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Semester 2
Module 5
Effect of Heat and pH on Proteins
Protein
Protein Occurrence
Polymers of some 20 different amino acids Joined together by peptide bonds Different proteins have different chemical properties Because of widely different secondary and tertiary structures
Amino Acids
Grouped on the basis of the chemical nature of the side chains Side chains my be polar or non-polar High levels of polar amino acid residues in a protein increase water solubility
Amino Acids
Amino acids are the building blocks (monomers) of proteins. 20 different amino acids are used to synthesize proteins. The shape and other properties of each protein is dictated by the precise sequence of amino acids in it.
Each amino acid consists of an alpha carbon atom to which is attached a hydrogen atom an amino group (hence "amino" acid) a carboxyl group (-COOH). This gives up a proton and is thus an acid (hence amino "acid") one of 20 different "R" groups. It is the structure of the R group that determines which of the 20 it is and its special properties.
Amino Acids
Most polar side chains are those of the basic amino acidic amino acids Present at high levels in soluble albumins and globulins Wheat proteins, gliadin and glutenin, have low levels of polar side chains and are quite insoluble in water Acidic amino acids may also be present in proteins in the form of their amides, glutamine and asparagine This increases the nitrogen content of the protein Hydroxyl groups in the side chains may become involved in ester linkages with phosphoric acid and phosphates Sulfur amino acids may form disulfide cross-links between neighbouring petide chains or between different parts of the same chain
Amino Acids
Joined together by peptide bonds: Form the primary structure of proteins The amino acid composition established the nature of secondary and tertiary structures These influence the functional properties of food proteins and their behavior during processing 20 Amino acids : Only about half essential for human nutrition Amount of essential amino acids present in a protein and their availability determine the nutritional quality of the protein Animal proteins are higher quality than plant proteins
Egg protein
One of the best quality proteins Biological value of 100 Widely used as a standard, Protein Efficiency Ratio (PER) sometimes use egg white as a standard
Amino Acids
When hydrolyzed by strong mineral acids or with the aid of certain enzymes, proteins can be completely decomposed into their component amino acids Simplest amino acid: Glycine, The R-group is H (Hydrogen)
Amino Acids
Sulfur-containing amino acids Cysteine Cystine Methionine Monoamino dicarboxylic amino acids Aspartic acid Glutamic acid Basic amino acids Lysine Arginine Histidine
Amino Acids
Aromatic amino acids Phenylalanine Tyrosine Tryptophan
Protein Classification
Classification of Proteins
Based mostly on the solubility of proteins in different solvents More recent criteria being used includes: Behaviour in the centrifuge Electrophoretic propterties
Proteins are divided into the following main groups Simple Proteins Conjugated Proteins Derived Proteins
Protein Classification
Simple Proteins
Yield only amino acids on hydrolysis and include the following classes Albumins Globulins Glutelins Prolamins Sclereproteins Histones Protamines
Protein Classification
Conjugated Proteins
Contain an amino acid part combined with a non-protein material such as a lipid, nucleic acid, or carbohydrate Some of the major conjugated proteins are as follows: Phosphoproteins Lipoproteins Nucleoproteins Glycoproteins Chromoproteins
Protein Classification
Derived Proteins
These are compounds obtained by chemical or enzymatic methods and are divided into primary and secondary derivatives Primary derivatives Slightly modified and are insoluble in water Ex. Rennet-coagulated casein Secondary derivatives Changed more extensively, include proteoses, peptones, and petides Difference between these breakdown products is in size and solubility All are soluble in water Not coagulated by heat Proteoses can be precipitated with saturated ammonium sulfate solution Peptides contain two or more amino acid residues
Protein Structures
Secondary Structure
Involves folding the primary structure Hydrogen bonds between amide nitrogen and carbonyl oxygen are the major stabilizing force Bonds may be formed between different areas of the same polypeptide chain, or adjacent chains The secondary structure may be either a-helix or sheet Helical structures are stabilized by intramolecular hydrogen bonds Sheet structures are stabilized by intermolecular hydrogen bonds
Protein Structures
Tertiary Structure
