TABLE OF CONTENTS EXPERIMENT I: NUTRITIONAL LABELING USING A COMPUTER PROGRAM …..……..3 HIS Workshop 2012 1 University of Puerto Rico Mayagüez Campus Technology and Food Science Food Chemistry Workshop Guides for Laboratories Practice Professor: Edna Negrón de Bravo Professor: María L. Plaza
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HIS Workshop 2012 1
University of Puerto Rico
Mayagüez Campus
Technology and Food Science
Department
TABLE OF CONTENTS
EXPERIMENT I: NUTRITIONAL LABELING USING A COMPUTER PROGRAM …..……..3
EXPERIMENT II: DETERMINATION OF WATER ACTIVITY (aw) USING THE DECAGON
AQUALAB ……………………………………………………………………………………..…....9
EXPERIMENT III: VITAMIN C DETERMINATION BY INDOPHENOL METHOD………...12
EXPERIMENT IV: TITRATABLE ACIDITY DETERMINATION BY TITRATION
METHOD………………………………………………………………………………….……......17
EXPERIMENT V: ACIDS, PROTEINS, AND MAILLARD BROWNING……………………..20
TITRATABLE ACIDITY DETERMINATION BY TITRATION METHOD
INTRODUCTION
The titratable acidity or total acidity of a food is determinate by acid-base titration to measure the
total concentration of acids. This acids are mostly organic acids (citric, malic, lactic, tartaric), but
phosphoric acid is an inorganic acid sometimes added to food. The organic acids present in foods
influence the flavor, color, microbial stability and quality. The titratable acidity of fruits is used,
along with sugar content, as an indicator of maturity. While organic acids may be naturally present
in the food, they also may be formed by fermentation or added during formulation and processing.
Increasing the acidity of foods, either through fermentation or the addition of weak acids, has been
used as a preservation method since ancient times. Organic acids are more effective as preservatives
in the undissociated state.
There are two fundamentally different conventions for expressing acidity: titratable acidity
and hydrogen ion concentration, or pH. The former expresses total acidity but does not measure the
strengths of the acid, while pH indicates acid strength. pH is defined as the logarithm of the
reciprocal of the hydrogen concentration. It may be also defined as the negative logarithm of the
molar concentration of hydrogen ions. Thus, a [H3O+] concentration of 1 x 10-6 is expressed simply
as pH 6. The [OH-] concentration is expressed as pOH and would be pOH 8 in this case.
It is important to understand that pH and tritatable acidity are not the same. To determine
the titratable acidity, a known volume (or weight) of a food sample is titrated with a standard base,
to either a pH or phenolphthalein endpoint. The volume of titrant used, along with the normality of
the base and the volume (or weight) of sample, are used to calculate the titratable acidity, expressed
in terms of the predominant organic acid.
MATERIALS
Caprisun juice and natural juice (lemon, grapefruit)
Pipettes 10 ml
Burette 50 ml
Burette clamp
Clamp support
Magnetic stirring plate
Erlenmeyer flask 125 ml
0.1N NaOH
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Phenolphtalein 1%
PROCEDURE
1. Pipette 10 ml juice sample into each three 125 ml Erlenmeyer flasks.
2. Measure the pH of the samples with a pH meter and record the value.
3. Add three drop of phenolphthalein and a magnetic stir to each flask.
4. Assemble the apparatus for carrying out the titration as shown in Figure 1.
5. Titrate each sample with the 0.1N NaOH solution until phenolphthalein endpoint.
6. Record the initial and final burette readings and calculate the difference to determine the
amount of NaOH used for each titration.
