cholesterol in milk cultures - (Apr/18/2012 )
I hope some advise from you. Cholesterol removal by lactic acid bacteria is well documented, and the cholesterol decomposition ability is widespread among microorganisms.
In some experiments carried out using commercial rehydrated milk I found this ability when bacteria were grown at 30ºC for 48 h (tubes with control milk -not inoculated- have more cholesterol amounts than fermented milk by bacteria ). However when the same microorganisms where grown at 10ºC for 1 week, I fdetected a higher cholesterol content into the bacterial milk cultures than in the control milk not inoculated. I repeat the essays at 10ºC and the cholesterol amounts are higher into the fermented milk. Why?
Can somebody help me to understand this, for me anomalous result?.
What assay are you using?
Total cholesterol in milk samples was determined by direct saponification (using 33% KOH + EtOH, 60ºC -->hexane extraction with water) coupled with HPLC (7:3:3, ACN:MeOH:Iso). The methods were developed using previous standard reports.
Saponficiation is pretty tndard as you say. Have you ibcluded any internal standards?
At the begining of experiments, IS and spiked cholesterol assays were perfromed in control milks (many times in fresh rehydr milks). Recoveries were between 100-110%. Probably not include an IS during experiments at 30/10ºC was a fault¿?. probably was also a fault do not made spiked experiemnts at 10ºc¿?Perhaps it would confirm recoveries in control milk at 10ºC (10d) not as good as in fermented milks at lower temperature 10d. However, this will not explain the results. Why worse recoveries in control milk (10ºC) than in fermented milk (10ºC)?
Try admix of internal cholesterol standard in fermented milk. Think it important to finish validating the method before presuming the difference is real.
ok. thanks I´ll try it
(but I think that is not the problem, perhaps some emulsion problem during saponification?: recoveries worked well during first steps)