Involves a pattern of folding of the chains into a compact unit Stabilized by hydrogen bonds van der Waals forces disulfide bridges hydrophobic interactions This structure results in the formation of a tightly packed unit with most polar amino acid residues located on the outside and hydrated Internal part with most of the apolar side chains and virtually no hydration
Large molecules of molecular weights above about 50 000 may form quaternary structures by association of subunits
These structures my be stabilized by hydrogen bonds, disulfide bridges and hydrophobic interactions
Denaturation
Denaturation
Heat denaturation is sometimes desirable The denaturation of whey proteins for the production of milk powder used in baking Proteins of egg white are readily denatured by heat and by surface forces when egg white is whipped to a foam Meat proteins are denatured in the temperature range 57 to 75C, which has a profound effect on texture, water holding capacity and shrinkage Denaturation may result in flocculation of globular proteins and may lead to the formation of gels Protein denaturation and coagulation are aspects of heat stability that can be related to the amino acid composition and sequence of the protein
Denaturation
DEFENITION: Denaturation is a major change in the native structure that does not involve alteration of the amino acid sequence
Effect of heat usually involves a change in the tertiary structure, leading to a less ordered arrangement of the polypeptide chains The temperature range in which denaturation and coagulation of most proteins takes place is about 55 to 75C Casein and gelatin are examples of proteins that can be boiled without apparent change in stability
Denaturation
The exceptional Stability of Casein
Makes it possible to boil, sterilize, and concentrate milk, without coagulation In the first place restricted formation of disulfide bonds due to low content of cystine and cysteine results in increased stability Casein with its extremely low content of sulfur amino acids are less likely to become involved in the type of sulfhydryl agglomeration The heat stability of casein is also explained by the restraints against forming a folded tertiary structure These restraints are due to the relatively high content of proline and hydroxyproline in the heat stable proteins
Protein Quality
Protein Quality
Protein poor diet supplies limited amino acids to the consumer Many processes actually leads to a further decline in the protein quality of food products These effects need to be monitored and, where necessary, controlled to ensure a safe and nutritious food supply to the population
Protein Quality
The Essential Amino Acids
Histidine Isoleucine Leucine Lysine Methionine (and/or cysteine) Phenylalanine (and/or tyrosine) Threonine Tryptophan Valine
Physical and Chemical environments that a protein is exposed to during processing can result in wide variety of changes
Changes in amino acid side chains Amino acid razemize and develop new cross-links in alkaline solution Losses in nutritional quality Significant changes in functionality Arginine, Cystine, Threonine, Serine, and Cysteine are destroyed Glutamine and Asparagine are deaminated under alkaline conditions In Acid Solutions, Tryptophan is rapidly destroyed, and Serine and Threonine are slowly destroyed Ultraviolet light destroys Tryptophan, Tyrosine and Phenylalanine
The Thermally related changes in proteins can be broken into four basic catagories
The next table and data represents breads baked at different temperatures, but clearly illustrate the loss in nutritive value as a result of intense heating
PER
2.50 1.02 0.42
Crumb
Crumb (higher baking temperature) Light Toast Medium Toast Dark Toast
1.47
0.90 0.64 0.45 0.32
Solubility
One of the most easily observed thermal changes in protein is the change in conformation which affects solubility Generally, protein solubility decreases with increases in the time and temperature of heat treatment Thermal denaturation of protein occurs when hydrogen and other non-covalent bonds, such as ionic and van der Waals bonds within the protein, are disrupted by the heating process Thus, the normal secondary, tertiary, and quaternary structure of the protein is disrupted, and the protein becomes denatured
Coaggregration Since food systems typically contain many different proteins, thermal processing can cause co-aggregation among proteins in the mixture Such co-aggregation can be important in determining the characteristics of the food The aggregations are also extremely difficult to study because they are chemically very stable When -casien is heated in the presence of -lactoglobulin, a disulfide link is formed between the two proteins, which reduces the thermal denaturation of the normally stable -lactoglobulin The functionality of milk powder for baking is significantly enhanced by heating the milk before drying to enhance the formation of the disulfide links between -casien and lactoglobulin
