HCMC INTERNATIONAL UNIVERSITY

SCHOOL OF BIOTECHNOLOGY

LABORATORY MANUAL

GENERAL BIOCHEMISTRY 2

Ho Chi Minh City, 2019

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 CONTENTS
********
Course title: GENERAL BIOCHEMISTRY 2
Number of credit: 2

Week Topic Page

1 Orientation
2 Pipette Calibration 3-9
3 Soluble Carbohydrate Quantification 10-12
4 Hydrolysis of Starch by Salivary Amylase 13-18
5 Protein Quantification 19-21
6 Enzymatic Activity of Bromelain 22-23
7 Midterm examination
8 Cheese Production From Milk 24-26
9 Calcium Quantification in Powdered Milk 27-28
10 Ascorbic Acid Quantification in Foods 29-30
11 Flavonoids Quantification In Different Sorts Of Tea. 31-32
12 Final examination

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WEEK 2: PIPETTE CALIBRATION

1. Objectives

Students should understand types and functions of some types of pipettes and know how to calibrate the
pipette

2. Introduction

How pipettes work?

There are two types of pipettes: air displacement and positive displacement pipettes. Air
displacement pipettes are meant for general use with aqueous solutions. Positive displacement pipettes are
used for highly viscous and volatile liquids. Both pipette types have a piston that moves in a cylinder or
capillary. In air displacement pipettes, a certain volume of air remains between the piston and the liquid.
In positive displacement pipetting, the piston is in direct contact with the liquid.
Air displacement pipetting is highly accurate for standard pipetting applications. However,
conditions, such as atmospheric pressure as well as the specific gravity and viscosity of the solution, may
affect the performance of air displacement pipettes.

How an air displacement pipette works?

1. The piston moves to the appropriate position when the volume is set.
2. When the operating button is pressed to the first stop, the piston expels the same volume of air
as indicated on the volume setting.
3. After immersing the tip into the liquid, the operating button is released. This creates a partial
vacuum, and the specified volume of liquid is aspirated into the tip.
4. When the operating button is pressed to the first stop again, the air dispenses the liquid. To
empty the tip completely, the operating button is pressed to the second stop (blow-out).

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Getting started
 Check your pipette at the beginning of your working day for dust and dirt on the outside. If needed,
wipe with 70% ethanol.
 Check that you are using tips recommended by the manufacturer. To ensure accuracy, use only
high quality tips made from contamination free polypropylene.
 Tips are designed for single use. They should not be cleaned for reuse, as their metrological
characteristics will no longer be reliable.
 Pre-rinsing (three to five times) the tip with the liquid to be pipetted improves accuracy. This is
especially important when pipetting volatile compounds since it prevents liquid from dripping out
of the tip.
 Pipette parallel samples in a similar way.
 Avoid turning the pipette on its side when there is liquid in the tip. Liquid might get into the interior
of the pipette and contaminate the pipette.
 Avoid contamination to or from hands by using the tip ejector.
 Always store pipettes in an upright position when not in use.

Pipetting techniques
Forward pipetting:

When pipetting and mixing a sample or reagent into another liquid. The forward technique is
recommended for aqueous solutions, such as buffers, diluted acids or alkalis.
Otherwise no: Formation of bubbles or foam in the tip or in the test tube or well.

1. Press the operating button to the first stop.

2. Dip the tip into the solution to a depth of 1 cm, and slowly release the operating button.
Withdraw the tip from the liquid, touching it against the edge of the reservoir to remove excess
liquid.
3. Dispense the liquid into the receiving vessel by gently pressing the operating button to the first
stop. After one second, press the operating button to the second stop. This action will empty the
tip. Remove the tip from the vessel, sliding it along the wall of the vessel.
4. Release the operating button to the ready position.

Repetitive pipetting:

Especially for adding reagents into tubes or into the wells of microplates. This technique is intended for
repeated pipetting of the same volume.
1. Press the operating button to the second stop.

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 2. Dip the tip into the solution to a depth of 1 cm, and slowly release the operating button.
Withdraw the tip from the liquid, touching it against the edge of the reservoir to remove excess
liquid.
3. Dispense the liquid into the receiving vessel by gently pressing the operating button to the first
stop. Hold the button in this position. Some liquid will remain in the tip, and this should not be
dispensed.
4. Continue pipetting by repeating steps 2 and 3.

Reverse pipetting:

For pipetting samples or reagents when no mixing into another liquid is required. Reverse pipetting avoids
the risk of splashing, and foam or bubble formation. The reverse technique is used for pipetting solutions
with a high viscosity or a tendency to foam. This method is also recommended for dispensing small
volumes. It can also be used with air displacement pipettes.

1. Press the operating button to the second stop.

2. Dip the tip into the solution to a depth of 1 cm, and slowly release the operating button. This
action will fill the tip. Withdraw the tip from the liquid, touching it against the edge of the
reservoir to remove excess liquid.
3. Dispense the liquid into the receiving vessel by depressing the operating button gently and
steadily to the first stop. Hold the button in this position. Some liquid will remain in the tip, and
this should not be dispensed.
4. The liquid remaining in the tip can be pipetted back into the original solution or thrown away
with the tip.
5. Release the operating button to the ready position.

Pipetting of heterogeneous samples:

When pre-rinsing the tip is not possible and the full sample should be dispensed for correct analysis. This
technique is used for pipetting heterogeneous samples, such as blood or serum.

1. Press the operating button to the first stop. Dip the tip into the sample. Make sure the tip is
sufficiently below the surface.
2. Release the operating button slowly to the ready position. This action will fill the tip with the
sample. Remove the tip from the solution by sliding it along the wall of the vessel. Dip the tip into
the target solution. Make sure the tip is sufficiently below the surface.
3. + 4. Press the operating button to the first stop and release it slowly to the ready position. Do not
remove the tip from the solution. Repeat this process until the interior wall of the tip is clear.

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 5. Remove the tip from the solution by sliding it along the wall of the vessel. Press the operating button
to the second stop, and completely empty the tip. 6. Release the operating button to the ready position.

