PRACTICAL FILE

Subject: Advanced Food Science

Subject code: MFT 501A

Submitted by

Rama Paudel

Roll no 7920013

Central Department of Food Technology

Central Campus of Technology

Tribhuvan University

Dharan-14, Sunsari
S.N. Name of practical Date Page Remarks
No.
1. Isolation of casein from milk by 2080-01-13 1-4
isoelectric precipitation by acidic
solution

2. Determination of chlorophyll of 2080-01-28 5-8

green chilly

3. Extraction of lycopene and its 2080-01-29 9-12

determination by
spectrophotometric method
4. Identification of unknown given 2080-03-08 13-21
samples of carbohydrates

5. Separation of the 2080-01-18 22-26

Peptides/Proteins in the Water-
Soluble Extract of the Cheese
Samples using Tricine-SDS-
PAGE

6. Isolation of Lactic acid bacteria 2080-02-10 27-29

from yoghurt

7. Detection of coliform in water by 2080-02-11 30-31

membrane filtration method

8. Staining of Gram positive or 2080-02-12 32-33

Grams negative Bacteria
 Experiment No 1

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 Electric PH meter

Heating arrangement Whattmann filter no 1

Steel vessel Muslin cloth

Citric vessel Hot air oven

95% alcohol Weighing balance

1
Procedure

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

 Cooled to at about 30˚c.
 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).
 Coagulated protein was filtered with the help of a vacuum filter through muslin cloth.
 Washed until acid was washed out.
 Moisture content of the obtained cake was determined.
 Percentage fat content of the obtained cake was determined.
 Final casein content was obtained subtracting cake weight with moisture content and
percentage fat content.
 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

2
 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%

3
Results and discussion

The % casein content of given sample of milk was found to be 3.72%. 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, and 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.

4
 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

5
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

6
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

7
 = 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 chilies 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.

8
 Experiment No 3

Spectrophotometric determination of lycopene content in tomatoes

Introduction
Carotenoids, such as beta-carotene and lycopene, are important components of antioxidant
defense against lipid peroxidation in living cells. Lycopene, an aliphatic hydrocarbon, has
received particular attention as a result of studies indicating that it has highly efficient
antioxidant and free radical scavenging capacity. This is the main reason that the development
of tomato varieties with increased lycopene content requires efficient selection and the ability to
measure lycopene in thousands of samples.

Lycopene belongs to the family of carotenoids. It has a structure that consists of a long

chain of conjugated double bonds, with two open end rings. The structure lycopene is the longest
of all carotenoids. Lycopene ([C40H56], molecular weight 536.85) is an unsaturated hydrocarbon
carotenoid containing 13 carbon-carbon double bonds, 11 of which are conjugated and arranged
in a linear array. These conjugated double bonds are responsible for the vibrant red color of
lycopene.
Lycopene is a lipophilic compound that is insoluble in water, but soluble in organic
solvents, and it has a quenching constant double that of beta-carotene and 10 times alpha
tocopherol (Hadley and others 2003). The quenching ability is directly related to the position of
excited state energy levels, which depend on the length of the conjugated carbon double bone
chain (Young and Lowe 2001)

Objective

9
The objective of this study was rapid accurate and reliable determination of lycopene in tomato
using spectrophotometer.

Principle

Lycopene is extracted in 80% acetone and the absorption at 503 nm are read in a
spectrophotometer. Using the absorption coefficients, the amount of lycopene is calculated using
the empirical formula:

3.1206∗OD of sample∗volume makeup∗dilution∗100

Mg of lycopene /100 gm sample =
1∗wt of sample∗1000

Requirements

80% acetone Petroleum ether

Separating funnel Spectrophotometer

Beaker Sodium sulphate

Procedure

i. Two tomato samples one well ripened and another medium ripened was taken.
ii. Grinding of tomato sample without water.
iii. 5 gm of sample including seed was taken.
iv. It was transferred to 100 ml beaker.
v. 100 ml of acetone was added.
vi. It was stirred with glass rod for 5 min.
vii. Extract was taken and transferred to separating funnel.
viii. Repeat the process 5,6,7 till the residue becomes white.
ix. 10-15 ml of petroleum ether was added in separating funnel.
x. Small amount of water (4-5 ml) containing 5% sodium sulphate was added. Phase
separation occurs;
xi. Lower phase was transferred to another separating funnel.
10
 xii. Upper phase (petroleum ether extract) containing carotenoids (lycopene) was transferred
in a brown ambered bottle.
xiii. Repeat the steps 9,10,11,12 until the acetone is colorless.
xiv. Transfer the petroleum ether extract in 50 ml volumetric flask and make the volume by
50 ml petroleum ether.
xv. Pinch amount of anhydrous sodium sulphate was added.
xvi. Take 5 ml petroleum ether extract into another clean dry 50 ml volumetric flask.
xvii. Volume was made up by adding petroleum ether.
xviii. Color was measured in 1 cm cubeet at 503 nm in a spectrometer using petroleum ether as
a blank.

Observation and Calculations

For sample A (Fully-ripened tomato sample)

O.D. of the sample = 0.158

mg lycopene/100g = (3.1206 × O.D. of the sample × volume make up of sample ×dilution ×

100) / (weight of sample × 1000)

= (3.1206 × 0.107 × 50 ×50 ×100)/ (10×1000)

=8.348

For sample B (matured tomato sample)

O.D. of the sample = 0.117

mg lycopene/100g = (3.1206 × O.D. of the sample × volume make up of sample × dilution ×

100) / (weight of sample × 1000)

= (3.1206 × 0.098 × 50 ×50 ×100)/ (10×1000)

=7.645

Results and discussion

11
For analysis of lycopene we used a fast and simple spectrophotometric method. The
lycopene concentration was expressed as mg/100g product. The results show that lycopene
content of samples of fresh tomatoes fully ripened was approximately 8.34mg/100g whereas
for matured tomatoes sample it was found to be 7.645mg/100g. Depending on several factors
related to the cultural practices, variety and maturity (Odriozola-Serrano et al. 2008)
lycopene content varies from one sample to another. Lycopene content of fresh tomatoes
ranged from 1.21 to 13.43 mg per 100 g, the average content of tomato pastes was 38.88
mg/100g, of ketchups – 11.12 mg per 100 g, and of tomato juices – 7.05 mg/100 g.
Lycopene intake was estimated at 1.93mg per person per day.

12
 Experiment No 4

Identification of unknown given samples of carbohydrates

Introduction
From chemical point of view carbohydrates are polyhydroxy aldehydes or polyhydroxy ketones
(or compounds thereof), or polymers that can liberate these compounds upon hydrolysis. With
some exceptions, carbohydrates have the general formula: C nH2nOn, where n refers to any
positive integer. Carbohydrates can also be represented by (CH 2O)n where n ≥ 3. This formula
indicates that the term ‘carbohydrate’ is derived from ‘carbon-hydrate’. Carbohydrates are a
major source of energy for man and many animals. In man’s diet, the chief carbohydrate is the
starch; it also constitutes the bulk of what he eats. For a child, lactose is of course the major
carbohydrate.

Classification of carbohydrates

Carbohydrate may be classified into three broad groups, namely:

1. Monosaccharide: glucose, fructose, galactose, ribose, xylose, arabinose, ribulose, etc

2. Oligosaccharide: sucrose, maltose, cellobiose, melibiose, lactose, raffinose, etc

3. Polysaccharide: starch, glycogen, dextrin, chitin, hyaluronic acid, pectin, etc

Monosaccharides

Monosaccharides are the simplest units of carbohydrate and cannot be hydrolyzed further into
smaller units under reasonably mild conditions. They are often called simple sugars or neutral
sugars. The term sugar in food chemistry implies carbohydrate that is soluble in water and sweet
in taste. In fact, not all monosaccharides are neutral, nor are they only made up of carbon,

13
hydrogen and oxygen. There is indeed a wide range of monosaccharides, for instance, osamines,
uronic acids, sialic acid (= neuraminic acid), etc. Neutral monosaccharides can be divided into
trioses, tetroses, pentoses, hexoses, etc., depending on the number of carbons present; and aldose
or ketose depending on whether the functional group is an aldehyde or a ketone.