DATA, CALCULATIONS AND RESULTS
Table 1. NaOH volume used during titration of the blank
BlankInitial lecture NaOH (ml)
Final lectura NaOH (ml)
Total vol. used in blank titration
1
2
3
4
Table 2. NaOH volume used during titration of the sample
Sample identityInitial lecture NaOH (ml)
Final lectura NaOH (ml)
Total Vol. used in simple titration
1
2
3
4
Formula
% tritable acidity = (Vsample – Vblank) L x NNaOH (eq.g/l) x Acid Factor (88.06g/eq.g) x 100
Sample weight (g)
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Figure 1: Titratable acidity
a. Titration setup
b. Titration end point
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EXPERIMENT V
ACIDS, PROTEINS, AND MAILLARD BROWNING
INTRODUCTION
Proteins are the most abundant macrocomponent in cells making more than 50% of the dry weight. They are very important in foods due to the nutritional properties and the functional properties. The challenge for food scientists is to develop new processes and products that maintain both properties: their nutritional value and functionality. Functional properties of proteins determine their behavior in foods and strongly affect their texture and quality. The amino acid composition of proteins influences the functional qualities of individual proteins. Some proteins may act as surfactants, have foam and emulsion-stabilizing ability; others have coagulating, foaming and leaving capacity, and others have good water binding or holding properties that allow them to coagulate and form gels under certain conditions; and some are important for their enzymatic activity.
Many foodstuffs possess distinctive color and odor characteristic as a result of reactions
between amino groups and reducing compounds (Maillard reaction or Strecker
degradation). Depending on the extent of formation, these pigments and odors may be
desirable or undesirable. In addition, free amino acids influence taste sensations.
Denaturation of proteins causes changes that might affect the quality and stability of food.
EXPERIMENT A: MAILLARD REACTION
INTRODUCTION AND OBJECTIVE
Under certain conditions, reducing sugars may react with compounds bearing a free amino group
and undergo a sequence of reactions known collectively as the Maillard reaction. As a part of this,
alpha-dicarbonyl compounds produced in the Maillard reaction can react with amino acids and
produce aromatic pyrazines. While a certain amount of browning and flavor generation is desirable
in many foods, excess browning and aroma are undesirable. The objective of this exercise is to
evaluate the aroma and color of heated amino acid-glucose solutions.
MATERIALS
D-Glucose- 50 mg
L-Aspartic acid- 50 mg
L-Lysine- 50 mg
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L-Phenylalanine
L-Valine- 50 mg
L-Methionine- 50 mg
L-Leucine- 50 mg
L-Proline- 50 mg
L-Arginine- 50mg
PROCEDURE
1. To 50 mg of D-glucose in a test tube add 50 mg of an amino acid; add 0.5 ml of distilled
water. Mix thoroughly.
2. Smell each mixture and record any sensations. Place a piece of heavy aluminum foil over
each test tube top and heat the solutions in a water bath at 100 °C for 45 minutes. Cool the
contents to about 25 °C in a water bath. Record the odor sensations for each solution
(e.g.,chocolate-like, popcorn-like). Record the color as 0 = none, 1 = light yellow, 2 = deep
yellow, 3 = brown. (Note: Color formation can be measured quantitatively if the solutions
are diluted to 5 ml, except for arginine and lysine, which need to be diluted to 500 and 1000
ml, respectively). The transfer the samples to colorimeter tuber and determine their
absorbance at 400 nm. At 400 nm, the pigmentation or degree of browning is measured.
What factors influence the degree of Maillard browning?
DATA AND RESULTS
SampleTreatment
Odor sensation
ColorAbsorbance @ 400 nm
1 L-Aspartic acid
2 L-Lysine
3 L-Phenylalanine
4 L-Valine
5 L-Methionine
6 L-Leucine
7 L-Proline 8
L-Arginine
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EXPERIMENT B: EFFECT OF HEAT ON PROTEINS
INTRODUCTION AND OBJECTIVE
Heat is one common denaturing agent of proteins in foods. Most proteins denature (uncoil) at a
specific temperature. Once denatured, they are susceptible to coagulation/gelation, and this can be
detected as turbidity in the dispersion. The objective of this exercise is to determine the effect of
heat on the denaturation of egg albumin in aqueous solution.
MATERIALS
Egg – 1
Water baths (set at 55, 60, 63, 65 and 68 °C)
Spectrophotometer
PROCEDURE
1. Prepare 100 ml of 10% dispersion (v/v) of egg white and distilled water. Filter to remove
opaque membranes.
2. Place 5 ml of the albumin solution in each of five test tubes. Place tubes in a water bath.
Save the remainder for unheated control.
3. Place one test tube at each of following temperatures: 55, 60, 63, 65, and 68 °C. Heat
samples for 30 minutes. Cool the samples in a tap water.
