kim pang protein 5

7/29/2019 Kim Pang Protein 5

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FOOD CHEMISTRY 3FCHE30

Semester 2

Module 5

Effect of Heat and pH on Proteins

Faculty of ScienceDept. of Horticulture and Food Technology


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Protein

Protein Occurrence Polymers of some 20 different amino acids

Joined together by peptide bonds

Different proteins have different chemical properties Because of widely different secondary and tertiary

structures

Amino Acids

Grouped on the basis of the chemical nature of the sidechains

Side chains my be polar or non-polar

High levels of polar amino acid residues in a proteinincrease water solubility


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Amino Acids

Amino acids are the building blocks (monomers) of proteins. 20different amino acids are used to synthesize proteins. Theshape and other properties of each protein is dictated by theprecise sequence of amino acids in it.

Each amino acid consists of an alpha carbon atom to which isattached

a hydrogen atom an amino group (hence "amino" acid) a carboxyl group (-COOH). This gives up a proton and is thus an

acid (hence amino "acid") one of 20 different "R" groups. It is the structure of the R group that

determines which of the 20 it is and its special properties.


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The amino acid shown here is Alanine.


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Amino Acids

Most polar side chains are those of the basic amino acidicamino acids Present at high levels in soluble albumins and globulins

Wheat proteins, gliadin and glutenin, have low levels of polarside chains and are quite insoluble in water

Acidic amino acids may also be present in proteins in theform of their amides, glutamine and asparagine This increases the nitrogen content of the protein

Hydroxyl groups in the side chains may become involved inester linkages with phosphoric acid and phosphates

Sulfur amino acids may form disulfide cross-links betweenneighbouring petide chains or between different parts of thesame chain


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Amino Acids

Joined together by peptide bonds: Form the primary structure ofproteins

The amino acid composition established the nature of secondaryand tertiary structures These influence the functional properties of food proteins and

their behavior during processing 20 Amino acids : Only about half essential for human nutrition Amount of essential amino acids present in a protein and their

availability determine the nutritional quality of the protein Animal proteins are higher quality than plant proteins

Egg protein One of the best quality proteins Biological value of 100 Widely used as a standard, Protein Efficiency Ratio (PER)

sometimes use egg white as a standard


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Amino Acids

When hydrolyzed by strong mineral acids or with the aid of certainenzymes, proteins can be completely decomposed into theircomponent amino acids

Simplest amino acid: Glycine, The R-group is H (Hydrogen)

Aliphatic monoamino monocarboxylic amino acids Glycine

Alanine

Valine

Leucine

Isoleucine Serine

Threonine

Proline


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Amino Acids

Sulfur-containing amino acids

Cysteine

Cystine

Methionine

Monoamino dicarboxylic amino acids

Aspartic acid

Glutamic acid

Basic amino acids Lysine

Arginine

Histidine


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Amino Acids

Aromatic amino acids

Phenylalanine Tyrosine

Tryptophan


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Protein Classification

Classification of Proteins Based mostly on the solubility of proteins in different solvents More recent criteria being used includes:

Behaviour in the centrifuge Electrophoretic propterties

Proteins are divided into the following maingroups

Simple Proteins

Conjugated Proteins

Derived Proteins


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Simple Proteins Yield only amino acids on hydrolysis and include the

following classes Albumins

Globulins

Glutelins

Prolamins

Sclereproteins Histones

Protamines


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Conjugated Proteins Contain an amino acid part combined with a non-protein

material such as a lipid, nucleic acid, or carbohydrate Some of the major conjugated proteins are as follows:

Phosphoproteins

Lipoproteins

Nucleoproteins

Glycoproteins Chromoproteins


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Derived Proteins These are compounds obtained by chemical or enzymatic methods

and are divided into primary and secondary derivatives Primary derivatives

Slightly modified and are insoluble in water Ex. Rennet-coagulated casein

Secondary derivatives Changed more extensively, include proteoses, peptones, and

petides Difference between these breakdown products is in size and

solubility All are soluble in water Not coagulated by heat Proteoses can be precipitated with saturated ammonium sulfate

solution Peptides contain two or more amino acid residues


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ProteinStructures

Proteins are macromolecules with differentlevels of structural organization Primary Structure

the peptide bonds between component amino acids

and also the amino acid sequences in the molecule Secondary Structure

Involves folding the primary structure Hydrogen bonds between amide nitrogen and carbonyl oxygen

are the major stabilizing force Bonds may be formed between different areas of the same

polypeptide chain, or adjacent chains The secondary structure may be either a-helix or sheet Helical structures are stabilized by intramolecular hydrogen

bonds Sheet structures are stabilized by intermolecular hydrogen

bonds


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ProteinStructures

Tertiary Structure Involves a pattern of folding of the chains into a compact unit Stabilized by

hydrogen bonds van der Waals forces disulfide bridges hydrophobic interactions

This structure results in the formation of a tightly packed unitwith most polar amino acid residues located on the outside and

hydrated Internal part with most of the apolar side chains and virtually no

hydration Large molecules of molecular weights above about 50 000

may form quaternary structures by association of subunits These structures my be stabilized by hydrogen bonds, disulfide

bridges and hydrophobic interactions


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Denaturation

The denaturation process Changes the molecular structure without breaking any peptide

bonds of a protein This process is peculiar to proteins and affects different proteins to

different degrees, depending on the structure of a protein Denaturation can be brought about by a variety of agents

Heat (most important), causes the destruction of enzymeacticvity

pH

Salts Surface effets

Denaturation usually involves loss of biological activity andsignificant changes in some physical or functional properties, suchas solubility

Usually non-reversible


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Denaturation

Heat denaturation is sometimes desirable The denaturation of whey proteins for the production of

milk powder used in baking

Proteins of egg white are readily denatured by heat andby surface forces when egg white is whipped to a foam Meat proteins are denatured in the temperature range 57

to 75C, which has a profound effect on texture, waterholding capacity and shrinkage

Denaturation may result in flocculation of globular proteins

and may lead to the formation of gels Protein denaturation and coagulation are aspects of heat

stability that can be related to the amino acid compositionand sequence of the protein


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Denaturation

DEFENITION: Denaturation is a major change inthe native structure that does not involvealteration of the amino acid sequence

Effect of heat usually involves a change in the tertiarystructure, leading to a less ordered arrangement of thepolypeptide chains

The temperature range in which denaturation andcoagulation of most proteins takes place is about 55 to 75C Casein and gelatin are examples of proteins that can be

boiled without apparent change in stability


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Denaturation

The exceptional Stability of Casein Makes it possible to boil, sterilize, and concentrate milk,

without coagulation

In the first place restricted formation of disulfide bonds due to low content

of cystine and cysteine results in increased stability Casein with its extremely low content of sulfur amino

acids are less likely to become involved in the type ofsulfhydryl agglomeration

The heat stability of casein is also explained by therestraints against forming a folded tertiary structure

These restraints are due to the relatively high content ofproline and hydroxyproline in the heat stable proteins


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Protein Quality

What is Protein Quality?

Proteins with a relatively high content of essential amino acidsare called first class proteins or high quality proteins

This type of proteins are quite expensive to produce Ex. Red meat, white meat, dairy products, eggs and a few

legumes (peas and soya beans) Animal proteins usually contain much more of the essential amino

acids than do plant proteins Intermediate quality proteins are those derived from plant material

Potatoes, rice, wheat etc. Poor quality proteins are derived from millet, sorghum, cassava and

other roots and tubers


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Protein Quality

Protein poor diet supplies limited amino acids to theconsumer

Many processes actually leads to a further decline in the

protein quality of food products These effects need to be monitored and, where necessary,

controlled to ensure a safe and nutritious food supply to thepopulation


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Protein Quality

The Essential Amino Acids

Histidine

Isoleucine

Leucine

Lysine

Methionine (and/or cysteine)

