Biology, Medicine, & Natural Product Chemistry  ISSN 2089-6514 (paper) 

Volume 13, Number 1, April 2024 | Pages: 235-238 | DOI: 10.14421/biomedich.2024.131.235-238 ISSN 2540-9328 (online) 
 

 

 

 

The Study of Biosurfactant Stability and The Effect on Lipase Activity 

 
Mieke Alvionita1,*, Rukman Hertadi2, Rahmat Rizki Fazli3, 

Andi Alfira Ratna Faradisa Dewi1, Tasyang Oktavia Rose1 
1Department of Chemistry, Faculty of Mathematics and Natural Sciences, State University of Malang, Indonesia. 

2Bandung Institute of Technology, Jalan Ganesa No 10 Coblong, West Java, Indonesia. 
3Faculty of Pedagogy and Education, Syiah Kuala University. Jalan Syech Abdurrauf No 4 23111 Banda Aceh, Aceh, Indonesia. 

 

 

Corresponding author* 
mieke.alvio.fmipa@um.ac.id 

 

 

 

 

Abstract 

 

Lipase is one of hydrolase enzyme that catalyzed hydrolysis reaction of triacylglycerols into free fatty acids and monoglycerides or 

glycerol. These biocatalysts are widely used in several industries, namely food and pharmaceutical industry. The activity of lipase can 

increase significantly if the substrate forms an emulsion. Since biosurfactant has been known to have emulsification characteristic, the 

effect of biosurfactant addition into lipase is necessary to be investigated. It is the first report that evaluate the effect of microbial 

surfactant on lipase activity. The purpose of this research is to analyze the stability of biosurfactant emulsion under various conditions, 

such as salinity and pH as well as their potential to enhance lipase activity. Biosurfactant used was Halomonas elongata BK-AG18 from 

the collection of Biochemistry Research Group ITB, Bandung, Indonesia. It was found that after the addition of NaCl, there was no 

significant decrease in the emulsification activity of the biosurfactant. The emulsification index (IE24) of biosurfactant with several NaCl 

concentrations (2, 5, 10, 15, 20, and 25%) was obtained around 50%. Stability test of biosurfactant at pH range 4-10 showed the highest 

IE24 of biosurfactant was obtained at pH 6. The effect of biosurfactants on lipase hydrolysis activity is also discussed in this article. 

Lipase hydrolysis activity was tested using p-nitrophenyl palmitate substrate. The highest lipase activity was obtained after the addition of 

70% biosurfactant (v/v) at 0.026 units. This study shows that biosurfactant from H. elongata BK-AG18 has the potential to increase lipase 

activity. 

 

Keywords: Biosurfactans; Bledug Kuwu; H. elongata; Lipase activity; Stability. 

 

 

INTRODUCTION 
 

Lipase is a type of water-soluble hydrolase enzyme that 

has the ability to hydrolyse triacylglycerols into free fatty 

acids and monoglycerides or glycerol (Yadav, et al., 

2017). This ability is utilised in the food and 

pharmaceutical industry, biodiesel, and biomederation 

(Melani, Tambourgi and Silveira, 2020). Guerrand 

(2017) reported that the dairy industry used lipase to 

break down milk fat and give cheese its unique flavour. 

Lipase can also modify fats and oils to produce biodiesel 

(Silveira, Tardioli and Farinas, 2016), while in the 

pharmaceutical industry lipase can synthesise chiral 

intermediate compounds to produce polixatel as an 

anticancer drugs (Fukaya, et al., 2016). Lipase can be 

isolated from plants, animals, fungi and bacteria. When 

bacteria produce lipase, intermediates (by-products) are 

also produced, namely surfactants and known as 

biosurfactants. 

There are two types of surfactants, synthetic 

(artificial) and derived from living things called 

biosurfactants. Biosurfactants are surface-active 

amphiphilic compounds that have surfactant properties, 

consisting of hydrophilic and hydrophobic parts (Xi, 

Ping and Alikhani, 2021). The two substances function to 

reduce surface tension and interference between two 

immiscible substances (Sharma and Oberoi, 2017) by 

forming interfacial micelles (Roy, 2017). In addition, 

biosurfactants can assist lipase to bind to the oil so that 

lipase can easily degrade the long chains of TAG. 

Biosurfactants are preferred over synthetic surfactants 

because of their environmental friendliness, low toxicity, 

and biodegradability (Santos, et al., 2016), whereas 

synthetic surfactants have properties that are opposite to 

surfactants derived from living things. Markande, et al. 
(2021) explained that biosurfactants are widely used 

environmentally (biomederation, wastewater treatment, 

soil health, biofilm); industrial sector (food, textile, 

chemical, petroleum); agricultural sector as antifitogenic, 

animal manure management; health as antitumor, 

antimicrobial, antioxidant, antiinflammatory. 

