Proteins

Proteins – basic concepts Role of proteins Nutrition Energy and essential amino acids Can possess anti-nutritional properties Trypsin inhibitors in soy = reduced digestibility Allergens – IgE mediated food allergy attributed to naturally occurring food proteins (negative immunological response to a protein) Toxins – α-amanitin a cyclic peptide found in a poisonous mushroom species Structure Provide structure in living organisms and also foods Collagen – main component of connective tissue Gelatin – hydrolyzed collagen – eg. Jello Proteins roughly contain 5-50 % C, 6-7 % H, 20-23 % O, 12-19 % N, and 0-3 % S (Barret, 1985 –Chemistry and Biochem of Amino Acids) Measuring N content is often used to estimate the protein content in foods Catalysts Enzymes (which are proteins) catalyze chemical reactions in living tissue and foods

Proteins – basic concepts Role of proteins Functional properties Gelation Emulsifiers Water bonding Increase viscosity Texture Browning Have amino acids that can react with reducing sugars Maillard Browning Acrylamide (produced by asparagine rxn with reducing sugars) Some enzymes can also cause browning Polyphenol oxidase - Apples

Typical protein contents of the edible portion of various foods Food or Beverage  Protein g/100g Apples, raw, with skin (09003)  0.26 Beer, regular (14003)  0.46 Milk, human, mature (01107) 1.03 Bananas, raw (09040) 1.09 Cabbage, raw (11109) 1.28 Potatoes, white, flesh and skin, raw (11354) 1.68 Potatoes, microwaved, flesh and skin (11675) 2.44 Corn, sweet, yellow, canned, whole kernel, drained solids (11172) 2.46 Rice, brown, long-grain, cooked (20037) 2.58 Soy milk, original and vanilla, with added Ca, Vitamins A & D (16139) 2.60 Milk, whole, 3.25% milk fat, with added vitamin D (01077) 3.15 Ice creams, vanilla (19095) 3.50 Yogurt, plain, low fat (01117) 5.25 Tofu, soft (nigari) (16127) 6.55 Cereals, ready-to-eat, cornflakes (08020) 6.61 Chocolate, dark, 70-85% cacao solids (19904) 7.79 Rice, brown, long-grain, raw (20036) 7.94  Lentils, mature seeds, boiled (16070)  9.02 Bread, white (18069)  9.15 Pasta, fresh-refrigerated, plain (20093)  11.31 Egg, whole, cooked, hard boiled (01129)  12.58 Cod, Pacific, raw (15019)  15.27 Cod, Pacific, cooked, dry heat (15192)  18.73 Almonds, dry roasted (12063)  21.06 Chicken, breast meat only, raw (05062)  21.23 Cheese, cheddar (01009)  24.90 Tuna, light, canned in water, drained solids (15121)  25.51 Lentils, raw (16069)  25.80 Chicken, breast meat only, roasted (05064)  31.02 Cheese, Parmesan, hard (01033)  35.75 Values obtained from the USDA National Nutrient Database, numbers in parentheses are the USDA 5 digit identifier

Proteins – basic concepts 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 = natural form) 20 essential amino acids – book has 21, includes selenol (contains Selenium) which was discovered in 2002 More amino acids exist in nature but are not genetically coded Differ by their side chains (R-groups) All have central α C, basic amino group, and a carboxyl group Amino acid charge behavior Neutral Acidic Basic α α

Proteins – basic concepts Amino acids are generally grouped into 3 classes Charged and polar Uncharged and polar These two classes of amino acids are found on the surface of proteins 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 structure Primary  Secondary  Tertiary  Quaternary 1. Primary structure Linear sequence of amino acids of the protein molecule (backbone) 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 Sequence always described N-terminal to C-terminal Amine partial (+), carboxyl partial (-) This backbone structure dictates rest of the structure (2°, 3°, etc. structure) Condensation reaction

Proteins – basic concepts 2. Secondary structure Refers to arrangement of the polypeptide backbone Random coil Helical and sheet Predictable arrangement of two main secondary structures (regular spatial arrangement) -helix -sheet a) -helix A coiled structure formed with internal H bonds (between C=O and N-H) Amphiphilic – both polar and non-polar surfaces Is the main structure in fibrous proteins (myosin is an ex.) – more often in hydrophilic proteins Less in globular proteins

