Effect of processing on protein content of fish and meat products

EFFECT OF PROCESSING ON THE PROTEIN CONTENT OF FISH AND MEAT PRODUCTS

Dissertation Submitted to the

LOYOLA COLLEGE (Autonomous)UNIVERSITY OF MADRAS

In partial fulfillment of theRequirement for the award of the

Degree ofMASTER OF SCIENCE

INFOOD CHEMISTRY & FOOD PROCESSING

By

M.ANBU MALAR(08-PFP-012)

Under the guidance of

Dr. V. KANNAPPANSupervisor

Professor in Food chemistry & Food processingLOYOLA COLLEGE (Autonomous)

Chennai-34

DEPARTMENT OF FOOD CHEMISTRY & FOOD PROCESSINGLOYOLA COLLEGE (Autonomous)

CHENNAI-342009-2010

CERTIFICATE

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

Place: Chennai-34Date:

CHAPTER CONTENTS PAGE NO.

I INTRODUCTION

STRUCTURE OF PROTEINS

STRUCTURAL HIERARCHY IN PROTEINS

FUNTIONAL PROPERTIES OF PROTEINS

NUTRITIONAL PROPERTIES OF PROTEINS

PROTEIN QUALITY EVALUATION

PROTEIN INTAKE AND HEALTH

PROTEIN DENATURATION

ANALYSIS OF PROTEIN

PROTEIN SEPARATIONAND CHARECTERISATION

II SCOPE AND OBJECTIVE

III MATERIALS & METHODS

EXPERIMENTS

METHOD

IV RESULTS & DISCUSSION

V SUMMARY

VI REFERENCES

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 wish to express my heartfelt gratitude and respect to my guide Dr. V.

KANNAPPAN, Department of food chemistry & food processing, Loyola College

(Autonomous) Chennai, for her excellent consistent and expert guidance.

I extend my gratitude to Rev. Dr. A. Albert muthumalai S.J., Principal Loyola

College, Chennai, for giving me this opportunity to study in this institution.

I wish to thank Rev.Dr.John Pragasam, Deputy Principal, Loyola college Chennai-

34 for his constant encouragement and support.

My heartfelt prayers and gratitude goes out to the staff whose support and guidance

has been a great source of inspiration.

I express my warm and heartfelt gratitude to my parents and their prayers, moral

support and care in achieving my aim fruitfully.

CHAPTER - 1:

INTRODUCTION:

Proteins are the most versatile macromolecules in living systems and serve crucial functions in essentially all biological processes. They function as catalysts, transport and store other molecules such as oxygen.They provide mechanical support and immune protection, generate movement, transmit nerve impulses, and control growth and differentiation.

Several key properties enable proteins to participate in such a wide range of functions:

1. 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.

The function of a protein is directly dependent on its three dimensional structure.

Remarkably, proteins spontaneously fold up into three-dimensional structures that are determined by the sequence of amino acids in the protein polymer.

Thus, 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.

2. Proteins contain a wide range of functional groups: These functional groups include alcohols, thiols, thioethers, carboxylic acids, carboxamides, and a variety of basic groups.

When combined in various sequences, this array of functional groups accounts for the broad spectrum of protein function.

For instance, the chemical reactivity associated with these groups is essential to the function of enzymes, the proteins that catalyze specific chemical reactions in biological systems.

3. 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.

These assemblies include macro-molecular machines that carry out the accurate replication of DNA, the transmission of signals within cells, and many other essential processes.

4. Some proteins are quite rigid, whereas others display limited flexibility: Rigid units can function as structural elements in the cytoskeleton

(the internal scaffolding within cells) or in connective tissue. Parts of proteins with limited flexibility may act as hinges, springs,

and levers that are crucial to protein function, to the assembly of proteins with one another and with other molecules into complex units, and to the transmission of information within and between cells.

1.1 Proteins Are Built from a Repertoire of 20 Amino Acids:

Amino acids are the building blocks of proteins. An -amino acid consists of a central carbon atom, called the carbon, linked to an amino group, a carboxylic acid group, a hydrogen atom, and a distinctive R group. The R group is often referred to as the side chain. With four different groups connected to the tetrahedral -carbon atom, -amino acids are chiral; the two mirror-image forms are called the l isomer and the d isomer.

Twenty kinds of side chains varying in size, shape, charge, hydrogen- bonding capacity, hydrophobic character, and chemical reactivity are commonly found in proteins. Indeed, all proteins in all species bacterial, archaeal, and eukaryotic are constructed from the same set of 20 amino acids. This fundamental alphabet of proteins is several billion years old.The remarkable range of functions mediated by proteins results from the diversity and versatility of these 20 building blocks.[29]

1.1.1. STRUCTURE OF PROTEIN:

1.1.2. 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 convention for the designation of the order of amino acids is that the N-terminal end (i.e. the end bearing the residue with the

free α-amino group) is to the left (and the number 1 amino acid) and the C-terminal end (i.e. the end with the residue containing a free α-carboxyl group) is to the right.

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). The formation of a dipeptide from two amino acids is accompanied by the loss of a water molecule. The equilibrium of this reaction lies on the side of hydrolysis rather than synthesis. Hence, the biosynthesis of peptide bonds requires an input of free energy.

Nonetheless, peptide bonds are quite stable kinetically; the lifetime of a peptide bond in aqueous solution in the absence of a catalyst approaches 1000 years.A series of amino acids joined by peptide bonds form a polypeptide chain, and each amino acid unit in a polypeptide is called a residue.

A polypeptide chain has polarity because its ends are different, with an -amino group at one end and an -carboxyl group at the other. By convention, the amino end is taken to be the beginning of a polypeptide chain, and so the sequence of amino acids in a polypeptide chain is written starting with the aminoterminal residue. Thus, in the pentapeptide Tyr-Gly-Gly-Phe-Leu (YGGFL), phenylalanine is the amino-terminal (N-terminal) residue and leucine is the carboxyl-terminal (C-terminal) residue. Leu-Phe-Gly-Gly-Tyr (LFGGY) is a different pentapeptide, with different chemical properties.

A polypeptide chain consists of a regularly repeating part, called the main chain or backbone, and a variable part, comprising the distinctive side chains.The polypeptide backbone is rich in hydrogen-bonding potential. Each residue contains a carbonyl group, which is a good hydrogen-bond acceptor and, with the exception of proline, an NH group, which is a good hydrogen-bond donor.

These groups interact with each other and with functional groups from side chains to stabilize particular structures. Most natural polypeptide chains contain between 50 and 2000 amino acid residues and are commonly referred to as proteins.

Peptides made of small numbers of amino acids are called oligopeptides or simply peptides. The mean molecular weight of an amino acid residue is about 110, and so the molecular weights of most proteins are between 5500 and 220,000. A protein with a molecular weight of 50,000 has a mass of 50,000 daltons, or 50 kd (kilodaltons).

1.1.3. Rotation About Bonds in a Polypeptide:

The structure of each amino acid in a polypeptide can be adjusted by rotation about two single bonds.

(A) Phi (f) is the angle of rotation about the bond between the nitrogen and the a-carbon atoms, whereas psi (y) is the angle of rotation about 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, showing how f is measured.

(C) A view down the bond between the a-carbon and the carbonyl carbon atoms, showing how y is measured.

1.1.4. Secondary Structure: Polypeptide Chains Can Fold Into Regular Structures Such as the Alpha Helix, the Beta Sheet, and Turns and Loops:

The ordered array of amino acids in a protein confer regular conformational forms upon that protein. These conformations constitute the secondary structures of a protein. In general proteins fold into two broad classes of structure termed, globular proteins or fibrous proteins.

Globular proteins are compactly folded and coiled, whereas, fibrous proteins are more filamentous or elongated. It is the partial double-bond character of the peptide bond that defines the conformations a polypeptide chain may assume. Within a single protein different regions of the polypeptide chain may assume different conformations determined by the primary sequence of the amino acids.

A. The α-Helix:

The α-helix is a common secondary structure encountered in proteins of the globular class. The formation of the α-helix is spontaneous and is stabilized by H-bonding between amide nitrogens and carbonyl carbons of peptide bonds spaced four residues apart. 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.

