Proteins by yaofenji


           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
   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
    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
 Primary  Secondary  Tertiary 
1. Primary structure
      Backbone of the protein molecule
      Described by the amino acid
       sequence that make up a
           Amino acids are linked to each   R-group
                                                       Condensation reaction
            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
      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
3. Tertiary structure
   Represents the secondary structure folding into a 3D
   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
   Hydrophobic groups on inside       Results
                                           Decrease solubility
                                           Increase viscosity
                                           Altered functional properties
             Heat pH Pressure              Loss of enzymatic activity
             Oxidation Salts Etc.          Sometimes increased
            Proteins – basic concepts
Factors causing protein denaturation
 pH                                                100

       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-

        covalent interactions holding the protein
        together causing it to eventually unfold
       Freezing can also denature due to ice
        crystals & weakening of hydrophobic
                                                       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)

   A. Isomerization (racemization)
           L- to D-amino acids
           We cannot digest D-amino acids
           Not a very serious problem in texturized vegetable protein
       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
      LAL formation is usually not a problem in                       H
       food processing but loss of lysine may occur                N C CO
                                                                   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
      Severe heat treatment drastically reduces protein solubility and functionality and
       may give decreased digestibility/bioavailability
  1. Degradation of cysteine
              H3C - CH2SH                                H3C - CH2OH + H2S(g)
      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
                                            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

 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,
The properties of food proteins are altered by environmental
conditions, processing treatments and interactions with other
         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
        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
       At low salt concentrations
        protein solubility increases
       At high salt concentrations
        protein solubility decreases

                        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
           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
        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
        Heat
        pH
        Pressure
        Shearing
    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
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
      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
                           Denaturation (%)


         Proteins – functional properties
   Factors influencing gel properties
    pH, salts, T, heating/cooling
 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
       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- Cl-               Loss of repulsion
               +                 +
                                                     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


                                            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

                  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
   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
       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



      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
       Type of protein
            To form a good emulsion the protein has to be able
              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
       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
    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
   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 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
            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
              The foam structure breaks down
              Proteins become too denatured
        Proteins – functional properties
   How do we measure foam formation and
     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
1. Chemical modification
       Reactive amino acids are chemically modified by adding a group to
            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
        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

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