Phenylalanine (and/or tyrosine)

Threonine

Tryptophan

Valine


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Environmental Effects onProtein Quality

The environment can exert profound changes on thefunctionality and nutritional quality of the protein

Degradative reactions can result from the processing or

storage environment which can cause undesirable changesin proteins As a result of these reactions protein can exhibit:

Losses in functionality Losses in nutritional quality Increased risk of toxicity Desirable and undesirable flavor changes


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Environment changes that can adversely affect proteins include Heat in the presence and absence of carbohydrate Extremes in pH (particularly alkaline)

Exposure to oxidative conditions Caused by light and Caused by oxidizing lipids

Nutrients are destroyed when foods are processed, largely becausethey are

Sensitive to pH of solvent Sensitive to oxygen, light, heat or combinations of these

The amino acid composition of food protein is of fundamentalimportance in determining nutritional quality and functionality


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Influences of Processing on Proteins Different proteins and food systems have very different

susceptibilities to damage resulting from processing In order to obtain measurable responses, experimental conditions

are frequently much harsher than those to which a food might beexposed during commercial processing

Most commercial processes such as dehydration, canning, bakingand domestic cooking have only small effects on nutritional qualityof proteins

There are the exceptions, for example, conditions where foods areexposed to Very high pH Extreme heat Peroxidizing lipids


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Physical and Chemical environments that a protein isexposed to during processing can result in wide variety ofchanges

Changes in amino acid side chains Amino acid razemize and develop new cross-links in alkaline

solution Losses in nutritional quality Significant changes in functionality Arginine, Cystine, Threonine, Serine, and Cysteine are destroyed

Glutamine and Asparagine are deaminated under alkalineconditions In Acid Solutions, Tryptophan is rapidly destroyed, and Serine and

Threonine are slowly destroyed Ultraviolet light destroys Tryptophan, Tyrosine and Phenylalanine


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Sulfur amino acids are damaged by reaction products from lipidoxidation or by the addition of bleaching or oxidizing agents

All amino acids, especially Lysine, Threonine, and Methionine, aresensitive to dry heat, browning, and radiations


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Influence of Heat on Protein

Susceptibility to heat damage varies among different proteinsources

Susceptibility is increased in the presence of various

carbohydrates and other food constituents Nutritional value of proteins can be significantly affectedeven when there is little or no apparent significant differencein amino acid composition

The Thermally related changes in proteins can be

broken into four basic catagories


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(1) Alteration in the tertiary structure of theprotein Requires only mild heating

Exerts no nutritional effect Tertiary changes can have significant influence on functionality

Ex. Loss in Solubility If the protein is an Enzyme, it is likely, but not inevitable, that

changes in tertiary structure will reduce or eliminate enzymatic

activity Thermal denaturation is of great significance in food technology

because of the changes in the chemical and physical properties ofthe proteins


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Globular proteins will exhibit changes (generally losses) in Solubillity Viscosity Osmotic properties Electrophoretic mobilty Immunosensitivity Chemical sensitivity

The changes are due to new reactive side chains of the proteinbeing exposed as a result of the increased random coil being

introduced to the protein Fibrillar proteins when heated will suffer changes in Elasticity Flexibility Fibrillar length


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Thermal changes will alter the properties of the food,improving or destroying the functional properties Enzyme inhibitors, such as Trypsin inhibitors, are

denaturated Avidin is denaturated by heat Egg albumin becomes insoluble (but in a useful form for

consumption) Gluten is an example of a Protein that losses its dough-

forming properties as a result of too much heat Most of these changes, however, have no measurable effect

on the nutritional quality of the proteins themselves.


