Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

Подождите немного. Документ загружается.

dihydrocapsaicin, hence do not taste pungent. It is not

known whether they are intermediates in the biosyn-

thesis of capsaicin and dihydrocapsaicin.

0023 Continuous light has been reported to induce for-

mation of capsaicinoids during postharvest ripening

of nonpungent C. annuum, and cells isolated from

sweet pepper stem callus will synthesize capsaicin in

vitro under appropriate conditions. The nonpungent

mutant, at least in C. annuum, may therefore involve

some change in a regulatory or switch mechanism

affecting the pathway of capsaicin biosynthesis,

rather than a simple block in this pathway.

0024 Although many secondary metabolites of the Sola-

naceae (such as nicotine) are produced wholly or

partly in the roots, this is not true of the capsaici-

noids. Experiments in which tomatoes were grafted

on to hot pepper rootstocks, and vice versa, showed

that capsaicinoids are produced only in the fruits.

They usually appear about 2 weeks after flowering

and reach a plateau 4 weeks after flowering. Tem-

perature, particularly night temperature, seems to

affect both the formation and accumulation of cap-

saicinoids. Capsaicinoids are synthesized in the epi-

dermal cells of the placenta, in the inner compartment

of the endoplasmic reticulum. They are translocated

across the cell membrane, through the cell wall, and

accumulate in cavities beneath the cuticle covering

the placenta. This may cause the cuticle to crack and

the capsaicinoids to spread over the seeds and inner

wall of the fruit, leading to the widespread, but erro-

neous, belief that pungent peppers have pungent seeds

and pungent fruit walls.

0025 For a long time, pungency of Capsicum was assessed

organoleptically by the Scoville test, which was simple

and convenient but not accurate or reproducible. A

specified weight of Capsicum was combined with a

specified volume of alcohol, diluted with sugar solu-

tion to the threshold of taste, and the reciprocal of the

dilution was the measure of pungency in Scoville heat

units (150 000 Scoville units ¼1% capsaicinoids). The

major techniques now used for separating and quanti-

fying the various capsaicinoids are gas chromatog-

raphy, high-performance liquid chromatography, and

high-performance thin-layer chromatography. There

is still no simple nonorganoleptic test that can be

applied in the field. (See Chromatography: Thin-layer

Chromatography; High-performance Liquid Chro-

matography; Gas Chromatography.)

Pharmacodynamic Properties

0026 Capsaicinoids are responsible for many pharmacody-

namic as well as organoleptic properties of Capsicum.

Hot peppers stimulate saliva flow and may overcome

loss of appetite. They also stimulate gastric juices,

thereby aggravating stomach ulcers. They increase

peristalsis, thus having a laxative action (which has

benefited archeologists studying food remains in pre-

historic feces). Hot peppers increase perspiration,

which may partially explain their popularity in hot

climates. Chilli-containing foods reputedly reduce

risks of thromboembolism and significantly lower

both liver and serum triglycerides. Capsaicinoids

induce coughing and sneezing, inflame the skin, and

have an irritating effect on mucous membranes of the

eyes and nose. This was exploited in the past by some

Amerindians, who punished their children by holding

them over the smoke of a fire on to which dried

chillies had been thrown, and exploited more recently

by manufacturers of certain aerosols sold for use

against ‘muggers.’

0027The burning and pain associated with hot peppers

result from pungent capsaicinoids acting on sensory

neurons known as nociceptors. Repeated exposure

leads to desensitization. Experimenters who desensi-

tized their tongues to capsaicin found that their taste

thresholds for other pungent compounds, such as

ginger and mustard, also increased, but their ability

to perceive tactile stimuli or basic tastes, such as

sweet, salt, sour, or bitter, was not affected. Capsaicin

apparently acts via a receptive site in the nociceptor.

This site seems also to be involved in the perception of

temperatures which are dangerously high (perhaps

explaining why pungent foods are perceived as

‘hot’). Capsaicin kills the nociceptor, or destroys its

peripheral terminals. This has been exploited in the

topical use of capsaicin as an analgesic to treat condi-

tions such as shingles and rheumatoid arthritis.

Nutritional Value

0028Hot peppers are traditionally valued for adding inter-

est to bland, starchy foods and for masking off-

flavors in meat and other stored products. Both hot

and sweet peppers are also excellent sources of vita-

mins, particularly vitamins A and C. Carotene (pro-

vitamin A) is deficient in many tropical diets. Green

peppers have a b-carotene equivalent of 180 mg per

100 g; red peppers contain 4770 mg of carotenoids

per 100 g. (See Retinol: Physiology.)