Proteins – basic concepts b) -sheet “Flat sheets” parallel or antiparallel structure These sheets are stabilized with regular bonding of C=O with NH (via H-bonds) between -sheets Antiparallel are more stable due to better alignment of hydrogen bonding atoms More stable than α-helix High amount in insoluble (hydrophobic) proteins, but more stable to denaturation c) Random coils Absence of secondary structure (order) Irregular random arrangement of a polypeptide chain -sheets

Proteins – basic concepts 3. Tertiary structure Represents the secondary structure folding into a 3D conformation/structure This is the highest degree structure of many proteins The type of tertiary structure formed is dictated by Amino acid sequence -helix/-sheet Proline content α-helix breaker Stabilizing forces H bonding Solvent conditions Dictates where amino acid residues are located Surface – interact w/ solvent Interior – interact w/ side chains (effects stability) β-lactoglobulin

Proteins – basic concepts 4. Quaternary structure A complex of two or more tertiary structures The units are linked together through non-covalent bonds β-lactoglobulin Milk (pH 6.8)– 37 kDa dimer Cheese (pH 4.5) – 144 kDa octamer Some proteins will not become functional unless they form this structure. Examples: Hemoglobin Myosin 2 heavy chains, 4 light chains (475 kDa)

Proteins – basic concepts Types of forces/bonds that stabilize the protein structure Solvent-solute interactions

Molecular forces involved in protein structure Type Bond Energy (KJ/mol) Functional groups from amino acid side chains involved Van der Waals interactions (dipole) 1-9 Permanent, induced and instantaneous dipoles Hydrophobic interactions 4-12 Aliphatic and aromatic side chains Hydrogen bond 8-40 Carboxyl, amide, imidazole, guanidino, amino, hydroxyl and phenolic groups Electrostatic interactions 42-84 Carboxyl and amino groups Covalent bond 330-380 Disulfide moiety

Proteins – basic concepts Proteins exist in two main states DENATURED STATE Loss of native conformation Altered secondary, tertiary or quaternary structure May be reversible or irreversible, partial or complete 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

Proteins – basic concepts Factors causing protein denaturation pH Too much charge can cause high electrostatic repulsion between charged amino acids and the protein structure unfolds As unfolds, hydrophobic interior is exposed. Unfavorable because of buried groups phenolic Alkyl etc. %Denatured 100 pH 12

Proteins – basic concepts 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 (water participation less) %Denatured 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 (e.g. SDS) 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 Hydrolysis Hydrolysis of proteins also referred to as proteolysis Cleaves peptide bond and adds H2O (reverse of peptide bond formation) 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.) Hydrolyzed protein usually listed as an ingredient on soy sauce label Modifies protein functional properties E.g. increased solubility Increases bioavailability of amino acids Excessive consumption of free amino acids is not good however (too much N)

Proteins – basic concepts Important reactions of proteins and effect on structure and quality Maillard reaction (carbonyl - amino browning) Can change functional properties of proteins Changes color (browning) Changes flavor (roasted, buttery, burnt etc.) Decreases nutritional quality (participating amino acid lost from a nutritional standpoint)

Proteins – basic concepts Important reactions of proteins and effect on structure and quality Alkaline reactions Soy protein concentrates (textured vegetable protein) 0.1 M NaOH for 1 hr @ 60°C or greater Denatures proteins by hydrolysis Some amino acids become highly reactive NH3 groups in lysine SH groups and S-S bonds become very reactive (e.g. cysteine) Loss of some aa as a result (cysteine, cystine, serine, and threonine), ↓ nutritional quality (minimal)

Proteins – basic concepts 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) Lysinoalanine formation (LAL) Lysine becomes highly reactive at high pH and reacts with dehydroalanine forming a cross-link = lysinolalanine Lysine, an essential amino acid, becomes unavailable (problem because is limiting aa in cereal grains) Lysinoalanine

Proteins – basic concepts Heat Mild heat treatments lead to alteration in protein structure and often beneficially effect digestibility or bioavailability (↓ solubility) However, severe (above 200 °C) heat treatment drastically reduces protein solubility and functionality and may give decreased digestibility/bioavailability Pyrolysis Degradation of cysteine Amide crosslinking (isopeptide bond formation) Transglutaminase enzyme also does the same thing Leads to terrible flavor problems  H2S(g) Need severe heat for this reaction - not very common

