Proteins

1. Proteins

2. 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

3. 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

4. Typical protein contents of the edible portion of various foods Values obtained from the USDA National Nutrient Database, numbers in parentheses are the USDA 5 digit identifier

5. 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) There are 20 common amino acids that are genetically coded – book has 21, includes selenol(contains Selenium) which was discovered in 2002 More (100s) amino acids exist in nature but are not genetically coded Differ by their side chains (R-groups) All have central αC, basic amino group, and a carboxyl group Amino acid charge behavior Neutral Acidic Basic Proteins – basic concepts α α

6. 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

7. Proteins – basic concepts Polar Amino Acids - Hydrophilic

8. Proteins – basic concepts Non-polar Amino Acids – Hydrophobic/Amphophilic

9. 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 eachother in a chain via a peptide bond • A covalent bond • Sequence always described N-terminal to C-terminal • N-H partial (+), carbonyl oxygen (C=O) partial (-) • This backbone structure dictates rest of the structure (2°, 3°, etc. structure) Condensationreaction

10. 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

11. 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 Proteins – basic concepts -sheets http://www.youtube.com/watch?v=wM2LWCTWlrE

12. 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) Proteins – basic concepts β-lactoglobulin

13. 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 kDaoctamer 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

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

15. Molecular forces involved in protein structure

16. 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

17. 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. 100 %Denatured 0 0 pH 12

18. 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) 100 %Denatured 0 0 100 T (C)

19. 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)

20. 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)

21. 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)

22. 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)

23. 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

24. 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) • Leads to terrible flavor problems  H2S(g) • Need severe heat for this reaction - not very common

25. 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 – basic concepts

26. 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

27. Example of protein functional properties in different food systems Proteins – functional properties

28. Example functional proteins

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

30. 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

31. 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) Proteins – functional properties %Solubility Salt concentration

32. 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

33. 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 More soluble Less soluble Centrifuge at 20,000g for 30 min Protein samples at different pH’s at 0.1M NaCl pellet Solubility (%) =

34. Proteins – functional properties Solution • 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

35. 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 Proteins – functional properties & usually cooling too

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

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

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

39. 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

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

41. 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” Proteins – functional properties The results can be related to the sensory properties of the gel

42. 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

43. 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

44. 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

45. Example how thermal denaturation may have an effect onwater 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

46. Salts/ionic strength This is highly protein dependent muscle proteins Proteins – functional properties NaCl Na+ Na+ Na+ Cl- Cl- Na+ Na+ Cl- Cl-

47. 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

48. 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 andprotein gels are a good example – direct interaction with H2O or form gel network pI

49. 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

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