Dissertation Submitted to the LOYOLA COLLEGE (Autonomous) UNIVERSITY OF MADRAS In partial fulfillment of the Requirement for the award of the Degree of MASTER OF SCIENCE IN FOOD CHEMISTRY & FOOD PROCESSING By M.ANBU MALAR (08-PFP-012) Under the guidance of Dr. V. KANNAPPAN Supervisor Professor in Food chemistry & Food processing LOYOLA COLLEGE (Autonomous) Chennai-34


This is to certify that the thesis entitled, “EFFECT OF PROCESSING ON THE PROTEIN CONTENT OF FISH AND MEAT PRODUCTS” submitted by M.ANBU MALAR (08-PFP-012) to LOYOLA COLLEGE (Autonomous, Chennai-34) in partial fulfillment of the requirements for the Degree of Master of Science in “Food chemistry & Food processing” is a record of work done under our supervision and guidance, during the academic year 2009-2010.

Signature of the supervisor DR. V. KANNAPPAN Professor in food chemistry Loyola college (Autonomous) Chennai-34. .

Signature of the HOD DR. V. KANNAPPAN Professor in food chemistry Loyola college (Autonomous) Chennai-34.

Place: Chennai-34 Date:


My heartfelt prayers and gratitude goes out to the staff whose support and guidance has been a great source of inspiration. Dr. Albert muthumalai S. V. I express my warm and heartfelt gratitude to my parents and their prayers. Chennai. Loyola college Chennai34 for his constant encouragement and support.Dr. Department of food chemistry & food processing.. moral support and care in achieving my aim fruitfully. Principal Loyola College. . for her excellent consistent and expert guidance. Deputy Principal. Loyola College (Autonomous) Chennai. for giving me this opportunity to study in this institution. A. I wish to thank Rev.John Pragasam. KANNAPPAN.J.ACKNOWLEDGEMENT I praise and thank the almighty god who is the source of all wisdom and for showering his blessings on me in bringing out this project successfully. I extend my gratitude to Rev. I wish to express my heartfelt gratitude and respect to my guide Dr.

1: INTRODUCTION: Proteins are the most versatile macromolecules in living systems and serve crucial functions in essentially all biological processes. proteins spontaneously fold up into three-dimensional structures that are determined by the sequence of amino acids in the protein polymer. Proteins contain a wide range of functional groups: These functional groups include alcohols. thioethers. They function as catalysts. proteins are the embodiment of the transition from the one dimensional world of sequences to the three-dimensional world of molecules capable of diverse activities. the chemical reactivity associated with these groups is essential to the function of enzymes. . and a variety of basic groups. Several key properties enable proteins to participate in such a wide range of functions: 1.  The function of a protein is directly dependent on its three dimensional structure. transmit nerve impulses.  When combined in various sequences. carboxamides.CHAPTER .They provide mechanical support and immune protection. carboxylic acids. and control growth and differentiation.  For instance. Proteins are linear polymers built of monomer units called amino acids: The construction of a vast array of macromolecules from a limited number of monomer building blocks is a recurring theme in biochemistry. this array of functional groups accounts for the broad spectrum of protein function. transport and store other molecules such as oxygen. 3. thiols. the proteins that catalyze specific chemical reactions in biological systems. 2. Proteins can interact with one another and with other biological macromolecules to form complex assemblies:  The proteins within these assemblies can act synergistically to generate capabilities not afforded by the individual component Proteins. generate movement.  Thus.  Remarkably.

and levers that are crucial to protein function. These assemblies include macro-molecular machines that carry out the accurate replication of DNA. The convention for the designation of the order of amino acids is that the N-terminal end (i. charge. to the assembly of proteins with one another and with other molecules into complex units. Twenty kinds of side chains varying in size.  Parts of proteins with limited flexibility may act as hinges. hydrophobic character.[29] 1. the end bearing the residue with the . springs. Indeed. and a distinctive R group. and chemical reactivity are commonly found in proteins. a carboxylic acid group. hydrogen. linked to an amino group. 1. called the carbon.1. An -amino acid consists of a central carbon atom. the two mirror-image forms are called the l isomer and the d isomer.e. With four different groups connected to the tetrahedral -carbon atom.1.1 Proteins Are Built from a Repertoire of 20 Amino Acids: Amino acids are the building blocks of proteins. and many other essential processes. Primary Structure: Amino Acids Are Linked by Peptide Bonds to Form Polypeptide Chains: The primary structure of peptides and proteins refers to the linear number and order of the amino acids present. the transmission of signals within cells. STRUCTURE OF PROTEIN: 1. Some proteins are quite rigid.2. 4. -amino acids are chiral. whereas others display limited flexibility:  Rigid units can function as structural elements in the cytoskeleton (the internal scaffolding within cells) or in connective tissue. and eukaryotic are constructed from the same set of 20 amino acids. The R group is often referred to as the side chain. shape. and to the transmission of information within and between cells.1. all proteins in all species bacterial.bonding capacity. This fundamental alphabet of proteins is several billion years old. archaeal. a hydrogen atom.The remarkable range of functions mediated by proteins results from the diversity and versatility of these 20 building blocks.

e. and so the molecular weights of most proteins are between 5500 and 220. which is a good hydrogen-bond acceptor and.000 daltons. A polypeptide chain consists of a regularly repeating part. or 50 kd (kilodaltons). Hence.A series of amino acids joined by peptide bonds form a polypeptide chain. the end with the residue containing a free α-carboxyl group) is to the right. The equilibrium of this reaction lies on the side of hydrolysis rather than synthesis.The polypeptide backbone is rich in hydrogen-bonding potential. the amino end is taken to be the beginning of a polypeptide chain. the biosynthesis of peptide bonds requires an input of free energy. peptide bonds are quite stable kinetically. Most natural polypeptide chains contain between 50 and 2000 amino acid residues and are commonly referred to as proteins. By convention. A protein with a molecular weight of 50. and so the sequence of amino acids in a polypeptide chain is written starting with the aminoterminal residue. phenylalanine is the amino-terminal (N-terminal) residue and leucine is the carboxyl-terminal (Cterminal) residue.000 has a mass of 50. comprising the distinctive side chains. with an -amino group at one end and an -carboxyl group at the other. and each amino acid unit in a polypeptide is called a residue. These groups interact with each other and with functional groups from side chains to stabilize particular structures. A polypeptide chain has polarity because its ends are different. The formation of a dipeptide from two amino acids is accompanied by the loss of a water molecule. called the main chain or backbone. and a variable part. with the exception of proline.000. The mean molecular weight of an amino acid residue is about 110. an NH group. Each residue contains a carbonyl group. Thus. Nonetheless. with different chemical properties. Leu-Phe-Gly-Gly-Tyr (LFGGY) is a different pentapeptide. the lifetime of a peptide bond in aqueous solution in the absence of a catalyst approaches 1000 years. in the pentapeptide Tyr-Gly-Gly-Phe-Leu (YGGFL). Peptides made of small numbers of amino acids are called oligopeptides or simply peptides.free α-amino group) is to the left (and the number 1 amino acid) and the C-terminal end (i. . which is a good hydrogen-bond donor. Proteins are linear polymers formed by linking the -carboxyl group of one amino acid to the -amino group of another amino acid with a peptide bond (also called an amide bond).

1.1. (C) A view down the bond between the a-carbon and the carbonyl carbon atoms. (B) A view down the bond between the nitrogen and the a-carbon atoms. Rotation About Bonds in a Polypeptide: The structure of each amino acid in a polypeptide can be adjusted by rotation about two single bonds. . showing how f is measured.3. showing how y is measured. whereas psi (y) is the angle of rotation about the bond between the a-carbon and the carbonyl carbon atoms. (A) Phi (f) is the angle of rotation about the bond between the nitrogen and the acarbon atoms.

the Beta Sheet. These conformations constitute the secondary structures of a protein. Secondary Structure: Polypeptide Chains Can Fold Into Regular Structures Such as the Alpha Helix. and Turns and Loops: The ordered array of amino acids in a protein confer regular conformational forms upon that protein. The α-Helix: The α-helix is a common secondary structure encountered in proteins of the globular class. This orientation of H-bonding produces a helical coiling of the peptide backbone such that the R-groups lie on the exterior of the helix and perpendicular to its axis. The formation of the α-helix is spontaneous and is stabilized by Hbonding between amide nitrogens and carbonyl carbons of peptide bonds spaced four residues apart. In general proteins fold into two broad classes of structure termed. whereas. It is the partial double-bond character of the peptide bond that defines the conformations a polypeptide chain may assume. . globular proteins or fibrous proteins.4.1. fibrous proteins are more filamentous or elongated. Within a single protein different regions of the polypeptide chain may assume different conformations determined by the primary sequence of the amino acids.1. A. Globular proteins are compactly folded and coiled.