B. β-Sheets:

An α-helix is composed of a single linear array of helically disposed amino acids, β-sheets are composed of 2 or more different regions of stretches of at least 5-10 amino acids. 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.

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 are said to be pleated. This is due to positioning of the α-carbons of the peptide bond which alternates above and below the plane of the sheet. β-sheets are either parallel or antiparallel.

In parallel sheets adjacent peptide chains proceed in the same direction (i.e. the direction of N-terminal to C-terminal ends is the same), whereas, in antiparallel sheets adjacent chains are aligned in opposite directions. β-sheets can be depicted in ball and stick format or as ribbons in certain protein formats.

1.1.5.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. 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-dimensional form of the protein. Secondary structures of proteins often constitute distinct domains.

Therefore, tertiary structure also describes the relationship of different domains to one another within a protein. The interactions of different domains is governed by several forces: These include hydrogen bonding, hydrophobic interactions, electrostatic interactions and van der Waals forces.

1.1.6. 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. Proteins with multiple polypetide chains are oligomeric proteins. The structure formed by monomer-monomer interaction in an oligomeric protein is known as quaternary structure.

Oligomeric proteins can be composed of multiple identical polypeptide chains or multiple distinct polypeptide chains. Proteins with identical subunits are termed homo-oligomers. Proteins containing several distinct polypeptide chains are termed hetero-oligomers.

Hemoglobin, the oxygen carrying protein of the blood, contains two α and two β subunits arranged with a quaternary structure in the form, α2β2. Hemoglobin is, therefore, a hetero-oligomeric protein.[1-2]

1.2. Forces Controlling Protein Structure:

1.2.1. Hydrogen Bonding:

Polypeptides contain numerous proton donors and acceptors both in their backbone and in the R-groups of the amino acids. The environment in which proteins are found also contains the ample H-bond donors and acceptors of the water molecule. H-bonding, therefore, occurs not only within and between polypeptide chains but with the surrounding aqueous medium.

1.2.2. Hydrophobic Forces:

Proteins are composed of amino acids that contain either hydrophilic or hydrophobic R-groups. It is the nature of the interaction of the different R-groups 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.

The hydrophobicity of certain amino acid R-groups tends to drive them away from the exterior of proteins and into the interior. This driving force restricts the available conformations into which a protein may fold.

1.2.3. Electrostatic Forces:

Electrostatic forces are mainly of three types; charge-charge, charge-dipole and dipole-dipole. Typical charge-charge interactions that favor protein folding are those between oppositely charged R-groups such as K or R and D or E. A substantial component of the energy involved in protein folding is charge-dipole interactions.

This refers to the interaction of ionized R-groups of amino acids with the dipole of the water molecule. The slight dipole moment that exist in the polar R-groups of amino acid also influences their interaction with water. It is, therefore, understandable that the majority of the amino acids found on the exterior surfaces of globular proteins contain charged or polar R-groups.

1.2.4. van der Waals Forces:

There are both attractive and repulsive van der Waals forces that control protein folding. 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.

Repulsive van der Waals forces involve the interactions that occur when uncharged non-bonded atoms come very close together but do not induce dipoles. The repulsion is the result of the electron-electron repulsion that occurs as two clouds of electrons begin to overlap.

Although van der Waals forces are extremely weak, relative to other forces governing conformation, it is the huge number of such interactions that occur in large protein molecules that make them significant to the folding of proteins.[3-5]

1.2.5. 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. The most common treatments are to use either 2-mercaptoethanol or dithiothreitol (DTT). Both of these chemicals reduce disulfide bonds. To prevent reformation of the disulfide bonds the peptides are treated with iodoacetic acid in order to alkylate the free sulfhydryls.

There are three major chemical techniques for sequencing peptides and proteins from the N-terminus. These are the Sanger, Dansyl chloride and Edman techniques.[6-8]

1.2.6. Sanger's Reagent

This sequencing technique utilizes the compound, 2,4-dinitrofluorobenzene (DNF) which reacts with the N-terminal residue under alkaline conditions. The derivatized amino acid can be hydrolyzed and will be labeled with a dinitrobenzene group that imparts a yellow color to the amino acid. Separation of the modified amino acids (DNP-derivative) by electrophoresis and comparison with the migration of DNP-derivative standards allows for the identification of the N-terminal amino acid.

1.2.8. Dansyl chloride

Like DNF, dansyl chloride reacts with the N-terminal residue under alkaline conditions. Analysis of the modified amino acids is carried out similarly to the Sanger method except that the dansylated amino acids are detected by fluorescence. This imparts a higher sensitivity into this technique over that of the Sanger method.

1.2.9. 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. Using this method it is possible to obtain the entire sequence of peptides. This method utilizes phenylisothiocyanate to react with the N-terminal residue under alkaline conditions.

The resultant phenylthiocarbamyl derivatized amino acid is hydrolyzed in anhydrous acid. The hydrolysis reaction results in a rearrangement of the released N-terminal residue to a phenylthiohydantoin derivative.

As in the Sanger and Dansyl chloride methods, the N-terminal residue is tagged with an identifiable marker, however, the added advantage of the Edman process is that the remainder of the peptide is intact. The entire sequence of reactions can be repeated over and over to obtain the sequences of the peptide. This process has subsequently been automated to allow rapid and efficient sequencing of even extremely small quantities of peptide.[9-10]

1.3. 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). This reagent causes specific cleavage at the C-terminal side of M residues. The number of peptide fragments that result from CNBr cleavage is equivalent to one more than the number of M residues in a protein.

The most reliable chemical technique for C-terminal residue identification is hydrazinolysis. A peptide is treated with hydrazine, NH2–NH2, at high temperature (90°C) for an extended length of time (20-100hr).

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. Due to the high percentage of hydrazine induced side reactions this technique is only used on carboxypeptidase resistant peptides.[11-15]

1.4. Functional Properties of Proteins

Food preferences by human beings are based primarily on sensory attributes such as texture, flavor, color, and appearance. The sensory attributes of a food are the net effect of complex interactions among various minor and major components of the food. The functional roles of food proteins in food given are summarized in the table 1.1;

TABLE 1.1 Functional Roles of Food Proteins in Food Systems

Function Mechanism Food Protein typeSolubility Hydrophilicity Beverages Whey proteinsViscosity Water binding ,

hydrodynamicsize and shape

Soups, gravies, and saladdressing s, desserts

Gelatin

Water binding Hydrogen bonding , ionicHydration

Meat sausages, cakes, andBreads

Muscle proteins, eggproteins

Gelation Water entrapment andimmobilization, networkformation

Meats, gels, cakes,bakeries, cheese

Muscle proteins, eggand milk proteins

Cohesion-adhesion

Cohesion adhesion Meats, sausages, pasta,baked goods

Muscle proteins, eggproteins, whey proteins

Elasticity Hydrophobic bonding ,disulfide cross-links

Meats, bakery Muscle proteins, cerealproteins

Emulsification Adsorption and film formationat interfaces

Sausages, bologna, soup,cakes, dressing s

Muscle proteins, eggproteins, milk proteins

Foaming Interfacial adsorption and filmFormation

Whipped topping s, icecream, cakes, desserts

Egg proteins, milkproteins

Fat and flavorbinding

Hydrophobic bonding ,Entrapment

Low-fat bakery products,Doughnuts

Milk proteins, eggproteins, cereal proteins

The physical and chemical properties that govern protein functionality include size; shape; amino acid composition and sequence; net charge and distribution of charges; Hydrophobicity/Hydrophilicity ratio; secondary, tertiary, and quaternary structures; molecular flexibility/rigidity; and ability to interact/react with other components. Since proteins possess a multitude of physical and chemical properties, it is difficult to delineate the role of each of these properties with respect to a given functional property.[34-35]

1.5.Nutritional Properties of Proteins: Proteins differ in their nutritive value. Several factors, such as content of essential amino acids and digestibility, contribute to these differences. The daily protein requirement therefore depends on the type and composition of proteins in a diet.

