Download Lecture 2 - Proteins_in_food

Classes:
Proteins
Enzymes
Structural proteins (collagen, keratin, elastin, …)
Contractile proteins (myosin, actin)
Hormones (insulin, growth hormone, …)
Transfer proteins (serum albumin, transferrin, haemoglobin, …)
Antibodies
Storage proteins (plant seed proteins, egg albumin, …)
Toxins, allergens, …
Receptors
All proteins can be used as food. Only those proteins that are easily
digestible, non-toxic, nutritionally adequate, functionally usable in food
products and available in abundance are considered as food proteins.
Major sources: milk, meat, eggs, cerials, legumes, and oil seeds.
Solubility
Proteins
Based on solubility characterisatics, proteins can be classified as:
1. Albumins
soluble in water at pH 6.6
examples: serum albumin, ovalbumin, -lactalbumin
2. Globulins
soluble in diluite salt solutions at pH 7.0
examples: glycinin, phaseolin, -lactoglobulin
3. Glutelins
soluble only in acid (pH 2) and alkali (pH 12)
examples: wheat glutelins
4. Prolamins
soluble in 70% ethanol
examples: zein, gliadins
Solubility is influenced by several solution conditions such as pH, ionic
strength, temperature, presence of organic solvents, …
Denaturation of proteins – can result in:
-loss of some functional properties
-insolubilization
-inactivation (examples: trypsin inhibitors, lectins, lipoxigenases)
-better foaming and emulsifying properties
-thermal denaturation
gel formation
Protein denaturing agents
Temperature:
Heat is most commonly used in food processing and preservation.
Increasing temperature
many proteins denature. However, some
proteins denature by decreasing the temperature !!
Examples: glycinin of soybeans aggregates and precipitates at 2°C;
-casein dissociates from casein micelles at low temperatures and this
alters the physicochemical properties of the micelles.
Tm (melting temperature): 50% native and 50% denatured.
Temperature destabilizes most non-covalent interactions.
Water facilitates thermal denaturation; dry protein powders are extremely
stable to thermal denaturation.
Some sugars (sucrose, lactose, glucose, …) stabilize proteins against
thermal denaturation.
For some proteins (-lactoglobulin, soy proteins, serum albumin) salts
(e.g. 0.5M NaCl) increase the Tm considerably.
Protein denaturing agents
Hydrostatic pressure:
At high pressure (between 1 and 11 kbar) proteins denature at 25°C.
Denaturation is due to compressablilty of most proteins.
Pressure-induced denaturation is highly reversible.
Used for microbial inactivation.
Pressure processing does not affect colour or damages amino acids,
flavour, does not generate toxic substances.
Disadvantage: expensive.
Shear:
High mechanical shear generated by shaking, kneading, whipping, etc.
causes denaturation.
Denaturation because of air trapping and adsorption of proteins at the
air-liquid interface (hydrophobic residues orient towards the gas phase).
During extrusion or high shear blending: high temperature and high shear
forces result in irreversible denaturation.
Protein denaturing agents
pH:
Degree of unfolding is greater in alkaline solution than at low pH.
pH-induced denaturation is often reversible.
Organic solvents:
Weaken hydrophobic interactions.
Detergents:
Example: SDS (sodium dodecyl sulfate).
SDS binds to denatured protein
shift in N  D equilibrium.
Because of strong binding: irreversible denaturation.
SDS induces an extended helix.
Protein denaturing agents
Organic solutes:
Examples: urea, guadinium hydrochloride.
Denaturation due to solubilization of hydrophobic amino acids in urea
and guadinium-HCl.
These denaturants bind to denatured proteins
shift from native to
denatured conformation.
Urea and guanidinium-HCl break down the hydrogen-bonded structure
of water.
In urea solutions: formation of cyanate and ammonia. Cyanate binds to
amino groups, changing the charge of the protein resulting in irreversible
denaturation.
Protein denaturing agents
Chaotropic salts:
Examples: NaSCN, NaClO4
In general, the ability of various anions to influence the structural stability
of proteins follows the Hofmeister series:
F– < SO42– < Cl– < Br– < I– < ClO4– < SCN– < Cl3CCOO–
Fluoride, sulfate and chloride salts are structure stabilizers, the salts of
the other anions are structure destabilizers.
Proteins in food and feed
Protein sources:
Plant origin (legumes, cerials)
Animal origin (meat, fish, milk, eggs)
Plant proteins are incomplete proteins.
Cerials: rich in cysteine, poor in lysine.
Legumes: deficient in methionine and cysteine.
In all human cultures, cerials and legumes are combined in the diet.
Examples: rice + beans; corn + beans.
Milk proteins
Caseins: phosphoproteins
Whey proteins (milk serum)
Minor proteins: milk fat globule membrane, enzymes, etc.
Caseins:
Precipitate at low pH (4.6 at 20°C).
In cow milk, 80% of the milk proteins are caseins.
Different classes: -, -, - and -caseins.
-casein = proteolytic fragment of -casein.