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(2) Non-enzymatic browning / Maillard reaction The reaction most of us think of first when considering damage to

proteins during food processing Has the most significance from the nutritional point of view (The denaturation of protein may be more significant in terms of

effect on protein functionality)

This reaction occurs primarily between the -amino group ofLysine and a carbohydrate

The lysine after the very earliest stages of the reaction, becomes

unavailable Therefore, with the resulting Maillard reaction product bound to the

protein The solubility of the protein changes Colour will change as the melanoidin pigments are formed


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Keep in mind that while the browning reaction can accountfor substantial losses in nutritional quality of proteins, it isalso critical to the development of flavor in foods

Environment of protein or food can have a substantial effect

on the nature and extent of the observed browning The Maillard reaction occurs during both

Storage Heat treatment

The reaction is slow at room temperature and increases with

temperature The loss of the essential amino acid Lysine serves as the

best single indicator of damage to the protein from thebrowning reaction


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The pH can also influence the browning reaction of protein Acidification inhibits the browning reaction Raising the pH above 7.0 greatly enhances browning

The Maillard reaction increases approximately linearlyfrom pH 3 to 8.0

This is also the region where most foods are subject toheat treatment


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The browning of bread during the baking process is essentialto the development of what we consider to be bread flavor Lysine is the first limiting amino acid in wheat and

therefore in bread This limiting factor can be aggravated by the baking

process

The next table and data represents breads baked at differenttemperatures, but clearly illustrate the loss in nutritive valueas a result of intense heating


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Table: PER (protein efficiency ratio) of Bread and Toast

Bread in Diet PER

Casein (control) 2.50

Whole bread 1.02

Crust 0.42

Crumb 1.47

Crumb (higher baking temperature) 0.90

Light Toast 0.64

Medium Toast 0.45

Dark Toast 0.32


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When looked at the influences of high-temperature short-time heating of pizza doughs on the amino acid profiles, theresults show

Lysine, and to a lesser extent cystine, tyrosine, andthreonine are lost in the crust after baking The losses range from 7.1% in whole-wheat pizza crust

to 19.4% in commercial pizza crust It was proposed that the losses in nutritive value of pizza

crust could be correlated with losses in lysine


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A major environmental factor which influences the extent ofbrowning in proteins is the Water Content of the System

Anhydrous protein is fairly stable to heat and storage in the

presence of carbohydrate At water activities of 0.4 0.7 the browning reactionproceeds rapidly

The reaction then slows as the protein is diluted Liquid milk, therefore, is more stable to heating effects

than powdered milk with residual moisture


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It is clear that heating and/or storage ofprotein in the presence of reducing sugars

and limited water is an environment thatwill facilitate rapid degradation of theprotein, particularly the amino group


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(3) More severe heat treatment Particularly lysine and cystine are sensitive to this type of

thermal decomposition.

Lysine and Arginine side chains react with the free acids ofglutamic and aspartic acid or with the amides to yieldisopeptide cross-links which can impede digestion and exibitmajor effects on functionality

Cystine is relatively sensitive and is converted to dimethyl

sulfide as well as other products at temperatures of 115C A lactone ring is formed between a terminal carboxyl group

and hydroxel amino acids


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(4) Heat damage on the outside surface ofroasted foods The result of roasting is racemization of amino acid residues

in the protein Or in the case of extensively heated material, complete

destruction of the amino acids Temperatures of 180 300C

Such as occur in roasted coffee, meat, fish and in thebaking of some biscuits These reactions also account for some of the flavor and

colour developed as a result of the roasting process


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Solubility One of the most easily observed thermal changes in protein

is the change in conformation which affects solubility

Generally, protein solubility decreases with increases in thetime and temperature of heat treatment

Thermal denaturation of protein occurs when hydrogen andother non-covalent bonds, such as ionic and van der Waalsbonds within the protein, are disrupted by the heating

process Thus, the normal secondary, tertiary, and quaternary

structure of the protein is disrupted, and the protein becomesdenatured

I fl f H t P t i


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Influence of Heat on ProteinSolubility

