Proteins - Principle of food chemistry

Proteins - Principle of food chemistry

Proteins are polymers of some 21 different

amino acids joined together by peptide

bonds Because of the variety of side chains

that occur when these amino acids are linked

together, the different proteins may have

dif-ferent chemical properties and widely

differ-ent secondary and tertiary structures The

various amino acids joined in a peptide chain

are shown in Figure 3-1 The amino acids

are grouped on the basis of the chemical

nature of the side chains (Krull and Wall

1969) The side chains may be polar or

non-polar High levels of polar amino acid

resi-dues in a protein increase water solubility

The most polar side chains are those of the

basic and acidic amino acids These amino

acids are present at high levels in the soluble

albumins and globulins In contrast, the wheat

proteins, gliadin and glutenin, have low levels

of polar side chains and are quite insoluble in

water The acidic amino acids may also be

present in proteins in the form of their

amides, glutamine and asparagine This

increases the nitrogen content of the protein

Hydroxyl groups in the side chains may

become involved in ester linkages with

phos-phoric acid and phosphates Sulfur amino

acids may form disulfide cross-links between

neighboring peptide chains or between

dif-ferent parts of the same chain Proline andhydroxyproline impose significant structurallimitations on the geometry of the peptidechain

Proteins occur in animal as well as ble products in important quantities In thedeveloped countries, people obtain much oftheir protein from animal products In otherparts of the world, the major portion ofdietary protein is derived from plant prod-ucts Many plant proteins are deficient in one

vegeta-or mvegeta-ore of the essential amino acids Theprotein content of some selected foods islisted in Table 3-1

AMINO ACID COMPOSITION

Amino acids joined together by peptidebonds form the primary structure of proteins.The amino acid composition establishes thenature of secondary and tertiary structures.These, in turn, significantly influence thefunctional properties of food proteins andtheir behavior during processing Of the 20amino acids, only about half are essential forhuman nutrition The amounts of these essen-tial amino acids present in a protein and theiravailability determine the nutritional quality

of the protein In general, animal proteins are

of higher quality than plant proteins Plant

Proteins

CHAPTER 3

Figure 3-1 Component Amino Acids of Proteins

Joined by Peptide Bonds and Character of Side

Chains Source: From Northern Regional

Re-search Laboratory, U.S Department of ture.

Agricul-proteins can be upgraded nutritionally byjudicious blending or by genetic modificationthrough plant breeding The amino acid com-position of some selected animal and vegeta-

ble proteins is given in Table 3—2.

Egg protein is one of the best quality teins and is considered to have a biologicalvalue of 100 It is widely used as a standard,and protein efficiency ratio (PER) valuessometimes use egg white as a standard.Cereal proteins are generally deficient inlysine and threonine, as indicated in Table

pro-Table 3-1 Protein Content of Some Selected

3-3 Soybean is a good source of Iysine but

is deficient in methionine Cottonseed

pro-tein is deficient in lysine and peanut propro-tein

in methionine and lysine The protein of

potato although present in small quantity

(Table 3-1) is of excellent quality and is

equivalent to that of whole egg

Table 3-3 Limiting Essential Amino Acids of

Some Grain Proteins

First Second Limiting Limiting Grain Amino Acid Amino Acid

Wheat Lysine Threonine

Corn Lysine Tryptophan

Rice Lysine Threonine

Sorghum Lysine Threonine

Millet Lysine Threonine

PROTEIN CLASSIFICATION

Proteins are complex molecules, and fication has been based mostly on solubility indifferent solvents Increasingly, however, asmore knowledge about molecular composi-tion and structure is obtained, other criteriaare being used for classification Theseinclude behavior in the ultracentrifuge andelectrophoretic properties Proteins are di-vided into the following main groups: simple,conjugated, and derived proteins

classi-Simple Proteins

Simple proteins yield only amino acids onhydrolysis and include the following classes:

• Albumins Soluble in neutral, salt-free

water Usually these are proteins of tively low molecular weight Examples

rela-Table 3-2 Amino AcJd Content of Some Selected Foods (mg/g Total Nitrogen)