4. Determine the absorbance of the samples with the spectrophotometer at 450 nm. Use the
unheated sample to zero the spectrophotometer. Be sure to mix samples prior to reading.
DATA AND RESULTS
Table 1. Absorbance of Albumin solution
TubeTemperature (°C)
OpticalDensity 450
1 0.00 Set at 0
2 55
3 60
4 63
5 65
6 68
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EXPERIMENT C: FOAMING
INTRODUCTION AND OBJECTIVE
Denaturation of the protein occurs when it unfolds, changing its nature. These changes might be mild or extensive and may be reversible or irreversible. Raw eggs are translucent because light is refracted and passed between individual proteins. When eggs denatures such as when it is beaten or whipped or cooked, fried, boiled it changes it appearance from translucent to opaque or white.
When eggs are beating vigorously denaturation occurs and its volume increases from the original volume. Factors that affect the extent of denaturation, the foaming capacity (FC) of the proteins and its stability (FS) are type and time of beating, pH, temperature, concentration and the presence of other ingredients such as salt, sugars, and fat.
In this experiment your will see how several factors affect FC and FS of eggs: temperature, dilution of water, pH and how certain ingredients such as fat, sugar, acid affect the foam formation, volume, surface area, and appearance.
Materials:
1 dozen of eggs 8 Funnels, 8 glass measuring cup, 8 100 ml graduate cylinder, 8 plastic spatulas, 8 Glass 8 measuring spoons 8 Mixers 1/8 tsp Cream of tartar 1tbs Distilled Water 2 tbs Sugar 1/8 Salt
PROCEDURE
1. Each group of two students should take one egg, one funnel, one measuring cup, and a graduate cylinder. A plastic spatula, a glass and a spoon.
2. Carefully break an egg and separate the egg white and yolk without breaking the yolk. 3. Beat the egg white with a mixer for 1 minute. This will be your control.4. Measure the foam capacity by reading how many cups the volume has increased. Pour the
recently formed foam to the funnel that will be on the top of the 100ml graduated cylinder.5. Each group will add one of the following: cream of tartar, or sugar, or water, or egg yolk to
the egg whites, cold eggs and do the same procedure. Then compare it to the control using the same method.
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DATA
Measurement Control Sugar Water Egg Yolk
Cream of
Tartar
Overbeating Salt Temperature
Volume (Cups)Foam
CapacityAppearanceBubbles size (Coarse or
Finer)Color
(Opaque or Shinny)Foam
Stability:Coalescence
time (sec)
Volume Drip (ml)
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EXPERIMENT D: COAGULATION OF PROTEINS
INTRODUCTION AND OBJECTIVE
While heat is a common coagulating agent in food, other food components may also affect the
extent to which proteins denature. Among these components are salts, sugars, and acids. The
objective of this exercise is to investigate some of the factor that affect the coagulation of proteins.
MATERIALS
Egg white – 1
Distilled water
0.1M sodium chloride solution – 5 ml
0.1M calcium chloride solution – 5 ml
0.1M ferric chloride solution– 5 ml
0.1M sucrose solution – 5 ml
1.0M sucrose solution – 5 ml
0.001M hydrochloride acid solution – 5 ml
0.1M hydrochloride acid solution – 5 ml
PROCEDURE
1. Dilute one egg (slightly beaten) with three volumes distilled water; stir slowly but
thoroughly and filter.
2. To each of a series of test tubes, add 10 ml of the albumin dispersion and 5 ml of each of
the solutions listed above, including distilled water.
3. Record the pH of the solutions containing distilled water and 0.01 M and 0.1 M HCl.
4. Place all of the tubes in a beaker of water, heat slowly, and note the temperature at which
opalescence (cloudiness) develops. Generally, samples will need to be heated for at least 20
minutes.
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DATA AND RESULTS
Table 1. Temperature at which cloudiness develop
TubeAlbumin solution mixed with pH Temperature
1 Distilled Water
2 0.1M sodium chloride solution -----
3 0.1M calcium chloride solution -----
4 0.1M ferric chloride solution -----
5 0.1M sucrose solution -----
6 1.0M sucrose solution -----
7 0.001 HCl acid solution
8 0.1M HCl acid solution
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EXPERIMENT VI
PIGMENTS
INTRODUCCION
Pigments contribute greatly to the aesthetic appeal of food. The chemical forms of some
pigments are easily altered under conditions that may also affect the structural integrity of the
tissue. Heating, pH changes, and oxidation reactions can affect pigment quality. The predominant
meat pigment is myoglobin. Reactions of myoglobin determine the color of fresh and cured meats.
