P1: SFK/UKS BLBS102-c01 P2: SFK BLBS102-Simpson March 21, 2012 11:8 Trim: 276mm X 219mm Printer Name: Yet to Come Introduction to Food Biochemistry FOOD PROTEIN BIOCHEMISTRY Properties of Amino Acids Proteins are polymers of amino acids joined by peptide bonds Twenty amino acids commonly exist and their possible combinations result in the potential for an incredibly large number of sequence and 3D structural protein variants Amino acids consist of a carbon atom (Cα ) that is covalently bonded to an amino group and a carboxylic acid group Thus, they have an N–Cα –C ‘backbone’ In addition, the Cα is bound to a hydrogen atom and one of 20 ‘R groups’ or ‘side chains’, hence the general formula: + NH3 − CHR − COO− The above general formula describes 19 of the 20 amino acids except proline, whose side chain is irregular, in that it is covalently bound to both the α-carbon and the backbone nitrogen The covalent bonds among different amino acids in a protein are called peptide bonds The 20 amino acids can be divided into three categories based on R-group differences: non-polar, polar and charged polar The functional properties of food proteins are directly attributable to the amino acid R-group properties: structural (size, shape and flexibility), ionic (charge and acid–base character) and polarity (hydrophobicity/ hydrophilicity) At neutral pH, most free amino acids are zwitterionic, i.e they are dipolar ions, carrying both a positive and negative charge, as shown in the general formula above Since the primary amino and carboxyl groups of amino acids are involved in peptide bonds within a protein, it is only the R groups (and the ends of the peptide chains) that contribute to charge; the charge of a protein being determined by the charge states of the ionisable amino acid R groups that make up the polypeptide, namely aspartic acid (Asp), glutamic acid (Glu), histidine (His), lysine (Lys), arginine (Arg), cysteine (Cys) and tyrosine (Tyr) The acidic amino acids are Tyr, Cys, Asp and Glu (note: Tyr and Cys require pH above physiologic pH to act as acids, and therefore, are less important charge contributors in living systems) Lys, Arg and His are basic amino acids The amino acid sequence and properties determine overall protein structure Some examples are as follows: Two residues of opposite charges can form a salt bridge For example, Lys and Asp typically have opposite charges under the same conditions, and if the side chains are proximate, then the negatively charged carboxylate of Asp can salt-bridge to the positively charged ammonium of Lys Another important inter-residue interaction is covalent bonding between Cys side chains Under oxidising conditions, the sulfhydryl groups of Cys side chains (–S–H) can form a thiol covalent bond (–S–S–) also known as a disulphide bond Lastly, hydrophobic (non-polar) amino acids are generally sequestered away from the solvent in aqueous solutions (which is not always the case for food products), since interaction with polar molecules is not energetically stable Protein Nutritional Considerations In terms of survival and good health, the contributions of protein in the diet are to provide adequate levels of what are referred to as ‘essential amino acids’ (Lys, methionine, phenylalanine, threonine, tryptophan, valine, leucine and isoleucine), amino acids that are either not produced in sufficient quantities or not at all by the body to support building/repairing and maintaining tissues as well as protein synthesis Single source plant proteins are referred to as incomplete proteins since they not have sufficient quantities of the essential amino acids in contrast to animal proteins, which are complete For example, cereals are deficient in Lys, while oilseeds and nuts are deficient in Lys as well as methionine In order for plant proteins to become ‘complete’, complementary sources of proteins must be consumed, i.e the deficiency of one source is complemented by an excess from another source, thus making the combined protein ‘complete’ Although some amino acids are gluconeogenic, meaning that they can be converted to glucose, proteins are not a critical source of energy Dietary protein breakdown begins with cooking (heat energy) and chewing (mechanical energy) followed by acid treatment in the stomach (chemical energy) as well as the mechanical actions of the upper GI The 3D structures of proteins are partially lost due to such forces and are said to ‘unfold’ or denature In addition to protein denaturation, the stomach and upper intestine produce two types of proteases (enzymes that hydrolyse peptide