B. in antiparallel sheets adjacent chains are aligned in opposite directions. The H-bonding residues are present in adjacently opposed stretches of the polypetide backbone as opposed to a linearly contiguous region of the backbone in the α-helix. β-sheets can be depicted in ball and stick format or as ribbons in certain protein formats. 1.5.e. β-sheets are either parallel or antiparallel. Included in this description is the spatial relationship of different secondary structures to one another within a polypeptide chain and how these secondary structures themselves fold into the three- . In parallel sheets adjacent peptide chains proceed in the same direction (i. The folding and alignment of stretches of the polypeptide backbone aside one another to form β-sheets is stabilized by H-bonding between amide nitrogens and carbonyl carbons.Tertiary Structure: Water-Soluble Proteins Fold Into Compact Structures with Nonpolar Cores: Tertiary structure refers to the complete three-dimensional structure of the polypeptide units of a given protein. the direction of N-terminal to C-terminal ends is the same). whereas. β-Sheets: An α-helix is composed of a single linear array of helically disposed amino acids.1. This is due to positioning of the αcarbons of the peptide bond which alternates above and below the plane of the sheet. β-sheets are composed of 2 or more different regions of stretches of at least 5-10 amino acids. β-sheets are said to be pleated.

6. The structure formed by monomer-monomer interaction in an oligomeric protein is known as quaternary structure. contains two α and two β subunits arranged with a quaternary structure in the form. Hemoglobin. hydrophobic interactions. a hetero-oligomeric protein. Secondary structures of proteins often constitute distinct domains. Forces Controlling Protein Structure: 1.1. Hydrogen Bonding: Polypeptides contain numerous proton donors and acceptors both in their backbone and in the R-groups of the amino acids.[1-2] 1. Hydrophobic Forces: Proteins are composed of amino acids that contain either hydrophilic or hydrophobic R-groups.2. It is the nature of the interaction of the different R-groups . Proteins with multiple polypetide chains are oligomeric proteins. The environment in which proteins are found also contains the ample H-bond donors and acceptors of the water molecule. tertiary structure also describes the relationship of different domains to one another within a protein.2. 1. Oligomeric proteins can be composed of multiple identical polypeptide chains or multiple distinct polypeptide chains. 1. H-bonding. Proteins with identical subunits are termed homo-oligomers. therefore.1. Quaternary Structure: Many proteins contain 2 or more different polypeptide chains that are held in association by the same non-covalent forces that stabilize the tertiary structures of proteins. Hemoglobin is. electrostatic interactions and van der Waals forces. Proteins containing several distinct polypeptide chains are termed hetero-oligomers. α 2β2. Therefore.2. The interactions of different domains is governed by several forces: These include hydrogen bonding. occurs not only within and between polypeptide chains but with the surrounding aqueous medium.dimensional form of the protein. the oxygen carrying protein of the blood.2. therefore.

therefore. This driving force restricts the available conformations into which a protein may fold. charge-dipole and dipole-dipole. The hydrophobicity of certain amino acid R-groups tends to drive them away from the exterior of proteins and into the interior. it is the huge number of such interactions that occur in large protein molecules that make them significant to the folding of proteins. 1.2. Typical charge-charge interactions that favor protein folding are those between oppositely charged R-groups such as K or R and D or E. The repulsion is the result of the electron-electron repulsion that occurs as two clouds of electrons begin to overlap. Electrostatic Forces: Electrostatic forces are mainly of three types. Repulsive van der Waals forces involve the interactions that occur when uncharged non-bonded atoms come very close together but do not induce dipoles. relative to other forces governing conformation. understandable that the majority of the amino acids found on the exterior surfaces of globular proteins contain charged or polar R-groups.4.2.3. This refers to the interaction of ionized R-groups of amino acids with the dipole of the water molecule. A substantial component of the energy involved in protein folding is charge-dipole interactions. 1. charge-charge. Attractive van der Waals forces involve the interactions among induced dipoles that arise from fluctuations in the charge densities that occur between adjacent uncharged non-bonded atoms. It is. The slight dipole moment that exist in the polar Rgroups of amino acid also influences their interaction with water.with the aqueous environment that plays the major role in shaping protein structure. The spontaneous folded state of globular proteins is a reflection of a balance between the opposing energetics of H-bonding between hydrophilic R-groups and the aqueous environment and the repulsion from the aqueous environment by the hydrophobic R-groups. van der Waals Forces: There are both attractive and repulsive van der Waals forces that control protein folding.[3-5] . Although van der Waals forces are extremely weak.

9. The resultant phenylthiocarbamyl derivatized amino acid is hydrolyzed in anhydrous acid. Using this method it is possible to obtain the entire sequence of peptides. The derivatized amino acid can be hydrolyzed and will be labeled with a dinitrobenzene group that imparts a yellow color to the amino acid.6. . dansyl chloride reacts with the N-terminal residue under alkaline conditions. Separation of the modified amino acids (DNP-derivative) by electrophoresis and comparison with the migration of DNPderivative standards allows for the identification of the N-terminal amino acid. This imparts a higher sensitivity into this technique over that of the Sanger method. Sanger's Reagent This sequencing technique utilizes the compound. To prevent reformation of the disulfide bonds the peptides are treated with iodoacetic acid in order to alkylate the free sulfhydryls.2. There are three major chemical techniques for sequencing peptides and proteins from the N-terminus. Amino-Terminal Sequence Determination: Several different chemical reactions can be used in order to permit separation of peptide strands and prevent protein conformations that are dependent upon disulfide bonds.4-dinitrofluorobenzene (DNF) which reacts with the N-terminal residue under alkaline conditions. The most common treatments are to use either 2-mercaptoethanol or dithiothreitol (DTT). This method utilizes phenylisothiocyanate to react with the N-terminal residue under alkaline conditions. Dansyl chloride and Edman techniques. 2. Dansyl chloride Like DNF. Analysis of the modified amino acids is carried out similarly to the Sanger method except that the dansylated amino acids are detected by fluorescence.2.[6-8] 1.2. 1. These are the Sanger. Both of these chemicals reduce disulfide bonds. 1. Edman degradation The utility of the Edman degradation technique is that it allows for additional amino acid sequence to be obtained from the N-terminus inward. The hydrolysis reaction results in a rearrangement of the released N-terminal residue to a phenylthiohydantoin derivative.

4. The functional roles of food proteins in food given are summarized in the table 1. The sensory attributes of a food are the net effect of complex interactions among various minor and major components of the food. This reagent causes specific cleavage at the C-terminal side of M residues. This process has subsequently been automated to allow rapid and efficient sequencing of even extremely small quantities of peptide.As in the Sanger and Dansyl chloride methods.1. The most reliable chemical technique for C-terminal residue identification is hydrazinolysis.[11-15] 1. the N-terminal residue is tagged with an identifiable marker. Due to the high percentage of hydrazine induced side reactions this technique is only used on carboxypeptidase resistant peptides. The entire sequence of reactions can be repeated over and over to obtain the sequences of the peptide.[9-10] 1. however. The number of peptide fragments that result from CNBr cleavage is equivalent to one more than the number of M residues in a protein. NH2–NH2. at high temperature (90°C) for an extended length of time (20-100hr). and appearance. the added advantage of the Edman process is that the remainder of the peptide is intact. flavor. A peptide is treated with hydrazine. This treatment cleaves all of the peptide bonds yielding amino-acyl hydrazides of all the amino acids excluding the C-terminal residue which can be identified chromatographically compared to amino acid standards. color. Chemical Digestion of Proteins: The most commonly utilized chemical reagent that cleaves peptide bonds by recognition of specific amino acid residues is cyanogen bromide (CNBr).3. . Functional Properties of Proteins Food preferences by human beings are based primarily on sensory attributes such as texture.

cakes. cakes. cereal proteins Sausages. and Breads Meats. cereal proteins .1 Functional Roles of Food Proteins in Food Systems Function Solubility Viscosity Water binding Gelation Cohesionadhesion Mechanism Hydrophilicity Water binding . egg proteins Muscle proteins. soup. and salad dressing s. bakery Elasticity Emulsification Hydrophobic bonding . ice cream. Doughnuts Egg proteins. gravies.TABLE 1. Entrapment Milk proteins. egg proteins. ionic Hydration Water entrapment and immobilization. whey proteins Muscle proteins. network formation Cohesion adhesion Food Beverages Soups. egg cakes. gels. pasta. desserts Meat sausages. Muscle proteins. cakes. sausages. bologna. bakeries. hydrodynamic size and shape Hydrogen bonding . milk proteins Foaming Fat and flavor binding Interfacial adsorption and film Formation Hydrophobic bonding . disulfide crosslinks Adsorption and film formation at interfaces Muscle proteins. desserts Low-fat bakery products. egg and milk proteins Meats. dressing s proteins. cheese Protein type Whey proteins Gelatin Muscle proteins. milk proteins Whipped topping s. egg proteins. baked goods Meats.

Some oilseed proteins. molecular flexibility/rigidity. The essential amino acids whose concentrations in a protein are below the levels of a reference protein are termed limiting amino acids. Several factors.5. are very low in lysine and rich in methionine.2 and 1. contribute to these differences. and ability to interact/react with other components.1. tertiary. Hydrophobicity/Hydrophilicity ratio. Table 1.[34-35] 1. secondary. shape.3. barley. Protein Quality The “quality” of a protein is related mainly to its essential amino acid composition and digestibility. such as rice.2 Essential Amino Acid Contents and Nutritional Value of Proteins from Various Sources (mg /g Protein) Property (mg Egg /g protein) Amino acid concentration (mg /g protein) His 22 Cow's Milk Beef Fish Wheat Soybean 27 34 35 21 30 . and quaternary structures. such as content of essential amino acids and digestibility. are deficient in both methionine and lysine contents.The physical and chemical properties that govern protein functionality include size. Since proteins possess a multitude of physical and chemical properties. such as peanut protein. The daily protein requirement therefore depends on the type and composition of proteins in a diet. wheat. those of legumes and oilseeds are deficient in methionine and rich or adequate in lysine. amino acid composition and sequence. The essential amino acid content in various food samples and recommended essential amino acid pattern are given in tables 1.Nutritional Properties of Proteins: Proteins differ in their nutritive value.5. net charge and distribution of charges. and maize. The nutritional quality of a protein or protein mixture is ideal when it contains all of the essential amino acids in proportions that produce optimum rates of growth and/or optimum maintenance capability. it is difficult to delineate the role of each of these properties with respect to a given functional property. While proteins of cereals. 1.