0029Fresh peppers are perhaps even more important

as a source of vitamin C. The Hungarian chemist

Szent-Gyo

rgyi won the Nobel Prize for his isolation

of vitamin C, first from the adrenal gland, later from

paprika and lemon. Fresh paprika fruits contain up to

340 mg per 100 g, i.e. more vitamin C per unit weight

than citrus. Much of this is lost when peppers are

dried (paprika powder contains only 30–60 mg per

100 g), but about two-thirds is retained in canned

Capsicum fruit. (See Ascorbic Acid: Physiology.)

PEPPERS AND CHILLIES 4465

Harvesting, Handling, and Processing

0030 Green bell peppers and fresh green chillies are picked

when the fruit has reached its full size but the seeds

are not yet fully mature. This is usually about 1 month

after flowering, or 70 days after planting. Fruits are

picked by hand at 7–14-day intervals over a harvest-

ing period of about 3 months. They are usually picked

with calyx and fruit stalk attached, since bacteria and

fungi may infect the scar left by removal of the calyx

and cause postharvest rots. Harvested fruits can be

stored for up to 14 days, preferably under cool humid

conditions (7–10



C and 95% relative humidity).

Ventilation may be advisable to remove accumulating

ethylene, which would accelerate fruit ripening. Ripe

peppers are harvested 2–3 weeks later than green

peppers but are handled in the same way. Since the

grower has to wait longer before harvest, ripe fruits

usually command higher prices than green peppers.

0031 Sweet peppers may also be marketed as dehydrated

flakes. Stalks, calyces, placentas, and seeds are re-

moved mechanically and the pericarp is diced, sprayed

with sulfite-bisulfite solution and dried by hot air.

Pimentos for canning have the tough outer cuticle

plus the underlying epidermis removed by roasting

or by treatment with lye, then are cored to remove

placenta and seeds and canned either whole or diced.

0032 The quality of Capsicum powders depends on their

color and flavor, which are influenced by how the

fruits are harvested and handled. Paprika is always

made from red-fruited cultivars. The genotype influ-

ences both the quantity of pigment present and how

well it is retained after harvest. Traditionally, fruits

are harvested when fully ripe and dried in the open air

for 3 weeks or more. The red pigments increase for

the first 25 days of after-ripening, then remain con-

stant until about 40 days after harvest, when they

start to break down. Mechanical harvesting requires

that 80–90% of the fruits mature simultaneously. In

Hungary, plants are cut by machine at ground level

and laid in the field to dry and after-ripen. Artificial

drying in hot air is also widely used, but if tempera-

tures exceed 80



C, fruit color starts to deteriorate.

Stalks, calyces, placentas, and seeds are separated

from the dried fruits because they would dilute the

color, but at least 5% of cleaned seeds are then re-

added to facilitate grinding (the precise percentage

depends on the intended grade of the final product).

The seeds contain fat, and carotenoids are fat-soluble,

so adding ground seeds helps to disperse the color

evenly, though the fat may become rancid during

storage. (See Drying: Theory of Air-drying.)

0033 Pungent fruits are handled in much the same way

as paprika. Grinding is done mechanically, formerly

in the importing countries, but costs are high and

grinding of chillies is particularly unpopular because

of the cough- and sneeze-provoking effects of the

dust, so grinding is increasingly left to the producing

countries. The ground powder is fumigated to control

microorganisms and stored under dry, cool, dark

conditions to minimize breakdown of the red pig-

ments. (See Fumigants.)

0034Oleoresins are extracted from dried and ground

fruits. If the seeds are ground with the fruits, fat

from the seeds dilutes both color and pungency, and

hence quality, of the oleoresin. However, if the seeds

are removed, the cost of production is increased and

the yield of oleoresin is decreased. Acetone and ethyl-

ene dichloride are frequently used solvents. The ex-

tract is distilled to remove the solvent, leaving the

concentrated oleoresin.

Global Distribution, Products, and

Commercial Importance

0035Capsicum is adapted to tropical or subtropical cli-

mates, but can be grown in temperate climates in

glasshouses or under protected cultivation. The Food

and Agriculture Organization (FAO) production

yearbooks do not distinguish between C. annuum

and other species, or between pungent and nonpun-

gent peppers. Over the last two decades, total world

production of chillies and green peppers (presumably

including also ripe fruits of sweet pepper and pi-

mento) increased from about 7 million to nearly

17 million tonnes. Over half of this is produced in

Asia. China alone produces over 40% of the world

total.