Based on configuration, monosaccharides can be further classified into two families (i) L-family
and (ii) D-family, taking the configuration of glyceraldehyde as standard. The sugars having the
same configuration as of D-glyceraldehyde at the asymmetric carbon most distant (= penultimate
carbon) from the carbonyl group are designated as D-sugars. Those with opposite configuration
(that of L-glyceraldehyde) are called L-sugars. The term configuration as used here refers to
chirality or the disposition of substituents in an asymmetric carbon. Configuration may be altered
only by severance of covalent bond. Another term of importance in carbohydrate chemistry is
conformation, which is defined as those changes in ring shape which are possible when rotating
substituents about a bond linking two carbon atoms. The two common ring sizes of the sugars
are the six membered pyranose ring and the five-membered furanose ring.

Oligosaccharides

Oligosaccharides are carbohydrate polymers of 2-10 sugar units. The oligosaccharides

containing two monosaccharide units are called disaccharides, and those containing three units,
trisaccharide. The oligosaccharides most frequently encountered in nature are disaccharides, e.g.,
sucrose, lactose, maltose, and melibiose. The trisaccharide of importance is raffinose.
Disaccharides yield two moles of monosaccharides on hydrolysis (by chemical or enzymatic
means). Some examples of oligosaccharides and their hydrolytic products are as follows:

Sucrose  glucose + fructose

Maltose  glucose + glucose

Lactose  galactose + glucose

Melibiose  galactose + glucose

Raffinose  galactose + glucose + fructose

14
Polysaccharides

Polysaccharides, as the term indicates, are polymers of monosaccharides. Polysaccharides can be

classified into two broad groups, viz., (i) homopolysaccharides, and (ii) heteropolysaccharides.

(i) Homopolysaccharides: They have the same monosaccharide as the repeating unit in their
molecule. Starch, cellulose, and dextran are polysaccharides of glucose. Inulin is a polymer of
fructose.

(ii) Heteropolysaccharides: They have widely varying numbers of monosaccharides or their

derivatives in their molecule. Pectin, alginic acid, and xanthan gum are some
heteropolysaccharides of importance.

Objective

To identify unknown sample of given carbohydrate using different tests.

Principle

15
 Fig. scheme for identification of an unknown carbohydrates.

A. Molisch's Test

This is a group test for carbohydrates, and is given by all carbo- hydrates whether free or bound
to such substances as proteins (glyco- or muco-proteins) and lipids (glycolipids).

Reagents

(i) Molisch's reagent: This is prepared by dissolving 1 gm of a-naphthol in 95% ethyl alcohol
and making the volume up to 100 ml.
(ii) Concentrated sulphuric acid.

Procedure

16
Take 3 ml of carbohydrate solution in a test tube. Add 1-2 drops of Molisch's reagent, mix,
incline the test tube and run 3 ml of conc. sulphuric acid along the wall of the test tube. Make the
test tube upright and rotate it between your palms.

Appearance of a reddish-violet ring at the junction of the two liquids indicates the presence
of a carbohydrate.

Principle

The polysaccharides and disaccharides are hydrolysed by concentrated sulphuric acid into mono-
saccharides. The monosaccharides are dehydrated by the acid to form furfural or one of its
derivatives.

B. Iodine Test:

This test is only given by starch. Starch reacts with iodine solution forms complex blue colour
solution. On heating the blue colour disappears and on cooling the blue colour reappears.

The chemical reaction is given below.

I3- + starch ------------------- I2 + I-

Procedure

1. Take the sample solution to be tested in a clean test tube.

2. Add 2-3 drops of iodine solution.

3. Observe the change in color.

4. If there is the appearance of a blue color then the presence of starch is confirmed.

C. Benedict's Test

This is also a test for the reducing carbohydrates.

Reagent

17
Benedict's reagent: This consists of the following

Copper sulphate, crystalline:17.3 gm

Sodium citrate: 173.0 gm

Sodium carbonate, anhydrous: 100.0 gm

Water to make:1,000 ml

Sodium citrate and sodium carbonate are dissolved in about 750 ml of water by heating. Copper
sulphate is separately dissolved in about 100 ml of water, and is then added to the solution of
sodium citrate and sodium carbonate with continuous stirring. Finally, the volume is made up to
1,000 ml with water.

Procedure

Take 5 ml of Benedict's reagent in a test tube. Add 8 drops of the carbohydrate solution, mix,
boil for 2 minutes. The appearance of a green, yellow, orange or red precipitate indicates that the
carbohydrate is a reducing one.

Principle

The principle is similar to that of Fehling's test. Copper sulphate hydrolyses to form cupric
hydroxide which is reduced to cuprous oxide on heating with a reducing carbohydrate. In
Benedict's test, alkaline medium is provided by sodium carbonate, and the precipitation of cupric
hydroxide is checked by sodium citrate.

