Isolation of biosurfactant/bioemulsifier from Saccharomyces cerevisiae by different treatment

Biosurfactant are amphiphilic compound have been increasingly attracting the attention of the scientific community as promising candidates for the replacement of a number of synthetic surfactants. The aim of this study is to extraction of Biosurfactant from Saccharomyces cerevisiae by using three treatment (absolute ethanol , ammonium sulphate and heating). The present study clearly showed that , the treatment with boiling to 30 min was very effect to extract the biosurfactant from S. cerevisiae best than other treatments with high emulsification index and surface activity where were 81.08% and 20 mm , respectively.


Introduction
Microorganisms have ability to produce a broad range of molecules. amongst them there are amphipathic molecules, known biosurfactants (Fracchia et al., 2012;Willem , 2012).
Surfactants are generally organic compounds that are amphiphilic, , concept they contain both hydrophobic groups (''tails'') and hydrophilic groups (''heads''), and that act preferably in the interface of fluid phases with various levels of polarity and bridges of hydrogen, like oil/water or air/water Interfaces (Luna et al., 2011). Biosurfactants were created by different groups of microorganisms (Sridhar et al., 2013). Due to their good properties like lower toxicity, higher foaming capacity , higher biodegradability and higher activity at extreme temperatures,salinity and pH levels (Prabakaran and Sumathi, 2014). biosurfactants have been increasingly attracting the interest of the scientific community as promising candidates for the instead of a number of synthetic surfactants (Vidhya et al., 2015). These compounds are biological molecules with remarkable surfactant characteristics identical to the well-known synthetic surfactants and they also include microbial compounds with surfactant characteristics (Trindade et al., 2008). the present study has carried out to test absolute ethanol , ammonium sulphate and heating treatments to obtain of biosurfactant/bioemulsifier from S. cerevisiae with high emulsification activity.

Isolation and identification of S. cerevisiae
Dry bakery yeasts which were imported from different origns were collected from local markets, Which included; AY isolate from Angel yeast (Chinese origin), TY isolate from Town-instant (Chinese origin), YY isolate from Yuva yeast (Turkish origin), GY isolate from Gloripan (Egypt origin) and SY from Saf-instant (France origin). All isolates streaked on Sabouraud dextrose agar and incubated for 24 hrs at 30 ºC, then it examind under microscope and made conventional biochemical tests according to (Herrero et al., 1999;Lodder, 1974;Barnett et al., 1985).

Cultivation of S. cerevisiae
Twenty Five ml of YEGP broth (10) g yeast extract, (20) g Glucose and (20) g Peptone in (1000) ml of distilled water) inoculated with a fresh culture of S. cerevisiae YY and incubated at 30 °C for 24 hrs. and further used as the seed culture for the production of biosurfactant (Dhivya , 2014).

Biosurfactant Production
200 ml of YPEG broth medium pepared into 500 ml of erlenmyer flask. After cooling to 50 °C , 2 mL of seed culture was added to each container. The containers were incubated and shaken (125) rpm at (28-30)°C for 48 h Use as crude biosurfactant (Farahnejad et al., 2004).

Oil-spreading test
To apply the oil-spreading test, oil was layered over water in a petri plate and a drop of supernatant and pellet was added to the surface of oil. The diameter of the clear zone on the oil surface was measured in 2 replications for each isolate. A water drop was used as a negative control (Shoeb et al.,2015)

Measurement of emulsification activity
The emulsification index test was done to method described by (Shoeb et al.,2015) used to detect activity of biosurfactant, as follows: Emulsifying capacity of isolates was evaluated by an emulsification index (E24) for kerosene oil. To do so, 1.5 mL of Kerosene was added to 1.5 ml of supernatant and pellet in a test tube, which was vortexed at high speed for (2) min and allowed to stand for 24 h. The percentage of the emulsification index was calculated using the following equation. E24 = Height of emulsion formed × 100/total height of solution.