Ensuring optimum performance

Error-free pipetting requires both precision and accuracy. A number of factors can affect these
specifications. These form the main quantitative parameters for evaluating pipette performance.
What are accuracy and precision?
• Accurate, but not precise: The mean volume is the correct (set) volume, but the separate
pipetting differ from the set volume.
• Precise, but not accurate: There is no variation between the separate pipetting, but the mean
volume differs from the set volume.
• Accurate and precise: The mean volume is the set volume and there is no variation between
the different pipetting

Factors affecting the accuracy of air displacement pipettes

Temperature
Temperature has many effects on pipetting accuracy. The factor that has the greatest effect is the
temperature difference between the used delivery device and liquid. The air gap (dead air volume) between
the liquid surface and the piston experiences thermal expansion effects according to the case. This either
reduces or increases the liquid amount aspirated into the tip along with other effects.
Density
The density (mass/volume ratio) affects the liquid volume that is aspirated into the tip. A smaller
dose of liquid with higher density than water is aspirated compared to similar operation with water. With
lower density liquids the effect is the opposite. This is caused by the flexible dead air volume along with
the earth gravity. The density of liquids also varies according to the temperature. Typically the density for
water is 0.998 kg/dm3, for ethanol 0.79 kg/dm3 and for sulfuric acid (95-98% H2SO4) 1.84 kg/dm3 (the
values apply at the temperature of 20°C).
Altitude

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 The geographic altitude affects the accuracy through air pressure. The air pressure decreases in
higher altitudes and the conversion factor Z decreases as well. Also, with some liquids the boiling point
decreases quite close to room temperature, which will increase the evaporation loss dramatically.

Principle
Under a constant temperature and atmospheric pressure, the density of distilled water is constant.
The volume of water can be determined by weighting dispensed water. The calibration of pipette is carried
out by gravimetric method. When determining the volume of water, the accuracy of measurements is
effected by ambient temperature, atmospheric pressure and relative humidity. These factors are usually
combined to give the Z factor, used in calculation of volume of water. Then the calculated volume of
water is compared with the theoretical volume to determine the accuracy and precision of the pipette.

3. Materials

 Pipette and tips

 50 ml beaker
 Distilled water
 Temperature meter (±0.1℃ )
 Analytical balance (±1.0 mg)

4. Procedure
1. Determine the water temperature and record it.
2. Place a beaker filled with distilled water into analytic balance and close the door of balance waiting
for equilibrium of inner vapor
3. Place a plastic medicine cup on the pan and adjust the weight to zero.
4. Put a tip onto the pipette and set the volume which is to be tested.
5. Pre-rinse the tip: aspirate and dispense the setting volume three times and press the push – button
on the second stop to remove any remaining liquid.
6. Press the push-button to the first positive stop. Hold the pipette vertically, immerse the tip so that
14 mm in the liquid and release the push button slowly and smoothly to aspirate the liquid.
7. Wait one second and withdraw the tip from the liquid.
8. Wipe any droplets away from the outside of the tip using a kimwipe.
9. Place the end of the tip against the inside wall of the plastic cup at an angle of 10-40 °.
10. Press the push-button smoothly to the first stop. Wait one second, change new site and press the
push-button on the second stop.
11. Keeping the push-button press to the end, remove the pipette by drawing the tip along the inside
surface of the plastic cup and release the push button.
13. Close the door of balance and record the value on the balance display after it has stabilized. 14.
Repeat step (6) to (12) 14 times (the weight display of balance should be adjusted to zero every time)
15. Eject the tip.
16. Calculation

Convert the weight unit of measured value into the volume unit of measured value using the following
formula:
Volume (ml or µl) = Weight (mg or µg) x Z
Z value: conversion factor, which is conversion of density (see table 1)
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Calculate the average (Mean), accuracy, standard deviation (S.D.) and imprecision (C.V.) using the
following formula:

Accuracy value must be 99-101 % and C.V. value must be less than 1 %.

Pipette Calibration Record

Pipette number:

Water temperature: ; Atmospheric pressure : ; Z value :

Operator: ; Date :
Weight record (mg):

Record:

Mean = ; Accuracy = ; C.V.=

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Table 1. Values for Z (µg/mg), as a function of temperature and atmospheric pressure, for distilled water

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WEEK 3: SOLUBLE CARBOHYDRATE QUANTIFICATION

1. Objectives

Students should be able to use spectrophotometer, create a standard curve and know how to test
concentration of soluble carbohydrate in natural products.

2. Introduction

Nelson's method is used to establish a standard cure for glucose. Samples containing accurately known
concentrations of glucose are subjected to this colorimetric assay, absorbance readings recorded, and the
data plotted as a standard curve. It should be borne in mind that this method is a general test for soluble
carbohydrate and does not distinguish between monosaccharides (such as glucose) and disaccharides
(such as maltose).
The soluble carbohydrate is heated with an alkaline solution of copper tartrate and cuprous oxide is
produced, which reacts with arsenomolybdate to give molybdenum blue, the intense blue color is then
measured in the colorimeter. Sodium sulphate is included in the reaction mixture to minimize the entry of
atmospheric oxygen into the solution, which would cause reoxidation of cuprous oxide.

3. Materials

 Nelson’s A reagent
 Nelson’s B reagent
 Arsenomolebdate reagent
 Stock sugar standards
 Alcohol
 Some natural products.
 Boiling water bath.
 Volumetric flasks (50mL and 100mL)
 Beakers (50mL and 100mL)
 Filter paper (11mm)
 Test tubes
 Pipettes (1mL and 10mL)
 Blender
 Spectrophotometer

4. Procedure

Preparation of glucose standards

- Obtain about 10 ml of 0.5 mM glucose.

- Transfer measured amounts of the glucose solution into one set of labelled test tubes according to
the protocol in Table 1. Add sufficient distilled water to each tube to bring the final volume up to
1.0 ml.
- There should now be one set of seven tubes each containing concentrations of glucose ranging
from 0 to 0.5 µmoles/ml.
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Nelson's test for glucose

- Add 1.0 ml of Nelson's reagent to each tube (this must be freshly prepared each day by mixing 25
parts of Nelson's reagent A with 1 part of Nelson's reagent B).
- Place an aluminium foil cap on the top of each tube and heat the tubes in a vigorously boiling water
bath for exactly 20 minutes. Cool the tubes in a beaker of cool tap water.
- After cooling, add 1.0 ml of arsenomolybdate reagent to each and shake occasionally over a five
minutes period to dissolve the Cu2O and reduce the arsenomolybdate.
- Allow the tubes to stand for a few minutes. Use a propipette to deliver this reagent. (See caution
below). Make the volume of each tube up to 10.0 ml with distilled water, mix, and determine the
absorbance at 510 nm.

