2. Proteins

Sources of proteins

Animal proteins

• Milk proteins

• Egg (white) proteins

• Animal by-products (blood/gelating)

Plant proteins

• Soy proteins

• Pea proteins

• Lupin proteins

• Sunflower proteins

Novel (alternative) proteins

• Algae proteins

• ‘leafy’ proteins

• Microbial proteins

Function of proteins in nature

• Nutrition: source of nitrogen and essential maino acids

• Structure: (collagen → gelatine)

• Metabolism: Homeostasis, hormones, enzymes, antibodies

Function of proteins in food

• Nutrition: meat, cheese, nuts

• Texture: gluten in bread, gelatin in sweets

• Taste: Maillard reaction 

Structure of amino acids

• Contains an amino group 

• And a carboxylic acid group

• Contain a different side chain

How are amino acids bonded together?

• Via peptide bonds

L and D amino acids

• As amino acids are chiral, they can have the same molecular formula but a different structure. 

• The L variant of amino acids occurs naturally

• During processing, amino acids can turn into D amino acids which are not digested well. 

Alpha and beta amino acids

• Alpha amino acids are common and beta amino acids only exist in plants. 

• Beta amino acids do not naturally occur in proteins

Polar charged amino acids

• When an amino acid has a carboxyl or amino group

• This causes different properties.

  • Hydrophilic

  • High solubility

  • Mainly present on the outside of the protein

  • Reactive

  • Charge depends on pH

Polar non charged amino acids

• Contain a side chain with: a hydroxyl group, a sulfhydryl group or an amide group 

• Properties:

  • Hydrophilic

  • High solubility

  • Mainly present on the outside of the protein

  • Reactive

Non-polar non-charged amino acids

• Contains aliphatic group, aromatic group, imino acid or thio-ether, CH3

• Properties:

  • Hydrophobic

  • Low solubility

  • Mainly present on the inside of the protein

Occurrence of peptides

In nature:

• Blood (buffering function)

• Plants (hormones)

In food:

• Bread (glutathione: breaks down gluten and helps to retain bread airyness)

In foods after hydrolysis:

• Microbial: fermentation products

• Enzymatic: protein hydrolysates 

Function of peptides

• Are more reactive than proteins

• Can have taste (often bitter)

  • Aspartame: a very sweet peptide

• Are more easily taken up by the body

• Are studied for their bio-functional properties

Number of amino acids in a peptide naming

Two different peptide bonds

• Naturally: Trans (side chains do not hinder each other, energetically favorable)

• Only after processing: cis

Nomenclature for peptides

• When the amino acid is not bonded via a peptide bond to the alpha carbon, then a gamma is written before the name. 

What changes the protein structure?

• Temperature

• pH and ionic strength

• Solvent

Results in changes in:

• Solubility

• Functionality

Structural forces in proteins

• Covalent bonds (atoms are really connected)

Not bonds but interactions:

• Hydrogen bonds

• Hydrophobic interaction

• Electrostatic interaction

What influences the structural forces in proteins?

• pH and isoelectric point

• Salt

• Temperature

• Solvent (water, ethanol)

Primary structure

• Sequence of amino acids

Secondary structure

• Known as alpha helix and beta sheet

• Alpha helix:

  • Stabilized by hydrogen bonds

• Beta sheets:

  • Stabilized by hydrogen bonds

Tertiary structure

• Stabilized by:

  • Electrostatic and hydrophobic interactions

  • Disulfide bonds

  • Van de Waals interactions

Quaternary structure

• Spatial arrangement of different polypeptide chains

• Stabilized by:

  • Electrostatic and hydrophobic interactions

  • Disulfide bonds

Classification of food proteins

• Protein structure

  • Important for solubility and functionality

• Protein solubility

  • Important for food applications

Different proteins and their solubility

• Globular proteins (milk, blood and soy): high solubility

  • Combination of a helices and B sheets alternated by random coils

• Fibrillar proteins (gelatin, meat): low solubility

  • The entire peptide is arranged within a single regular secondary structure

• Random coil proteins (caseins): high solubility

  • Can turn into micelles

• Other proteins (gluten): low solubility

Classification based on solubility

Osborne fractionation

• Can help to identify different amino acids in protein. 

