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

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are formed. A very small fraction of the 1,2- or 2,3-

diacylglycerols is expected to undergo acyl migration

to the 1,3-diacylglycerol, which is probably hydro-

lyzed by lipases. Polyunsaturated fatty acids are poor

substrates for pancreatic lipase, but carboxyl ester

hydrolase hydrolyzes them effectively. When the fatty

acids and 2-monoacylglycerol are absorbed into the

enterocyte, the 2-monoacylglycerol is reacylated by

monoacylglycerol transferaseto 1,2-diacyl-sn-glycerol

and 2,3-diacyl-sn-glycerol. The fatty acyl-CoAs are

similar for acylation of the position 1 or the position

3. The activated fatty acids used in the acylation come

from newly synthesized fatty acids in the cell and the

fatty acids absorbed from lumen after bile phospho-

lipid and fat digestion. The formed 1,2-diacyl-sn-

glycerol and 2,3-diacyl-sn-glycerol are acylated by

diacylgycerol acyl transferase to triacylglycerols.

Since the acyl migration is expected be slow at



C, the synthesized triacylglycerols retain the ori-

ginal fatty acid at the sn-2 position. The reacylation

of absorbed 2-monoacylglycerols is the predominant

pathway during active fat absorption. When the

absorption 2-monoacylglycerols from the gut is

minimal, the acylation of free glycerol through the

3-glycerophosphate by activated fatty acids proceeds

to form 1,2 diacyl-sn-glycerol phosphate. The unsat-

urated fatty acids acylate mainly the sn-2 position

and saturate the sn-1 position. Triacylglycerol is

formed through dephosphorylation and acylation at

the sn-3 position. Triacylglycerols formed by the

3-glycerophosphate pathway do not maintain the ori-

ginal fatty acid of the diet at the sn-2 position. How-

ever, during fat absorption, the 2-monoacylglycerol

pathway dominates.

0027 The synthesized mucosal triacylglycerols are pack-

aged to chylomicrons and intestinal very-low-density

lipoproteins, and secreted into the lymph. They are

hydrolyzed in the capillary bed. Finally, the fatty acids

and partial acylglycerols are reesterified in adipose or

muscle cells. If the monoacylglycerol pathway is

active in the tissue, where the triacylglycerols are

stored, the original composition of 2-monoacylgly-

cerol is retained. However, it is more likely that a

different distribution of fatty acids occurs in adipose

tissue triacylglycerols.

See also: Chromatography: Principles; Cocoa:

Production, Products, and Use; Coconut Palm; Eggs:

The Use of Fresh Eggs; Fish Oils: Production;

Composition and Properties; Dietary Importance; Infants:

Nutritional Requirements; Peanuts; Triglycerides:

Structures and Properties; Characterization and

Determination; Vegetable Oils: Types and Properties; Oil

Production and Processing; Composition and Analysis;

Dietary Importance

Further Reading

Balca

o VM and Malcata FX (1998) Lipase catalyzed

modification of milkfat. Biotechnology Advances 16:

309–341.

Christie WW (1986) The positional distribution of fatty

acids in triglycerides. In: Hamilton RJ and Rossel JB

(eds) Analysis of Oils and Fats. London: Elsevier.

Fox PF (ed.) (1995) Advanced Dairy Chemistry, Volume 2:

Lipids. London: Chapman & Hall.

Gunstone FD, Harwood JL and Padley FB (eds) (1994) The

Lipid Handbook, 2nd edn. London: Chapman & Hall.

Hartel RW (1996) Applications of milk-fat fractions in

confectionery products. Journal of American Oil

Chemists’ Society 73: 945–953.

Jensen RG, Ferris AM and Lammi-Keefe CJ (1991) Sympo-

sium: Milk fat – composition, function, and potential for

change. The composition of milk fat. Journal of Dairy

Science 74: 3228–3243.

Kuksis A (1992) Yolk lipids. Biochimica et Biophysica Acta

1124: 205–222.

Laakso P (1996) Analysis of triacylglycerols – Approaching

the molecular composition of natural mixtures. Food

Reviews International 12: 199–250.

Marangoni AG and Narine SS (eds) (2002) Physical Prop-

erties of Lipids. New York: Marcel Dekker, Inc.

Moran DPJ and Rajah KK (eds) (1994) Fats in Food

Products. Glasgow: Blackie Academic & Professional.

Small DM (1991) The effects of glyceride structure on

absorption and metabolism. Annual Review of Nutri-

tion 11: 413–434.

Xu X (2000) Production of specific-structured triacylgly-

cerols by lipase-catalyzed reactions: a review. European

Journal of Lipid Science and Technology 2000:

287–303.

Characterization and

Determination

E W Hammond, Greenisle-Consulting, Kettering, UK

Fatty Acid Composition

0001The fatty acid (FA) composition of triacylglycerides

(TAG) is dependent upon the origin of the TAGs, but

the FAs will normally be of even carbon number for

vegetable fats and oils. Exceptions are TAG contain-

ing FA originating from bacterial (and some other

microflora) origins. Thus, the TAG from ruminants

such as cattle and sheep contain odd-chain and

branched-chain fatty acids in small amounts. Typical

fatty acid chain lengths for all TAGs are generally

between C14 and C22, with the major fatty acids

being of the C16 and C18 types. There are exceptions

5868 TRIGLYCERIDES/Characterization and Determination

here also. Some plants major in other chain lengths

such as palm kernel and coconut fats (C8–C14), as

well as cuphea, which has mostly C8 and C10, and

wild-type rapeseed, which has half its FAs as a C22

chain length. Fish and marine animals have longer-

chain FAs in the C18–C22 range. Amongst these

FAs are the unsaturated series. These have one to

six double bonds at specific positions in the chain

because of their biochemical origin and species origin.

