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Proteins
Proteins – basic concepts
Role of proteins
1. Nutrition 4. Functional properties
Energy and essential Gelation
amino acids
Emulsifiers
May cause allergies and
Water bonding
be toxic/carcinogenic
Increase viscosity
2. Structure
Texture
Provide structure in living
organisms and also foods 5. Browning
Have amino acids that can
3. Catalysts
react with reducing sugars
Enzymes (which are
Some enzymes can also
proteins) catalyze
cause browning
chemical reactions in
living tissue and foods
Proteins – basic concepts
Proteins are biological polymers that fold into a 3D
structure with amino acids being their basic structural
unit
20 amino acids common to proteins (L-amino acids)
They differ by their side chains (R-groups)
Amino acid charge behavior
Neutral
Acidic
Basic
Proteins – basic concepts
Amino acids are generally grouped into 3
classes
1. Charged and polar
2. Uncharged and polar
These two classes of amino acids are found on the surfaces
of proteins
3. Non-polar and hydrophobic
These are found more in the interiors of proteins where there
is little or no access to water
You are expected to be able to identify which amino
acids are polar or non-polar
Proteins – basic concepts
Polar Amino Acids - Hydrophilic
Proteins – basic concepts
Non-polar Amino Acids – Hydrophobic/Amphophilic
Proteins – basic concepts
Four levels of protein
structure
Primary Secondary Tertiary
Quaternary
1. Primary structure
Backbone of the protein molecule
Described by the amino acid
sequence that make up a
polypeptide
R-group
chain
Amino acids are linked to each R-group
Condensation reaction
other
in a chain via a peptide bond
A covalent bond
This backbone structure dictates
rest of the structure
Proteins – basic concepts
2. Secondary structure
Refers to arrangement of protein in space
Predictable arrangement of two main
secondary structures
-helix
-sheet
a) -helix
A coiled structure formed with internal H bonds
(between C=0 and N-H)
High amount in soluble (hydrophilic) proteins
Is the main structure in fibrous proteins
Less in globular proteins
Proteins – basic concepts
b) -sheet
“Flat” parallel or antiparallel
structure
These sheets are stabilized with
regular bonding of C=O with NH (via
H-bonds) between -sheets
High amount in insoluble
(hydrophobic) proteins
-sheets
c) Random coils
Absence of secondary structure
Irregular random arrangement of a
polypeptide chain
Proteins – basic concepts
3. Tertiary structure
Represents the secondary structure folding into a 3D
conformation/structure
This is the end structure of many proteins
The type of 3D structure formed is
dictated by
Amino acid sequence
-helix/-sheet
Proline content
Stabilizing forces
Solvent conditions
This structure folds up to bury its hydrophobic amino acids primarily
on the inside and expose its hydrophilic groups on the outside
2 general groups
Fibrous proteins
Globular proteins
Proteins – basic concepts
4. Quaternary structure
A complex of two or more
tertiary structures
The units are linked together
through non-covalent bonds
Some proteins will not
become functional unless
they form this structure.
Examples:
Hemoglobin
Myosin
Proteins – basic concepts
Types of forces/bonds that stabilize the protein structure
Proteins – basic concepts
Proteins exist in two main states
NATIVE STATE DENATURED STATE
Usually most stable Loss of native confirmation
Usually most soluble Altered secondary, tertiary or
Polar groups usually on the quaternary structure
outside
Hydrophobic groups on inside Results
Decrease solubility
Increase viscosity
Altered functional properties
Heat pH Pressure Loss of enzymatic activity
Oxidation Salts Etc. Sometimes increased
digestibility
Proteins – basic concepts
Factors causing protein denaturation
pH 100
%Denatured
Too much charge can cause high
electrostatic repulsion between charged
amino acids and the protein structure is
broken up
A charge is very unfavorable in the
hydrophobic protein interior 0
0 pH 12
Temperature 100
High temperature destabilizes the non-
%Denatured
covalent interactions holding the protein
together causing it to eventually unfold
Freezing can also denature due to ice
crystals & weakening of hydrophobic
interactions
0 0 100
T (C)
Proteins – basic concepts
Detergents
Prefer to interact with the hydrophobic part of the protein (the
interior) thus causing it to open up
Lipids/air (surface denaturation)
The hydrophobic interior opens up and interacts with the
hydrophobic air/lipid phase (e.g. foams and emulsion)
Shear
Mechanical energy (e.g. whipping) can physically rip the protein
apart or introduce the protein to a hydrophobic phase (air or lipid
– foaming and emulsification)
Proteins – basic concepts
Important reactions of proteins and effect on
structure and quality
1. Hydrolysis
Proteins can be hydrolyzed (the peptide bond) by acid or enzymes to give
peptides and free amino acids (e.g. soy sauce, fish sauce etc.)