1.5.1. Protein Quality

The “quality” of a protein is related mainly to its essential amino acid composition and digestibility. While proteins of cereals, such as rice, wheat, barley, and maize, are very low in lysine and rich in methionine, those of legumes and oilseeds are deficient in methionine and rich or adequate in lysine. Some oilseed proteins, such as peanut protein, are deficient in both methionine and lysine contents. The essential amino acids whose concentrations in a protein are below the levels of a reference protein are termed limiting amino acids. 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. The essential amino acid content in various food samples and recommended essential amino acid pattern are given in tables 1.2 and 1.3;

Table 1.2 Essential Amino Acid Contents and Nutritional Value of Proteins from Various Sources (mg /g Protein)

Property (mg /g protein) Amino acid concentration(mg /g protein)

Egg Cow'sMilk

Beef Fish Wheat Soybean

His 22 27 34 35 21 30

Ile 54 47 48 48 34 51Leu 86 95 81 77 69 82Lys 70 78 89 91 23 68Met+Cys 57 33 40 40 36 33Phe+Tyr 93 102 80 76 77 95Thr 47 44 46 46 28 41

Trp 17 14 12 11 10 14Val 66 64 50 61 38 52Total essential amino acids

512 504 480 485 336 466

Protein content (%)

12 3.5 18 19 12 40

Chemical score (%)

100 100 100 100 40 100

PER 3.9 3.1 3.0 3.5 1.5 2.3BV 94 84 74 76 65 73NPU 94 92 67 79 40 61

Table 1.3 Recommended Essential Amino Acid Pattern for Food Proteins (mg/g protein)

Amino acid Infant(2–5 years)

Preschool child(10–12 years)

Preschool child

Adult

Histidine 26 19 19 16Isoleucine 46 28 28 13Leucine 93 66 44 19Lysine 66 58 44 16Met + Cys 42 25 22 17Phe + Tyr 72 63 22 19Threonine 43 34 28 9

Try35ptophan 17 11 9 5Valine 55 35 25 13Total 434 320 222 111

1.5.2. Digestibility

Although the content of essential amino acids is the primary indicator of protein quality, true quality also depends on the extent to which these amino acids are utilized in the body. Thus, digestibility of amino acids can affect the quality of proteins.

Digestibility, the proportion of food protein which is absorbed, is defined from measurements of the nitrogen content of foods and faeces, with “true” digestibility taking into account the extent to which faecal nitrogen is “endogenous”, which in turn is measured as faecal nitrogen loss on a protein free diet

where I = nitrogen intake, F = faecal nitrogen loss on the test diet,and Fk = faecal nitrogen loss on a protein-free diet.

1.5.3. Protein conformation

The structural state of a protein influences its hydrolysis by proteases. Native proteins are generally less completely hydrolyzed than partially denatured ones. Generally, insoluble fibrous proteins and extensively denatured globular proteins are difficult to hydrolyze.

1.5.4. Antinutritional factors

Most plant protein isolates and concentrates contain trypsin and chymotrypsin inhibitors (Kunitz type and Bowman-Birk type) and lectins. Lectins, which are glycoprotein, bind to intestinal mucosa cells and interfere with absorption of amino acids. Plant proteins also contain other antinutritional factors, such as tannins and phytate. Tannins, which are condensed products of

polyphenols, covalently react with -amino groups of lysyl residues. This inhibits trypsin-catalyzed cleavage of the lysyl peptide bond.[16-17]

1.6. 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. protein utilization is generally discussed in terms of digestibility, a measure of the dietary intake which is made available to the organism after digestion and absorption, and biological value, a measure of how well the absorbed amino acid profile matches that of the requirement. Overall protein utilization, i.e. net protein utilization (NPU), will therefore reflect both digestibility and biological value.

Bioavailability comprises digestibility, chemical integrity, and freedom from interference in metabolism, highlighting those aspects of amino acid utilization that may be important with specific foods and food processing methods. 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%.

1.6.1. Evaluation of Protein Nutritive Value

Since the nutritional quality of proteins can vary greatly and is affected by many factors, 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, and (b) monitoring changes in the nutritive value of proteins during food processing, so that processing conditions that minimize quality loss can be devised. The nutritive quality of proteins can be evaluated by several biological, chemical, and enzymatic methods.[18]

1.6.2. Biological value

The amino acid profile is assumed to determine the effectiveness with which absorbed dietary nitrogen can be utilized, which is usually defined in terms of biological value.Biological value, BV, is calculated as follows:

where U = urinary nitrogen loss on the test diet,and UK = urinary nitrogen loss on a protein-free diet

1.6.3. 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. 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.

If biological value is determined solely by the amino acid profile, thenPDCAAS should predict biological value

1.6.4. Protein intake and health

Protein supplements are the most widely consumed ergogenic aid, whereas single amino acids are consumed for a wide variety of reasons, most of which have little or no secure scientific foundation. The possibility of toxicity resulting from consuming very large amounts of individual amino acids has been examined in various publications. In the United Kingdom, in the context of guidance on high intakes, 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.e. 1.5 g protein/kg). A second issue is the possibility that protein intakes in excess of recommended intakes may confer health benefits, i.e. it may be

that optimum protein intakes are greater than a recommended intake derived as in this report. This section therefore examines the relationship between protein intakes and long term health in relation to a number of specific disease states, and also whether it is possible to identify a maximum level of protein that can be consumed without adverse effects.

1.6.5. 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). However, 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. This concept arose from studies in rats, in which low-protein diets were shown to delay the developmentof chronic renal failure (9, 10). However, it seems unlikely that this mechanism would operate in humans, in whom the decline in kidney function occurs through a fall in filtration by non-sclerotic nephrons, 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, hypertension, or polycystic kidney disease.

1.6.6. Bone health

The relationship between protein intake and bone health appears to be more complex than was previously believed. Thus the potential negative effect of protein on calcium balance is a function of the overall dietary acid–base balance. In addition, protein seems to have direct anabolic effects on the bone matrix. It is well documented that diets containing high protein can result in an increase in urinary calcium excretion (17–20), amounting to a 50% increasein urinary calcium for a doubling of protein intake (19). Bone mineral balance is very sensitive to acid–base balance, and calcium can be mobilized from bone in response to the need to buffer the acid load produced by oxidation of the sulfur-containing amino acids, methionine and cysteine (21). Accordingly, increased resorption of bone has been shown to occur as a consequenceof increased protein intake (20, 21). This raises the controversial issue of whether this process might lead to a decrease in bone calcium (19, 22, 23). Heaney (24, 25) has suggested thatthis is unlikely, as low protein intake itself leads to bone loss, and higher protein intake generally leads to a higher calcium intake. However, it is now clear that net

renal acid excretion predicts calcium excretion, and that this can be predicted from the ratio of dietary protein to potassium, since the dietary intake of potassium occurs mainly as salts of weak organic acids and thereforehas an alkalizing effect (26). In women, lower intakes of endogenous non-carbonic acid (i.e. a lower protein intake but a higher potassium intake) were related to better measures of bone health (27, 28). This probably explains the beneficial influence of fruit and vegetables, the major dietary source of potassium, on bone health (29, 30). It may also explain why calcium citratemalate supplements are more effective for bone health than other calcium salts (31). 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, protein can exert an independently beneficial effect through its insulin-like growth factor-1 (IGF-1)-mediated anabolic influences on bone. 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). The magnitude and importance of the bone protein pool are such that a positive effect of protein on bone is not surprising. Apart from any IGF-1-mediated effects there is considerable evidence for a limitation on the synthesis of glycine (36), which accounts for 25% of collagen, so that competition for glycine between collagen and its other important metabolic demands might prevent its reutilization during bone collagen turnover.It does appear that dietary protein as part of a well-balanced diet is most likely to be beneficial for bone, possibly at dietary levels in excess of the recommended intake.