Milk

399 782 450 156 434 396 278 463 160 214 255 424 1151 144 514 342

Egg

393 551 436 210 152 358 260 320 428 381 152 370 601 796 207 260 478

Wheat

204 417 179 94 159 282 187 183 276 288 143 226 308 1866 245 621 281

Peas

267 425 470 57 70 287 171 254 294 595 143 255 685 1009 253 244 271

Com

230 783 167 120 97 305 239 225 303 262 170 471 392 1184 231 559 311

are egg albumin, lactalbumin, and serum

albumin in the whey proteins of milk,

leucosin of cereals, and legumelin in

legume seeds

• Globulins Soluble in neutral salt

solu-tions and almost insoluble in water

Examples are serum globulins and

(3-lac-toglobulin in milk, myosin and actin in

meat, and glycinin in soybeans

• Glutelins Soluble in very dilute acid or

base and insoluble in neutral solvents

These proteins occur in cereals, such as

glutenin in wheat and oryzenin in rice

• Prolamins Soluble in 50 to 90 percent

ethanol and insoluble in water These

proteins have large amounts of proline

and glutamic acid and occur in cereals

Examples are zein in corn, gliadin in

wheat, and hordein in barley

• Scleroproteins Insoluble in water and

neutral solvents and resistant to enzymic

hydrolysis These are fibrous proteins

serving structural and binding purposes

Collagen of muscle tissue is included in

this group, as is gelatin, which is derived

from it Other examples include elastin,

a component of tendons, and keratin, a

component of hair and hoofs

• Histories Basic proteins, as defined by

their high content of lysine and arginine

Soluble in water and precipitated by

ammonia

• Protamines Strongly basic proteins of

low molecular weight (4,000 to 8,000)

They are rich in arginine Examples are

clupein from herring and scombrin from

mackerel

Conjugated Proteins

Conjugated proteins contain an amino

acid part combined with a nonprotein

mate-rial such as a lipid, nucleic acid, or

carbohy-drate Some of the major conjugatedproteins are as follows:

• Phosphoproteins An important group

that includes many major food proteins.Phosphate groups are linked to thehydroxyl groups of serine and threonine.This group includes casein of milk andthe phosphoproteins of egg yolk

• Lipoproteins These are combinations of

lipids with protein and have excellentemulsifying capacity Lipoproteins occur

in milk and egg yolk

• Nucleoproteins These are combinations

of nucleic acids with protein Thesecompounds are found in cell nuclei

• Glycoproteins These are combinations

of carbohydrates with protein Usuallythe amount of carbohydrate is small, butsome glycoproteins have carbohydratecontents of 8 to 20 percent An example

of such a mucoprotein is ovomucin ofegg white

• Chromopmteins These are proteins with

a colored prosthetic group There aremany compounds of this type, includinghemoglobin and myoglobin, chlorophyll,and flavoproteins

Derived Proteins

These are compounds obtained by cal or enzymatic methods and are dividedinto primary and secondary derivatives, de-pending on the extent of change that hastaken place Primary derivatives are slightlymodified and are insoluble in water; rennet-coagulated casein is an example of a primaryderivative Secondary derivatives are moreextensively changed and include proteoses,peptones, and peptides The differencebetween these breakdown products is in sizeand solubility All are soluble in water and

chemi-not coagulated by heat, but proteoses can be

precipitated with saturated ammonium

sul-fate solution Peptides contain two or more

amino acid residues These breakdown

prod-ucts are formed during the processing of

many foods, for example, during ripening of

cheese

PROTEIN STRUCTURE

Proteins are macromolecules with different

levels of structural organization The primary

structure of proteins relates to the peptide

bonds between component amino acids and

also to the amino acid sequence in the

mole-cule Researchers have elucidated the amino

acid sequence in many proteins For

exam-ple, the amino acid composition and

se-quence for several milk proteins is now well

established (Swaisgood 1982)

Some proteolytic enzymes have quite

spe-cific actions; they attack only a limited

num-ber of bonds, involving only particular amino

acid residues in a particular sequence This

may lead to the accumulation of well-defined

peptides during some enzymic proteolytic

reactions in foods

The secondary structure of proteins

in-volves folding the primary structure

Hydro-gen bonds between amide nitroHydro-gen and

car-bonyl oxygen are the major stabilizing force

These bonds may be formed between

differ-ent areas of the same polypeptide chain or

between adjacent chains In aqueous media,

the hydrogen bonds may be less significant,

and van der Waals forces and hydrophobic

interaction between apolar side chains may

contribute to the stability of the secondary

structure The secondary structure may be

either the oc-helix or the sheet structure, as

shown in Figure 3-2 The helical structures

are stabilized by intramolecular hydrogen

bonds, the sheet structures by intermolecular

hydrogen bonds The requirements for mum stability of the helix structure wereestablished by Pauling et al (1951) Thehelix model involves a translation of 0.54 nmper turn along the central axis A completeturn is made for every 3.6 amino acid resi-dues Proteins do not necessarily have tooccur in a complete a-helix configuration;rather, only parts of the peptide chains may

