Hi, I am currently working on extracting EPS from lactic acid bacteria and would like to ask some questions,
My procedure is to mix 80% TCA [15% (v/v)] to the incubation medium. And I will centrifuge the solution (medium+TCA) after 30 minutes incubation, remove the precipitate part, and mix the supernatant with 2 times volume of ethanol for 24 hours. The next day I will remove the supernatant, take the precipitate and mix it again with 2 times volume of ethanol. In the end, I will centrifuge down the EPS, dilute the pellete with deionized water and dialyze it for 48 hours, and freeze-dry.
My question are,
There are so many version and procedure to extract EPS from bacteria and it make me confuse, I will be glad if someone can help me with my questions. Thank you :)
Lactic acid bacteria ( LAB ) are able to produce exopolysaccharides ( EPS ) in the surrounding medium as a slime or on the surface of bacterial cells to fo form a capsule ..........of the EPS demonstrated a highly compact structure with a smooth surface, facilitating formation of film by the polymer, the EPS was composed of many different sizes of spherical lumps with tendency to form molecular aggregates. To extract the EPS can be estimated by the phenol - sulfuric acid method using glucose as standard and purified by fractionation with an anion exchange chromatography on DEAE - cellulose column ( 26 x 40 cm ) , eluted with deionized water, , subsequently with 0.2 and 0.5 mol/L NaCl solutions at a flow rate of 1 ml/ min. The pooled fraction containing EPS are dialyzed and lyophilized for further purification by gel permeation chromatography on a Sepharose CL - 6B column ( 25 x 50 cm ). The purity o f the purified EPS sample can then be checked by Ultraviolet - visible ( UV - vis ) spectroscopy using UV- vis spectrophotometer ( U - 3900, Hitachi Ltd. , Japan). For further details consult Pol. J. Food Nutr. Sci. 2015, Vol. 65, No.4. pp269 -279.
The exopolysaccharides were extracted from the treated bacterial strains by the method described by (Hebber et al., 1992). Bacterial strains were grown separately on nitrogen deficient selective media, at 28ºC for 5-7 days, then the biomass was harvested by centrifugation at 15000 for 15 min. The obtained pellets were re-suspended in a bottle (250 ml capacity) containing 5ml of 0.85% KCl sterilized solution and 10ml of selective medium. Bottle was incubated on orbital shaker (150 rpm) for 3-5 days at 28ºC pellets were dehydrated in alcohol and dried at 35ºC for extracting hydro soluble slime . Exopolysaccharides were hydrolyzed by heating at 100ºC with 1N HCL for 7h. The acid hydrolyzate was reported under vacuum to dryness. Analysis of hydrolyzed bacterial exopolysaccharides was carried out using thin-layer chromatography (TLC) on cellulose plate F-254 (Merck). The solvent system used was ethyl acetate: pyridine: water: acetic acid: propionic acid (50:30:10:5:5) (v/v), (Bellogin et al., 1984). The plates were developed by aniline hydrogen phthalate (Partridge, 1949).
Dear Dr. Monika Nanda,
The answers for your questions are:
(1) You need to find out that the suitable 'carbon source' for your bacterial strain regarding EPS production (Different bacterial strains requires different sugar as 'carbon source' such as Glucose, Maltose, Sucrose etc. ), subsequent to which you can modify your MRS culture medium by adding sugar supplement additionally. Since, EPS production requires more carbon source in the form of monosaccharide units, therefore it is required to maintain sufficient amount of carbon source in your culture medium additionally.
(2) Different peoples have used 80% TCA from 15 - 20% V/V for the precipitation of protein content. Certain EPS polymers are degradable by the TCA. It depends upon the structure stability of the EPS.
You can precipitate EPS using 3 volume of ice cold ethanol, with and without TCA protein-precipitation step, and see the difference. In both the sample you can estimate the protein as well as EPS/carbohydrate content and find out whether your EPS is destroyed using TCA.
(3) You can check the protein content using bradford. The traces of protein can also be removed by using Chloroform:Isoamylalcohol (24:1) solution as well. However, the purity of the EPS can be best checked using HPLC equipped with GPC column. Presence of additional peaks, shoulders indicates impurity.
With best regards,
Dr. A. K. Dubey
Ashwini Kumar Dubey Thank you for your answer, Sir. If you do not mind, I have other questions regarding the protein and purity check of EPS.
1. What is the best way to prepare the EPS sample for that experiments? The final form of my extracted EPS is a white cotton candy-like texture. To check the carbohydrate and protein content of EPS, should I dilute the EPS to certain concentration or can I use the after-dialysis liquid form of EPS (which is before freeze-drying) as a sample for checking the protein and carbohydrate content?
2. And also regarding checking the protein and carbohydrate content, is there any reference number of what consider as 'pure' EPS?
Thank you.
Dear Monika,
You should use the freeze dried sample of EPS. For example: you dissolve 10 mg freeze dried EPS in 10 mL of double distilled water (i.e., 1 mg/mL solution of EPS). Now, in this solution, estimate what is the amount of protein and/or carbohydrate. If your EPS (carbohdrate) is 100% pure then it will have only sugar moieties and would not have any protein content. The presence of any protein content in the EPS is impurity. For spectral studies such as NMR etc for its structure elucidation, it is necessary to have >99% purity of your sample.
Dear Dr.Monyika, Try to use pure ethanol or chloroform: butanol for precipitation of EPS from the broth. Then collect the precipitate and dilute in 2D water. Once again precipitate the EPS from the 2D water.
Check my Paper: Isolation and characterization of exopolysaccharide from Leuconostoc lactis KC117496 isolated from idli batter
What if, if I dont conduct dialysis step for my EPS? I do not have dialysis bag for now.
I wanna conduct NMR analysis, do you think there will be any drawbacks?
Thanks
Best
Dear Miss Monika, U can try chloroform-butanol method or ethanol method and i preferred to go with ethanol. For the purity you can try DEAE-cellulose chromatography with the dialysed EPS and check the outcome with UV SPECTROPHOTOMETRY. All the best.
Check My Paper.
dear Monika Nanda , i have the same question with you and im curious how you finally determine the percentage of EPS purity ? what the method that you actually follow ? thanks
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