Digestibility and Nutritional value of heat-damaged Proteins It has been known for some time that heat induces a number of changes in the physical properties of proteins and that such changes can influence the digestibility and hence nutritional value of proteins The reduction in the nutritional quality of heated proteins is attributed to the isopeptide cross-links formed as a result of the heat treatment Homogenates of the small intestine showed considerable activity toward the isopeptides Glutamyllysine will pass the gut wall The decreased protein digestibility reduces the apparent bioavailability of most of the amino acid residues in the protein AS the intensity of heating increases, the level of isopeptides also increases The severity of damage to the remainder of the protein increases with increasingly intense heat treatment
Thermal decomposition
Several amino acids has been studied Free radicals are formed in protein or Lysine heated at 200C for 22 minutes It is of concern that these free radicals appear to be stable in water and in digestive juices An aspect of thermal decomposition that must be considered is the possible formation of toxic products Mutagenic activity on flame-broiled fish and beef Several mutagens are of protein and amino acid origin Two of the most toxic mutagens are derived from tryptophan It is important to note that these compounds are only formed at temperatures in excess of 300C
Photo-oxidation of Proteins
Photo-chemical reactions
Amino acid side chains that are readily modified by photooxidation are Sulhydryl Imidazole Phenoxyindole Thiol ether Data indicated that there are losses in the oxidizable amino acids, but that aspartic acid and valine are stable to photooxidation
Photo-oxidation of Proteins
Photo-oxidation of Proteins
The damage results from short wavelengths Glycine is not damaged at wavelengths above 2265 The precise changes and pathways of destruction are influenced by Irradiation wavelength Irradiation dose Reaction conditions Individual amino acid being irradiated The sulfur amino acids exhibit more measurable photodecomposition than the aliphatic amino acids The aromatic amino acids can act as photosensitizers in the protein, particularly sensitizing the sulfur amino acids
Photo-oxidation of Proteins
Two of the potent photosensitizers in foods are riboflavin and chlorophyll Generally, two classes of reactions are initiated when a photosensitizer, oxygen, and a protein are present and light of the proper wavelength activates the system FIRST TYPE: the photosensitezer is excited to the triplet state by light of the proper wavelength The excited sensitizer then univalently oxidazes an amino acid side chain of the protein initiating a free radical destruction of the amino acid
Photo-oxidation of Proteins
SECOND TYPE: The excited photosensitizer excites a ground-state oxygen which forms a highly reactive singlet oxygen The singlet oxygen can the attack lipids or reactive side chains of amino acids
Maximum interaction or degradation of the protein takes place when the lipid oxidation is at the stage of maximum peroxide formation
Losses in available lysine appeared to take place in the initial induction period and during the induction of peroxides Oxidizing lipids or peroxides in the environment of the protein clearly cause significant change in the protein Oxidations and cross-links generated tend to adversely affect Solubility Enzyme activity Nutritive quality
Chlorine
Another environmental oxidizer which can damage protein quality The initial side of attack of the chlorine is the sulfur of methionine First intermediate formed is chlorosulfonium Second step is the formation of a carbonium ion intermediate and cleavage of the carbon sulfur bond The splitting yields a trichloroamino acid porduct Nutritional impact not likely to be significant, since foods produced from chlorinated flour are not generally consumed as sole sources of protein The loss of small amounts of methionine would not be significant
Racemization
Observed in severely alkaline-treated proteins This reaction occurs via removal of the -methine hydrogen Forming a carbanion intermediate Carbanion then reacts rapidly with proton with an equal likelihood of readdition of the proton Forming either the normal L form or the D form of amino acid residue Protein sequence may have some mediating influence on this at moderately alkaline pH, but as pH increases, the randomness of the protein structure would also be expected to increase The rate of racemization is proportional to the hydroxide ion concentration above pH 8.0 Below pH 8.0 racemization is dependent on electron withdrawing ability or the amino acid side chain