1.6.7. Kidney stones

A second potential consequence of high-protein diets, which has been extensively discussed in the literature, is an increased occurrence of renal stones. Renal stones occur very commonly, and have been estimated to affect 12% of the United States population at some time (38). Initial studies showed that an increase in dietary animal protein resulted in an elevation of urinary calcium and oxalate, which was estimated to increase therisk of forming stones by 250% (39, 40). Moreover, 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 a higher intake of animal protein increased, the risk of stones (41, 42). Although some studies suggest that high animal protein intake might increase the risk of kidney stones, particularly in those subjects who are classified as idiopathic calcium stone formers, as yet no clear conclusions can be drawn since dietary effects are apparent only in studies with very large differences in protein intakes (i.e. >185 g/day

compared with 80 g/day). It is recommended that in order to minimize the risk of kidney stones in patients who are at risk, the diet should ideally provide at least the safe level (0.83 g/kg per day), but not excessive amounts (i.e. less than 1.4 g/kg per day), preferably from vegetable sources.

1.7. 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. The native state (of a single protein molecule) is thermodynamically the most stable with lowest feasible free energy at physiological conditions. Any change in its environment, such as pH, ionic strength, temperature, solvent composition, etc., will force the molecule to assume a new equilibrium structure. Subtle changes in structure, which do not drastically alter the molecular architecture of the protein, are usually regarded as “conformational adaptability,” whereas major changes in the secondary, tertiary, and quaternary structures without cleavage of backbone peptide bonds are regarded as “denaturation.” 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.

Often denaturation has a negative connotation, because it indicates loss of some properties. For example, many biologically active proteins lose their activity upon denaturation. In the case of food proteins, denaturation usually causes insolublization and loss of some functional properties.However, from a food application stand point protein denaturation during processing is not always undesirable. In fact in some cases it is highly desirable. For instance, partial denaturation of proteins at the air-water and oil-water interfaces improves their foaming and emulsifying properties, whereas excessive thermal denaturation of soy proteins diminishes their foaming and emulsifying properties.

1.7.1. Temperature and Denaturation

Heat is the most commonly used agent in food processing and preservation. Proteins undergo varying degrees of denaturation during processing. When a protein solution is gradually heated above a critical temperature, it undergoes a sharp transition from the native state to the denatured state. The temperature at the transition midpoint, where the concentration ratio of native and denatured states is known either as the melting temperature Tm or the denaturation temperature.

Thermal stability of proteins from thermophilic and hyperthermophilic organisms, which can with stand extremely high temperatures, is also attributed to their unique amino acid composition. The Cys, Met, and Trp contents which can be oxidized easily at high temperatures, are also low in hyperthermostable proteins.On the other hand, thermostable proteins have high levels of Ile and Pro. The high Ile content is belived to help in better packing of the interior core of the protein, 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. Water greatly facilitates thermal denaturation of proteins [37,95]. Dry protein powders are extremely stable to thermal denaturation. The value of Td decreases rapidly as the water content is increased from 0 to 0.35 g water/g protein. An increase in water content from 0.35 to 0.75 g water/g protein causes only a marginal decrease in Td. Above 0.75 g water/g protein, the Td of the protein is the same as in a dilute protein solution.

1.7.2. pH and Denaturation

Proteins are more stable against denaturation at their isoelectric point than at any other pH. At neutral pH, most proteins are negatively charged, and a few are positively charged. Since the net electrostatic repulsive energy is small compared to other favorable interactions, most proteins are stable at around neutral pH. The degree of unfolding is greater at extreme alkaline pH values than it is at extreme acid pH. pH-induced denaturation is mostly reversible.

1.7.3. Detergents and Denaturation

Detergents, such as sodium dodecyl sulfate (SDS), are powerful protein denaturing agents. SDS at 3–8 mM concentration denatures most globular proteins. The mechanism involves preferential binding of detergent to the denatured protein molecule. This causes a shift in equilibrium between the native and denatured states.[30]

1.8. ANALYSIS OF PROTEINS

Proteins are important constituents of foods for a number of different reasons. They are a major source of energy, as well as containing essential amino-acids, such as lysine, tryptophan, methionine, leucine, isoleucine and valine, which are essential to human health, but which the body cannot synthesize. Isolated proteins are often used in foods as ingredients because of their unique functional properties, i.e., their ability to provide desirable appearance, texture or stability. Typically, proteins are used as gelling agents, emulsifiers, foaming agents and thickeners. Many food proteins are enzymes which are capable of enhancing the rate of certain biochemical reactions. These reactions can have either a favorable or detrimental effect on the overall properties of foods. Food analysts are interested in knowing the total concentration, type, molecular structure and functional properties of the proteins in foods.

1.8.1 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. The amount of protein present is then calculated from the nitrogen concentration of the food. The same basic approach is still used today, although a number of improvements have been made to speed up the process and to obtain more accurate measurements. It is usually considered to be the standard method of determining protein concentration. 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. A conversion factor of 6.25 (equivalent to 0.16 g nitrogen per gram of protein) is used for many applications, however, this is only an average value, and each protein has a different conversion factor depending on its amino-acid composition.

The Kjeldahl method is widely used internationally and is still the standard method for comparison against all other methods. Its universality, high precision and good

reproducibility have made it the major method for the estimation of protein in foods.

However it does not give a measure of the true protein, since all nitrogen in foods is not in the form of protein. Different proteins need different correction factors because they have different amino acid sequences. The use of concentrated sulfuric acid at high temperatures poses a considerable hazard, as does the use of some of the possible catalysts The technique is time consuming to carry-out.

1.8.2 Enhanced Dumas method:

Recently, an automated instrumental technique has been developed which is capable of rapidly measuring the protein concentration of food samples. This technique is based on a method first described by a scientist called Dumas over a century and a half ago. It is beginning to compete with the Kjeldahl method as the standard method of analysis for proteins for some foodstuffs due to its rapidness.

It is much faster than the Kjeldahl method (under 4 minutes per measurement, compared to 1-2 hours for Kjeldahl). It doesn't need toxic chemicals or catalysts. Many samples can be measured automatically. It is easy to use. But it has high initial cost. It does not give a measure of the true protein, since all nitrogen in foods is not in the form of protein. Different proteins need different correction factors because they have different amino acid sequences. The small sample size makes it difficult to obtain a representative sample.

1.8.3 UV-visible spectroscopy:

A number of methods have been devised to measure protein concentration, 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, or they chemically or physically modify proteins to make them absorb (or scatter) light in this region. The basic principle behind each of these tests is similar. First of all a calibration curve of absorbance (or turbidity) versus protein concentration is prepared using a series of protein solutions of known concentration. The absorbance (or turbidity) of the solution being analyzed is then measured at the same wavelength, and its protein concentration determined

from the calibration curve. The main difference between the tests are the chemical groups which are responsible for the absorption or scattering of radiation, e.g., peptide bonds, aromatic side-groups, basic groups and aggregated proteins.

Tryptophan and tyrosine absorb ultraviolet light strongly at 280 nm. The tryptophan and tyrosine content of many proteins remains fairly constant, and so the absorbance of protein solutions at 280nm can be used to determine their concentration. The advantages of this method are that the procedure is simple to carry out, it is nondestructive, and no special reagents are required. 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. Even so, methods have been developed to overcome this problem, e.g., by measuring the absorbance at two different wavelengths.

Biuret Method:

A violet-purplish color is produced when cupric ions (Cu2+) interact with peptide bonds under alkaline conditions. The biuret reagent, which contains all the chemicals required to carry out the analysis, can be purchased commercially. It is mixed with a protein solution and then allowed to stand for 15-30 minutes before the absorbance is read at 540 nm. The major advantage of this technique is that there is no interference from materials that adsorb at lower wavelengths, and the technique is less sensitive to protein type because it utilizes absorption involving peptide bonds that are common to all proteins, rather than specific side groups. However, it has a relatively low sensitivity compared to other UV-visible methods.

Lowry Method:

The Lowry method combines the biuret reagent with another reagent (the Folin-Ciocalteau phenol reagent) which reacts with tyrosine and tryptophan residues in proteins. This gives a bluish color which can be read somewhere between 500 - 750 nm depending on the sensitivity required. 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. This method is more sensitive to low concentrations of proteins than the biuret method.