0 74 67 3.3 Recommended Essential Amino Acid Pattern for Food Proteins (mg/g protein) Amino acid Infant (2–5 years) 26 46 93 66 42 72 43 Histidine Isoleucine Leucine Lysine Met + Cys Phe + Tyr Threonine Preschool child (10–12 years) 19 28 66 58 25 63 34 Preschool child 19 28 44 44 22 22 28 Adult 16 13 19 16 17 19 9 .3 73 61 Table 1.9 94 94 3.5 65 40 2.5 76 79 1.1 84 92 3.Ile Leu Lys Met+Cys Phe+Tyr Thr 54 86 70 57 93 47 47 95 78 33 102 44 48 81 89 40 80 46 48 77 91 40 76 46 34 69 23 36 77 28 51 82 68 33 95 41 Trp Val Total essential amino acids Protein content (%) Chemical score (%) PER BV NPU 17 66 512 14 64 504 12 50 480 11 61 485 10 38 336 14 52 466 12 3.5 18 19 12 40 100 100 100 100 40 100 3.

such as tannins and phytate. Generally. Digestibility. Thus.5. F = faecal nitrogen loss on the test diet. 1. Native proteins are generally less completely hydrolyzed than partially denatured ones.Try35ptophan 17 Valine 55 Total 434 1. is defined from measurements of the nitrogen content of foods and faeces. bind to intestinal mucosa cells and interfere with absorption of amino acids.3. Tannins. Antinutritional factors Most plant protein isolates and concentrates contain trypsin and chymotrypsin inhibitors (Kunitz type and Bowman-Birk type) and lectins.5. and Fk = faecal nitrogen loss on a protein-free diet. Digestibility 11 35 320 9 25 222 5 13 111 Although the content of essential amino acids is the primary indicator of protein quality. digestibility of amino acids can affect the quality of proteins.2. which in turn is measured as faecal nitrogen loss on a protein free diet where I = nitrogen intake. which are condensed products of . Protein conformation The structural state of a protein influences its hydrolysis by proteases. true quality also depends on the extent to which these amino acids are utilized in the body. the proportion of food protein which is absorbed.5. which are glycoprotein. Lectins.4. 1. Plant proteins also contain other antinutritional factors. with “true” digestibility taking into account the extent to which faecal nitrogen is “endogenous”. insoluble fibrous proteins and extensively denatured globular proteins are difficult to hydrolyze.

-amino groups of lysyl residues. The adult requirement value for good-quality protein determined in nitrogen balance studies appears to be about twice the value of the obligatory nitrogen loss implying a net protein utilization of only about 50%. The nutritive quality of proteins can be evaluated by several biological. a measure of how well the absorbed amino acid profile matches that of the requirement.6. and enzymatic methods.6. Protein quality evaluation Protein quality evaluation aims to determine the capacity of food protein sources and diets to satisfy the metabolic demand for amino acids and nitrogen. and (b) monitoring changes in the nutritive value of proteins during food processing. 1.[16-17] 1.e.1. will therefore reflect both digestibility and biological value. Overall protein utilization. This inhibits trypsin-catalyzed cleavage of the lysyl peptide bond. i. and freedom from interference in metabolism. a measure of the dietary intake which is made available to the organism after digestion and absorption. it is important to have procedures for evaluating quality. Quality estimates are useful for (a) determining the amount required to provide a safe level of essential amino acids for growth and maintenance. Bioavailability comprises digestibility. Evaluation of Protein Nutritive Value Since the nutritional quality of proteins can vary greatly and is affected by many factors. net protein utilization (NPU). highlighting those aspects of amino acid utilization that may be important with specific foods and food processing methods. chemical integrity.[18] . so that processing conditions that minimize quality loss can be devised. protein utilization is generally discussed in terms of digestibility. chemical. and biological value.

it may be . BV.6.2. several potential adverse effects were identified and it was concluded that it was prudent for adults to avoid protein intakes of more than twice the reference dietary amount (i. whereas single amino acids are consumed for a wide variety of reasons. Biological value The amino acid profile is assumed to determine the effectiveness with which absorbed dietary nitrogen can be utilized.6. 1.6. then PDCAAS should predict biological value 1.e. Protein intake and health Protein supplements are the most widely consumed ergogenic aid. in the context of guidance on high intakes. This is achieved by a comparison of the content of the limiting amino acid in the protein or diet with its content in the requirement pattern: amino acid score = mg of amino acid in 1 g test protein --------------------------------------------------mg of amino acid in requirement pattern then PDCAAS = digestibility × amino acid score. i. If biological value is determined solely by the amino acid profile. Biological value. A second issue is the possibility that protein intakes in excess of recommended intakes may confer health benefits. Amino acid score The amino acid score determines the effectiveness with which absorbed dietary nitrogen can meet the indispensable amino acid requirement at the safe level of protein intake. The possibility of toxicity resulting from consuming very large amounts of individual amino acids has been examined in various publications. which is usually defined in terms of biological value.e. and UK = urinary nitrogen loss on a protein-free diet 1. most of which have little or no secure scientific foundation.3. is calculated as follows: where U = urinary nitrogen loss on the test diet.5 g protein/kg).4.1. In the United Kingdom.

Bone health The relationship between protein intake and bone health appears to be more complex than was previously believed. and also whether it is possible to identify a maximum level of protein that can be consumed without adverse effects. This concept arose from studies in rats. Heaney (24. in whom the decline in kidney function occurs through a fall in filtration by non-sclerotic nephrons. 23). in which lowprotein diets were shown to delay the development of chronic renal failure (9. However. increased resorption of bone has been shown to occur as a consequence of increased protein intake (20. rather than by glomerular sclerosis as occurs in the rat (14). Wasler (14) suggested protein restriction on the grounds of renal function is justifiable and prudent only in subjects who are likely to develop kidney failure owing to diabetes. 21). However. However. In addition.that optimum protein intakes are greater than a recommended intake derived as in this report. Thus the potential negative effect of protein on calcium balance is a function of the overall dietary acid–base balance. This raises the controversial issue of whether this process might lead to a decrease in bone calcium (19. Bone mineral balance is very sensitive to acid–base balance. amounting to a 50% increase in urinary calcium for a doubling of protein intake (19).6. and higher protein intake generally leads to a higher calcium intake. 10). 25) has suggested that this is unlikely. the suggestion that the decline of glomerular filtration rate that occurs with advancing age in healthy subjects (13) can be attenuated by reducing the protein in the diet appears to have no foundation. 1. 1. and calcium can be mobilized from bone in response to the need to buffer the acid load produced by oxidation of the sulfurcontaining amino acids. protein seems to have direct anabolic effects on the bone matrix. methionine and cysteine (21). 22.5. hypertension.6. it seems unlikely that this mechanism would operate in humans. it is now clear that net . This section therefore examines the relationship between protein intakes and long term health in relation to a number of specific disease states. It is well documented that diets containing high protein can result in an increase in urinary calcium excretion (17–20). or polycystic kidney disease. Accordingly. as low protein intake itself leads to bone loss.6. Renal function There is clear evidence that high intakes of protein by patients with renal disease contribute to the deterioration of kidney function (8–12).

e. Although some studies suggest that high animal protein intake might increase the risk of kidney stones. Apart from any IGF-1-mediated effects there is considerable evidence for a limitation on the synthesis of glycine (36). which has been extensively discussed in the literature. Kidney stones A second potential consequence of high-protein diets. 1. lower intakes of endogenous non-carbonic acid (i. This probably explains the beneficial influence of fruit and vegetables.renal acid excretion predicts calcium excretion. It may also explain why calcium citrate malate supplements are more effective for bone health than other calcium salts (31). 30).e. The magnitude and importance of the bone protein pool are such that a positive effect of protein on bone is not surprising. Renal stones occur very commonly. and that this can be predicted from the ratio of dietary protein to potassium. a lower protein intake but a higher potassium intake) were related to better measures of bone health (27. on bone health (29. prospective studies of the effect of dietary calcium and other nutrients on the risk of kidney stones showed that a higher intake of calcium decreased. and have been estimated to affect 12% of the United States population at some time (38). Moreover. particularly in those subjects who are classified as idiopathic calcium stone formers. More recent studies have shown that in elderly populations protein supplements increase serum IGF-1 levels and attenuate proximal femur bone loss in patients with recent hip fracture (35). is an increased occurrence of renal stones.7. 28). so that competition for glycine between collagen and its other important metabolic demands might prevent its reutilization during bone collagen turnover. the major dietary source of potassium. protein can exert an independently beneficial effect through its insulin-like growth factor-1 (IGF-1)-mediated anabolic influences on bone. and a higher intake of animal protein increased. The importance of achieving low net renal acid excretion is that once the potential acidifying influence of dietary protein is balanced by the alkalizing effect of the dietary potassium intake. It does appear that dietary protein as part of a well-balanced diet is most likely to be beneficial for bone. which accounts for 25% of collagen.6. 42). >185 g/day . In women. the risk of stones (41. 40). which was estimated to increase therisk of forming stones by 250% (39. since the dietary intake of potassium occurs mainly as salts of weak organic acids and therefore has an alkalizing effect (26). Initial studies showed that an increase in dietary animal protein resulted in an elevation of urinary calcium and oxalate. possibly at dietary levels in excess of the recommended intake. as yet no clear conclusions can be drawn since dietary effects are apparent only in studies with very large differences in protein intakes (i.