bonds) that act on dietary proteins Endopeptidases are proteases that cleave interior peptide bonds of polypeptide chains, while exopeptidases are proteases that cleave at the ends of proteins exclusively Pepsin, an acid protease that functions optimally at extremely low pH of the stomach, releases peptides from muscle and collagen proteins In the upper intestine, serine proteases trypsin and chymotrypsin further digest peptides, yielding free amino acids for absorption into the blood (Champe et al 2005) An important consideration regarding the nutritional quality of proteins is the effect of processing Heat and chemical treatments can serve to unfold proteins, thereby aiding to increase enzymatic hydrolysis, i.e unfolded proteins have a larger surface area for enzymes to act This may increase the amino acid bioavailability, but it can also lead to degradative/transformative reactions of amino acids, e.g deamidation of asparagine and glutamine, reducing these amino acids as nutrient sources Animal Protein Structure and Proteolysis in Food Systems Animal tissues have similar structures despite minor differences between land and aquatic (fish and shellfish) animal tissues Post-mortem, meat structure breaks down slowly, resulting in desirable tenderisation and eventual undesirable degradation/spoilage Understanding meat structure is critical to understanding these processes, and Table 1.4 lists the location and major functions of myofibrillar proteins associated with the contractile apparatus and cytoskeletal framework of animal tissues Individual muscle fibres are composed of myofibrils, which are the basic units of muscular contraction The skeletal muscle of fish differs from that of mammals, in that the fibres arranged between the sheets of connective tissue are much shorter The connective tissue appears as short, transverse sheets P1: SFK/UKS BLBS102-c01 P2: SFK BLBS102-Simpson March 21, 2012 10 11:8 Trim: 276mm X 219mm Printer Name: Yet to Come Part 1: Principles/Food Analysis Table 1.4 Locations and Major Functions of Myofibrillar Proteins Associated with Contractile Apparatus and Cytoskeletal Framework Location Contractile apparatus A-band M-line I-band Cytoskeletal framework GAP filaments N2 -line By sarcolemma Z-line Protein Major Function Myosin c-Protein F-, H-, I-Proteins M-Protein Myomesin Creatine kinase Actin Tropomyosin Troponins T, I, C β-, γ -Actinins Muscle contraction Binds myosin filaments Binds myosin filaments Binds myosin filaments Binds myosin filaments ATP synthesis Muscle contraction Regulates muscle contraction Regulates muscle contraction Regulates actin filaments Connectin (titin) Nebulin Vinculin α-Actinin Eu-actinin, filamin Desmin, vimmentin Synemin, Z-protein, Z-nin Links myosin filaments to Z-line Unknown Links myofibrils to sarcolemma Links actin filaments to Z-line Links actin filaments to Z-line Peripheral structure to Z-line Lattice structure of Z-line Source: Eskin 1990, Lowrie 1992, Huff-Lonergar and Lonergan 1999, Greaser 2001 (myocommata) that divide the long fish muscles into segments (myotomes) corresponding in numbers to those of the vertebrae A fine network of tubules, the sarcoplasmic reticulum, separates the individual myofibrils, and within each fibre is a liquid matrix referred to as the sarcoplasm-containing enzymes, mitochondria (cellular powerhouse), glycogen (carbohydrate storage form in animals), adenosine triphosphate (energy currency), creatine (part of energy transfer in muscle) and myoglobin (oxygen transport molecule) The basic unit of the myofibril is the sarcomere, which is made up of a thick set of filaments consisting mainly of myosin, a thin set containing primarily of F-actin and a filamentous ‘cytoskeletal structure’ composed of connectin and desmin Meat tenderisation is a very complex, multi-factorial process involving glycolysis and the actions of both endogenous proteases (e.g cathepsins and calpains) as well as intentionally added enzymes Table 1.5 lists some of the more common enzymes used in meat tenderisation Papain, ficin and bromelain are proteases of plant origin that efficiently break down animal proteins applied in meat tenderisation industrially or at the household/restaurant levels Enzymes such as pepsins, trypsins and cathepsins cleave animal tissues at various sites of peptide chains, while enteropeptidase (enterokinase) is also known to activate trypsinogen by cleaving its Lys6-Ile7 peptide bond Plasmin, pancreatic elastase and collagenase are responsible for the breakdown of animal connective tissues Chymosin (rennin) is the primary protease critical