-alpha, and interleukin beta or IL-1β), prostaglandin E2, monocyte chemoattractant protein-1, NO, and reactive oxygen species (ROS), and suppresses the expression of MMP-9 (matrix metallopeptidase 9), COX-2 (cyclooxygenase-2), and iNOS (inducible nitric oxide synthase) [175].


## 5.4. Antibacterial Activity

The demand for new antimicrobial agents significantly increases due to the resistance of pathogenic microorganisms towards already present antimicrobial drugs. Surfactin, a lipopeptide, was reported to exert antibacterial activity against various pathogenic bacteria. Beside surfactin, otherBacillus-related lipopeptides were also reported to possess well-known inhibitory activity towards the growth of pathogenic bacteria [176–179].Huang et al. [178] reported that surfactin and fengycin produced by the strain B. subtilis fmbj effectively inactivate endospores of B. cereus. The lipopeptide mainly damages the surface structure of the spores. B. velezensis strain H3-isolated surfactin isoforms were reported to active against P. aeruginosa, St. aureus, Klebsiella pneumoniae, and Mycobacterium [179].In another study, fengycin isoforms isolated from marineBacillus strain markedly inhibited the growth of various bacteria such as K. aerogenes, Citrobacter fruendii, Micrococcus flavus, Proteus vulgaris, Alcaligenes faecalis, E. coli, and Serratia marcescens [180].Lipopeptide antibiotic subtulene A isolated from the culture filtrate ofB. subtilis SSE4 was reported to inhibit the growth of Gram-positive and Gram-negative bacterial strains such as Stenotrophomonas maltophilia, Enterobacter cloacae, and Xanthomonas campestris [181]. Fengycin and surfactin lipopeptides containing culture filtrate of the endophytic B. amyloliquefaciens was reported to potently inhibited the growth of all tested Gram-negative ones except Ochrobactrum anthropi and all Gram-positive bacteria tested except B. [157].Recently, a study conducted by Lv et al. [177] has also reported that B. amyloliquefaciens C-1 fermentation supernatant contains a mixture containing surfactin and fengycin, which inactivate the growth of Clostridium difficile (bacteria that can infect the bowel and cause diarrhoea). Iturin analog isolated from Bacillus strain was reported to inactivate the growth of Xanthomonas arboricola and Pseudomonas syringae [176].


## 5.5. Antifungal and Biocontrol

It has been documented thatBacillus lipopeptides exert a wide array of antifungal activities. Briefly, iturin markedly inhibits the growth of nematophagous fungi, wood-staining fungi, Aspergillus ﬂavus, Penicillium roqueforti, and Colletotrichum demiatium [19, 182–186], whereas fengycin was reported to inhibit the Fusarium graminearum, Botrytis cinerea, and Podosphaera fusca [187, 188].More detailed investigations conducted by various researchers reported that lipopeptides exert morphological changes such as hyphal swellings, changed organization of mitochondria, decreased intracellular pH, esterases, and mitochondria activities, and decreased hydrophobicity of the hyphae [48, 189].Desmyttere et al. [190] conducted a study to explore the antifungal activities of lipopeptides isolated from B. subtilis against apple scab disease causing Venturia inaequalis strains. Their study results revealed that Bacillus subtilis lipopeptide mixtures containing (fengycin, surfactin, and mycosubtilin) markedly inhibited the growth of V. inaequalis S755 and V. inaequalis rs552.Han et al. [191] documented that B. amyloliquefaciens L-H15-derived peptides (iturin A with C15β-amino fatty acid and cyclic peptide with a molecular weight of 852.4 Da) exhibited strong antagonism against Fusarium oxysporum, Rhizoctonia solani, and Phytophthora capsici.Dimkić et al. [176] studied five different lipopeptide-producing strains of Bacillus (SS-10.7, SS-12.6, SS-13.1, SS-27.2, and SS-38.4), and their extracts were further tested against Pseudomonas syringae pv. aptata (P16) and Xanthomonas arboricola pv. juglandis (301, 311, and 320). The results revealed that Bacillus strains mostly produced kurstakins, iturins, surfactins, and fengycins lipopeptides. Moreover, they reported that ethyl acetate extracts exert more favorable effect on phytopathogens.Botrytis cinerea is a necrotrophic fungi, which infects more than 200 plant species including fruits and vegetables. Toral et al. [48] conducted a study to determine anti-B. cinerea activity of lipopeptides isolated from Bacillus XT1 CECT 8661. They observed that lipopeptide-rich extract mainly containing surfactin, bacillomycin, and fengycin potently inhibits the growth of B. cinerea. What’s more, SEM (scanning electron microscope) and TEM (transmission electron microscope) analysis revealed that lipopeptides alter the morphology of the phytopathogen.


## 5.6. Antiviral Activity

It has been well documented that lipopeptides such as surfactin possess a broad spectrum of antiviral activity against SARS-CoV-2, herpes simplex virus (HSV-1 and HSV-2), Newcastle disease virus, Semliki Forest virus, murine encephalomyocarditis virus, Simian immunodeficiency virus, vesicular stomatitis virus, transmissible gastroenteritis virus, porcine parvovirus feline calicivirus, pseudorabies virus, and bursal disease virus, porcine epidemic diarrhoea virus, and viral hemorrhagic septicemia virus. The chemical structure of surfactin lipopeptide, for example, length of the carbon chain, makes it fit for the inactivation of various viruses [51, 192–194]. Moreover, it was also observed that surfactin more significantly inactivates the enveloped viruses such as herpes viruses and retroviruses compared with nonenveloped viruses [195]. This may be due to the physicochemical interaction among membrane active property of surfactin and the virus lipid membrane [196]. Surfactin permeates into the lipid bilayer and results in the complete disintegration of the envelope containing the viral proteins involved in virus adsorption and penetration to the target cells [195].