There is also a range of FAs which have other

chemically functional groups such as hydroxyl (e.g.,

ricinoleic acid from castor bean), epoxide (e.g.,

epoxyoleic acid from Vernonia anthelmintica) and

cyclopropene (e.g., sterculic acid from Sterculia

foetida).

0002 The reason for mentioning the wide range of FAs is

to point out that confident and accurate analysis of a

sample of TAGs requires some knowledge of its

origin. However, for most purposes, the analysis of

the FA composition of TAGs from food is relatively

simple, since the fats used have fairly uncomplicated

fatty acid compositions. (See Fats: Classification;

Fatty Acids: Properties.)

0003 FA analysis of TAGs is done by gas chromatog-

raphy after derivatization to methyl esters. (See

Fatty Acids: Analysis.)

Positional Analysis

0004 The FA composition of the three positions of the

glycerol in TAGs is not entirely random: there is

positional order. Lipase enzymes associated with

TAG synthesis and hydrolysis are positionally specific

in their action. This specificity is partly responsible

for the differences in TAG composition between the

many natural vegetable and animal fats and also for

the consistency of fat structure within a fat species.

The specific TAG structure of some fats is very im-

portant to their use in foods. For instance, cocoa

butter has a high proportion of symmetrical TAGs,

where position 2 is occupied by oleic acid, while

positions 1 and 3 are occupied by a saturated fatty

acid, typically palmitic and stearic acids. This struc-

ture produces a fat with very specific melting and

crystallisation properties that are responsible for the

particular physical qualities of chocolate products.

0005 During research on such fats, it can be necessary to

determine the positional composition of the FAs in

the TAGs. This is achieved very conveniently using

the specificity of some lipases. Usually, pig pancreatic

lipase is used, which, in its purified form, is 1:3-

specific. It hydrolyzes the esters on the 1 and 3 pos-

itions of TAGs, leaving the 2 position unchanged. The

2-monoacylglyceride obtained yields the composition

of position 2. By examining the diacylglycerides

(DAGs), product information on positions 1 and 3

can be obtained. If the DAG is chemically phos-

phorylated and then reacted with phospholipase A,

information that is more specific is obtained about

the sn-1 and sn-3 positions. (See Cocoa: Production,

Products, and Use; Enzymes: Uses in Analysis; Vege-

table Oils: Composition and Analysis.)

0006Recent significant advances in the interpretation

C nuclear magnetic resonance spectra of pure

TAGs show a more powerful way forward in deter-

mining TAG structure. However, at this time, the

interpretation of spectra from natural vegetable oils

is complex, because these oils are complex mixtures

of different TAGs. In addition, the equipment used

is not generally available to all laboratories that

might be doing such an analysis. Alternative ap-

proaches using enzymes combined with different

stereospecific derivatives and other column chroma-

tographic techniques have been used with some suc-

cess. These new approaches increase the possibility

that such stereospecific positional analysis of natural

TAG mixtures can become routine.

Methods

Isolation of TAGs

0007The sample must be greater than 99% TAGs for these

methods to produce accurate results. If it is not, seven

drops (100–120 mg) of melted and well-mixed fat are

added to 2 ml of diethyl ether. This solution is applied

to a small column of basic alumina (2.0 g, Brockmann

Grade 1 basic alumina) in a glass filter tube, the

column is washed with 2 5 ml diethyl ether, and

the eluate is then collected in a screw-cap vial. The

sample in the vial is then evaporated with a stream of

nitrogen. This procedure normally provides 80–

100 mg of pure TAGs. The method can be scaled up,

but it is better to do multiple preparations in parallel.

Lipolysis – 2 Position Only

0008Approximately 100 mg of melted and well-mixed

TAG is weighed into a screw cap vial (10 ml size).

Two milliliters of tris(hydroxymethyl)aminomethane

(Tris) buffer (1.0 M, pH 8.0) is added to the vial to-

gether with 0.2 ml of 22% aqueous calcium chloride

(made up from the hexahydrate) and 10 drops of

10% aqueous Biosolve detergent/emulsifier (Beck-

mann solubilizer for scintillation counting). (Note:

0.5 ml of 0.1% sodium cholate may be substituted

for the Beckmann biosolve, but emulsification will

be less efficient, and yields are variable in our experi-

ence.) Other emulsifiers may be substituted, but

require testing. The vial is placed in a water bath

(set at 40



C) for 15 min, and a solution of pure

TRIGLYCERIDES/Characterization and Determination 5869

monoheptadecanoin internal standard in diethyl

ether (5 mg ml

1

) is prepared and stored in a closed

vessel. The sample is sonicated for 1 min using an

MSE ultrasonic sample preparator (MSE Soniprep

150) and then replaced in the water bath. This step

needs to be done efficiently with a probe-type soni-

cator, particularly when the sample has a high melting

point. The sample temperature is stabilized in the

water bath for 2 min. An amount of the pancreatic

lipase enzyme (Sigma catalogue number L3126), ap-

proximately equal to the weight of TAGs used, is

added to the sample (the amount of enzyme is import-

ant). The vial is immediately capped and shaken vig-

orously in a wrist-action shaker for exactly 3.5 min

(or the time determined for each batch of enzyme

using standard TAG). Without delay, approximately

1.5 ml of cold, 2 M hydrochloric acid is added, and

the vial transferred to an ice bath and shaken. Exactly

2.0 ml of the monoheptadecanoin solution and a

further 2.5 ml diethyl ether are added and the sample

quickly inverted a few times but without vigorous

shaking. The vial is replaced in the ice bath, and the

separated upper ether layer is transferred to another

vial in the ice bath. The reaction mixture is extracted

with a further 5 ml of ether and combined with the

first on the ice bath. The combined ether extracts are

washed twice with 5-ml portions of water, and then

the ether extract is transferred to a vial containing

approximately 3.0 g of anhydrous sodium sulfate.