Plant pigments may be categorized as carotenoids, chlorophylls, and flavonoids. Included in the
flavonoid group are the phenolic compounds, which are the substrates in the enzymatic browning of
fruits and vegetables. Preservation of desirable color, flavor, and textural qualities present at harvest
of ripe fruits and vegetable depends greatly on control of the deteriorative changes caused by
endogenous enzymes. Sometimes colorants are added to foods to enhance their marketability.
EXPERIMENT A: THE EFFECTS OF HEAT AND pH ON PLANT PIGMENTS
INTRODUCTION AND OBJECTIVE
Many plant pigments, especially chlorophyll and the anthocyanins, are sensitive to heat and changes
in pH. Under favorable acid conditions, these pigments will exhibit their correct colors, but when
the pH is increased or decreased, the pigment may change to an undesirable color. This presents a
sensory defect in the food. The objective of this exercise is to determine the effect of heat and pH
on plant pigments.
MATERIALS
Frozen peas - 25g
Canned peas – 25g
Vinegar – 10ml
Grape juice- 10ml
Cramberry juice – 50ml
1N NaOH – 100ml
PROCEDURE: CHLOROPHYLL
1. Heat 150 ml deionized water to boiling.
2. Add approximately 25 g frozen peas.
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3. When the water returns to a boil, time for 7 min.
4. Remove the sample from the water and place in a beaker.
5. Add 10 ml vinegar to about 150ml deionized water and determine the pH of the solution.
Boil the solution and repeat Steps 2 to 4.
6. Add 10 ml 1N NaOH to 150 ml deionized water and determine the pH of the solution. Boil
the solution and repeat Steps 2 to 4.
7. Expose a fourth 25 g sample of peas to a cold mixture of 10 ml vinegar and 150 ml
deionized water for 7 min without cooking.
8. Expose a fifth 25 g sample of peas to a cold mixture of 2 g NaHCO 3 and 150 ml deionized
water without cooking.
9. Set up a sixth beaker with canned peas.
10. Compare all the samples for color and texture.
RESULTS
Table 1. pH, Color and texture of sweet peas
Beaker TreatmentpH
solutionColor Texture
1 frozen ------
2 Boiled ------
3 Boiled + Vinegar
4 Boiled + NaOH
5 Cold H2O + vinegar
6 Cold H2O + NaHCO3
7 canned ------
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Figure 1. Sweet peas color
a. Fresh b. canned
PROCEDURE: ANTHOCYANINS
1. Mix 10 ml of grape juice and 90 ml of distilled water.
2. Determine the pH of the solution.
3. Remove 5 to 10 ml of this solution to a test tube.
4. Adjust the remaining solution to pH 5.0 with 1N NaOH.
5. Remove 5 to 10 ml of this solution to a test tube.
6. Adjust the remaining solution to pH 7.0
7. Remove 5 to 10 ml of the solution to a test tube.
8. Adjust the remaining solution to pH 10.0
9. Remove 5 to 10ml to a test tube.
10. Compare all the samples, especially noting the color.
11. Mix 50 ml cranberry juice and 50 ml of distilled water.
12. Repeat steps 2 to 10 with cranberry juice
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DATA AND RESULTS
Table 2. pH and color of cranberries and grape juices
Sample Treatment pH solution Color
1 Grape juice + H2O
2 Grape juice, pH 5.0
3 Grape juice, pH 7.0
4 Grape juice, pH 10.0
5 Cranberry juice + H2O
6 Cranberry juice, pH 5.0
7 Cranberry juice, pH 7.0
8 Cranberry juice, pH 10.0
EXPERIMENT B: ENZIMATIC BROWINING
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INTRODUCTION AND OBJECTIVE
Some plant tissues contain phenolic compounds associated with their cells walls. Some of these also
contain polyphenoloxidase (PPO), an enzyme that will convert the phenolic to a quinone, which
will eventually be transformed into a brown melanoidin pigment. This reaction is generally
undesirable when it occurs in tissues of fruit such as apple, banana, or pear. It is thus important to
know how to control this browning reaction. The objective of this exercise is to assess the effect of
various treatments on enzymatic discoloration of Red Delicious apples.