## 5.7. Antiadhesion and Antibiofilm

Surface adhesion and biofilm formation are the mechanisms by which most of bacteria are used for their survival. Lipopeptides have the potential to decrease the interfacial tension and surface of biofilms. In numerous ways, lipopeptides disrupt the membrane structure. For example, surfactin gets inserted into the lipid bilayers, chelates monovalent and divalent cations, solubilizes the fluid phospholipid phase, and modifies the membrane permeability [197]. Surfactin may form voltage-independent channels in biofilms, and these channels disturb the membrane integrity and permeability, leading to membrane disruption [13, 198]. Iturin isoform (mycosubtilin) produced by B. subtilis interacts with membranes via its sterol alcohol group and exhibits resistance to fungi [199]. B. circulans strain showed antiadhesive property towards many bacteria species [200]. Similarly, various Bacillus strains inhibit biofilm formation [6, 78–80].


## 5.8. Others

Bacillus lipopeptides were also reported in wound healing and oral care products [65]. Zouari et al. [29] documented how the B. subtilis SPB1 biosurfactant supplementation improves the liver function, hyperlipidemia, and hypertriglyceridemia in high-fat-high-fructose (HFHF) diet-fed rats. In another study, the same research group also observed that B. subtilis SPB1 biosurfactant treatment improves the renal functions and inhibits angiotensin I-converting enzyme (ACE) in HFHF diet-fed rats [39]. Moreover, B. subtilis strain containing surfactin was shown to effectively kill the larval and pupal stages of mosquito species, for example, Aedes aegypti, Culex quinquefasciatus, and Anopheles stephensi [195].


## 6. Lipopeptide Applications in Food

Lipopeptides have well-defined antiadhesive, antibacterial, antiviral, and anticancer properties, ensuring their role in the cosmetics, pharmaceutical, and even food industries. Lipopeptides are mainly employed as surfactants in the food industry. Moreover, rhamnolipids and surfactins are positively exploited in the baking industry, providing good texture, volume, and product stability. They are also used to promote the emulsification process in the fat tissue to regulate fat globule agglomeration. Certain lipopeptides derived fromEnterobacter cloacae have recently been presented into the food market with their high emulsifying characteristics owing to the potential to improve viscosity even at extreme acidic conditions. In terms of economic growth, the most significant increases in food additives have been seen in emulsifiers and hydrocolloids that were up to 10.5% and 6.0%, respectively.The vulnerability of biologically active peptides as an antimicrobial agent in the food preservation is rare due to their limits to proteases. The usage of ring-structured peptides like lipopeptides, on the other hand, can prevent this susceptibility. There are two types of lipopeptides: a cyclic heptapeptide acylated withβ-amino fatty acids that have a chain length of C14–C16, and the fengycin group containing a β-hydroxy fatty acid with uncommon amino acids including allo-threonine and ornithine. They also consisted of cyclic heptapeptide that makes a lactone linkage with β-hydroxy fatty acids. They are enzyme-insensitive (particular protease), suppressing the development of a broad variety of pathogenic fungi (Fusarium graminearum, Rhizoctonia solani, and Aspergillus flavus) as well as postharvest pathogens such as Botrytis cinerea and Penicillium expansum.Gandhi and his coworkers revealed that the rhamnolipid emulsifier with a concentration of 0.10% significantly improved the texture, moisture content, and appearance of muffins for longer periods. Surfactin inclusion in many fermented food products, like natto, a Japanese soybean meal, is extremely favorable for acceptance as an ingredient or addition. Juola et al. [201] determined the surfactin content of various natto types. Notably, the greatest concentrations discovered were close to 2.2 mg/g, which corresponds to 80–100 mg surfactin per 50 g natto. Additional research is required to establish the surfactant’s recommended daily intake (RDI) to pronounce it harmless and is usually considered to be a generally recognized as safe (GRAS) organism. Therefore, surfactin has strong potential to be used in the food sector.Zouari et al. [202] prepared the cookies using sesame peel flour partially replaced with white wheat flour. When additional sesame peel flour was employed, the characteristics such as toughness, water content, and spread factor had been degraded. Interestingly, adding 0.1% B. subtilis SPB1 biosurfactant significantly enhanced the textural profile, even when compared to the standard surfactant glycerol monostearate [202]. In another research, the possible lipopeptides from Bacillus spp. reduced the Ochratoxin and A. carbonarius that were found in the processing of wine-making [203]. In the wine-making process, the concentration of Ochratoxin should not surpass 2.0 μg/L as it is a carcinogenic mycotoxin. Additionally, this compound has a detrimental effect on yeast fermentation behaviour. Lipopeptides also had higher antifungal capabilities than SO2 and stimulated yeast growth as well as the generation of esters and acids that are involved in the olfactory profile [203].


## 7. Conclusion

Lipopeptides are very useful molecules due to their multiple applications. MostBacillus lipopeptides have been applied in food, cosmetic, biotechnology, pharmaceutical industries, where emulsifying, antimicrobial, and surfactant properties are used. The application and production of lipopeptides are very promising trend; however, the high cost of production makes them unfit for large-scale synthesis. Furthermore, even though there are many reports displaying the thrombolytic, antitumor, and anti-inflammatory activity of lipopeptides, the few numbers of clinical trials warrant more significant efforts. In future, extensive studies should be carried out to verify previously published author findings, which further help with the utilization of these miracle compounds. In summary, Bacillus lipopeptides have strong potential application in various fields and a lot of work will be needed to formulate strategies for improved large-scale biosynthesis of lipopeptides.



---

*Source: 3930112-2022-04-27.xml*