maxi-be helical, with other areas of the chain in amore or less unordered configuration Pro-teins with a-helix structure may be eitherglobular or fibrous In the parallel sheetstructure, the polypeptide chains are almostfully extended and can form hydrogen bondsbetween adjacent chains Such structures aregenerally insoluble in aqueous solvents andare fibrous in nature

The tertiary structure of proteins involves apattern of folding of the chains into a com-pact unit that is stabilized by hydrogen bonds,van der Waals forces, disulfide bridges, andhydrophobic interactions The tertiary struc-ture results in the formation of a tightlypacked unit with most of the polar amino acidresidues located on the outside and hydrated.This leaves the internal part with most of theapolar side chains and virtually no hydration.Certain amino acids, such as proline, disruptthe a-helix, and this causes fold regions withrandom structure (Kinsella 1982) The nature

of the tertiary structure varies among proteins

as does the ratio of a-helix and random coil.Insulin is loosely folded, and its tertiary struc-ture is stabilized by disulfide bridges Lyso-zyme and glycinin have disulfide bridges butare compactly folded

Large molecules of molecular weightsabove about 50,000 may form quaternarystructures by association of subunits Thesestructures may be stabilized by hydrogenbonds, disulfide bridges, and hydrophobicinteractions The bond energies involved in

Figure 3-2 Secondary Structures of Proteins, (A) Alpha Helix, (B) Antiparallel Sheet

3rd turn

2nd turn

1st turn Rise per

residue

forming these structures are listed in Table

3-4

The term subunit denotes a protein chain

possessing an internal covalent and

noncova-lent structure that is capable of joining with

other similar subunits through noncovalent

forces or disulfide bonds to form an

oligo-meric macromolecule (Stanley and Yada

1992) Many food proteins are oligomeric

and consist of a number of subunits, usually

2 or 4, but occasionally as many as 24 A

list-ing of some oligomeric food proteins is

given in Table 3-5 The subunits of proteins

are held together by various types of bonds:

electrostatic bonds involving carboxyl,

amino, imidazole, and guanido groups;

hy-drogen bonds involving hydroxyl, amide,

and phenol groups; hydrophobic bonds

in-volving long-chain aliphatic residues or

aro-matic groups; and covalent disulfide bonds

involving cystine residues Hydrophobic

bonds are not true bonds but have been

described as interactions of nonpolar groups

These nonpolar groups or areas have a

ten-dency to orient themselves to the interior of

the protein molecule This tendency depends

on the relative number of nonpolar amino

Table 3-4 Bond Energies of the Bonds Involved

Van der Waals bond 1 -2

These refer to free energy required to break the

bonds: in the case of a hydrophobic bond, the free

energy required to unfold a nonpolar side chain from

the interior of the molecule into the aqueous medium.

acid residues and their location in the peptidechain Many food proteins, especially plantstorage proteins, are highly hydrophobic—somuch so that not all of the hydrophobic areascan be oriented toward the inside and have to

be located on the surface This is a possiblefactor in subunits association and in somecases may result in aggregation The hydro-phobicity values of some food proteins asreported by Stanley and Yada (1992) arelisted in Table 3-6

The well-defined secondary, tertiary, andquaternary structures are thought to arisedirectly from the primary structure Thismeans that a given combination of aminoacids will automatically assume the type ofstructure that is most stable and possiblegiven the considerations described by Paul-ing etal (1951)

Table 3-5 Oligomeric Food Proteins

Molecular Protein Weight (d) Subunits

Lactoglobulin 35,000 2 Hemoglobin 64,500 4 Avidin 68,300 4 Lipoxygenase 108,000 2 Tyrosinase 128,000 4 Lactate 140,000 4 dehydrogenase

7S soy protein 200,000 9

Invertase 210,000 4 Catalase 232,000 4 Collagen 300,000 3 11S soy protein 350,000 12 Legumin 360,000 6 Myosin 475,000 6

Source: Reprinted with permission from D.W Stanley

and R.Y Yada, Thermal Reactions in Food Protein

Sys-tems, Physical Chemistry of Foods, H.G Schwartzberg

and R.H Hartel, eds., p 676, 1992, by courtesy of cel Dekker, Inc.