During solubility changes the protein goes through stagesand some changes are observable

Interactions with different functional groups become moreprevalent as the protein unfolds Sulfhydryl Dislufide Tyrosyl

These changes lead to a diverse and complicated series ofreactions that ultimately lead to the precipitation of the

protein The interaction of water along with heat causes various ionic

and polar groups in the protein to exert considerableinfluence on the folded conformations of the proteins

I fl f H t P t i


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Influence of Heat on ProteinSolubility

Moist heat frequently exerts more involved reactions in theprotein than dry heat and is influenced greatly by pH andionic stregth

Dry proteins denature at different rates, but minimum

solubility was reached for most proteins by 153C The effects of moist heat were found more complex than theeffects of dry heat

Proteins receiving dry heat exhibited a linear loss in solubilityas function of increasing temperature from 110C to 115C

Wet-heated samples showed a sigmoidal curve (minimum of120C) with the solubility plot over the same temperaturerange The solubility then decreased sharply after 145C the 115C sample being nearly as insoluble as the dry-

heated protein


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Coaggregration

Since food systems typically contain many different proteins,thermal processing can cause co-aggregation amongproteins in the mixture

Such co-aggregation can be important in determining the

characteristics of the food The aggregations are also extremely difficult to study

because they are chemically very stable When -casien is heated in the presence of

-lactoglobulin, a disulfide link is formed between the two

proteins, which reduces the thermal denaturation of thenormally stable -lactoglobulin The functionality of milk powder for baking is significantly

enhanced by heating the milk before drying to enhance theformation of the disulfide links between -casien and -lactoglobulin


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Influence of Heat on Protein Digestibility and Nutritional value of heat-damaged Proteins

It has been known for some time that heat induces a numberof changes in the physical properties of proteins and thatsuch changes can influence the digestibility and hencenutritional value of proteins

The reduction in the nutritional quality of heated proteins is

attributed to the isopeptide cross-links formed as a result ofthe heat treatment Homogenates of the small intestine showed considerable

activity toward the isopeptides Glutamyllysine will pass the gut wall

The decreased protein digestibility reduces the apparent bio-availability of most of the amino acid residues in the protein

AS the intensity of heating increases, the level of isopeptidesalso increases

The severity of damage to the remainder of the proteinincreases with increasingly intense heat treatment


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Thermal decomposition Several amino acids has been studied Free radicals are formed in protein or Lysine heated at

200C for 22 minutes

It is of concern that these free radicals appear to be stable inwater and in digestive juices An aspect of thermal decomposition that must be considered

is the possible formation of toxic products Mutagenic activity on flame-broiled fish and beef Several mutagens are of protein and amino acid origin Two of the most toxic mutagens are derived from

tryptophan It is important to note that these compounds are only formed

at temperatures in excess of 300C


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Photo-oxidation of Proteins

Photo-chemical reactions Amino acid side chains that are readily modified by photo-

oxidation are

Sulhydryl Imidazole Phenoxyindole Thiol ether

Data indicated that there are losses in the oxidizable aminoacids, but that aspartic acid and valine are stable to photo-oxidation


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Mechanisms of free radical initiation andsubsequent damage to protein Free radicals plays an important role in photolytic reactions

both in direct dissociations and in photosensitized oxidations Several factors influence the pathway and extent of free

radical damage The nature of the sensitizer The nature of the substrate and redox potential of the

substrate Concentration of sensitizer and oxygen in the system The nature of the medium (potential for diffusion)

The aliphatic amino acids, although they absorb light only toa small extent, can be damaged significantly by the radiation

in the absence of a photosensitizer


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The damage results from short wavelengths Glycine is not damaged at wavelengths above 2265

The precise changes and pathways of destruction areinfluenced by Irradiation wavelength Irradiation dose Reaction conditions Individual amino acid being irradiated