Classification based on in-solubility

Amide group

Is not an amino acid!!

Where is the C-terminal?

Electrostatic interactions between proteins

Consequence of charge for functional properties

• At the isoelectric point proteins are slightly attracted to each other due to hydrophobic forces

• Can aggregate

• Solubility is low at isoelectric point.

• Charged proteins can have interactions with other charged molecules.

Isoelectric point

The pH at which a molecule, such as a protein or amino acid, has no net electrical charge

Chemical reactions in proteins during processing

• Hydrolysis of peptide bonds

• Formation of lysino-alanyl derivatives

• Formation or reshuffling of S-S bridges

• Deamination of decarboxylation

• Maillard reaction (works better at extreme high pH or extreme low)

Effect of processing proteins

• Loss of essential amino acids

• Crosslinking of proteins → decreased digestibility

• Change in texture

• Change of allergenic potential

• Formation of aroma compounds/off-flavors

• Change of charge

Hydrolysis of peptide bonds

Lysino-analyl derivatives step 1

Lysino-analyl derivatives step 2

Lysino analnyl derivatives and boiling

• The more boiling, the more lysinoalanine is formed

• Therefore the derivatives are an indication of heat damage

Reactions with disulfide bridges

1. Oxidation or reduction of sulfhydryl groups

2. Reshuffling of disulphide bonds

Decarboxylation

• Indicator of microbial spoilage as volatile compounds are formed (aroma and off-flavors)

Deamination

• Indicator of microbial spoilage as volatile compounds are formed (aroma and off-flavors)

• Side group needs to have an OH on the beta carbon atom (if its enzymatic, de-amination is possible for all amino acids)

Strecker degradation

• combination of decarboxylation and deamination

• Removal of a-carboxylic acid and a amino gorup:

• results in:

  • Formation of aldehydes (aroma compounds) 

Free amino acids vs amino acids

Requirements for an isopeptide bond formation

The side group needs to contain a -COOH

Disadvantage of reactions that can cause crosslinking op peptides

Can decrease digestibility

Disadvantage of amino acids with reactive side groups

Can cause loss of essential amino acids

Physical changes caused by heat

• When heated above denaturation temperature (60-80C)

• At timescales of seconds - minutes

• Increased mobility of protein molecule

• Stress on the stabilizing interactions

• Exposure of hidden hydrophobic groups

• Exposure of S-S/SH groups

• Leads to: unfolding and/or aggregation

When can aggreagtion in proteins also happen if there is no denaturation?

At the iso-electric point

Reversible vs irreversible denaturation/unfolding

• Changing the folding of a protein is reversible

• Aggregation and gelling is not reversible

Effect of denaturation on solubility

Aggregation at low and high concentration

• Precipitation at low concentration

• Gelation at high concentration

Important effects of protein denaturation

• Decreased solubility

• Aggregation

• Increased accessibility of peptide bonds to proteolytic enzymes

• Loss of biological activity

• Increased reactivity

Aggregation at high ionic strength/low ionic strength

• At high ionic strength/close to the isoelectric point, aggregation and gelation is easy

• At low ionic strength, or far from the isoelectric point, the electrostatic repulsion prevents aggregation, therefore more proteins will refold. 

What reactions cause hydrolysis of proteins that leads to changes in taste:

Enzymatic reaction:

• Fermentation products (e.g. cheese)

• infant formula/clincal food (for reduced allergenicity)

• sports nutrition

Heating (high temperatures/acidic conditions)

• animal nutrition (fish meal)

Example of a sweet tasting peptide

Aspartame

• Sweetening dipeptide (Asp-Phe-CH3)

• Sweetening power is 180 times that of sucrose

• The sweet taste is probably related to the polarity of the molecule

Taste of amino acids

• The amino acid reacts with the taste receptor on your tongue in three different places. 