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 isoelectric point). The proteins form an insoluble complex with the dye because of the electrostatic attraction between the molecules, but the unbound dye remains soluble. The anionic dye binds to cationic groups of the basic amino acid residues (histidine, arganine and lysine) and to free amino terminal groups. The amount of unbound dye remaining in solution after the insoluble protein-dye complex has been removed (e.g., by centrifugation) is determined by measuring its absorbance. The amount of protein present in the original solution is proportional to the amount of dye that bound to it: dyebound = dyeinitial - dyefree.(ref.no.

1.9 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. Protein type is usually determined by separating and isolating the individual proteins from a complex mixture of proteins, so that they can be subsequently identified and characterized. Proteins are separated on the basis of differences in their physicochemical properties, such as size, charge, adsorption characteristics, solubility and heat-stability. The choice of an appropriate separation technique depends on a number of factors, including the reasons for carrying out the analysis, the amount of sample available, the desired purity, the equipment available, the type of proteins present and the cost.

1.9.1 Methods Based on difference in solubility:

Salting out:

Proteins are precipitated from aqueous solutions when the salt concentration exceeds a critical level, which is known as salting-out, because all the water is "bound" to the salts, and is therefore not available to hydrate the proteins. Ammonium sulfate [(NH4)2SO4] is commonly used because it has a high water-solubility, although other neutral salts may also be used, e.g., NaCl or KCl. Generally a two-step procedure is used to maximize the separation efficiency. In

the first step, the salt is added at a concentration just below that necessary to precipitate out the protein of interest. The solution is then centrifuged to remove any proteins that are less soluble than the protein of interest. The salt concentration is then increased to a point just above that required to cause precipitation of the protein. This precipitates out the protein of interest (which can be separated by centrifugation), but leaves more soluble proteins in solution. The main problem with this method is that large concentrations of salt contaminate the solution, which must be removed before the protein can be resolubilzed, e.g., by dialysis or ultrafiltration.

Isoelectric Precipitation:

The isoelectric point (pI) of a protein is the pH where the net charge on the protein is zero. Proteins tend to aggregate and precipitate at their pI because there is no electrostatic repulsion keeping them apart. Proteins have different isoelectric points because of their different amino acid sequences (i.e., relative numbers of anionic and cationic groups), and thus they can be separated by adjusting the pH of a solution. 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. As the dielectric constant of a solution decreases the magnitude of the electrostatic interactions between charged species increases. This tends to decrease the solubility of proteins in solution because they are less ionized, and therefore the electrostatic repulsion between them is not sufficient to prevent them from aggregating. The dielectric constant of aqueous solutions can be lowered by adding water-soluble organic solvents, such as ethanol or acetone. The amount of organic solvent required to cause precipitation depends on the protein and therefore proteins can be separated on this basis. The optimum quantity of organic solvent required to precipitate a protein varies from about 5 to 60%. 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.

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. 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.

1.9.2 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. This technique is the most commonly used chromatographic technique for protein separation. A positively charged matrix is called an anion-exchanger because it binds negatively charged ions (anions). A negatively charged matrix is called a cation-exchanger because it binds positively charged ions (cations). The buffer conditions (pH and ionic strength) are adjusted to favor maximum binding of the protein of interest to the ion-exchange column. 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.g., different pH or ionic strength).

Affinity Chromatography

Affinity chromatography uses a stationary phase that consists of a ligand covalently bound to a solid support. The ligand is a molecule that has a highly specific and unique reversible affinity for a particular protein. The sample to be analyzed is passed through the column and the protein of interest binds to the ligand, whereas the contaminating proteins pass directly through. The protein of interest is then eluted using a buffer solution which favors its desorption from the column. This technique is the most efficient means of separating an individual

protein from a mixture of proteins, but it is the most expensive, because of the need to have columns with specific ligands bound to them.

Both ion-exchange and affinity chromatography are commonly used to separate proteins and amino-acids in the laboratory. They are used less commonly for commercial separations because they are not suitable for rapidly separating large volumes and are relatively expensive.

1.9.3 Separation Due to Size Differences

Proteins can also be separated according to their size. Typically, the molecular weights of proteins vary from about 10,000 to 1,000,000 daltons. In practice, separation depends on the Stokes radius of a protein, rather than directly on its molecular weight. The Stokes radius is the average radius that a protein has in solution, and depends on its three dimensional molecular structure. For proteins with the same molecular weight the Stokes radius increases in the following order: compact globular protein < flexible random-coil < rod-like protein.

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, but prevent the passing of larger molecules. 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. Low molecular weight solutes flow through the bag, but the large molecular weight protein molecules remain in the bag. Dialysis is a relatively slow method, taking up to 12 hours to be completed. It is therefore most frequently used in the laboratory. Dialysis is often used to remove salt from protein solutions after they have been separated by salting-out, and to change buffers.

Ultrafiltration

A solution of protein is placed in a cell containing a semipermeable membrane, and pressure is applied. Smaller molecules pass through the membrane, whereas the larger molecules remain in the solution. The separation principle of this technique is therefore similar to dialysis, but because pressure is applied separation

is much quicker. Semipermeable membranes with cutoff points between about 500 to 300,000 are available. That portion of the solution which is retained by the cell (large molecules) is called the retentate, whilst that part which passes through the membrane (small molecules) forms part of the ultrafiltrate. Ultrafiltration can be used to concentrate a protein solution, remove salts, exchange buffers or fractionate proteins on the basis of their size. Ultrafiltration units are used in the laboratory and on a commercial scale.

Size Exclusion Chromatography

This technique, sometimes known as gel filtration, also separates proteins according to their size. 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). Molecules larger than the pores in the beads are excluded, and move quickly through the column, whereas the movement of molecules which enter the pores is retarded. Thus molecules are eluted off the column in order of decreasing size. Beads of different average pore size are available for separating proteins of different molecular weights. Manufacturers of these beads provide information about the molecular weight range that they are most suitable for separating. Molecular weights of unknown proteins can be determined by comparing their elution volumes Vo, with those determined using

1.9.4 Separation by Electrophoresis

Electrophoresis relies on differences in the migration of charged molecules in a solution when an electrical field is applied across it. It can be used to separate proteins on the basis of their size, shape or charge. In non-denaturing electrophoresis, a buffered solution of native proteins is poured onto a porous gel (usually polyacrylamide, starch or agarose) and a voltage is applied across the gel. The proteins move through the gel in a direction that depends on the sign of their charge, and at a rate that depends on the magnitude of the charge, and the friction to their movement:

Proteins may be positively or negatively charged in solution depending on their isoelectic points (pI) and the pH of the solution. A protein is negatively charged if the pH is above the pI, and positively charged if the pH is below the pI. The magnitude of the charge and applied voltage will determine how far proteins migrate in a certain time. In non-denaturing electrophoresis the native proteins are separated based on a combination of their charge, size and shape.

In denaturing electrophoresis proteins are separated primarily on their molecular weight. Proteins are denatured prior to analysis by mixing them with mercaptoethanol, which breaks down disulfide bonds, and sodium dodecyl sulfate (SDS), 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. Each protein molecule binds approximately the same amount of SDS per unit length. Hence, the charge per unit length and the molecular conformation is approximately similar for all proteins. 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. This type of electrophoresis is commonly called sodium dodecyl sulfate -polyacrylamide gel electrophoresis, or SDS-PAGE.

To determine how far proteins have moved a tracking dye is added to the protein solution, e.g., bromophenol blue. This dye is a small charged molecule that migrates ahead of the proteins. 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. The relative mobility of each protein band is calculated:

Electrophoresis is often used to determine the protein composition of food products. The protein is extracted from the food into solution, which is then separated using electrophoresis. SDS-PAGE is used to determine the molecular weight of a protein by measuring Rm, and then comparing it with a calibration

curve produced using proteins of known molecular weight: a plot of log (molecular weight) against relative mobility is usually linear. Denaturing electrophoresis is more useful for determining molecular weights than non-denaturing electrophoresis, because the friction to movement does not depend on the shape or original charge of the protein molecules. In addition iso electric focusing electrophoresis and two dimensional electrophoresis are also employed.

