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Critical Reviews in Food Science and Nutrition

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Authenticity Assessment of Dairy Products


Miguel Angel De La Fuente & Manuela Jurez

Instituto del Fro (CSIC), Jos Antonio Novais 10, Ciudad Universitaria s/n , Madrid, Spain
Published online: 18 Jan 2007.

To cite this article: Miguel Angel De La Fuente & Manuela Jurez (2005) Authenticity Assessment of Dairy Products, Critical
Reviews in Food Science and Nutrition, 45:7-8, 563-585, DOI: 10.1080/10408690490478127
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Critical Reviews in Food Science and Nutrition, 45:563585 (2005)

C Taylor and Francis Inc.
ISSN: 1040-8398
DOI: 10.1080/10408690490478127

Authenticity Assessment
of Dairy Products

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Instituto del Fro (CSIC), Jose Antonio Novais 10, Ciudad Universitaria s/n, Madrid, Spain

The authenticity of dairy products has become a focal point, attracting the attention of scientists, producers, consumers,
and policymakers. Among many others, some of the practices not allowed in milk and milk products are the substitution
of part of the fat or proteins, admixtures of milk of different species, additions of low-cost dairy products (mainly whey
derivatives), or mislabeling of products protected by denomination of origin. A range of analytical methods to detect frauds
have been developed, modified, and continually reassessed to be one step ahead of manufacturers who pursue these illegal
activities. Traditional procedures to assess the authenticity of dairy products include chromatographic, electrophoretic, and
immunoenzymatic methods. New approaches such as capillary electrophoresis, polymerase chain reaction, and isotope ratio
mass spectrometry have also emerged alongside the latest developments in the former procedures. This work intends to
provide an updated and extensive overview since 1991 on the principal applications of all these techniques together with
their advantages and disadvantages for detecting the authenticity of dairy products. The scope and limits of different tools
are also discussed.

cheese, fraud, milk, protected denomination of origin

The authenticity of foods is currently of major concern for researchers, consumers, industries, and policymakers at all levels
of the production process. An authentic raw material or finished
product has to comply with labeling, principally in terms of
ingredients, production technology, and genetic identity. Dairy
products are of particular interest, because they are a group of
foods that play an important role in feeding the population and
are essential for certain groups of consumers (women, children,
and the elderly). Milk is a fairly expensive raw material, and
from an economic point of view it could, therefore, be attractive
to modify its composition and replace part of it with other dairy
or non-dairy ingredients. European regulations are strict on this
matter: only skimming and some additions (minerals, vitamins,
and milk proteins) are legally allowed in milk. For instance, replacing milk fat or protein with another of different origin is a
practice that is not allowed, even though this substitution could
enhance the nutritional value of the final product.
Ramos and Juarez1 and Juarez2 reviewed the main possible
frauds in dairy products and the analytical procedures for detecting them. Since then, progress in dairy chemistry and technology
Address correspondence to Miguel Angel de la Fuente, Instituto del Fro
(CSIC), Jose Antonio Novais 10, Ciudad Universitaria s/n, 28040 Madrid, Spain.

has led to the manufacture of specialized milk products. Unfortunately, this progress has also provided opportunities for very
sophisticated types of manipulations that are difficult to detect.
One specific current challenge is to control the labeling claims
on high-quality dairy products. Protected Denomination of Origin (PDO) cheeses, for instance, have distinct characteristics and
enhanced quality and are defined according to their geographical area of production, as well as in terms of the materials and
technology used in their manufacturing. The determination of
origin is a key component of PDO. Criteria and procedures for
assessing the authenticity of these high-quality products need to
be developed.
Lees3 reported the legislation and analytical techniques currently available for controlling the authenticity of milk products
in Europe. However, while strict standards and criteria for product definition exist, practical means for judging product authenticity are not always available. The main approach for solving
these kinds of problems is to look for a specific marker or indicator in the product, which could be a component (complex
molecule, nucleic acid), or determine the ratio between some
chemical constituents and assume that these ratios are a constant
component of the particular dairy product. From this perspective, it seems to make sense that the addition of any substance to
milk products will modify the value of these ratios or highlight
an anomaly in their chemical composition. In this area, many
pattern classification procedures can be applied to the dataset to


