Not every polysacchride is precipitating with ethanol, some of them will precipitate also with aceton or different alcohols (as you used isoprop)....I would suggest to do the precipitation not at ambiant temperature, but in a fridge (4°C) or even a freezer (-20°). As the alcohol won't freeze, this often enhances the formation of a precipitate...

I agree with Sascha and would also suggest to store the samples at -20°C. If colouration of the samples is a problem, you might use PVPP as a fining agent in advance, which will remove some of the color - but might also remove some of the polysaccharide :-( .

I would also ask you to keep in mind that must is a much more complex matrix than finished wine. Just think about sugars and proteins, which get fermented and decomposed during fermentation. So do not expect the must samples to go smoothly through your preparation procedure.

Thank you both for your prompt response..

I forgot to mention that when I replaced ethanol with isopropanol I also changed the temperature. I carried out  the precipitation at 4°C.

I also thought to remove anthocyanins with PVPP but I did not do it because I was afraid that it might also remove the polysaccharides.

Of what i understand through the observations made is that my real problem is my sample after i dissolve it in water. I have a huge precipitate and I don't know what is it.

I have to take 40uL of my sample for the  Dubois analysis but if I the solution has some form of precipitate or aggragate, the analysis might not be accurate as the proptocol calls for using a homogenous solution ...

I understand that must is a much more complex matrix than finished wine, but is their a way to ensure that the precipitate in the final solution would not give me innacurate results, also, how do i know if the aggregate/precipitate i have in the solution is or is not a polysaccharide?

Thanking you.

Digest the "polysaccharide" and get it analyzed on its sugar constituents. This will give you an idea, which polysaccharide you have isolated. There is quite some literature out there on polysaccarides in grapes/wine, which you should consult first.

Take a look at Nunan et al. Plant Physiol. (1998) 118: 783–792 for details on grape polysaccharides. There are many subsequent papers as well. Also, for constituent sugar analysis we use high-performance anion  exchange chromatography (see Nep et al. Food Hydrocolloids 53 (2016) 134-140) for an example.

It is quite possible that ice-cold alcohol will cause tartrate crystals to precipitate very common in both musts and wine, in addition to polysaccharides, proteins and polyphenols; which may explain your 'huge' precipitate... in the end it may be better to change your analysis procedure so as not require a 'homogenous' solution at the end... science is challenging indeed...best wishes

Thanks for your answers everybody.

I am using an old but easy and fast method for the total polysaccharide content in wine of Ludwig et Goldberg, 1956, based on  anthrone-sulfuric acid method. I realize the first step of precipitation at 4 degrees and it's better at least to triplicate every sample.

Thank you. I will look for it.