# Elucidating the biosurfactant potential of Bacillus strains: a study of structure and function in food applications

Houda Gharsallah, Cédric Guerin, Ahlem Eddehech, Sami Boufi, Mecit Halil Öztop, Rafik Balti, Zied Zarai

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Houda Gharsallah, Cédric Guerin, Ahlem Eddehech, Sami Boufi, Mecit Halil Öztop, et al.. Elucidating the biosurfactant potential of Bacillus strains: a study of structure and function in food applications. International Journal of Food Science and Technology, 2025, 60 (1), 10.1093/ijfood/vvaf116. hal-05225630

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# Elucidating the biosurfactant potential of *Bacillus* strains: a study of structure and function in food applications

Houda Gharsallah<fnref n="1" />, Cedric Guerin<fnref n="2" />, Ahlem Eddehech<fnref n="1" />,<fnref n="3" />, Sami Boufi<fnref n="4" />, Mecit Halil Öztop<fnref n="5" />,*, Rafik Balti<fnref n="2" />, and Zied Zarai<fnref n="1" />,<fnref n="3" />

## Abstract

This study explores the isolation and characterisation of biosurfactant-producing *Bacillus* strains. Four β-hemolytic isolates *Bacillus velezensis* (H6), *Bacillus subtilis* (S15 and S4), and *Bacillus cereus* (S32) were identified as potential biosurfactant producers. Among them, strain H6 showed the highest activity, reducing surface tension by 35 mN/m. This surface activity was further supported by FTIR analysis, which confirmed the lipopeptide nature of the compounds. In addition, thermogravimetric analysis indicated the thermal stability up to 140–190 °C. The biosurfactants also exhibited low critical micelle concentration values (0.3–0.5 g/L) outperformed sodium dodecyl sulphate and confirming their strong surface-active potential. Liquid chromatography-tandem mass spectrometry revealed diverse lipopeptide families, including surfactins, iturins, and fengycins, with strain H6 producing the highest levels. Overall, the biosurfactants displayed excellent functional properties, achieving up to 90% emulsification and foaming activity, which highlights their applicability in industrial sectors, particularly in food processing.

**Keywords:** biosurfactant, lipopeptides, *Bacillus*, functional properties, Liquid chromatography-tandem mass spectrometry

<footnote n="1">¹Food Technology Department, High Institute of Biotechnology of Sfax, University of Sfax, Sfax, Tunisia</footnote>

<footnote n="2">²Laboratoire de Genie des Procédés et Matériaux, Centre Européen de Biotechnologie et de Bioéconomie (CEBB), Université Paris-Saclay, CentraleSupélec, Pomacle, France</footnote>

<footnote n="3">³Laboratory of Biochemistry and Enzymatic Engineering of Lipases, Engineering National School of Sfax (ENIS), University of Sfax, Sfax, Tunisia</footnote>

<footnote n="4">⁴LMSE, Faculty of Science, University of Sfax, Sfax, Tunisia</footnote>

<footnote n="5">⁵Department of Food Engineering, Faculty of Engineering, Middle East Technical University, Ankara, Turkiye</footnote>

<footnote>*Corresponding author: Department of Food Engineering, Middle East Technical University, 06800 Ankara, Turkey. Email: mecit@metu.edu.tr</footnote>
**Graphical abstract**

<figure><img src="image_3.png" /></figure>

**Introduction**

One promising avenue is the use of biosurfactants, surface-active compounds produced by microorganisms, as alternatives to chemically synthesised surfactants. As concerns over environmental impact and food safety continue to rise, biosurfactants, particularly lipopeptides, have emerged as essential molecules with potential applications across multiple sectors, notably in the food industry (Passos et al., 2023; Ribeiro et al., 2020; Roy et al., 2024). In food processing, biosurfactants play a vital role due to their ability to stabilise emulsions, disperse oils, and enhance the texture of food products. Their amphiphilic nature, consisting of both hydrophilic and hydrophobic moieties, allows them to reduce surface and interfacial tension between liquids, solids, and gases, making them ideal for applications such as emulsification, foaming, and wetting in various food products (Thakur et al., 2024). This is particularly relevant in the formulation of stable emulsions used in sauces, mayonnaise, creams, and other processed foods, where consistency and texture are critical (Sondhi, 2023). Lipopeptides, a specific class of biosurfactants produced primarily by *Bacillus* species, have gained attention for their multifunctionality. Surfactins, fengycins, and iturins are the three major families of lipopeptides, each contributing unique functionalities that are critical in food product development. For example, surfactins exhibit superior surface tension reduction, making them useful in improving the quality and mouthfeel

of food emulsions (Chen et al., 2021). Fengycins and iturins are recognised for their strong antimicrobial and antifungal properties, which can extend the shelf life of food products by preventing microbial contamination (Zhang et al., 2022). One of the most advantageous aspects of lipopeptides is their natural emulsifying capability, which can improve the texture, stability, and overall sensory quality of food products. In products, such as whipped creams, dressings, and beverages, lipopeptides help maintain homogeneity and prevent phase separation over time. This is particularly important in modern food processing, where clean-label ingredients are favoured over synthetic additives (Sharma & Sharma, 2021). Their ability to stabilise oil-in-water emulsions without chemical stabilisers or preservatives is a critical advantage in developing natural and health-conscious food products.

Beyond their emulsification properties, lipopeptides' antimicrobial activity has drawn considerable interest for food preservation. Their ability to inhibit a wide range of foodborne pathogens and spoilage organisms makes them ideal candidates for replacing chemical preservatives (Kourmentza et al., 2021). Surfactins, for instance, are potent antimicrobial agents effective against both Gram-positive and Gram-negative bacteria, while fengycins are especially effective against moulds and yeasts. These characteristics make them suitable for applications in baked goods, dairy products, and beverages, where spoilage microorganisms
pose significant challenges (Shi & Maktabdar, 2022). The pro-
duction of biosurfactants also aligns with the food industry's
growing interest in sustainability. Their biodegradability and low
environmental impact are critical attributes, particularly as the
industry moves towards more eco-friendly manufacturing pro-
cesses. Additionally, the use of agro-industrial waste as a sub-
strate for microbial biosurfactant production reduces production
costs and provides a sustainable waste management solution.
For example, waste from sugar, dairy, and vegetable oil industries
has been successfully used to cultivate biosurfactant-producing
microorganisms (Santos et al., 2023). These residues are typically
rich in carbon and nitrogen sources, making them ideal low-cost
substrates for microbial fermentation. Their valorisation not only
addresses waste disposal issues but also contributes to the devel-
opment of a circular bioeconomy by converting waste streams
into value-added biomolecules. This strategy supports the reduc-
tion of environmental pollution and dependence on synthetic,
petroleum-based surfactants, aligning with global sustainability
goals.

Despite their advantages, the widespread use of biosurfac-
tants in the food industry is still limited by production costs
and scalability challenges (Santos et al., 2023; Thakur et al.,
2024). Nonetheless, recent progress in microbial strain engineer-
ing, process optimisation, and fermentation technology has led
to enhanced yields and economic viability. When combined with
low-cost agro-industrial substrates, these advancements offer a
promising pathway to the large-scale, sustainable production of
biosurfactants suitable for food applications.

Additionally, advances in metabolic engineering are enhancing
microbial strains to produce higher yields of biosurfactants with
tailored properties suited for specific food applications (Chab-
hadiya et al., 2024; Phulpoto et al., 2023).

This study aims to isolate and characterise biosurfactants with
exceptional emulsifying, foaming, and thermal stability proper-
ties, ideal for food processing applications. These biosurfactants
can improve texture, consistency, and shelf life in food products.
As research advances in optimising their production, they offer
a sustainable, high-performance alternative to synthetic surfac-
tants, paving the way for innovation in food formulation and
industry practices.

Materials and methods

Screening of biosurfactant-producing bacteria

Five soil samples (biological replicates) were aseptically collected
from a triglyceride-contaminated olive mill wastewater retention
basin in Sfax, Tunisia, at a depth of 10 cm using sterile bags. Sam-
ples were transported to the laboratory under refrigerated condi-
tions for immediate processing. To isolate lipid-degrading bacte-
ria, 1 g of soil was mixed with 9 ml of sterile saline (0.85% NaCl),
heat-treated (80 °C for 10 min) to select for sporulating bacteria,
with particular emphasis on biosurfactant-producing *Bacillus spp*.,
known for their lipopeptide-mediated triglyceride emulsification
capacity (Mnif & Ghribi, 2015). Serial dilutions were plated onto
Luria Bertani (LB) agar using the spread plate method (100 µl
per plate), with three technical plating replicates per dilution for
each biological replicate. Isolates were functionally screened for
emulsification activity on triglyceride-supplemented media. After
incubation at 30 °C for 24 hr, colonies were selected based on
morphology, Gram staining, and extracellular enzyme production
(Bezza & Chirwa, 2015; Sharma et al., 2023). Biosurfactant produc-
tion was preliminarily screened using the hemolytic activity assay,

where isolates were cultured on blood agar (BA) and incubated at
37 °C for 24–48 hr. β-hemolysis, indicated by clear zones around
colonies, suggested biosurfactant production (Lamilla et al., 2018;
Shoeb et al., 2015). Four strains (H6, S15, S32, and S40) showing
hemolytic activity were selected for further analysis. This assay is
a reliable qualitative method for detecting biosurfactant produc-
tion, as supported by previous studies (Clarridge, 2004; Rodrigues
& Teixeira, 2010; Shoeb et al., 2015; Thavasi et al., 2011).