The sulfur amino acids exhibit more measurable photo-decomposition than the aliphatic amino acids The aromatic amino acids can act as photosensitizers in the

protein, particularly sensitizing the sulfur amino acids


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Two of the potent photosensitizers in foods are riboflavin andchlorophyll

Generally, two classes of reactions are initiated when aphotosensitizer, oxygen, and a protein are present and lightof the proper wavelength activates the system

FIRST TYPE: the photosensitezer is excited to the triplet state by light

of the proper wavelength

The excited sensitizer then univalently oxidazes anamino acid side chain of the protein initiating a free radical destruction of the amino acid


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SECOND TYPE: The excited photosensitizer excites a ground-state

oxygen which forms a highly reactive singlet oxygen

The singlet oxygen can the attack lipids or reactive sidechains of amino acids


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Interaction of Protein with Lipids

Lipid hydroperoxides cause a number of interestingreactions with various reactive amino acid residues in protein

These various reactions all help account for thepolymerization of proteins

Lipid peroxidation free radicals serve as initiators of thepolymerization Substantial losses in amino acids when proteins were

exposed to peroxidizing lipids Methionine, histidine, cystine and lysine were the most

vulnerable to damage Losses in digestibility and biological value of the proteins

after oxidation


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Considerable conversion of methionine to methioninesulfoxides when methionine is exposed to oxidizing lipid

Loss of methionine in casein which was exposed toautoxidizing methyl linoleate in a model system

Observed considerable browning takes place in the modelsystem as the reaction continues

Malonaldehyde, an intermediate in the decomposition oflipids, has been shown to react with sulfur amino acids, with

enamine and imine linkages being proposed in the products When malonaldehyde reacts with collagen, lysine and

tyrosine are the principal amino acids damaged


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Maximum interaction or degradation of theprotein takes place when the lipid oxidation is atthe stage of maximum peroxide formation

Losses in available lysine appeared to take place in theinitial induction period and during the induction of peroxides Oxidizing lipids or peroxides in the environment of the

protein clearly cause significant change in the protein Oxidations and cross-links generated tend to adversely

affect Solubility Enzyme activity Nutritive quality


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Chlorine Another environmental oxidizer which can damage protein

quality The initial side of attack of the chlorine is the sulfur of

methionine First intermediate formed is chlorosulfonium Second step is the formation of a carbonium ion

intermediate and cleavage of the carbon sulfur bond The splitting yields a trichloroamino acid porduct

Nutritional impact not likely to be significant, since foodsproduced from chlorinated flour are not generally consumedas sole sources of protein The loss of small amounts of methionine would not be

significant


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Influence of alkaline conditions

Proteins are frequently exposed to a highpH environment Even brief exposure of protein to an extreme in pH can result

in significant changes in protein Alkaline treatment advantages Improving solubility of protein Destroying toxins Improving flavor or texture

Undesirable aspect of alkaline Particularly at high temperatures

Racemization Cross-links such as isopeptides or lysinoalanine


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Racemization Observed in severely alkaline-treated proteins This reaction occurs via removal of the -methine hydrogen

Forming a carbanion intermediate Carbanion then reacts rapidly with proton with an equal

likelihood of readdition of the proton Forming either the normal L form or the D form of amino

acid residue Protein sequence may have some mediating influence on

this at moderately alkaline pH, but as pH increases, therandomness of the protein structure would also be expected

to increase The rate of racemization is proportional to the hydroxide ionconcentration above pH 8.0

Below pH 8.0 racemization is dependent on electronwithdrawing ability or the amino acid side chain


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In addition to high pH, increases in length of alkalinetreatment and temperature increases racemization

Heat at slightly acid pH can induce significant racemization

Biological effects of racemization

Decreases in the vitro digestibility of alkaline-treated proteins Alkaline-treated casein was much more resistant to

enzymatic hydrolysis than untreated casein

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