0036Data in the FAO trade yearbooks combine, under

the heading pimento, information for capsicum, cay-

enne, chillies, paprika, and red pepper. The confusion

surrounding the name chilli has already been dis-

cussed. Capsicum is a similarly confusing term,

which may be used for the nonpungent vegetable

peppers, but may also be used for some mildly pun-

gent dried fruits, e.g. Ancho, that are larger than most

fruits known as chillies. Applied to the oleoresin,

capsicum signifies the most pungent, rather than the

least pungent, of the commercial grades. Cayenne is

usually made from small-fruited peppers, grown in

various parts of the world, and is usually very pun-

gent. However, in the USA cayenne is made from

locally grown, large-fruited cultivars and is less pun-

gent. Paprika comes mainly from Hungary, Spain,

and the USA and is usually, though not always, non-

pungent. Ground red pepper and crushed red pepper

have similar names, but ground red pepper is usually

milder than crushed red pepper. Red pepper may also

refer to ripe fruits of some sweet peppers.

4466 PEPPERS AND CHILLIES

0037 The FAO trade yearbook shows that, in 1997,

North America and Europe were net importers of

over 100 000 t of ‘pimento.’ Asia and Africa were

net exporters. China contributed about 40% of

Asia’s exports, thereby earning over US$72 million,

while India contributed a further 30% and earned

approximately US$40 million.

0038 Oleoresins are valued by some commercial food

processors for their greater hygiene (less contam-

ination by microorganisms, insects, or rodent drop-

pings) and more precise standardization of pungency.

They are used mainly in the USA and the UK, but

elsewhere do not seem to be replacing pepper

powders. Three different oleoresins are produced

commercially: oleoresin paprika, oleoresin red

pepper (or oleoresin chilli), and oleoresin capsicum.

Oleoresin capsicum is used mainly in the pharmaceut-

ical industry but also in the production of some foods

and beverages, such as ginger ale. One kilogram of

oleoresin capsicum replaces approximately 20 kg of

cayenne. Oleoresin red pepper is made from less pun-

gent fruits, such as the long, thin peppers much grown

in Asia. One kilogram of oleoresin red pepper re-

places about 10 kg of red pepper. Oleoresin paprika

is used for coloring salad dressings and oleomargar-

ines, partly because it retains its color longer than

annatto. One kilogram of oleoresin paprika replaces

12–15 kg of paprika powder.

0039 Probably less than 10% of total global production

of hot peppers is for export. The remainder is grown

for local use. Mexico leads the world in chilli con-

sumption at 15 g per head per day. Sweet peppers are

eaten less regularly than hot peppers but are con-

sumed in larger quantities (up to 20 g per meal). At

these levels of intake, Capsicum adds considerably

more than spice to the diet of some of the world’s

poorest people.

See also: Ascorbic Acid: Physiology; Chlorophyl;

Chromatography: Thin-layer Chromatography; High-

performance Liquid Chromatography; Gas

Chromatography; Colorants (Colourants): Properties

and Determination of Natural Pigments; Drying: Theory of

Air-drying; Fumigants; Retinol: Physiology; Ripening of

Fruit; Sensory Evaluation: Aroma

Further Reading

Andrews J (1984) Peppers: The Domesticated Capsicums.

Austin, Texas: University of Texas Press.

Camara B, Hugueney P, Bouvier F, Kuntz M and Mone

ger R

(1995) Biochemistry and molecular biology of chromo-

plast development. International Review of Cytology

163: 175–247.

Caterina MJ, Schumacher MA, Tominaga M, Rosen TA,

Levine JD and Julius D (1997) The capsaicin receptor: a

heat-activated ion channel in the pain pathway. Nature

389: 816–824.

Govindarajan VS (1985) Capsicum – production, technol-

ogy, chemistry and quality. Part I: history, botany, culti-

vation and primary processing. CRC Critical Reviews

in Food Science and Nutrition 22: 109–176.

Govindarajan VS (1986) Capsicum – production, technol-

ogy, chemistry and quality. Part II: processed products,

standards, world production and trade. CRC Critical

Reviews in Food Science and Nutrition 23: 207–288.

Govindarajan VS (1986) Capsicum – production, technol-

ogy, chemistry and quality. Part III: chemistry of the

color, aroma and pungency stimuli. CRC Critical

Reviews in Food Science and Nutrition 24: 245–355.

Govindarajan VS and Sathyanarayana MN (1991) Cap-

sicum – production, technology, chemistry and quality.

Part V: impact on physiology, pharmacology, nutrition

and metabolism; structure, pungency, pain and desensi-

tization. CRC Critical Reviews in Food Science and

Nutrition 29: 435–474.

Govindarajan VS, Rajalakshmi D and Chand N (1987)

Capsicum – production, technology, chemistry and

quality. Part IV. Evaluation of quality. CRC Critical

Reviews in Food Science and Nutrition 25: 185–282.

