June - summer training report

Department of Microbiology
Submitted by – Dinkar Batta
 Food Biochemistry laboratory Test

• Contents
 Qualitative and quantitative analysis of food samples:-
a) Ash determination & Moisture determination
b)Test for carbohydrates and its quantitative estimation by
different methods.
c)Protein detection and its estimation
d)Fat detection and its estimation
 Hands on experience with some instruments

 High pressure liquid chromatography(shimadzu)

 Kjeldhal appratus
 Soxhlet Appratus
 Moisture analyzer
 PCR
Ash and moisture determination of a bakery sample
ASH
Principle:- Ash refers to the inorganic (oxides, sulphates, phosphates, silicates and
chlorides)residue remaining after either ignition or complete oxidation of organic
matter in a foodstuff.
Incinerating of the sample is done in a muffle furnace at 500±50°C as such a high
temperature burns off all organic materials.

Materials:-

Porcelain Crucible
Muffle furnace
Weighing balance
Tongs
Spatula
Dessicator

Sample preparation:- weigh a cake sample and Dry crucible separately on weighing
balance.
Place a sample in hot air oven at 130 °C for 1hour
For complete removal of moisture from cake.
Procedure:- Weigh a 1-4g moisture free cake sample and this weight is denoted as initial weight
(W).
Transfer the sample to previously weighed crucible which is denoted as (T).
Place the crucible in a muffle furnace at a
temperature of 400°C and incinerated for about 6 hours until a white ash will be obtained.
Take out the sample from hot air oven with the help of gloves and Tongs.
Cool the crucible in desiccators.
Take weight of crucible after cooling and denoted it as Final weight (R)
Measure the ash content by applying formula.

Formula

Ash % = (R-T) X100

(W-T)
T= tare weight of crucible
R= weight of crucible + residue
W= weight of crucible + weight of sample
Moisture Analysis
Principle :- One of the most fundamental and important analytical procedures that
can be performed on a food product is an assay for the amount of moisture.
Knowing and controlling the water content in food is vital to ensure consistently high
standards of products. The water content in food can affect several attributes of food
products including:
•Taste
•Texture
•Appearance
Water content in food can also alter the weight of the product.
Materials Needed :-
•Petri plate
•Sample
•Weighing balance
•Hot air Oven
•Spatula
Procedure:-
1. Wash and complete dry the Petri plates.
2. Weigh the dry plates and samples weight 1-5g.
3. Place a sample of cakes in Petri plates and note the total weight.
4. Set the temperature of hot air oven to 130°C .
5. Place sample in hot air oven for 60 minutes.
6. Weigh the dry sample weight of plate.
7. Calculate moisture percentage by applying formula.
Moisture %
= Initial weight(W1)–final weight(W2)/final weight(W2)X100
 Qualitative Test for carbohydrate
detection in food sample

 1) Molisch’s Test:
It is a sensitive chemical test for all carbohydrates, and some compounds containing
carbohydrates in a combined form, based on the dehydration of the carbohydrate by
sulfuric acid to produce an aldehyde (either furfural or a derivative), which then
condenses with the phenolic structure resulting in a red or purple-colored compound.
Procedure
• Apply this test two different carbohydrate solutions of your own choice,
• preferably to one monosaccharide and one polysaccharide. –
• Place 2 mL of a known carbohydrate solution in a test tube, add 1 drop of
Molisch’s reagent (10% α-naphthol in ethanol). –
• Pour 1-2 mL of conc. H2SO4 down the side of the test tube, so that it forms a
layer at the bottom of the tube. –
• Observe the color at the interface between two layers and compare your result
with a control test.
• A brown color due to charring must be ignored and the test should be repeated
with a more dilute sugar solution.
• Fehling’s Test
Fehling’s Solution (deep blue colored) is used to determine the presence of
reducing sugars and aldehydes. Perform this test with fructose, glucose, maltose
and sucrose.
Procedure
• To 1 mL of Fehling’s solution A (aqueous solution of CuSO4) add 1 mL of Fehling
solution B (solution of potassium tartrate).
• Add 2 mL of the sugar solution, mix well and boil.
• Try to see the red precipitate of cuprous oxide that forms at the end of the
reaction
 Quantitative Estimation of carbohydrates in
food sample

Quantitative estimation of sugar content in bakery products by Anthrone Method.

