Quantitative Analysis of Gelation in Egg Protein Systems

1
Quantitative Analysis of Gelation in Egg Protein
Systems

• Food Technology. Vol. 38, Num. 5. May 1984. Pp.
  67-96.

2
ABSTRACT

• Important function of proteins in food systems is
  gelation
• This involves the formation of a
  three-dimensional matrix
• Through inter-protein hydrogen bonding
• Allows the immobilization of water within the gel
  structure
• Success of certain cooking products can be
  controlled by the
• Coagulation gelation of proteins
• Irreversible, heat-induced coagulation or egg
  proteins

3
ABSTRACT

• Understanding of of the gelation process will
  permit manipulation of variables to obtain a gel
  of desired textural characteristics and
  functional properties
• Coagulation of proteins can be brought about by
• Heat, pressure, salts, acids, alkalies, alcohols
  or denaturation agents such urea

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OBJECTIVE

• This article will concentrate on the quantitative
  analysis of heat induced coagulation or gelation
  of egg-albumen on proteins
• Factors affecting the kinetic parameters of
  gelation and the rheological properties of the
  gel will be discussed as well as methods used to
  measure gelation

5
TERMINOLOGY

• DENATURATION
• Is the process in which a protein or polypeptide
  is transformed from an ordered to a disordered
  state without rupture of covalent bonds
• or
• Any process, except chemical modification, not
  involving rupture of peptide bonds which causes a
  change in the three-dimensional structure of a
  protein from its native in vivo form
• Denaturation involves protein-solvent
  interactions and leads to changes in physical
  properties, such as loss of solubility of the
  protein
• Sometimes unfolding of the protein structure is
  considered part of denaturation

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TERMINOLOGY

• Aggregation
• Is a general term reffering to protien-protein
  interactions, with formation of complexes of
  higher molecular weights
• Aggregation is usually governed by a balance
  between attractive and repulsive forces
• Attractive forces can be involve hydrogen bonds,
  covalent bonds such as disulfide linkages, and
  hydrophobic associations whereas repulsive forces
  can involve coloumbic forces which are affected
  by the net charge of the protein molecule or the
  ionic strength of the solution

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TERMINOLOGY

• Coagulation
• Is the random aggregation of already denatured
  protein molecules, in which polymer-polymer
  interactions are favored over polymer-solvent
  reactions
• The coagulum is often turbid, and the formation
  of the coagulum is usually thermally irreversible
• A coagulum may settle out of solution

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TERMINOLOGY

• Gelation
• Is an orderly aggregation of proteins, which may
  or may not be denatured
• Forming a three dimensional netword
• Polymer-polymer and polymer-solvent interactions,
  as well as attractive and repulsive forces, are
  balanced such that a well-ordered matrix can be
  formed
• The gel may be turbid or translucent, in the
  latter case the gel may be thermoreversible
• (The term gelation is also used in another
  context with respect to egg-yolk proteins. The
  phenomenon of egg-yolk gelation refers to the
  formation of an irreverisble gelled product upon
  freezing the yolk)

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Theory of Gelation

• NATIVE PROTEIN
• DENATURED PROTEIN
• AGGREGATED PROTEIN

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Theory of Gelation

• The first step is considered a denaturation
  process and the second step an aggregaton process
• Comparison of the rate of the denaturation step
  vs that of the aggregation step helps determine
  gel characteristics
• For a given rate of denaturation the rate of
  aggregation will be slow if the attractive forces
  between the denatured protein chain are small
• The resulting gel will be a finer network of
  protien chains, will be less opaque, and will
  exhibit less syneresis than one with a faster
  rate of aggregation

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Theory of Gelation

• Conditions favoring denaturation , such as high
  or low pH, have the opposite effect on
  aggregation of globuler proteins
• Due to the fact that a high net charge,
  protein-solvent interactions such as
  denatureation are favored
• Rather than protein-protein interactions such as
  aggregation
• The kinetics of the denaturation step relative to
  the aggregation step appear to be important in
  determining the type of gel produced

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Theory of Gelation

• KINETIC TERMS
• Reaction Rate Constant
• Z value
• Describes the temperature dependency of reaction
• Which is defined as the necessary rise in heating
  temperature, C, needed for a 10-fold increase in
  the reaction rate

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Factors Affecting Gelation

• Electrostatic Charge
• Is one of the most commonly investigated factors
• The pH as well as the ionic strength of the
  protein environment can alter the charge
  distribution among the amino acid side chains and
  can either decrease or increase the
  protein-protein interaction
• The main factor contributing to the heat-induced
  aggregation of ovalbumin is the degree of
  electrostatic repulsion among the denatured
  protein molecules