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compare similarities or differences in the sample data with the

original data.
Analytical dairy science has advanced significantly over the
last decade, the main reason being the availability and improvements in advanced techniques, such as chromatography, immunoenzymatic assays, and electrophoresis. In addition to new
developments in these techniques, the interdisciplinary and dynamic nature of milk product analysis is being enhanced by
the application of disciplines already used to analyze other
foodstuffs. Among them, capillary electrophoresis (CE), polymerase chain reaction (PCR), and isotope ratio mass spectrometry (IRMS) are just gaining popularity for solving dairy authenticity problems. The aim of this article is to provide an updated
review (19912003) of the methodologies that have been identified as showing potential for addressing the issues related to
the authenticity of dairy products and also to collate information on ongoing research into this matter. In line with European
regulations, we will focus primarily on the detection of foreign
fat or protein in milk products, admixtures of the milk of different species, measurement of the presence of whey derivatives
in milks, determination of geographical origin of high-quality
dairy products, and the study of the contents of heated/dried
milks added to milk.

Fatty Acid Composition

Analytical methods for assessing the authenticity of milk fat
based on fatty acid composition have a long tradition2,15 and
were adopted by the International Dairy Federation (IDF) as the
basis for the quantification of milk fat in fat mixtures.16 These
methods are supported by the fact that the presence of butyric
acid is rather unique to milk fat. To increase the reliability of
these methods, the content of other fatty acids was also included
in the calculation. For example, the amount of butyric acid or
some ratios involving butyric and other fatty acids (caproic, lauric or oleic acids) determined by analysis of fatty acid methyl
esters are still used as parameters to analyze admixtures of vegetable fats to milk fat.17,18 However, low percentage (<10%)
admixtures of vegetable oils with fatty acid composition similar
to milk fat render standard fatty acid methyl ester analysis difficult. This approach is also limited by the natural variability of
the fatty acid composition of milk fat due to variations in feeding depending on season and region, to genetic factors, and to
the nutritional status of the cattle. Thus, statistical evaluation of
large data sets by linear discriminant or multivariate regression
analyses is often required.19,20

Analysis of Triglycerides
Milk fat is a valuable fat for human nutrition and represents
an expensive raw material. Of all milk fat products, butter is the
most important one for economic reasons, and it must, therefore, have high standards of quality. In this respect, the E.U. has
developed strict standards of identity for butter and has established that this dairy product must be obtained exclusively from
milk or cream. In spite of this, butter has always been subjected
to adulteration by the addition of less expensive vegetable or
animal fats. As a result, the detection of non-milk fats in butter
is far more important today than ever before, and a number of
investigations have been carried out by several research groups
to develop analytical methods for this purpose. In recent years,
different reviews have also appeared with updated information
on the detection of non-milk fats in butter.49
Even though certain techniques of physical analysis such as
scanning calorimetry have been successfully applied,1012 most
current analytical approaches to authenticity issues in milk fat
constituents are essentially based on the Gas Chromatography
(GC) separation of its components. Nowadays, the best way of
revealing the presence of foreign fats in milk fat includes the
study of the fatty acid composition, the triglyceride (TG) profile, and the different fractions of other minor lipid constituents,
mainly from the unsaponifiable fraction. Other recent works
have considered the use of complementary techniques in the
identification of milk fat such as pyrogram fingerprints obtained
by pyrolysis GC with Flame Ionization Detector (FID), as well as
the determination of compounds contributing to milk fat aroma
by headspace GC.13,14

Alternatively, the analysis of intact TG is regarded as advantageous, because the genetically controlled specific distribution
of the fatty acid moieties on the glycerol backbone is preserved;
the information content is, thus, higher. Although milk fat TG
composition is also affected by variations in feeding, depending
on the season and region, determination by GC of classes of
milk fat into groups of identical numbers of acyl-C atoms (C24C54) has been reported to be a more effective criterion than fatty
acid composition for determining their origin.21,22 Based on the
original idea of Timms,23 a mathematical transformation was
performed in order to stabilize the natural variability of the TG
profile of cows milk fat. On the basis of 76 Australian milk fat
samples, pure milk fat was characterized by an equation that included terms for the percentages of the TG C40, C42, and C44,
and levels of foreign fat as low as 5%23 were detected. More
exhaustive and accurate studies carried out by Precht21,2426
on 755 samples of pure milk and on samples of animal and
vegetable fat by multiple regression analysis modified the equation increasing the procedure sensitivity to detect different foreign fats in milk fat. The limit of detection varied according to
the source of the added fat, but was generally <5%. Alternative computational models (artificial neural networks, principal
component regression, and partial least-squares regression) were
equally effective in detecting lower levels of adulteration.2730
The method based on these equations has been officially accepted by the E.U. (European Union)31 for the determination of
the purity of milk fat, and limits are given to assess the presence of different foreign fats. Its applicability is confirmed in
one Belgian and one Italian report32,33 and in another report,28