Modifies protein functional properties
E.g. increased solubility
Increases bioavailability of amino acids
Excessive consumption of free amino acids is not good however
2. Maillard reaction (carbonyl - amine browning)
Changes functional properties of proteins
Changes color
Changes flavor
Decreases nutritional quality (amino acids less available)
Proteins – basic concepts
3. Alkaline reactions
E.g. used in soy processing (textured vegetable protein)
0.1 M NaOH for 1 hr @ 60°C
Denatures proteins
Opens up its structure due to electrostatic repulsion
The peptide bond may also be hydrolyzed
Some amino acids become highly reactive
NH3 groups in lysine
SH groups and S-S bonds become very reactive (e.g. cysteine)
Reactions:
A. Isomerization (racemization)
L- to D-amino acids
We cannot digest D-amino acids
Not a very serious problem in texturized vegetable protein
production
Proteins – basic concepts
B. Lysinoalanine formation (LAL)
Lysine becomes highly reactive at high pH and reacts with
dehydroalanine forming a cross-link
Lysine, an essential amino acid, becomes unavailable
Problem
Lysine is the limiting amino acid in cereal foods
The essential amino acids of least quantity
Lysinoalanine can lead to kidney toxicity in rats, and possibly
humans
LAL formation is usually not a problem in H
food processing but loss of lysine may occur N C CO
H
(CH2)4
NH
CH2
N C CO
H H
Proteins – basic concepts
4. Heat
Mild heat treatments lead to alteration in protein structure and often beneficial
effect on function and digestibility/bioavailability
Example: heating can denature digestive protease inhibitors, e.g. soybean trypsin
inhibitor
Severe heat treatment drastically reduces protein solubility and functionality and
may give decreased digestibility/bioavailability
Examples:
1. Degradation of cysteine
Heat
H3C - CH2SH H3C - CH2OH + H2S(g)
H2O
Leads to terrible flavor problems H2S(g)
2. Amide crosslinking
Need severe heat for this reaction - not very common
ASN or GLY + LYS LYS unavailable + NH3
Proteins – basic concepts
5. Oxidation
Lipid oxidation
Aldehyde, ketones react with lysine making it unavailable
Usually not a major problem
Methionine oxidation (no major concern)
Sulfoxide or sulfone
Oxidized by; H2O2, ROOH etc.
NH2 O O
HC C C S CH3 S CH3 + S CH3
H2 H2
O
COOH
Met Sulfoxide Met Sulfone
Met sulfoxide still active as an essential amino acid
Met sulfone is not good – no or little amino acid activity
Proteins – functional properties
Functional properties defined as:
“those physical and chemical properties of proteins that affect
their behavior in food systems during preparation, processing,
storage and consumption, and contribute to the quality and
organoleptic attributes of food systems”
Many food products owe their function to food proteins
It is important to understand protein functionality to
develop and improve existing products and to find new
protein ingredients
Proteins – functional properties
Example of protein functional properties in different food
systems
Functional Property Food System
Solubility Beverages, Protein concentrates/isolates
Water-holding ability Muscle foods, cheese, yogurt
Gelation Muscle foods, eggs, yogurt, gelatin, tofu,
baked goods
Emulsification Salad dressing, mayonnaise, ice cream, gravy
Foaming Meringues, whipped toppings, angel cake,
marshmallows
The properties of food proteins are altered by environmental
conditions, processing treatments and interactions with other
ingredients
Proteins – functional properties
1. Solubility
Many functional properties of
proteins depend on their solubility
The solubility of the protein is
affected by the balance of
hydrophobic and hydrophilic amino
acids on its surface
Charged amino acids play the most
important role in keeping the protein
soluble
The proteins are least soluble at
their isoelectric point (no net charge)
The protein become increasingly
soluble as pH is increased or
decreased away from the pI
Proteins – functional properties
Salt concentration (ionic
strength) is also very
important for protein
solubility
At low salt concentrations
protein solubility increases
(salting-in)
At high salt concentrations
protein solubility decreases
(salting-out)
%Solubility
Salt concentration
Proteins – functional properties
Denaturation of the protein can both increase or
decrease solubility of proteins
E.g. very high and low pH denature but the protein is
soluble since there is much repulsion
+ +
Low pH +
+ + + +
+ +
+ + +
Very high or very low temperature on the other hand will
lead to loss in solubility since exposed hydrophobic
groups of the denatured protein lead to aggregation
(may be desirable or undesirable in food products)
Insoluble complex
Proteins – functional properties
How do we measure solubility?
