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

Подождите немного. Документ загружается.

components. In recent years, the production of filled

milks has become increasingly important. In this case,

the milk fat is replaced by locally available and

cheaper vegetable fats.

Storage and Packing

0041 An adequate keeping quality of up to 12 months can

be achieved for both sweetened condensed milk and

evaporated milk, but this largely depends on the stor-

age conditions. At a storage temperature above 25



the aging process and related physical, as well as

organoleptic, defects appear more rapidly. For recom-

bined products, the normal shelf-life stability, without

major deviations from the original aspect, is reduced

by about half of a fresh-milk product. (See Storage

Stability: Parameters Affecting Storage Stability.)

0042 The most commonly used packing is the tin plate

can, which offers absolute protection against light, is

basically crash proof, and has advantages for handling

and storage. Recycling is also an important factor. As

alternative packing, glass containers or paper/plastic

laminates can be used; however, depending on the

packing material, a considerable shelf-life reduction

should be taken into account. For paper/plastic lamin-

ates, aseptic filling techniques are used, especially for

evaporated milk. (See Canning: Principles.)

Nutritional Considerations

0043 Owing to the concentrated form, both sweetened

condensed milk and evaporated milk have an in-

creased compositional analysis compared with milk

(Table 1). For sweetened condensed milk, the figure

for carbohydrate includes the sucrose content neces-

sary for product conservation; the carbohydrate in

evaporated milk and unconcentrated milk is lactose

only. The rather high energy value of sweetened con-

densed milk is due mainly to the amount of carbohy-

drates present, whereas the protein and fat contents

compare more or less with evaporated milk. (See

Milk: Dietary Importance.)

Production and Usage

0044 World production amounts to over 4.5 million

tonnes. About one-third is produced as sweetened

condensed milk and two-thirds as evaporated milk.

As recently as 35–40 years ago, infant feeding was

still a major application for either sweetened or un-

sweetened concentrated milk. Owing to the nature of

the products, somewhat distinct usages are common

practice.

Sweetened Condensed Milk

0045Owing to the high sucrose content of > 40% and its

viscous consistency, this product is frequently used as

a jam-like bread spread.

0046An early, but no longer popular, use of sweetened

condensed milk was to dilute it with water and con-

sume it as a drink. Presently, coffee or tea whitening

and sweetening is the major use of sweetened con-

densed milk. In many countries, it is also used in

combination with cocoa or other milk modifiers in

the preparation of homemade drinks.

0047A growing application is in sweet dessert prepar-

ations like icecreams, cakes, cookies, etc. Specific to

the Latin American continent is its use for Dulce de

Leche, basically a caramelized sweetened condensed

milk obtained by boiling the can in water for 2–3h.

Different applications vary from country to country

and from one geographical region to another,

according to traditional consumption habits.

Evaporated Milk

0048A large area of application is coffee and tea whitening

and the preparation of milk-based beverages. The

other main field for general usage is in the culinary

sector to enhance the taste and texture of mashed

potatoes, pasta, quiches, soups, and a wide variety

of savory and sweet recipes. The main uses can also

vary specifically from one country to another.

Manufacturing Principles

0049The raw material used for both products is usually

cows’ milk, although in certain regions, it may be a

mixture of cows’ and buffalos’ milk. (See Buffalo:

Milk.)

Sweetened Condensed Milk

0050A flow diagram for the manufacture of sweetened

condensed milk is shown in Figure 1.

tbl0001 Table 1 Typical comparative figures for standard concentrated products and milk (all values in grams per 100 g)

Product Protein Fat Carbohydrates Minerals (ash) Calcium Phosphorus

Sweetened condensed milk 7.8 8.0 55.2 1.8 0.28 0.23

Evaporated milk 6.5 7.5 9.8 1.4 0.24 0.19

Milk 3.2 3.5 4.6 0.7 0.12 0.09

1576 CONDENSED MILK

0051 Raw milk is collected and selected according to the

usual quality criteria. The milk is analyzed for fat and

solids-nonfat (SNF) content and the ratio between

both components is adjusted according to the fat/

SNF ratio of the final product to be manufactured

by the addition of either cream or skim milk.

0052 If the final product should have a composition of

8.0% fat and 21% SNF, the raw milk should be

adjusted to the same fat/SNF ratio (see Table 2).

0053 Sugar is dissolved in the cold milk, in principle by

recirculation through a dissolving vat. The amount of

sugar is determined by the quantity of milk prepared

for a batch standardization, its fat content, and the

compositional requirement of the sucrose content of

the final product, e.g.,

10 000 kg of milk  3:25% fat

100

 sugar factor

sugar content of finished product

Fat



After standardization, the milk/sugar mixture under-

goes a heat treatment, which consists of a tempera-

ture/time combination usually above 100



C and

holding for seconds or minutes. This heat treatment

eliminates nearly all the bacteriological flora of the

milk, but the choice of temperature/time combination

greatly influences final product characteristics such

as the consistency and shelf-life stability. (See Heat

Treatment: Chemical and Microbiological Changes.)