D. Barfoed's Test

This is a test for monosaccharides.

Reagent

Barfoed's reagent: This consists of:

Copper acetate, crystalline 13.3 gm
Water to make 200 ml
Glacial acetic acid 1.8 ml

18
Procedure

Take 3 ml of Barfoed's reagent and 1 ml of carbohydrate solution in a test tube. Boil for 30
seconds. Allow to cool.

A red precipitate at the bottom of the test tube indicates that the carbohydrate under test is a
monosaccharide.

Principle

This test differs from the previous two tests in that the reduction of cupric ions is carried out in a
mildly acidic medium. Since acidic medium is unfavourable for reduction, only the strongly
reducing carbohydrates, ie. monosaccharides," give a positive test.

E. Seliwanoff's Test

This is a test for ketohexoses, eg. fructose.

Reagent:

Seliwanoff's reagent: This consists of:

Resorcinol: 0.05 gm

Conc. hydrochloric acid: 33 ml

Water to make: 100 ml

The final concentration of hydrochloric acid is about 12% as the strength of conc. hydrochloric
acid is 36%.

Procedure

Take 3 ml of Seliwanoff's reagent and 1 ml of the carbohydrate solution in a test tube. Boil for
30 seconds. The appearance of a red or pink colour indicates the presence of a ketohexose.

Principle

19
 The carbohydrates are converted into furfural derivatives by conc. hydrochloric acid. The
furfural derivatives of ketohexoses condense with resorcinol to form a red complex.

F. Osazone Test

This is a test for reducing carbohydrates. Reducing disaccharides and many monosaccharides can
be identified by this test.

Reagents

(i) Osazone mixture: This is prepared by mixing thoroughly one part of phenylhydrazine
hydrochloride and two parts of sodium acetate by weight.
(ii) Glacial acetic acid. Procedure: It is convenient to carry out this test simultaneously.

On all the reducing carbohydrates detected by the earlier tests. Take 5 ml each of glucose,
fructose, maltose and lactose solutions in correspondingly labelled test tubes. Add about 0.3 gm
of osazone mixture and 5 drops of glacial acetic acid into each test tube. Warm gently to dissolve
the solids. Keep all the test tubes in a boiling water-bath. Yellow crystals will appear in the tubes
containing glucose and fructose within 5 minutes. Remove these tubes from the water-bath and
allow them to cool spontaneously. Remove tubes containing maltose and lactose after 45 minutes
and let them cool. With the help of a glass rod, take out some crystals on a glass slide, cover
them with a cover-slip and observe under a microscope.

Glucose and fructose form needle-shaped osazone crystals lying singly or in sheaves.
Maltosazone crystals are sunflower- shaped, and lactosazone crystals are puff-shaped.

Principle

When the reducing carbohydrates are treated with phenylhydrazine at 100°C and pH 4.3, a series
of reaction take place resulting in the formation of osazones of the respective carbohydrates.

Procedure

i. Four unknown sample were taken.

ii. Molisch’s test was conducted to identify whether the given sample was carbohydrate or not a
carbohydrate.

20
iii. Iodine test was conducted to determine polysaccharide or mono or disaccharides.
iv. Benedict test was performed to identify reducing or non-reducing sugar.
v. Barfoed’s test was performed to separate monosaccharide and reducing disaccharide.
vi. Finally; seliwanoff’s test was conducted for monosaccharides and Osazone test for reducing
disaccharides.
vii. Given samples were identified and tabulated.

Observations

Tests samples A B C D

Molisch’s Test +ve +ve +ve +ve

Iodine Test -ve -ve +ve -ve
Benedict’s Test +ve +ve +ve
Barford Test +ve -ve +ve
Seliwanoff Test -ve +ve
Osazone Test Sunflower
shaped crystal

Conclusions

From the above observations it was concluded that all the samples show positive Molisch’s Test
giving purple ring which supports that all the given samples are carbohydrates. Iodine test was
conducted to all the sample. Sample C shows positive iodine test which confirms that sample C
was starch. Benedict test was conducted then to determine reducing and non-reducing sugar. All
the samples show positive Benedict test which confirms that all were reducing sugars. Barford
test was conducted to separate monosaccharides and disaccharide s reducing sugars. Sample A
and D shows positive Barford test which confirm that sample A and sample D were
monosaccharide whereas sample B shows negative Barford test which confirms sample B was
reducing disaccharide. For sample A and D Seliwanoff’s test was conducted to distinguish
whether the given sample was fructose or glucose. From Seliwanoff’s test sample A shows
negative Seliwanoff’s test which confirm that sample A was Glucose while sample D gives

21
positive Seliwanoff’s test which confirm that sample D was Fructose. For sample B Osazone test
was conducted which gives sunflower shaped crystals which confirm sample B was Maltose.