This is then capped and shaken to dehydrate the

extract.

0009 Isolation of monoacylglyceride (MAG) for FA an-

alysis is carried out using the following procedure.

Using either a pipette or a thin-layer chromatography

(TLC) sample applicator, the ether solution is applied

as a continuous streak 1 cm above the bottom of a

TLC plate. The plate should be 20 cm 20 cm glass

coated with a 0.5-mm-thick layer of kieselgel ’G’

(plates supplied by Anachem). This streak is focused

by developing the plate twice to 3 cm above the base

in 100% diethyl ether and air-drying the plate

between developments. The plate is fully developed

in a solvent of hexane/diethyl ether/formic acid

(60:40:1, by v/v/v). The plate is air-dried and lightly

oversprayed with 0.05% methanolic dichlorofluores-

cein. When the plate is dry, it is examined under

ultraviolet light (350 nm), and the MAG, which will

be at or just above the focused origin, is marked. (See

Chromatography: Thin-layer Chromatography.)

0010 The marked MAG band is scraped off and placed

in a 20 ml Q/Q tube, to which 7 ml of acid methano-

lysis reagent is added. After thorough mixing, this

is heated to 60



C for 1 h with occasional mixing.

Refluxing temperatures are discouraged here as vapor

locks can form in the silica bulk, causing localized

hot spots and possible decomposition of the fatty

acid methyl esters (FAMEs). Water (7 ml) and diethyl

ether (5 ml) are added, and the mixture is shaken after

cooling. The top layer is transferred to a vial contain-

ing anhydrous sodium sulfate. The sample is then

analyzed by gas chromatography. The MAG yield

can then be estimated from the internal standard

peak. The yield should be between 20 and 30% for

reliable results that will reflect the true composition

of FA on the 2 position of the TAG. (See Chromatog-

raphy: Gas Chromatography.)

Lipolysis – 1:2 and 2:3 Positions

0011The DAG is required for this procedure. It is rare for

good results for both MAG and DAG to be achieved

from a single lipolysis. This is because the technique

has to be optimized for the best yield of either MAG

or DAG with zero or the least possible amount of acyl

migration. Therefore, the lipolysis technique reported

above for 2-MAG is repeated with the following

changes:

0012The weight of lipase enzyme is reduced to one-

quarter the weight of sample TAG (e.g., 25 mg of

enzyme to 100 mg of TAG).

0013The reaction time is increased to 6 min or that time

found for each batch of enzyme using standard

TAG.

0014The MAG internal standard is not used.

0015After reaction, the sample is cooled on ice and

carefully acidified to pH 2.0, then rapidly ex-

tracted into diethyl ether. The extract is washed

twice with 5-ml aliquots of cold water, keeping it

on ice as much as possible. The ether extract is

then transferred to a vial containing anhydrous

sodium sulfate, prior to TLC.

0016TLC is done as above, except that the formic acid

is replaced by 2% ammonia. The 1,2/2,3 DAG

will be found as a single band about 3 cm above

the MAG. If lipolysis has been too protracted, acyl

migration may have taken place, and two bands

will be apparent (1,2/2,3 (lower) and 1,3 (upper)

DAG). If the 1,3 DAG level is seen to be major

component, the sample lipolysis must be repeated.

The 1,2(2,3) DAG is recovered by elution from the

silica with diethyl ether and concentrated with a

stream of nitrogen.

0017The pure DAGs have to be chemically phosphoryl-

ated prior to reaction with phospholipase A, this is

done according to the Lands et al. procedure using

DAG kinase.

0018Twenty-five milligrams of the sn-1,2(2,3) DAG is

weighted into a tube; the following are then added in

sequence: 100 ml of bile salts solution (200 mg ml

1

5870 TRIGLYCERIDES/Characterization and Determination

water); 600 ml of 0.05 M adenosine triphosphate;

250 ml of 1.0 M magnesium chloride; 500 mlof

0.05 M sodium phosphate buffer, pH 7.95; 5.0 mg

of DAG kinase in 500 ml of cysteine phosphate buffer.

0019 After mixing and incubating at 37



C with continu-

ous shaking for 60 min, 1 ml of 1 M hydrochloric acid

is then added.

0020 The lipids are extracted from the reaction mixture

with 15 ml of chloroform/methanol (2:1, v/v), shak-

ing carefully, and then adding 3 ml of 1% sodium

chloride solution. The lower chloroform layer is

then transferred to another tube, and a second extrac-

tion is made with a further 6 ml of chloroform. Two

drops of triethylamine are added to the combined

chloroform layers, and the solvents are then evapor-

ated. The phosphatidate products may be purified by

TLC in a developing solvent of chloroform/methanol/

water (65:36:8, v/v/v).

0021 Stereospecific hydrolysis of the phosphorylated

DAG is the final stage and is achieved with phospho-

lipase A (Crotalus adamanteus venom – Sigma cata-

logue No. P0790). This enzyme is specific for the 2

position FA in an sn-1,2-diacyl 3-phosphatide but

does not attack the sn-2,3-diacyl 1-phosphatide.

