I think your guess of salting out is right. Conc. of PC might have crossed CMC. Another reason may be improper sonication parameters (10 mins is very short period unless you are using high intensity sonication).  Stability of micelle can be confirmed by DLS study or by quantification of leakage during storage period.

Hi Aadhinath,

Thankyou for those helpful hints.

What do you mean by leakage during storage period? 

For stability study, encapsulate some dye into the micelle and study how much of dye is leaking out of micelle during storage period. Usually this is carried for studying the stability of liposomes. 

To see the difference between liposome and micelle, you can use cross polarizing filters. Between them, liposome solution will give a streaming birefringent texture, while, micelle solution will give isotropic.    

for the precipitate I would suspect Ca Taurodeoxycholate or something associated with it.

Sonication likely induces vesicle formation associated with very slow kinetics of restructuring. For reproducible results you therefore will need strict control of intensity of sonication (power, duration, volume treated) and of time regime. Long aging times might be necessary to get consistent results.

Thanks everyone for your ideas! Titus, I agree with you, as sonicating one volume over another for the same periods of time contribute to the varying results. When you say aging times, do you mean sonication time? or time after sonication before carrying out any assay?

aging after sonication (means after being prepared)

Ok, thanks again :)

Hi again everyone

I understand that bile salts and phospholipids are detergents and emulsifiers and, as such, aggregate with each other and form micelles in water. And micelles can form also with mono-acyl glycerols and fatty acids. These are optically clear in solution if detergent is at concentration above its CMCs, Am I right so far?

Can someone clarify for me - can small chain triglycerides form micelles with bile salts/phospholipids? What about medium and long chain triglycerides? Or would they be called "emulsions" rather than micelles? And is it normal for these to be turbid, no matter the concentration of triglyceride?

Many thanks

Elena

Dear Elena,

micelles are able to take up hydrophobic compounds, e.g. small, medium and long chain triglycerides. This is named micellar solubilization. Capacity to do this is limited. In best case a 1/1 molar ratio can be obtained. For the triglycerides maybe only one molecule per micelle. So increasing concentration of triglycerides will end up as emulsion in equilibrium with solubilized triglycerides. So in your case concentration to obtain a clear solution (only solubilized oil) might be very low.

Thanks Titus, that is very helpful.

Hi Titus,

In one of your comments above you mention the possibility of Ca-bile salt salting out. What about phospholipids and Ca2+? Can they form insoluble aggregates?

The literature gives me information about lower CMC and different aggregate formations due to increased ionic strength, but nothing about salting out. I have found little information on the conditions needed for Ca2+ binding of phospholipids.

I am also unsure if any sedimentation is favorable or hindering to lipolytic enzyme activity.

Any ideas?

Thanks for all expert advice.

I am not an expert on this. Maybe this article on calcium ions binding to phospholipids is helpful.

http://www.jlr.org/content/33/7/985.full.pdf

Thanks Titus :)