• Principle:- Carbohydrates are important components and structural materials in plants. They exist
as free sugars and polysaccharides. The basic units of carbohydrates are the monosaccharide
which cannot be split further
• The carbohydrate content is measured by hydrolysing the polysaccharides into simple sugars.
• Complex Sugars are first hydrolyzed into simple sugars using dilute Hydro chloric acid.
• In hot acidic medium glucose is dehydrated to hydroxy methyl Furfural.This further compound
forms with anthrone a green coloured product with an absorption maximum at 620 nm.
•
• Materials :-
• 2.5N HCL
• Anthrone Reagent : dissolve 0.2g of anthrone reagent in 100ml conc sulphuric acid.
• Glucose stock:- dissolve 100mg of glucose in 100ml distilled water
• Working stock :- 10ml of stock glucose diluted in 100ml distelled water.

• Sample prepration :-
• Add a sufficient amount of bakery sample in a beaker put the beaker on hot plate. heat the
sample on low heat and stir it continously so that moisture and oil which is present in sweet can
be easily removed and it became dry.
• Dry sample was grinded in pestle mortar to make it powdery.
Procedure:-
• Weigh 100mg of sample in a boiling tube.
• Hydrolyse the sample by keeping it in a boiling water bath for three hours with 5ml
of 2.5 N HCl and cool to room temperature
• Neutralise it by adding solid sodium carbonate until effervescence ceases.
• Make up to the volume of 100 ml by adding distilled water.
• Transfer the diluted sample in eppendofs tubes
and centrifuge the
sample.
• Collect the supernatant and take 0.5ml and 1ml aliquots.transfer the aliquots for
further analysis.
• Prepare the standards by taking 0,5ppm,10ppm,15ppm,20ppm,25ppm,30ppm of
working standard. 0 serves as blank.
• Make up to the volume of 1ml by adding distilled water in each test tubes.
• Add 4ml of Anthrone reagent.
• Heat the test tubes for 8min in boiling water bath.
• Cool rapidly and Od was noted at 630 nm.
• Plot a standard graph by noting the concentration on the X-axis and absorbance on
the Y-axis.
• From the graph calculate the amount of sugar content present in the sample tube.
• Estimation of total proteins in cake by lowry Method
• Principle:-
• The lowry method combines the biuret reaction with the reduction of the
folin-ciocalteu phenol reagent (phosphomolybdic-phosphotungstic acid) by
tyrosine and tryptophan residues in the proteins. The bluish color developed
is read at 750nm(high sensitivity for low protein concentration)or 500nm(low
sensitivity for high protein concentration).the original procedure has been
modified by Miller and Hartree to improve the linearity of the colour response
to protein concentration and replace the use of two unstable reagents with
one stable reagent.