Bacterial identification

The molecular identification of strains H6, S15, S32, and S40 was conducted using 16S rRNA sequencing, a widely recognised method for bacterial identification due to its accuracy and conservation across prokaryotes (Li et al., 2024a). Genomic DNA was extracted using the QIAamp DNA Mini Kit (QIAGEN GmbH, Germany) (Gürtler & Stanisich, 1996), and PCR amplification was performed with universal primers Fd1 (5'-AGAGTTTGATCC TGGCTCAG-3') and Rd1 (5'-AAGGAGGTGATCCAGCC-3'), targeting conserved regions of the 16S rRNA gene (Patel, 2001). These primers are commonly used for *Bacillus* species identification, amplifying approximately 1.5 kb of the 16S rRNA gene (Barrow et al., 2007). The PCR conditions and reagent concentrations followed established protocols (Sun et al., 2020). Amplified products were purified using the QIAquick PCR Purification Kit (QIAGEN GmbH) and sequenced on an Avant Genetic Analyser (Model 3100) (Altschul et al., 1990). Sequences were compared with the GenBank database using BLASTN (NCBI) for identification (Thompson et al., 1994).

Phylogenetic analysis involved multiple sequence alignments
using ClustalW, and trees were constructed using the Neighbour-
Joining method with the Kimura two-parameter model (Nishi-
maki & Sato, 2019). Bootstrap analysis with 1,000 replicates val-
idated the tree topology, ensuring robust phylogenetic relation-
ships (Mukherjee et al., 2006). This approach is consistent with
standard methods for bacterial taxonomy and classification.

Culture condition for biosurfactant production
and extraction

The bacterial strains were cultured in 2 L flasks containing 500 ml of an optimised medium (OM) designed to enhance bioactive compound production. The OM composition (g/L) included peptone (20), sucrose (25), yeast extract (4.5), KH₂PO₄ (2), MnSO₄ (0.006), and MgSO₄ (0.6), consistent with formulations used to promote biosurfactant production in *Bacillus* species (Datta et al., 2018; Liu et al., 2021). Flasks were incubated at 30 °C for 5 days on an orbital shaker at 150 rpm, conditions known to maximise yield (Habib et al., 2020; Youssef et al., 2004).

To extract extracellular proteins, the culture medium was cen-
trifuged at 6,000 rpm for 20 min to separate cells from the super-
natant. Biosurfactants were precipitated by adjusting the super-
natant pH to 2.0 using 6 N HCl, a method validated for effective
precipitation (Vigneshwaran et al., 2021). After overnight storage
at 4 °C, samples were centrifuged (6,000 rpm, 4 °C) for 10 min. The
precipitate was suspended in distilled water, neutralised to pH 7.0
with 1 N NaOH to preserve bioactivity, (Mukherjee et al., 2006) and
lyophilised for storage at -20 °C.

Fourier transform infrared spectroscopy

The extracted biosurfactant was characterised using Fourier
transform infrared (FTIR) spectroscopy with a Perkin-Elmer
FTIR/NIR Frontier instrument. Infrared absorption spectra were
recorded in the medium infrared range, extending from 400 to 3,800 cm⁻¹.

## Determination of critical micelle concentration

To determine the critical micelle concentration (CMC), the dried extracted biosurfactants were dissolved in Milli-Q water at increasing concentrations (0–10 mg ml⁻¹). The surface tension was then measured using a digital K12-Kruss tensiometer with the Du Noüy ring method. The CMC was deduced as the inflection point on the surface tension versus biosurfactant concentration curve.

## Thermal stability of the biosurfactant

To assess the thermal stability of the biosurfactant, thermogravimetric analysis (TGA) was conducted on the extracted biosurfactants using Perkin-Elmer TGA-800. A sample weighing ten milligrams was placed in an aluminium pan and subjected to a temperature ramp from 30 to 500 °C at a heating rate of 10 °C/min under air atmosphere. The thermostability of the extracted biosurfactant was evaluated based on the thermal degradation profiles obtained from this analysis.

## Mass spectrometry and liquid chromatography analysis

A non-targeted analysis of biosurfactants was performed using an ultra-high performance liquid chromatography system (UHPLC) (Elute, Bruker) coupled with trapped ion mobility spectrometry and quadrupole time-of-flight mass spectrometry (TIMS-ToF, Bruker). Chromatographic separation was achieved with a Waters Acquity UPLC HSS T3 column (2.1 x 150 mm, 1.8 µm), which served as the stationary phase and was maintained at 35 °C. The mobile phases consisted of eluent A (Milli-Q water) and eluent B (acetonitrile). Elution was carried out at a flow rate of 0.3 ml/min, starting with 10% eluent B for 1 min. The proportion of eluent B was gradually increased to 100% over 19 min and held at 100% for an additional 10 min. The gradient was then returned to the initial conditions and stabilised for 10 min, resulting in a total analysis time of 40 min. The injection volume was 1 µl.

High-resolution mass spectrometry (HRMS) data were acquired at a rate of 10 Hz, covering a mass range of 150–2,000 m/z in auto MS/MS mode. The electrospray ionisation source operated in negative ion mode, with a capillary voltage of 4 kV, a desolvation temperature of 220 °C, a gas flow rate of 10 L/min, and a nebuliser pressure of 2.2 bar.

Data processing was conducted using Metaboscape 2023b, TASQ 2023b, and Python 3.12.2 (Bruker). The analysis was performed in technical triplicates to ensure accuracy and reproducibility. Identification was done as the following: chemical formulas were guessed from the mass of the monoisotopic peak and the isotope profile, database such as Chemspider or Pubchem were checked for molecule matching the chemical formula. Then in-silico fragmentation was done the matching list and compared to the experimental MS/MS spectrum acquired.

## Interfacial properties of the biosurfactant

### Emulsifying properties

The emulsion activity index and the emulsion stability index of biosurfactants were determined according to the method (Zarai et al., 2016). The biosurfactants solution was prepared by dissolving the dry extract at 60 °C for 30 min. Then, 30 ml of 1% (wt/vol) biosurfactants solution was homogenised with 10 ml

of soybean oil for 1 min at room temperature, using the ultraturrax homogeniser. Aliquots of the emulsion (5 µl) were taken from the bottom of the container at time zero and after 10 min of homogenisation. Then, these aliquots were diluted 100 times with 0.1% sodium dodecyl sulphate solution and were thoroughly mixed for 10 s using a vortex mixer. Finally, the absorbance of these diluted samples was measured at 500 nm using a spectrophotometer (T70, UV/VIS spectrometer, PG Instruments Ltd., China).

### Foaming properties

The foaming capacity (FC) and the foaming stability (FS) of biosurfactants solution were tested, according to the method of Zarai et al. (2016). Homogenisation was carried out using an ultra-turrax to incorporate air for 1 min at room temperature. The whipped sample was then immediately transferred to a 50 ml graduated cylinder, and the total volume was measured at time $t_0$ after 30 and 60 min to record (FC) and (FS), respectively.

## Results and discussion

### Screening and identification of biosurfactant producing bacteria

A large bacterial collection was isolated from an olive oil-contaminated industrial site in Sfax, Tunisia, using an enrichment culture technique. Pure colonies obtained on LB agar plates were screened for biosurfactant production using hemolytic activity testing on BA. As shown in Figure 1A, only four strains coded H6, S15, S40, and S32 exhibited clear β-hemolysis, characterised by a distinct translucent halo around the colonies. This observation suggests their potential to produce biosurfactants, as β-hemolysis has been frequently associated with the secretion of surface-active compounds capable of disrupting red blood cell membranes.

Although several methods have been proposed to screen for biosurfactant production, including drop-collapse, oil spreading, and emulsification index assays, hemolytic activity remains a widely accepted preliminary indicator. Bamba et al. (2024) emphasised the reliability of this approach, demonstrating that precise measurement of hemolytic zones correlates with biosurfactant yield and activity. Furthermore, they noted that, to date, there are no documented cases of biosurfactant production by strains that completely lack hemolytic activity. This reinforces the relevance of hemolysis screening as a cost-effective and rapid first-line method for identifying promising biosurfactant-producing isolates with potential pharmaceutical applications, such as anti-coagulant or antiadhesive agents.