Caution: The arsenomolybdate reagent contains sodium arsenate, a deadly poison, you must not mouth
pipette this reagent! Use a propipette for this reagent.

Table 1. Protocol for glucose standard curve

Calculation

- Plot the standard curve with the absorbance (Y axis) against concentration of glucose (X axis).
- Draw the best straight line through the origin and all of the points. The concentration of unknown
carbohydrate solution can be determined from the curve.

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Determine the concentration of unknown samples

- Prepare your test tube of your samples. Repeat all the steps in part 2.
- Triplicate your experiment. Determine the concentration of your sample. Express the concentration
in M and mg/ml.

The concentration of standard glucose solution:

After conducting the Nelson’s test, fill the following table:

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WEEK 4: HYDROLYSIS OF STARCH BY SALIVARY AMYLASE
1. Objectives

Students are expected to be able to evaluate activity of salivary amylase and its inhibitors

2. Introduction

Amylase, an enzyme present in saliva, catalyzes the hydrolysis of the glycosidic linkages in starch.
The effect of enzyme concentration, substrate concentration, temperature, pH, and heavy metal cations on
the activity of this enzyme will be investigated.
Metabolism
The chemical reactions that go on in living organisms, and which are essential for growth,
reproduction, movement, and all other vital functions, are individually and collectively referred to as
metabolism. Many thousands of such metabolic processes are occurring constantly in even the simplest
forms of life.
Enzymes are proteins that act as catalysts for metabolic reactions. They increase the rate of the
reaction, but do not influence the kind or amount of products formed. In general, each metabolic reaction
has to be catalyzed in the living organism by its own special enzyme.
The existence of enzymes in biological materials can be demonstrated by the effects they bring about.
In this experiment, digestive enzymes capable of breaking down such polysaccharides as starch will be
studied.
Food materials must be broken down by digestion before they can be absorbed and utilized by the
body. Consequently, if an organism has no enzyme capable of attacking a particular type of carbohydrate,
protein, or fat, that particular substance will have no food value for the organism. Carbohydrates and fats
can be broken down by strong acids or bases. However, digestive enzymes can accomplish the same
breakdown under conditions compatible with life, that is, at moderate temperatures and physiological pH
ranges.
Starch
The nutritional reservoir in plants is starch, which is actually a mixture of two polysaccharides.
Amylose, the unbranched type of starch, consists of glucose residues in α-l,4 linkage

Amylopectin, the branched form, has about one α-l,6 1inkage per thirty α-l,4 linkages.
Amylases
Hydrolysis of starch is the process of digestion. Enzymes called amylases catalyze only the
hydrolysis of α-1,4 glycosidic linkages in the amylose and amylopectin components of starch. The
hydrolysis does not proceed directly from polysaccharides to monomer units; rather, partial hydrolysis
products of intermediate size are obtained. These products are maltose and dextrin. Maltose consists of
two glucose units in α-1,4 linkage; dextrin is made up of several glucose units joined by α-1,6 linkage in
addition to α-1,4 linkages.

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 Amylase enzymes are present in saliva, so the digestion of carbohydrates begins in the mouth.
Digestion continues briefly in the stomach until the pH drops too low, and then is completed in the
intestines by the attack of another amylase.
The chemical conversion of starch to dextrins and maltose is a key part of the brewing step in the
production of beer. The amylase enzymes are added to the grain mixture as brewer's malt. Approximately
one third of the calories in regular beer are contributed by carbohydrate remaining in the beer after
fermentation. The carbohydrates remain because amylase is unable to break down the α-l,6 glycosidic
linkages in the amylopectin portion of the starch. To make "light beer," less starch is used in the initial
brewing step and a second enzyme, amyloglucosidase, which can cleave the α-1,6 glycosidic linkages in
amylopectin, is added in the fermentation step. The light beer will have a lower carbohydrate composition
because of the added amyloglucosidase, and a lower alcohol content because of the reduced starch in the
initial fermentation mixture.
Study of Enzyme Activity
To follow the action of an enzyme, it is necessary to test for the appearance of a product or the
disappearance of a reactant over a measured period of time. In the reactions of amylase with starch, you
will test for the disappearance of starch by reacting samples of the reaction mixtures with iodine. Initially,
an unhydrolyzed starch solution reacts with iodine to give a blue-black color. The amylase catalyzes the
hydrolysis of the α-1,4 glycosidic linkages, forming maltose and dextrin. After the starch is hydrolyzed,
the blue-black color produced with iodine is no longer observed, and only the red or gold color of the
iodine solution is seen. The faster the amylase hydrolyzes the starch, the sooner the failure of the blue-
black color to appear is observed. If the blue-black color continues to appear each time it is tested, it can
be concluded that the enzyme is no longer active and that no hydrolysis of starch has occurred.
Enzyme activity depends on several factors, including enzyme concentration, substrate
concentration, pH, temperature, and presence of heavy metal cations. Your own saliva containing the
amylase enzyme will be used for this experiment, although the levels of amylase vary considerably from
one person to another. Each experiment must be timed. As you proceed with each experiment, you will
check enzyme activity by reacting a few drops of the reaction mixture with iodine. The time at which the
blue-black color of starch does not appear will be noted in each experiment.
The time required for the hydrolysis of starch will be correlated to the relative enzyme activity.
When enzyme activity is high, the time for the starch to hydrolyze will be very short. When the enzyme
is operating poorly or not at all, the activity is low, and more time will be required for the starch to
hydrolyze. In some cases, the enzyme will be completely inactivated and the blue-black color of the starch
and iodine will persist throughout the entire experiment. Graphs will be prepared showing the effects of
concentration, pH, temperature, and heavy metal cations on the relative enzyme activity.

3. Materials

 Test tubes
 Plastic pipette droppers
 Water bath
 Paraffin
 Alcohol
 Marker
 Distilled water
 Tissue paper
 Beaker
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  Pasteur pipette (1mL)
 Starch solution
 Iodine
 Benedict's reagent
 Barfoed’s reagent
 AgNO3
 NaCl

4. Procedure

A. Effect of Enzyme Concentration

- Collect approximately 5 mL of saliva in a medium test tube.