Isolation of biosurfactant
The biosurfactant was extracted from S. cerevisiae by using three different treatments ( absolute ethanol , ammonium sulphate and boiling ). And use broth without any treatment .
Crude biosurfactant was prepared as previous and treated with following treatments: First treatment ; 25 ml of previous culture was treated with five volume of absolute ethanol for 30 and 60 min in an ice bath , then centrifuged at 10000 rpm at 4ºC for 15min, then the pellet was taken and dissolved in an amount of PBS [(8) g NaCl, (0.2)g KCL, (1.44)g Na 2 HPO 4 , (0.27)g KH 2 PO 4 were dissolved in 800 ml of sterile distilled water .pH 7.4] after then assay the biosurfactant activity .
Second treatment ; 25 ml of culture was treated with heating to 100 c for 30 and 60 min , then centrifuged at 10000 rpm at 4ºC for 15min, then the supernatant was taken and test biosurfactant activity to determine the best time to heating. Third treatment ; a specific weight of ammonium sulfate crystals was added to the (100 ml)of crude biosurfactant gradually in an ice bath with continuous stirring for 30 min to get saturation percentage of (0-25 , 25-50 , 50-75 and 75-100 %), then centrifuged at 10000 rpm at 4ºC for 15min, then the pellet was taken and dissolved in an amount of PBS . then dialyzed over night against the same buffer at 4 ºC. and assay biosurfactant activity to determine the best saturation percentage.

Results and disscusion
We obtained five isolates of S. cerevisiae from dry baker yeast where were blue color , oval to round with bud under microscope . After incubatation at 35c for 24 hrs, the colonies on Sabouraud agar were small, convex, regular margins and distinct odor. They were fermented to carbohydrates sources except (Lactose) because they don't have the enzyme lactase, Also It was produced ester odor on YPD agar , for While it can not hydrolysed the urea because they do not possed urease enzyme therefore the indicator color did not change (Lodder,1970).

Screening of S. cerevisiae for biosurfactant Production
Five S. cerevisiae isolates were screened for cell-bound and excreted biosurfactant production by two methods; Surface activity by Oil spreading test and Emulsification index (E24).
Oil spreading assay is a reliable method to detect biosurfactant production by diverse microorganisms based on the ability of the biosurfactants present in the supernatant of isolate solutions capable of spreading the oil and producing a clear zone.This clearing zone on the oil surface correlates to surfactant activity, also called oil displacement activity (Walter et al., 2010).
All isolates of S. cerevisiae were positive for the oil-spreading assay , but S. cerevisiae YY isolate showed the highest oil spreading activity (table-1).

Table 1: The clear zone of biosurfactant on oil surface layer
The diameter of the clearing zone formed by biosurfactant-containing solution has been shown to be directly proportional to the concentration of biosurfactant (Morikawa et al., 2000) . Results are displayed in Figure (

Biosurfactant Production
The selected biosurfactant-producing isolate S. cerevisiae (YY) was grown in yeast extract glucose pepton (YPEG) production medium, pH 5.6 incubated at 30 o C for 24 hrs in shaking incubator was centrifuged under refrigeration condition. After incubation period , the flasks exhibited turbidity concomitant biomass and biosurfactant production.

Isolation of biosurfactant
In this present study , all treatments included precipitate the proteins .
We observed the boiling mehod for 30 min was the best treatment for biosurfactant extract from S. cerevisiae , That may be due to digestion of large portion of cell wall glucan and release the types of mannoprotein. Where many studies showed that the biosurfactant/ bioemulsifier was mannoprotein ( Alcantara et al., 2012 ;Dikit, et al., 2010 ;Walenckaa et al.,2007). Alcantara, et al., (2011) reported that, the isolation of the biosurfactant by heat treatment for 5 min from S. cerevisiae 2031 cells effectively solubilised the extracellular biosurfactant. Where The height emulsification index and oil displacement activity calculated as 81.08% and 20 mm , respectively These result approach to The result of study of (Alcantara, et al., 2011), where declare that boiling of S. cerevisiae cells gave the highest E24 of 79.44%.
while boiling for 60 min effected on activity of biosurfactant where led to lowing it.
Absolute ethanol possibly react with component and product of the cell and led to damage it and lowing the activity of biosurfactant .
The biosurfactant was precipitated by ammonium sulphate with different percet according to saturation percents. the results showed that the best range of the biosurfactant was precipitated in the saturation range between (50 75%) . The E24 and surface activity was 42% and 10 mm respectively.
Ammonium sulfate is a salt, which is often used for the precipitation of proteins because of its high solubility (Burgess, 2009 ). The result was shown in table 3

Conclusion
Boiling method for 30 min for Isolation of biosurfactant from S. cerevisiae YY was best than other treatmens where activity of biosurfacant was 81.08 % and 20 mm by emulsification index and surface activity , respectively. Where these method was very easy and do not Consume tools and spend short time .