0054 The viscosity of the product is an important

factor. It is rather difficult to indicate precise heat

treatment conditions, since they depend on the equip-

ment used and the characteristics of the raw material

milk which can produce different behavior through

regional, climatic, and seasonal influences. Constant

observation of viscosity and age thickening is neces-

sary to counteract any negative effect. In general,

lower heat treatments favor increased viscosities,

and higher, intensive heating tends to give lower vis-

cosities. The viscosity should be sufficient to avoid

phase separation during storage, and age thickening

should be moderate in order to keep the product

pourable during its storage life.

0055Concentration after heat treatment takes place

under vacuum and usually in a multiple-effect

evaporator.

0056Sweetened condensed milk is not a sterilized or

sterile product; its conservation is assured by the

bacteriostatic effect of the high sucrose concentra-

tion. The sugar-in-water ratio in the concentrated

product must be above 61%. However, osmophilic

organisms can develop in this medium, so it is import-

ant that the equipment after heat treatment and up to

filling is of a hygienic design and is operated under the

best hygienic conditions to avoid reinfection. Strict

controls for equipment cleaning and sterilization are

essential.

0057After evaporation, the concentration must be as

close as possible to the final product’s total solids

content; an adjustment by, for example, water should

be omitted because of the risk of contamination.

0058After evaporation, the product is cooled to

normally 20–25



C, and at the final concentration,

part of the lactose contained in the product is

Fresh milk Sugar

Lactose

Cooling/seeding

Storage/fillingHoldingEvaporationHeat treatment

123

Standardization

fig0001 Figure 1 Basic flow diagram for the manufacture of sweetened condensed milk (continuous process). Reproduced from Condensed

Milk, Encyclopaedia of Food Science, Food Technology and Nutrition, Macrae R, Robinson RK and Sadler MJ (eds), 1993, Academic Press.

tbl0002Table 2 Example adjustment of the fat and SNF content of raw

milk to achieve a final product composition of 8.0% fat and 21%

SNF

Finalproduct Ratio Standardized

milk

Ratio

Fat (%) 8.0

0.381

3.25

0.381

SNF (%) 21.0 8.52

CONDENSED MILK 1577

oversaturated. To avoid autocrystallization, fine-

milled and pasteurized dry lactose crystals are added

to the concentrate to initiate instant and controlled

crystallization. During storage, product stirring

should be maintained for several hours to finalize

the crystallization process.

0059 Filling, usually into metallic tin plate cans, is the

last delicate operation. Cans and lids must be steril-

ized, and this is often achieved by passing them

through a flaming installation. Air in the filling area

must be filtered and of an excellent bacteriological

quality; the air space in the can must be as low as

possible to restrict mold growth. It is not necessary to

perform an aseptic operation, but excellent hygienic

conditions must prevail.

Evaporated Milk

0060 A flow diagram for the manufacture of evaporated

milk is given in Figure 2.

0061 In principle, this product is poststerilized, which is

a conventional process, whereby after filling and

closing operations, the product is sterilized in the

final container (e.g., tin plate cans, glass bottles, etc.).

0062 For stability reasons, the product contains a small

percentage of stabilizing salts (either one or a com-

bination of the earlier-mentioned salts). Where legis-

lation allows, the hydrocolloid, carrageenan, may

be added. Stabilizing salts are indispensable as manu-

facturing and technological aids for adjusting or

regulating the heat stability of the product during

the poststerilization process. Carrageenan influences

phase stability (creaming and protein sedimentation)

during the product’s storage life.

0063 During the last 20 years, aseptically filled evapor-

ated milks, mainly in soft packs, have been developed

and commercialized. To realize aseptic filling, the

product has to be heat-treated in-line for final steril-

ization. Ultrahigh temperature (UHT)-type installa-

tions are used for this purpose. (See Heat Treatment:

Ultra-high Temperature (UHT) Treatments.)

0064The processing steps for milk standardization, heat

treatment, and concentration/evaporation are similar

or identical to those described for sweetened con-

densed milk, with the exception of the addition of

sugar.

0065After concentration, evaporated milk is homogen-

ized, cooled for intermediate storage, and sterilized

after filling.

0066Heat treatment/heat stability The ability of concen-

trated milk to withstand high-temperature steriliza-

tion is essential. This is commonly described as heat

stability and refers to the resistance of milk concen-

trate to coagulation during sterilization in containers.

0067Milk has a natural heat stability, which is influ-

enced mainly by compositional factors, like mineral

salt content, protein content, degree of acidity, etc.

The natural heat stability also varies according to

season and lactation period. In order to obtain milk

sterilizable at different levels of concentrations, this

natural heat stability must be improved and adapted.

In practice, this is achieved by temperature/time com-

bination of preliminary heat treatments on the milk

before concentration and the addition of certain

mineral salts. Precise indications are rather difficult,

owing to the various influencing factors. The optimal

heat stability has to be found by an empirical ap-

proach. In principle, more intensive heat treatments

increase the heat stability, but above optimum condi-

tions, a reverse effect is attained.

