Proteins

1. Proteins

2. 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

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 • Some enzymes can also cause browning

4. 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 Proteins – basic concepts

5. 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 surfaces of proteins

6. Proteins – basic concepts • 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

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 • Backbone of the protein molecule • Described by the amino acid sequence that make up apolypeptide chain • Amino acids are linked to eachother in a chain via a peptide bond • A covalent bond • This backbone structure dictates rest of the structure R-group R-group Condensationreaction

10. 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 Proteins – basic concepts

11. 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 Proteins – basic concepts -sheets

12. 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 Proteins – basic concepts

13. 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 Proteins – basic concepts

14. 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 Proteins – basic concepts

15. Proteins – basic concepts Types of forces/bonds that stabilize the protein structure

16. Proteins – basic concepts 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

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 is broken up • A charge is very unfavorable in the hydrophobic protein interior 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 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 • 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 • 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

21. Proteins – basic concepts 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)

22. Proteins – basic concepts Important reactions of proteins and effect on structure and quality • Alkaline reactions • Soy processing (textured vegetable protein) • 0.1 M NaOH for 1 hr @ 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)

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 • Not a very serious problem in texturized vegetable protein production

24. Proteins – basic concepts 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

25. Proteins – basic concepts 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

26. Proteins – basic concepts • 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

27. Proteins – basic concepts • Heat • Degradation of cysteine • Leads to terrible flavor problems  H2S(g) • Amide crosslinking • Need severe heat for this reaction - not very common

28. 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 – basic concepts

29. 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

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

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

32. 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 Proteins – functional properties

33. 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

34. Proteins – functional properties • 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

35. 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. • It is of much importance to standardize methods for solubility • One standard assay: More soluble Less soluble Centrifuge at 20000g for 30 min Protein samples at different pH’s at 0.1M NaCl Solubility (%) = protein left in supernatant *100 total protein

36. Proteins – functional properties Sol • 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

37. 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 Proteins – functional properties

38. Proteins – functional properties • 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) cooling Denaturation (%) Gel strength/Viscosity heating heating T

39. Proteins – functional properties • 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 cooling Denaturation (%) Gel strength/Viscosity heating heating T

40. Proteins – functional properties • 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) cooling Denaturation (%) Gel strength/Viscosity heating heating T

41. Proteins – functional properties • 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)

42. Factors influencing gel properties pH Salts T heating/cooling scheme Proteins – functional properties

43. Proteins – functional properties • 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

44. Proteins – functional properties • 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)

45. Proteins – functional properties • 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 + + Cl- NaCl + + + + Cl- Cl- Loss of repulsion Loss of gel strength Loss of water-holding Cl- + +

46. Proteins – functional properties • 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 50-80 mM) Max gel strength seen at (a) pH 3.5 and 30 mM NaCl; (b) pH 7.5 and 50 mM NaCl

47. 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 Proteins – functional properties

48. 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 = More product yield = More $ • Product quality may also be better, more juiciness

49. 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 in a food product • This is the water we are interested in when it comes to water binding

50. Proteins – functional properties • 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