for the initial milk clotting step in cheese making and is traditionally obtained from calf stomach Lactic acid bacteria (starter) gradually acidify milk to the pH 4.7, the optimal pH for coagulation by chymosin Most lactic acid starters have limited proteolytic activities, i.e product proteins are not degraded fully as in the case of GI tract breakdown of dietary proteins The proteases and peptidases breakdown milk caseins to smaller protein molecules that, combined with milk fat, provide the cheese structure Other enzymes such as decarboxylases, deaminases and transaminases are responsible for the degradation of amino acids into secondary amines, indole, α-keto acids and other compounds that give the typical flavour of cheeses (see Table 1.6 for enzymes and their reactions) Germinating seeds also undergo proteolysis, although in a much lower amount relative to meat Aminopeptidase and carboxypeptidase A are the main, known enzymes (Table 1.7) here that produce peptides and amino acids needed in the growth of the plant In beer production, a small amount of protein is dissolved from the wheat and malt into the wort The protein fraction extracted from the wort may precipitate if present in the resulting beer due to its limited solubility at lower temperatures, resulting in hazing Proteases of plant origin such as papain, ficin and bromelain break down such proteins to reduce this ‘chill-haze’ problem in the brewing industry Protein Modifications A protein’s amino acid sequence is critical to its physicochemical properties, and it follows that changes made to individual amino acids may alter its functionality In addition to the many chemical alterations that may occur to amino acids during P1: SFK/UKS BLBS102-c01 P2: SFK BLBS102-Simpson March 21, 2012 11:8 Trim: 276mm X 219mm Printer Name: Yet to Come 11 Introduction to Food Biochemistry Table 1.5 Proteases in Animal Tissues and Their Degradation Enzyme Aspartic proteases Pepsin A (pepsin, EC 3.4.23.1) Gastricsin (pepsin C, EC 3.4.23.3) Cathepsin D (EC 3.4.23.5) Serine proteases Trypsin (a- and b-trypsin, EC 3.4.21.4) Chymotrypsin (Chymotrypsin A and B, EC 3.4.21.1) Chymotrysin C (EC 3.4.21.2) Pancreatic elastase (pancreato-peptidase E, pancreatic elastase I, EC 3.4.21.36) Plasmin (fibrinase, fibrinolysin, EC 3.4.21.7) Enteropeptidase (enterokinase, EC 3.4.21.9) Collagenase Thio/cysteine proteases Cathepsin B (cathepsin B1, EC 3.4.22.1) Papain (EC 3.4.22.2) Fiacin (ficin, EC 3.4.23.3) Bromelain (EC 3.4.22.4) γ -Glutamyl hydrolase (EC 3.4.22.12 changed to 3.4.1.99) Cathepsin H (EC 3.4.22.16) Calpain-1 (EC 3.4.22.17 changed to 3.4.22.50) Metalloproteases Procollagen N-proteinase (EC 3.4.24.14) Reaction Preferential cleavage, hydrophobic, preferably aromatic, residues in P1 and P’1 positions More restricted specificity than pepsin A; high preferential cleavage at Tyr bond Specificity similar to, but narrower than that of pepsin A Cleavage to the C-terminus of Arg and Lys Preferential cleavage: Tyr-, Trp-, Phe-, LeuPreferential cleavage: Leu-, Tyr-, Phe-, Met-, Trp-, Gln-, AsnHydrolysis of proteins, including elastin Preferential cleavage: Ala Preferential cleavage: Lys >Arg; higher selectivity than trypsin Activation of trypsinogen by selective cleavage of Lys6-Ile7 bond Hydrolysis of collagen into smaller molecules Broad speicificity, Arg–Arg bond preference in small peptides Broad specificity; preference for large, hydrophobic amino acid at P2; does not accept Val at P1 Similar to that of papain Broad specificity similar to that of pepsin A Hydrolyses γ -glutamyl bonds Protein hydorlysis; acts also as an aminopeptidase and endopeptidase (notably cleaving Arg bond) Limited cleavage of tropinin I, tropomyosin, myofibril C-protein, cytoskeletal proteins; activates phosphorylase, kinase, and cyclic-nucleotide-dependent protein kinase Cleaves N-propeptide of pro-collagen chain α1(I) at Pro+Gln and α1(II), and α2(I) at Ala+Gln Source: Eskin 1990, Haard 1990, Lowrie 1992, Huff-Lonergan and Lonergan 1999, Gopakumar 2000, Jiang 2000, Simpson 2000, Greaser 2001, IUBMB-NC website (www.iubmb.org) food processing, e.g deamidation, natural, enzymatic protein modifications collectively known as post-translational modifications may also occur upon their expression in cells Some examples are listed in Table 1.8 Protein Structure Protein folding largely occurs as a means to minimise the energy of the system where hydrophobic groups are maximally shielded from aqueous environments and while the exposure of hydrophilic groups to aqueous environments is maximised Protein structures follow a hierarchy: primary, secondary, tertiary and quaternary structures Primary structure refers to the amino acid sequence; secondary structures are the structures formed by amino acid sequences (e.g α-helix, β-sheet, random coil); tertiary structures are the 3D structures made up of secondary structures (the way that helices, sheets and random coils pack together) and quaternary structure refers to the association of tertiary structures (e.g two subunits of an enzyme) in oligomeric proteins The overall shapes of proteins fall into two general types: globular and fibrous Enzymes, transport proteins and receptor proteins are examples of globular proteins having a compact, spherical shape Hair keratin and muscle myosin are examples of fibrous proteins having elongated structures that are simple compared to globular proteins Oxidative Browning Oxidative browning, also called enzymatic browning, involves the actions of a group of enzymes generally referred to as polyphenol oxidase (PPO) or phenolase PPO is normally compartmentalised in tissue such that oxygen is unavailable Injury or cutting of plant material, especially apples, bananas, pears and lettuce, results in decompartmentalisation, making O2 available P1: SFK/UKS BLBS102-c01 P2: SFK BLBS102-Simpson March 21, 2012 11:8 Trim: 276mm X 219mm 12 Printer Name: Yet to Come Part 1: Principles/Food Analysis Table 1.6 Proteolytic Changes in Cheese Manufacturing Enzyme Reaction Coagulation Chymosin (rennin, EC 3.4.23.4) κ-Casein → Para-κ-casein + Glycopeptide, similar to pepsin A Proteolysis Proteases Amino peptidases, dipeptidases, tripeptidases Proteases, endopeptidases, aminopeptidases Proteins → High-molecular-weight peptides + Amino Acids Low molecular weight peptides → Amino acids High-molecular weight peptides → Low molecular weight peptides Decomposition of amino acids Aspartate transaminase (EC 2.6.1.1) Methionine γ -lyase (EC 4.4.1.11) Tryptophanase (EC 4.1.99.1) Decarboxylases l-Asparate + 2-Oxoglutarate → Oxaloacetate + l-Glutamate l-Methionine → Methanethiol + NH3 + 2-Oxobutanolate l-Tryptophan + H2 O → Indole + Pyruvate + NH3 Lysine → Cadaverine Glutamate → Aminobutyric acid Tyrosine → Tyramine Tryptophan → Tryptamine Arginine → Putrescine Histidine → Histamine Alanine → Pyruvate Tryptophan → Indole Glutamate → α-Ketoglutarate Serine → Pyruvate Threonine → α-Ketobutyrate Deaminases Source: Schormuller 1968, Kilara and Shahani 1978, Law 1984a, 1984b, Grappin et al 1985, Gripon 1987, Kamaly and Marth 1989, Khalid and Marth 1990, Steele 1995, Walstra et al 1999 (www.iubmb.org) Table 1.7 Protein Degradation in Germinating Seeds Enzyme Reaction Neutral or aromatic aminoacyl-peptide + H2 O → Neutral or aromatic amino acids + Peptide Release of a C-terminal amino acid, but little or no action with -Asp, -Glu, -Arg, -Lys or -Pro Aminopeptidase (EC 3.4.11.xx) Carboxypeptidase A (EC 3.4.17.1) Source: Stauffer 1987a, 1987b, Bewley and Black 1994, IUBMB-NC website (www.iubmb.org) Table 1.8 Amino Acid Modifications Amino Acid Arginine Glutamine Asparagine Various Serine Histidine Tyrosine Threonine Asparagine Serine Threonine Modification Deamination Deamination Deamination C-terminal amidation Phosphorylation Phosphorylation Phosphorylation Phosphorylation Glycosylation Glycosylation Glycosylation Product Citrulline Glutamic acid Aspartic acid Amidated amino acid/protein Phosphoserine Phosphohistidine Phosphotyrosine Phosphothreonine Various; N-linked glycoprotein Various; O-linked glycoprotein Various; O-linked glycoprotein P1: SFK/UKS BLBS102-c01 P2: SFK BLBS102-Simpson March 21, 2012 11:8 Trim: 276mm X 219mm Printer Name: Yet to Come 13 Introduction to Food Biochemistry to PPO for subsequent action on the phenolic ring of Tyr Phenolics are hydroxylated, thus producing diphenols that are then subsequently oxidised to quinones: Tyrosine O2 +BH2 → Dihydroxylphenylamine + O2 → o-Benzoquinone + 2H2 O The action of PPO can be desirable in various food products, such as raisins, prunes, dates, cider and tea; however, the extent of browning needs to be controlled The use of reducing compounds is the most effective control method for PPO browning The most widespread anti-browning treatment used by the food industry was the addition of sulfiting agents; however, due to safety concerns (e.g allergenic-type reactions), other methods have been developed, including the use of other reducing agents (ascorbic acid and analogues, Cys, glutathione), chelating agents (phosphates, EDTA), acidulants (citric acid, phosphoric acid), enzyme inhibitors, enzyme treatment and complexing agents (e.g copolymerised β-cyclodextrin