0022 Ten milligrams of the phosphatidate is dissolved in

3 ml diethyl ether, to which 0.1 ml of 0.5 M Tris

buffer (pH 8.5) containing 0.002 M calcium chloride

and 0.5 mg of Ophiophagus hannah venom is added.

This mixture is shaken gently for 16–20 h (overnight).

Then, 5 ml of isobutanol and 20 ml of acetic acid are

added and the whole reduced to dryness. The lipid in

the residue is dissolved in a small amount of chloro-

form/methanol (2:1, v/v) and applied as a streak to a

TLC plate and the application area thoroughly dried.

The plate is first developed in hexane/diethyl ether/

formic acid (50:50:1, v/v/v) to the top. The top third

is sprayed with dichlorofluorescein (0.1%) in metha-

nol. The free FA band is located under ultraviolet

light (320 nm) and recovered. The TLC plate is then

redeveloped in chloroform/methanol/14 M ammo-

nium hydroxide (90:8:2, v/v/v), dried, and sprayed

with rhodamine 6 G (0.1%) in toluene. The lyso-

phosphatide and unhydrolyzed phosphatidate are

located as above and recovered. All three of the

recovered materials are prepared for analysis of

their component FA by gas–liquid chromatography

(GLC).

Results

0023 The initial pancreatic lipase technique gives a reliable

composition of the FAs present at the 2 position of

a TAG. The phosphorylation and phospholipase A

techniques produce a free FA fraction, a lysophospha-

tide, and unreacted phosphatidate. The FA fraction is

typical of the sn-2 position and should confirm the

result from the 2-position lipolysis, although there are

often discrepancies in this comparison, and the pri-

mary (pancreatic lipase) result should be regarded

as more reliable. The lysophosphatide gives the FA

composition of the sn-1 position of the TAG. The

unreacted phosphatidate gives the FA composition

of the sn-2 and sn-3 positions, combined, of the

TAG. The difference provides a complete TAG

positional composition.

Cis

trans

Ratios

0024The analysis of the total trans content by infrared (IR)

techniques is covered in detail elsewhere in this En-

cyclopedia, which includes a full IR method. A cis/

trans ratio is obtained in this case by the ratio of the

percentage total unsaturates (determined by GLC)

minus the percentage trans value (determined by IR

analysis). (See Fatty Acids: Analysis; Spectroscopy:

Infared and Raman.)

0025Trans unsaturated fats have been a part of the

human diet at least since meat and fat derived from

ruminant animals began to be consumed. However,

the amount of such trans unsaturated FAs consumed

was relatively small. The advent of chemically

hardened fats for the manufacture of margarine and

shortenings dramatically changed the level of trans

unsaturated FAs consumed. The hardening process

involves catalytic addition of hydrogen across the

unsaturated centers. However, during the process, a

proportion of the double bonds isomerize to the trans

geometry, typically 30–70% depending on the type of

catalyst and the conditions of the hardening process.

The trans isomerization process is accompanied by

double bond movement along the FA chain, so that a

complex mixture is formed. This mixture is very dif-

ficult to analyze with which to obtain accurate cis/

trans ratios. GLC of FAMEs derived from the fat has

proved the best technique, but even here, there are

considerable problems. (See Margarine: Methods of

Manufacture.)

0026For GLC of FAMEs, the official AOCS method

prescribes the cyanopropylpolysiloxane phase

SP2340. The column should be 60 m 0.25 mm

fused silica with a 1-m retention gap of silanized fused

silica with a film thickness of 0.2 mm. Such columns

provide adequate separation of cis and trans FAME

for most situations involving food fats (except fats

containing hydrogenated fish oils). However, there

will be peak overlap, and for exacting analyses,

where information is required on the isomeric nature

of the trans FAME, even these columns are not

adequate. For separation that is more complete, a

column of SP2560, 0.2-mm film on 100 m 0.25 mm

fused silica should be used. In both column cases, the

TRIGLYCERIDES/Characterization and Determination 5871

carrier gas can be helium (linear velocity 20 cm s

1

)or

hydrogen (40 cm s

1

). The column temperature

should be optimized, using standards, between 175

and 200



C isothermal. Otherwise, faster results can

be obtained by programming the temperature be-

tween 100 and 225



C with some sacrifice to opti-

mum resolution. The samples should be injected

using the on-column technique in preference to split

injection.

0027 Cis/trans ratios calculated from this type of chro-

matography are generally more accurate than those

obtained via IR techniques. However, this is true only

below 10% trans content. Above 10% trans content,

the IR technique gives more accurate results.

TAG Analysis in Foods

0028 TAG is readily analyzed by chromatography after

extraction from the food matrix. The extraction may

be the most difficult part of the whole method and

should be given careful attention. Many foods lend

themselves to Soxhlet extraction with petroleum or

chloroform solvents. Other foods containing spray-

dried, or encapsulated fats have to be hydrated first,

or fat yields will be low. Cereal flours require extrac-

tion with water-saturated n-butanol followed by a

mixture of chloroform/methanol in order to obtain

all their lipids. Once a total lipid extract is obtained,

it can be analyzed complete for class composition.

0029 If the TAG is less than 95% of the total lipid,

it should be ’cleaned up’ as above. This TAG can

be analyzed via three routes; high-temperature gas

chromatography (HTGC), nonaqueous reversed-

phase high-performance liquid chromatography, or

argentation high-performance liquid chromatog-

raphy. (See Chromatography: High-performance

Liquid Chromatography.)

0030 HTGC can be applied in two forms: nonpolar-

phase- and polar-phase gas chromatography (GC).