- Place 2 droppers full of 1 % starch solution in each of 5 medium test tubes. Number the test tubes
1-5.
- Place the tubes in a 37-40 °C water bath. After 5 minutes, add the following amounts of saliva to
the test tubes as quickly as possible, mixing each solution thoroughly, and then returning the tubes
to the water bath. Do not overheat the bath or you will inactivate the enzyme.
Test tube Number of drops of saliva
1 1
2 2
3 5
4 10
5 20
- Prepare a spot plate for testing for the presence of starch in the samples by placing one drop of
iodine reagent in each of five depressions on the spot plate.
- Two minutes after the addition of saliva, transfer one drop from each test tube (using a different
pipet for each tube) to a separate drop of iodine in the spot plate. Note the color produced for each.
Remember that the complex formed by starch and iodine is an indigo blue. If the color of the iodine
solution remains red or gold after adding the starch solution, the starch has been completely
hydrolyzed.
- As soon as one of your starch solutions has hydrolyzed, use it to begin preparing the solutions for
Parts B and C below. Continue to test the remaining starch solutions that have not yet hydrolyzed.
- Clean the spot plate and then prepare it for the next testing by placing one drop of iodine reagent
in each of five depressions.
- Repeat the testing at 5 minutes after the addition of saliva, and at 5 minute intervals thereafter.
Continue testing for 20 minutes, or until the blue-black color no longer appears for each sample.
- Record the time required for the hydrolysis of starch in each sample.

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 Graph: Enzyme Activity (Time for Starch Hydrolysis) vs. Enzyme Concentration (Drops of Saliva)

Questions:
1. How does the activity of the enzyme change as its concentration increases?

2. What are some reasons for this change in activity?

B. Benedict's Test for Easily Oxidized Groups

The Benedict's reagent is a mild oxidizing agent consisting of the Cu+2 ion and the poly
atomic citrate ion (C6H6O7-2) in basic solution. The Cu+2 ion forms a complex with citrate; it is
wrapped by and protected within the citrate ion. This protection allows Cu+2 ion to remain
dissolved in the basic solution (otherwise, the extremely insoluble Cu(OH)2 would form). When
an easily oxidized compound is added to Benedict's reagent, and the solution is warmed, Cu+2 ion
is reduced to Cu+1 ion. These ions cannot be protected from OH− by citrate, and precipitate as
copper(I) oxide (Cu2O). The Benedict's reagent has a bright blue color caused by the Cu+2 ion, but
Cu2O is a brick-red solid. Visible evidence of a positive Benedict's test is the disappearance of the
blue color of the solution, and the appearance of a brick-red precipitate. Monosaccharides and
disaccharides that contain aldehydes or α-hydroxy ketones will give a positive Benedict's test, as
these groups are easily oxidized to carboxylic acids.

Carbohydrates to be tested: hydrolyzed starch and fresh starch solutions.

- In each of two medium test tubes place 2 droppers full of Benedict's reagent and 1 mL of either
fresh starch solution or the hydrolyzed starch from Part A.
- Shake the contents of each tube well, and place both tubes at the same time in an actively boiling
water bath. Heat in the water bath for 3 minutes. On the report sheet, record any changes in the
colors or transparencies of the solutions, and in the formation and color of precipitates.

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C. Barfoed's Test for Monosaccharides

As is true of the Benedict's test, the Barfoed's test depends on the presence of easily
oxidized groups to reduce Cu+2 ion to Cu+1 ion to form the brick-red precipitate of Cu2O.
However, the Barfoed's reagent is slightly acidic rather than basic; this difference allows for
selectivity between the monosaccharides and disaccharides. Monosaccharides will test positive
within two to three minutes. Disaccharides with oxidizable groups require ten minutes or more
before they show the positive result.

Carbohydrates to be tested: hydrolyzed starch and fresh starch solutions.

- In each of two medium test tubes place 1 dropper full of Barfoed’s reagent and 1 mL of either
fresh starch solution or the hydrolyzed starch from Part A.
- Shake the contents of each tube well, and place both tubes at the same time into an actively
boiling water bath. Heat in the water bath for 5 minutes (no longer). During this period, observe
the tubes closely and record any change in color or clarity of the solutions.

Questions:
Are the results of the Benedict’s and Barfoed’s tests what you had expected? Explain your answer
by giving the structures and names of the products of the complete enzymatic hydrolysis of starch.

D. Inhibition of Enzyme Activity

- Place 2 droppers full of 1% starch solution in each of 4 medium test tubes. Add the following to
the test tubes:
Test tube:
1 1 mL of 0.1 M AgNO3 + 4 mL water
2 1 mL of 0.1 M NaCl + 4 mL water
3 5 mL of ethanol
4 5 mL of water
- Place the tubes in a 37-40 °C water bath. After 5 minutes, add 5 drops of saliva to each of the test
tubes as quickly as possible, mixing each solution thoroughly, and then returning the tubes to the
water bath. Do not overheat the bath or you will inactivate the enzyme.
- Prepare a spot plate for testing for the presence of starch in the samples by placing one drop of
iodine reagent in each of four depressions on the spot plate.

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- Five minutes after the addition of saliva, transfer one drop from each test tube (using a different
pipet for each tube) to a separate drop of iodine in the spot plate. Note the color produced for each.
Questions:

1. Which of the compounds added to the reaction tubes inhibited enzyme activity?

2. Explain how the compounds you mentioned above inhibit enzyme activity.

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WEEK 5: PROTEIN QUANTIFICATION
1. Objectives

Students are expected to be able to quantify concentration of protein in natural products.

2. Introduction

Protein quantification is highly essential in biochemical research. Several assays have been
developed; however, each has limitation due to biochemical separation process and purposes of
experimenters. Basing on differences in amino acid content of distinct proteins, currently experimental
condition, experiences of experimenters, protein of interest and their amounts, suitable assays will be
applied to obtain the best outcome with acceptable error. Before deciding which assay will be used,
considering its sensitivity, accuracy, interfering substances and available timing are highly
recommended.
The purified source of the protein to be quantified should be chosen as the standard one to reduce
result’s errors. In case of the proteins of interest whose sources have not been isolated, purified or
commercially sold, the other proteins having similar structures or coming from the same family protein
should be chosen as alternatives, instead to obtain a high similar color yield.
In this practical, Hartree-Lowry assay is used for protein quantification. Standard protein of known
concentration is used to construct calibration curve. Folin- Ciocalteu reagent is added to the protein
solutions to develop a color whose intensity is measured colorimetrically. Albumin solution is selected
as an appropriate standard. Various known concentration of albumin solution are mixed well with
reagent to enhance color development. Two below reactions account for intensely blue color
development:
+The coordination of peptide bonds with alkaline copper (biuret assay)
+The reduction of the Folin-Ciocalteu reagent by tyrosine and tryptophan residues in protein.
The advantage of Hartree-Lowry assay is its sensitivity, which is up to 100 times greater than of
the Biuret assay; however, more time is required when applying Hartree-Lowry assay. Since extracted
protein solution is a mixture of different proteins whose tyrosine and tryptophan contents are variable,
the color development may changes even though their concentration are the same.