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where butter samples from different E.U. Member States are

Although the official E.U. method for the determination of the
purity of milk fat is founded on TG analysis by packed column
GLC, the original E.U. regulation31 and its revised versions34,35
foresee capillary columns as an alternative technique, provided
the same results are obtained. Different works carried out to assess the authenticity of milk fat using short capillary (less than
5 m) column GC have been described.22,3641 This abundance
of studies is due to the development of better columns with high
thermal stability and selectivity of the stationary phase. In addition, short capillary column analysis reduces the analysis time,
consuming less carrier gas, and providing similar levels of accuracy than packed columns. However, there has been some controversy about the quantitative aspects of TG profiling of milk
fat by capillary column GC. Although capillary GC was equivalent to the packed column in terms of analytical precision and
all the samples tested fulfilled the purity criteria (S-values), irrespective of the chromatographic technique used, Ulberth et al.41
observed that S-values obtained by capillary GC deviated to
some extent from those of the packed column, and differences
exceeded the value for reproducibility laid down in the European
rules. A later study39 was carried out by testing the purity of 50
widely varying samples of milk fat in accordance with E.U.
rules. For all the samples, the differences between S-values obtained from packed and capillary column data did not exceed
the reproducibility limits stipulated by E.U. rules. However, in
order to guarantee comparable results, these authors39 recommended rechecking by each laboratory wishing to use a capillary instead of a packed column to monitor the purity of milk
In the quantitative analysis of milk fat TG, the introduction
technique is critical, because the sample consists of a mixture
of TG with a wide range of volatilities, and relatively high temperatures are required for complete vaporization. Classic hot
split injection was by far the least suitable technique since it
causes strong discrimination and decomposition. Although oncolumn injection has been the most frequently used approach to
reduce these undesirable effects22,37,38,40,41 Programmed Temperature Vaporizer (PTV) Injection has also been assayed.40,42
Banfi et al.42 demonstrated that PTV injection gives results comparable to those obtained with the on-column injector. The repeatability and reproducibility obtained with PTV widely fulfill
the requirements of the official E.U. method, demonstrating that
this configuration can be applied as a valid alternative to the
on-column injection system.
Owing to differences with cows milk fat TG profiles
(Figure 1), the equations proposed by E.U. rules are not suitable to monitor goats or ewes milk fat. To detect foreign fat
in goats and ewes milk fat, new multiple regression equations based on TG contents of the goats and ewes fats have
been proposed.43,44 Fontecha et al.43 applied these mathematical equations to provide a fast and highly sensitive means of
determining mixtures of non-milk fats in goats milk. Although
this procedure could also be very useful for specific detection


of mixtures of non-goat milk fat in goats milk, the limit of

detection reached was not very low. The method for determination of TG proposed by Precht25,26 was also applied for analyzing different types of cheeses to detect the presence of non-dairy
fat.45 In cheeses with extensive lipolysis, the Precht formula erroneously indicated the presence of non-dairy fat; this was attributed to the decrease in low-molecular weight TG produced
by the lipases. In unlipolyzed samples, or those with only slight
lipolysis, this equation, however, successfully highlighted the
presence of undeclared non-dairy fat. It was concluded that a
new formula should be developed for cheeses with extensive