Hence, it was concluded that sample A was Glucose, sample B was Maltose, sample C was
starch and sample D was Fructose.

Precautions

1. Handle the acids like concentrated sulfuric acid with care.

2. Always use droppers to take reagents from the reagent bottles.

3. While heating the reaction mixture do it carefully.

Experiment No 5

Separation of the Peptides/Proteins in the Water-Soluble Extract of the

Cheese Samples using Tricine-SDS-PAGE

Tricine-Sodium Dodecyl Sulphate-Poly Acrylamide Gel Electrophoresis (Tricine-SDS-PAGE)

procedure of Schagger (2006) with some modification was used to separate the peptides/proteins

22
of water-soluble extracts. All the chemical of analytical grade were used. HPLC grade water was
used for all solution described below.

Acrylamide solution

Acrylamide: 48.0 g

N,N'-Methylene-bis-acrylamide: 1.5 g

Dissolved in water to a final volume of 100 ml.

Gel Buffer

Tris: 36.34g (3M)

Sodium Dodecyl Sulfate (SDS): 0.30 g

Dissolved in 60 ml water, pH adjusted to 8.45 with concentrated HCI and made final volume to
100 ml with water.

A. SDS stock Solution (20%)

SDS: 10.0 g

Dissolved in 50 ml water. Warmed gently for complete dissolution.

B. Tris-HCI, 1M, pH 6.8

C. Tris: 12.1g

Dissolved in 80 ml water and adjusted pH to 6.8 with concentrated HCI and made the volume to
100ml with water.

C. Sample Buffer:

Prepared by combining;

20% SDS : 4.0 ml

Glycerol : 2.4 ml

23
2-Mercaptoethanol : 0,4 ml

Brilliant blue G (Carmoissine): 2.0 mg (0.002g)

TrisHCl, 1M, pH 6.8 : 1.0 ml

Ammonium Per sulfate (APS) Solution: (10%)

Dissolved 100 mg Ammonium Persulfate in 1.0 ml water

D. Anode Buffer:

Tris: 121.1g

Dissolved in 1.0 litre of water. Adjusted pH to 8.9 with concentrated HCI and final volume made
to 5 litre with water.

E. Cathode Buffer:

Prepared by combining:

Tris: 12.11g

Tricine: 17.92 g

SDS: 1.0g

Dissolved in 1 litre water. The pH of the solution should be approximately 8.2

F. Fixative Solution

Prepared solution by combining:

Methanol: 50 ml

Glacial acetic acid: 10 ml

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Made final volume up to 100 ml.

Staining Solution

1. Separeting gel (15%)

Acrylamide solution: 9ml

Gel Buffer: 10ml

Glycerol: 3.2 ml

water 7.8 we

10% Aps → 200 μL

TEMED: 15 μL

2. Stacking gel (7%)

Acrylamide solution: 1 ml

Gel Boffer: 3 ml

Water: 8.4 ml

10% Aps →300 μL

TEMED: 20 μL

Sample preparation

Protein sample (water soluble extracts of cheddar cheese) 15 µl after passing through 0.2 pm
filter paper, were mixed with 45 µl of sample buffer and heated at boiling water for 5 minutes.
Molecular weight marker (45 KDa to 3.5 KDa) was also prepared in the same manner.

Electrophoresis

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Anode buffer was filled in the bottom of electrophoretic unit. Then the plate was inserted into the
unit. Well, were rinsed with cathode buffer. Then protein samples and Molecular weight marker
were loaded into the well. The cathode buffer was then carefully layered over the applied
samples. Electrophoresis was then carried out maintaining initial voltage of 30 V. When the
samples entered into spacer gel, the voltage is increased to 90 V till it approaches to stacking gel
and then voltage was increased to 270 V. The current was maintained constantly 80 mA
throughout the run and was applied till marker dye reach 1 cm of the anodic end of the gel was
removed carefully and kept deep in a fixative solution for 60 minutes and stained in Coomassie
staining for 2 hours. It was removed after staining and kept into distaining solution for 2 hours
renewing the solution every 30 min.