Most methods are highly empirical as results vary greatly with
protein concentration, pH, salt, mixing conditions, temperature
etc.
It is of much importance to standardize methods for solubility
One standard assay:
Centrifuge at 20000g for 30 min More Less
soluble soluble
Protein samples at different pH’s
Solubility (%) = protein left in supernatant *100
at 0.1M NaCl
total protein
Proteins – functional properties
2. Gelation Sol
Texture, quality and sensory attributes of
many foods depend on protein gelation on
processing
Sausages, cheese, yogurt, custard…..
Gel; a continuous 3D network of proteins
that entraps water
Protein - protein interaction and protein -
water (non-covalent)
A gel can form when proteins are denatured
by
Heat
pH
Pressure
Shearing
Gel
Proteins – functional properties
Thermally induced food gels (the most common)
Involves unfolding of the protein structure by heat which exposes
its hydrophobic regions which leads to protein aggregation to form
a continuous 3D network
This aggregation can be irreversible or reversible
Proteins – functional properties
A) Thermally irreversible gels
The thermally set gel (called thermoset) will form
irreversible cross-links and not revert back to solution
on cooling
Examples; Muscle proteins (myosin), egg white proteins
(ovalbumin)
Balance of forces is critical
Gel strength/Viscosity
in gel formation:
Denaturation (%)
- If the attractive forces cooling
between the proteins are too
weak they will not form gels
-If the attractive forces are
too strong the proteins will
precipitate heating
heating
T
Proteins – functional properties
B) Thermally reversible gels
These gels (called thermoplastic) will form gels on
cooling (after heating) and then revert fully or
partially back to solution on reheating (“melt”)
Example; Collagen (gelatin)
Gel strength/Viscosity
cooling
Denaturation (%)
heating
heating
T
Proteins – functional properties
Factors influencing gel properties
pH, salts, T, heating/cooling
scheme……….
pH pH close to or at pI
Highly protein dependent
Some protein form better gels at pI
No repulsion, get aggregate type gels
Softer and opaque
Others give better gels away from pI
More repulsion, string-like gels
Stronger, more elastic and transparent
pH away from pI
Too far away from pI you may get no gel
too much repulsion
By playing with pH one can therefore
play with the texture of food gels and
thus produce different textures for
different foods
Proteins – functional properties
Salt concentration (ionic
strength)
Again, highly protein dependent
Some proteins “need” to be 0.5M NaCl
solubilized with salt before being
able to form gels, e.g. muscle
proteins (myosin) Heat
Some proteins do not form good
gels in salt because salt will
minimize necessary electrostatic
interactions between the proteins
+ +
Cl-
NaCl
+ + +
+Cl- Cl- Loss of repulsion
Cl-
+ +
Loss of gel strength
Loss of water-holding
Proteins – functional properties
Example of the effect of pH and salt
Ovalbumin (one of the most important egg proteins)
(pH is >7 and < 3; salt <20 mM) (pH is 4.7 (pI); salt 50-80 mM)
Max gel strength seen at (a) pH 3.5 and 30 mM NaCl; (b) pH 7.5 and 50 mM NaCl
Proteins – functional properties
How do we measure gel quality?