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Factors Affecting Gelation

• Electrostatic Charge
• When the heat-denatured protein concentration is
  high (gt0.5) the aggregate size decreases as the
  pH increases from 5.8 to 10.0
• This is due to increased repulsive forces among
  the protein molecules at the alkaline pH levels
• Decreasing the pH or adding cations decreases the
  negative charge and accelerates aggregate
  formation, as does increasing the ionic strength

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Factors Affecting Gelation

• Protein Concentration
• Is also a factor affecting aggregation
• Almost all of the protein aggregates, regardless
  of concentration, when ovalbumin is heated at 80C
• About 80 of the protein (pH 6.2) heated at 75C
  for 5 min aggregates, regardless of albumin
  concentration
• But at 70C, a concentration of at least 1 is
  required for aggregation.
• A higher protein concentration is probably needed
  to allow a closer association of molecules for
  aggregate formation at the lower temperatures

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Factors Affecting Gelation

• The Formation of Disulfide Bonds
• And the exposure of hydrophobic amino acid
  residues are thought to be involved in the first
  step of coagulation
• Proteins with higher percentages of hydrophobic
  amino acids are classified as coagulation-type
  proteins and concentration dependent
• While proteins with a lower percentage are
  gelation-type proteins and concentration
  independent

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Factors Affecting Gelation

• Further heating causes egg albumin to polymerize
  by intermolecular sulhydryl-disulfide exchange,
  forming a network
• Many globular proteins with differing sulfhydryl
  convents can form heat induced gels
• No correlation between disulfide or sulthydryl
  content and gel-forming ability can be found

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Factors Affecting Gelation

• Composition
• Of the albumin mixture also affects the
  aggregation of the proteins
• Denaturation temperatures of globulins, ovalbumin
  and lysozyme are 72.0, 71.5, and 81.5C,
  respectively
• The gel strength of the lysozyme gel is the
  highest, followed by globulin
• In binary mixtures of albumin proteins,
  aggregation occurs near the denaturation
  temperature of the least heat-stable protein
• The lysozyme gel is the firmest

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Measurement of Heat-Induced Gelation

• GEL STRENGTH
• Measure the gel strength or firmness of the gel
  after various heating times
• The rigidity of heat-induced 8.2 ovalbumin gels,
  as in measured by the initial slope of a
  force-distance curve given by a compression test
  varies with the pH of the solution, with maximum
  strength exhibited at either side of the
  isoelectric point
• The increase in firmness is not a linear function
  of temperature the higher temperatures produce
  gels considerably firmer then those at lower
  temperature

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Measurement of Heat-Induced Gelation

• GEL STRENGTH
• they found that gel strength increases with
  increasing pH and with increasing heating
  temperature (Table 2)

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Measurement of Heat-Induced Gelation

• SOLUBILITY
• The change in solubility of a heated protein
  system has also been monitored as an indicator of
  the coagulation or gelation process
• Egg albumin was shown to increase rapidly within
  the first minute, then slowly
• optical density of egg white at 550 nm increases
  with time heating (58C, 0 60 min)
• And increases more rapidly with decreasing pH
  (8.0 10.5)

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Measurement of Heat-Induced Gelation

• GRAVIMETRIC ANANLYSIS
• Of the coagulum or the supernatant after
  centrifugation has been another method of
  following the coagulation process
• Recovering or separating the precipitate or
  coagulum from the solution can be difficult
  because the precipitates can vary from rapidly
  sedimenting flocculates to nonsedimenting,
  sol-like opaque aggregates

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Measurement of Heat-Induced Gelation

• FORMATION OF DISULFIDE LINKAGES
• Chemical properties such as the formation of
  disulfide linkages can be determined as
  coagulation proceeds
• Measured the number of free sulfydryl groups on
  solutions of dissolved albumin coagulums formed
  by heating 4.5 solutions at 80C
• The number of moles of SH groups of protein
  decreased from 5 to 3.5 after 1 min of heating
  and reached a plateau of 3 after 5 min

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Measurement of Heat-Induced Gelation

• ELECTROPHORESIS
• The disappearance or appearance of different
  polyacrylamide gel electrophoretic (PAGE) bands
  of proteins with different heating time and
  temperatures has been observed

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Measurement of Heat-Induced Gelation

• STRUCTURAL CHANGES
• The denaturation step of the gelation process has
  been studied by observing structural changes of
  egg protein solutions upon heating
• The reduced viscosity of a 1.8 ovalbumin
  solution increases with temperature of heating
  above 65C and with time of heating
• Removal of the aggregate by centrifugation yields
  supernatants with reduced viscosities

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NEW METHOD DEVELOPED

• The above methods for measuring heat-induced
  coagulation do have certain limitations, one of
  which is the need to make single-point
  measurements on many samples at different time
  intervals throughout the process to obtain a
  continuous