• Materials :-
– 2% Sodiumcarbonate in 0.1 N Sodium Hydroxide in 100ml distilled water.
– 0.5% Copper suphate pentahydrate in 1% potassium tartarate in 100ml distilled
water.
– Alkaline copper solution = 50ml ofa + 1ml of B
– Folin reagent- 1part folin:water
• Standard prepration :-
• Bovine albumin serum 50mg in 50ml distilled water.
Sample prepration :- Weigh a sample and grind with pestle mortar in 5-10ml of phosphate
buffer. centrifuge it and use the supernatant for protein determination.
Procedure :-
Pipette out
10ppm,15ppm,20ppm,25ppm,30ppm,35ppm of the working standard in a series of test tubes.
Pipette out 0.1ml and 0.2ml samples in a test tubes.
Make upto the volume of 1ml in each test tubes by adding distilled water.
A tube with 1ml of distilled water serve as the blank.
Add 5ml of alkaline reagent in each test tube including blank.
Mix well with the help of cyclomixer and allow to stand for a 10min.
Then add 0.5ml of folin phenol reagent.
Mix well and incubate at room temprature in dark for 30min. Blue colour is developed.
Take readings at 660nm.
Draw the standard graph from readings and calculate the proteins amount in a sample.
Fat determination in different flavored fruit cakes by soxhlet method
Theory :-
The method described by Soxhlet in 1879 is the most commonly used example of a semi-continuous method
applied to extraction of lipids from foods.
According to the Soxhlet’s procedure, oil and fat from solid material are extracted by repeated washing
(percolation) with an organic solvent, usually hexane or petroleum ether, under reflux in a special glassware.
In this method the sample is dried, ground into small particles and placed in a porous cellulose thimble. The
thimble is placed in an extraction chamber, which is suspended above a flask containing the solvent and
below a condenser. The flask is heated and the solvent evaporates and moves up into the condenser where it
is converted into a liquid that trickles into the extraction chamber containing the sample.
when the solvent surrounding the sample exceeds a certain level it overflows and trickles back down into the
boiling flask. At the end of the extraction process, which lasts a few hours, the flask containing the solvent
and lipid is removed.
the solvent in the flask is then evaporated and the mass of the remaining lipid is measured. The percentage
of lipid in the initial sample can then be calculated.

Materials:-
Weighing balance
Hot air oven
Dessicator
Thimble
Beakers
Soxhlet apparatus
Sample preparation:-
• Cake samples was taken in a petri-plates and placed it into a hot air oven at 130 °C for 30min.
Moisture was removed and each sample weighed 5g in a beaker

Procedure:-
1. Rinse all the beakers and place them in oven with the temperature about 100°C
2. If all moisture were removed from beakers , place them in dessicators about 5 to 10 minutes
to bring them into room temperature.
3. Now weigh the empty beaker and the let the weight be w 1. This is initial beaker weight (IBW).
4. Now insert the thimble in the thimble holder and place it on the beaker.
5. Weigh the samples of cake.
 Instrumentation
 High pressure liquid chromatography(shimadzu)
 Kjeldhal appratus
 Soxhlet Appratus
 Moisture analyzer
 PCR
 • High-performance liquid
High pressure liquid
chromatography chromatography or
commonly known as
HPLC, is an analytical
technique used to separate,
identify or quantify each
component in a mixture.
• The mixture is separated
using the basic principle of
column chromatography an
d then identified and
quantified by spectroscopy.
 Standard operating procedure

• Read the SDS for all materials.

• Prepare the analyte solution in a fume hood.
• Make sure the solvent reservoirs are filled before using the instrument.
• Make sure that you are using the correct mobile phase.
• a. If you are changing the mobile phase, completely wash all lines and columns
before use.
• Ensure that the pump lines have been purged of air bubbles and check the system
for leaks before beginning the analysis.
• Turn on the HPLC and equilibrate the mobile phase solution.
• Make sure that the pressure is behaving normally and is well below the maximum
pressure for the HPLC system.
• Constantly monitor the solvent levels in the solvent mobile phase bottles, NEVER
let them run dry.
• Click the ‘injection’ icon button (or the equivalent on your instrument). Inject the
background into the instrument.
• Once the background has stabilized, then inject your sample into the instrument.
• The HPLC column should be washed for at least 30 minutes after each
run to ensure that it is properly cleaned. The procedure for washing
the column and inject will vary depending on the instrument and on
the sample that is being analyzed.
• Turn off the HPLC.
• Dispose of all waste in the appropriate hazardous waste containers.
  The Kjeldahl method was developed in 1883
Kjeldhal appratus by a brewer called Johann Kjeldahl. A food is
digested with a strong acid so that it
releases nitrogen which can be determined
by a suitable titration technique. The
amount of protein present is then
calculated from the nitrogen concentration
of the food. The Kjeldahl method can
conveniently be divided into three steps:
 Digestion
 Neutralization
 Titration
 Standard operating procedure