many biologically active proteins lose their activity upon denaturation. ionic strength. partial denaturation of proteins at the air-water and oil-water interfaces improves their foaming and emulsifying properties. In the case of food proteins.83 g/kg per day). .” whereas major changes in the secondary. Proteins undergo varying degrees of denaturation during processing. 1. but not excessive amounts (i. because it indicates loss of some properties. less than 1. For example. are usually regarded as “conformational adaptability. and quaternary structures without cleavage of backbone peptide bonds are regarded as “denaturation.7.compared with 80 g/day). temperature. preferably from vegetable sources. Often denaturation has a negative connotation. tertiary.” Denaturation is a phenomenon wherein a well defined initial state of a protein formed under physiolosical conditions is transformed into an ill defined final state under non physiological conditions using a denaturing agent. denaturation usually causes insolublization and loss of some functional properties. such as pH. Protein Denaturation The native structure of a protein is the net result of various attractive and repulsive interactions emanating from various intramolecular forces as well as interaction of various protein groups with surrounding solvent water. Temperature and Denaturation Heat is the most commonly used agent in food processing and preservation. from a food application stand point protein denaturation during processing is not always undesirable.e. In fact in some cases it is highly desirable. the diet should ideally provide at least the safe level (0. it undergoes a sharp transition from the native state to the denatured state. When a protein solution is gradually heated above a critical temperature. where the concentration ratio of native and denatured states is known either as the melting temperature Tm or the denaturation temperature. whereas excessive thermal denaturation of soy proteins diminishes their foaming and emulsifying properties. It is recommended that in order to minimize the risk of kidney stones in patients who are at risk. However. Any change in its environment.. solvent composition.7. will force the molecule to assume a new equilibrium structure. The native state (of a single protein molecule) is thermodynamically the most stable with lowest feasible free energy at physiological conditions. etc.4 g/kg per day). which do not drastically alter the molecular architecture of the protein. Subtle changes in structure. For instance.1. The temperature at the transition midpoint. 1.

are also low in hyperthermostable proteins.7.On the other hand. thermostable proteins have high levels of Ile and Pro.Thermal stability of proteins from thermophilic and hyperthermophilic organisms. Met. This causes a shift in equilibrium between the native and denatured states. Detergents and Denaturation Detergents. Dry protein powders are extremely stable to thermal denaturation. Since the net electrostatic repulsive energy is small compared to other favorable interactions. pH-induced denaturation is mostly reversible. pH and Denaturation Proteins are more stable against denaturation at their isoelectric point than at any other pH. and Trp contents which can be oxidized easily at high temperatures. which reduces buried cavities or viod spaces can reduce mobilityof the polypeptide chain at high temperatures and this minimizes the increase in the configurational entropy of the polypeptide chain at high temperatures. SDS at 3–8 mM concentration denatures most globular proteins. most proteins are stable at around neutral pH. such as sodium dodecyl sulfate (SDS). An increase in water content from 0. is also attributed to their unique amino acid composition. At neutral pH. and a few are positively charged. most proteins are negatively charged. The high Ile content is belived to help in better packing of the interior core of the protein. the Td of the protein is the same as in a dilute protein solution.35 to 0.35 g water/g protein.3. 1.75 g water/g protein causes only a marginal decrease in Td. are powerful protein denaturing agents. which can with stand extremely high temperatures. The value of Td decreases rapidly as the water content is increased from 0 to 0.95]. The mechanism involves preferential binding of detergent to the denatured protein molecule. The Cys.7.[30] . Above 0.75 g water/g protein. Water greatly facilitates thermal denaturation of proteins [37.2. 1. The degree of unfolding is greater at extreme alkaline pH values than it is at extreme acid pH.

Typically. The same basic approach is still used today.e. this is only an average value.1. Many food proteins are enzymes which are capable of enhancing the rate of certain biochemical reactions. although a number of improvements have been made to speed up the process and to obtain more accurate measurements.25 (equivalent to 0. such as lysine. proteins are used as gelling agents. type. 1. and each protein has a different conversion factor depending on its amino-acid composition.16 g nitrogen per gram of protein) is used for many applications. leucine. methionine. Because the Kjeldahl method does not measure the protein content directly a conversion factor (F) is needed to convert the measured nitrogen concentration to a protein concentration. their ability to provide desirable appearance. It is usually considered to be the standard method of determining protein concentration. however. i. These reactions can have either a favorable or detrimental effect on the overall properties of foods. but which the body cannot synthesize. The amount of protein present is then calculated from the nitrogen concentration of the food.8.. A conversion factor of 6. emulsifiers. ANALYSIS OF PROTEINS Proteins are important constituents of foods for a number of different reasons. which are essential to human health. Food analysts are interested in knowing the total concentration. tryptophan. as well as containing essential aminoacids. foaming agents and thickeners. high precision and good .1 Kjeldahl method: In Kjeldahl method food is digested with a strong acid so that it releases nitrogen which can be determined by a suitable titration technique. They are a major source of energy. texture or stability. The Kjeldahl method is widely used internationally and is still the standard method for comparison against all other methods. isoleucine and valine. molecular structure and functional properties of the proteins in foods. Isolated proteins are often used in foods as ingredients because of their unique functional properties.8. Its universality.

It is beginning to compete with the Kjeldahl method as the standard method of analysis for proteins for some foodstuffs due to its rapidness. But it has high initial cost. It doesn't need toxic chemicals or catalysts. The absorbance (or turbidity) of the solution being analyzed is then measured at the same wavelength.8. which are based on UV-visible spectroscopy. These methods use either the natural ability of proteins to absorb (or scatter) light in the UV-visible region of the electromagnetic spectrum. The basic principle behind each of these tests is similar. It does not give a measure of the true protein. an automated instrumental technique has been developed which is capable of rapidly measuring the protein concentration of food samples. First of all a calibration curve of absorbance (or turbidity) versus protein concentration is prepared using a series of protein solutions of known concentration.3 UV-visible spectroscopy: A number of methods have been devised to measure protein concentration. Different proteins need different correction factors because they have different amino acid sequences. since all nitrogen in foods is not in the form of protein. It is much faster than the Kjeldahl method (under 4 minutes per measurement. The use of concentrated sulfuric acid at high temperatures poses a considerable hazard. and its protein concentration determined .reproducibility have made it the major method for the estimation of protein in foods.8. as does the use of some of the possible catalysts The technique is time consuming to carry-out. Many samples can be measured automatically. However it does not give a measure of the true protein. It is easy to use. 1. compared to 1-2 hours for Kjeldahl). or they chemically or physically modify proteins to make them absorb (or scatter) light in this region. since all nitrogen in foods is not in the form of protein. 1. The small sample size makes it difficult to obtain a representative sample. This technique is based on a method first described by a scientist called Dumas over a century and a half ago.2 Enhanced Dumas method: Recently. Different proteins need different correction factors because they have different amino acid sequences.

The advantages of this method are that the procedure is simple to carry out. . basic groups and aggregated proteins. The major advantage of this technique is that there is no interference from materials that adsorb at lower wavelengths. and so the absorbance of protein solutions at 280nm can be used to determine their concentration. aromatic side-groups. e. rather than specific side groups. However. Biuret Method: A violet-purplish color is produced when cupric ions (Cu2+) interact with peptide bonds under alkaline conditions. and the technique is less sensitive to protein type because it utilizes absorption involving peptide bonds that are common to all proteins.. It is mixed with a protein solution and then allowed to stand for 15-30 minutes before the absorbance is read at 540 nm. This method is more sensitive to low concentrations of proteins than the biuret method. The main difference between the tests are the chemical groups which are responsible for the absorption or scattering of radiation. methods have been developed to overcome this problem. Even so. The tryptophan and tyrosine content of many proteins remains fairly constant..g. e. Tryptophan and tyrosine absorb ultraviolet light strongly at 280 nm. The major disadvantage is that nucleic acids also absorb strongly at 280 nm and could therefore interfere with the measurement of the protein if they are present in sufficient concentrations. it is nondestructive. Lowry Method: The Lowry method combines the biuret reagent with another reagent (the FolinCiocalteau phenol reagent) which reacts with tyrosine and tryptophan residues in proteins. can be purchased commercially.g. peptide bonds. There is a small peak around 500 nm that can be used to determine high protein concentrations and a large peak around 750 nm that can be used to determine low protein concentrations. The biuret reagent.from the calibration curve. This gives a bluish color which can be read somewhere between 500 750 nm depending on the sensitivity required. it has a relatively low sensitivity compared to other UV-visible methods. which contains all the chemicals required to carry out the analysis. and no special reagents are required. by measuring the absorbance at two different wavelengths.