Analysis of Minor Components

Analysis of minor components, mainly constituents of the
unsaponifiable matter, can be an indispensable tool for authentication purposes. Traditionally, sterol analysis has been used for
the detection of admixtures of animal fats and vegetable oils by
determination of cholesterol and phytosterols. This determination is the most sensitive method for differentiating vegetable
and animal fat. Animal fats, such as milk fat, primarily contain
cholesterol; phytosterols are not detectable or only present at
trace levels. Among the different sterols present in vegetable
oils, -sitosterol is usually the main constituent, therefore, a
suitable marker for the detection of the addition of vegetable oil
to milk fat.
The Provisional IDF method46 to determine sterols in milk
fats involves saponification of the lipids, extraction of the unsaponifiable matter, pre-separation by Thin-Layer Chromatography (TLC), derivatization of the sterols, and subsequent GC
analysis (Figure 2). Presently, it is still published as a provisional
standard, due to the absence of repeatability and reproducibility,
even though several interlaboratory tests have been performed
over the last few years. The high variability should be attributed
to the steps required. Contarini et al.47 showed that the accuracy
of determining cholesterol in milk fat by this method strictly
depended on evaluating the correction factor for the internal
standard; therefore, it should be done at the same time as the
sample analysis. A detection limit of 45% for vegetable fat
was determined in milk fat.
Apart from its analytical disadvantages, the IDF method46 is
tedious and time-consuming and thus, not advisable as a routine procedure. In order to carry out rapid and reliable determinations for sterols and simplify the analyses, different alternatives have been developed over the last 10 yr (Figure 2). The
use of fused silica columns GC4850 meant that the sterol fraction derivatization step could be eliminated, thereby reducing
the analysis time and improving sensitivity. The unsaponifiable
matter was also directly analyzed by GC as free sterols51 or
trimethylsilyl derivatives.52 In this way, isolation of the sterols
by preparative TLC could be avoided. Alonso et al.53 analyzed
the sterol profile after sample alkali-catalyzed transesterification
with KOH/methanol by GC. This method requires little analysis


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Figure 1

Gas chromatographic profiles of triglycerides of goat (A) and cow (B) milk fat samples using short capillary column. (Reproduced with permission

time and eliminates the need for saponification, extraction, and

derivatization steps (Figure 2). It is, therefore, suitable for routine use to detect possible additions of vegetable oils in milk

Although most of the procedures used to assess the sterols

contents include a GC as a final step, determination by HPLC
has also been reported.54,55 Kamm et al.56 developed a method
combining HPLC and GC that involves transesterification of the

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Figure 2 Scheme of the analytical procedures to determine sterol fraction

composition in milk fat by gas chromatography.

fat, pre-separation of the sterol fraction from other lipid constituents, and on-line transfer to the capillary GC system. The
on-line approach eliminated time-consuming sample preparation steps prior to GC analysis, and meant that it was possible
to detect adulterations at low levels of cotton and rapeseed oil
in milk fat using -sitosterol as a marker.
Other minor components have been reported to establish the
criteria for assessing the authenticity of milk fat and derivatives.
Milk fat diglycerides C30-C36 showed characteristic fingerprints compared with other fat sources, especially with tallow.57
The 3,5-cholestadiene, a derivative from partial dehydroxylation
of cholesterol during refining treatment, was found to be an index
for the addition of refined beef tallow to butter.58 These markers
could be used in addition to the most common ones described in
the previous paragraphs. Povolo et al.57 reported an interesting
approach based on the combination of data from the determination of different compounds (diglycerides, 3,5-cholestadiene
and TG) by GC to detect the presence of beef tallow in milk
fat. When the results were evaluated separately, the methods did
not seem to be able to decrease the limit of detection below 5%.
The application of multivariate statistical techniques increased
the power of these analyses, and the statistical model calculated
was able to detect the presence of 2% of beef tallow; it could be
applied when the results obtained by the Official Method35 are
close to their limit of detection.


Identification of the species of origin for milk products is
important from a theoretical standpoint and useful in practice,
since it allows the detection of fraud by substituting a less costly
type of milk for one of a higher quality. In comparison with cows
dairy products, goats, ewes, and buffalos products are more
expensive, and authentication is, therefore, of great economical