Proteins – basic concepts Oxidation Lipid oxidation Aldehyde, ketones as a result of lipid oxidation react with lysine making it unavailable Usually not a major problem Methionine oxidation (no major concern) Produces sulfoxide, sulfone also possible 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: “physical and chemical properties of proteins that affect the behavior of molecular constituents in food systems. Relates to: Preparation Processing Storage Consumption Quality Organoleptic (sensory) attributes Many food products have functionality because of food proteins Protein functionality plays a key role in the (1)improvement of existing products (2) new product development (3) protein waste products utilized as new ingredients

Proteins – functional properties 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, surimi Gelation Muscle foods, custards, eggs, yogurt, gelatin, tofu, baked goods, surimi Emulsification Salad dressing, mayonnaise, ice cream, gravy, frozen desserts Foaming Meringues, whipped toppings, angel cake, sponge cake, marshmallows, yeast-leavened breads

Example functional proteins Product Major functional protein(s) Representative protein ingredient Cereals Glutenin, gliadin Wheat gluten Legumes 11S Globulin, 7S Globulin Soy protein concentrates or isolates Meat, Poultry Myosin Surimi Fish Collagen Gelatin Eggs Ovalbumin Dried egg white Milk Casein Caseinates α-lactalbumin, β-lactoglobulin Whey protein concentrates (50-80 % protein) or isolates (90 % protein)

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

Proteins – functional properties Solubility Functional properties of proteins depend on their solubility Affected by the balance of hydrophobic and hydrophilic amino acids on its surface Hydrophilic surface = good water solubility 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

Proteins – functional properties 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

Proteins – functional properties Denaturation of the protein can both increase or decrease solubility of proteins – condition dependent pH - very high and low pH denature but the protein is soluble since there is much repulsion Temperature (very high or very low) 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

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 etc. Generally, the assay consists of putting the protein in samples of different pH and centrifuging The more protein that stays in solution (supernatant), the more soluble the protein is The bigger the pellet the less soluble the protein is Protein samples at different pH’s at 0.1M NaCl Centrifuge at 20,000g for 30 min More soluble Less soluble pellet Solubility (%) = protein left in supernatant total protein 𝐱 𝟏𝟎𝟎

Proteins – functional properties Gelation Gel; a continuous 3D network of proteins that entraps water Works by protein - protein interaction and protein - water (non-covalent) Texture, quality and sensory attributes of many foods depend on protein gelation on processing Sausages, cheese, yogurt, custard A gel can form when proteins are denatured by Heat, pH, pressure, shearing, solvent Gel Solution

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, which forms a cross-linked network This aggregation can be irreversible or reversible & usually cooling too

Proteins – functional properties Thermally irreversible gels (also known as thermoset) Thermoset gels form chemical bonds that will not break during reheating of the gel (remains rigid even if reheated) Examples - Muscle proteins (myosin), egg white proteins (ovalbumin) Balancing act 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

Proteins – functional properties Thermally reversible gels (thermoplastic) Gels form on cooling (after heating) and then revert fully or partially back to solution on reheating (“melt”) Collagen breakdown product gelatin is this type of gel Denaturation (%) cooling T Gel strength/Viscosity heating

Proteins – functional properties Factors influencing gel properties pH Salts (ionic strength) Temperature (final) heating/cooling scheme

Proteins – functional properties Factors influencing gel properties pH Highly protein dependent Some protein form better gels at pI No repulsion, get aggregate type of gels Soft 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 (stays soluble) By playing with pH one can therefore play with the texture of food gels producing different textures for different foods

Proteins – functional 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) 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

Proteins – functional properties 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 – called a “torsion test” The results can be related to the sensory properties of the gel

Proteins – functional properties Water binding The ability of foods to take up and/or hold water is of paramount importance to the food industry More H2O = higher weight = More $$ Product quality may also be better, more juiciness

Proteins – functional properties 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 water 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 in a food system? 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 Temperature - if you form a gel on heating (which denatures the proteins) then you would get more water binding Salt type & concentration

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 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