Kobata K, Todo T, Yazawa S, Iwai K and Watanabe T

(1998) Novel capsaicinoid-like substances, capsiate and

dihydrocapsiate, from the fruits of a non-pungent culti-

var, CH-19 Sweet, of pepper (Capsicum annuum L.).

Journal of Agricultural and Food Chemistry 46:

1695–1697.

Purseglove JW, Brown EG, Green CL and Robbins SRJ

(1981) Spices, vol. 1. London: Longman.

Somos A (1984) The Paprika. Budapest: Akade

miai Kiado

Suzuki T and Iwai K (1984) Constituents of red pepper

species: chemistry, biochemistry, pharmacology and

food science of the pungent principle of Capsicum

species. In: Brossi A (ed) The Alkaloids, vol. 23, pp.

227–299. Orlando, Florida: Academic Press.

Peptic Ulcer See Colon: Structure and Function; Diseases and Disorders; Cancer of the Colon

PEPPERS AND CHILLIES 4467

PEPTIDES

D Gonza

lez de Llano, Centro Nacional de

Biotecnologia, Cantoblanco, Madrid, Spain

C Polo Sa

nchez, Instituto de Fermentaciones

Industriales (CSIC), Madrid, Spain

Introduction

0001 Peptides consist of chains of amino acids linked to

each other by amide bonds, also known as peptide

bonds. They form a heterogeneous group of com-

pounds, because of the large number of different

naturally occurring amino acids and the high poten-

tial variability in the order and number (chain length)

of residues making up the peptide.

0002 The number of amino acids that a peptide contains

is indicated by a prefix, e.g., di-, tri-, tetrapeptide; the

term ‘oligopeptides’ or ‘low-molecular-weight pep-

tides’ refers to peptides with 10 or fewer residues,

while ‘polypeptides’ is used for peptides with higher

molecular weights. Although the transition point

from polypeptide to protein is not well defined, pro-

teins are normally considered to have at least 100

residues (mol wt > 10 000). By convention, peptides

are referred to by the first three letters of the amino

acids making up the chain or by one-letter abbrevi-

ations. Also by convention, the amino acid with the

free amino group is referred to as the N-terminus

and is represented on the left, while the amino acid

with the free carboxyl group is referred to as the

C-terminus and is represented on the right. (See

Amino Acids: Properties and Occurrence.)

0003 The N-terminal amino group and the C-terminal

carboxyl group on peptides react chemically in the

same way as the a-amino and a-carboxyl groups on

free amino acids, and they can be used for detection

and quantification purposes.

0004 The acid–base behavior of peptides is dependent

upon the free a-amino group on the N-terminal

residue, the free a-carboxyl group on the C-terminal

residue, and the ionizable R groups located at inter-

mediate positions.

0005 In the presence of carbonyl compounds, certain

peptides take part in the Maillard reaction, leading

to the formation of melanoid pigments that con-

tribute to the development of undesirable coloration

in foodstuffs. Conversely, there are also lysine-

containing peptides that retard browning reactions

with glucose, and these may be appropriate for forti-

fying the lysine content of sugar-containing foods to

be cooked. (See Browning: Nonenzymatic.)

0006Peptides also play a very important role in deter-

mining the rheological properties of foods. Hydro-

lysis of proteins to peptides during processing

(fermentation, ripening, and cooking) of foods may

modify food texture.

Peptides in Foods

0007Peptides are naturally present in foodstuffs. Most

simple peptides are the result of partial hydrolysis of

protein polypeptide chains. On the other hand, non-

protein peptides have also been recorded in foods.

Such peptides usually differ in structure from the

peptides derived from proteins, and these structural

variations may protect them from the action of pepti-

dases.

0008The tripeptide glutathione, present in the cells of all

higher animals, is an example of a nonprotein pep-

tide. It contains a glutamic acid residue linked by an

unusual peptide bond involving its g-carboxyl group.

Glutathione plays a role in the active transport of

amino acids, acts as an antioxidant for lipids and as

an activator for certain enzymes, and is a coenzyme

of glyoxalase. Other nonprotein peptides include

the dipeptides carnosine, anserine, and balenine,

which are present in vertebrate muscle and contain a

b-amino acid (b-alanine) bound to l-histidine or

1-methyl-l-histidine. These histidine dipeptides are

present in differing proportions in the muscles of

different species and therefore are a mean of deter-

mining the source of the meats used in meat or of

identifying marine fish species.

0009Peptides may also be present in foods because they

have been used as additives (e.g., sweeteners, flavor

enhancers, or bulking agents for light beverages) to

improve food quality. Some peptides produced by the

enzymatic hydrolysis of proteins possess better func-

tional properties than the parent proteins and conse-

quently are used by the food industry for a variety of

purposes.