Total protein
acidify to pH 4.6 & filter
Precipitate: caseins
Filtrate: serum or whey proteins
50% saturated ammonium sulfate
Precipitate: lactoglobulin
Supernatant: lactalbumin
Milk proteins
Skimmed milk:
Milk from which fat has been removed.
Serum or whey:
Milk without fat globules and casein micelles.
Casein micelle: Ø 130-600 nm
contains 104–105 casein molecules.
Casein micelles are built up of sub-micelles (25-30 molecules of -, and -caseins.
Why do caseins form micelles? N-terminal ends of - and -caseins are
rich in phosphoserine (bind Ca2+); -caseins are not phosphorylated but
glycosylated and stabilize the casein micelles (proteolysis of -casein by
chymosin
casein precipitates).
Sub-micelles are linked together with Ca2+ ions or through chains of
colloidal Ca-phosphate particles.
Casein micelles: high concentration of protein, calcium and phosphate
without risk of forming Ca-phosphate crystals.
(a) cross-section of a typical
submicelle
(b) cross-links
between
submicelles
through
colloidal Caphosphate
[Ca9(PO4)6]
particles
(c) Formation
of full-size
micelles
-N-acetylneuraminyl(2-4)--galactosyl-(1-6)N-acetylgalactosamine
Milk proteins
Serum proteins:
-lactalbumin
-lactoglobulin
immunoglobulins
enzymes: lactoperoxidase (anti-bacterial lactoperoxidase system),
phosphatase, lipase (sets fatty acids free
rancid flavour)
Egg proteins
Egg yolk: triglycerides, phospholipids, cholesterol, proteins
(phosphoproteins, lipoproteins and IgY)
Egg white (albumen): 85% water, 10% protein, some lipid and
carbohydrate.
Egg white proteins:
ovalbumin (54%), conalbumin (12%), ovomucoid (11%), lysozyme
(3.4%), avidin, ovoglobulin, flavoproteins.
Ovalbumin: a phosphoglycoprotein (GlcNAc2–Man4);
readily denatured by heating;
contains 4 –SH and 1 S–S
when the protein unfolds, S–S
exchange
new intra- and interdisulfide bridges
gel formation;
denatures at air-water and fat-water interfaces
foaming when
water is displaced by air or fat.
Conalbumin: a glycoprotein; metal-binding (iron
antimicrobial);
foaming stabilized by Cu2+ ions.
Ovomucoid: trypsin inhibitor; glycoprotein responsible for viscosity.
Lysozyme: antimicrobial.
Avidin: binds biotin
antimicrobial.
Proteases and
Protease Inhibitors
Protein protease inhibitors have been found in many plant tissues,
particularly in legume seeds and other storage organs, but also in
animal fluids (serum) and micro-organisms.
Major classes of proteases:
Serine proteases
Thiol proteases
Aspartyl proteases
Metalloproteases
Proteases – some examples
Kunitz soybean trypsin inhibitor
Soybean Bowman-Birk
trypsin-chymotrypsin inhibitor
Soybean Bowman-Birk
trypsin-chymotrypsin inhibitor
Action of trypsin inhibitors
Protein protease inhibitors are tightly folded molecules that keep
their structure even after cleavage of the specific peptide bond.
The fragments generated remain bound in the active site
no regeneration of active enzyme
Are protein protease inhibitors antinutritive factors?
Early observations: unless cooked for several hours, soybeans do not support
the normal growth of rats and other small experimental animals (Osborne and
Mendel, 1917).
Soybeans contain a heat-labile protein that inhibits the proteolytic activity of
trypsin (Bowman, 1944).
The nutritive quality of soybean flour heated at various temperatures increased
in proportion to the destruction of trypsin inhibitor
is trypsin inhibitor the cause of the poor utilization of raw soybean?
Lots of experiments ……
Conclusion: protease inhibitors cause pancreatic enlargement.
According to Yehudith Birk, the evidence that the inhibitors constitute a hasard
to health is only presumptive and should be placed in perspective in relation to
the level of total protease inhibitors in the overall diet. Most of the in vivo
research has been done with small animals that consumed large quantities of a
particular food component over a relatively long period of time, a situation quite
remote from the eating patterns of humans.
Plant protease inhibitors have been shown to be potential cancer chemopreventive agents as shown of a wide variety of in vitro & in vivo model systems.
Mechanism of action is not fully understood.
Plant protein protease inhibitors are involved in protection against insects.
Problem: development of resistance.
Meat proteins
Muscle: 70% structural or fibrillar protein – 30% water-soluble proteins
Myosin: most abundant of the muscle proteins
has enzyme activity (ATP
ADP + Pi)
Actin (actin/myosin ratio = 1/3)
G-actin: globular – F-actin: fibrous actin
Actomyosin: complex between actin and myosin
Structure of skeletal muscle
Skeletal muscle
consists of parallel
bundels of muscle
fibers. Each fiber is
a single, very large
multinucleated cell,
20-100µm in
diameter, often
spanning the length
of the muscle. Each
fiber contains about
1,000 myofibrils,
2µm in diameter.
