*2.4.3. Estimation of fat content*

(1) A mixture of 50 ml of methanol and diethyl ether were taken in 1:1 concentration. (2) From 30 ml of stock solution, 1 ml of test sample was added in the mixture and few drops of phenolphthalein as an indicator. (3) The solution was titrated with 0.1 N NaOH. (4) The change in color was observed from light yellow to a brownish coffee color.

Calculation

Calculation

$$\text{Acid value (mg/L)} = \frac{(Test - Blank)X0.1X254}{Sample used (ml)}$$

#### *2.4.4. Estimation of crude protein*

The biuret test is a chemical test used to detect the presence of peptide bonds. In the presence of peptides (–CO-NH- groups), a copper (II) ion forms violate coordination complexes in an alkaline solution. Copper (II) reduces to copper (I). The intensity of the color complex is measured by colorimetrically at 520 nm.

**Reagents:** (1) Biuret reagent: Dissolve 3 g of CuSO4.5H2O and 9 g of sodium potassium tartrate in 500 ml of 0.2 N NaOH solutions. To this solution, add 5 g of potassium iodide and make about 1 L with 0.2 N NaOH. (2) Standard protein solution: 5 mg of bovine serum albumin/ml water. Prepare fresh.

**Method:** (1) From 30 ml of stock solution, take 1 ml of sample and make volume of up to 4 ml with distilled water. (2) Add 6 ml of biuret reagent to it and mix well. (3) Heat the mixture at 37°C for 10 min. (4) Cool the tubes and read the absorbance at 520 nm against a reagent blank. (Prepare similarly with 4 ml of distilled water). (5) Draw the standard graph by pipetting out 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 ml, of standard protein solution into a series of test tubes and make volume of up to 4 ml with distilled water in each. Carry out steps 2 to 4. (6) Calculate the protein content in the sample using a standard graph.