0010Peptides have lower molecular weight and less sec-

ondary structures, as well as higher number of ioniz-

able groups and exposure of hydrophobic groups

than native proteins. These facts imply that solubility,

surface activity, foaming, and emulsifying properties

may be different from that of the intact protein.

0011Enzymatic hydrolysis has been reported to enhance

the emulsifying capacity of different proteins. Pep-

tides with an inhibitory effect on autoxidation of

linolenic acid and hence potentially useful as antioxi-

dants have been found in fermented foods and in

protein hydrolysates. Moderately large peptides have

4468 PEPTIDES

pronounced bulking properties and large peptides act

as foam stabilizers; because of these interesting func-

tional properties, enzymatic hydrolysates of soya

bean proteins have been used in beverages. Hydro-

lyzed hydrophobic proteins from gluten (zeins, glia-

dins) have been prepared with a view to increase

solubility and diversify their functional properties.

0012 Analysis of the peptides of enzymatic hydrolysates

of trypsin can be used to confirm the presence of

nonmeat proteins such as soyabean proteins in meat

products. Tryptic peptides of the caseins from milks

of different species are distinct and may be used to

detect blends of milks. The presence of the casein

glycomacropeptide is also useful in detecting adulter-

ation of powdered milks with rennet whey.

Organoleptic Properties

0013 Peptides are tasteless or bitter, with the exception of

certain dipeptides of glutamic acid and aspartic

acid, which are sweet. Others, like the lower alkane

members in the series of methyl esters of l-aspartyl-a-

amino-cycloalkane carboxylic acid and a series of l-

aspartyl-d-alanyl tripeptides, are sweet but turn bitter

or tasteless when the size of the C-terminal amino

acid ring increases. There is, thus, an important rela-

tionship between size and flavor in these peptides.

Peptides belonging to l-aspartyl d-alanyl amides are

strong sweeteners and may be used as good sugar

substitutes. (See Sensory Evaluation: Practical Con-

siderations.)

0014 Foods may also contain peptides with a flavor

similar to that of monosodium glutamate, termed

umami taste or relish, which are able to mask the

bitter taste produced in foods by bitter peptides or

other bitter substances. This group includes hydro-

philic peptides, in particular acid peptides that

balance flavor in foods.

0015 Warmed-over flavor in cooked–stored–recooked

meat has also been related to the higher hydrophobic

peptide content in such meats as compared to cooked

meats. However, low-molecular-weight peptides play

an important role in the flavor formation and inten-

sity of meat, beef broth, and dry cured ham.

0016 Bitter peptides form as a result of proteolytic reac-

tions that take place in foods. These bitter peptides

have been identified in soyabean and casein hydro-

lysates as well as in foods, like cheese, that undergo

fermentation of ripening processes with marked pro-

teolytic activity. The bitter taste has been related to

the hydrophobic amino acid content and to chain

length. An overly high proportion of hydrophobic

amino acids gives rise to bitterness; however, above

a given molecular weight bitter flavors are no longer

perceptible, even though hydrophobic amino acids

are present. Proteins do not have a bitter taste even

when they contain hydrophobic amino acids, yet

hydrolysis of such proteins may indeed yield bitter

peptides. Bitter flavors in di- and tripeptides have

been observed to increase when the hydrophobic

amino acid is located on the C-terminal residue and

the basic amino acid is on the N-terminal residue.

Enzymatic activity is extremely important in the

development of bitter flavors in foods. Trypsin does

not degrade bitter peptides, although peptidases

may break down bitter peptides to nonbitter lower-

molecular-weight peptides and amino acids.

0017Bitterness is the most frequent flavor defect

limiting cheese acceptability. It has been attributed

to the formation and accumulation of bitter peptides

derived from casein. During cheese manufacture

rennet or rennet substitutes and microbial proteases

are the principal agents hydrolyzing the caseins and

are thus thought to be responsible for the occasional

appearance of bitter taste. The use of rennet sub-

stitutes may give rise to the appearance of bitter

peptides; for instance, commercial chicken pepsin

produces an objectionable level of bitterness in cheese.

On the other hand, cheeses made from goats’ or ewes’

milk are less susceptible to bitter flavor defects than

cheeses made from cows’ milk.

0018Aged cheeses, in particular hard cheeses, require

long ripening periods in which to acquire the desired

organoleptic characteristics. Proteases have therefore

been used to accelerate the ripening process and lower

costs. However, the proteases give rise to bitter pep-

tides that limit cheese quality. In recent years a con-

siderable amount of work has been carried out on

peptidases that break down the bitter peptides de-

rived from the caseins to smaller, nonbitter peptides.