0068It must be understood also that processing oper-

ations, such as concentration and homogenization,

Stabilizing

salts

Filling Sterilization

Homogenization/cooling

StorageEvaporationHeat treatment

123

Standardization

Standardized

milk

Carrageenan

fig0002 Figure 2 Basic flow diagram for the manufacture of evaporated milk. Reproduced from Condensed Milk, Encyclopaedia of Food Sci-

ence, Food Technology and Nutrition, Macrae R, Robinson RK and Sadler MJ (eds), 1993, Academic Press.

1578 CONDENSED MILK

have a destabilizing effect on the concentrate. The

various operations must be balanced so as to ensure

a product of optimum quality.

0069 Concentration After preheating, the milk is evapor-

ated under vacuum. It is of utmost importance that

the evaporator works under optimal hygienic condi-

tions, despite the fact that the product is sterilized

afterwards. In general, falling film evaporators are

commonly used today.

0070 Homogenization In order to obtain a satisfactory

homogenization effect, the homogenizer and, in par-

ticular, the homogenizing valves must be kept con-

stantly in the best mechanical conditions.

0071 The homogenization temperature should prefer-

ably be around 65



0072 A homogenizing pressure of 200–250 bar is usu-

ally applied, and the best results are obtained by a

two-valve system in series. In principle, the second

valve is adjusted to 20–25% of the total pressure.

Excessive pressure has a destabilizing effect, which

is irreversible. Following homogenization, the

product is cooled for intermediate storage.

0073 Pilot sterilization It is advantageous to fill some

cans and to sterilize them under the given conditions

in order to verify the heat stability of the concentrate.

Corrective actions are further possible by addition

of stabilizing salt and adaptation of the sterilizing

conditions.

0074 Sterilization After filling into containers (cans), the

product is sterilized. The purpose of sterilization

is to obtain physical and bacteriological stability.

According to practical application, one speaks of

commercial sterility.

0075 The effect of sterility is expressed by the F value

as an integral function of the lethality of the micro-

organisms present, related to C. botulinum, at the

specific destruction temperature of 121



C. Usually,

an F value of 4 is considered satisfactory.

0076 Today, modern instruments are available for

measuring the time/temperature profile within any

type of sterilizer.

0077 Usually, rotary and continuous sterilizers are used.

Major Product Defects

Bacteriological Problems

0078 For evaporated milk, the sterilization processes

usually applied are proven and sufficiently safe to

guarantee product safety and commercial sterility. If

problems arise, they are mainly traced back to an

insufficient can integrity. Faulty soldering, welding

of can bodies, or closure seams are the weakest

points. However, the presence of excessive numbers

of thermophilic spores can cause severe spoilage.

Appropriate line and finished product controls must

be put in place. (See Spoilage: Bacterial Spoilage.)

0079Sweetened condensed milk is less vulnerable to

spoilage because of its high sugar content, but it is

not protected against osmophilic organisms. Plant

and manufacturing hygiene are the keys to success.

Physical Instability

0080Instability may be of various individual defects, some

of which may be interrelated. Separation problems

can, and mainly are, a result of inadequate product

viscosities. Certain texture problems may be related

to nonoptimal heat treatments, but for sweetened

condensed milk, it may also be a result of coarse

lactose crystallization.

0081Age thickening is a constant problem for sweetened

condensed milk. The increase of viscosity over the

storage time is always present and, from a manufac-

turing point of view, can be corrected only by an

optimal heat treatment of the milk prior to concen-

tration. Considerable practical experience is required

to keep this phenomenon under control.

0082Age gelation of evaporated milk is a similar phe-

nomenon to age thickening for sweetened condensed

milk but often appears only and suddenly after long

storage times. The product can remain with normal

physical properties up to 10 months or more and can

then thicken within a few weeks. This gelation is

strictly a storage defect and should not be confused

with thickening or coagulation during the steriliza-

tion process or with coagulation resulting from

microbiological activity. The main factors influencing

age gelation are, as above, insufficient and nonopti-

mal preheating conditions of the milk before evapor-

ation, marginal sterilization conditions, and an

insufficient stabilizing salt level.

0083Inferior raw milk quality and final product storage

conditions can also play an important role within the

complex context of age gelation or thickening for

either evaporated or sweetened condensed milk.

Products Manufactured by Recombining

0084As already mentioned, traditional dairy products like

sweetened and unsweetened condensed milk are

made by recombination using skim milk powder

and anhydrous milk fat for the dairy components.

The following is a brief description of these processes.

(See Recombined and Filled Milks.)

0085When recombining sweetened condensed milk (see

Figure 3), according to a process using only flash

CONDENSED MILK 1579

cooling evaporation, the skim milk solids have to be

dissolved at a concentration of 40%, where some

formation of lumps is practically unavoidable. How-

ever, these lumps are easily dispersible by mechanical

force, as obtained through a colloid mill. A recircula-

tion system has a beneficial effect on skim milk

powder hydration.