In a secondary quantitative screening, the bacterial strains were cultured in standard nutrient broth, and the surface tension of the medium was measured on the third day to confirm biosurfactant production. As presented in Figure 1B, all four strains reduced surface tension compared to pure water, though their effectiveness varied. Strain H6 was the most efficient, reducing the surface tension by up to 35 mN/m. Contaminated soils, particularly those tainted with olive oil or other hydrocarbons, serve as a natural selection environment for microorganisms with specialised metabolic capabilities. Bacillus species, renowned for their remarkable adaptability, thrive in such environments by utilising hydrocarbons as a carbon source. These bacteria secrete biosurfactants, which facilitate the solubilisation of oils, making them more bioavailable and allowing the bacteria to assimilate the compounds more effectively for growth and development.
<figure><img src="image_4.png" /><figcaption>**Figure 1.** Screening of biosurfactant producing bacteria and analytic characterisation of the extracted biosurfactants (A) hemolysis assay, (B) surface tension measurement, and (C) determination of critical micelle concentration (CMC) of the extracted biosurfactants.</figcaption></figure>

The biosurfactants produced by *Bacillus* strains, particularly in oil-contaminated soils, are not only essential for their survival and metabolic processes but also exhibit valuable properties for various biotechnological applications, including bioremediation and industrial processes.

## Phylogenetic analysis for taxonomic identification

The biosurfactant-producing isolates were further identified by partial sequencing of their 16S rRNA gene. The results revealed that the selected bacteria belonged to the *Bacillus* genus. Isolate S40 (access number: PQ539064) was closely related to *B. subtilis* with 99.70% identity. Isolate S32 (access number: PQ541341) had a 100.00% similarity with *B. cereus* strain. Strain S15 (access number: PQ541200 showed 98.84% similarity to *B. subtilis* strain, while strain H6 (access number: PQ539041) exhibited 98.84% identity to *B. velezensis*.

A comparative analysis of these sequences with database entries (Blastn) revealed a high homology (99%) with species

belonging to the genus *Bacillus*. This genus is a heterogeneous group, with its members sharing numerous morphological and biochemical similarities. Based on these sequences, a phylogenetic tree (Figure 2) was constructed using the MEGA 11 software. This tree included the four studied strains alongside reference strains of the genus *Bacillus* to refine their phylogenetic classification (Barghouthi, 2011).

In summary, the phylogenetic tree offers valuable insights into the evolutionary relationships among *Bacillus* strains. It enables the identification of closely related strain clusters and highlights the genetic diversity within this genus, providing a deeper understanding of their taxonomic and evolutionary contexts.

## Fourier transform infrared spectroscopy

To further characterise the biosurfactants produced by the four *Bacillus* strains, we analysed their chemical structures using FTIR spectroscopy. The FTIR spectra of the four biosurfactant samples are presented in Figure 3A. These spectra provided valuable insights into the functional groups present in the
<figure><img src="image_5.png" /><figcaption>**Figure 2.** Phylogenetic tree of *Bacillus* species based on 16S rRNA gene sequences through neighbour-joining methods using MEGA version 6.0.</figcaption></figure>

biosurfactants, confirming their identity as lipopeptides and revealing distinct structural features that are characteristic of the different strains.

Results show nearly identical bands, with only minor differences in intensity. The observed patterns were highly similar to the FTIR spectra of lipopeptide biosurfactants produced by *Bacillus* species, exhibiting several characteristic absorption bands (Youssef et al., 2004). The spectra revealed characteristic bands of peptides, carboxylic acids, and aliphatic hydrocarbon chains. In the region between 2,800–3,600 cm⁻¹, the three bands at 2,850, 2,920, and 2,960 cm⁻¹ were assigned to the C–H stretching vibrations of the hydrocarbon chains which is crucial for their emulsifying properties (Suthar & Nerurkar, 2016). Broad bands peaking between 3,100–3,600 cm⁻¹ corresponded to N–H and carboxylic groups (Marchut-Mikolajczyk et al., 2021). The region between 600–1,800 cm⁻¹ displayed prominent features of peptide and carboxylic acid groups, with two strong bands at 1,640 cm⁻¹ and 1,530 cm⁻¹ attributed to Amide I and Amide II, respectively. The Amide I band is mainly due to C=O stretching vibrations, while the Amide II band arises from N–H bending and C–N stretching vibrations of the peptide backbone. The carboxylic group is evident from the bands at 1,700 cm⁻¹ and 1,220 cm⁻¹, assigned to C=O and C–O stretching, respectively. This is in line with literature reporting similar peptide linkages in lipopeptides, reinforcing the idea that these compounds possess structural motifs critical for their biological activity (Gomaa et al., 2021; Zargar et al., 2022). Further confirmation of carboxylic group presence is seen in the evolution of the FTIR spectra in the 600–1,800 cm⁻¹ region for samples neutralised to pH 8 and dried to remove water. As shown in Figure 3B, the bands at 1,700 and 1,220 cm⁻¹ disappeared, with new bands emerging at 1,560 and 1,300 cm⁻¹, assigned to carboxylate groups.

## Thermal stability profile of the extracts biosurfactants

The thermal stability of various lipopeptide samples was studied by TGA to assess the onset of thermal degradation, and establish the maximum temperature at which the biosurfactant remains stable. As shown in Figure 3C, the degradation process appears to be multi-stepped, reflecting the decomposition of distinct fragments within each sample. However, the onset temperature of thermal decomposition (Tonset) varies according to the preparation route. The highest T-onset was observed for samples S32 and S15, reaching up to 190 °C, whereas samples H6 and S40 showed much lower Tonset values, not exceeding 140 °C. This lower thermal stability limits their applicability in processes requiring sterilisation or autoclaving (Patel et al., 2022). The significant differences in thermal stability among the samples are likely attributed to variations in their chemical composition, particularly the presence of thermally unstable chemical moieties in H6 and S40. However, the exact chemical structures responsible for this reduced stability remain unclear. These findings highlight the importance of optimising the chemical composition of lipopeptides to enhance their thermal stability for specific applications. Further studies, including detailed structural analysis, could provide insights into the specific chemical moieties that influence thermal stability and guide the development of more robust lipopeptides for high-temperature applications (Moldes et al., 2023).

## Determination of the CMC

Establishing the CMC of biosurfactants is critical for several applications, as it provides an index to evaluate their surface activity (Wu et al., 2022). Properties such as solubilisation and detergency
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>**Figure 3.** FTIR spectra of the different biosurfactant (A) biosurfactant collected after precipitation at pH 2 and (B) comparison of the FTIR spectra at pH 2 and pH 8. Thermogravimetric analysis (TGA) of the *bacillus* biosurfactants extract (C).</figcaption></figure>

<figure><img src="image_8.png" /></figure>

can be significantly influenced by the presence of micelles in solution (Dini et al., 2024). The CMC, defined as the minimum concentration of biosurfactant required to achieve maximum surface tension reduction in water and initiate micelle formation, was assessed from the plot of surface tension (γ) versus biosurfactant concentration. As shown in Figure 1C, the surface tension of water decreased gradually with increasing lipopeptide concentration from 63 to 30–34 mN/m, and then remained constant over a critical lipopeptide concentration ranging from 0.3 to 0.5 g/L, consistent with findings previously reported by Abdel-Mawgoud et al. (2010) and Das et al. (2009). The CMC values of the different lipopeptides were 0.3, 0.35, 0.4, and 0.5 g/L for strains S15, H6, S32, and S40, respectively.

The CMC values of these lipopeptides are lower than those typically observed for crude lipopeptides obtained from *B. subtilis* fermentation strains (Al-Sulaimani et al., 2012; Sharma & Pandey, 2020; Wu et al., 2022). Compared to synthetic surfactants like sodium dodecyl sulphate, which has a CMC of approximately

2.3 g/L (Zhao et al., 2021), these biosurfactants demonstrate superior surface activity and significantly lower CMC values. Additionally, the low toxicity and high biodegradability of these natural biomolecules make them environmentally friendly and sustainable alternatives to conventional chemical surfactants.

## Mass spectrometry and liquid chromatography analysis

To elucidate the composition of the partially purified biosurfactants from the screened *Bacillus* strains, the extracts were subjected to liquid chromatography-mass spectrometry (LC-MS/MS) analysis. This analytical approach selectively highlights peaks predominantly associated with cyclic lipopeptides. Distinct families of lipopeptides are resolved, including iturins around 10 min, fengycins around 15 min, and surfactins around 20 min (Zhang et al., 2020; Zong et al., 2019). In this preliminary analysis, notable inter-sample variability is observed, particularly within the surfactin family, where the H6 strain displays a unique
chromatographic profile when compared to the other strains. These differences in lipopeptide composition could have significant implications for the bioactivity and potential industrial applications of these strains.

The plot of a Kendrick graph using a CH2 unit repetition shows the different families of molecules. Groups having the same Kendrick default and a difference of 14 m/z are most likely homologues with a longer carbon chain. In microorganisms, there is most often a variety of homologues of lipopeptides.