The combined use of proteases and peptidases may be

an effective means of shortening ripening times, while

preventing the development of bitter flavors. (See

Cheeses: Chemistry and Microbiology ofMaturation.)

0019Bitter peptides that contribute to bread flavor form

during the fermentation and baking of bread dough

as a result of the action proteolytic enzymes on the

protein fraction in the flour (gliadins and glutelins).

Analytical Procedures

Spectrophotometric Methods

0020The N-terminal a-amino groups on peptides react

quantitatively with reagents such as ninhydrin to

form colored derivatives or with o-phthalaldehyde

and fluorescamine to form fluorescent derivatives.

These reactions are useful in making quantitative

determinations of peptides in foodstuffs and for de-

tection during chromatographic analysis. Due to the

PEPTIDES 4469

peptide bond, peptides also take part in reactions

which free amino acids do not undergo, like the classic

biuret reaction. This reaction consists of the forma-

tion, in an alkaline medium, of a colored complex with

a transition metal (e.g., copper or nickel) that can be

measured quantitatively by spectrophotometry. This

reaction is utilized in quantifying peptides. (See Spec-

troscopy: Visible Spectroscopy and Colorimetry.)

0021 The peptide content can also he determined by

assaying the aromatic amino acids tyrosine and tryp-

tophan, which is readily achieved by measuring

absorbance at 280 nm or by measuring the color re-

actions of these amino acids with certain reagents like

Folin-Ciocalteu’s reagent. Another analytical method

for determining the high-molecular-weight peptide

content is by precipitating the peptides with acid

dyes (amino black, orange G) and measuring the

excess dye remaining in solution. These methods are

useful in determining the total peptide contents of

foodstuffs, but their drawback is that they provide

no information on how many or which peptides are

present. For this, other techniques such as chroma-

tography or electrophoresis are needed.

Chromatographic Methods

0022 Paper chromatography and thin-layer chromatog-

raphy have conventionally been used to separate pep-

tides, particularly for the fractions isolated by gel

permeation chromatography (GPC) and ion exchange

chromatography (IEC), but they are lengthy, time-

consuming procedures with low resolving power

and low reproducibility.

0023 Gel filtration chromatography on open Sephadex

columns with different pore sizes (G-10, G-25, and G-

50) using water or saline or acid buffers as solvents is

used extensively in the fractionation of peptides. De-

tection is performed by measuring absorbance of the

column eluate with an ultraviolet detector at 280 nm,

the absorbance wavelength for tyrosine and trypto-

phan, or at 214 nm, the absorbance wavelength for

most amino acids and the peptide bond. Peptides elute

in fractions according to their molecular weight. Small

peptides must normally be separated from the amino

acids; gel filtration on columns prepared

by hydroxypropylation (Sephadex LH-20), ion

exchange on diethylaminoethyl (DEAE)-cellulose or

DEAE-Sephadex with Cu

2þ

complexes, or ligand ex-

change columns with a stationary phase modified with

2þ

are used for this purpose. Ion exchange chroma-

tography on DEAE-cellulose or on Aminex A-5 is

normally used for the separation of high-molecular-

weight peptides, employing saline buffers as eluent.

Detection is carried out at 280 nm or in the visible

region after derivatization with ninhydrin.

0024These methods are now most often used as pre-

paratory techniques for subsequent high-performance

liquid chromatographic (HPLC) or electrophoretic

analysis of the peptide fractions. (See Chromatog-

raphy: Principles.)

High-performance Liquid Chromatography

0025Because of its versatility, short analysis times, high

resolution, and effective separations, and because it

is well suited to automation procedures, HPLC is the

most widely used method of peptide analysis. Practic-

ally all known mechanisms have been employed in

the chromatographic separation of peptides, e.g.,

separation based on molecule size (GPC), on charge

(IEC), on hydrophobicity (reversed-phase and inter-

action chromatography), and even on combinations

thereof; of these, however, reversed-phase chroma-

tography is most commonly used to separate mixtures

of peptides from foodstuffs (Figure 1). It affords the

possibility of changing the stationary phase, the pH,

the ionic strength of the aqueous buffer, or the type of

organic modifier, of using different gradient shapes,

and of working at a variety of temperatures, making

this method suitable for analyzing highly different

peptides. Moreover, the option of using microcol-

umns makes it possible to detect quantities on the

order of picomols and has been an important step

toward HPLC–mass spectrometry coupling. (See

Chromatography: High-performance Liquid Chro-

matography; Combined Chromatography and Mass

Spectrometry.)

0026Reversed-phase chromatography Reversed-phase

columns with pore sizes of 6–10 nm are highly appro-

priate for separating small peptides. Columns with

pore sizes of 30–50 nm give the best results when

separating large peptides (mol wt 4000), because the

molecules are able to pass freely through the pores,

thus permitting greater access to the alkyl chains and

increasing column efficiency and load capacity.