1.9.5 Amino Acid Analysis

Amino acid analysis is used to determine the amino acid composition of proteins. A protein sample is first hydrolyzed (e.g. using a strong acid) to release the amino acids, which are then separated using chromatography, e.g., ion exchange, affinity or absorption chromatography.[31]

1.10. Nutritional Profile

Tuna is an excellent source of niacin, selenium, and protein. Tuna is also a very good source of vitamin B6 and thiamin. In addition, it is a good source of omega-3 fatty acids, phosphorus, potassium, and magnesium.

1.10.1. In-Depth Nutritional Profile

In addition to the nutrients highlighted in our ratings chart, an in-depth nutritional profile for Tuna is also available. This profile includes information on a full array of nutrients, including carbohydrates, sugar, soluble and insoluble fiber, sodium, vitamins, minerals, fatty acids, amino acids and more.

1.10.2.Sampling technique:

Tuna is sold in many different forms. It is available fresh as steaks, fillets or pieces. Tuna is probably best known in its canned form.

Just as with any seafood, it is best to purchase fresh tuna from a store that has a good reputation for having a frequent supply of fresh fish. Get to know a fishmonger (the person who sells the fish) at the store, so you can have a trusted resource from whom you can purchase your fish with confidence.

Fresh whole tuna should be displayed buried in ice, while fillets and steaks should be placed on top of the ice. Try to avoid purchasing tuna that has dry or brown spots.

Smell is a good indicator of freshness. Since a slightly "off" smell cannot be detected through plastic, if you have the option, purchase displayed fish as opposed to pieces that are prepackaged. Once the fishmonger wraps and hands you the fish that you have selected, smell it through the paper wrapping and return it if it has a truly strong fishy odor.

Canned tuna is available either solid or in chunks, and is packaged in oil, broth or water. Although the tuna packed in oil is usually the moistest, it also has the highest fat content, and the oils in which it is packed are high in omega-6 fats.

Since omega-6s and omega-3s compete for the same enzymes that activate them for use in the body, and most Americans already consume too many omega-6 fats in comparison to omega-3s, it is best to purchase tuna packed in water or broth. Oftentimes, 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).

When storing all types of fresh seafood, including tuna, it is important to keep them cold since fish spoils quickly and is very sensitive to temperature. Therefore, after purchasing tuna or other fish refrigerate it as soon as possible. If the fish is going to accompany you during a day full of errands, keep a cooler in the car where you can place your tuna to make sure it stays cold and does not spoil.

The temperature of most refrigerators is slightly warmer than ideal for storing fish. To ensure maximum freshness and quality, it is important to use special storage methods to create the optimal temperature for holding the fish.

One of the easiest ways to do this is to place fish, which has been well wrapped, in a baking dish filled with ice. The baking dish and fish should then be placed on the bottom shelf of the refrigerator, which is its coolest area. Replenish the ice one or two times per day.

The length of time that tuna can stay fresh stored this way depends upon how fresh it is, i.e. when it was caught. Fish that was caught the day before you purchased it can be stored for about four days, while fish that was caught the week before can only be stored for about one or two days.

You can extend the shelf life of tuna by freezing it. To do so, 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]

CHAPTER 2

2.1. Scope and objective:-

Proteins play multiple role as enzymes, hormones, catalyst, etc., The consumption of protein rich food is essential for human being to meet the daily requirement. Protein deficiency leads to malnutrition in children which is common in our country according to WHO (world health organization). The quality and quantity of the protein changes with the processing and also depends on the technique used.

The quality of protein depends on the level at which it provides the nutritional amounts of essential amino acids needed for overall body health, maintenance, and growth. Animal proteins, such as eggs, cheese, milk, meat, and fish, are considered high-quality, or complete, proteins because they provide sufficient amounts of the essential amino acids. Plant proteins, such as grain, corn, nuts, vegetables and fruits, are lower-quality, or incomplete proteins because many plant proteins lack one or more of the essential amino acids, or because they lack a proper balance of amino acids.

In order to make out the difference in the protein content in stable processed foods from raw foods, and to know the effect of processing on protein content of the food. Protein analysis would be carried out on the following raw as well as processed samples.

S.No. Raw Samples Processed Samples 1. Raw Sardines Sardines in Brine (canned) 2. Raw Tuna (yellow fin) Tuna in Brine (canned) 3. Raw Mackerel Mackerel in Brine (canned) 4. Raw Chicken (Broiler) Minced Chicken (Broiler canned) 5. Raw Beef Minced Beef (canned) 6. Raw Pork Minced Pork (canned) 7. Raw Mutton -

Therefore, to improve the quality of processed foods with respect to the protein content to reach the essential needs of the human being. 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, rich in protein can be incorporated and maintain the food without any loss of protein from it.

CHAPTER-3METHODS AND MATERIALS:

METHOD: KJELDAHL NITROGEN METHOD:

3.1 Principle of Method:-

The Kjeldahl procedure measures the nitrogen content of a sample. The protein content then, can be calculated assuming a ratio of protein to nitrogen for the specific food being analyzed. The Kjeldahl procedure can be basically divided into three parts: (1) digestion, (2) distillation, (3) titration. In the digestion step, organic nitrogen is converted to an ammonium in the presence of a catalyst at approximately 370°C.

In the distillation step, the digested sample is made alkaline with NaOH and the nitrogen is distilled off as NH3. This NH3 is “trapped” in a boric acid solution. The amount of ammonia nitrogen in this solution is quantified by titration with a standard HCl solution. A reagent blank is carried through the analysis and the volume of HCl titrant required for this blank is subtracted from each determination.

3.2. Chemicals:

a) Hazards Boric acid (H3BO3) b) Bromocresol green c) Ethanol, 95% pured) Methyl red e) Sodium hydroxide (NaOH) Corrosive f) Sulfuric acid, conc. (H2SO4) Corrosiveg) Kjeldahl digestion tablets Irritant h) Potassium sulfate (K2SO4) i) Cupric sulfate j) Oxalic acid dehydrate purified (Harmful)

3.3. Reagents:

Sulfuric Acid concentrated, 0.1N, (Fisher scientific Thermic electron LLS

India Pvt Ltd.)

Catalyst/Salt Mixture, 5% of potassium sulfate and 1% of cupric sulfate

(Kjeldahl digestion tablets) Contains potassium sulfate, cupric sulfate,

(Merck specialties Pvt Ltd., Qualigens fine chemicals Ltd.).

Sodium Hydroxide Solution, 40%, w/v, NaOH in deionized distilled (dd)

water, Dissolve 400g sodium hydroxide (NaOH) pellets in 1L dd water.

Cool. Add dd water to make up to 1.0L (Fisher chemic Ltd.).

Boric Acid Solution 4% ** In a 500ML flask, dissolve 20g boric acid in ca.

Cool to room temperature under tap water.

100 mg bromocresol green/100 ml ethanol and 100 mg methyl red/100 ml

ethanol.

3.4. Procedure:(Instructions are given for analysis in triplicate. Follow manufacturer’s instructions for specific Kjeldahl digestion and distillation system used. Some instructions given here may be specific for one type of Kjeldahl system.)

I. Digestion:

.

1. Turn on digestion block and heat to appropriate temperature.

2. Accurately weigh approximately 0.1 g corn flour. Record the weight. Place corn flour in digestion tube. Repeat for two more samples.

3. Add one catalyst tablet and appropriate volume (e.g., 7 ml) of concentrated sulfuric acid to each tube with 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).

4. Place rack of digestion tubes on digestion block. Cover digestion block with exhaust system turned on.

5. Let samples digest until digestion is complete. The samples should be clear (but neon green), with no charred material remaining.

6. Take samples off the digestion block and allow to cool with the exhaust system still turned on.

7. Carefully dilute digest with an appropriate volume of dd water. Swirl each tube.

II. Distillation:

1.Follow appropriate procedure to start up distillation system. 2. Dispense appropriate volume of boric acid solution into the receiving flask. Place receiving flask on distillation system. Make sure that the tube coming from the distillation of the sample is submerged in the boric acid solution.

3. Put sample tube in place, making sure it is seated securely, and proceed with the distillation until completed. In this distillation process, 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.

