Experiment no: 1

Name of the experiment: Isolation of casein from milk by isoelectric precipitation by acidic
solution

Objective:

 To perform the isoelectric precipitation of casein present in milk.

 To determine % casein protein by dehydration.

Principle

Milk protein consists of 80% casein and 20% whey protein. There are four major types of casein
molecules: alpha-s1, alpha-s2, beta, and kappa. Casein, like proteins, are made up of many
hundreds of individual amino acids. Each may have a positive or a negative charge, depending
upon the pH of the (milk) system. Milk, in its natural state, is negatively charged. The negative
charge permits the dispersion of casein in the milk. When an acid is added to milk, the H+
concentration neutralizes the negatively charged casein micelles. When milk is acidified to pH
4.7, the isoelectric point (the point at which all charges are neutral) of casein, an isoelectric
precipitate known as acid casein is formed. Cottage cheese and cream cheese manufacture
involves an acid precipitation of casein with lactic acid or lactic acid-producing microorganisms.
During the addition of acid to milk, the negative charges on the outer surface of the micelle are
neutralized (the phosphate groups are protonated), and the neutral protein precipitates.

Requirements

Milk, Heating arrangement, Steel vessel, citric acid, 95 % alcohol, electric pH meter, Whatman
No 1 filter paper, Hot air oven or dryer, Weighing balance

Procedure

1. 500 ml of milk was taken in a beaker and boiled in a steel vessel.

2. Cooled to at about 30˚c.
3. Acidification was done with drop wise addition of citric acid and stirred with glass rod. (If
excess acid is added neutralize with 0.1 N NaCl).
4. Coagulated protein was filtered with the help of a vacuum filter through muslin cloth.
5. Washed until acid was washed out.
6. Moisture content of the obtained cake was determined.
7. Percentage fat content of the obtained cake was determined.
8. Final casein content was obtained subtracting cake weight with moisture content and
percentage fat content.
9. Final casein content (%) was determined.

Observation and calculation:

Weight of sample (milk): 522 gm

Ph of the milk: 6.3

Heating temperature: 80℃

Temperature of milk after cooling: 35℃

Weight of muslin cloth: 13.75 gm

Weight of curd: 47 gm

47
% curd: ∗100 = 9%
522

For % moisture content:

Wt. of sample (initial): 10 gm

Wt. of sample (final): 4.68 gm

initial wt −final wt
% moisture content = ∗100
initial wt

10−4.68
= ∗100
10

=53.2%

Total moisture including pretreatment heating:

Weight of sample (before pretreatment): 23.55 gm

Weight of sample (after pretreatment): 23.19 gm

23.55−23.19
% moisture content= ∗100 = 1.5%
23.55
⸫ total moisture content=53.2%+1.5%= 54.7%

For fat content (%)

weight of sample taken (S): 5.1 gm

weight of dry beaker (W1): 125 gm

weight of beaker + sample (W2): 125.20 gm

W 2−w 1
% fat content= 100 %
s

125.20−125
= ∗100
5.1

=3.92%

For % casein

Fat content in curd: 3.92% of 47= 1.84 gm

Moisture content in curd: 54.7% of 47= 25.7 gm

⸫Casein content in curd=47-1.84-25.7 = 19.46 gm

Also;

19.46
% casein= ∗100 %
522

=3.72%

Results and discussion

The casein % was found to be 3.72%. Isoelectric point (PI) of a protein is the pH at which the
protein carries no net electrical charge, as it exists in its neutral or zwitterionic form. At the pI,
the number of positive and negative charges on the protein molecule is equal, this lack of charge
leads to several important changes in the protein’s behavior, including its solubility and stability,
which ultimately result in coagulation. Proteins coagulate at pI due to minimum repulsion,
Reduced solubility, change in protein structure leading to precipitation. Isoelectric point (PI) of
most proteins is in the PH range of 4-6. At a solution PH that is above the PI the surface of the
protein is predominantly negatively charged and therefore like-charged molecules will exhibit
repulsive forces.
Experiment No. 2 Determination of chlorophyll of green chilly.

Objectives

 To determine chlorophyll a, chlorophyll b and total chlorophyll of given sample (green

chilly).

