3.7.

3 Determination of lipolytic activity

Free fatty acids content was determined by the method of Deeth et al. (1975).

About 3.to 5 ml of inoculated milk was pipetted into a 35 ml test tube. 10 ml

extraction mixture (isopropanol: petroleum ether: 4N H2SO4, 40:10:1) was added
and mixed. Six ml petroleum ether and 4 ml water were added, shaken vigorously for
15 sec. Allow the contents to separate in to two layers and volume of the upper layer
was recorded. Portion of the upper layer was transferred into a 50 ml conical flask
and 2 drops of 1 per cent methanolic α-naphtholphthalein and titrated with 0.002N
methanolic potassium hydroxide solution. Blank determination was made by
replacing the milk with water.

Free fatty acid (µ Eq/ml) = TN/PV x 103

Where,
T - is the net titration volume,
N - is the normality of the methanolic KOH,
P - is the proportion of the upper layer titrated and
V - is the volume of milk
Increase in FFA was used as an index of lipolysis.