Proteins
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Proteins. Proteins – basic concepts. Role of proteins Nutrition Energy and essential amino acids May cause allergies and be toxic/carcinogenic Structure Provide structure in living organisms and also foods Catalysts

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Proteins

Proteins


Proteins basic concepts

Proteins – basic concepts

  • Role of proteins

    • Nutrition

      • Energy and essential amino acids

      • May cause allergies and be toxic/carcinogenic

    • Structure

      • Provide structure in living organisms and also foods

    • Catalysts

      • Enzymes (which are proteins) catalyze chemical reactions in living tissue and foods


Proteins basic concepts1

Proteins – basic concepts

  • Role of proteins

    • Functional properties

      • Gelation

      • Emulsifiers

      • Water bonding

      • Increase viscosity

      • Texture

    • Browning

      • Have amino acids that can react with reducing sugars

      • Some enzymes can also cause browning


Proteins basic concepts2

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


Proteins basic concepts3

Proteins – basic concepts

  • Amino acids are generally grouped into 3 classes

    • Charged and polar

    • Uncharged and polar

      • These two classes of amino acids are found on the surfaces of proteins


Proteins basic concepts4

Proteins – basic concepts

  • Amino acids are generally grouped into 3 classes

    • 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 concepts5

Proteins – basic concepts

Polar Amino Acids - Hydrophilic


Proteins basic concepts6

Proteins – basic concepts

Non-polar Amino Acids – Hydrophobic/Amphophilic


Proteins basic concepts7

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 apolypeptide chain

    • Amino acids are linked to eachother in a chain via a peptide bond

      • A covalent bond

  • This backbone structure dictates

    rest of the structure

R-group

R-group

Condensationreaction


Proteins basic concepts8

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


Proteins basic concepts9

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

c) Random coils

Absence of secondary structure

Irregular random arrangement of a polypeptide chain

Proteins – basic concepts

-sheets


Proteins basic concepts10

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

Proteins – basic concepts


Proteins basic concepts11

3. Tertiary structure

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


Proteins basic concepts12

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


Proteins basic concepts13

Proteins – basic concepts

Types of forces/bonds that stabilize the protein structure


Proteins basic concepts14

Proteins – basic concepts

Proteins exist in two main states

DENATURED STATE

  • Loss of native confirmation

    • Altered secondary, tertiary or quaternary structure

  • Results

    • Decrease solubility

    • Increase viscosity

    • Altered functional properties

    • Loss of enzymatic activity

    • Sometimes increased digestibility

NATIVE STATE

  • Usually most stable

  • Usually most soluble

  • Polar groups usually on the outside

  • Hydrophobic groups on inside

  • Heat

  • pH

  • Pressure

  • Oxidation

  • Salts


Proteins basic concepts15

Proteins – basic concepts

Factors causing protein denaturation

  • pH

    • 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

100

%Denatured

0

0

pH

12


Proteins basic concepts16

Proteins – basic concepts

Factors causing protein denaturation

  • Temperature

    • High temperature destabilizes the non-covalent interactions holding the protein together causing it to eventually unfold

    • Freezing can also denature due to ice crystals & weakening of hydrophobic interactions

100

%Denatured

0

0

100

T (C)


Proteins basic concepts17

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 concepts18

Proteins – basic concepts

Important reactions of proteins and effect on structure and quality

  • 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


Proteins basic concepts19

Proteins – basic concepts

Important reactions of proteins and effect on structure and quality

  • Maillard reaction (carbonyl - amine browning)

    • Changes functional properties of proteins

    • Changes color

    • Changes flavor

    • Decreases nutritional quality (amino acids less available)


Proteins basic concepts20

Proteins – basic concepts

Important reactions of proteins and effect on structure and quality

  • Alkaline reactions

    • 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)


Proteins basic concepts21

Proteins – basic concepts

Important reactions of proteins and effect on structure and quality

  • Alkaline reactions

    • 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 concepts22

Proteins – basic concepts

Important reactions of proteins and effect on structure and quality

  • Alkaline reactions

    • 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


Proteins basic concepts23

Proteins – basic concepts

Important reactions of proteins and effect on structure and quality

  • Alkaline reactions

    • Lysinoalanine formation (LAL)

      • Problem

        • Lysine is the limiting amino acid in cereal foods

          • Essential amino acid of least quantity

        • Lysinoalanine can lead to kidney toxicity in rats, and possibly humans

        • LAL formation is usually not a problem in food processing but loss of lysine is


Proteins basic concepts24

Proteins – basic concepts

  • 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


Proteins basic concepts25

Proteins – basic concepts

  • Heat

    • Degradation of cysteine

      • Leads to terrible flavor problems  H2S(g)

    • Amide crosslinking

      • Need severe heat for this reaction - not very common


Proteins basic concepts26

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.