or polyvinylpolypyrrolidone; Sapers et al 2002) Application of these PPO activity inhibitors is strictly regulated in different countries (Eskin 1990, Gopakumar 2000, Kim et al 2000) Oxidative browning is one of three types of browning reactions important in food colour, the other two being non-oxidative/Maillard browning and caramelisation (covered above and extensively in food chemistry texts; see Damodaran et al 2008) Enzymatic Texture Modifications Transglutaminase (TGase, EC 2.3.2.13, protein-glutamine-yglutamyltransferase) catalyses acyl transfer between R group carboxyamides of glutamine residues in proteins, peptides and various primary amines; the ε-amino group of Lys acts as acyl acceptor, resulting in polymerisation and inter- or intra-molecular cross-linking of proteins via formation of ε-(-y-glutamyl) Lys linkages via exchange of the Lys ε-amino group for ammonia at the carboxyamide group of a glutamine residue Formation of covalent cross-links between proteins is the basis for TGasebased modification of food protein physical properties The primary applications of TGase in seafood processing have been for cold restructuring, cold gelation of pastes and gel-strength enhancement through myosin cross-linking Quality Index Trimethylamine and its N-oxide have long been used as indices for freshness in fishery products Degradation of trimethylamine and its N-oxide leads to the formation of ammonia and formaldehyde with undesirable odours The pathway on the production of formaldehyde and ammonia from trimethylamine and its Noxide is shown in Figure 1.3 Most live pelagic and scombroid fish (e.g tunas, sardines and mackerel) contain an appreciable amount of His in the free state In post-mortem scombroid fish, the free His is converted by the bacterial enzyme His decarboxylase into free histamine Histamine is produced in fish caught 40–50 hours after death when Trimethylamine Trimethylamine N-oxide reductase Trimethylamine dehydrogenase + H O, NADH - NAD + + H O, flavoprotein Trimethylamine N-oxide Trimethylamineoxidealdolase Formaldehyde Dimethylamine Dimethylamine dehydrogenase + H O, FAD - FADH Methylamine Amine dehydrogenase Formaldehyde + H2 O - Formaldehyde Ammonia Figure 1.3 Degradation of trimethylamine and its N -oxide Trimethylamine N -oxide reductase (EC 1.6.6.9), trimethylamine dehydrogenase (EC 1.5.8.2), dimethylamine dehydrogenase (EC 1.5.8.1), amine dehydrogenase (EC 1.4.99.3) (From Haard et al 1982, Gopakumar 2000, Stoleo and Rehbein 2000, IUBMB-NC website (www.iubmb.org).) fish are not properly chilled Improper handling of tuna and mackerel after harvest can produce enough histamine to cause food poisoning (called scombroid or histamine poisoning), resulting in facial flushing, rashes, headache and gastrointestinal disorder These disorders seem to be strongly influenced by other related biogenic amines, such as putrescine and cadaverine, produced by similar enzymatic decarboxylation (Table 1.9) The presence of putrescine and cadaverine is more significant in shellfish, such as shrimp The detection and quantification of histamine is fairly simple and inexpensive; however, the detection and quantification of putrescine and cadaverine are more complicated and expensive Despite the possibility that histamine may not be the main cause of poisoning (histamine is not stable under strong acidic conditions such as the stomach), it is used as an index of freshness of raw materials due to the simplicity of histamine analysis (Gopakumar 2000) Urea is hydrolysed by the enzyme urease (EC 3.5.1.5), producing ammonia, which is one of the components measured by total volatile base (TVB) TVB nitrogen has been used as a quality index of seafood acceptability by various agencies (Johnson and Linsay 1986, Cadwallader 2000, Gopakumar 2000) Live shark contains relatively high amounts of urea, thus under improper handling urea is converted to ammonia, giving shark meat an ammonia odour, which is a quality defect ... Deaminases Source: Schormuller 19 68, Kilara and Shahani 19 78, Law 1 984 a, 1 984 b, Grappin et al 1 9 85 , Gripon 1 987 , Kamaly and Marth 1 989 , Khalid and Marth 1990, Steele 19 95, Walstra et al 1999 (www.iubmb.org)... BLBS102-Simpson March 21, 2012 11 :8 Trim: 276mm X 219mm Printer Name: Yet to Come 11 Introduction to Food Biochemistry Table 1 .5 Proteases in Animal Tissues and Their Degradation Enzyme Aspartic... reactions important in food colour, the other two being non-oxidative/Maillard browning and caramelisation (covered above and extensively in food chemistry texts; see Damodaran et al 20 08) Enzymatic Texture