4. Upon completing distillation of one sample, proceed with a new sample tube and receiving flask.

5. After completing distillation of all samples, follow manufacturer’s instructions to shut down the distillation unit.

III. Titration:

1. Record the normality of the standardized HCl solution as determined by the teaching assistant.

2. If using an automated pH meter titration system, follow manufacturer’s instructions to calibrate the instrument. Put a magnetic stir bar in the receiver flask and place it on a stir plate. Keep the solution stirring briskly while titrating, but do not let the stir bar hit the electrode. Titrate each sample and blank to an endpoint pH of 4.2. Record volume of HCl titrant used.

3. If using a colorimetric endpoint, put a magnetic stir bar in the receiver flask, place it on a stir plate, and keep the solution stirring briskly while titrating. Titrate each sample and blank with the standardized HCl solution to the first faint gray color.Record volume of HCl titrant used.

3.7. Calculations

Calculate the percent nitrogen and the percent protein for each of your duplicate or triplicate corn flour samples, and then determine average values. The corn flour sample you analyzed was not a dried sample. Report percent protein results on a wet weight basis (wwb) and on a dry weight basis (dwb). Assume moisture content of 10% (or use the actual moisture content if previously determined on this corn flour sample). Use 6.25 for the nitrogen to protein conversion factor.

Normality is in mol/1000 mLCorrected acid vol. = (ml std. acid for sample) − (ml std. for blank)% Protein = % N × Protein Factor

CHAPTER-4RESULTS AND DISCUSSION:

This chapter contains the results obtained in the protein analysis of certain raw and processed animal food samples.

SAMPLE-1a: SARDINES FISH (RAW):-

FORMULA FOR % OF NITROGEN = 1.4* N H2SO4 * (T.V – B.V) / SAMPLE WEIGHT

FORMULA FOR % OF PROTEIN = % OF NITROGEN * 6.25

S.NO SAMPLE WEIGHT IN

GRAM

NORMALITYOF H2SO4

VOLUME OF H2SO4 IN ml

% OFNITROGEN

% OFPROTEIN

1. BLANK 0.0979 0.3 0.00 0.002. 0.304 0.0979 8.8 3.8 23.73. 0.555 0.0979 13.0 3.13 19.564. 0.740 0.0979 17.8 3.24 20.255. 1.069 0.0979 24.2 3.06 19.126. 1.532 0.0979 32.8 2.907 18.17

SAMPLE-1b: SARDINES FISH (RAW):-

S.NOSAMPLE

WEIGHT IN GRAM

NORMALITY OF H2SO4


% OF NITROGEN

% OF PROTEIN

1. BLANK 0.0979 0.3 0.00 0.002. 0.332 0.0979 7.2 2.848 17.83. 0.550 0.0979 11.2 2.716 16.94. 0.718 0.0979 15.7 2.939 18.375. 1.024 0.0979 24.1 3.185 19.96. 1.514 0.0979 36.7 3.295 20.59

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 weights were taken. 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.

SAMPLE-2a: SARDINES PROCESSED IN BRINE SOLUTION:(ANCHOR SEA FOOD PRODUCTS)

SAMPLE WEIGHT IN

NORMALITY OF H2SO4


% OF NITROGEN

% OF PROTEIN

S.NO GRAM

1. BLANK 0.0994 0.2 0.00 0.002. 0.325 0.0994 6.8 2.82 17.623. 0.556 0.0994 12.1 2.97 18.564. 0.715 0.0994 9.9 1.88 11.755. 1.058 0.0994 21.3 2.77 17.346. 1.512 0.0994 30.9 2.82 17.62

SAMPLE-2b: SARDINES PROCESSED IN BRINE SOLUTION:(ANCHOR SEA FOOD PRODUCTS)


FORMULA FOR % OF PROTEIN = % OF NITROGEN * 6.25.

S.NOSAMPLE

WEIGHT IN GRAM

NORMALITY OF H2SO4


% OF NITROGEN

% OF PROTEIN

1. BLANK 0.0994 0.2 0.00 0.002. 0.319 0.0994 6.8 2.87 17.933. 0.538 0.0994 8.6 2.17 13.564. 0.738 0.0994 8.2 1.50 9.375. 1.088 0.0994 22.2 2.81 17.566. 1.545 0.0994 20.5 1.82 11.3

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. SAMPLE-3a: TUNA FISH (RAW):-

S.NOSAMPLE

WEIGHT IN GRAM

NORMALITY OF H2SO4


% OF NITROGEN

% OF PROTEIN

1. BLANK 0.1082 0.2 0.00 0.002. 0.355 0.1082 10.0 4.181 26.133. 0.523 0.1082 15.7 4.460 27.874. 0.733 0.1082 21.1 4.319 26.995. 1.021 0.1082 23.2 3.412 21.3

6. 1.533 0.1082 42.7 4.189 26.18

SAMPLE-3b: TUNA FISH (RAW):-

S.NOSAMPLE

WEIGHT IN GRAM

NORMALITY OF H2SO4


% OF NITROGEN

% OF PROTEIN

1. BLANK 0.1082 0.2 0.00 0.002. 0.325 0.1082 9.6 4.381 27.383. 0.513 0.1082 15.0 4.370 27.314. 0.715 0.1082 22.6 4.745 29.665. 1.098 0.1082 32.2 4.414 27.596. 1.532 0.1082 44.5 4.380 27.37

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. 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.

SAMPLE-4a: TUNA FISH PROCESSED IN BRINE SOLUTION:-(ANCHOR SEA FOOD PRODUCTS)

S.NOSAMPLE

WEIGHT IN GRAM

NORMALITY OF H2SO4


% OF NITROGEN

% OF PROTEIN

1. BLANK 0.0957 0.2 0.00 0.002. 0.341 0.0957 7.6 2.907 18.173. 0.531 0.0957 10.1 2.497 15.614. 0.706 0.0957 15.1 2.827 17.675. 1.017 0.0957 23.6 3.082 19.26

6. 1.531 0.0957 42.8 3.727 23.29

SAMPLE-4b: TUNA FISH PROCESSED IN BRINE SOLUTION:-(ANCHOR SEA FOOD PRODUCTS)

S.NOSAMPLE

WEIGHT IN GRAM

NORMALITY OF H2SO4


% OF NITROGEN

% OF PROTEIN

1. BLANK 0.0957 0.2 0.00 0.002. 0.312 0.0957 8.6 3.607 22.543. 0.580 0.0957 16.1 3.672 22.954. 0.718 0.0957 19.4 3.582 22.395. 1.055 0.0957 28.6 3.606 22.546. 1.527 0.0957 41.8 3.650 22.81

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.

SAMPLE-5a: MACKEREL FISH (RAW):-

S.NOSAMPLE

WEIGHT IN GRAM

NORMALITY OF H2SO4


% OF NITROGEN

% OF PROTEIN

1. BLANK 0.1138 0.2 0.00 0.002. 0.341 0.1138 6.8 3.083 19.273. 0.551 0.1138 12.0 3.411 21.324. 0.705 0.1138 15.4 3.434 21.465. 1.032 0.1138 18.2 2.778 17.36

6. 1.563 0.1138 30.2 3.057 19.11

SAMPLE-5b: MACKEREL FISH (RAW):-

S.NOSAMPLE

WEIGHT IN GRAM

NORMALITY OF H2SO4


% OF NITROGEN

% OF PROTEIN

1. BLANK 0.1138 0.1 0.00 0.002. 0.333 0.1138 6.6 3.109 19.433. 0.531 0.1138 11.8 3.510 21.944. 0.709 0.1138 14.1 3.145 19.665. 1.056 0.1138 20.9 3.138 19.616. 1.538 0.1138 32.9 3.397 21.23

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. 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.