0086 In principle, dissolving sugar in a low-temperature

medium does not pose a major problem but is some-

what time-consuming. Therefore, the circulated

solution is heated after powder dosage in order to

make the sugar dissolve rapidly.

0087 A slightly simplified process for recombining can

be used for evaporated milk (see Figure 4).

0088 Dissolving skim milk powder at a low concentration

generally poses less problems. Skim milk powder is

certainly the most critical raw material and, depending

on its characteristics, directly affects the physical

properties of the final recombined product. This influ-

ence depends to a large extent on the heat treatment

given to the liquid skim milk before drying. Seasonal

and regional influences may also play a role.

0089Since milk proteins are sensitive to heat, the extent

of their denaturation reflects the heat treatment ap-

plied and is used for classifying skim milk powders.

The latter have been classified by the American Dairy

Products Institute into three groups according to the

level of undenatured whey protein nitrogen present in

the powder after manufacture. In general terms, this

is expressed as the whey protein nitrogen index (WPN

index) (see Table 3). Whereas the high-heat-type

powders are, in principle, the most appropriate for

sterilized products, the low-heat range powders are

usually more suitable for nonsterilized recombined

products such as sweetened condensed milk.

0090Anhydrous milk fat is the principal source of fat

used in recombined dairy products. It contributes

significantly to the taste and milky character of the

product. This is a clear indication for the quality

requirements for this raw material. In certain cases,

Mixing vessel Mixing vessel

Butteroil

Sugar

Colloid mill Colloid mill

Pasteurization

Flash cooling

evaporation

Lactose seeding

Storage and

filling

Without heating

Heating to 658 C

Intermediate storage

(308 C)

Water

1ST PHASE 2ND PHASE

Skim milk

powder

Intermediate storage

(508 C)

fig0003 Figure 3 Flow chart for the recombination of sweetened con-

densed milk. Reproduced from Condensed Milk, Encyclopaedia of

Food Science, Food Technology and Nutrition, Macrae R, Robinson

RK and Sadler MJ (eds), 1993, Academic Press.

Water

Mixing vessel

total solids 25−27%

Heating

45−558 C

Colloid mill

Butteroil

Deaeration

Pasteurization

Homogenization

Cooling

Storage before

filling/sterilization

Skim milk

powder

fig0004Figure 4 A flow chart for the recombination of evaporated milk.

Reproduced from Condensed Milk, Encyclopaedia of Food Science,

Food Technology and Nutrition, Macrae R, Robinson RK and Sadler

MJ (eds), 1993, Academic Press.

1580 CONDENSED MILK

vegetable oils are used as butterfat substitutes, pri-

marily for economic reasons. Such products are inter-

nationally recognized as ‘filled milks.’ The vegetable

oils used in filled milks should be double-refined and

deodorized and have a low peroxide value.

0091 Water is a basic ingredient of all recombined dairy

products. In general, a good drinking water quality is

sufficient and acceptable. However, as water taste,

odor, and possibly also color may influence the final

product, special monitoring of water quality is

important.

0092 Buttermilk powder can be and is used in recom-

bined products. From the technological point of view,

buttermilk powder is an emulsifying aid for the fat,

since it contains a relatively large amount of the

phospholipids that are lost in the separation process

of skim milk and anhydrous milk fat. For most appli-

cations, only sweet buttermilk is suitable; buttermilk

powder obtained from acidified cream for butter pro-

duction is not suitable. (See Emulsifiers: Uses in Pro-

cessed Foods.)

0093 As the natural vitamin content of fresh milk is

usually reduced slightly during processing of the raw

materials for recombining, vitamins A, D, and B are

usually added. For filled milks, further addition of

vitamin E is carried out and is recommended for

nutritional purposes.

0094Various processing aids, mainly phosphates but

also other emulsifiers or stabilizers, are added to

achieve specific product characteristics and consist-

encies.

0095All additives must be in accordance with legal pre-

scriptions or comply with the Food and Agriculture

Organization/World Health Organization standard

(Code of Principles). (See Legislation: Additives.)

See also: Buffalo: Milk; Canning: Principles; Heat

Treatment: Ultra-high Temperature (UHT) Treatments;

Chemical and Microbiological Changes; Legislation:

Additives; Milk: Dietary Importance; Recombined and

Filled Milks; Spoilage: Bacterial Spoilage; Storage

Stability: Parameters Affecting Storage Stability

Further Reading

Codex Alimentarium (1984) Rome: Food and Agriculture

Organization of the United Nations/World Health

Organization.

Hunziker OF (1935) Condensed Milk and Milk, 5th edn.

La Grange, IL: Hunziker.

Pizzoferrato L, Manzi P, Vivanti V et al. (1998) Mailland

reaction in milk-based foods: nutritional consequences.