3. Materials

 Test tubes
 Pipettes (1mL, 2mL, and 5mL) and pumps
 Volumetric flasks (50mL, 100mL)
 Beakers (50mL, 100mL)
 Graduated cylinder (50mL)
 Falcons (50mL)
 Filter paper (11mm)
 Spectrophotometer
 Centrifuges
 Albumin solution
 Solution A
 Solution B
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  Solution C
 Folin-Ciocalteu reagent

Solution A: Get 2g of Na2CO3 and dissolve it in 0.1 M NaOH to make 100mL.

Solution B: Get 0.5g of CuSO4.5H2O and dissolve it in 1% Sodium Citrate to make 100mL
Solution C: This solution can only be used within a hour, and it is the mixture of solution A and
solution B at a rate 49:1. When the solution C loses its pale-blue color, it cannot be used any more.

4. Procedure

Sample preparation
- Get soybeans and pulverize them by blender or stone mortar.
- Take an exact amount of 5g pulverized soybeans and put it into a stone mortar.

Extraction
1st time:
- Get about 40mL of distilled water and put small amount of water into the stone mortar and grind
down the sample.
- Then put the rest of water to the stone mortar and grind down the sample carefully.
- Put the extract solution into a beaker. The grounds of the soybean is still kept in the stone mortar.
2nd time:
- Do the same as the 1st time with about 30 mL of distilled water.
3rd time:
- Do the same as the 1st time with about 30 mL of distilled water.

- After 3 times of extraction, centrifuge (5000rs/m for 10 minutes) to remove the remained grounds
out of the extract solution.
- After removing remained grounds, extracted protein solution will be poured into volumetric flask
of 100mL.
- Then, distilled water is added into the volumetric flask to reach the marked level. This is your
original protein solution or 100-diluted solution
- You need to make 10,000-diluted solution by dissolving 1mL of 100-diluted solution in 99mL of
distilled water.

Making standard curve and quantifying protein content of sample

- 6 test tubes are numbered respectively from 1 to 6.
- The 0.1% albumin solution is diluted with the different amount of water to make the protein
solutions with different concentrations (0, 50, 100, 150, 200, and 250μg/mL).
- To get the expected result, we must follow the procedure in the table below.

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Tube Number 1 2 3 4 5 6
Step 1 0.1% Albumin solution (mL) 0 0.5 1.0 1.5 2.0 2.5
Step 2 Distilled water (mL) 10 9.5 9.0 8.5 8.0 7.5
Step 3 Shake the tube well
The concentration of each tube (μg/mL) 0 50 100 150 200 250

- After finishing making test tubes of protein solution followed the table above, you continue to
make test tubes of standard protein solutions followed the table below.
- 10 test tubes are numbered respectively from 1’ to 10’.

Tube number (mL) 1’ 2’ 3’ 4’ 5’ 6’ 7’ 8’ 9’ 10’

Protein solution 0.4 0.4 0.4 0.4 0.4 0.4
Original solution
0.4 0.4
(100-diluted)
Sample solution
0.4 0.4
(10,000-diluted)
Solution C 2 2 2 2 2 2 2 2 2 2
Shake each tube well and also keep them for 10 minutes
Folin-Ciocalteu 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2
Shake each tube well and also keep them for 10 minutes
Distilled water 2.4 2.4 2.4 2.4 2.4 2.4 2.4 2.4 2.4 2.4
Shake the tubes well, keep them for 5 minutes and measure the A750nm

- Add your results in the table below

Tube number 1’ 2’ 3’ 4’ 5’ 6’ 7’ 8’ 9’ 10’

OD
ΔOD
Protein concentration
0 50 100 150 200 250 x y
(μg/mL)
- Draw a standard curve
- Calculate amount of protein that contains in 100 gram of soybean
21 | P a g e
WEEK 6: ENZYMATIC ACTIVITY OF BROMELAIN
1. Objectives

Students are expected to be able to determine enzymatic activity of bromelain from natural products.

2. Introduction

Bromelain is an enzyme classified in group of protease, and it is present much in pineapple. To

determine the enzymatic activity of bromelain, experimenter often uses this enzyme to catalyze the
degradation of substrate such as hemoglobin and casein. This reaction produces peptides and amino acids,
including tyrosine. Tyrosine can be quantified colorimetrically after adding Folin-Ciocalteu reagent.

3. Materials

 Test tubes
 Pipettes (1mL, 2mL, and 5mL) and pump
 Beakers (500mL, 250mL and 100mL)
 Falcons (50mL)
 Filter paper (11mm)
 Spectrophotometer
 Centrifuges
 5M NaOH solution
 0.2M HCl solution
 5% Trichloroacetic acid solution
 2% Casein solution
 Tyrosine solution
 Folin-Ciocalteu reagent

4. Procedure

Preparation of sample

- Choose fresh, moderately ripe pineapple to discard, cut into the pieces and then pulverize it by
blender or stone mortar.
- Centrifuge (5000rs/m for 10 minutes) to remove the remained grounds out of the pineapple juice.

Explore the enzymatic activity

- Perform the experiment following the table below.

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 Tube of tyrosine Tube of sample Tube of water
(mL) (mL) (mL)
2% Casein in the
2.5 2.5 2.5
phosphate buffer
Keep the tubes at 350 C for 5 minutes
5% Trichloroacetic acid 5 0 5
0.2M HCl 0 0.5 0.5
Tyrosin solution 0.5 0 0
Pineapple solution
0 0.2 0
containing bromelain
Shake the tubes well at 350C for 10 minutes
5% Trichloroacetic acid 0 5 0
Pineapple solution
0.2 0 0.2
containing bromelain

- Shake each tube well and keep them still for 10 minutes at 250C, then filter the tubes.
- Transfer 2mL of filtered solution of each tube into new tubes
- Add 5mL of 0.5M NaOH solution and 1mL of Folin-Ciocalteu reagent
- Shake the tubes well, then put the tubes at the stand for 15 minutes and measure the A578nm
or A620nm.
- The activity of bromelain enzyme is determined depending on quantity (μg) of tyrosine
produced from casein degradation under catalysis of enzyme in 1mL of solution or 1mg of
bromelain mixture for 1 minute.
- The enzymatic activity is calculated by the formula below:

x1: Tyrosine solution (mL)

x2: Enzyme solution (mL)

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WEEK 8: CHEESE PRODUCTION FROM MILK

1. Objectives

Students are expected to be able to demonstrate the use of enzymes in casein coagulation.

