Proteins.

Name: Proteins.
Uploaded: 2017-06-28T16:20:29+00:00
Duration: PTM32S50
Channel: Branden Dalton
Description: Proteins.

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

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 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

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

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

Polar Amino Acids - Hydrophilic

Non-polar Amino Acids – Hydrophobic/Amphophilic

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 a polypeptide chain Amino acids are linked to each other in a chain via a peptide bond A covalent bond This backbone structure dictates rest of the structure Condensation reaction R-group

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

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 -sheets

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

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

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

Types of forces/bonds that stabilize the protein structure

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

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 %Denatured 100 pH 12

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 %Denatured 100 T (C)

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)

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

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)

Important reactions of proteins and effect on structure and quality Alkaline reactions Soy processing (textured vegetable protein) 0.1 M NaOH for 1 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)

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

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

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

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

Heat Degradation of cysteine Leads to terrible flavor problems  H2S(g) Amide crosslinking Need severe heat for this reaction - not very common Transglutaminase enzyme also does the same thing

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 – 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

Example of protein functional properties in different food systems 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, marshmallows

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

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

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) Salt concentration %Solubility

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

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: Protein samples at different pH’s at 0.1M NaCl Centrifuge at 20000g for 30 min Solubility (%) = protein left in supernatant * total protein More soluble Less soluble

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 Sol

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

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) Denaturation (%) Gel strength/Viscosity cooling T heating

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 Denaturation (%) Gel strength/Viscosity cooling T heating

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) Denaturation (%) cooling T Gel strength/Viscosity heating

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)

Factors influencing gel properties pH Salts T heating/cooling scheme

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

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)

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 + NaCl Cl- Loss of repulsion Loss of gel strength Loss of water-holding

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 mM) Max gel strength seen at (a) pH 3.5 and 30 mM NaCl; (b) pH 7.5 and 50 mM NaCl

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

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 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

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

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

Salts/ionic strength This is highly protein dependent muscle proteins Na+ Cl- NaCl

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

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 and protein gels are a good example pI

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

Emulsification Proteins can be excellent emulsifiers because they contain both hydrophobic and hydrophilic groups LOOP TRAIN + ENERGY

Emulsification Whey protein stabilized emulsion Both phases Lipid phase removed (protein matrix showing)

Whey protein stabilized emulsion Both phases Lipid phase removed (protein matrix showing)

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

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)

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

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)

Foaming FOAM FORMATION FOAM BREAKDOWN

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

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

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)

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

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)

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

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 % of original biological value Often used in very small amounts due to possible toxicity Not the method of choice for food proteins

Protein modification to improve function Chemical modification Example of types of chemical groups that can be added to proteins

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

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

Proteins.

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