Journal of Food Protection 61: 235–239.

Ramonaityte DT (2001) Copper, zinc, tin and lead in

canned evaporated milk, produced in Lithuania: the

initial content and its change at storage. Food Additives

and Contaminants. 18(1): 31–37.

Webb BH, Johnson AH and Alford JA (1974) Fundamen-

tals of Dairy Chemistry. Westport, CT: AVI.

Confectionery See Sweets and Candies: Sugar Confectionery

CONJUGATED LINOLEIC ACID

C E Fernie, Scottish Crop Research Institute,

Invergowrie, Dundee, UK

What is Conjugated Linoleic Acid (CLA)?

0001 Conjugated Linoleic Acid (CLA) is the collective name

given to a group of 18:2 free fatty acid positional and

geometric isomers, containing two conjugated double

bonds (Figure 1). Most fatty acids commonly occur-

ring in nature contain methylene-interrupted double

bonds, e.g., cis-9, cis-12 18:2 (linoleic acid). However,

conjugated dienes have been known for more than 50

years. They are seen as the products of both autoox-

idation and of partial hydrogenation of polyunsatur-

ated fatty acids, as well as being minor components of

dairy and ruminant lipids. There is increasing interest

in CLA, as in animal trials, it has shown potentially

beneficial effects on a variety of medical conditions

such as cancer and type 2 diabetes, as well as affecting

body composition parameters such as bone compos-

ition and lean body mass. As a result, this is an area of

tbl0003 Table 3 WPN index (values are in milligrams of nitrogen per

gram of powder)

Classification WPNindex

High heat 1.5

Medium heat 1.51–5.99

Low heat 6.0

CONJUGATED LINOLEIC ACID 1581

continuing growth in research in a wide variety of

scientific fields. This review will attempt to give an

overview of the current state of knowledge in this

developing area of interest.

Occurrence in Natural Products

0002 The most common source of natural CLA in the

human diet is from the meat and milk products of

ruminant animals. CLA isomers are present in these

materials at levels of approximately 0.5% of total

fat on average, although seasonal variations are

observed. The predominant isomer is the cis-9,

trans-11 acid, at levels of 80–90% of the total CLA,

with minor amounts of a wide number of other

isomers present, ranging from the 6,8 through to the

13,15 positional isomers, in almost all of the different

geometric configurations possible (i.e., cis, cis; cis,

trans/trans, cis and trans, trans), particularly in milk

fat and cheese. Biohydrogenation of linoleic acid is

thought to be the main mechanism of formation of

natural CLA. The process involves the action of the

ruminant bacterium Butyrivibrio fibrisolvens on diet-

ary linoleic acid (via linoleic acid isomerase) to give

CLA as an intermediate in the formation of trans-11

18:1 (vaccenic acid) and subsequently 18:0 (stearic

acid). As levels of CLA produced by this mechanism

do not seem to be high enough to account for the

amounts of CLA in ruminant tissues, a second mech-

anism has been proposed, involving the action of the

D-9 desaturase enzyme on vaccenic acid within adi-

pose and mammary tissues to give the cis-9, trans-11

CLA isomer (Figure 2).

0003 The variety of conjugated isomers in ruminant milk

and tissue is thought to be due to a combination of

double bond migration, and the action of specific cis,

trans isomerases in the rumen.

0004 Altering the feeding regime of the animals can vary

the levels of CLA in ruminant fats. Pasture-fed

animals have higher levels of CLA than those given

commercial feed. However, feed enriched with unsat-

urated fatty acids can also increase the CLA levels in

milk and meat. There can be wide variations in CLA

production between animals of different breeds, and

at different stages of lactation. This may relate to the

D-9 desaturase level of the individual animals.

0005CLA has been reported in fish, and its occurrence in

farmed fish is probably due to the CLA content of

vegetable oil in fish food. However, there is concern

that CLA can be misidentified in fish tissues, as it

elutes on some gas chromatographic columns in the

same region as the naturally occurring 18:4 isomers.

This may have been the case in early reports of low

levels of CLA in trout, shrimp, and mussels, as cur-

rently no mechanisms are known to account for CLA

formation in marine foods.

0006CLA is also present in small amounts, as a result of

the hydrogenation process, in margarines, cooking

oils and other partially hydrogenated fats.

Commercial Production

0007Mixtures of CLA isomers are available commercially,

for use in scientific studies and as reference standards,

and also as dietary supplements. These products,

which are usually in the free fatty acid form, are

typically produced by the alkaline isomerisation of a

910

Linoleic acid

1614

1312

109

Cis-9, trans-11 con

ated linoleic acid

fig0001 Figure 1 Linoleic acid is a methylene-interrupted fatty acid, i.e.,

it has a CH

group between the two double bonds, at position 11.

CLA is a conjugated fatty acid as there are no groups between the

double bonds in the carbon chain.

