Experiment No: Date:23/10/2019

Production of Bioemusifier
Aim: Study the bioemulsifier producing Yeast cell by drop collapse method.
Introduction:
Two immiscible liquids can form an emulsion which may be described as unstable
dispersion system of these two liquids by the action an emulsifier. Emulsifiers are a large
category of compounds considered as surface active agents or surfactants. An emulsifier acts
by reducing the speed of chemical reactions, and enhancing its stability. Bioemulsifiers are
known as surface active biomolecule materials, due to their unique features over chemical
surfactants, such as non-toxicity, biodegradability, foaming, biocompatibility, efficiency at
low concentrations, high selectivity in different pH, temperatures and salinities.
As it is clear, oil and water are incompatible. The mixture of oil and water lead to the
production of an emulsion. When an emulsion stays still for a while, oil droplets begin to
separate from water. In this regards, emulsifiers are used to stop this process. In fact,
emulsifiers are used to prevent the emulsion from breaking. Examples of emulsions currently
used in the food industry include milk, butter, margarine, mayonnaise and ice cream.
Emulsifiers are a large category of compounds also known as surface active agents or
surfactants. The word surfactant is used for molecules that migrate to the surface between
phases. Bioemulsifiers are higher in molecular weight compared to biosurfactants since they
are complex mixtures of heteropolysaccharides, lipopolysaccharides, lipoproteins and
proteins. Emulsifiers have double lipophilic and hydrophilic properties. Adding an emulsifier
to an unmixable compound, reduces surface tension between the two phases and prevents it
from separating. Therefore, the two liquids are able to form an emulsion. Since an emulsion
consists of water-soluble and oil-soluble fragments, an emulsifier is placed on the surface of
the area where the two liquids (water and oil) are connected.
Bioemulsifiers derived from various Yeast and Fungi:
1. Mannoprotein:- Mannoprotein bioemulsifier is a glycoprotein. Within the cellular wall
of Saccharomyces spp. and Kluyveromyces marxianus of yeast, mannoprotein molecules
are present in glucan, networks, and released from the cell wall of yeast using pressurized
heat treatments. This bioemulsifier is able to stabilize oil-inwater emulsions (O/W).

2. Liposan:- Liposan bioemulsifier is a water-soluble emulsifier obtained from extracting

organic solvents fermented by Candida lipolytica yeast. Liposan is produced in the
 extracellular layer and consists of 83% carbohydrate and 17% protein. The presence of
protein fractions. Liposan is resistant to temperatures less than 70°C, but after heating at
100 °C for one hour, 60% of its emulsifiable strength reduces.
Bioemulsifiers derived from bacteria
1. Lauryl fructose:- This bioemulsifier is produced by the lipase enzyme obtained from
Pseudomonas spp.60 in a culture media containing dry pyridine. This bioemulsifier has
emulsification properties for a variety of hydrocarbons, edible oils, and oil based oils such
as margarine and shortening.

2. Emulsion:- Emulsan is an extracellular poly-anionic bioemulsifier produced by

Acinetobacter calcoaceticus RAG 1 bacteria. In fact, emulsan is a
lipoheteropolysaccharide polymer containing D-galactose-amine produced during the
stationary phase. This bioemulsifier is a poly-anionic and amphiphilic compound which is
able to stabilize the hydrocarbon emulsion in water by creating a very thin layer between
the hydrocarbon droplets and water. Another type of emulsan considered as bioemulsion
is produced by Acinetobacter calcoaceticus, which is used in the formulation and
production of soft cheese and ice creams as well as creams and skinprotecting materials.
Functional Properties:
Functional properties of bioemulsifiers are namely emulsification, wetting, foaming,
cleaning, phase separation, surface activity, heavy metal binding, bacterial pathogenesis,
antibacterial activity, antiviral activity, antifungal activity and reduction in the viscosity of
the heavy liquids such as crude oil enables them to be utilized for many industrial and
domestic application.
Conventional Methods:
Conventional screening methods for detection of bioemulsifiers producers are as follows:
1. Modified Drop collapse method:
Mocrotitre plates thinly coated with Pennzoil. A sample of 5µl (culture broth) is added
to the center of the well and observations are carried out for 1mi. If the drop of the sample
collapses from the coated oil it is the indication of BS in the culture broth.
2. Oil spread method:
In this method of detection, 20µl of crude oil is added to 50ml of distilled water
(DW) petri plate. Ten microliter of culture broth is added and oil coated water surface.
Colony surrounded by an emulsified halo is considered positive for BS production.
 Industrial and environmental potentials of bioemulsifier:
 Bioemulsifier has a potential application in biological field as anti-viral, anti-fungal and anti-
bacterial agents
 They have proved to be a potent applicator in environmental bioremediation, cleaning the
environment polluted with the crude oil or polyaromatic hydrocarbons, in industrial
emulsifications and stabilization process,
 Bioremediation: Treatment of hydrocarbon polluted soil can be done by using
biosurfactant/bioemulsifier. The emulsification properties of bioemulsifier make them utilize
hydrocarbon degrade it efficiently.
Requirements
1. Sterile Yeast Malt broth
2. Yeast culture
3. Glass slide
4. Polystyrene strip
5. Syringe.
Procedure
1. The yeast culture was grown in 250ml Erlenmeyer flask containing 50ml of yeast malt
broth and was incubated for 48 hours at 28°C on a rotatory shaker at 150r/min.
2. After incubation place a drop of cell suspension on polystyrene strip coated glass
slide.
3. Observe the drop collapse which contain bioemulsifier.
4. Observe the non-emulsifier containing drop to remain stable.
5. Use water in Drop collapse method.
6. 20µl of supernatant and 20µl of control solution (water) can be added on slide by the
syringe at an angle of 45°
7. After 1 min, the drops can be examined visually.
Observation

Result
On a polystyrene coated glass slide surface a drop of yeast cell suspension was collapsed
after 1 min, when compared with a control drop.
Conclusion
Bioemulsifier was produced by yeast cells and confirmed by a drop collapse method.