Polysaccharide purificaion from cheese? - (Nov/06/2013 )
I am currently working on cheese with an exopolysaccharide-producing LAB adjunct. Previously in our lab we have used an EPS+ strain in yogurt and had no problems in the isolation & quantification of EPS in the product following the method of Amatayakul et al. (2006). However, the cheese matrix, assumably due to such high fat and protein levels, is proving much harder to work with. Below is the adapted protocol with which I had attemted the isolation. I believe the problem lies with the very first steps and if any of you can give me advice on the matter then that would be greatly appreciated. The only material I have found of relevance is a german paper from 1975 (Polysaccharide additives in foods rich in proteins. i Sample preparation for estimation, especially in fresh cheese, Henning Klostermeyer, Ulrike Popp, Irene Schmitz) which I don't have access to and is problem well out of date.
Isolation of EPS:
- Homogenise 1g of cheese in 45ml sterile dH2O in stomacher on high for 5min.
- Transfer the contents to a conical flask and add TCA to a final concentration of 12% (v/v) & agitate at room temperature for 30min.
- Centrifuge at 3313 x g for 30min at 4ºC to remove precipitated protein and cells.
- Add 1ml of Carrez solution 1. Mix well.
- Add 1ml of Carrez solution 2. Mix well.
If no precipitate appears, the clarification has to be repeated in a second sample using a five-fold volume of Carrez solutions (5 ml each).
- Adjust the pH to 7.5-8 with 0.1M NaOH.
- Transfer to a 100ml graduated cylinder and fill to mark with dH2O.
- Boil samples in sealed containers for 30min to denature whey proteins & centrifuge.
- Precipitate EPS with 2 vol. ethanol, O/N at 4°C with agitation.
- Centrifuge to pellet precipitated crude EPS.
- Resuspend in 10ml sterile dH2O and dialyse (12-14kDa MWCO) against dH2O for 7 days with twice daily water changes.
Quantification by phenol sulphuric acid method.
my first idea would be to remove the fat by a non-polar solvent and the mentioned paper suggests the same: the abstract says that they removed fat by a mix of ethanol, diethyl ether and petroleum ether before they digested the proteins with a mix of several proteases (Pepsin, etc).
(I can send you the papers, but they are except the abstract in german)