Background

Chlorophylls are found in virtually all photosynthetic organisms. They occur in several distinct
forms: chlorophylls a and b are the major types found in higher plants and green algae;
chlorophylls c and d are found, often with a, in different algae; chlorophyll e is a rare type found
in some golden algae; and bacterio-chlorophyll occurs in certain bacteria. In green plants
chlorophyll occurs in chloroplasts, approximately in the ratio 3a:1b. The determination of
chlorophylls has great importance in food technology as well as other disciplines. In food
technology, chlorophyll is related to esthetic quality of fruits and vegetables. It is also
extensively used as a parameter in studying maturation and ripening of fruits and vegetables. In
aquatic ecology, chlorophyll determination is used for biomonitoring. Chlorophylls are very
sensitive pigments. They are readily degraded by oxygen and acid. Any activity (e.g., grinding)
that frees the intracellular chlorophyllase enzyme also leads to rapid destruction of chlorophylls.
This warrants great care in sample preparation. The quantitative analysis of chlorophyll is
universally carried out by spectrophotometric method. Depending on the purpose of analysis and
the nature of sample, some variations, particularly with respect to absorbance wavelength and
extraction solvent, are also observed. Different investigators have used wavelengths of 600, 630,
642.5, 645, 663, 660, 665 and 750nm. The solvents commonly used are 80% acetone, acetone-
methanol (90% acetone), or 100% acetone. The centrifugation speed ranges from 2,500 to 5,000
rpm.

Principle

Chlorophyll is extracted in 80% acetone and the absorption at 663 and 645nm are read in a
spectrophotometer. Using the absorption coefficients, the amount of chlorophyll is calculated
using the empirical formula:

Chl a, mg/g tissue = 12.7 (A663)-2.69 (A645) *V/1000*W

Chl b, mg/g tissue = 22.9 (A645)-4.68 (A663) *V/1000*W

Total chlorophyll, mg/g tissue = Chl a + Chl b

Where; A = absorbance at specific wavelengths;

V = final volume of chlorophyll extract;

W = fresh weight of tissue extracted

Requirements

 Green vegetable sample  Spectrophotometer  Centrifuge  Weighing arrangement

 Refrigerator  Measuring cylinder, 100ml  Volumetric flask, 50ml  Beakers  Knife, mortar-
pestle  80% acetone

Procedure

1. Cut the sample into small pieces (or thin slices)

2. Take 1 g of sample and grind it to a fine pulp in mortar and pestle with about 10ml of 80%
acetone

3. Centrifuge the pulp at 5000 rpm for 5 min

4. Transfer the green supernatant to a 50-ml volumetric flask

5. Scrap the sediment in the centrifuge tube and grind it again in the same mortar and pestle with
a small amount of 80% acetone to extract the residual chlorine

6. Centrifuge the mixture as done earlier and pool the extract in the 50-ml volumetric flask
(containing the previous supernatant)

7. Repeat the extraction until no perceptible green color in the residue

8. Add 80% acetone to the pooled extract to make up the volume (i.e;50ml)

9. Stand the extract in refrigerator for 10 min to lower the temperature

10. Read the absorbance of the extracts in spectrophotometer at 663 and 645 nm using 80%
acetone as the blank
Calculation

Weight of sample taken (W) = 1 gm

Blank (80% acetone reading) = 0.043

Absorbance of sample B

At 645 nm At 663 nm
0.082 0.172
0.092 0.223
0.092 0.217
Avg 0.095 Avg 0.204

Absorbance of sample A

Blank (80% acetone reading) = 0.007

At 645 nm At 663 nm
0.091 0.213
0.097 0.211
0.109 0.211
Avg 0.099 Avg 0.211

Chlorophyll for sample A

i. Chl a, mg/g tissue = 12.7 (A663)-2.69 (A645) *V/1000*W

=12.7 (0.211)-2.69 (0.099) *50/1000*1

=2.66 mg/g

ii. Chl b, mg/g tissue = 22.9 (A645)-4.68 (A663) *V/1000*W

= 22.9 (0.099)-4.68 (0.211) *50/1000*1

 =2.218 mg/g

iii. Total chlorophyll, mg/g tissue = chl a +chl b

= 2.66 + 2.218
= 4.87 mg/g

Chlorophyll for sample B

i. Chl a, mg/g tissue = 12.7 (A663)-2.69 (A645) *V/1000*W

=12.7 (0.204)-2.69 (0.095) *50/1000*1

=2.578 mg/g

ii. Chl b, mg/g tissue = 22.9 (A645)-4.68 (A663) *V/1000*W

= 22.9 (0.095)-4.68 (0.204) *50/1000*1

=2.127 mg/g

iii. Total chlorophyll, mg/g tissue = chl a +chl b

= 2.578 + 2.217
= 4.705 mg/g

Result and Discussion

The total chlorophyll content of green chilly for sample A was found to be 4.87 mg/g and
for sample B was found to be 4.705 mg/g using spectrophotometric instruments.
Chlorophyll is green Pigment found in plants and is responsible for their green color.
Chlorophyll content in green chilly can vary depending on several factors, including
specific variety of chilly, its maturity and growing conditions. As chilly mature, they can
change in color from green to yellow, orange and red and this change is associated with a
decrease in chlorophyll content. Therefore, green chillies harvested while still green and
immature have relatively high chlorophyll content. The chlorophyll content can vary
widely among different types of green chilly and individual specimens.