because all the water is "bound" to the salts. The amount of unbound dye remaining in solution after the insoluble protein-dye complex has been removed (e. including the reasons for carrying out the analysis. charge. adsorption characteristics.1 Methods Based on difference in solubility: Salting out: Proteins are precipitated from aqueous solutions when the salt concentration exceeds a critical level. 1. the amount of sample available. such as size. Protein type is usually determined by separating and isolating the individual proteins from a complex mixture of proteins. the equipment available. The anionic dye binds to cationic groups of the basic amino acid residues (histidine. so that they can be subsequently identified and characterized.. arganine and lysine) and to free amino terminal groups. the type of proteins present and the cost.g.dyefree. solubility and heat-stability.. NaCl or KCl.no. the desired purity. 1.g. The choice of an appropriate separation technique depends on a number of factors.Dye binding methods: A known excess of a negatively charged (anionic) dye is added to a protein solution whose pH is adjusted so that the proteins are positively charged (i. e. The proteins form an insoluble complex with the dye because of the electrostatic attraction between the molecules. although other neutral salts may also be used. but the unbound dye remains soluble. In . < the isoelectric point).9.e. Ammonium sulfate [(NH4)2SO4] is commonly used because it has a high watersolubility.(ref. which is known as salting-out. and is therefore not available to hydrate the proteins. The amount of protein present in the original solution is proportional to the amount of dye that bound to it: dyebound = dyeinitial . Proteins are separated on the basis of differences in their physicochemical properties.9 Protein Separation and Characterization: Food analysts are often interested in the type of proteins present in a food because each protein has unique nutritional and physicochemical properties. by centrifugation) is determined by measuring its absorbance. Generally a two-step procedure is used to maximize the separation efficiency.

The optimum quantity of organic solvent required to precipitate a protein varies from about 5 to 60%. This tends to decrease the solubility of proteins in solution because they are less ionized. which must be removed before the protein can be resolubilzed. The dielectric constant of aqueous solutions can be lowered by adding water-soluble organic solvents. and therefore the electrostatic repulsion between them is not sufficient to prevent them from aggregating. .g. Proteins have different isoelectric points because of their different amino acid sequences (i. but leaves more soluble proteins in solution. Isoelectric Precipitation: The isoelectric point (pI) of a protein is the pH where the net charge on the protein is zero.the first step. Proteins tend to aggregate and precipitate at their pI because there is no electrostatic repulsion keeping them apart. As the dielectric constant of a solution decreases the magnitude of the electrostatic interactions between charged species increases. Solvent fractionation is usually performed at 0oC or below to prevent protein denaturation caused by temperature increases that occur when organic solvents are mixed with water. The main problem with this method is that large concentrations of salt contaminate the solution. by dialysis or ultrafiltration. The amount of organic solvent required to cause precipitation depends on the protein and therefore proteins can be separated on this basis. This precipitates out the protein of interest (which can be separated by centrifugation). e. and thus they can be separated by adjusting the pH of a solution..e.. The salt concentration is then increased to a point just above that required to cause precipitation of the protein. relative numbers of anionic and cationic groups). The solution is then centrifuged to remove any proteins that are less soluble than the protein of interest. the salt is added at a concentration just below that necessary to precipitate out the protein of interest. such as ethanol or acetone. When the pH is adjusted to the pI of a particular protein it precipitates leaving the other proteins in solution Solvent Fractionation: The solubility of a protein depends on the dielectric constant of the solution that surrounds it because this alters the magnitude of the electrostatic interactions between charged groups.

2 Methods Based on selective Adsorption Ion Exchange Chromatography Ion exchange chromatography relies on the reversible adsorption-desorption of ions in solution to a charged solid matrix or polymer network. Contaminating proteins bind less strongly and therefore pass more rapidly through the column. The protein of interest is then eluted using another buffer solution which favors its desorption from the column (e. This technique is the most commonly used chromatographic technique for protein separation. different pH or ionic strength). The buffer conditions (pH and ionic strength) are adjusted to favor maximum binding of the protein of interest to the ion-exchange column. The ligand is a molecule that has a highly specific and unique reversible affinity for a particular protein.g. The sample to be analyzed is passed through the column and the protein of interest binds to the ligand. The protein of interest is then eluted using a buffer solution which favors its desorption from the column.. A positively charged matrix is called an anion-exchanger because it binds negatively charged ions (anions). Proteins that are stable at high temperature or at extremes of pH are most easily separated by this technique because contaminating proteins can be precipitated while the protein of interest remains in solution. 1. whereas the contaminating proteins pass directly through.9. Affinity Chromatography Affinity chromatography uses a stationary phase that consists of a ligand covalently bound to a solid support.Denaturation of Contaminating Proteins Many proteins are denatured and precipitate from solution when heated above a certain temperature or by adjusting a solution to highly acid or basic pHs. This technique is the most efficient means of separating an individual . A negatively charged matrix is called a cation-exchanger because it binds positively charged ions (cations).

the molecular weights of proteins vary from about 10. Dialysis is a relatively slow method. rather than directly on its molecular weight. Ultrafiltration A solution of protein is placed in a cell containing a semipermeable membrane. Dialysis Dialysis is used to separate molecules in solution by use of semipermeable membranes that permit the passage of molecules smaller than a certain size through. and depends on its three dimensional molecular structure. but it is the most expensive. Both ion-exchange and affinity chromatography are commonly used to separate proteins and amino-acids in the laboratory. whereas the larger molecules remain in the solution. separation depends on the Stokes radius of a protein. but the large molecular weight protein molecules remain in the bag. It is therefore most frequently used in the laboratory.protein from a mixture of proteins. The Stokes radius is the average radius that a protein has in solution.3 Separation Due to Size Differences Proteins can also be separated according to their size. and pressure is applied.000 daltons. but prevent the passing of larger molecules. They are used less commonly for commercial separations because they are not suitable for rapidly separating large volumes and are relatively expensive. In practice. Dialysis is often used to remove salt from protein solutions after they have been separated by salting-out. Typically. taking up to 12 hours to be completed. because of the need to have columns with specific ligands bound to them. The separation principle of this technique is therefore similar to dialysis.9.000. but because pressure is applied separation . 1. For proteins with the same molecular weight the Stokes radius increases in the following order: compact globular protein < flexible random-coil < rod-like protein.000 to 1. Smaller molecules pass through the membrane. A protein solution is placed in dialysis tubing which is sealed and placed into a large volume of water or buffer which is slowly stirred. and to change buffers. Low molecular weight solutes flow through the bag.

Molecular weights of unknown proteins can be determined by comparing their elution volumes Vo. In non-denaturing electrophoresis. It can be used to separate proteins on the basis of their size. Ultrafiltration units are used in the laboratory and on a commercial scale. whilst that part which passes through the membrane (small molecules) forms part of the ultrafiltrate. Manufacturers of these beads provide information about the molecular weight range that they are most suitable for separating. Beads of different average pore size are available for separating proteins of different molecular weights. Semipermeable membranes with cutoff points between about 500 to 300. and move quickly through the column. exchange buffers or fractionate proteins on the basis of their size. a buffered solution of native proteins is poured onto a porous gel (usually polyacrylamide. Molecules larger than the pores in the beads are excluded. and at a rate that depends on the magnitude of the charge. and the friction to their movement: . whereas the movement of molecules which enter the pores is retarded. A protein solution is poured into a column which is packed with porous beads made of a cross-linked polymeric material (such as dextran or agarose).4 Separation by Electrophoresis Electrophoresis relies on differences in the migration of charged molecules in a solution when an electrical field is applied across it. Ultrafiltration can be used to concentrate a protein solution. That portion of the solution which is retained by the cell (large molecules) is called the retentate. Thus molecules are eluted off the column in order of decreasing size. Size Exclusion Chromatography This technique. starch or agarose) and a voltage is applied across the gel. with those determined using 1.is much quicker. remove salts.000 are available. shape or charge. The proteins move through the gel in a direction that depends on the sign of their charge. also separates proteins according to their size. sometimes known as gel filtration.9.

This type of electrophoresis is commonly called sodium dodecyl sulfate -polyacrylamide gel electrophoresis. As proteins travel through a gel network they are primarily separated on the basis of their molecular weight because their movement depends on the size of the protein molecule relative to the size of the pores in the gel: smaller proteins moving more rapidly through the matrix than larger molecules. The protein is extracted from the food into solution. In denaturing electrophoresis proteins are separated primarily on their molecular weight. which breaks down disulfide bonds. To determine how far proteins have moved a tracking dye is added to the protein solution. and positively charged if the pH is below the pI. Hence. The relative mobility of each protein band is calculated: Electrophoresis is often used to determine the protein composition of food products. the charge per unit length and the molecular conformation is approximately similar for all proteins. SDS-PAGE is used to determine the molecular weight of a protein by measuring Rm.Proteins may be positively or negatively charged in solution depending on their isoelectic points (pI) and the pH of the solution. In non-denaturing electrophoresis the native proteins are separated based on a combination of their charge. A protein is negatively charged if the pH is above the pI. which is then separated using electrophoresis. The magnitude of the charge and applied voltage will determine how far proteins migrate in a certain time. Each protein molecule binds approximately the same amount of SDS per unit length. which is an anionic surfactant that hydrophobically binds to protein molecules and causes them to unfold because of the repulsion between negatively charged surfactant head-groups. After the electrophoresis is completed the proteins are made visible by treating the gel with a protein dye such as Coomassie Brilliant Blue or silver stain. or SDS-PAGE. and sodium dodecyl sulfate (SDS). This dye is a small charged molecule that migrates ahead of the proteins.g. and then comparing it with a calibration .. bromophenol blue. e. size and shape. Proteins are denatured prior to analysis by mixing them with mercaptoethanol.