Nonpolar phase GC is the choice for accurate basic

information about composition, which, when con-

sidered together with FAME analysis, will normally

confirm the type of fat. The data provided are a

separation based upon ’carbon number’ (essentially

molecular weight), but it does not discriminate be-

tween saturated and unsaturated species of TAGs. A

7–10 m 0.53 mm fused-silica column with a bonded

phase of SP2100, OV1, or equivalent at a thickness of

0.1 mm gives excellent results (a 1-m silanized reten-

tion gap must be fitted). The injection technique

should be on-column (Carlo Erba) with helium or

hydrogen as the carrier gas. The carrier gas flow is

high at 10 ml min

1

. For nonlauric fats, the initial

column temperature is set at 100



C, held for 2 min,

and then programmed at 25



Cmin

1

to 310



C. For

lauric fats or fats containing butter, the initial tem-

perature is set to 50



C with a hold of 4 min, and then

programmed at 25



C min

1

to 310



C. The TAG

sample is dissolved in iso-octane (or, if solubility is a

problem, chloroform) to a concentration of about

5mgml

1

(5 mgin5ml). The sample volume loaded

is 0.5 ml, equivalent to about 2 mg of TAGs. Figure 1

shows typical data.

0031The polar-phase GC technique provides more in-

formation on the isomeric composition of TAGs and

discriminates between the saturated and unsaturated

TAGs. However, there are quantitative problems in

the application of this method, requiring trained

analysts with good interpretive skills.

See also: Chromatography: Thin-layer Chromatography;

High-performance Liquid Chromatography; Gas

Chromatography; Cocoa: Production, Products, and Use;

Enzymes: Uses in Analysis; Fats: Classification; Fatty

Acids: Properties; Analysis; Margarine: Methods of

Manufacture; Spectroscopy: Infrared and Raman;

Vegetable Oils: Composition and Analysis

Further Reading

Buteau GH, Jr. and Fairbairn D (1969) Experimental Para-

sitology 25: 265.

Damiani P, Santinelli F, Simonetti MS, Castellini M and

Rosi M (1994) Comparison between two procedures

for stereospecific analysis of triacylglycerols from

vegetable oils – 1: Olive Oil. Journal of the American

Oil Chemists’ Society 71(10): 1157–1162.

Duchateau GSMJE, van Oosten HJ and Vasconcellos MA

(1996) Analysis of cis- and trans-fatty acid isomers in

hydrogenated and refined vegetable oils by capillary

gas–liquid chromatography. Journal of the American

Oil Chemists’ Society 73(3): 275–282.

2.0

600

800

1000

1200

1400

4.0

C24

C26

C28

C30

C32

C34

C36

C38

C40

C42

C44

C46

C48

C50

C52

C54

C56

C58

6.0

Time (min)

Intensity (mV)

8.0 10.0 12.0

fig0001Figure 1 Triacylglyceride carbon number analysis by HTGC

using cold on-column injection and a Carlo Erba gas chromato-

graph. See text for further details. The sample is a mixture of

cocoa butter and butter fat. The peak names are the sum of the

carbons in the FA chains and exclude those in glycerol (e.g., C54

might equal 3 C18 or C16 þC18 þC20).

5872 TRIGLYCERIDES/Characterization and Determination

Hammond E (1989) Chromatographic techniques for lipid

analysis. Trends in Analytical Chemistry 8(8): 308–313.

Hammond EW (1993) Chromatography for the Analysis of

Lipids. Boca Raton, FL: CRC Press.

Lands WEM, Pieringer RA, Slakey PM and Zschocke A

(1966) A micro method for the stereospecific determin-

ation of triglyceride structure. Lipids 1: 444–448.

Litchfield C (1972) Analysis of Triglycerides. New York:

Academic Press.

Mannina L, Luchinat C, Emanuele MC and Segre A (1999)

Acyl positional distribution of glycerol tri-esters in vege-

table oils: a

C NMR study. Chemistry and Physics of

Lipids 103: 47–55.

Ratnayake WMN and Beare-Rogers JL (1990) Problems of

analysing c9 cis and trans fatty acids of margarine on the

SP2340 capillary column. Journal of Chromatographic

Science 28: 633–639.

Tripe See Offal: Types of Offal

TRITICALE

K Lorenz, Colorado State University, Fort Collins,

CO, USA

Introduction

0001 Triticale, the first synthetic cereal, is the product of a

cross between wheat (Triticum spp.) and rye (Secale

cereale L.) and has been of both practical and theor-

etical interest. This cross is an attempt to combine in

one strain the high yield and good bread-baking qual-

ity of wheat and the winter hardiness, high lysine and

protein, drought tolerance, and disease resistance of

rye. In all soils presently unsatisfactory for wheat but

acceptable for rye, it would be desirable to be able to

grow a cereal which is equal to wheat in both yielding

capacity and in nutritional, and bread-baking quality.

0002 While it was simple to outline this task, many diffi-

culties had to be overcome before successful crosses

and fertile hybrids could actually be produced. From

the 1930s to the 1950s, research on triticale never

really progressed past the botanical curiosity stage,

despite considerable efforts. Many of the goals that

had been set eluded the plant breeders. Recent research

efforts, however, have resulted in triticales of accept-

able quality and characteristics which are grown com-

mercially in many countries around the world.

