Review lecture 2
1. What is ash content?
1. What is moisture content?
2. What is principle of ash
content analysis?
2. What is principle of
moisture content analysis?
PROTEIN ANALYSIS
3 Methods
3.
M h d ffor ddetermination
i i
3. Methods
3
M h d ffor ddetermination
i i
of ash content?
of moisture content?
4. How to calculate the ash
content?
Assoc. Prof. PHAM VAN HUNG
Department of Food Technology
4. How to calculate the
moisture content?
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In this lecture
„
Introduction to Protein
„
Importance of protein analysis
„
Principle
p of total p
protein analysis
y
„
Procedure of total protein analysis
„
Protein fraction analysis
Introduction
„
Definition: “Proteins are a sequence
(polymers) of amino acids joined together by
peptide bonds”.
„
Different proteins have different chemical
properties and structures.
1
Introduction
„
Introduction
Protein is one of nutritional component of
food.
‰
‰
‰
‰
‰
Proteins
Lipids
Carbohydrates
Minerals
Vitamins
„
Three main group of proteins
‰
‰
‰
„
Classification of proteins (protein fractions)
‰
„
What is difference between proteins and
other nutritional components?
Simple Proteins
Conjugated Proteins
Derived Proteins
Based mostly on the solubility of proteins in
different solvents
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Introduction
„
Protein fractions in foods:
‰
Four classes
„ Albumins
„ Globulins
„ Gliadins (Prolamins)
„ Glutelins
Importance of protein analyses
„
Nutrition labeling
„
Pricing: The cost of certain commodities is based on the protein
content as measured by nitrogen content (e.g., cereal grains; milk for
making certain dairy products, e.g., cheese).
„
Functional property investigation: Proteins in various types of
food have unique food functional properties: for example,
example gliadin and
glutenins in wheat flour for breadmaking, casein in milk for
coagulation into cheese products, and egg albumen for foaming.
„
Biological activity determination: Some proteins, including
enzymes or enzyme inhibitors, have actions on food quality: for
instance, the proteolytic enzymes in the tenderization of meats,
pectinases in the ripening of fruits, and trypsin inhibitors in legume
seeds are proteins.
2
Importance of protein analyses
„
Methods for protein analysis
Protein analysis is required when you want to
know:
„
Total protein
‰
1. Total protein content
‰
2. Protein content during isolation and purification of
a protein
3. Profile of a particular protein in a mixture
„
Total protein of food
Total protein of each protein fractions
Amino acid composition
4. Amino acid composition
5. Nonprotein nitrogen
6. Nutritive value of a protein
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Total Protein Determination Methods
1.
Kjeldahl Method.
2
Dye Binding Method.
3.
Biuret Method.
4.
Lowry Method.
5.
Ultraviolet Method.
Kjeldahl Method - Principle
• is a method for the quantitative determination of nitrogen in
foods developed by Johan Kjeldahl in 1883.
• The method consists of heating a sample with sulphuric
acid, which decomposes the organic substance by oxidation
o liberate
be a e thee reduced
educed nitrogen
oge as aammonium
o u su
sulphate.
p a e.
to
Then the solution is then distilled with a small quantity of
sodium hydroxide, which converts the ammonium salt to
ammonia. The amount of ammonia present, and thus the
amount of nitrogen present in the sample, is determined by
back titration.
3
Procedure
Conversion Factors from Nitrogen to Protein for Foods
1. Step 1: Sample preparation
2. Step 2: Digestion
3. Step 3: Neutralization and Distillation
[2]
The protein content in foodstuffs is estimated by multiplying
the determined nitrogen content by a nitrogen-to-protein
conversion factor.
C d protein
Crude
i (%) = Nitrogen
i
content (%) x CF
C
4. Step 4: Titration
Why do we need conversion factor?
5. Step 5: Calculations
[5]
A factor is used to convert percent N to percent crude protein.
Most proteins contain 16% N, so the conversion factor is 6.25
(100/16 = 6.25).
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Conversion Factors from Nitrogen to Protein for Foods
6.25
Corns
6.38
5.83
5.70
Milk Whole wheat Wheat flour
Eggs
Barley
Peas
Oats
Meat
Rye
Beans
Millet
Apparatus
5.30
Nuts
4
Apparatus
Apparatus
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Application
Advantages:
1. Applicable to all types of foods
2. Inexpensive (if not using an automated system)
3. Accurate; an official method for crude protein
content
4. Has been modified (micro Kjeldahl method) to
measure microgram quantities of proteins
Disadvantages:
1. Measures total organic nitrogen, not just protein
nitrogen
2. Time consuming (at least 2 h to complete)
3. Poorer precision than the biuret method
4. Corrosive reagent
Analysis of protein fractions
„
Separation of protein fractions
‰
Using different solvents: water, alcohol, Alkaline
„
Determine total protein
„
Determine molecular weight of protein
5
Analysis of protein fractions
Analysis of protein fractions
Sample
Residue
Add distilled water
Add NaCl
Supernatant (Albumin)
Centrifuge
SDS-PAGE
Stirring
SDS-PAGE
Centrifuge
Supernatant (Globulin)
Kjeldah
Kjeldah
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Analysis of protein fractions
Analysis of protein fractions
Residue
Residue
Extracted 3 times with
70% (v/v) ethanol
Extracted 2 times with TrisHCl (pH 8.8) + SDS + DTT
Shaking
SDS-PAGE
Centrifuge
Supernatant (Glutenin)
Kjeldah
SDS: Sodium dodecyl sulfate
DTT: Dithiothreitol
Shaking
SDS-PAGE
Centrifuge
Supernatant (Gliadin)
Kjeldah
6
Analysis of protein fractions
The end!
Alb i
Albumin
Gliadin
Gl b li
Globulin
Glutenin
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