17151311 9 7 5 3

16141210 8 6 4 21

171513 11

171513

9753

16141210

181614

1210

86421

18642

97 5 3

18642

18:0

(stearic acid)

Trans-11 18:1

(vaccenic acid)

Cis-9, trans-11 18:2

(Conjugated linoleic acid)

Cis-9, cis-12 18:2

(linoleic acid)

∆-9 desaturase

fig0002Figure 2 Biohydrogenation of linoleic acid to produce CLA,

both as an intermediate towards stearic acid and by desaturation

of vaccenic acid.

1582 CONJUGATED LINOLEIC ACID

linoleate-rich vegetable oil, such as sunflower or saf-

flower oil. The CLA produced from this process in

aqueous solution generally contains four main CLA

isomers: trans-8, cis-10; cis-9, trans-11; trans-10,

cis-12, and cis-11, trans-13. Under harsh reaction

conditions, these isomers can be present in approxi-

mately equal proportions. It is possible, however, to

manipulate the reaction temperature and time to give

a mixture that is greater than 90% of the cis-9, trans-

11 and trans-10, cis-12 isomers, in approximately

equal proportions. This is more desirable, particu-

larly for supplement usage. By using propylene glycol

instead of water, with an appropriate basic catalyst

(e.g., potassium hydroxide), the amounts of the minor

isomers can be reduced to around 1%, but small

amounts of isomerization will always occur.

0008 The processes described above all give mixtures of

CLA isomers. It is also possible to obtain CLA

enriched in one particular isomer. Enzymes derived

from fungi such as Candida rugosa and Geotrichum

candidum show a preference for the cis-9, trans-11

isomer, and, by preferentially esterifying this CLA

isomer, give an unesterified fraction that is rich in

the trans-10, cis-12 isomer. This has been demon-

strated on a laboratory scale, but has yet to be opti-

mized for commercial production. Alternatively, a

series of selective crystallisations can be used to frac-

tionate the cis-9, trans-11 and trans-10, cis-12

isomers, to give material of high isomeric purity. In

the future, the production of CLA is likely to expand

to include production of CLA-containing triacylgly-

cerols, as this is the form of lipid most generally

present in foodstuffs.

Analysis of CLA

0009 The primary concern in analyzing CLA is the com-

plete separation and quantitative identification of

the individual isomers in the sample. The form

of the sample will also determine the approach to

analysis. Samples are generally either in the free fatty

acid form, or present as esterified CLA as a compon-

ent in a biological matrix, from which the CLA needs

to be extracted first. A number of analytical tech-

niques have proven useful.

C NMR allows identifi-

cation and quantification of all the positional and

geometrical isomers in a single analysis, but has the

drawback of requiring a comparatively large amount

of sample, so it is not helpful for analyzing biological

extracts. Gas chromatography (GC), gas chroma-

tography–mass spectrometry (GC-MS) and high-

performance liquid chromatography (HPLC) can all

give large amounts of information regarding sample

composition, but for these methods, the sample must

be extracted and also derivatized prior to analysis.

A combination of the above techniques is often re-

quired to give a comprehensive analysis.

0010As CLA is prone to isomerization, care must be

taken in the extraction method chosen to obtain

CLA from tissue samples. A mild alkaline hydrolysis

can be used, for instance, to give the CLA as free fatty

acid, which can then be derivatized for analysis using

one of the methods described below.

0011Prior to GC analysis, fatty acids must be converted

to derivatives (usually methyl esters). Although there

are standard methods used to derivatize methylene-

interrupted fatty acids, it has become apparent over

the last few years that some of these methods, in

particular those using an acidic catalyst, are unsuit-

able for use with conjugated free fatty acids. Under

vigorous conditions, they lead to isomerization of

the sample and artefact formation. Though base-

catalyzed methods do not normally esterify methyl-

ene-interrupted free fatty acids, esterification of free

CLA can be achieved, but some of the methods used

have also shown artefact formation on reaction with

CLA. Phase transfer methods using methyl iodide in a

basic buffer solution have been utilized on a small

scale with consistent results. Despite the drawbacks,

acidic methods can give good quantitative results

providing that the reaction is carried out under mild

conditions with fresh reagent. Esterified CLA can be

converted to the methyl esters by the standard

method of transesterification with sodium methoxide

in methanol solution. The most useful GC columns

for resolving complex mixtures of CLA isomers are

longer, more polar columns such as the 100-m Cp-Sil

88 or the 120-m BPX-70 capillary columns. Neither

of these columns will completely resolve a complex

mixture of CLA isomers, although they will give good

results on most commercial samples. For this reason,

complex mixtures of biological CLA should also be

analyzed by either HPLC, or GC-MS, or both.

0012HPLC has proved especially useful in the analysis

of conjugated dienes, such as CLA. A UV detector is

usually employed as conjugated dienes show a strong

absorbance in the region of 230–235 nm. Silver-ion

HPLC, employing more than one column, can be used

to separate all the positional and geometric isomers

in a commercial CLA mixture, as methyl esters.