Many studies on biosurfactants isolated from 
microbes have been done in the past including 

Ochrobactrum intermedium (Zarinviarsagh, 

Ebrahimipour and Sadeghi, 2017), Serratia (Oliveira, et 
al., 2021), Burkholderia sp. (Carvalho-Gonçalves and 

Manuscript received: 04 April, 2024. Revision accepted: 14 June, 2024. Published: 28 June, 2024. 

https://doi.org/10.14421/biomedich.2024.131.235-238


 

 
 

236 Biology, Medicine, & Natural Product Chemistry 13 (1), 2024: 235-238 

 

 

Gorlach-Lira, 2018). It was reported by Spreb, et al. 

(2017) that the correlation of emulsification activity of 

Aspergillus strains with the biosurfactants produced was 

very high. Rahayu, et al. (2019) also reported linear 

lipase activity with biosurfactant activity isolated from 

keratinolytic bacteria. Lipase activity can increase 

significantly if the substrate forms an emulsion, so lipase 

activity depends on hydrophobic and hydrophilic regions. 

Therefore, lipase is defined as a carboxylesterase that 

reacts with the substrate to form an emulsion. It is known 

that biosurfactants have emulsification activity, therefore 

this study aims to determine whether lipase activity 

decreases or increases due to the addition of 

biosurfactants. 

 

 

MATERIALS AND METHODS 
 

Study area  

Bledug Kuwu is a crater mud located in Semarang, 

Central Java Province, Indonesia. Sampling was 

conducted in sample point 7°07'04"S 111°07'17"E.  

 

Procedures 

Biosurfactant Stability and Emulsification Index 

(IE24)  

The stability of biosurfactant was conducted by 

performing biosurfactant emulsification test on various 

concentrations of NaCl and buffer with pH range 4-10. 

NaCl solution was made with various concentrations (2, 

5, 10, 15, 20, and 25% (b/v)) Then 2 mL of NaCl 

solution was put into a test tube, added to each tube 2 mL 

oil and 2 mL biosurfactant. Homogenised using a vortex 

for 2 minutes. After 24 hours, the IE24 value was 

determined.  

Glycine buffer was prepared with a pH range of 4-10. 

The IE24 value in the pH range of 4-10 was carried out 

in the same way as the emulsification test at various 

NaCl concentrations. 

 
Lipase Activity Test  

The standard curve of p-nitrophenol was prepared with a 

concentration variation of (0, 10, 20, 30, 40, 50, 60, 70) 

120 μg/mL. Afterwards, biosurfactant solution (CMC = 

275 mg/L) with concentrations of 0%, 10%, 30%, 50%, 

70%, and 100% of the total volume of glycine buffer was 

prepared.  

Lipase activity test was carried out by preparing 

substrate solution (pH = 7.0) for blank, sample, and 

control made in a centrifuge tube by: 960 μL buffer and 

100 μL ethanol (blank); 950 μL buffer, 100 μL ethanol, 

10 μL pNPP (sample); 950 μL buffer, 100 μL ethanol, 10 

μL pNPP (control). 

To determine the occurrence of the enzymatic 

reaction, each substrate solution was mixed and added 

with the mixture as follows: 900 μL of substrate solution 

and 300 μL of soluble buffer (blank); 900 μL of substrate 

solution, 300 μL of crude porcine extract, biosurfactant 

solution of various concentrations (0, 10, 30, 50, 70, 

100%) (Sample); 900 μL of substrate solution and 300 

μL of crude porcine extract that had been boiled at 80°C 

for 30 minutes. If it was homogeneous, the incubation 

process was continued for 15 minutes in a 70°C 

incubator. Afterwards, centrifuged at 12000 rpm for 10 

minutes. The absorbance of blank, sample, and control 

was measured at a wavelength of 405 nm in duplo. 

 

Data analysis 

IE24 value was determined by the following equation. he 

stands for height of emulsion layer and hs for total height 

of the solution and emulsion mixture 

 

𝐼𝐸24 =  ℎ𝑒/ℎ𝑠 × 100% 

 

 

RESULTS AND DISCUSSION 
 

Results 

Stability of Biosurfactant in NaCl Solution 

 
Figure 1. Emulsification activity of biosurfactants at various NaCl 

concentrations. 

 

Stability of Biosurfactant at pH Change 

 
Figure 2. Emulsification activity of biosurfactants at various pH. 

 

Effect of Biosurfactants on Lipase Activity 

 
Figure 3. Lipase activity after the addition of Biosurfactant (v/v) 

 



 

 
 

 Alvionita et al. – The Study of Biosurfactant Stability 237 
 

 

Discussion 

The stability of biosurfactant in certain temperature, pH, 

and salinity range determines the application of the 

biosurfactant. In this study, only the influence of salinity 

and pH conditions on the stability of biosurfactant was 

conducted. The stability of biosurfactant produced by H. 

elongata BK-AG18 in this study was measured by 

looking at its emulsification activity (IE24) in various 

NaCl levels, specifically 2, 5, 10, 15, 20 and 25%. 

Biosurfactant is said to have low emulsification activity 

if it has a low IE24 value, and vice versa. The IE24 value 

of biosurfactant at several salt concentrations is shown in 

Figure 1. The emulsification index of biosurfactant 

without NaCl addition has a percentage of 53.9%. 