MATERIALS
1 Red Delicious apple
1% thiourea – 60 ml
Ascorbic acid – 10 mg
Sodium sulfite – 10 mg
Dipotassium phosphate – 120 mg
Spectophotometer
Buchner funnel and filter flask
Whatman No. 1 filter paper
Blender
PROCEDURE
Note: It is important to work quickly once the apple tissue is cut the slices are placed into solution.
Have the beakers labeled and solutions prepared in advance.
1. Peel and pare apples, cut into uniform thin slices, and divide into four lots of 30.0g each.
Place one lot into a beaker containing 60 ml 1% thiourea, which will stop the browning
reaction and serve as the control. Place the second lot into a beaker containing 60ml
deionized water. Place the third lot into a beaker containing 60 ml deionized water with
0.01 g ascorbic acid. Place the fourth lot into a beaker containing 0.01 g sodium sulfite in
60 ml water, and after 45 seconds decant the solutions and replace with a solution
containing 0.12g dipotassium phosphate in 60 ml water.
2. After the apple tissue has been in solution for 30 min, homogenize the contents of each
beaker in a blender, and filter through a Buchner funnel into a filtration flask under
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aspirator vacuum using Whatman No. 1 filter paper. Attach the funnel to the filtration flask,
turn on the aspirator, wet a piece of filter paper using a squeeze bottle of deionized water,
and carefully place the filter paper in the Buchner funnel. Pour apple tissue from the
blender into the Buchner funnel and continue filtration until several milliliters of filtrate
have been collected.
3. Place 1 ml of each filtrate in four different test tubes each containing 5 ml water and mix.
4. Transfer contents of each test tube to a cuvette and read optical density at 475 nm using a
spectrophotometer. Use deionized water to set the instrument to 0% T.
DATA AND RESULTS
Table 1. Absorbance of filtrate
Sample Apple Treatment Optical Density (475 nm)
1 1% thiourea (control)
2 Deionized water
3 0.01 g Ascorbic acid
4 0.01 g sodium sulfite + 0.12 g dipotassium phosphate
EXPERIMENT C: PEROXIDASE ASSAY TO DETERMINE ADEQUACY OF
BLANCHIG INTRODUCTION AND OBJECTIVE
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Blanching is a heating process performed prior to freezing that inactivates enzymes that would
otherwise cause deterioration in palatability, color and ascorbic acid content during storage.
Polyphenol oxidase (PPO), which catalyzes the enzymatic browning reaction studied in experiment
3, is one target enzyme to inactive during blanching. Catalase and peroxidase are two endogenous
enzymes that are frequently used as indices of the adequacy of blanching treatment because they are
more heat resistant than PPO. It is assumed that if these enzymes are inactivated, enzymes that
catalyze undesirable color, flavor, and texture changes will also be inactivated. The objective of this
exercise is to test for the presence of peroxidase in raw and blanched samples of a vegetable.
MATERIALS
Broccoli-1 head
Guaicol-1 ml
0.08% H2O2 (2.7 ml of 3% H2O2 diluted to 100 ml with deionized water)-10ml
Watch glasses-2
Funnel
Glass wool
PROCEDURE
PREPARATION OF SAMPLES
1. Divide broccoli into two lots. Remove large leaves and lignified parts of stalks. Cut
broccoli lengthwise into uniform pieces. Blanch one lot by immersing in boiling water for 3
min. Remove from boiling water and plunge into cold water until cool. Let drain.
2. For each lot, blend one part vegetable with three parts distilled water (by weight) in a
blender for 3 min. Filter through a funnel with a glass wool plug.
ASSAY FOR PEROXIDASE
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1. Put several milliliters of filtrate from the raw broccoli on a watch glass.
2. Add several drops of guiacol and several drops of 0.08% H2O2. A brick-red color indicates
peroxidase activity.
3. Repeat Steps 2a and b for the blanched broccoli.
DATA AND RESULTS
Table 1. Observation and Conclusion of enzyme presence