Reversed-phase HPLC has been used successfully to

concentrate the CLA methyl esters of biological

samples for further analysis.

0013For GC-MS, a different type of derivative, usually

containing nitrogen, is required to give good-quality

spectra of CLA. Both dimethyloxazoline (DMOX)

derivatives and 4-methyl-1,2,4-triazoline-3,5-dione

(MTAD) adducts are commonly used and can be

used to locate the conjugated double bond positions

in both CLA and its metabolites.

CONJUGATED LINOLEIC ACID 1583

Biological Activity

0014 As mentioned above, CLA has exhibited biological

effects in a number of major medical conditions.

However, many of the CLA mixtures used for early

trials were poorly characterized. As a result, there is

little conclusive information as to which CLA isomers

are the most active and under which circumstances. It

is only recently that individual pure cis-9, trans-11

and trans-10, cis-12 isomers have become available

for clinical trials. Consequently, the discussions

below assume that studies were carried out with com-

mercial isomer mixtures of free fatty acids, although

where a particular isomer has demonstrated a repro-

ducible effect this is indicated.

Anticarcinogenicity

0015 The interest in CLA as a biologically active material,

rather than as a minor lipid component or an oxida-

tion product, began in the early 1980s, when re-

searchers at the University of Wisconsin reported the

discovery of an extract from grilled minced beef,

which was found to inhibit mutagenesis. Since then,

many studies have been performed, both in vivo using

a number of different animals, and in vitro with a

variety of animal and human cell lines. Although other

lipids, such as fish oil, have been shown to have anti-

carcinogenic effects, CLA appears to be potent at

much lower concentrations (e.g., from 0.1% w/w in

rat tumors). A number of studies have concluded that

the cis-9, trans-11 isomer is the active CLA isomer

against cancer, but this remains to be proven.

0016 In vivo, dietary supplementation with CLA de-

creased the number and size of mammary tumors in

rats and mice, independent of the amount and types

of other fat in the diet. However, no effect was seen in

trials using tumor cells from hormone nonresponsive

cell lines such as the WAZ-2T (-SA) and MDA-MB-

231 cell lines. Further studies are required in this

field, as this may be relevant to elucidating the mech-

anism by which CLA acts on tumors. In tests on skin

carcinogenesis in mice, topically applied CLA was

found to be effective in reducing the size and number

of skin tumors, although dietary supplementation

with CLA only affected the number and not the size

of the tumors. Fewer data have been published on

the effect of CLA on prostate cancer and on fore-

stomach and colon cancers, with conflicting data

being obtained in different mouse and rat models.

0017 Much of the in vitro work on carcinogenesis has

been done on the estrogen-responsive MCF-7 human

breast cancer cell line. In the majority of these

studies, CLA has been shown to reduce cell growth

in a dose-dependent manner. Again, however, estro-

gen-unresponsive cell lines failed to show tumor

inhibition on treatment with CLA. Limited numbers

of studies on a variety of other human cancer cell lines

have also shown tumor inhibition in colorectal, liver,

prostate, and lung cells.

0018A number of studies have been conducted to try

and elucidate the mechanism by which CLA acts on

cancer cells. Initial suggestions that CLA acts as an

antioxidant have recently been contradicted by a

number of trials. CLA can block the formation of

carcinogen DNA adducts in rats in certain organs.

Another potential mode of action would be by

affecting the production of eicosanoids implicated in

tumorigenesis. CLA is incorporated into both neutral

lipids and phospholipids, at varying levels, depending

on the tissue type, and can be metabolized to give

18:3, 20:3, and 20:4 fatty acids containing conju-

gated double bonds. Dietary supplementation with

CLA has also been shown to reduce the synthesis of

the eicosanoid prostaglandin E

in mouse epidermis.

However, the direct method of action has still to be

determined.

0019There appears to be a direct link between levels of

CLA in breast tissue during maturation and the

limiting of lateral branching of the terminal end bud

cells of the developing breast, leading to a decreased

risk of breast cancer in later life. Again, the mechan-

ism for this effect has yet to be shown. Interestingly, a

number of studies examining human dietary CLA

intake and breast cancer risk world-wide have failed

to show any consistent positive or negative effect of

dietary CLA on cancer development. This may be due

to limitations of the accuracy of the dietary surveys

conducted.

Body Composition

0020In animal studies, CLA reduced body fat and

increased lean body mass in growing animals, for

a number of species, including mice, rats, and pigs.

Other physiological factors connected with these

effects in different studies were a reduction in lipopro-

tein lipase activity, an increase in the enzyme carnitine

palmitoyl transferase (associated with b-oxidation of

lipids), and reduction in adipocyte size (but not cell

number). Reduced body fat and increased body pro-

tein and water have been directly associated with the

trans-10, cis-12 isomer. Potential mechanisms for

these effects include alteration of membrane structure,

effects on eicosanoid and cytokine production, and

changes in peroxisome proliferator-activated receptor

(PPAR) activity.