Many different methods available
Gel texture and gel water-holding capacity are the methods most
commonly used
One of the better texture methods is to twist a gel in a modified
viscometer (torsion meter) and measure its response (stress and
strain) until it breaks
The results can be related to the
sensory properties of the gel
Proteins – functional properties
3. Water binding
The ability of foods to take up and/or hold water is of paramount importance
to the food industry
More H2O = More product yield = More $
Product quality may also be better, more juiciness
Water is associated with protein at several levels (as discussed in the
Water part)
Surface monolayer
Very small amount of water that is tightly bound to charged groups on proteins
Vicinal water
Several water layers that interact with the monolayer, slightly more mobile
Bulk phase water
Water that is as mobile as free water but is
a) Trapped mostly by capillary action
b) Freely flowing in a food product
This is the water we are interested in when it comes to water binding
Proteins – functional properties
What factors influence water binding?
1. Protein type
More hydrophobic = less water uptake/binding
More hydrophilic = more water uptake/binding
2. Protein concentration
More protein concentration = more water uptake
3. Protein denaturation
Depends
E.g. if you form a gel on heating (which denatures the
proteins) then you would get more water binding
water would be physically trapped in the gel matrix
Example how thermal denaturation may have an effect on water binding
SPS = Soy protein isolate forms gel on heating
Caseinate = Milk proteins (casein) does not gel on heating
WPC = Whey protein concentrate forms gel on heating
Proteins – functional properties
4. Salts/ionic strength
This is highly protein dependent
E.g. muscle proteins
NaCl
Na+ Na+
Na+ Cl- Cl- Na+
Na+ Cl- Cl-
Phosphate salts (in combination with NaCl) are frequently used in
food processing to make food proteins bind and hold more water
Na-tripolyphosphate O
O O O
=
=
=
=
NaO - P - O - P - O - P -ONa Na - [O - P - O]13 - Na
O O O
Na Na NA O
Na-hexametaphosphate
Salt brine Salt brine
some phosphate phosphate
Cook Cook Cook
10% reduction 30% reduction 100% reduction
Proteins – functional properties
5. pH (protein charge)
Has a great influence on the
water uptake and binding of
proteins
Water binding is the lowest at
pI since there is no effective pI
charge and proteins typically
aggregate (i.e. don’t like to
be in contact with water)
Water binding increases
greatly away from pI - +
+ - + -
Muscle proteins and protein + - - + + -
gels are a good example pH pH
- - - -
- -
- - - - -
- -
- - - - - - - -
More repulsion and more water uptake/binding
Proteins – functional properties
How do we measure water binding and
uptake?
Most common methods are:
A) Water-uptake
Measuring the water uptake of a protein or protein food (e.g.
protein gel) by adding it to different solutions and then
draining and measuring water content of protein/food vs.
the original water content
B) Water-binding (also called water-holding capacity)
Subject your sample to an external force (centrifuge it or
add pressure to it) and then measure how much water is
squeezed out
Proteins – functional properties
4. Emulsification
Proteins can be
excellent emulsifiers
because they contain
both hydrophobic and
hydrophilic groups
+
ENERGY
LOOP
TRAIN
Proteins – functional properties
Whey protein stabilized emulsion Whey protein stabilized emulsion
Both phases Lipid phase removed
(protein matrix showing)
Proteins – functional properties
Factors that affect protein-based
emulsions
Type of protein
To form a good emulsion the protein has to be able
to:
a) Rapidly adsorb to the oil-water interface
b) Rapidly and readily open up and orient its hydrophobic
groups towards the oil phase and its hydrophilic groups to
the water phase
c) Form a stable film around the oil droplet
Proteins – functional properties
To follow the above the following are important for
the protein
Distribution of hydrophobic vs. hydrophilic amino acids
Need a proper balance
Increased surface hydrophobicity will increase emulsifying
properties
Structure of protein
Globular is better than fibrous
Flexibility of protein
The more flexible it is the easier it opens up
Solubility of protein
If insoluble it will not form a good emulsion (will not migrate well)
pI is not good
Increasing solubility will increase emulsification ability (up to a point)
Proteins – functional properties
How do we measure emulsifying properties?