2. Introduction

Cow's milk is rich in a wide range of chemical compounds that can be processed into
various dairy products such as cheese, butter, and yogurt. Specifically the milk component
involved in cheese production is a soluble protein called casein. The enzyme rennet can be used
to catalyze the conversion of casein in milk to para-casein by removing a glycopeptide from the
soluble casein. Para-casein further clots, i.e. coagulates, in the presence of calcium ions to form
white, creamy lumps called the curd, leaving behind the supernatant called the whey.

rennet Ca++
casein -------> para-casein (aq.) --------> para-casein (ppt)

The precipitate is soft at this point and can be separated from the whey by the use of
cheese cloth. Filtration does not work very well; filter paper clogging is a recurrent problem.
There is no standard method of cheese making; limitless variations exist for all stages of the
process: pre-ripening, curdling, addition of artificial ingredients and salt for flavor, and aging.
This variation in processing accounts for the wide range of cheeses commercially available,
differing in texture and flavor. The curd can also be processed with other techniques to make a
variety of desserts. However, all processes have one thing in common: the separation of the curd
from the whey.
Three ways of preparing milk for curdling will be introduced in this experiment.
Industrially, the lactic acid level in the milk is increased by adding a starter culture of Streptococci
or Lactobacilli to the milk and fermenting at 32ºC for 10 to 75 minutes. In addition to biologically
converting the lactose present in the milk to lactic acid, these strains of microorganisms also
greatly affect the flavor of the final product. Thus, the selection of a suitable strain, the amount
of starter culture, and the length of pre-ripening, is of the utmost importance in creating the subtle
differences in the final color and aroma that distinguishes an expensive cheese from a cheap one.
If one has not yet acquired a keen palate for cheeses, the second approach should suffice.
In this approach, one takes advantage of the existing Lactobacillus culture in buttermilk and uses
it as the starting culture. One ml of buttermilk is added per 100 ml of milk, and the mixture is
then fermented at room temperature for 4-12 hours. At the end of fermentation, the temperature
of the mixture is raised to 32ºC, and artificial coloring is added to the mixture prior to curdling.
The third way to prepare the milk in a short time frame is to add acid (HCl) and to heat to
32ºC. Of course, there leaves much to be desired in this method if you are a cheese connoisseur.
After rennet is added to the pre-cured milk, the coagulation process is started. In cheese making,
as coagulation comes to completion, the temperature is gradually raised to about 38ºC. This
slightly elevated temperature facilitates the separation of the curd from the whey. A higher
temperature also hardens the curd. The curd may be hardened further by cooking it for a longer
period of time, either with or without the whey.

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 After the curd is separated from the whey, salt, seasoning, and other curing and flavoring
ingredients are added. The curd is wrapped in cheese cloth and pressed for 12 to 18 hours to
remove the additional whey soaked in the curd. The curd hardens and forms a cheese block in the
shape of the press as the whey is squeezed out. Finally, the cheese block is dried for 6 hours.
It is now ready for consumption, or it may be left to age in a controlled cool environment
(2-13ºC). Although a higher temperature promotes faster curing, there is also a higher chance of
spoilage due to undesirable microbial activities at elevated temperatures. Prior to aging, the
cheese block is usually wrapped tightly to exclude air and microbial contaminants from entering
and spoiling the cheese. One way to accomplish this is to dip the cheese block in a pot of melted
wax. During the aging process, many complicated microbial and chemical actions continue to
take place in the cheese block. Thousands of techniques exist to develop various distinctive
flavors. These reactions are not well characterized; thus, cheese making is still an art rather than
a science. Depending on the technique employed, this final aging process takes anywhere from 2
weeks to 6 months.
It is generally quite straight forward to locate the sources of enzymes; the natural sources
of enzymes are where nature needs them. It comes as no surprise that rennet had traditionally
been isolated from the fourth stomach of young calves because digestion by suckling calves is
nature's primary way of processing cow milk. However, the ways of nature are not always the
most economical from man's perspective when a process is adapted to different uses to benefit
mankind in ways not originally intended by the nature. Thus enters the study of biochemical
engineering.
Chemical engineering is the study of how to make a large quantity of chemicals in an
economical fashion. For example, frequently a chemical engineer must devise a process to mass
produce a polymer that is totally different from the one originally used by an organic chemist
working with small test tubes and beakers in a laboratory. Biochemical engineering, being a
subfield of chemical engineering, also deals with the same kinds of problems facing chemical
engineering, except that the chemicals are not synthetic (manmade) but biological (naturally
existing) in nature. Although rennet is naturally excreted from a calf's stomach lining, extracting
it from its natural source is not economical. Other proteases can also convert casein to paracasein,
but their action does not stop there. They further degrade the curd to soluble subunits. Fortunately,
large quantities of rennet of consistent quality can now be produced easier and cheaper in a well-
controlled environment by microbial fermentation.
A word of caution is in order here. Enzymes as a class of chemicals are not generally
considered as dangerous, toxic, nor poisonous; they do not cause skin irritations or burns as acids
or bases. Some exceptions are proteases that catalyze the breakdown of protein molecules to
amino acids components. Because meat is mainly protein, protease can digest the soft moist
sections of the skin. We can all imagine how that is going to feel. Thus, you should exercise the
same caution with enzymes as you do with any other chemicals.