Breeding

0003 Triticales are produced by crossing either tetraploid

wheat (Triticum durum L.: genomes AABB) or

hexaploid wheat (Triticum aestivum L.: genomes

AABBDD) with diploid rye (Secale cereale L.:

genomes RR) and doubling the resulting haploid by

treatment with colchicine, which produces a fertile

amphidiploid. These crosses produce primary hexa-

ploid (genomes AABBRR) and octoploid (genomes

AABBDDRR) triticales, respectively. The production

of triticales is outlined in Table 1. Intercrossing

primary triticales within their own ploidy levels was

practised from the 1930s until the 1950s and resulted

in improvements in plant type, but yields and

stabilities of the best lines remained significantly

below those of wheat. In the mid-1960s, hexaploid

triticales and hexaploid wheat were intercrossed, pro-

ducing significant improvement. Dwarfing and dis-

ease resistance genes from hexaploid wheat were

added to the hexaploid triticale gene pool. In the

1980s, crossing of octoploid triticales with hexaploid

triticales, which allows substitution of D-genome

chromosomes and chromosome segments from

wheat for those of the A and B genomes, became the

main approach to triticale germ plasm expansion.

Perhaps the most important aspect of triticale breed-

ing for the future, especially for hybrid production, is

the use of doubled-haploid technology.

World Production and Yields

0004Triticale production figures, given in Table 2, are

obtained from communications with plant breeders.

Areas of production have been increasing annually

for several years: the increase is occurring mainly on

marginal soils, such as the sandy soils of Poland and

Russia. In many countries, triticale offers farmers

an alternative crop which is not controlled by any

TRITICALE 5873

marketing board nor required to meet quality stand-

ards of other grain crops. Recent cultivars also have

a dual purpose by providing green feed for animals

before developing into a grain crop.

0005The yield potential of triticales under optimum

crop production environments has reached nearly

the same level as that of wheat while outperforming

wheat under marginal environments. Yield capacity

has improved because of the addition of genes for

tillering, additional dwarfing, and better grain test

weight. Yields will vary, however, depending on the

triticale variety, location at which it is grown, the date

of seeding, and the amount of available water during

the growing season.

0006Machinery designed for the harvest of wheat can be

used for triticale. No special harvest equipment is

needed.

Agronomic Factors

0007The influence of agronomic variables on yield, com-

position, and other triticale characteristics has been

described in many scientific publications. In general,

the ability to adapt still seems to be limited for some

varietiesof triticale.Theirperformance is influencedby

such factors as latitude, daylength, elevation, tempera-

ture, availability of moisture, and nutrients in the soil.

Some triticales still have the tendency to produce few

tillers whichlimits yield. Triticale still lacks the benefits

of many generations of natural selection which wheat

and rye have gone through in order to perform well

under many different agronomic conditions.

0008Any disease that attacks wheat and rye also

attacks triticale. The cross does not exhibit improved

tbl0001 Table 1 Development of primary and secondary triticales and examples of substitutional triticales

Primary triticale

Common wheat Rye !

Octoploid triticale

Triticum aestivum L. Secale cereale L.

AABBDD (2n ¼ 42) RR (2n ¼ 14) AABBDDRR (2n ¼ 56)

Durum wheat  Rye !

Hexaploid triticale

T. turgidum L. S. cereale L.

AABB (2n ¼ 28)  RR (2n ¼ 14) AABBRR (2n ¼ 42)

Secondary triticale

Triticale  Triticale

AABBDDRR  AABBDDRR !AABBDDRR

AABBDDRR  AABBRR !AABBDDRR or AABBRR

AABBRR  AABBRR !AABBRR

Triticale  Wheat

AABBDDRR or AABBRR  AABBDD

AABBDDRR or AABBRR  AABB

Triticale  Rye

AABBDDRR or AABBRR  RR

Substitutional triticales

(examples with 2n ¼ 42 only)

Example 1 A

Example 2 A

Example 3 A

Example 4 A

(B/R)

Example 5 A

(single gene transfer from rye)

Colchicine treatment is given to hybrid plants to double chromosome number.

Subscripts indicate the number of chromosomes present from each genome. B/R indicates a translocation of B and R chromosomes.

tbl0002 Table 2 Estimates of world production of triticale (area under

cultivation: ha)

Country 1986 1996

Poland 309 000 659 300

Russia 250 000 500 000

Australia 140 000 300 000

Germany 36 000 300 000

US 100 000 180 000

France 200 000 162 000

Bulgaria 10 000 100 000

South Africa 15 000 95 000

Brazil 16 000 90 000

Portugal 7 000 90 000

Spain 35 000 80 000

Sweden n.a. 45 000

Italy 15 000 30 000

Romania n.a. 20 000

Argentina 25 000 16 000

Tunisia 5 000 16 000

UK 16 000 16 000

n.a. not available.

1 acre ¼ 0.405 ha.

Data from Gustafson JP, Bushuk W and Dera AR (1990) Triticale:

production and ulitization. In: Lorenz K and Kulp K (eds) Handbook of

Cereal Science and Technology, pp. 373–399. New York: Marcel Dekker; and

Varughese G (1997) Triticale: an overview. In: Proceedings of Satellite

Meeting of the International Triticale Association on Triticale Quality. All Africa

Crop Science Congress, Pretoria, South Africa, pp. 10–15.

5874 TRITICALE

resistance to many wheat diseases, as was hoped. Some

genes for resistance in either parental species are not

fully expressed in triticale, which resulted in suscepti-

bility to disease for which wheat or rye show resist-

ance. There are no explanations. Obviously, complex

reactions between genes of wheat and rye take place.