Myofibrils consist of
thin filaments
(contain proteins
actin, tropomyosin &
troponin) and thick
filaments (myosin).
Relaxed muscle
Contracted muscle
Thick filaments: bipolar structures created by the association of many myosin molecules.
Muscle contraction (a): occurs by sliding of thick & thin filaments past each other so that
the Z disks in neighbouring I bands approach each other.
(b) Thick and thin filaments are arranged within a fiber in such a way that each thick
filament is surrounded by six thin filaments.
Muscle contraction/decontraction
Nerve impuls on sarcolemma provokes release of Ca2+ ions from the
sarcoplasmic reticulum
Ca2+ binds to troponin of the thin filaments, thereby causing a change in the
shape of the troponin molecules, and cause tropomyosin to move and expose
“active sites” on the actin molecules
The activated actin molecules react with myosin of the thick filaments and ATP
is bound to the site of the head of myosin
Hydrolysis of ATP causes movement of the two filaments with respect to each
other and contraction occurs
When nerve impulses cease: Ca2+ is pumped back into the sarcoplasmic
reticulum and tropomyosin binds to actin
no interaction between actin and
myosin
muscle is relaxed and returns to its extended state by the action of
other muscles
Thus: ATP is required for contraction and for the active pumping of Ca2+
tropomyosin
troponin
actin
Molecular mechanism of muscle contraction
Principle: conformational changes in the myosin head that are coupled to stages in the ATP
hydrolytic cycle cause successive dissociation from/reassociation with the actin subunits.
Reaction sequence in actomyosin-catalyzed
ATP hydrolysis
ADP
ATP
Pi
Actin
4
Actin – Myosin
Actin – Myosin – ADP - Pi
3
Myosin – ATP
2
Myosin – ADP – Pi
Actin
1
H2O
Rigor mortis
During life: ATP derived from respiration and glycolysis (depending on the
activity of the muscle)
Upon death: only glycolysis
formation of lactic acid – pH ↓↓ to 5-5.5
also glycolysis stops
no ATP formed
no pumping of Ca2+
ions
permanent link between thick & thin filaments,
i.e.: RIGOR MORTIS
The drop in pH prevents outgrowth of putrefactive and pathogenic bacteria
and also causes moisture release (“dripping”)
During conditioning (hanging) of the carcas: rigor mortis disappears because
of the action of a specific protease that is only active arounds pH 5 and
requires Ca2+ for its activity
The role of this enzyme in the living animal is obscure
The protease causes the breakdown of the thin filaments at a place where
they are joining the Z-line filaments
meat becomes tender
Wheat proteins
Can be classified according to their solubility properties in different solvents:
Two of them, i.e. gliadins and glutenins,
are of major importance for us as
components of “gluten”.
Gluten forms a network that gives dough its visco-elastic properties:
Gliadins and glutenins are very different proteins.
Gliadins: ● monomeric proteins
● low molecular weight
● only intramolecular disulfide bridges
● soluble in alcohol
Glutenins:
● polymeric proteins
● high molecular weight
● subunits linked together with intermolecular disulfide bridges
The visco-elastic properties of wheat gluten is the result of the
structural differences between the main components:
Glutenins:
● highly elastic
● give strength to dough
Gliadins: ● not elastic
● upon hydration they become very viscous and extensible
● allow gluten to expand during leavening
Wheat proteins exhibit no anti-nutritional properties and are highly digestible.
They find applications in feed for calves, fish, piglets, pets, ...
Some people are suffering from “gluten intolerance” or coeliac disease.
Coeliac disease:
caused by a chronic reaction to gluten and result in destruction of the villi in the
small intestine
malabsorption of nutrients
AVOID gluten (wheat is replaced by corn, rice, ...).
Texture of gluten-free baked products can be provided by xanthan gum and
guar gum.
Functional properties of proteins
Functionality of food proteins is defined as: “those physical and chemical
properties that affect the behaviour of proteins in food systems during
processing, storage, preparation and consumption”.
Functional roles of food proteins in food systems:
solubility, viscosity, water-binding, gelation, cohesion – adhesion, elasticity,
emulsification, foaming, fat- and flavour-binding.
Examples: egg white possesses multiple functionalities such as gelation,
emulsification, foaming, water-binding and heat-induced coagulation. The
multiple functionalities of egg white result from complex interactions between
ovalbumin, conalbumin, lysozyme, ovomucin and other albumin proteins.
Physical and chemical properties that govern functionality:
size and shape; amino acid composition and sequence; net charge and
charge distribution; hydrophobicity/hydrophilicity ratio; secondary, tertiary
and quaternary structure; molecular flexibility/rigidity; ability to interact with
other food components.
Functional properties related to hydrodynamic properties:
viscosity; gelation; texturization.
Functional properties related to the chemical and topographic properties of
the protein surface: wettability; dispersibility; solubility, foaming;
emulsification; fat- and flavour-binding.
Attempts to predict functional properties from physicochemical
properties have not been successful.