interest. Some dairy products should contain defined proportions

of milks from different ruminant species, whereas others, such as
PDO cheeses, with a higher market value can only be made with
pure milk from cow, goat, ewe, or buffalo, and milk addition
from another species or even breed is not allowed. Adequate
control methods are required to check the presence of cheaper
milk in PDO cheeses. Apart from economic loss, correct species
identification is important for the consumer for other reasons:
medical requirements, food allergies, or religious practices. This
situation has prompted research to find methods for detecting the
species of origin of dairy products.
Although analysis of selected fatty acid by GC59,60 and TG
by NMR61 to distinguish milks from different ruminant species
can be found in the literature, most of the works on this issue
involve protein fraction studies. The present European Community reference method62 for the detection of cows milk in
ewes and goats milk products is based on isoelectric focusing (IEF) of -caseins, peptides originated from -casein after
enzymatic proteolysis by plasmin. A sample is judged as being positive if both bovine 2 - and 3 -caseins are equal to or
greater than the level in the 1% standard (ovine or caprine). For
a more accurate analytical result, samples must be analyzed simultaneously in an IEF gel with two reference standards of milk
with 0 and 1% cows milk, calculating the bovine milk present
by comparing the intensity of the bovine 2 - or 3 -caseins with
1% bovine milk reference. Although the scope of the qualitative
detection of cows milk in milk and cheese of other species is
covered by this method, the quantitative determination of cows,
ewes, and goats milk in ternary mixtures remains a subject for
further study, because ewes and goats milk could not be distinguished. Moreover, the IEF method is time-consuming and
requires special equipment, as well as specially trained technical
staff. Several alternative methods (chromatographic, immunological, and electrophoretical) have been published to authenticate the species of origin of milk products, and they might be
applicable to routine analysis. Identification procedures based
on DNA extraction and amplification are also now emerging.

Procedures based on different types of electrophoresis
abound in the literature. Different studies6365 have tried to improve the sensitivity of the IEF reference method introducing
detection by immunoblotting with polyclonal antibodies (PAB)
against bovine milk proteins. Mayer et al.66 complement IEF
with cation-exchange HPLC to determine para--caseins. As
these molecules were not substantially affected by the degree of
ripening, the method could be suitable for determination of the
percentages of cows, goats, and ewes milk in mixed cheese.
However, quantitative results in cheeses had to be regarded as
approximate values. This is because the estimated percentage
of cows milk in mixed cheese is greatly affected by the casein
content of the milks used for cheese making and the different
technological procedures applied.

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Traditional methods such as polyacrylamide gel electrophoresis (PAGE) have been used to identify milk from goat,
ewe, and cow in their mixtures67,68 to detect bovine milk
in ovine or caprine cheeses,6971 or to ensure the authenticity of ovine yogurts and guarantee that they are accurately
labeled.72 Another study65 detected denatured bovine whey proteins in cheeses from ewes and/or goats milk combining protein separation by PAGE with western blotting detection using
commercially available polyclonal anti-bovine -lactoglobulin
In comparison with IEF, PAGE is less costly and requires
fewer technical skills. However, in a routine examination, it
is still time-consuming and labor intensive. Furthermore, it
also presents the disadvantage of poor visualization of small
molecules. Presently, capillary electrophoresis (CE), with its
high resolving power, rapid method development, easy sample preparation, and low operation cost, has been shown to
have the greatest potential for different foods and also dairy
products.73,74 Capillary zone electrophoresis (CZE) has been
used to detect adulteration of goats and ewes milk, with cows
milk, on the basis of the different migration time of the S1 casein fractions of the different species. Cattaneo et al.75 demonstrated the capability of CZE to analyze milk mixtures and to
determine up to 8% of cows milk content with coated silica capillary. The use of uncoated capillary CZE meant that
costs were reduced, and that the addition of raw or reconstituted cows milk in goats milk of as little as 1%76 could be
The differences between the CE profiles at low pH of the casein fraction from 100% cows, ewes, or goats milk provided
the basis for the selection of different peaks, characteristic of the
presence of these milk types in ternary mixtures. Molina et al.77
used the Principal Component Regression (PCR) and Partial
Least-Square regression (PLS) to predict the percentages of milk
of each species in the mixture. Thereafter, they characterized78,79
the casein fractions of cheeses made from goats, ewes, and
cows milk to identify the major protein components of the casein fraction and their breakdown products. Caprine para-casein and bovine -casein peaks were indicators of the presence of milk of these species. These peaks could be seen in
cheeses made from mixtures and could be potential indicators
for identifying and quantifying these mixtures. Other authors80
showed that a CZE electropherogram of the ethanol-water protein fraction in isoelectric acidic buffers contains enough information to classify cheeses according to both, genetic origin
and ripening time. On the basis of the electropherograms, and
using PLS multivariate regression, the contents of cows milk
in presumably pure goat and ewe cheeses, as well as in binary
and ternary mixtures, was predicted with low relative standard
The study of whey protein profile by CZE could also be used
as a tool to determine the content of cows milk in mixed dairy
products. A method using a methyl silanized capillary column in
borate buffer (pH 9.2) based on establishing different ratios between the whey proteins present in the acid soluble fraction was