Get to know a fishmonger (the person who sells the fish) at the store.10.curve produced using proteins of known molecular weight: a plot of log (molecular weight) against relative mobility is usually linear. it is a good source of omega-3 fatty acids. It is available fresh as steaks. ion exchange. fillets or pieces. A protein sample is first hydrolyzed (e. an in-depth nutritional profile for Tuna is also available. Denaturing electrophoresis is more useful for determining molecular weights than non-denaturing electrophoresis. . sugar. 1. In addition iso electric focusing electrophoresis and two dimensional electrophoresis are also employed. which are then separated using chromatography.10. vitamins. it is best to purchase fresh tuna from a store that has a good reputation for having a frequent supply of fresh fish.g. Tuna is probably best known in its canned form. including carbohydrates.5 Amino Acid Analysis Amino acid analysis is used to determine the amino acid composition of proteins. In-Depth Nutritional Profile In addition to the nutrients highlighted in our ratings chart.[31] 1. because the friction to movement does not depend on the shape or original charge of the protein molecules. phosphorus.g. In addition.10. soluble and insoluble fiber.Sampling technique: Tuna is sold in many different forms. and magnesium. using a strong acid) to release the amino acids.9.1. 1. e. Just as with any seafood. so you can have a trusted resource from whom you can purchase your fish with confidence. amino acids and more. fatty acids. 1. potassium. minerals. affinity or absorption chromatography. and protein. Tuna is also a very good source of vitamin B6 and thiamin.2. selenium.. sodium. Nutritional Profile Tuna is an excellent source of niacin. This profile includes information on a full array of nutrients.

while fish that was caught the week before can only be stored for about one or two days. and most Americans already consume too many omega-6 fats in comparison to omega-3s. Canned tuna is available either solid or in chunks. Try to avoid purchasing tuna that has dry or brown spots. Fish that was caught the day before you purchased it can be stored for about four days.e. which has been well wrapped. while fillets and steaks should be placed on top of the ice. after purchasing tuna or other fish refrigerate it as soon as possible. The length of time that tuna can stay fresh stored this way depends upon how fresh it is. broth or water. purchase displayed fish as opposed to pieces that are prepackaged. when it was caught. The baking dish and fish should then be placed on the bottom shelf of the refrigerator. When storing all types of fresh seafood. If the fish is going to accompany you during a day full of errands. i. Smell is a good indicator of freshness. it is best to purchase tuna packed in water or broth. One of the easiest ways to do this is to place fish. Oftentimes. if you have the option. . keep a cooler in the car where you can place your tuna to make sure it stays cold and does not spoil. smell it through the paper wrapping and return it if it has a truly strong fishy odor. To ensure maximum freshness and quality. Once the fishmonger wraps and hands you the fish that you have selected. it is important to use special storage methods to create the optimal temperature for holding the fish. in a baking dish filled with ice. Since a slightly "off" smell cannot be detected through plastic. The temperature of most refrigerators is slightly warmer than ideal for storing fish. including tuna. canned tunas do not distinguish which specific species was used except to note that it is either light tuna (bluefin or yellowfin) or white tuna (usually albacore). Since omega-6s and omega-3s compete for the same enzymes that activate them for use in the body. and is packaged in oil. Therefore.Fresh whole tuna should be displayed buried in ice. it also has the highest fat content. and the oils in which it is packed are high in omega-6 fats. which is its coolest area. Although the tuna packed in oil is usually the moistest. Replenish the ice one or two times per day. it is important to keep them cold since fish spoils quickly and is very sensitive to temperature.

wrap it well in plastic and place it in the coldest part of the freezer where it will keep for about two to three weeks.[34-60] .You can extend the shelf life of tuna by freezing it. To do so.

and fish. 5. proteins because they provide sufficient amounts of the essential amino acids. Plant proteins. and growth. vegetables and fruits. The consumption of protein rich food is essential for human being to meet the daily requirement. or incomplete proteins because many plant proteins lack one or more of the essential amino acids. are lower-quality. catalyst. The quality of protein depends on the level at which it provides the nutritional amounts of essential amino acids needed for overall body health. Protein analysis would be carried out on the following raw as well as processed samples.1. 1..CHAPTER 2 2. etc. maintenance. 7. such as grain. nuts. The quality and quantity of the protein changes with the processing and also depends on the technique used. 3. or because they lack a proper balance of amino acids. Raw Samples Raw Sardines Raw Tuna (yellow fin) Raw Mackerel Raw Chicken (Broiler) Raw Beef Raw Pork Raw Mutton Processed Samples Sardines in Brine (canned) Tuna in Brine (canned) Mackerel in Brine (canned) Minced Chicken (Broiler canned) Minced Beef (canned) Minced Pork (canned) - . and to know the effect of processing on protein content of the food. Scope and objective:Proteins play multiple role as enzymes. milk. In order to make out the difference in the protein content in stable processed foods from raw foods. Protein deficiency leads to malnutrition in children which is common in our country according to WHO (world health organization). hormones.No. are considered high-quality. 2. S. 6. 4. such as eggs. corn. cheese. or complete. meat. Animal proteins.

rich in protein can be incorporated and maintain the food without any loss of protein from it. To recommend protein fortification in processed foods and to maintain the protein content and to increase the food quality to incorporating the new processing techniques to maintain the constant levels of protein content of foods during processing. The loss of protein content during processing can be value added with other kinds of food materials. to improve the quality of processed foods with respect to the protein content to reach the essential needs of the human being.Therefore. .

The amount of ammonia nitrogen in this solution is quantified by titration with a standard HCl solution. (3) titration. (2) distillation. A reagent blank is carried through the analysis and the volume of HCl titrant required for this blank is subtracted from each determination. This NH3 is “trapped” in a boric acid solution.1 Principle of Method:The Kjeldahl procedure measures the nitrogen content of a sample. organic nitrogen is converted to an ammonium in the presence of a catalyst at approximately 370°C. . In the distillation step.CHAPTER-3 METHODS AND MATERIALS: METHOD: KJELDAHL NITROGEN METHOD: 3. the digested sample is made alkaline with NaOH and the nitrogen is distilled off as NH3. The Kjeldahl procedure can be basically divided into three parts: (1) digestion. The protein content then. In the digestion step. can be calculated assuming a ratio of protein to nitrogen for the specific food being analyzed.

Add dd water to make up to 1. . Cool. 95% pure Methyl red Sodium hydroxide (NaOH) Corrosive Sulfuric acid. 40%.  Sodium Hydroxide Solution.. Cool to room temperature under tap water. w/v. Qualigens fine chemicals Ltd.).  100 mg bromocresol green/100 ml ethanol and 100 mg methyl red/100 ml ethanol. (Fisher scientific Thermic electron LLS India Pvt Ltd. conc.0L (Fisher chemic Ltd. (H2SO4) Corrosive Kjeldahl digestion tablets Irritant Potassium sulfate (K2SO4) Cupric sulfate Oxalic acid dehydrate purified (Harmful) 3.1N. 0.3. NaOH in deionized distilled (dd) water. 5% of potassium sulfate and 1% of cupric sulfate (Kjeldahl digestion tablets) Contains potassium sulfate. cupric sulfate.3. Reagents:  Sulfuric Acid concentrated. dissolve 20g boric acid in ca. Dissolve 400g sodium hydroxide (NaOH) pellets in 1L dd water.  Boric Acid Solution 4% ** In a 500ML flask.2.)  Catalyst/Salt Mixture. (Merck specialties Pvt Ltd. Chemicals: a) b) c) d) e) f) g) h) i) j) Hazards Boric acid (H3BO3) Bromocresol green Ethanol.).

The samples should be clear (but neon green).) I. 7 ml) of concentrated sulfuric acid to each tube with corn flour. 4. Turn on digestion block and heat to appropriate temperature. 1. Follow manufacturer’s instructions for specific Kjeldahl digestion and distillation system used. .1 g corn flour. Prepare duplicate blanks: one catalyst tablet + volume of sulfuric acid used in the sample + weigh paper (if weigh paper was added with the corn flour samples).. Accurately weigh approximately 0.g. Record the weight. Procedure: (Instructions are given for analysis in triplicate. 2. 5. Some instructions given here may be specific for one type of Kjeldahl system. Add one catalyst tablet and appropriate volume (e. Place rack of digestion tubes on digestion block. Digestion: . with no charred material remaining.3. Repeat for two more samples. Let samples digest until digestion is complete. Cover digestion block with exhaust system turned on. Place corn flour in digestion tube.4. 3.

Take samples off the digestion block and allow to cool with the exhaust system still turned on. .6. Make sure that the tube coming from the distillation of the sample is submerged in the boric acid solution. In this distillation process. Put sample tube in place. proceed with a new sample tube and receiving flask. a set volume of NaOH solution will be delivered to the tube and a steam generator will distill the sample for a set period of time. Swirl each tube. 2. 3. 4. Upon completing distillation of one sample.Follow appropriate procedure to start up distillation system. Dispense appropriate volume of boric acid solution into the receiving flask. Place receiving flask on distillation system. Distillation: 1. 7. making sure it is seated securely. Carefully dilute digest with an appropriate volume of dd water. and proceed with the distillation until completed. II.