In the LC-MS data, when a mass filter is applied to keep only values between 800 and 2000 m/z and, those families appear clearly in the Kendrick plot. In doing so, the strains can be compared in their profile and the relative concentrations. As in the base peak chromatogram, surfactins seem to be the main source of variation between strains. A closer look at the surfactin family reveals distinct differences in the homologues produced by each strain. Specifically, strain H6 produces a broader range and higher concentration of surfactin compounds compared to the other strains, indicating its superior biosurfactant synthesis capabilities. This diversity in surfactin homologues across the strains suggests that the biosynthetic pathways for surfactin vary, resulting in strain-specific biosurfactant profiles. The LC-MS data highlights the relative abundance of surfactin, iturin, and fengycin families and underscores their role in contributing to the strain-dependent differences observed in biosurfactant production. Other families can be identified such as the iturins or fengycins while some remain unknown. Table 1 provides a detailed breakdown of the lipopeptide composition, with the primary peaks corresponding to surfactin family compounds. These peaks, occurring at varying retention times, were identified as surfactin homologues with acyl chains of different lengths, including Surfactin C11, C12, C13 for Surfactin A, C14 for Surfactin C, and C15 for Surfactin B. The MS/MS spectra confirmed the presence of these homologues, showcasing the diversity in fatty acid chain branching. Surfactins, cyclic lipopeptides comprising β-hydroxy fatty acids and a cyclic peptide ring linked via a lactone bridge, are well-known for their wide range of biological activities, including antibacterial, antifungal, antiviral, anti-inflammatory, anti-mycoplasma, and thrombolytic effects. Surfactin B isoforms were distinguished by their specific fragmentation patterns and structural features. Additionally, an isomer of surfactin C12 (C50H87N7O13) was detected. Similar fragmentation patterns for surfactin variants have been previously reported, suggesting that longer acyl chains enhance bioactivity and stability (Dini et al., 2024; Saikia et al., 2021). The analysis also confirmed the presence of iturins, specifically Iturin A2, A4, and A7, which vary in molecular structure due to differences in their lipid tail lengths. These variations were reflected in their retention times: 10.46 min for Iturin A2, 11.05 min for A4, and 11.80 min for A7, demonstrating that longer lipid chains result in increased retention time which is typical in reversed phase chromatography. The iturin family (comprising Iturin A, C, D, and E) features a cyclic peptide structure made up of seven α-amino acids and one β-amino acid, with variations in the fatty acid chain length and composition. These biosurfactants are recognised for their bioactive properties, making them promising candidates for biocontrol and antifungal applications (Saiyam et al., 2024; Araguppi et al., 2023).

Interestingly, LC-MS analysis also revealed the presence of fengycin compounds, though their peaks were sparse, appearing between retention times of 14.97 and 16.56 min. The MS/MS spectra identified these peaks as fengycin B lipopeptides with saturated lipid tails of C13, C14, C15, C16, and C17.

Fengycins, which are lipodecapeptides, are cyclised via a lactone bridge formed between the phenolic hydroxyl group of Tyr3 and the carboxylic acid group of the C-terminal Ile10. These compounds are known for their strong antifungal activity, especially against filamentous fungi (Adetunji et al., 2022; Li et al., 2024b).

Following the identification of the various lipopeptide families and their isomers, a comparative analysis of their relative abundances was performed by integrating and comparing peak areas, as presented in Table 2. The data revealed significant differences in lipopeptide composition and abundance among the four strains (H6, S40, S32, and S15), indicating distinct metabolic profiles and biosynthetic capacities.

Strain H6 stood out, producing the highest concentrations of Surfactin B and Surfactin A. Additionally, this strain showed elevated levels of a Surfactin B isomer and Surfactin C, suggesting an optimised biosynthetic pathway for these compounds. In contrast, strains S40, S32, and S15 exhibited considerably lower concentrations of these lipopeptides. Iturin A homologues were produced at comparable levels in strains H6 and S40, but were less abundant in strains S32 and S15. Notably, strain H6 also had the highest production of Iturin A4.

In terms of fengycin production, strain H6 again demonstrated the highest levels, particularly for the C14 variant, indicating a robust biosynthetic capability. Strain S40 followed, though fengycin C12 levels were markedly lower. Strain S32 exhibited reduced fengycin concentrations, indicating a lower efficiency in its biosynthetic pathway, while strain S15 had the lowest levels, likely due to differences in its metabolic profile or environmental factors.

Overall, although all strains are *Bacillus*-related, strain H6 exhibited a notably diverse and prolific lipopeptide profile in contrast, strains S40, S32, and S15 demonstrated reduced lipopeptide diversity and abundance, indicating less efficient biosynthetic pathways. This can be attributed to several factors, as highlighted in the literature. Certain *Bacillus* strains exhibit significant variability in lipopeptide production due to genetic polymorphisms, the presence of supplementary biosynthetic gene clusters, and optimised regulatory pathways. These strains typically possess enhanced regulatory networks that upregulate the expression of critical biosynthetic enzymes involved in lipopeptide synthesis. Moreover, horizontal gene transfer, including the acquisition of plasmids, contributes substantially to the expansion of the lipopeptide repertoire in these strains (Trappe et al., 2016; Yasmin et al., 2022).

## Interfacial properties of the biosurfactant

### Emulsifying properties

The stability of water-in-oil emulsions is a key factor in food product viability, as these emulsions are naturally thermodynamically unstable due to the large interfacial area between the phases (Liu et al., 2016). Biosurfactants, with their unique amphiphilic structure, act as efficient emulsifiers, enhancing the stability of such systems by interacting with oils, proteins, and carbohydrates in food products (Zhao et al., 2018).

The emulsifying performance of four lipopeptide biosurfactants was assessed using crude oil as a substrate, with all strains demonstrating strong emulsification properties (Table 3). Table 3 summarises the emulsification index after 24 hr (E24) and the corresponding emulsion stability. These quantitative values reflect the ability of each biosurfactant to form and maintain oil-in-water emulsions, which is crucial in industrial applications requiring stable formulations.
<table><caption>**Table 1.** LC-MS data of lipopeptides in the *Bacillus* strains investigated.</caption><thead><tr><th colspan="8">Negative mode</th></tr><tr><th>Retention time (min)</th><th>Compound name</th><th>Molecular formula</th><th>Ion</th><th>Experimental mass (m/z)</th><th>Theoretical mass (m/z)</th><th>Error (ppm)</th></tr></thead><tbody><tr><td>14.45</td><td>Fengycin C C13</td><td>C<sub>71</sub>H<sub>108</sub>N<sub>12</sub>O<sub>20</sub></td><td>[M-2H]<sup>2-</sup></td><td>723.3817</td><td>723.3829</td><td>1.66</td></tr><tr><td>14.97</td><td>Fengycin C C14</td><td>C<sub>72</sub>H<sub>110</sub>N<sub>12</sub>O<sub>20</sub></td><td>[M-2H]<sup>2-</sup></td><td>730.3916</td><td>730.3907</td><td>-1.23</td></tr><tr><td>15.84</td><td>Fengycin C C15</td><td>C<sub>73</sub>H<sub>112</sub>N<sub>12</sub>O<sub>20</sub></td><td>[M-2H]<sup>2-</sup></td><td>737.3977</td><td>737.3985</td><td>1.08</td></tr><tr><td>15.72</td><td>Fengycin C C16</td><td>C<sub>74</sub>H<sub>114</sub>N<sub>12</sub>O<sub>20</sub></td><td>[M-2H]<sup>2-</sup></td><td>744.4111</td><td>744.4063</td><td>-6.45</td></tr><tr><td>16.56</td><td>Fengycin C C17</td><td>C<sub>75</sub>H<sub>116</sub>N<sub>12</sub>O<sub>20</sub></td><td>[M-2H]<sup>2-</sup></td><td>751.4147</td><td>751.4142</td><td>-0.67</td></tr><tr><td>10.46</td><td>Iturin A2</td><td>C<sub>48</sub>H<sub>74</sub>N<sub>12</sub>O<sub>14</sub></td><td>[M-H]<sup>-</sup></td><td>1041.5372</td><td>1041.5375</td><td>0.29</td></tr><tr><td>11.05</td><td>Iturin A4</td><td>C<sub>49</sub>H<sub>76</sub>N<sub>12</sub>O<sub>14</sub></td><td>[M-H]<sup>-</sup></td><td>1055.5525</td><td>1055.5531</td><td>0.57</td></tr><tr><td>11.8</td><td>Iturin A7</td><td>C<sub>50</sub>H<sub>78</sub>N<sub>12</sub>O<sub>14</sub></td><td>[M-H]<sup>-</sup></td><td>1069.5685</td><td>1069.5688</td><td>0.28</td></tr><tr><td>19.33</td><td>Surfactin C11</td><td>C<sub>49</sub>H<sub>85</sub>N<sub>7</sub>O<sub>13</sub></td><td>[M-H]<sup>-</sup></td><td>978.6107</td><td>978.6133</td><td>2.66</td></tr><tr><td>19.94</td><td>Surfactin C12</td><td>C<sub>50</sub>H<sub>87</sub>N<sub>7</sub>O<sub>13</sub></td><td>[M-H]<sup>-</sup></td><td>992.6288</td><td>992.6289</td><td>0.10</td></tr><tr><td>19.03</td><td>Surfactin C12 isomer</td><td>C<sub>50</sub>H<sub>87</sub>N<sub>7</sub>O<sub>13</sub></td><td>[M-H]<sup>-</sup></td><td>992.6268</td><td>992.6289</td><td>2.12</td></tr><tr><td>19.64</td><td>Surfactin A</td><td>C<sub>51</sub>H<sub>89</sub>N<sub>7</sub>O<sub>13</sub></td><td>[M-H]<sup>-</sup></td><td>1006.6444</td><td>1006.6446</td><td>0.20</td></tr><tr><td>20.63</td><td>Surfactin B</td><td>C<sub>52</sub>H<sub>91</sub>N<sub>7</sub>O<sub>13</sub></td><td>[M-H]<sup>-</sup></td><td>1020.6609</td><td>1020.6602</td><td>-0.69</td></tr><tr><td>20.45</td><td>Surfactin B isomer</td><td>C<sub>52</sub>H<sub>91</sub>N<sub>7</sub>O<sub>13</sub></td><td>[M-H]<sup>-</sup></td><td>1020.6609</td><td>1020.6602</td><td>-0.69</td></tr><tr><td>21.23</td><td>Surfactin C</td><td>C<sub>53</sub>H<sub>93</sub>N<sub>7</sub>O<sub>13</sub></td><td>[M-H]<sup>-</sup></td><td>1034.6767</td><td>1034.6759</td><td>-0.77</td></tr></tbody></table>