0027The mobile phase is commonly a mixture of water

and an organic solvent, normally acetonitrile, metha-

nol, or 2-propanol, although other organic solvents

such as methoxyethanol, ethanol, butanol, or tetra-

hydrofuran are also used. Gradient elution is nor-

mally required for reversed-phase HPLC of peptides.

0028Because of their high polarity, peptides do not

interact sufficiently with the hydrophobic chains of

the stationary phase in reversed-phase chromatog-

raphy. On the other hand, they do interact with any

free silanol groups in these phases. Salts or strong

acids must be added to the stationary phase to block

the silanol groups. In order to reduce the polar nature

of the peptides, chromatography should be carried

4470 PEPTIDES

out at a pH of less than 3, so that the carboxyl groups

on the acids amino acids (aspartic acid and glutamic

acid) are not in their dissociated form. However, at

this pH the basic amino acids histidine, lysine, and

arginine are charged and must be blocked by forming

ion pairs. The selectivity of the chromatographic

system can be modified depending on the pH and on

the type of ion-pairing reagent used. Trifluoroacetic

acid has been widely employed as the ion-pair reagent

for peptides.

0029 Ion exchange chromatography IEC has also been

used in peptide separations. Two types of ion exchan-

gers are utilized: cationic exchangers to separate

neutral and basic peptides, and anionic exchangers

to separate neutral and acid peptides. Peptide separ-

ations have been carried out on polymeric cationic

exchange resins (polystyrene/divinylbenzene copoly-

mers), and indeed automatic peptide analysers, simi-

lar to those used in amino acid analysis, have been

designed, with postcolumn detection using ninhydrin.

0030Size exclusion chromatography (SEC) High-

performance SEC has most commonly been applied

to protein separations, though on occasion it has also

been used to separate peptides from foodstuffs. The

stationary phase may be inorganic, such as silica or

alumina. The disadvantage of inorganic materials is

that their active surfaces adsorb charged molecules

and hence they must first be deactivated, for instance

using organosilanes. Organic materials such as

glycerol methacrylate, sulfonated styrene divinyl-

benzene, and polyesters with surface hydroxyl groups

have also been used.

0031The different separation mechanisms referred to

above (reversed-phase HPLC with small or large

pore sizes, IEC, and SEC) are complementary, and

the best separations are sometimes achieved by com-

bining the various methods.

0032Detection Peptides are detected in amounts ranging

from 100 to1000 ng by absorbanceat between200 and

230 nm. Detection of peptides containing aromatic

Absorbance at 220 nm

0.4

0.3

0.2

0.1

0 20406080100

(a)

Absorbance at 220 nm

0.4

0.3

0.2

0.1

0 20406080100

(c)

Retention time (min)

(b)

Absorbance at 220 nm

0 20 40 60 80 100

(d)

Retention time (min)

Absorbance at 220 nm

0.4

0.3

0.2

0.1

0.4

0.3

0.2

0.1

0 20406080100

Trp

Tyr

Trp

Tyr

Trp

Tyr

Phe

Trp

fig0001 Figure 1 Chromatogram of the peptides of the water-soluble fraction from commercial (a) Vidiago, (b) Cabrales, (c) Beyos, and

(d) Peral cheeses. Column: Ultraspher ODS. Solvent: TFA/acetonitrile/water. Gradient elution. Detection at 220 nm. From Gonza

lez de

Llano D, Polo C and Ramos M (1995) Study of proteolysis in artisanal cheeses: high performance liquid chromatography of peptides.

Journal of Dairy Science 78(5): 1018–1024 with permission.

PEPTIDES 4471

amino acids (phenylalanine, tyrosine, and trypto-

phan) may be performed at 254 nm; tyrosine- or tryp-

tophan-containing peptides can also be detected at

280 nm. Rapid-scanning ultraviolet/visible detectors

based on diode array technology have proved an

extremely useful aid in identifying peptides that con-

tain aromatic residues. Wavelengths of the spectrum

maxima, convexity interval, and wavelength of the

second derivative spectrum maxima, all over in the

190–340 nm range, allow identification of the aro-

matic amino acids that form the peptides, as well as

identification of HPLC coeluted compounds such as

the cinnamic derivatives detected in the peptide frac-

tions of wines.