3. Materials

 1/2 gallon whole milk

 1/4 cup lemon juice or vinegar
 1/4 to 1/2 teaspoon salt
 4-quart saucepan
 Slotted spoon
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  Strainer or colander
 Mixing bowl
 Cheesecloth, nut bag, or other cloths for straining
 Dinner plates
 Weights, like a 32-ounce can of tomatoes

4. Procedure

- Heat the milk: Pour the milk into the saucepan and set over medium heat. Bring the milk
to a bare simmer — just below the boil at around 200°F. Stir the milk occasionally;
scraping the bottom of the pot to make sure the milk doesn't scald. When ready, the milk
will look foamy and steamy.
- Add the lemon juice: Remove the milk from heat and stir in the lemon juice. The milk
should begin to curdle immediately, but it's ok if it doesn't.
- Let the milk stand for 10 minutes: Cover the milk and let stand for 10 minutes to give the
acid time to completely separate the curds and whey. At the end of 10 minutes, the curds
should be completely separated and the liquid should look yellow and watery. If the milk
hasn't separated, try adding another tablespoon of acid. If it still won't separate, check
your milk and be sure you are using non-UHT milk; this kind of milk won't separate.
- Strain the curds: Set a strainer or colander over a mixing bowl and line it with cheesecloth,
a nut bag, or other straining cloth. Carefully scoop or pour the curds into the strainer,
letting the whey collect in the bowl beneath.
- Squeeze the curds: Gather the cheesecloth in your hand and gently squeeze to remove the
excess whey.
- Salt the curds: Open the cheesecloth and sprinkle 1/4 teaspoon of salt over the curds. Stir
gently and taste. Add more salt if desired.
- Press the curds: Transfer the curds (still in the cheesecloth) to a large dinner plate. Shape
them into a rough square and then fold the cheesecloth tightly around the curds to form a
neat rectangular package. Set a second plate on top of the package and weigh it down.
Press for at least 15 minutes or up to 1 hour.

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WEEK 9: CALCIUM QUANTIFICATION IN POWDERED MILK
1. Objective

Students are expected to be able to determine concentration of calcium in milk products.

2. Introduction
Oxalate ammonium will precipitate all calcium ion in any solution when the
experimenter set up all following conditions severely:
+ pH of solution environment is greater than 4
+ The hot, saturated (COONH4)2 solution is filled only one time into the sample containing
calcium ion.
+ Freeze the solution immediately after the solution has heated up for 1 minute
This method can be used for calcium quantification in milk, blood sample, urine, food,
etc. and the reactions are performed as following:
2+
Ca + (COONH4)2  2NH4+ + Ca(COO)2 (1)
Ca(COO)2 + H2SO4  CaSO4 + (COOH)2 (2)
5(COOH)2 + 2KMnO4 + 3H2SO4  K2SO4 + 2MnSO4 + 10CO2 + 8H2O (3)

3. Materials
 Racemic crucibles
 Muffle furnace
 Desiccator
 Burette
 Erlenmeyer flasks (250mL)
 Beakers (100mL)
 pH meter
 Filter papers (11mm)
 Powdered milk
 Saturated (COONH4)2
 Concentrated HCl
 Methyl red
 0.1M NH4OH
 Acetic acid
 Saturated Calcium chloride
 1N H2SO4
 0.02N KMnO4

27 | P a g e
4. Procedure
Preparation of sample
- Pretreat milk sample of 0.5g by burning them with absolute ethanol or dehydrate them
with concentrated sulfuric acid.
- After the milk sample is almost completely burned, it turns into black color.
- Put the pretreated milk samples contained in racemic crucibles into the muffle furnace to
heat with temperature of 500oC for 15 minutes, counted from the time that temperature
of the muffle furnace reach 500oC.
- Let the milk ash sample cool down and put them into desiccator over night before
performing experiment.

Calcium quantification
- Take three milk ash samples contained in three separated racemic crucibles out of the
desiccator and add 5mL of distilled water, and then 5 drops of concentrated chlohydric
acid.
- Mix well and transfer these solutions separately into 3 different beakers of 250mL to adjust
pH.
- Add 10-15 drops of methyl red and carry out the neutralization by 0.1 ammonium solution.
- Adjust the pH of the solution to from 5 to 5.2 by acetic acid of weak concentration. At that
point, the solution color is orange-pink. Due to the instrumental error, each pH meter will
give a different value of pH. Therefore, observe the color of solution is also very
important. Do not over-rely on any type of machine.
- While heating theses beakers by water bath, stirring these solutions and fill 2-3mL of
saturated (COONH4)2 solution.
- Continue to provide heat to these beakers and then mix these solution well for 30 seconds.
- Put these beakers in cool basin of water immediately.
- Keep the beaker in the basin of water for about 30 minutes.
- Use filter paper to collect all the precipitate.
- Use distilled water to wash the filter paper to know whether (COO)22- ions are all
eliminated or not. To check it, we use saturated calcium chloride solution.
- Collect precipitate retained by filter paper and put them into Erlenmeyer flask
- Add 20mL of 1N sulfuric acid solution into each Erlenmeyer flask and heat them in water
bath with temperature of 70oC for 1 minute.
- Titrate the solution with 0.02N potassium permanganate solution to determine the
concentration of (COO)22- ion in the solution.
- Calculate the amount of calcium ion in the solution and in the sample.

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WEEK 10: ASCORBIC ACID QUANTIFICATION IN FOODS
1. Objective
Student are expected to be able to determine concentration of vitamin C in some foods by
titration method