Proteins – functional properties 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

Proteins – functional properties How do we measure water binding and uptake? Usually designed for a specific product or application, most common methods are: Water-uptake (sorption) - Measuring water uptake of a protein or protein food (e.g. protein gel) by adding it to a sorbent (usually a dry powder), then remove and measure the change in water content of the sorbent Water-binding (also called water-holding capacity or expressible moisture) - Subject your sample to an external force (centrifuge or pressure) and then measure how much water is squeezed out Test needs to be carefully designed so that the actual internal structure of the gel or food is not destroyed when the pressure is applied

Proteins – functional properties Emulsification Proteins can be excellent emulsifiers because they contain both hydrophobic and hydrophilic groups that decrease the interfacial tension which allows for stability LOOP TRAIN + ENERGY

Proteins – functional properties Emulsification Whey protein stabilized emulsion Both phases Lipid phase removed (protein matrix showing)

Proteins – functional properties Factors that affect protein-based emulsions Type of protein To form a good emulsion the protein must be able to: Rapidly migrate to the oil-water interface Rapidly and readily open up and orient polar and non-polar side chains into the proper phases Form a stable film around the oil droplet

Proteins – functional properties Factors that affect protein-based emulsions The following are important for the protein emulsifiers Solubility of protein Insoluble will not form a good emulsion (can’t migrate well) If at pI is not good Increasing solubility increase emulsification ability (up to a point) Distribution of hydrophobic vs. hydrophilic amino acids Need a proper balance Generally increased surface hydrophobicity will increase emulsifying properties Shape of protein Globular is better than fibrous Flexibility of protein More flexible it is, easier it opens up

Proteins – functional properties How do we measure emulsifying properties? Most are highly empirical Two common methods Emulsification capacity - Oil titrated into a emulsion that is using protein as the emulsifier with mixing and the max amount of oil that can be added to the protein solution is measured Emulsification stability - Emulsion formed, then monitor breakdown (separation of water and oil phase) with time

Proteins – functional properties 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 producing bubbles (sparging) Mechanically agitate a protein solution (whipping) Gas release in food, e.g. leavened breads (a special case)

Proteins – functional properties Foaming FOAM FORMATION FOAM BREAKDOWN

Proteins – functional properties Factors that affect foam formation and stability Type of protein is important Good foaming proteins exhibit: High rates of diffusion/adsorption at the interface Ability to unfold/denature at the interface Ability to form intermolecular associations with other molecules (that results in film formation) Increased surface hydrophobicity is good Partially denaturing the protein often produces better foams Globular is better than fibrous

Proteins – functional properties 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) addition of cream of tartar  increases stability

Proteins – functional properties Factors that affect foam formation and stability Salt Very protein dependent Egg albumins, soy proteins, gluten Increasing salt usually improves foaming (stability) since the net charge is decreased (proteins lose solubility  salting-out) Whey proteins Increased salt negatively affect foaming (they get more soluble  salting in)

Proteins – functional properties 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 saturated fat stabilizes foam Cold coalesced fat droplets surround protein encapsulated air bubbles

Proteins – functional properties 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)

Proteins – functional properties 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 Proteins become too denatured The foam structure breaks down

Proteins – functional properties How do we measure foam formation and stability? Overrun (foam formation) – Start with a known volume of protein solution (e.g. 100 mL) foam it (usually by whipping), then measure the volume of foam vs. that of the liquid: % Overrun = Foam stability (drainage) – Using a special cylinder measure the amount of liquid that drains from the foam on storage to get a mL/min or mL/hr 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 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 50-100% of original biological/nutritive value Often used in very small amounts due to possible toxicity Not the method of choice for food proteins

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

Proteins – functional properties Protein modification to improve function Enzymatic modification Protein hydrolysis Proteins broken down by enzymes to peptides (smaller) Improved solubility and biological value Protein cross-linking Some enzymes (transglutaminase) can covalently link proteins together Great improvement in gel strength Amino acid modification Peptidoglutamiase converts Glutamine  glutamic acid (negatively charged) Asparagine  aspartic acid (negatively charged) Can convert an insoluble protein to a soluble protein

Proteins – functional properties 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 CPKelco former NutraSweet subsidiary) 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 (protein based so can denature) – limits its use in processing