Kernel Development and Sprouting

0009 Kernels of some triticales have a tendency to be shriv-

eled. Triticales developed from the late 1960s up to the

mid-1970s almost always showed shriveled grain. The

causes still need to be elucidated today. Many poten-

tial causes for grain shriveling have been examined

and rejected. There seems to be a genetic component

influencing this condition. Shriveled kernels have a

higher production of aberrant nuclei in the coenocytic

endosperm and higher levels of acid phosphatase. Ap-

plication of selection pressure for plump grain be-

tween 1977 and 1992 produced triticale advanced

lines with high test weights and high flour yield.

These lines have plump to only slightly shriveled grain.

0010 Preharvest sprouting associated with late maturity

has been a major problem, inhibiting the commercial-

ization of triticale in several countries. Development

of early-maturing varieties, capable of escaping the

high-moisture period during harvest, has decreased

chances of preharvest sprouting. New sources of

genetic resistance to this condition in wheat parents

are being utilized in present breeding efforts.

Composition

0011 Average percentage composition of triticales is shown

in Table 3. Slightly higher protein values compared to

wheat are frequently cited as an advantage of triticale

over wheat. However, high protein values are often

misleading. Nitrogen values for shrivelled kernels, in

which ratios of endosperm to bran plus germ are low

compared to normal kernels, are higher than those

for plump grains. Germ, scutellum, and aleurone

are proportionately higher in nitrogen than the

endosperm. Furthermore, triticale proteins are often

calculated using a conversion factor of N 6.25,

expressed on a dry-weight basis, which gives 30%

higher values than N 5.7 at 14% moisture.

Decreased protein values have accompanied the

improvement of triticale’s agronomic qualities. This

is not surprising, since improvements in grain size

result in a decrease in the percentage of protein in

cereals owing to a disproportionate increase in starch

content. (See Cereals: Contribution to the Diet.)

0012Lysine, which is the first limiting amino acid in

wheat, is often, but not always, present in larger

amounts in triticale than in wheat. Lysine is still the

first limiting amino acid in triticale. Essential amino

acid content of triticale compared to wheat and rye is

shown in Table 4. Expressed as yield per hectare, the

differences in lysine between wheat and triticale do

not seem significant since lysine depends on the

productivity of the variety. (See Amino Acids: Proper-

ties and Occurrence.)

0013The starch and fat (ether extract) contents of triti-

cale are not significantly different from those of the

parental species.

0014Except for lower amounts of nicotinic acid in triti-

cales compared to wheat, the vitamin composition of

triticales is comparable to that of wheat and better

that that of rye, as illustrated in Figure 1. Average

mineral composition of triticales compared to that of

wheat shows no major differences, with the possible

exception of iron, which appears to be present in

higher amounts in triticales than in wheats, as

shown in Table 5.

0015Toxic substances and nutritional inhibitors in triti-

cale, which may interfere with the digestibility and

availability of certain nutrients to humans and/or

animals, include ergot, resorcinols, phytates, and

enzyme inhibitors.

0016Ergot results from an infection of cereal grains

by the fungus Claviceps purpurea which produces

tbl0003 Table 3 Average percentage composition of triticales

Average composition (%)

(rangesinparentheses)

Dry matter 87.0 (86.7–90.0)

Moisture 12.0 (10.0–13.3)

Crude protein (N  6.25) 13.2 (11.3–18.5)

Ether extract 1.8 (1.3–2.7)

Crude fibre 2.4 (1.4–3.8)

N-free extract 67.8 (65.0–80.0)

Ash 1.8 (1.4–2.4)

Starch 50.7 (46.5–54.4)

tbl0004Table 4 Essential amino acid content (g per 100 g protein) of

triticale compared to wheat and rye

Amino acid Triticale (Yoreme) Wheat (INIA) Rye (Snoopy)

Lysine 3.44 2.83 4.02

Threonine 3.55 2.98 4.06

Methionine 1.28 1.42 1.35

Isoleucine 3.45 2.68 3.70

Leucine 7.20 7.22 775

Phenylalanine 4.94 3.77 4.74

Valine 4.48 3.73 5.10

Cultivar name in parentheses. Data adapted from Gustafson JP, Bushuk

W and Dera AR (1990) Triticale: production and utilization. In: Lorenz K and

Kulp K (eds) Handbook of Cereal Science and Technology, pp. 373–399. New

York: Marcel Dekker.

TRITICALE 5875

alkaloid-containing sclerotia. Relatively high levels of

infections of ergot on triticales have occurred in some

years. However, no statistics are available about the

extent of infections in the various triticale-growing

areas of the world.

0017Resorcinols seem to be partially responsible for

reduced food intake and rate of weight increase of

cattle, sheep, pigs, poultry, and horses. Triticales

contain higher amounts of resorcinols than wheat.

Milling of triticales will channel most of these

resorcinols into the feed fractions. Phytic acid forms

insoluble compounds with minerals, making

these elements unavailable. Phytic acid, trypsin, and

chymotrypsin inhibitors have been extracted from

triticale in amounts intermediate between wheat

and rye. (See Phytic Acid: Nutritional Impact.)

Food Uses

0018The prediction of the 1970s of a rapid expansion of

the acceptance of triticale by millers and bakers

for cereal-based products has not been realized. It

appears that the success of triticale today depends

on its ability to substitute to a greater or lesser extent

for traditional wheat products.

0019Triticale grain can be milled into flour by the same

milling process as used for wheat or rye. Because of

the wide variation in kernel hardness and the degree

of kernel shriveling, there is no standard procedure

for triticale. Research has shown that breads can be

baked from triticale flours provided that adjustments

in formulation, mixing, and fermentation are made

from those used in the production of white bread.