7. If using an automated pH meter titration system. The corn flour sample you analyzed was not a dried sample. follow manufacturer’s instructions to shut down the distillation unit. Report percent protein results on a wet weight basis (wwb) and on a dry weight basis (dwb). Use 6. place it on a stir plate. Calculations Calculate the percent nitrogen and the percent protein for each of your duplicate or triplicate corn flour samples.2. 3.25 for the nitrogen to protein conversion factor. Keep the solution stirring briskly while titrating. Put a magnetic stir bar in the receiver flask and place it on a stir plate. = (ml std. Record volume of HCl titrant used. If using a colorimetric endpoint.Record volume of HCl titrant used. III. Record the normality of the standardized HCl solution as determined by the teaching assistant. acid for sample) − (ml std.5. Titrate each sample and blank with the standardized HCl solution to the first faint gray color. for blank) % Protein = % N × Protein Factor . 2. Normality is in mol/1000 mL Corrected acid vol. After completing distillation of all samples. and then determine average values. 3. Titration: 1. and keep the solution stirring briskly while titrating. follow manufacturer’s instructions to calibrate the instrument. Assume moisture content of 10% (or use the actual moisture content if previously determined on this corn flour sample). but do not let the stir bar hit the electrode. put a magnetic stir bar in the receiver flask. Titrate each sample and blank to an endpoint pH of 4.

12 18.332 0.718 1.59 The amount of protein in sardines fish was estimated by kjeldhal method batch method was employed and in each batch one blank and five samples with different .1 36.NO 1.0979 0.8 13.304 0. SAMPLE-1a: SARDINES FISH (RAW):FORMULA FOR % OF NITROGEN = 1.00 23. 0.0979 0.907 0. 5.56 20.2 15.514 0. SAMPLE WEIGHT IN GRAM BLANK NORMALITY OF H2SO4 VOLUME OF H2SO4 IN ml % OF NITROGEN % OF PROTEIN 0.00 2.2 32.7 24.NO 1.17 SAMPLE-1b: SARDINES FISH (RAW):SAMPLE WEIGHT IN GRAM BLANK NORMALITY OF H2SO4 VOLUME OF H2SO4 IN ml % OF NITROGEN % OF PROTEIN S.0979 0.069 1.0979 0.0979 0.9 18.V) / SAMPLE WEIGHT FORMULA FOR % OF PROTEIN = % OF NITROGEN * 6.532 0.0979 0.7 0.2 11. 4.0979 0.555 0.8 0. 5.00 3.8 3.3 8.V – B. 4.0 17.8 16.7 19.24 3.0979 0.740 1. 3.CHAPTER-4 RESULTS AND DISCUSSION: This chapter contains the results obtained in the protein analysis of certain raw and processed animal food samples.8 24.37 19.00 17. 6.0979 0.0979 0.25 19.06 2.295 0.3 7. 2.25 S.024 1.0979 0.0979 0.848 2. 3.13 3.185 3.4* N H2SO4 * (T.716 2.9 20. 2. 6.939 3.550 0.

0.NO 1.058 1.17 1. 2.97 1. 2.9 0.715 1.NO 1.9 21. SAMPLE WEIGHT IN GRAM BLANK NORMALITY OF H2SO4 VOLUME OF H2SO4 IN ml % OF NITROGEN % OF PROTEIN S.2 22.62 18.088 1.0994 0.81 1.2 6. 6.82 0.V) / SAMPLE WEIGHT FORMULA FOR % OF PROTEIN = % OF NITROGEN * 6. The percentage of protein in the fish sample are listed in tables 1a and 1b it is found that this fish sample contains about 20% protein. 4.88 2.00 17.0994 0.325 0.0994 0. 3.538 0.82 2.50 2.00 2.75 17.0994 0.00 17.0994 0.weights were taken.1 9.93 13.8 12.512 0.56 11.62 SAMPLE-2b: SARDINES PROCESSED IN BRINE SOLUTION: (ANCHOR SEA FOOD PRODUCTS) FORMULA FOR % OF NITROGEN = 1.3 In order to determine the effect of processing on protein content in sardines fish the processed food sample containing the same type of fish was used in the analysis the values obtained are presented in table 2a and2b this values indicate that the processed fish sample contains less amount of protein then the raw fish.34 17.37 17.6 8.8 8.0994 0.00 2.25. 0. 5.77 2.0994 0.0994 0.3 30. . SAMPLE-2a: SARDINES PROCESSED IN BRINE SOLUTION: (ANCHOR SEA FOOD PRODUCTS) SAMPLE WEIGHT IN GRAM BLANK NORMALITY OF H2SO4 VOLUME OF H2SO4 IN ml % OF NITROGEN % OF PROTEIN S.56 11.82 0.0994 0.2 20. 3.2 6. 4.556 0.V – B. 5.0994 0.545 0.0994 0. 6.4* N H2SO4 * (T.319 0.738 1.87 2.56 9.0994 0.5 0.

412 4.37 The amount of protein in tuna fish was estimated by kjeldhal method batch method was employed and in each batch one blank and five samples with different weights were taken.098 1.1082 0.3 26.99 21.1082 0. 5.18 SAMPLE-3b: TUNA FISH (RAW):S.59 27.1 23.1082 0.NO SAMPLE WEIGHT IN GRAM BLANK NORMALITY OF H2SO4 VOLUME OF H2SO4 IN ml % OF NITROGEN % OF PROTEIN 1.13 27. 2.189 0.38 27. 0.1082 0.00 26.1082 0. 6.87 26.31 29. 6.7 21.715 1. 4.1082 0.523 0.66 27.513 0.6 32.00 4.0 15.2 42.370 4.460 4.5 0.1082 0.181 4.1082 0.325 0.2 9.319 3.532 0. .00 27.1082 0.NO 1. 5.7 0.381 4.2 44.355 0.1082 0. 0.733 1. 3.414 4.745 4.00 4.0 22. The percentage of protein in the fish sample are listed in tables 3a and 3b it is found that this fish sample contains about 27% protein. 2.380 0.533 0.021 1.SAMPLE-3a: TUNA FISH (RAW):SAMPLE WEIGHT IN GRAM BLANK NORMALITY OF H2SO4 VOLUME OF H2SO4 IN ml % OF NITROGEN % OF PROTEIN S. 3.1082 0.2 10.1082 0. 4.6 15.

582 3.6 41.54 22.312 0.0957 0.0957 0. 5.67 19.531 0.055 1.531 0.29 SAMPLE-4b: TUNA FISH PROCESSED IN BRINE SOLUTION:(ANCHOR SEA FOOD PRODUCTS) SAMPLE WEIGHT IN GRAM BLANK NORMALITY OF H2SO4 VOLUME OF H2SO4 IN ml % OF NITROGEN % OF PROTEIN S.SAMPLE-4a: TUNA FISH PROCESSED IN BRINE SOLUTION:(ANCHOR SEA FOOD PRODUCTS) SAMPLE WEIGHT IN GRAM BLANK NORMALITY OF H2SO4 VOLUME OF H2SO4 IN ml % OF NITROGEN % OF PROTEIN S.61 17.6 42. 2.4 28.17 15.0957 0. 0.26 23.8 0.1 15. 2.497 2.00 22.0957 0. 3.39 22. 6. 5. 6.NO 1.017 1. .6 16. 4.527 0.6 10.0957 0.8 0.607 3.827 3.672 3.706 1.727 0.580 0.082 3.00 18.1 19.54 22.2 8.00 3.606 3.00 2.0957 0.0957 0.81 In order to determine the effect of processing on protein content in tuna fish the processed food sample containing the same type of fish was used in the analysis the values obtained are presented in table 4a and 4b this values indicate that the processed fish sample contains less amount of protein then the raw fish.650 0.341 0. 0. 4.907 2.0957 0.718 1.0957 0.NO 1.2 7.0957 0.95 22.1 23.0957 0. 3.0957 0.

056 1. 4. 3. 2.1138 0.1138 0.0 15.083 3.2 30.1138 0.4 18.9 0.8 14. 5.66 19.11 SAMPLE-5b: MACKEREL FISH (RAW):SAMPLE WEIGHT IN GRAM BLANK NORMALITY OF H2SO4 VOLUME OF H2SO4 IN ml % OF NITROGEN % OF PROTEIN S.1 20.23 The amount of protein in mackerel fish was estimated by kjeldhal method batch method was employed and in each batch one blank and five samples with different weights were taken.36 19.057 0.00 19.6 11.1138 0. 4.NO 1.341 0.551 0.538 0.709 1.1138 0. 0.333 0.27 21.SAMPLE-5a: MACKEREL FISH (RAW):SAMPLE WEIGHT IN GRAM BLANK NORMALITY OF H2SO4 VOLUME OF H2SO4 IN ml % OF NITROGEN % OF PROTEIN S.43 21.2 0. . 0.510 3. 5.109 3.2 6.778 3.1138 0.705 1.1 6.00 3. 6.9 32.411 3.94 19.434 2.531 0. The percentage of protein in the fish sample are listed in tables 5a and 5b it is found that this fish sample contains about 21% protein.145 3.1138 0.8 12.1138 0.00 3.1138 0.1138 0. 2.00 19.397 0.32 21.563 0.1138 0.NO 1.138 3.032 1. 6.61 21.46 17.1138 0. 3.