Denaturation is a process that changes the

molecular structure without breaking any of

the peptide bonds of a protein The process is

peculiar to proteins and affects different

pro-teins to different degrees, depending on the

structure of a protein Denaturation can be

brought about by a variety of agents, of

which the most important are heat, pH, salts,

and surface effects Considering the

com-plexity of many food systems, it is not

sur-prising that denaturation is a complex

pro-cess that cannot easily be described in simple

terms Denaturation usually involves loss of

biological activity and significant changes in

some physical or functional properties such

as solubility The destruction of enzyme

activity by heat is an important operation in

food processing In most cases, denaturation

is nonreversible; however, there are some

Table 3-6 Hydrophobicity Values of Some Food

Proteins

Hydrophobicity Protein cal/residue

Source: Reprinted with permission from D.W Stanley

and R.Y Yada, Thermal Reactions in Food Protein

Sys-tems, Physical Chemistry of Foods, H.G Schwartzberg

and R.H Hartel, eds., p 677, 1992, by courtesy of

Mar-cel Dekker, Inc.

exceptions, such as the recovery of sometypes of enzyme activity after heating Heatdenaturation is sometimes desirable—forexample, the denaturation of whey proteinsfor the production of milk powder used inbaking The relationship among temperature,heating time, and the extent of whey proteindenaturation in skim milk is demonstrated inFigure 3-3 (Harland et al 1952)

The proteins of egg white are readily tured by heat and by surface forces when eggwhite is whipped to a foam Meat proteinsare denatured in the temperature range 57 to750C, which has a profound effect on tex-ture, water holding capacity, and shrinkage.Denaturation may sometimes result in theflocculation of globular proteins but mayalso lead to the formation of gels Foods may

dena-be denatured, and their proteins destabilized,during freezing and frozen storage Fish pro-teins are particularly susceptible to destabili-zation After freezing, fish may becometough and rubbery and lose moisture Thecaseinate micelles of milk, which are quitestable to heat, may be destabilized by freez-ing On frozen storage of milk, the stability

of the caseinate progressively decreases, andthis may lead to complete coagulation.Protein denaturation and coagulation areaspects of heat stability that can be related tothe amino acid composition and sequence ofthe protein Denaturation can be defined as a

major change in the native structure that does not involve alteration of the amino acid sequence The effect of heat usually involves

a change in the tertiary structure, leading to aless ordered arrangement of the polypeptidechains The temperature range in whichdenaturation and coagulation of most pro-teins take place is about 55 to 750C, as indi-cated in Table 3-7 There are some notableexceptions to this general pattern Casein andgelatin are examples of proteins that can be

boiled without apparent change in stability.

The exceptional stability of casein makes it

possible to boil, sterilize, and concentrate

milk, without coagulation The reasons for

this exceptional stability have been discussed

by Kirchmeier (1962) In the first place,restricted formation of disulfide bonds due tolow content of cystine and cysteine results inincreased stability The relationship betweencoagulation temperature as a measure of sta-

Figure 3-3 Time-Temperature Relationships for the Heat Denaturation of Whey Proteins in Skim

Milk Source: From H.A Harland, S.T Coulter, and R Jenness, The Effects of Various Steps in the Manufacture on the Extent of Serum Protein Denaturation in Nonfat Dry Milk Solids / Dairy ScL 35:

bility and sulfur amino acid content is shown

in Tables 3-7 and 3-8 Peptides, which are

low in these particular amino acids, are less

likely to become involved in the type of

sulf-hydryl agglomeration shown in Figure 3-4

Casein, with its extremely low content of

sulfur amino acids, exemplifies this

behav-ior The heat stability of casein is also

explained by the restraints against forming a

folded tertiary structure These restraints are

due to the relatively high content of proline

and hydroxyproline in the heat stable

pro-teins (Table 3-9) In a peptide chain free of

proline, the possibility of forming inter- and

intramolecular hydrogen bonds is better than

in a chain containing many proline residues

(Figure 3-5) These considerations show

how amino acid composition directly relates

to secondary and tertiary structure of

pro-teins; these structures are, in turn,

responsi-ble for some of the physical properties of the

protein and the food of which it is a part

NONENZYMIC BROWNING

The nonenzymic browning or Maillard

reaction is of great importance in food

man-ufacturing and its results can be either

desir-Table 3-7 Heat Coagulation Temperatures of

Some Albumins and Globulins and Casein

Coagulation

Egg albumin 56

Serum albumin (bovine) 67

Milk albumin (bovine) 72

The browning reaction can be defined as thesequence of events that begins with the reac-tion of the amino group of amino acids, pep-tides, or proteins with a glycosidic hydroxylgroup of sugars; the sequence terminates withthe formation of brown nitrogenous polymers

or melanoidins (Ellis 1959)