Yeast level is increased, the fermentation temperature

is lowered, and fermentation and proofing times are

shortened. The high a-amylase activity of most triti-

cale flours requires fermentation adjustments. Dough

mixing is critical, since triticale flours do not have the

same quality of gluten as wheat flour. The production

of breads of low specific volume from triticale flours,

as they are produced in many developing countries,

creates less of a problem than production of the high-

volume white bread.

0020The overall bread-making quality of newer triticale

cultivars is considerably better than that of earlier

ones, but it is still somewhat inferior to that of

bread wheat of the same protein content due to a

deficiency in protein quality, as reflected by a lower

percentage of gluten compared to bread wheat.

0021Triticale flour can be used to some extent in the

production of cakes, biscuits, tortillas, and other soft

wheat products since triticale basically performs like

a soft wheat. Layer cakes of acceptable quality can be

produced from 100% triticale flour after proper

chlorine treatment of the flour. Formulations of

layer cakes from blends of triticale–wheat flour,

ranging from 20% to 50% triticale, and additional

emulsifier in the formulation produced cakes equal to

or significantly larger than the soft wheat control

cakes without additional emulsifier.

Thiamin

Pantothenic

acid

Nicotinic acid

Spring wheat

Spring triticale

Spring rye

1.0

0.8

0.6

0.4

0.2

Concentration (mg g

−1

)Concentration (mg g

−1

)

complex

Riboflavin

Folic

acid

Biotin

fig0001 Figure 1 Vitamins in triticale, wheat, and rye. Reproduced from

Triticale. Encyclopaedia of Food Science, Food Technology and Nu-

trition, Macrae R, Robinson RK and Sadler MJ (eds), 1993, Aca-

demic Press.

tbl0005 Table 5 Mineral composition of triticale and wheat

Mineral Triticale Wheat

K (mg 100 g

1

) 400–500 330–494

Mg (mg 100 g

1

) 110–190 180–190

Ca (mg 100 g

1

) 30–150 34–40

P (mg 100 g

1

) 240–487 230–370

Na (ppm) 45–200 30–50

Zn (ppm) 13–26 17–30

Fe (ppm) 51–100 40–49

Cu (ppm) 3–7 4–5

Mn (ppm) 10–55 12–38

Data from Lorenz K (1982) Triticale processing and utilization: comparison

with other cereal grains. In: Wolff IA (ed.) Handbook of Processing and

Utilization in Agriculture, pp. 277–327. Boca Raton, FL: CRC Press; and

Kulshrestha K and Usha MS (1992) Biochemical composition and

nutritional quality of triticale. Journal of Food Science and Technology (India)

29: 109–115.

5876 TRITICALE

0022 Triticale flours gave significantly smaller biscuit

diameters and top-grain scores than biscuits baked

with soft wheat flours. The biscuit-baking perform-

ance of flours from certain triticale cultivars may be

improved, however, by increasing emulsification in

the dough system, to equal or exceed soft wheat

standards without additives.

0023 Triticale pancake and waffle mixes have appeared

on supermarket shelves. They are indistinguishable in

appearance from those made with wheat flour, but

differ in flavor and taste. Protein concentrates and

starch have been prepared from triticale. Whole-

grain triticale has been used to make bulgur.

Malting and Brewing

0024 Compared to barley, triticales show relatively large

malting losses, but yields of malt extracts are gener-

ally higher with triticales than with barley. Triticale

malts are high in nitrogenous material, diastatic

power, and a- and b-amylase. (See Malt: Malt Types

and Products; Chemistry of Malting.)

0025 Beers from triticale worts are darker in color and

have a higher pH than beers from barley. Most triti-

cales produced beers with satisfactory clarity–stabil-

ity and gas stability. Triticale beers generally contain

less alcohol and more nitrogenous compounds than

barley beers. (See Beers: Wort Production.)

Feed Grain

0026 Most triticale grown in the world today is used as

feed grain. Triticale feeding trials in general have been

encouraging. The overall conclusion from tests on

weaned piglets, growing-finishing pigs, and broiler

chicken was that triticale rated equal to wheat and

corn. Protein digestibility was significantly higher

with triticale than with sorghum in rations for cattle

and sheep. Triticale was found to be comparable to

durum wheat fed to turkeys. There are, however, also

triticale feeding studies which reported poor feeding

efficiency and reduction in weight gain. Reasons for

such inconsistencies are as varied as the triticale

varieties themselves. Variations in protein content,

amino acid composition, nutritional availability, and

antinutritional factors can all have an effect upon

triticale’s performance as a feed grain.

0027 It appears, however, that ergot-free triticale is at

least equal to wheat when used as a partial replace-

ment in animal rations.

Forage

0028 Triticale has been used as a forage from Palouse silt

loam of the US Pacific Northwest, to loam soils of

Guelph, Ontario, Canada, to the fine sandy loam of

Texas; although not originally intended for its use, it

has gained acceptance in most areas as an alternative

forage. Both winter and spring varieties may be util-

ized as a nutritious food supply for livestock. Forage

yields have been reported from as low as 4.98 tons

1

to 12.55 tons ha

1

with different varieties.

0029In southern New South Wales, Australia, dual-

purpose triticales have almost completely replaced

oats as a forage crop. In China, triticale has been

identified as a promising silage crop compared to

barley. In many parts of the world triticale is being

used successfully as a forage or in mixtures with

legumes for grazing, cut forage, hay, or as whole

crop silage.

See also: Bread: Breadmaking Processes; Chemistry of

Baking; Dietary Importance; Cakes: Nature of Cakes;

Methods of Manufacture; Cereals: Contribution to the

Diet; Dietary Importance; Malt: Chemistry of Malting