0021Feeding of CLA also leads to a loss of monounsatu-

rated fatty acids, which is an effect seen in treatment to

reduce obesity. It has recently been demonstrated that

this effect is due to repression of the expression of the

stearoyl-coenzyme A desaturase gene in fat cells by the

1584 CONJUGATED LINOLEIC ACID

trans-10, cis-12 isomer, leading to smaller lipid drop-

lets containing lower levels of monounsaturated fatty

acids. This is not seen with the cis-9, trans-11 isomer.

0022 A few studies of the influence of CLA on human

body composition have been conducted. The amounts

of CLA fed were variable (between 1.7 and 7 g per

day), and a number of the subjects studied were either

athletes or body builders. In general, although there

was little effect on body weight, in most of the sub-

jects, body fat was either reduced or stayed constant,

and lean body mass was either constant or increased.

However, these variations were in most cases not sig-

nificant. The existing data are not consistent enough

to show a dose-dependent response, and the effects of

exercise should be taken into consideration. Also,

studies in humans were conducted on adults, whereas

animal data were collected from growing subjects. It

has been speculated that feeding CLA affects plasma

leptin levels and thus acts as a control on food intake.

However, recent data in humans showed that, al-

though supplementation decreased leptin levels in

the short term, after approximately 7 weeks levels

returned to baseline values, and no alterations in

either appetite or body composition were observed.

Further studies are required with individual isomers,

in particular the trans-10, cis-12 isomer to assess fully

the effects and the potential to treat obesity.

Immunity

0023 In mice, dietary CLA is incorporated into liver lipids

by displacing linoleic and arachidonic acids, thus

modifying the n-6 fatty acid composition of the phos-

pholipids. Subsequent experiments in rats have shown

that this phospholipid modification affects eicosanoid

production. For instance, feeding 1% CLA for 2 weeks

reduced serum prostaglandin E

levels by approxi-

mately 50%. CLA can also lower the levels of some

leukotrienes (e.g., LTB

) in rats. This may then affect

cytokine production, but further studies are required

in this area. It has effects on immunoglobulin produc-

tion, specifically increasing levels of IgA and IgG,

which are both associated with antiallergic effects.

0024 Few data are currently available from human stud-

ies, but a recent trial on women showed no changes in

immune function during the course of the study. This

may be due to a dose effect, as the levels of CLA per

weight in animal models tend to be much greater than

the levels fed in the human studies.

Atherosclerosis

0025 Experiments on atherosclerosis have been conducted

on both rabbits and hamsters. Feeding CLA at 0.1–

0.5% of the diet in rabbits for 22 weeks showed a

reduction in plasma triacylglycerol, total cholesterol,

and LDL-cholesterol levels. There was also an effect

on plaque formation, with a reduction in the CLA fed

animals. In hamsters, CLA feeding at similar levels

was found to reduce total cholesterol and fatty streak

area, although linoleic acid feeding showed similar

effects. An isomer effect was seen in the hamster

model, with only the trans-10, cis-12 isomer decreas-

ing triacylglycerol and cholesterol. Contradictory

data have been obtained in C57BL/6 mice, in which

an increase in aortic fatty streaks was reported.

Bone Metabolism

0026Lipids play an important part in bone metabolism as

they are known to influence both bone modeling and

remodeling. Both CLA and n-3 fatty acids modulate

the production of prostaglandin E

in chicks and rats,

leading to increased bone formation and decreased

resorption. CLA is thought to act on prostaglandin

production by influencing the cyclooxygenase

enzymes, which produce prostaglandin E

from ara-

chidonic acid. This may occur through the production

of competitive 20:4 conjugated CLA metabolites. As

also seen in other studies, CLA reduces the level of

LTB

, which is a strong bone resorption factor. CLA

may influence cartilage functions in growing animals.

As yet, few experiments have been conducted with

CLA, and further data are required to elucidate its

mechanism of action.

Diabetes

0027In type 2 diabetes, treatment aims to reduce insulin

resistance and improve glucose uptake. In in vitro

tests, chemicals such as thiazolidinediones show

these effects by directly affecting PPARg expression.

CLA, in particular the cis-9, trans-11 isomer, has also

been shown to bind to, and activate, PPARg, as have

other unsaturated fatty acids. In Zuker diabetic fatty

(fa/fa; ZDF) rats, a diet containing 1.5% CLA pro-

duced normal glycemic responses to tolerance tests,

as well as reduced hyperinsulinemia.

Enhanced CLA Levels and Food

Production

0028In animals, there are two different reasons to try to

increase the levels of CLA in tissue and/or milk.

Firstly, CLA has effects on body composition, leading

to, for instance, increased lean meat and lowered

levels of fat in pigs and cows. This is becoming more

desirable as public concern increases due to suggested

links between increased dietary levels of fat and con-

ditions such as heart disease and obesity. The second

reason is because of the potentially beneficial effects

of CLA itself in a number of diseases. Although

CONJUGATED LINOLEIC ACID 1585