<table><caption>**Table 2.** Quantitative analysis of lipopeptides in the *Bacillus* strains investigated.</caption><thead><tr><th>Retention time (min)</th><th>Compound name</th><th>Area H6</th><th>Ecart type H6</th><th>Area 40</th><th>Ecart type 40</th><th>Area 32</th><th>Ecart type 32</th><th>Area 15</th><th>Ecart type 15</th></tr></thead><tbody><tr><td>14.45</td><td>Fengycin C C13</td><td>15,040</td><td>515</td><td>23,620</td><td>2,639</td><td>18,446</td><td>2028</td><td>17,321</td><td>772</td></tr><tr><td>14.97</td><td>Fengycin C C14</td><td>197,232</td><td>4,117</td><td>187,123</td><td>4,347</td><td>152,202</td><td>9,829</td><td>136,126</td><td>14,808</td></tr><tr><td>15.84</td><td>Fengycin C C15</td><td>89,586</td><td>7,527</td><td>80,723</td><td>1733</td><td>51,227</td><td>10,689</td><td>66,537</td><td>2,870</td></tr><tr><td>15.72</td><td>Fengycin C C16</td><td>50,619</td><td>2,876</td><td>53,550</td><td>3,926</td><td>37,275</td><td>2,829</td><td>36,582</td><td>2,538</td></tr><tr><td>16.56</td><td>Fengycin C C17</td><td>35,855</td><td>4,139</td><td>31,824</td><td>2,302</td><td>20,744</td><td>1924</td><td>21,931</td><td>2,340</td></tr><tr><td>10.46</td><td>Iturin A2</td><td>174,483</td><td>583</td><td>183,929</td><td>4,415</td><td>135,944</td><td>5,873</td><td>135,073</td><td>2,126</td></tr><tr><td>11.05</td><td>Iturin A4</td><td>187,858</td><td>3,622</td><td>147,131</td><td>3,259</td><td>95,512</td><td>1,358</td><td>92,692</td><td>2,836</td></tr><tr><td>11.8</td><td>Iturin A7</td><td>12,697</td><td>221</td><td>11,743</td><td>2,706</td><td>8,448</td><td>744</td><td>11,333</td><td>230</td></tr><tr><td>19.33</td><td>Surfactin C11</td><td>25,297</td><td>3,673</td><td>2,980</td><td>305</td><td>711</td><td>223</td><td></td><td></td></tr><tr><td>19.94</td><td>Surfactin C12</td><td>270,981</td><td>3,365</td><td>13,471</td><td>2,664</td><td>2,597</td><td>655</td><td>2,834</td><td>531</td></tr><tr><td>19.03</td><td>Surfactin C12 isomer</td><td>116,621</td><td>14,648</td><td>34,871</td><td>6,395</td><td>9,571</td><td>2024</td><td>10,935</td><td>1987</td></tr><tr><td>19.64</td><td>Surfactin A</td><td>718,623</td><td>29,156</td><td>88,021</td><td>26,309</td><td>23,021</td><td>4,155</td><td>31,059</td><td>2,373</td></tr><tr><td>20.63</td><td>Surfactin B</td><td>844,813</td><td>67,931</td><td>347,401</td><td>14,965</td><td>171,370</td><td>2,270</td><td>217,527</td><td>8,869</td></tr><tr><td>20.45</td><td>Surfactin B isomer</td><td>403,737</td><td>20,700</td><td>71,545</td><td>11,607</td><td>47,641</td><td>2,334</td><td>59,183</td><td>1,143</td></tr><tr><td>21.23</td><td>Surfactin C</td><td>451,829</td><td>38,120</td><td>110,923</td><td>7,188</td><td>60,944</td><td>6,362</td><td>81,218</td><td>2,782</td></tr></tbody></table>

<table><caption>**Table 3.** The emulsion activity index (EAI) and the emulsion stability index (ESI) of lipopeptide solutions (LP-H6, LP-S15, LP-S32, and LP-S40).</caption><thead><tr><th>Bacillus strains</th><th>Concentration (%. wt/vol)</th><th>Emulsification index (%)</th><th>Emulsion stability (%)<br>24 hr</th></tr></thead><tbody><tr><td rowspan="3">B. velezensis (H6)</td><td>0.5</td><td>91.03</td><td>47.76</td></tr><tr><td>1</td><td>94.15</td><td>47.81</td></tr><tr><td>2</td><td>97</td><td>73.16</td></tr><tr><td rowspan="3">B. subtilis (S15)</td><td>0.5</td><td>74.63</td><td>46.29</td></tr><tr><td>1</td><td>77.94</td><td>45.58</td></tr><tr><td>2</td><td>86.43</td><td>45.80</td></tr><tr><td rowspan="3">B. subtilis (S40)</td><td>0.5</td><td>76.11</td><td>44.78</td></tr><tr><td>1</td><td>82.60</td><td>44.91</td></tr><tr><td>2</td><td>92.66</td><td>50.66</td></tr><tr><td rowspan="3">B. cereus (S32)</td><td>0.5</td><td>70.13</td><td>46.25</td></tr><tr><td>1</td><td>88.23</td><td>44.11</td></tr><tr><td>2</td><td>73.33</td><td>44.99</td></tr></tbody></table>

Strain H6 exhibited the highest emulsification capacity, with an E24 of 97% at 2% (wt/vol) and 73% stability after 24 hr, making it ideal for microbial enhanced oil recovery (MEOR) and food

emulsions (Ravi et al., 2020). The strain S15 followed with an E24 of 86.4% and moderate stability, making it suitable for cosmetic formulations, where moderate stability is required (Mina et al.,
<table><caption>**Table 4.** Microscopic observation (40×) of the emulsion droplets formed by lipopeptide solutions (LP-H6, LP-S15, LP-S32, and LP-S40).</caption><thead><tr><th>Control</th><th>Water</th><th>E493</th><th>E495</th><th>E496</th><th>E497</th></tr><tr><th></th><th>LP_H6</th><th>LP_S15</th><th>LP_S32</th><th>LP_S40</th></tr></thead><tbody><tr><td>0.5 mg/ml</td><td></td><td></td><td></td><td></td></tr><tr><td>1 mg/ml</td><td></td><td></td><td></td><td></td></tr><tr><td>2 mg/ml</td><td></td><td></td><td></td><td></td></tr><tr><td>5 mg/ml</td><td></td><td></td><td></td><td></td></tr></tbody></table>

2021). The strain S40 demonstrated a high E24 of 92.6%, with good stability, indicating its potential for pharmaceutical applications where stable emulsions are critical (Ma et al., 2020), and the strain S32 had an E24 of 88% at 1%, with moderate stability, suitable for cost-effective food industry applications (Zhu et al., 2017). These results reveal that while all tested biosurfactants surpass the 50% E24 threshold commonly used to define effective emulsifiers, their performance varies depending on concentration and strain-specific lipopeptide composition. Higher E24 values generally indicate stronger emulsification ability, whereas higher stability percentages indicate the emulsion's resistance to breakdown over time.