1. Binding spot for proton acceptor: carboxylic group

2. Proton donor: Free amino group

3. Rest of the receptor interacts with the side chain. 

Effect of stereochemistry on taste of amino acids.

• This is because receptors are stereoselective

• If amino acid has a big apolar side group residue: then we have a bitter taste for L-amino acids but we have a very sweet taste for D-amino acids

• In proteins mostly L-amino acids occur and usually are bitter

• If side chain is polar or small apolar, the L-amino acid is sweet/neutral and neutral for D-amino acid.

Bitterness of amino acids

• Is related to their hydrophobicity

Rules of thumb on taste of peptides

1. Amino acids in a peptide are more bitter than free amino acids

2. Non-terminal amino acids are more bitter than terminal amino acids

3. A peptide is bitter when the average hydrophobicity >1400 cal/mole

4. Peptides > 6000 Da are not bitter

What does solubility influence?

• Visual appearance

• Texture (particles in product)

• Digestibility

• Foam/emulsion properties

Properties determining protein solubility

Balance between hydrophobic attraction and electrostatic repulsion

• Charge (depends on pH and ionic strength)

• Hydrophobic exposure (depends on folding state)

• Presence of polar groups (constant)

Contribution of amino acid side chains ((non)polar,(non)charged)

Non polar non charged: low solubility

Polar non charged: high solubility

Polar charged: high solubility

Where are amino acids usually found on a protein and why + two exceptions

Polar (hydrophilic): predominantly at the surface

Non-polar (hydrophobic): predominantly in the interior

This gives minimum free energy of the system

Exceptions: Cys and Pro

Solubility as a function of pH

At isoelectric point, proteins usually aggregate, and that is usually the minimum solubility level

Solubility as function of ionic strength

• The more salt added the more soluble it becomes

• Because charges on the protein are neutralized by salt ions

Effect of low salt concentration with protein

• There is more interaction of the protein with water molecules

• Protein solvent interactions become stronger

• Known as salting in of protein

Effect of high salt concentration on protein

• Known as salting out

• Less water molecules are available to interact with the protein

• Protein-protein interactions become stronger than protein-solvent interactions

Analysis technique for (in)soluble proteins

• Dumas/Kjedahl methods

• Determines N content

Analysis technique for soluble proteins

• Direct or indirect

• Indirect: proteins in a solution, in addition we put a reagent, a dye.

  • Dyes: biureet, phenolreagent, coomassie reagent

  • These dyes form a colored complex in the presence of a protein

  • More color = more protein

Kjeldahl/Dumas

Determination of total N content:

• All amino acids contain nitrogen.

• Kjeldahl:

  • Uses a powder of concentrated sulfuric acid, boil it a 350C

  • This destroys the protein and liberates the free nitrogen atoms

• Dumas:

  • Total destruction of system, amino acids are burned without acid at 1200C

  • N gas is released

Conversion from N content to protein content

g N/100g DM * conversion factor = g protein/100g DM

Calculate based on amino acid composition

Typically used value: 6.25 (g protein/g N), in most cases this is actually wrong.

Instead, use conversion factor

Spectrophotometry

• Soluble protein: amino acid with aromatic side chain → conjugated system absorbs UV light

• You can use lambert beer law

Advantages and disadvantages of total N content vs spectrophotometric

How to find which proteins are present?

SDS polyacrylamide gel electrophoresis (SDS-PAGE):

1. Unfolding of protein and dissociate complexes

2. Separation of proteins on a gel under influence of an electric field

3. Separation distance on gel relates with molecular weight

4. Calculate molecular weight by comparison with marker (contains protein of known Mw)

All methods used to determine information about proteins