1. 2. 3. 4. 5. 6.

0.312 0.515 0.721 1.036 1.506

0.1082 0.1082 0.1082 0.1082 0.1082 0.1082

0.1 5.8 9.9 15.7 20.9 29.7

0.00 2.767 2.882 3.277 3.041 2.977

0.00 17.29 18.01 20.48 19.00 18.6



1. 2. 3. 4. 5. 6.

0.349 0.553 0.712 1.014 1.580

0.0957 0.0957 0.0957 0.0957 0.0957 0.0957

0.2 6.8 13.4 16.0 19.3 30.5

0.00 2.456 3.198 2.973 2.523 2.569

0.00 15.35 19.98 18.58 15.77 16.05

In order to determine the effect of processing on protein content in mackerel fish the processed food sample containing the same type of fish was used in the analysis the values obtained are presented in table 6a and 6b this values indicate that the processed fish sample contains less amount of protein then the raw fish.



1. 2. 3. 4. 5. 6.

0.309 0.531 0.781 1.024 1.513

0.1035 0.1035 0.1035 0.1035 0.1035 0.1035

0.2 8.4 14.6 20.3 27.2 39.4

0.00 3.845 3.929 3.729 3.820 3.754

0.00 24.03 24.55 23.30 23.87 23.46



1. 2. 3. 4. 5. 6.

0.316 0.527 0.749 1.023 1.558

0.1035 0.1035 0.1035 0.1035 0.1035 0.1035

0.2 7.1 12.2 22.8 28.2 46.8

0.00 3.163 3.299 4.372 3.965 4.333

0.00 19.77 20.62 27.32 24.78 27.08

The amount of protein in broiler chicken was estimated by kjeldhal method batch method was employed and in each batch one blank and five samples with different weights were taken. The percentage of protein in the chicken sample are listed in tables 7a and 7b it is found that this chicken sample contains about 27% protein.



1. 2. 3. 4. 5. 6.

0.308 0.517 0.734 1.062 1.580

0.1035 0.1035 0.1035 0.1035 0.1035 0.1035

0.2 4.6 7.4 10.5 15.2 22.0

0.00 2.07 2.017 2.033 2.046 1.999

0.00 12.93 12.61 12.70 12.79 12.49



1. 2. 3. 4. 5. 6.

0.316 0.533 0.754 1.057 1.560

0.1035 0.1035 0.1035 0.1035 0.1035 0.1035

0.2 4.8 8.2 11.7 14.8 21.3

0.00 2.109 2.174 2.210 2.001 1.959

0.00 13.18 13.59 13.81 12.50 12.24

In order to determine the effect of processing on protein content in broiler chicken the processed food sample containing the same type of chicken was used in the analysis the values obtained are presented in table 8a and 8b this values indicate that the processed chicken sample contains less amount of protein then the raw chicken.

180 2. SAMPLE WEIGHT IN GRAM BLANK NORMALITY OF H2SO4 VOLUME OF H2SO4 IN ml % OF NITROGEN % OF PROTEIN S.62 14.SAMPLE-9a: MUTTON (RAW):FORMULA FOR % OF NITROGEN = 1.736 1.25. 4.1035 0.1035 0.325 0.1035 0.8 27. 3.0 8.NO 1.1035 0.6 20.4* N H2SO4 * (T. .1035 0.517 0.546 0. 0.00 2.536 0.220 2. The percentage of protein in the mutton sample are listed in tables 9a and 9b it is found that this mutton sample contains about 19% protein.018 1.799 18. 0.26 19.NO 1.4 0. SAMPLE WEIGHT IN GRAM BLANK NORMALITY OF H2SO4 VOLUME OF H2SO4 IN ml % OF NITROGEN % OF PROTEIN S.282 3. 2.1035 0.94 17.031 2.788 1. 4.511 0.V – B.053 1.1035 0.60 SAMPLE-9b: MUTTON (RAW):FORMULA FOR % OF NITROGEN = 1.1035 0.567 3.00 13.006 1.25.4* N H2SO4 * (T.7 6. 3.8 17.0 15.9 0.53 9.V) / SAMPLE WEIGHT FORMULA FOR % OF PROTEIN = % OF NITROGEN * 6.1035 0.45 16. 6.1035 0. 5.00 2.53 13.656 0. 6. 2.V – B.V) / SAMPLE WEIGHT FORMULA FOR % OF PROTEIN = % OF NITROGEN * 6. 5.87 16.5 27.793 2.598 0.126 2.23 The amount of protein in mutton was estimated by kjeldhal method batch method was employed and in each batch one blank and five samples with different weights were taken.00 12.319 0.1035 0.2 4.1035 0.2 15.2 5.

1 21.008 1. 4.58 18.1035 0.1035 0.93 20.1035 0.009 1.3 36.00 19.889 3.NO 1. 0.1035 0.SAMPLE-10a: BEEF (RAW):SAMPLE WEIGHT IN GRAM BLANK NORMALITY OF H2SO4 VOLUME OF H2SO4 IN ml % OF NITROGEN % OF PROTEIN S.4 8.92 20. 5.6 9.188 3. 2.12 16.522 0.1035 0. 4.NO 1.516 0.509 0.0 5.0 0.00 3.9 20. The percentage of protein in the beef sample are listed in tables 10a and 10b it is found that this beef sample contains about 20% protein.242 3.305 0.09 20.653 2.1035 0.421 0.1 11.1035 0.38 SAMPLE-10b: BEEF (RAW):SAMPLE WEIGHT IN GRAM BLANK NORMALITY OF H2SO4 VOLUME OF H2SO4 IN ml % OF NITROGEN % OF PROTEIN S. 2. 5.1035 0.337 0. 6.1035 0. 3.65 The amount of protein in beef was estimated by kjeldhal method batch method was employed and in each batch one blank and five samples with different weights were taken.00 18.311 0.1035 0.552 0.1035 0.1035 0.8 13.9 29.26 18.030 3. 6.22 2. 0.00 2.05 21.706 1.00 20. 3. .748 1.214 3.2 7.0 0.880 3.

362 0.0 6.56 14. 6.489 2.1 21.1035 0.269 1. 6. 3.052 1. 0. 4.016 2.97 14.1035 0.388 0.9 29.00 2. 2.1035 0.0 5.777 2.NO 1. 5.9 13.0948 0.549 0.1035 0.0948 0.1 8.777 0.236 2.3 18. 0.348 2.67 15.4 8.2 33.296 2.0948 0.1035 0.10 18.00 12.60 14.50 In order to determine the effect of processing on protein content in minced beef the processed food sample containing the same type of beef was used in the analysis the values obtained are presented in table 11a and 11b this values indicate that the processed beef sample contains less amount of protein then the raw beef.NO 1.62 17.35 17.00 2.503 0.00 13. 4.513 0.18 11. 5.0 0.SAMPLE-11a: MINCED BEEF PROCESSED IN BRINE SOLUTION:(COSTA FOOD PRODUCTS) SAMPLE WEIGHT IN GRAM BLANK NORMALITY OF H2SO4 VOLUME OF H2SO4 IN ml % OF NITROGEN % OF PROTEIN S.0948 0.0948 0.6 9.0948 0.1035 0.742 1.564 0.0 0. .800 0. 2.979 2.709 1.065 1.35 SAMPLE-11b: MINCED BEEF PROCESSED IN BRINE SOLUTION:(COSTA FOOD PRODUCTS) SAMPLE WEIGHT IN GRAM BLANK NORMALITY OF H2SO4 VOLUME OF H2SO4 IN ml % OF NITROGEN % OF PROTEIN S. 3.

. cereals and pulses as showed from the results. Putrefaction is a main cause of protein degradation by microorganisms during fermentation that involves in the food processing. smoking and chilling carried out in fish and meat products for processing causes the denaturation of protein and decreases the protein in the food samples Results explained that processed animal foods also can be up to extent to reach the recommended dietary allowances (RDA) as like raw foods. Certain processing techniques like freezing. Protein stability in the foods depends on the temperature. During thermal processing there can be degradation of protein conformation. chicken and other meat products like mutton and beef contains higher protein content than the processed food samples as well as vegetable sources. salting.DISCUSSION:Food processing is the field of advanced techniques to convert one form to another form. Raw food samples like fish. corn. Protein analysis carried out to describe the different in protein content in raw foods from processed foods. Proteins are important macromolecules that causes muscle mass and plays important metabolism in human being.

2. smoking. Animal proteins are higher in protein content than plant proteins but. It is found that the percentage of protein in tuna fish is much greater than sardines and mackerel. Sardines fish processed along with tomato sauce and baked beans. may be due to higher fat content in these food materials. Future recommendations can be followed to overcome the loss of protein in animal sources: 1. Baked beans minced along with beef meat and preserved with brine solution. . It strengthens the quality of food because of multipurpose and benefits of protein in the biological system. Chicken is greater in protein content than beef and mutton. 3. salting and chilling. these animal proteins are decreased by various processing technique like freezing. The loss of protein content in animal food samples during processing can be regained by certain value addition.CHAPTER-5 SUMMARY Protein analysis benefits to recommended protein rich foods to regular diet. Processed food samples contain less amount of protein than raw food samples. The protein content in six samples of raw animal products and five samples of processed food from animal source were determined. Tuna fish processed along with garlic-ginger paste and oil.

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