The reaction velocity and pattern are enced by the nature of the reacting aminoacid or protein and the carbohydrate Thismeans that each kind of food may show adifferent browning pattern Generally, Iysine

influ-is the most reactive amino acid because ofthe free £-amino group Since lysine is thelimiting essential amino acid in many foodproteins, its destruction can substantiallyreduce the nutritional value of the protein.Foods that are rich in reducing sugars arevery reactive, and this explains why lysine inmilk is destroyed more easily than in other

Table 3-8 Cysteine and Cystine Content of

Some Proteins (g Amino Acid/100 g Protein)

Cysteine Cystine Protein (%) (%)

Egg albumin 1.4 0.5 Serum albumin 0.3 5.7 (bovine)

Milk albumin 6.4 — p-Lactoglobulin 1.1 2.3 Fibrinogen 0.4 2.3 Casein — 0.3

Figure 3-4 Reactions Involved in Sulfhydryl

Polymerization of Proteins Source: From O.

Kirchmeier, The Physical-Chemical Causes of

the Heat Stability of Milk Proteins,

Milchwis-senschaft (German), Vol 17, pp 408-412, 1962.

foods (Figure 3-6) Other factors that

influ-ence the browning reaction are temperature,

pH, moisture level, oxygen, metals,

phos-phates, sulfur dioxide, and other inhibitors

The browning reaction involves a number

of steps An outline of the total pathway ofmelanoidin formation has been given byHodge (1953) and is shown in Figure 3-7.According to Hurst (1972), the followingfive steps are involved in the process:

1 The production of an Af-substitutedglycosylamine from an aldose orketose reacting with a primary aminogroup of an amino acid, peptide, orprotein

2 Rearrangement of the glycosylamine

by an Amadori rearrangement type ofreaction to yield an aldoseamine orketoseamine

3 A second rearrangement of the amine with a second mole of aldose toform a diketoseamine, or the reaction

ketose-Table 3-9 Amino Acid Composition of Serum Albumin, Casein, and Gelatin (g Amino Acid/100 g Protein)

10.9 16.5 12.8 5.9 4.0

Casein

1.9 3.1 6.8 9.2 5.6 5.3 4.4 0.3 1.8 5.3 5.7 13.5 7.6 24.5 8.9 3.3 3.8

Gelatin

27.5 11.0 2.6 3.3 1.7 4.2 2.2 0.0 0.9 2.2 0.3 16.4 14.1 6.7 11.4 4.5 8.8 0.8

of an aldoseamine with a second mole

of amino acid to yield a diamino sugar

4 Degradation of the amino sugars with

loss of one or more molecules of water

to give amino or nonamino

com-pounds

5 Condensation of the compounds

formed in Step 4 with each other or

with amino compounds to form brown

pigments and polymers

The formation of glycosylamines from the

reaction of amino groups and sugars is

reversible (Figure 3-8) and the equilibrium

is highly dependent on the moisture level.The mechanism as shown is thought toinvolve addition of the amine to the carbonylgroup of the open-chain form of the sugar,elimination of a molecule of water, and clo-sure of the ring The rate is high at low watercontent; this explains the ease of browning indried and concentrated foods

The Amadori rearrangement of the sylamines involves the presence of an acidcatalyst and leads to the formation of ketose-amine or 1-amino-1-deoxyketose according

glyco-Figure 3-5 Effect of Proline Residues on Possible Hydrogen Bond Formation in Peptide Chains (A)

Proline-free chain; (B) proline-containing chain; (C) hydrogen bond formation in proline-free and

pro-line-containing chains Source: From O Kirchmeier, The Physical-Chemical Causes of the Heat bility of Milk Proteins, Milchwissenschaft (German), Vol 17, pp 408-412, 1962.

Sta-A

B

C