SAMPLE-6a: MACKEREL FISH PROCESSED IN BRINE SOLUTION:-(ANCHOR SEA FOOD PRODUCTS)

S.NOSAMPLE

WEIGHT IN GRAM

NORMALITY OF H2SO4


% OF NITROGEN

% OF PROTEIN

1. BLANK 0.1082 0.1 0.00 0.002. 0.312 0.1082 5.8 2.767 17.293. 0.515 0.1082 9.9 2.882 18.014. 0.721 0.1082 15.7 3.277 20.485. 1.036 0.1082 20.9 3.041 19.00

6. 1.506 0.1082 29.7 2.977 18.6

SAMPLE-6b: MACKEREL FISH PROCESSED IN BRINE SOLUTION:-(ANCHOR SEA FOOD PRODUCTS)

S.NOSAMPLE

WEIGHT IN GRAM

NORMALITY OF H2SO4


% OF NITROGEN

% OF PROTEIN

1. BLANK 0.0957 0.2 0.00 0.002. 0.349 0.0957 6.8 2.456 15.353. 0.553 0.0957 13.4 3.198 19.984. 0.712 0.0957 16.0 2.973 18.585. 1.014 0.0957 19.3 2.523 15.776. 1.580 0.0957 30.5 2.569 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.

SAMPLE-7a: BROILER CHICKEN (RAW):-

S.NOSAMPLE

WEIGHT IN GRAM

NORMALITY OF H2SO4


% OF NITROGEN

% OF PROTEIN

1. BLANK 0.1035 0.2 0.00 0.002. 0.309 0.1035 8.4 3.845 24.033. 0.531 0.1035 14.6 3.929 24.554. 0.781 0.1035 20.3 3.729 23.305. 1.024 0.1035 27.2 3.820 23.87

6. 1.513 0.1035 39.4 3.754 23.46

SAMPLE-7b: BROILER CHICKEN (RAW):-

S.NOSAMPLE

WEIGHT IN GRAM

NORMALITY OF H2SO4


% OF NITROGEN

% OF PROTEIN

1. BLANK 0.1035 0.2 0.00 0.002. 0.316 0.1035 7.1 3.163 19.773. 0.527 0.1035 12.2 3.299 20.624. 0.749 0.1035 22.8 4.372 27.325. 1.023 0.1035 28.2 3.965 24.786. 1.558 0.1035 46.8 4.333 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.

SAMPLE-8a: MINCED CHICKEN PROCESSED IN BRINE SOLUTION:-(ZWAN FOOD PRODUCTS-HOLLAND)

S.NOSAMPLE

WEIGHT IN GRAM

NORMALITY OF H2SO4


% OF NITROGEN

% OF PROTEIN

1. BLANK 0.1035 0.2 0.00 0.002. 0.308 0.1035 4.6 2.07 12.933. 0.517 0.1035 7.4 2.017 12.614. 0.734 0.1035 10.5 2.033 12.705. 1.062 0.1035 15.2 2.046 12.79

6. 1.580 0.1035 22.0 1.999 12.49

SAMPLE-8b: MINCED CHICKEN PROCESSED IN BRINE SOLUTION:-(ZWAN FOOD PRODUCTS-HOLLAND)

S.NOSAMPLE

WEIGHT IN GRAM

NORMALITY OF H2SO4


% OF NITROGEN

% OF PROTEIN

1. BLANK 0.1035 0.2 0.00 0.002. 0.316 0.1035 4.8 2.109 13.183. 0.533 0.1035 8.2 2.174 13.594. 0.754 0.1035 11.7 2.210 13.815. 1.057 0.1035 14.8 2.001 12.506. 1.560 0.1035 21.3 1.959 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.

SAMPLE-9a: MUTTON (RAW):-



S.NOSAMPLE

WEIGHT IN GRAM

NORMALITY OF H2SO4


% OF NITROGEN

% OF PROTEIN

1. BLANK 0.1035 0.2 0.00 0.00

2. 0.319 0.1035 5.0 2.180 13.623. 0.546 0.1035 8.8 2.282 14.264. 0.788 0.1035 17.2 3.126 19.535. 1.018 0.1035 15.8 2.220 13.876. 1.511 0.1035 27.9 2.656 16.60

SAMPLE-9b: MUTTON (RAW):-



S.NOSAMPLE

WEIGHT IN GRAM

NORMALITY OF H2SO4


% OF NITROGEN

% OF PROTEIN

1. BLANK 0.1035 0.2 0.00 0.002. 0.325 0.1035 4.7 2.006 12.533. 0.536 0.1035 6.0 1.567 9.7994. 0.736 0.1035 15.6 3.031 18.945. 1.053 0.1035 20.5 2.793 17.456. 1.517 0.1035 27.4 2.598 16.23

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. 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.

SAMPLE-10a: BEEF (RAW):-

S.NOSAMPLE

WEIGHT IN GRAM

NORMALITY OF H2SO4


% OF NITROGEN

% OF PROTEIN

1. BLANK 0.1035 0.2 0.00 0.002. 0.311 0.1035 7.1 3.214 20.093. 0.522 0.1035 11.8 3.22 20.12

4. 0.748 0.1035 13.9 2.653 16.585. 1.008 0.1035 20.3 2.889 18.056. 1.516 0.1035 36.0 3.421 21.38

SAMPLE-10b: BEEF (RAW):-

S.NOSAMPLE

WEIGHT IN GRAM

NORMALITY OF H2SO4


% OF NITROGEN

% OF PROTEIN

1. BLANK 0.1035 0.0 0.00 0.002. 0.337 0.1035 5.4 2.880 18.003. 0.509 0.1035 8.6 3.188 19.924. 0.706 0.1035 9.1 3.242 20.265. 1.009 0.1035 21.9 3.030 18.936. 1.552 0.1035 29.0 3.305 20.65

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. 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.

SAMPLE-11a: MINCED BEEF PROCESSED IN BRINE SOLUTION:-(COSTA FOOD PRODUCTS)

S.NOSAMPLE

WEIGHT IN GRAM

NORMALITY OF H2SO4


% OF NITROGEN

% OF PROTEIN

1. BLANK 0.1035 0.0 0.00 0.002. 0.388 0.1035 5.4 2.016 12.603. 0.549 0.1035 8.6 2.269 14.18

4. 0.742 0.1035 9.1 1.777 11.105. 1.065 0.1035 21.9 2.979 18.626. 1.513 0.1035 29.0 2.777 17.35

SAMPLE-11b: MINCED BEEF PROCESSED IN BRINE SOLUTION:-(COSTA FOOD PRODUCTS)

S.NOSAMPLE

WEIGHT IN GRAM

NORMALITY OF H2SO4


% OF NITROGEN

% OF PROTEIN

1. BLANK 0.0948 0.0 0.00 0.002. 0.362 0.0948 6.1 2.236 13.973. 0.503 0.0948 8.9 2.348 14.674. 0.709 0.0948 13.3 2.489 15.565. 1.052 0.0948 18.2 2.296 14.356. 1.564 0.0948 33.0 2.800 17.50

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.

DISCUSSION:-

Food processing is the field of advanced techniques to convert one form to another

form. Proteins are important macromolecules that causes muscle mass and plays

important metabolism in human being. Protein analysis carried out to describe the

different in protein content in raw foods from processed foods. Raw food samples

like fish, chicken and other meat products like mutton and beef contains higher

protein content than the processed food samples as well as vegetable sources, corn,

cereals and pulses as showed from the results. Protein stability in the foods

depends on the temperature.

During thermal processing there can be degradation of protein conformation.

Putrefaction is a main cause of protein degradation by microorganisms during

fermentation that involves in the food processing.

Certain processing techniques like freezing, salting, 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.

CHAPTER-5

SUMMARY

Protein analysis benefits to recommended protein rich foods to regular diet.

It strengthens the quality of food because of multipurpose and benefits of protein in

the biological system. The protein content in six samples of raw animal products

and five samples of processed food from animal source were determined. It is

found that the percentage of protein in tuna fish is much greater than sardines and

mackerel. Chicken is greater in protein content than beef and mutton, may be due

to higher fat content in these food materials. Processed food samples contain less

amount of protein than raw food samples. Animal proteins are higher in protein

content than plant proteins but, these animal proteins are decreased by various

processing technique like freezing, smoking, salting and chilling. The loss of

protein content in animal food samples during processing can be regained by

certain value addition.

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.

2. Sardines fish processed along with tomato sauce and baked beans.

3. Tuna fish processed along with garlic-ginger paste and oil.

CHAPTER-6

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Effect of processing on protein content of fish and meat products

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