• Procedure
Digestion
1. Rinse all glassware once with 1:1 HCl, and then three times with deionized water. Do
not use commercial detergents. 7.1.2 Using an automatic pipet with disposable tips,
withdraw a 10 mL aliquot of sample.
2. Discard this first portion. Withdraw another 10 mL aliquot and transfer to a digestion
tube.
3. Add 2.0 mL of digestion solution (6.3) and several (two to three) boiling chips.
4. Prepare all samples, calibration standards, blanks, control standards, spikes, and
duplicates in the same manner.
5. Place the rack of tubes in a pre-heated block digester at 200EC for 60 minutes. Be
sure to place the end plates in position on the racks so heating occurs evenly.
6. Transfer the rack to a pre-heated high temperature block. Heat at 370EC for 30
minutes.
7. Remove the tubes from the block and allow to cool for about 15 minutes.
8. Add a 10 mL aliquot of deionized water to each tube. Mix the samples well using a
Vortex mixer. Transfer to 13 x 100 mm test tubes for analysis. Samples may also be
Analysis of Digested Samples
1. Allow at least 15 minutes for the heating unit to warm up to 60EC.
2. If the salicylate reagent is merged with a sample containing sulfuric acid in the absence of
the buffer solution, the salicylate reagent will precipitate. To prevent this, prime the system
by first placing the buffer transmission line in the buffer. Pump until the air bubble
introduced during the transfer reaches the "T" fitting on the manifold. Then place all other
lines in the proper containers. If precipitation does occur, all teflon tubing should be
replaced.
3. It is very important that all reagents be purged thoroughly with helium before beginning
analysis. Usually two to three minutes will suffice for each reagent.
4. Follow the Lachat Procedural SOP for the remainder of the analysis.
5. The diluents in the auto dilutor is reagent
6. Not demonized water.
7. In normal operation, the digested blank will result in a slight peak. This is due to the acid in
the digest and is present in every injection. Since this blank is constant for all samples and
standards it will not effect data quality.
1. Turn on the power to the Rapid Still II. Confirm that it is connected to a tap water source,
and that the water is flowing vigorously
2. Visually inspect the 1 L water supply to the steam source and verify that it is filled to the
indicator mark with tap water. If it is not, press the fill button on the front of the Rapid Still
II until the fill level is reached. When the reservoir is full, turn on the power to the steam
source.
3. Verify that the 50% NaOH supply is attached to the Rapid Still II, and that there is sufficient
solution in the supply to run the samples to be analyzed. It will require approximately 50
mL of NaOH per digested sample.
4. Add 100 mL of indicating boric acid solution to a sufficient number of 250 mL Erlenmeyer
flasks to distill each sample, standard or blank that has been digested. The graduated
markings on the side of the flasks are sufficiently accurate for this purpose.
5. Place a filled 250 mL Erlenmeyer flask on the receiving station of the Rapid Still II. Verify
that the ball of the receiving tube is below the surface of the receiving solution
6. Carefully place the first sample to be distilled on the Rapid Still II. Turn the digestion tube ¼
to ½ turn while pressing upward toward the rubber seat to assure a vapor tight fit of the
digestion tube to the still.
7. Verify that the power is on, the steam source is boiling, and the distillation timer is set to 0
minutes.
8.Using the volume indicator on the back of the Rapid Still II, estimate the volume of liquid in
the digestion tube. Within 10 mL is sufficiently accurate for the estimate.
9. “Tap” the NaOH addition switch on the front of the still to add NaOH in small increments.
Violent reaction will occur, which is minimized by slow addition. After a few mLs are added,
the solution may begin to turn dark and cloudy.
10.This is normal and will not affect the analysis. Continue addition until 50 mL of NaOH have
been added, or until the addition of continued amounts of the solution does not result in a
vigorous reaction within the vessel, whichever comes.