Interestingly, the higher concentration of NaCl added did 

not cause a significant decrease in biosurfactant activity. 

This is shown from the IE24 value of 45.3% at 25% 

NaCl concentration. The stability of biosurfactants at 

various salt concentrations was proven by several 

researchers. The findings by Purwasena, et al. (2019) 

stated that salinity does not significantly affect the 

stability of biosurfactants. In addition, the emulsification 

activity of biosurfactants also did not change drastically 

when adding NaCl up to 10% (Saikia, et al. 2012). 

According to Bognolo, G. (1999) biosurfactants have a 

better ionic strength tolerance than synthetic surfactants 

that deactivate at 2-3% salt concentration. This ability of 

biosurfactants is needed especially for Microbial 

Enhanced Oil Recovery (MEOR) and bioremediation of 

oil spills in oceans that have extreme salinity conditions 

(Prieto, et al., 2008). 

Figure 2 shows the effect of pH value between 4-10 

on the emulsification activity of biosurfactant from H. 

elongata BK-AG18. Starting from pH 4-6, there was a 

slight increase in biosurfactant emulsification activity 

until it reached its peak where almost 50% emulsification 

activity at pH 6. At pH 7-9, there was a slight decrease, 

followed by a very significant decrease at pH 10 where 

only 34.4% emulsification activity occurred. According 

to Ahmad, et al., (2021), the emulsification activity of 

biosurfactants decreases at high pH levels. Different 

from the study conducted by Zambir, et al. (2017), where 

biosurfactant from Streptomyces sp. R1 bacteria showed 

relatively the same stability over a wide pH range (2-12) 

as proven by the relatively constant surface tension value 

at that pH. 

This study investigates the effect of biosurfactant 

addition on lipase activity using the p-NPP method. So 

far, there have been many studies on the effect of 

surfactant addition on lipase activity. However, this study 

is the first to use crude biosurfactant from H. elongata 

BK-AG18 to test its effect on lipase activity from 

Burkholderia sp. Surfactants and lipase can interact with 

each other, either in solution or at the interface. Several 

types of surfactant interactions with lipase enzymes can 

form micelle aggregates in solution or compete with 

lipase in the adsorption process. At low concentrations, 

surfactants can form complexes with enzymes, yet at 

high concentrations surfactants can also cause unfolding 

of the enzyme structure. The type and concentration of 

surfactant significantly determines the consequences of 

its interaction with lipase (Delorme, 2011). This shows 

that surface active agents can have both positive and 

negative effects on the lipase itself. Therefore, the effect 

of biosurfactants on lipase is very interesting to study. 

In this study, it has been discovered that 

biosurfactants have the ability to emulsify palm oil up to 

50%. The activity of emulsifying insoluble substrates 

such as oil or fat may affect the interaction between the 

substrate and lipase. Based on Figure 3, lipase activity 

without the addition of biosurfactant showed a value of 

0.023 units. After the addition of 10% biosurfactant, 

there was a slight increase in lipase until it reached its 

peak (0.026 units) as a result of the addition of 70% 

biosurfactant. A decrease in lipase activity began to 

occur after the addition of 90% biosurfactant and the 

lowest activity (0.022 units) was shown in the addition of 

100% biosurfactant. This shows that crude biosurfactant 

at certain concentrations can increase lipase activity 

despite the increase is not very significant. Some 

previous studies proved that surfactant can have a 

positive effect on lipase activity. A study conducted by 

Oliveira, et al. (2017) proved that the addition of Triton 

X-100 surfactant slightly increased the catalytic activity 

of lipase from Rhizomucor miehei (RML). 

 

 

CONCLUSIONS 
 

Therefore, from this study can be conclude that the 

emulsification activity of biosurfactant from H. elongata 

BK-AG18 was not significantly decreased by the addition 

of NaCl concentration up to 25%. This indicates that the 

biosurfactant has good stability in high salinity 

conditions. In contrast, pH has a more significant effect 

on emulsification activity than salt concentration. In this 

study, the biosurfactant was most stable at pH 6. In 

addition, this study also showed that the addition of 

biosurfactant at different concentrations showed different 

effects on lipase activity. The best lipase activity was 

shown by the addition of 70% biosurfactant. 

 

 

Acknowledgements: This research is supported by 

funding from PNBP Universitas Negeri Malang 2021 

with contract number: 5.3.523/UN32.14.1/2021.   

 

Authors’ Contributions: Rukman Hertadi designed the 

study. Mieke Alvionita carried out the laboratory work & 

analyzed data. Mieke Alvionita, Rahmad Rizki Fazli, 

Andi Alfira Ratna Faradisa Dewi, and Tasyang Oktavia 

Rose wrote the manuscript. All authors read and 

approved the final version of the manuscript. 

 

Competing Interests: The authors declare that there are 

no competing interests. 



 

 
 

238 Biology, Medicine, & Natural Product Chemistry 13 (1), 2024: 235-238 

 

 

Funding: This research is supported by funding from 

PNBP Universitas Negeri Malang 2021 with contract 

number: 5.3.523/UN32.14.1/2021. 

 

 

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