Met sulfoxide still active as an essential amino acid

Met sulfone – no or little amino acid activity

Proteins – basic concepts


Proteins functional properties

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 properties1

Example of protein functional properties in different food systems

Proteins – functional properties


Proteins

The properties of food proteins are altered by environmental conditions, processing treatments and interactions with other ingredients


Proteins functional properties2

Solubility

Functional properties of proteins depend on their solubility

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


Proteins functional properties3

Solubility

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)

Proteins – functional properties

%Solubility

Salt concentration


Proteins functional properties4

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

  • 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)

+

+

+

Low pH

+

+

+

+

+

+

+

+

+

Insoluble complex


Proteins functional properties5

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:

More soluble

Less soluble

Centrifuge at 20000g for 30 min

Protein samples at different pH’s

at 0.1M NaCl

Solubility (%) = protein left in supernatant *100 total protein


Proteins functional properties6

Proteins – functional properties

Sol

  • Gelation

  • 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 properties7

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


Proteins functional properties8

Proteins – functional properties

  • 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)

cooling

Denaturation (%)

Gel strength/Viscosity

heating

heating

T


Proteins functional properties9

Proteins – functional properties

  • Thermally irreversible gels

  • Balance of forces is critical in gel formation:

    • - If the attractive forces between the proteins are too weak they will not form gels

    • -If the attractive forces are too strong the proteins will precipitate

cooling

Denaturation (%)

Gel strength/Viscosity

heating

heating

T


Proteins functional properties10

Proteins – functional properties

  • 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)

cooling

Denaturation (%)

Gel strength/Viscosity

heating

heating

T


Proteins functional properties11

Proteins – functional properties

  • 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)


Proteins functional properties12

Factors influencing gel properties

pH

Salts

T

heating/cooling scheme

Proteins – functional properties


Proteins functional properties13

Proteins – functional properties

  • Factors influencing gel properties

    • pH

    • 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

      • 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 producing different textures for different foods


Proteins functional properties14

Proteins – functional properties

  • Factors influencing gel properties

    • Salt concentration (ionic strength)

      • Again, highly protein dependent

      • Some proteins “need” to be solubilized with salt before being

        able to form gels, e.g. muscle proteins (myosin)


Proteins functional properties15

Proteins – functional properties

  • Factors influencing gel properties

    • Salt concentration (ionic strength)

      • Again, highly protein dependent

      • 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

Loss of gel strength

Loss of water-holding

Cl-

+

+


Proteins functional properties16

Proteins – functional properties

  • Factors influencing gel properties

    • pH

    • Salt concentration (ionic strength)

      • 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 properties17

How do we measure gel quality?

Many different methods available

Gel texture and gel water-holding capacity 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

Proteins – functional properties


Proteins functional properties18

Proteins – functional properties

  • 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


Proteins functional properties19

Proteins – functional properties

  • Water binding

    • Water is associated with protein at several levels (Back to Water)

    • Surface monolayer

      • Very small amount of water tightly bound to charged groups on proteins

    • Vicinal water

      • Several water layers that interact with the monolayer, slightly more mobile

    • Bulk phase water

      • Mobile water like free water but

        • Trapped mostly by capillary action

      • Freely flowing in a food product

      • This is the water we are interested in when it comes to water binding


Proteins functional properties20

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 concentrated = more water uptake

      3. Protein denaturation

    • Depends - 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


Proteins

Example how thermal denaturation may have an effect onwater 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 properties21

Salts/ionic strength

This is highly protein dependent

muscle proteins

Proteins – functional properties

NaCl

Na+

Na+

Na+

Cl-

Cl-

Na+

Na+

Cl-

Cl-


Proteins

Phosphate salts (in combination with NaCl) are frequently used in food processing to make food proteins bind and hold more water

Salt brine

Salt brine

 phosphate

some phosphate

Cook

Cook

Cook

30% reduction

10% reduction

100% reduction


Proteins functional properties22

Proteins – functional properties

  • pH (protein charge)

  • Great influence on the water uptake and binding of proteins

  • Water binding lowest at pI since there is no effective 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 andprotein gels are a good example

pI


Proteins functional properties23

Proteins – functional properties

  • How do we measure water binding and uptake?