Result and discussion

Whey contains seven different types of proteins with different molecular weight viz; β-
lactoglobulin (18 kDa), α-lactalbumin (14 kDa), Glycomacropeptide (8.6 kDa), Bovine serum
albumin (66 kDa), Immunoglobulin (150kDa), Lactoferrin (77kDa) and lactoperoxidase
(78kDa). In the above performed experiment we observed six different bands of the whey protein
they are β-lactoglobulin, α-lactalbumin, Glycomacropeptide, Bovine serum albumin,
Immunoglobulin and Lactoferrin. The different bands are formed due to their different molecular
weight.

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27
 Experiment No 6

Isolation of Lactic acid bacteria from yoghurt

Objective

 To identify lactic acid bacteria in yoghurt.

Theory

Lactic acid bacteria are a wide group of grams positive non spore forming, catalase negative, and
aerotolerant bacteria that includes a large number of genera (rod and cocci). LAB are
microorganisms which cause fermentation of foodstuffs. They are classified based on their
morphology, mode of glucose fermentation, growth at various temperature, and tolerance to salt.
In addition, fatty acid composition and motility are used for identification of LAB. The
fermentation of fermented foods is commonly brought on by naturally occurring, wide varieties
of LAB that are derived from the raw material, the processing equipment or the environment and
that start the fermentation process in the absence of a commercial starter.

Identification of lactic acid bacteria

Identification of each colony which is considered as LAB can be conducted by conventional

method based on morphological, biochemical and physiological characteristics of isolate. The
morphological test based on the formation of colony, size, color, edge form and texture of
colony, then the selected colony can be stained gram to find out whether gram positive or gram
negative and continued by looking the formation of cell using 100-magnified microscope. Some
of test that can be used to identify LAB are Catalase test, Growth test on different concentration
of salt, Motility test, Morphological observation of LAB Cells.

Requirements

 Sample
 Dilution blank
 Nutrient agar media/plate count agar media
 Sterile Petri dishes/pipette
 Test tube rack

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Procedure

 Six test tube was taken and labeled as A, B, C, D, E, F

 9ml distilled water was poured to these test tube.
 1ml of stock solution was poured to test tube A and mixed well.
 1ml of solution from test tube A was poured to test tube B and mixed well.
 1ml of solution from test tube B was poured to test tube C and mixed well.
 Process was repeated up to 10-6 dilution.
 Molten agar (Mann Rogosa Sharp Gar) was prepared in sterilized petri-plate and was
autoclaved and cooled to room temperature around 50℃.
 From the consecutive dilution, (10-2, 10-4, 10-6) 1ml sample was transferred to molten agar
plate and spreaded above the agar medium.
 Plated were incubated at 37℃ for 24-48hr, after required incubation period, colonies were
observed.
 Colonies were observed using colony counter meter.
 Colonies observed were taken from loop and transferred to sterilized test tube containing
3% H2O2.

Observation and Calculation

No of colony ×dilution factor

Colony forming unit=
Volume of culture plate

Cfu/gm in 10-2 plate = 220×10-2

Cfu/gm in 10-4 plate = 210×10-4

Cfu/gm in 10-6 plate = 200×10-6

Catalase test

No bubble formation takes place when treated with 3% H2O2 which indicates presence of lactic
acid bacteria in yoghurt.

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Results and discussion

The observed colony of consecutive dilution 10-2, 10-4, 10-6 were found to be 220× 10-2, 210×10-4
and 200×10-6. As bubble doesn’t appear on doing catalase test presence of lactic acid bacteria
was confirmed.

Experiment No 7

Detection of coliform in water by membrane filtration method

Objectives

 To become familiar with and use membrane filtration apparatus.

 To prepare M- Endo agar, a selective-cum-differential medium for coliforms.
 To carry out counting of typical coliform colonies and report the coliforms load in water.
 To carry out the calculation of microbial load in the sample.