Microscopic observations of the emulsions are presented by Table 4 and confirmed the consistency of these results, revealing well-dispersed, small-sized oil droplets, which were uniformly distributed within the aqueous phase. Table 4 provides qualitative micrographic evidence that complements the quantitative data in Table 3. It highlights the droplet size distribution and morphology for each strain-based emulsion. The emulsions from strains H6 and S15 exhibited a stable, fine droplet structure, consistent with their high emulsification capacity and stability, while S32 showed slightly larger, more dispersed droplets, correlating with its moderate emulsification performance and stability. These observations align with the E24 results, supporting the effectiveness of the biosurfactants in stabilising oil-water emulsions.

Taken together, Tables 3 and 4 provide a comprehensive understanding of both the physical performance and microstructural integrity of the biosurfactant-stabilised emulsions. These properties highlight the importance of biosurfactants in sectors like oil recovery, food, cosmetics, and pharmaceuticals, where fine-tuning emulsification performance can enhance product stability and functionality (Alves et al., 2020; Singh et al., 2020).

## Foaming properties

The foaming ability test was conducted on four biosurfactant isolates using increasing concentrations of CL (0.01–10 mg/L). As presented in Figure 4, the bar graphs display both FC, measured as the foam height ratio (HR), and FS, evaluated after a given period. Each bar represents the mean value of triplicate experiments with standard deviation. The results reveal significant variability in foaming behaviour, reflecting differences in the types and concentrations of biosurfactants present. Maximum HR values ranged from 92% to 242%, with S40 exhibiting the highest foaming properties.

The strong foaming ability of S40 can be attributed to the presence of surface-active lipopeptides, such as surfactins and fengycins, which lower surface tension and stabilise foam by forming elastic films at the air-liquid interface. However, due to variations in testing methods, direct comparisons remain challenging. S40 demonstrated the highest FC (242%) and stability, making it suitable for applications requiring persistent foam, such as enhanced oil recovery (EOR). B. subtilis-derived biosurfactants, such as surfactin, have been shown to improve oil extraction by mobilising trapped oil and enhancing recovery efficiency under harsh conditions (Zhang et al., 2018). Furthermore, in wastewater treatment, its high foaming ability aids in emulsifying and biodegrading hydrophobic pollutants, improving treatment efficiency (Mukherjee et al., 2006).

S32 exhibited excellent foaming properties (FC = 186%, FS = 117%) at low concentrations. These characteristics make it ideal for the detergent and cosmetic industries, where stable foam improves cleaning efficacy and enhances user experience. Bacillus-derived lipopeptides like surfactin are often incorporated into such formulations due to their FS and emulsification properties (Coutte et al., 2017).
<figure><img src="image_9.png" /><figcaption>**Figure 4.** The foaming capacity (FC) (A) and the foaming stability (FS) (B) of lipopeptides solution (LP-H6, LP-S15, LP-S32, and LP-S40).</figcaption></figure>

H6, with the lowest foaming potential (FC < 90%, FS = 30%), is more suitable for applications requiring minimal foam, such as the food, pharmaceutical, and paint industries. In food and pharmaceuticals, excessive foam can interfere with processing and product quality, while in paints, low foaming properties prevent surface defects and ensure uniform application. Biosurfactants like those derived from *Bacillus amyloliquefaciens* are favoured in these sectors for their low foaming tendencies (Jahan et al., 2020).

Overall, Figure 4 demonstrates that the foaming profiles of different isolates can guide their industrial application: S40 and S32 for high-foam needs, and H6 for low-foam formulations. Such interpretation underscores the practical significance of screening for both foaming intensity and stability. Thus, the foaming characteristics of Bacillus-derived lipopeptides should be optimised according to the specific industrial needs, with S40 and S32 being ideal for applications requiring strong foam, while H6 is better suited for industries that demand minimal foam. Fine-tuning

these properties is crucial for maximising their industrial effectiveness.

## Conclusion

This study conducted a screening of *Bacillus* strains (H6, S40, S32, and S15) for biosurfactant production. The extracted biosurfactants displayed low CMC values, indicating their high surfactant efficiency at low concentrations a highly desirable characteristic for large-scale industrial applications. Additionally, biosurfactants are proved thermally resilient, positioning them as promising sustainable alternatives to conventional synthetic surfactants. Strain H6 demonstrated exceptional foaming properties, with high FC and stability, making it particularly suitable for applications in oil recovery and cleaning, where stable foam layers are essential for performance. Furthermore, the emulsifying properties of the biosurfactants were outstanding, with excellent
stability in oil-water emulsions. This makes them suitable for a variety of applications, including the food industries, where stability and enhancement are critical. This study highlights the potential of *Bacillus*-derived lipopeptides as sustainable, efficient alternatives to synthetic surfactants, offering a broad range of applications across various food industrial sectors.

## Supplementary material

Supplementary material is available at *International Journal of Food Science and Technology* online.

## Data availability

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

## Author contributions

Houda Gharsallah (Conceptualisation, Data curation, Formal analysis, Methodology, Writing—original draft, Investigation, Resources), Cedric Guerin (Data curation, Formal analysis, Methodology, Software), Ahlem Eddehech (Writing—original draft), Sami Boufi (Formal analysis, Methodology, Data curation, Writing—review and editing), Mecit Halil Öztop (Funding acquisition, Project administration, Resources, Writing—review & editing, Methodology, Supervision), Rafik Balti (Conceptualisation, Methodology, Project administration, Supervision, Validation, Writing—review & editing), and Zied Zarai (Conceptualisation, Methodology, Project administration, Validation, Visualisation, Writing—original draft, Writing—review & editing, Resources, Supervision, Funding acquisition)

All authors have read and agreed to the published version of the manuscript.

## Funding

This work was supported by the European Union's Horizon 2020—Partnership for Research and Innovation in the Mediterranean Area (PRIMA) Section I Program under grant agreement No. 2032 for the project Functionalized Tomato Products (FunTomP).

## Conflicts of interest

All authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## References

* Abdel-Mawgoud, A. M., Lépine, F., & Déziel, E. (2010). Rhamnolipids: Diversity of structures, microbial origins, and roles. *Applied Microbiology and Biotechnology*, 86, 1323–1336. https://doi.org/10.1007/s00253-010-2498-2

* Adetunji, C. O., Anani, O. A., Olaniyan, O. T., Inobeme, A., Oloke, J. K., Palnam, W. D., & Ali, S. (2022). *Green sustainable process for chemical and environmental engineering and science*. Amsterdam: Elsevier.

* Al-Sulaimani, H., Al-Wahaibi, Y., Al-Bahry, S., Elshafie, A., Al-Bemani, A., Joshi, S., & Ayatollahi, S. (2012). Residual-oil recovery through injection of biosurfactant, chemical surfactant, and mixtures of both under reservoir temperatures: Induced-wettability and interfacial-tension effects. *SPE Reservoir Evaluation & Engineering*, 15, 210–217. https://doi.org/10.2118/158022-PA

* Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. *Journal of Molecular Biology*, 215, 403–410. https://doi.org/10.1016/S0022-2836(05)80360-2

* Alves, C. L., Oliveira, D., Lima, F. D., & Teixeira, J. A. (2020). Biosurfactants produced by *Bacillus cereus* UCP1615: Evaluation of emulsifying activity and stability for various vegetable oils. *Journal of Surfactants and Detergents*, 23, 421–432.

* Araguppi, D. A., Bagewadi, Z. K., Patil, N. R., & Mantri, N. (2023). Iturin: A promising cyclic lipopeptide with diverse applications. *Biomolecules*, 13, 1515. https://doi.org/10.3390/biom13101515

* Bamba, T., Aoki, R., Hori, Y., Ishikawa, S., Yoshida, K., Taoka, N., Kobayashi, S., Yasueda, H., Kondo, A., & Hasunuma, T. (2024). High-throughput evaluation of hemolytic activity through precise measurement of colony and hemolytic zone sizes of engineered *Bacillus subtilis* on blood agar. *Biology Methods and Protocols*, 9, bpae044. https://doi.org/10.1093/biomethods/bpae044

* Barghouthi, S. A. (2011). A universal method for the identification of bacteria based on general PCR primers. *Indian Journal of Microbiology*, 51, 430–444. https://doi.org/10.1007/s12088-011-0122-5

* Barrow, P. A., Jones, M. A., Smith, A. L., & Wigley, P. (2007). The long view: *Salmonella*—The last forty years. *Molecular Microbiology*, 9, 415–428.