Most are highly empirical
Two common methods
1. Emulsification capacity
Oil titrated into a protein solution with mixing and the max
amount of oil that can be added to the protein solution
measured
2. Emulsification stability
Emulsion formed and its breakdown (separation of water and
oil phase) monitored with time
Proteins – functional properties
5. Foaming
Foams are very similar to emulsion where air is the
hydrophobic phase instead of oil
The principle of foam formation by proteins is similar to
that of emulsion formation (most of the same factors are
important)
Foams are typically formed by
Injecting gas/air into a solution through small orifices
Mechanically agitate a protein solution (whipping)
Gas release in food, e.g. leavened breads (a special case)
FOAM FORMATION
FOAM BREAKDOWN
Proteins – functional properties
Factors that affect foam formation and stability
Type of protein is important
Increased surface hydrophobicity is good
Partially denaturing the protein often produces better foams
Globular is better than fibrous
pH
Foam formation is often better slightly away from the pI
Foam stability is often better at pI
The farther from pI the more repulsion and the foam breaks
down
Example; Egg foams (meringue) and cream of tartar
increases stability
Proteins – functional properties
Salt
Very protein dependent
Egg albumins, soy proteins, gluten
Increasing salt usually improves foaming since charges are
neutralized (they lose solubility salting-out)
Whey proteins
Increased salt negatively affect foaming (they get more soluble
salting in)
Lipids
Lipids in food foams usually inhibit foaming by adsorbing to the
air-water interface and thinning it
E.g. only 0.03% egg yolk (which has about 33% lipids)
completely inhibits foaming of egg white!
Cream an exception where very high level of fat stabilizes foam
Proteins – functional properties
Stabilizing ingredients
Ingredients that increase viscosity of the liquid phase stabilize
the foam (sucrose, gums, polyols, etc.)
We add sugar to egg white foams at the later stages of foam
formation to stabilize
Addition of flour (protein, starch and fiber) to foamed egg white to
produce angel cake (a very stable cooked foam)
Energy input
The amount of energy (e.g. speed of whipping) and the time
used to foam a protein is very important
To much energy or too long whipping time can produce a poor
foam
The foam structure breaks down
Proteins become too denatured
Proteins – functional properties
How do we measure foam formation and
stability?
Two widely used methods
1. Overrun (foam formation)
You start with a known volume of protein solution (e.g. 100
mL) and foam it and then measure the volume of foam vs. that
of the liquid:
%Overrun = foam volume – initial liquid volume * 100
initial liquid volume
2. Foam drainage (foam stability)
Using a special cylinder measure the amount of liquid that
drains from the foam on storage to get a mL/min or mL/hour
drain value (the smaller the value the more stable the foam)
Proteins – functional properties
Protein modification to improve function
Some proteins don’t exhibit good functional properties and have to
be modified to do so
Other proteins are excellent in one functional aspect but may be
poor in another but can be modified to have a broader range of
function
1. Chemical modification
Reactive amino acids are chemically modified by adding a group to
them
Lysine, tyrosine and cysteine
Increases solubility and gel-forming abilities
Modified protein has to be non-toxic and digestible
Retain 50-100% of original biological value
Often used in very small amounts due to possible toxicity
Not the method of choice for food proteins
Example of types of chemical groups that can be added
to proteins
Proteins – functional properties
2. Enzymatic modification
a) Protein hydrolysis
Proteins broken down by enzymes to smaller peptides
Improved solubility and biological value
b) Protein cross-linking
Some enzymes (transglutaminase) can covalently link proteins
together
Great improvement in gel strength
c) Amino acid modification
Peptidoglutamase converts
Glutamine glutamic acid (negatively charged)
Asparagine aspartic acid (negatively charged)
Can convert an insoluble protein to a soluble protein
Proteins – functional properties
3. Physical modification
Most of the methods involve heat to partly denature
the proteins
Texturized vegetable proteins – TVP (e.g. soy meat)
A combination of heat (above 60C), pressure, high pH (11) and
ionic strength used to solubilize and denature the proteins
which then arrange into 3D gel structures with meat like texture
Good water and fat holding capacity
Cheaper than muscle proteins often used in meat product
Protein based fat substitutes (e.g. SimplesseTM by
Nutrasweet Co.)
Milk or egg proteins heat denatured and mechanically sheared
and on cooling they form small globular particles that have the
same mouthfeel and juiciness as fat
SimplesseTM is very sensitive to high heat – limits its use in
processing