Background

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Although water is indispensable for life, the same water can be cause of illness due to
contamination by pathogens like Salmonella, Shigella, hepatitis virus, Cholera etc. The most
common contamination is however due to fecal materials, which potentially harbors pathogens
like Salmonella (Zero tolerance organism) that causes food borne illness called salmonellosis),
Shigella causes dysentery. However, the determination of these pathogens is not very practicable
for routine work because their detection and enumeration are not simple and straightforward.
Instead, a group of microorganisms called coliforms is used for routine analysis of food and
water as a presumptive test for the presence of above pathogens.

Principle

Coliform determination in water by membrane filtration is based on the principle of filtering

water sample through a hydrophobic membrane filter with a specific pore size (0.45µ), which
allows for the retention of bacteria while allowing water to pass through. The retained bacteria
on the filter can then be cultured in a selective cum differential medium such as m-Endo agar,
which is specific for coliform bacteria.

Requirements

 Water sample
 Membrane filtration unit, Buchner flask
 Incubator
 Membrane filter (47mm dia, 0.45µ pore size)
 Sterile tweezer
 M-Endo agar, 10ml
 Petri-plates

Procedure

1. Medium of M-endo agar was prepared by dissolving 1.04gm M-endo agar in 20ml water.
2. The different part of the membrane filtration unit was sterilized in an autoclave at 15psi
for 15 min.
3. The sterilized membrane filtration unit was aseptically assembled.
4. A pair of tweezers was disinfected by dipping it in 70% alcohol.

31
 5. Sterilized Millipore filter membrane on the filter holder with a pair of sterile tweezers and
the funnel was placed over the filter holder.
6. 100ml of water was aseptically poured and vacuum was applied to filter the water
rapidly.
7. Small amount of m-Endo agar prepared was poured in sterile Petri plate.
8. Clamp from the filtration unit was removed and membrane filtration was aseptically
taken out with the help of sterile pair of tweezers.
9. Membrane was placed on the m -endo agar plate with the help of tweezer.
10. Membrane was pressed gently on m-endo agar so that maximum surface will be fully
contact the medium.
11. Plate was inverted and incubated at 35-37℃ for 22-24hr.

Observation and Calculation

Sample A (TAP water) Sample B (Drinking water) Sample C (Ekdhare Water)

Appearance of coliform No appearance of colony No appearance of colony

No of colony: TNTC No of colony= 0 No of colony = 0

Result and Discussion

We detect coliform in sample A whereas coliform was not found in sample B and C.

Experiment No 8

Staining of Gram positive or Grams negative Bacteria

Objective

 To stain Grams positive or Grams negative Bacteria.

Theory

In microbiology and bacteriology, Gram stain, is a method of staining used to classify bacterial
species into two large groups: gram positive or grams negative bacteria based on their physical

32
properties of their cell walls. Lactic acid bacteria are a wide group of grams positive non spore
forming, catalase negative, and aerotolerant bacteria that includes a large number of genera (rod
and cocci). LAB are microorganisms which cause fermentation of foodstuffs. They are classified
based on their morphology, mode of glucose fermentation, growth at various temperature, and
tolerance to salt. In addition, fatty acid composition and motility are used for identification of
LAB. The fermentation of fermented foods is commonly brought on by naturally occurring, wide
varieties of LAB that are derived from the raw material, the processing equipment or the
environment and that start the fermentation process in the absence of a commercial starter.

Requirements

 Sample
 Glass plate
 Crystal violet
 Iodine solution
 Burner
 95% alcohol
 Saffron solution

Procedure

1. Glass plate was washed with 70% alcohol solution.

2. One loop of colony from Lactic acid bacteria growth medium was taken and spread on a
glass plate.
3. Glass plate was heated above burner.
4. 2 drops of crystal violet was added to heated glass plate.
5. Glass plate was allowed to cool for 1 min.
6. Glass plate was washed with DW.

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 7. Glass plate was dried by using burner
8. 2-3 drops of iodine was poured and leaved for 1 min.
9. Again, it was washed with DW and dried using burner.
10. 2-3 drops of 95% alcohol was poured and washed with DW then dries using burner.
11. Glass plate was washed with saffron solution followed by water then dried using burner.
12. Glass plate containing colony was observed through microscope.

Observation

Purple color colony was observed through the microscope which indicates the presence of lactic
acid bacteria.

Result and Discussion

We observed purple color colony under microscope that indicates the presence of gram-positive
lactic acid bacteria.

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