0033 Since numerous solvents and even other sample

components may absorb light in the range 200–

230 nm, formation of derivatives detectable at higher

wavelengths that are more specific for given chromo-

phores is commonly employed. To this end, phenyl

isothiocyanate and dansyl chloride are frequently

used inpeptide analysis as derivatizing agents.Peptides

may also be detected using a fluorescence detector to

detect either the natural fluorescence of certain amino

acids in the peptide sequence or the fluorescence of

derivatives formed artificially using such reagents as

fluorescamine and o-phthalaldehyde. Other reagents

used lately for derivatization of peptides are naphtha-

lene-2,3-dicarboxaldehyde, 3-(4-carboxy-benzoyl)

2-quinolinecarboxaldehyde, and 6-aminoquinoyl-

N-hydroxysuccimidylcarbamate.

0034 Following the significant advances made in liquid

chromatography/mass spectroscopy coupling in

recent years, mass spectrometry has turned into a

detection system that holds great promise for peptide

analysis, since it is capable of furnishing structural

information and quantitative data that are otherwise

difficult to obtain.

0035 The greatest advance in the analysis of peptides has

been the coupling with spectrometric techniques,

both for identification and for characterization.

Their high accuracy, sensitivity, and, in some in-

stances, tolerance to solvents make mass spectrom-

eters ideal detectors for analyses of HPLC-separated

peptides. Presently, electrospray ionization and

matrix-assisted laser desorption/ionization time-

of-flight (MALDI-TOF) mass spectrometry – soft ion-

ization methods – provide the suitable means to

determine accurate mass on peptides and proteins

with high sensitivity (subpicomol range). Further-

more, both methods are suitable for the creation of

peptide ions for analysis using tandem mass spec-

trometry and collision-induced dissociation methods.

Mass spectrometry in conjunction with database

searching plays an increasingly important role in the

characterization of food peptides.

Electrophoretic Techniques

0036Despite the important advantages afforded by HPLC

in the analysis of hydrophobic peptides, electrophor-

esis can be useful, because insoluble peptides can be

solubilized with detergents. Although a number of

different supports have been employed, polyacryla-

mide gel electrophoresis (PAGE) and sodium dodecyl

sulfate–PAGE (SDS-PAGE) have been widely used in

the study of peptide formation from proteins during

food processing. Another developed method of pep-

tide analysis that may be used either an alternative or

a complement to HPLC is capillary electrophoresis.

This technique has extremely high resolving power

and allows the separation of peptides differing in only

a single amino acid; it is simple, fast, and appropriate

for quantitative analysis, and only small amounts

of sample are required. Peptide separation by CE is

now almost a routine technique in food analysis.

Moreover the application of two analytical tech-

niques (HPLC-CE, SEC-CE) and coupling mass spec-

trometry to HPLC have acquired great importance

for characterization of complex peptide samples.

(See Electrophoresis: General Principles.)

Determination of Amino Acid Composition and

Sequence in Peptides

0037The amino acid composition of peptides is generally

assayed by carrying out acid hydrolysis with 6 mol l

1

hydrochloric acid, followed by determination of the

individual amino acids by HPLC. Various types of

HPLC have been employed to separate the amino

acids, but reversed-phase chromatography on C

columns is most commonly used. Detection of the

amino acids normally involves derivatization, since

maximum absorbance of amino acids is located in a

region of the spectrum (214 nm) in which many other

compounds also absorb. The most frequently used

derivatizing agents are dansyl chloride, phenyl iso-

thiocyanate and o-phthalaldehyde.

0038Peptide sequencing is normally performed by deg-

radation of the N-terminal amino acid using phenyl

isothiocyanate, Edman’s reagent. The terminal amino

groups react with the isothiocyanate, forming a phe-

nylthiocarbamylic derivative. When treated with acid

in an organic solvent, cyclization takes place and

phenylthiohydantoin amino acid is formed; this can

be separated from the rest of the chain, which remains

intact. The process can be repeated.

0039The usefulness of Edman degradation in the

sequencing of low-molecular-weight peptides is

limited, because of the low repetitive yield in the

successive sequencing cycles. For such peptides the

best results are obtained by coupling sequential

Edman degradation with detection by means of

4472 PEPTIDES

dansylation or by the double coupling method using

4-N,N-dimethylaminoazobenzene-4

-isothiocyanate

and phenyl isothiocyanate.

0040 Mass spectrometry has also become a powerful

tool for the sequencing of small amounts of peptides

and proteins. Tandem mass spectrometry and ion

source collision-induced dissociation produce infor-

mation specific to the amino acid sequence and the

specific covalently modified amino acid. A new

method, referred to as protein ladder sequencing,

for the N-terminal sequencing of peptides utilizes

multiple steps of partial Edman degradation chemis-

try prior to the analysis of the reaction mixture by

MALDI-TOF mass spectrometry.

See also: Amino Acids: Properties and Occurrence;

Determination; Chromatography: Principles; Thin-layer

Chromatography; High-performance Liquid

Chromatography