* Bezza, F. A., & Chirwa, E. M. N. (2015). Production and applications of lipopeptide biosurfactant for bioremediation and oil recovery by *Bacillus subtilis* CN2. *Biochemical Engineering Journal*, 101, 168–178. https://doi.org/10.1016/j.bej.2015.05.007

* Chabhadiya, S., Acharya, D., Mangrola, A., Shah, R., & Pithawala, E. A. (2024). Unlocking the potential of biosurfactants: Innovations in metabolic and genetic engineering for sustainable industrial and environmental solutions. *Biotechnology Notes*, 5, 111–119. https://doi.org/10.1016/j.biotno.2024.07.001

* Chen, Y., Kou, Y., Wang, L., Chen, D., Luo, J., Zhang, F., & Chen, G. (2021). High efficiency emulsifier of complex surfactant with authigenic viscosity. *Earth and Environmental Science*, 859, 012110. 10.1088/1755-1315/859/1/012110

* Clarridge, I. E. (2004). Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases. *Clinical Microbiology Reviews*, 17, 840–862. https://doi.org/10.1128/CMR.17.4.840-862.2004

* Coutte, F., Gudiña, E. J., Teixeira, J. A., & Oliveira, R. (2017). Production of lipopeptides by *bacillus* strains: Biosynthesis, regulation, and roles. *Biotechnology Advances*, 35, 107–120.

* Das, P., Mukherjee, S., & Sen, R. (2009). Biosurfactant of marine origin exhibiting heavy metal remediation properties. *Bioresource Technology*, 100, 4887–4890. https://doi.org/10.1016/j.biortech.2009.05.028

* Datta, P., Tiwari, P., & Pandey, L. M. (2018). Isolation and characterization of biosurfactant-producing and oil-degrading *Bacillus subtilis* MG495086 from formation water of Assam oil reservoir and its suitability for enhanced oil recovery. *Bioresource Technology*, 270, 439–448. https://doi.org/10.1016/j.biortech.2018.09.047

* Dini, S., Bekhit, A. E. D., Roohinejad, S., Vale, J. M., & Agyei, D. (2024). The physicochemical and functional properties of biosurfactants: A review. *Molecules*, 29, 2544. https://doi.org/10.3390/molecules29112544

* Gomaa, O. M., Selim, N., Fathy, R., Maghrawy, H. H., Gamal, M., Abd El Kareem, H., Kyazze, G., & Keshavarz, T. (2021). Characterization of a biosurfactant-producing electroactive *Bacillus* sp. for enhanced microbial fuel cell dye decolorization. *Enzyme and Microbial Technology*, 147, 109767. https://doi.org/10.1016/j.enzmictec.2021.109767
Gürtler, V., & Stanisich, V. A. (1996). New approaches to typing and identification of bacteria using the 16S-23S rDNA spacer region. *Microbiology*, 142, 3–16. https://doi.org/10.1099/13500872-142-1-3

Habib, S., Ahmad, S. A., Wan Johari, W. L., Abd Shukor, M. Y., Alias, S. A., Smykla, J., Saruni, N. H., Abdul Razak, N. S., & Yasid, N. A. (2020). Production of lipopeptide biosurfactant by a hydrocarbon-degrading Antarctic *Rhodococcus*. *International Journal of Molecular Sciences*, 21, 6138. https://doi.org/10.3390/ijms21176138

Jahan, R., Ahmad, W., & Ahsan, A. (2020). Biosurfactants, natural alternatives to synthetic surfactants: Physicochemical properties and applications. *Advances in Colloid and Interface Science*, 275, 102061. https://doi.org/10.1016/j.cis.2019.102061

Kourmentza, K., Gromada, X., Michael, N., Degraeve, C., Vanier, G., Ravallec, R., Coutte, F., Karatzas, K. A., & Jauregi, P. (2021). Antimicrobial activity of lipopeptide biosurfactants against foodborne pathogens and food spoilage microorganisms and their cytotoxicity. *Frontiers in Microbiology*, 11, 561060. https://doi.org/10.3389/fmicb.2020.561060

Lamilla, C., Braga, D., Castro, R., Guimarães, C., Va De Castilho, L., Freire, D. M., & Barrientos, L. (2018). Streptomyces *ludus* So3.2 from Antarctic soil as a novel producer of compounds with bioemulsification potential. *PLoS One*, 13, e0196054. https://doi.org/10.1371/journal.pone.0196054

Li, M. N., Han, Q., Wang, N., Wang, T., You, X. M., Zhang, S., Zhang, C. C., Shi, Y. Q., Qiao, P. Z., Man, C. L., Feng, T., Li, Y. Y., Zhu, Z., Quan, K. J., Xu, T. L., & Zhang, G. F. (2024a). 16S rRNA gene sequencing for bacterial identification and infectious disease diagnosis. *Biochemical and Biophysical Research Communications*, 739, 150974. https://doi.org/10.1016/j.bbrc.2024.150974

Li, Q., Wang, Y., Chen, C., Zeng, M., Jia, Q., Ding, J., Zhang, C., Jiao, S., Guo, X., Wu, J., Fan, C., Chen, Y., & Hu, Z. (2024b). Isolation of a novel *Bacillus subtilis* HF1 strain that is rich in lipopeptide homologs and has strong effects on the resistance of plant fungi and growth improvement of broilers. *Frontiers in Microbiology*, 15, 1433598. https://doi.org/10.3389/fmicb.2024.1433598

Liu, Q., Niu, J., Yu, Y., Wang, C., Lu, S., Zhang, S., Lv, J., & Peng, B. (2021). Production, characterization, and application of biosurfactant produced by *Bacillus licheniformis* L20 for microbial enhanced oil recovery. *Journal of Cleaner Production*, 307, 127193. https://doi.org/10.1016/j.jclepro.2021.127193

Liu, Y., Zhan, X., Chen, X., & Xue, X. (2016). Biosurfactants from *Bacillus* strains: Production, characterization, and applications in oil recovery. *Bioresource Technology*, 211, 303–309.

Ma, S., Zhang, Z., Zhu, L., & Zhang, L. (2020). Biosurfactants as drug delivery systems in pharmaceutical applications. *European Journal of Pharmaceutics and Biopharmaceutics*, 151, 102–110.

Marchut-Mikołajczyk, O., Drożdżyński, P., Polewczyk, A., Smułek, W., & Antczak, T. (2021). Biosurfactant from endophytic *Bacillus pumilus* 2A: Physicochemical characterization, production and optimization and potential for plant growth promotion. *Microbial Cell Factories*, 20, 1–11. https://doi.org/10.1186/s12934-021-01533-2

Mina, M. M., Faid, M. R., & Tabatabai, M. (2021). Biosurfactants in cosmetic formulations: A review of their applications and properties. *Journal of Cosmetic Science*, 72, 315–323.

Mnif, I., & Ghribi, D. (2015). Microbial-derived surface-active compounds: Properties and screening concept. *World Journal of Microbiology and Biotechnology*, 31, 1001–1020. https://doi.org/10.1007/s11274-015-1866-6

Moldes, A., Álvarez-Chaver, P., Vecino, X., & Cruz, J. (2023). Purification of lipopeptide biosurfactant extracts obtained from a complex residual food stream using Tricine-SDS-PAGE electrophoresis.

Frontiers in Bioengineering and Biotechnology, 11, 1199103. https://doi.org/10.3389/fbioe.2023.1199103

Mukherjee, S., Das, P., & Sen, R. (2006). Towards commercial production of microbial surfactants. *Trends in Biotechnology*, 24, 509–515. https://doi.org/10.1016/j.tibtech.2006.09.005

Nishimaki, T., & Sato, K. (2019). An extension of the Kimura two-parameter model to the natural evolutionary process. *Journal of Molecular Evolution*, 87, 60–67. https://doi.org/10.1007/s00239-018-9885-1

Passos, T. F., De Camargo Bertuso, P., & Nitschke, M. (2023). Biosurfactants as food additives: New trends and applications. *Biosurfactants Research and Development: Foundations and Frontiers in Enzymology*, 5, 85–106. https://doi.org/10.1016/j.b978-0-323-91697-4-00005-3

Patel, J. B. (2001). 16S rRNA gene sequencing for bacterial pathogen identification in the clinical laboratory. *Molecular Diagnostics*, 6, 313–327.

Patel, S., & Kharawala, K. (2022). Biosurfactants and Their Biodegradability: A Review and Examination. *International Journal of Engineering and Advanced Technology*, 11(3), 4–11. https://doi.org/10.35940/ijeat.b3319.0211322

Phulpoto, I. A., Yu, Z., Qazi, M. A., Ndayisenga, F., & Yang, J. (2023). A comprehensive study on microbial-surfactants from bioproduction scale-up toward electrokinetics remediation of environmental pollutants: Challenges and perspectives. *Chemosphere*, 311, 136979. https://doi.org/10.1016/j.chemosphere.2022.136979

Ravi, S. S., Chandra, R., & Venugopalan, R. (2020). Enhanced oil recovery using biosurfactants: Challenges and opportunities. *Journal of Petroleum Science and Engineering*, 194, 107401.