2. Introduction
Vitamin C, also known as L-ascorbic acid, is a water-soluble vitamin that is naturally
present in some foods, added to others, and available as a dietary supplement. Humans, unlike
most animals, are unable to synthesize vitamin C endogenously, so it is an essential dietary
component.
Vitamin C is required for the biosynthesis of collagen, L-carnitine, and certain
neurotransmitters; vitamin C is also involved in protein metabolism. Collagen is an essential
component of connective tissue, which plays a vital role in wound healing. Vitamin C is also an
important physiological antioxidant and has been shown to regenerate other antioxidants within
the body, including alphatocopherol (vitamin E). Ongoing research is examining whether vitamin
C, by limiting the damaging effects of free radicals through its antioxidant activity, might help
prevent or delay the development of certain cancers, cardiovascular disease, and other diseases in
which oxidative stress plays a causal role. In addition to its biosynthetic and antioxidant functions,
vitamin C plays an important role in immune function [4] and improves the absorption of nonheme
iron, the form of iron present in plant-based foods. Insufficient vitamin C intake causes scurvy,
which is characterized by fatigue or lassitude, widespread connective tissue weakness, and
capillary fragility.
The intestinal absorption of vitamin C is regulated by at least one specific dose-dependent,
active transporter. Cells accumulate vitamin C via a second specific transport protein. In vitro
studies have found that oxidized vitamin C, or dehydroascorbic acid, enters cells via some
facilitated glucose transporters and is then reduced internally to ascorbic acid. The physiologic
importance of dehydroascorbic acid uptake and its contribution to overall vitamin C economy is
unknown.
Oral vitamin C produces tissue and plasma concentrations that the body tightly controls.
Approximately 70%–90% of vitamin C is absorbed at moderate intakes of 30–180 mg/day.
However, at doses above 1 g/day, absorption falls to less than 50% and absorbed, unmetabolized
ascorbic acid is excreted in the urine. Results from pharmacokinetic studies indicate that oral doses
of 1.25 g/day ascorbic acid produce mean peak plasma vitamin C concentrations of 135
micromol/L, which are about two times higher than those produced by consuming 200–300
mg/day ascorbic acid from vitamin C-rich foods. Pharmacokinetic modeling predicts that even
doses as high as 3 g ascorbic acid taken every 4 hours would produce peak plasma concentrations
of only 220 micromol/L.
The total body content of vitamin C ranges from 300 mg (at near scurvy) to about 2 g. High
levels of vitamin C (millimolar concentrations) are maintained in cells and tissues, and are highest
in leukocytes (white blood cells), eyes, adrenal glands, pituitary gland, and brain. Relatively low
levels of vitamin C (micromolar concentrations) are found in extracellular fluids, such as plasma,
red blood cells, and saliva.
The method is based on the oxidation of ascorbic acid by blue reagent 2,6 -
dichlorphenolindophenol (DCIP). The products of this reaction are dehydroascorbic acid and light
pink reduced form of DCIP.

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3. Materials

 Analytical balance
 Burettes
 Erlenmeyer flasks (250mL)
 Graduate cylinder (50mL)
 Beakers (50mL, 100mL)
 Foods
 2% HCl
 2,6-dichlorphenolindophenol
 Alcohol
 Distilled water

4. Procedure
- Prepare an extract of vitamin C
- Measure out the necessary extract volume in a flask, add 1mL of 2% HCl to the diluted
extract and titrate with 2,6-dichlorphenolindophenol until the appearance of light pink
colour. If the extract contains chlorophyll and has green colour, titrate this extract,
comparing its colour with the colour of the same extract stored without titration, because
green colour masks light pink colour. In this case titrate the content of the flask until the
change of the colour of the titrated extract in comparison with a control sample (without
titration).
- Account. 1 mL of 2,6-dichlorphenolindophenol corresponds to 0.088 mg of ascorbic acid
(titer of ascorbic acid for 2,6-dichlorphenolindophenol).
- The account of the ascorbic acid content is carried out for 100 g of the dense foodstuff or
for 100 mL of the liquid foodstuff (in mg%). For account it is convenient to use the formula:

Where:
X - the content of ascorbic acid in the foodstuff (in mg/dl),
А - the result of the titration (the volume of 2,6-dichlorphenolindophenol),
B - the volume of the extract for the titration (ml) without the account of water,
C - the weight of a dense sample (g) or the volume of a liquid sample (ml) of a product
for the analysis,
D - the total volume of an extract (ml), 100 - the figure necessary for recalculation of the
content of ascorbic acid in 100 g (ml) of the food-stuff.

- Calculate the quantity of the product needed for satisfaction of the daily requirement of
vitamin С (in grams or ml).

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WEEK 11: FLAVONOIDS QUANTIFICATION IN DIFFERENT SORTS OF TEA.

1. Objective
Students are expected to be able to determine concentration of flavonoids in tea by titration method

2. Introduction

Vitamin P, more commonly known as bioflavonoids, or flavonoids, is not strictly true

vitamins, though they possess vitamin-like properties. They are a vast array of compounds
found in plants, and are classified as plant pigments. These pigments, over 4000 of which have
been identified, are responsible for the dazzling colors of fruits and flowers. This speaks
volumes for its broad range of attributes. Biofavonoids address most health conditions and
come from multiple natural sources.
Vitamin P was discovered by Nobel Price winning scientist Albert Szent-Gyorgyi in1936.
Vitamin P is also known as flavonoids. Scientists have identified over 4,000 flavonoids.
Flavonoids belong to the larger group of beneficial plant compounds known as polyphenols.
Heat, acidity, boiling and processing can cause significant loss of flavonoid content in food, as
much as 50% or more. Overcooking of vegetables is especially detrimental.
Vitamin P fuels your body with life-changing botanical elements. Biofavonoids are the
most natural concentrated source of nutrients linked to the function of every cell in your body.
These are a few of the bioflavonoids: quercetin, rutin, myricetin, apigenin, hesperin,
hesperidin, luteolin, catechin, eriodictyol, cyaniding and others. Each has an effect on human
health, which is why vitamin P’s range of health benefits is so broad.
For a start flavonoids are powerful antioxidants that scavenge the harmful free radicals that
damage our cells, and that alter genetic DNA, accelerate the aging process, and contribute to
development of many diseases. Free radicals occur naturally in the body as a result of
conversion of food to energy, but more is added by toxins such as cigarette smoke, UV rays,
radiation, and air and water pollution. Antioxidants are important as they neutralize these free
radicals, and reduce the damage they cause.
The method is based on the ability of vitamin Р to be oxidized by potassium permanganate.
Indigocarmine is used as an indicator. It reacts with potassium permanganate after the complete
oxidation of vitamin P. In this experiment, we will attempt to determine the amounts of vitamin
P in three different types of tea: Black, green, and oolong tea. The group of flavonoids present
in tea is the Flavan-3-ols

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3. Materials
 Dry tea
 Distilled water
 Alcohol
 Indigo carmine
 Analytical balance
 Burettes
 Erlenmeyer flasks (250mL)
 Graduate cylinder (50mL)
 Beakers (50mL, 100mL)

4. Procedure

- Put 100 mg of dry tea in a glass and add 50 ml of boiling water.

- Extract it for 5 minutes.
- Measure out 10 ml of extract of tea in another clean and dry 100 ml Erlenmeyer flask.
- Add 5 ml of distilled water of room temperature and 5 drops of indigo carmine.
- Titrate until yellow color appears and stays for 30 sec or more.
- Make the account of the content of vitamin P in the tea by the formula:

X - the content of vitamin Р in dry tea (mg%),

A - the result of the titration (volume of potassium permanganate) (ml).
- Then calculate the quantity of dry tea needed for satisfaction of the daily requirement
of vitamin P (in grams).
- Record the results in the table below

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