    • Most common methods are:

      • Water-uptake - Measuring water uptake of a protein or protein food (e.g. protein gel) by adding it to different solutions, then draining and measuring water content of protein/food vs. the original water content

      • Water-binding (also called water-holding capacity) - Subject your sample to an external force (centrifuge or pressure) and then measure how much water is squeezed out


Proteins functional properties24

Proteins – functional properties

  • Emulsification

  • Proteins can be excellent emulsifiers because they contain both hydrophobic and hydrophilic groups

+

ENERGY

LOOP

TRAIN


Proteins functional properties25

Proteins – functional properties

  • Emulsification

Whey protein stabilized emulsion

Lipid phase removed

(protein matrix showing)

Whey protein stabilized emulsion

Both phases


Proteins

Proteins – functional properties

Whey protein stabilized emulsion

Lipid phase removed

(protein matrix showing)

Whey protein stabilized emulsion

Both phases


Proteins functional properties26

Proteins – functional properties

  • Factors that affect protein-based emulsions

    • Type of protein

      • To form a good emulsion the protein has to be able to:

        • Rapidly adsorb to the oil-water interface

        • Rapidly and readily open up and orient its hydrophobic groups towards the oil phase and its hydrophilic groups to the water phase

        • Form a stable film around the oil droplet


Proteins functional properties27

Proteins – functional properties

  • Factors that affect protein-based emulsions

    • Type of protein

      • 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

          • More flexible it is, easier it opens up

        • Solubility of protein

          • Insoluble will not form a good emulsion (can’t migrate well)

          • pI is not good

          • Increasing solubility increase emulsification ability (up to a point)


Proteins functional properties28

Proteins – functional properties

  • How do we measure emulsifying properties?

    • Most are highly empirical

      • Two common methods

        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

        Emulsification stability - Emulsion formed and its breakdown (separation of water and oil phase) monitored with time


Proteins functional properties29

Proteins – functional properties

  • Foaming

  • Foams are very similar to emulsion where air is the hydrophobic phase instead of oil

  • Principle of foam formation 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)


Proteins functional properties30

Proteins – functional properties

  • Foaming

FOAM FORMATION

FOAM BREAKDOWN


Proteins functional properties31

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


Proteins functional properties32

Proteins – functional properties

  • Factors that affect foam formation and stability

    • pH

      • Foam formation is often better slightly away from 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 properties33

Proteins – functional properties

  • Factors that affect foam formation and stability

    • 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)


Proteins functional properties34

Proteins – functional properties

  • Factors that affect foam formation and stability

    • Lipids

      • Lipids in food foams usually inhibit foaming by adsorbing to the air-water interface and thinning it

        • 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 properties35

Proteins – functional properties

  • Factors that affect foam formation and stability

    • 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)


Proteins functional properties36

Proteins – functional properties

  • Factors that affect foam formation and stability

    • 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 properties37

Proteins – functional properties


Proteins functional properties38

Proteins – functional properties

  • Protein modification to improve function

    • Some proteins don’t exhibit good functional properties and must be modified

    • Other proteins are excellent in one functional aspect but poor in another but can be modified to have a broader range of function

    • 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


Proteins functional properties39

Proteins – functional properties

  • Protein modification to improve function

    • Chemical modification

      • Example of types of chemical groups that can be added to proteins


Proteins functional properties40

Proteins – functional properties

  • Protein modification to improve function

    • Enzymatic modification

      • Protein hydrolysis

        • Proteins broken down by enzymes to smaller peptides

        • Improved solubility and biological value

      • Protein cross-linking

        • Some enzymes (transglutaminase) can covalently link proteins together

        • Great improvement in gel strength

      • 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 properties41

Proteins – functional properties

  • 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 rearrange into 3D gel structures with meat like texture

        • Good water and fat holding capacity

        • Cheaper than muscle proteins  often used in meat products

      • 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


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