Ribeiro, B. G., Guerra, J. M., & Sarubbo, L. A. (2020). Biosurfactants: Production and application prospects in the food industry. *Biotechnology Progress*, 36, e3030. https://doi.org/10.1002/ptpr.3030

Rodrigues, L. R., & Teixeira, J. A. (2010). Biomedical and therapeutic applications of biosurfactants. In Brar, S. K. & Dhillon, G. S. (Eds.), *Biosurfactants* (pp. 75–87). Berlin: Springer. https://doi.org/10.1007/978-1-4419-5979-9_6

Roy, A., Khan, M. R., & Mukherjee, A. K. (2024). Recent advances in the application of microbial biosurfactants in food industries: Opportunities and challenges. *Food Control*, 163, 110465. https://doi.org/10.1016/j.foodcont.2024.110465

Saikia, R. R., Deka, S., & Sarma, H. (2021). Biosurfactants from bacteria and fungi: perspectives on advanced biomedical applications. In *Biosurfactants for a sustainable future: Production applications in the environment and biomedicine* (pp. 293–315). Amsterdam: Elsevier.

Saiyam, D., Dubey, A., Malla, M. A., & Kumar, A. (2024). Lipopeptides from *Bacillus*: Unveiling biotechnological prospects—Sources, properties, and diverses applications. *Brazilian Journal of Microbiology*, 55, 281–295. https://doi.org/10.1007/s42770-023-01228-3

Santos, B. L. P., Vieira, I. M. M., Ruzene, D. S., & Silva, D. P. (2023). Unlocking the potential of biosurfactants: Production, applications, market challenges, and opportunities for agro-industrial waste valorization. *Environmental Research*, 244, 117879.

Sharma, S., & Pandey, L. M. (2020). Production of biosurfactant by *Bacillus subtilis* RSL-2 isolated from sludge and biosurfactant-mediated degradation of oil. *Bioresource Technology*, 307, 123261. https://doi.org/10.1016/j.biortech.2020.123261

Sharma, D., & Sharma, D. (2021). Industrial applications of biosurfactants. In Sharma, D. & Kumar, A. (Eds.), *Biosurfactants: Greener surface active agents for sustainable future: Microbial surfactants* (pp. 115–143). Singapore: Springer.

Sharma, D., Singh, D., Sukhbir-Singh, G. M., Karamchandani, B. M., Aseri, G. K., Banat, I. M., & Satpute, S. K. (2023). Biosurfactants:
Forthcomings and regulatory affairs in food-based industries. *Molecules*, 28, 2823. https://doi.org/10.3390/molecules28062823

Shi, C., & Maktabdar, M. (2022). Lactic acid bacteria as biopreservation against spoilage molds in dairy products – A review. *Frontiers in Microbiology*, 12, 819684. https://doi.org/10.3389/fmicb.2021.819684

Shoeb, E., Ahmed, N., Akhter, J., Badar, U., Siddiqui, K., Ansari, F., Waqar, M., Imtiaz, S., Akhtar, N., & Shaikh, Q. U. A. (2015). Screening and characterization of biosurfactant-producing bacteria isolated from the Arabian Sea coast of Karachi. *Turkish Journal of Biology*, 39, 210–216. https://doi.org/10.3906/biy-1405-63

Singh, S., Banat, I. M., & Cameotra, S. S. (2020). Biosurfactants in biotechnology: Applications in pharmaceuticals and environmental clean-up. *Journal of Biotechnology*, 321, 1–16.

Sondhi, S. (2023). *Applications of next-generation biosurfactants in the food sector*. In Inamuddin & Adetunji, C.O. (Eds.), *Applications of Next-Generation Biosurfactants in the Food Sector* (pp. 43–56) Amsterdam: Elsevier.

Sun, J., Xu, J., Tang, Y., Zhou, Q., & Xiao, L. (2020). Advances in molecular detection methods for bacteria. *Frontiers in Microbiology*, 11, 2378.

Suthar, H., & Nerurkar, A. (2016). Characterization of biosurfactant produced by *Bacillus licheniformis* TT42 having potential for enhanced oil recovery. *Applied Biochemistry and Biotechnology*, 180, 248–260. https://doi.org/10.1007/s12010-016-2096-6

Thakur, V., Baghmare, P., Verma, A., Verma, J. S., & Geed, S. R. (2024). Recent progress in microbial biosurfactant production strategies: Applications, technological bottlenecks, and future outlook. *Bioresource Technology*, 408, 131211. https://doi.org/10.1016/j.biortech.2024.131211

Thavasi, R., Sharma, S., & Jayalakshmi, S. (2011). Evaluation of screening methods for the isolation of biosurfactant-producing marine bacteria. *Journal of Petroleum and Environmental Biotechnology*, S1, 1–7.

Thompson, J. D., Higgins, D. G., & Gibson, T. J. (1994). CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties, and weight matrix choice. *Nucleic Acids Research*, 22, 4673–4680. https://doi.org/10.1093/nar/22.22.4673

Trappe, K., Marschall, T., & Renard, B. Y. (2016). Detecting horizontal gene transfer by mapping sequencing reads across species boundaries. *Bioinformatics*, 32, i595–i604. https://doi.org/10.1093/bioinformatics/btw423

Vigneshwaran, C., Vasantharaj, K., Krishnanand, N., & Sivasubramanian, V. (2021). Production optimization, purification, and characterization of lipopeptide biosurfactant obtained from *Brevibacillus* sp. AVN13. *Journal of Environmental Chemical Engineering*, 9, 104867. https://doi.org/10.1016/j.jece.2020.104867

Wu, B., Xiu, J., Yu, L., Huang, L., Yi, L., & Ma, Y. (2022). Biosurfactant production by *Bacillus subtilis* SL and its potential for enhanced oil

recovery in low permeability reservoirs. *Scientific Reports*, 12, 7785. https://doi.org/10.1038/s41598-022-12025-7

Yasmin, A., Aslam, F., & Fariq, A. (2022). Genetic evidences of biosurfactant production in two *Bacillus subtilis* strains MB415 and MB418 isolated from oil contaminated soil. *Frontiers in Bioengineering and Biotechnology*, 10, 855762. https://doi.org/10.3389/fbioe.2022.855762

Youssef, N. H., Duncan, K. E., Nagle, D. P., Savage, K. N., Knapp, R. M., & McInerney, M. J. (2004). Comparison of methods to detect biosurfactant production by diverse microorganisms. *Journal of Microbiological Methods*, 56, 339–347. https://doi.org/10.1016/j.mimet.2003.11.001

Zarai, Z., Sila, A., Ben Ali, Y., & Gargouri, Y. (2016). *Helix aspersa* gelatin as an emulsifier and emulsion stabilizer: Functional properties and effects on pancreatic lipolysis. *Food Function*, 7, 326–336. https://doi.org/10.1039/C5FO00963D

Zargar, A. N., Lymperatou, A., Skiadas, I., Kumar, M., & Srivastava, P. (2022). Structural and functional characterization of a novel biosurfactant from *Bacillus* sp. IITD106. *Journal of Hazardous Materials*, 423, 127201. https://doi.org/10.1016/j.jhazmat.2021.127201

Zhang, L., Hu, X., Wang, X., Zhao, H., Sun, Y., & He, J. (2020). Structural diversity and biological activity of cyclic lipopeptides in *Bacillus* species: A comprehensive analysis. *Frontiers in Microbiology*, 11, 1997.

Zhang, B., Xu, L., Ding, J., Wang, M., Ge, R., Zhao, H., Zhang, B., & Fan, J. (2022). Natural antimicrobial lipopeptides secreted by *Bacillus* sp. and their application in food preservation: A critical review. *Trends in Food Science & Technology*, 127, 26–37. https://doi.org/10.1016/j.tifs.2022.06.009

Zhang, Y., Zhang, L., & Sun, J. (2018). Enhanced oil recovery performance of a biosurfactant produced by *Bacillus subtilis* and its synergistic effect with chemical surfactants. *Journal of Petroleum Science and Engineering*, 165, 111–119.

Zhao, L., Zhang, H., Yu, B., & Yang, Y. (2018). Biosurfactants in food formulations: New insights into food and emulsions. *Food Control*, 90, 92–100.

Zhao, F., Zhu, H., Cui, Q., Wang, B., Su, H., & Zhang, Y. (2021). Anaerobic production of surfactin by a new *Bacillus subtilis* isolate and the in situ emulsification and viscosity reduction effect towards enhanced oil recovery applications. *Journal of Petroleum Science and Engineering*, 201, 108508. https://doi.org/10.1016/j petrol.2021.108508

Zhu, Y., Jiang, S., & Liu, X. (2017). Cost-effective production of biosurfactants from *Bacillus* strains and their applications in food and agricultural industries. *International Journal of Food Science & Technology*, 52, 2054–2062.

Zong, Z., Zhang, S., Wu, H., Zhao, X., Li, W., Yang, L., Li, L., & Li, H. (2019). Characterization and comparative profiling of cyclic lipopeptides produced by *Bacillus* strains. *Journal of Applied Microbiology*, 127, 795–805.