applied to detect bovine milk in buffalos,81 ewes,82 and goats83

milk and cheese; amounts of between of 0.5% and 5% were detected. The qualitative analysis of ternary mixtures has also been
reported,83 but the loss of resolution between the -lactalbumin
and -lactoglobulin caprine and the overlap between goat and
ewe -lactalbumins did not allow accurate quantitative analysis of triple mixtures and double mixtures of goat-ewe milk.
Herrero-Martnez et al.84 developed a simple CE method for
the separation of bovine, caprine, and ovine whey proteins in
binary and ternary cheeses giving limits of detection as low as
1% in very short times. They used acidic isolectric buffers supplemented with a surfactant (Tween 20) that allowed the separation of bovine -lactalbumin and -lactoglobulin from small
peptides arising from casein degradation during cheese ripening. An additional advantage was that the methodology protocol
did not require coated capillaries that are necessary in methods
adopting alkaline buffers, and the analysis costs were, therefore,
substantially lower.
To summarize, although important advances have been made,
CE applications for solving problems of authenticity still have a
long way to go before they are complete. We could safely say that
CE, accompanied by instrumental development (MS detection,
new injection devices to enhance precision, microfabricated systems for routine protein separation), could become much more
popular in the future.

Chromatographic Methods
High performance liquid chromatography (HPLC) is very
automated and has proved to be a valuable quantitative technique for protein analysis in dairy products. Reversed-phase
HPLC (RP-HPLC)8589 and ion-exchange chromatography90
were used to separate whey proteins from milks of different species. Separation of caseins from bovine, ovine, and
caprine milk using various HPLC procedures has also been
reported.9193 However, separation time is usually within the
3050 min range, which is rather long for some routine quality control purposes. Moreover, some of these studies revealed
complex chromatograms,87 while others did not detect the presence of goats milk in triple mixtures of goat-ewe-cow milk and
binary combinations of goat-ewe milk.88
In a comparative study94 of caseins fraction of bovine, ovine
and caprine milks, results obtained by HPLC matched the ureaPAGE method. However, although HPLC was more efficient
for quantitative results, electrophoresis was shown to be more
sensitive to detecting adulterations.
In general terms, chromatographic and electrophoretic techniques based on physicochemical properties of mixed native
proteins could become complicated after the proteolysis that
occurs during ripening processes, since intermediate protein
and peptide products appear. Moroever, methods based on
the detection of heat-sensitive proteins such as whey proteins could become ineffective if heat-treated milk is used for



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Immunological Methods
Immunological procedures based on antigen-antibody precipitation reactions are suitable to authenticate dairy products
because of their sensitivity to differentiating milk proteins from
different species. The main characteristic of these methods is
their high specificity derived from the antigen-antibody reaction. In addition, they have the advantage of being inexpensive,
quick, sensitive and specific compared with conventional detection methods. Immunoassay kits are easy to use in the laboratory
for detecting the authenticity of dairy products and they can successfully be applied to the food sector. The most used immunological technique for testing the authenticity of dairy products
is enzyme immunoassay using Enzyme Linked Immunosorbent
Assay (ELISA) format. The main advantages of ELISA, apart
from the ones mentioned above, are that it provides quantitative
results and can be widely used as a routine procedure.
Antibodies used in immunoassays, depending on the production method, can be polyclonal or monoclonal (MAB). The
generation of PAB is less expensive and time-consuming than
MAB, which could explain the higher number of studies on the
former in the literature. Numerous ELISA methods with PAB for
the detection of bovine proteins in goats and ewes milk,95104
and goats milk in ovine milk products98,105108 based on different antigens (caseins and native and denatured whey proteins)
and varying techniques have been reported. Details about the
most recent approaches are shown in Table 1.
In the immunological detection of interespecific adulterations
of dairy products, the main problem lies in the production of antibodies exclusively specific for milk proteins from one species.
A strategy to enhance specificity was based on the generation
of antibodies against short specie-specific peptides located in
the primary sequence of caseins. PAB raised in rabbits against
the bovine S1 -casein sequence 140149100 appeared monospecific for bovine S1 -casein, since no antibody-antigen complex
was formed with homologous ovine or caprine proteins. It was
suggested101 that only one amino acid, glutamic acid residue
in position 148, was essential for the antigenic character of the

Table 1

bovine peptide. Similarly, Richter et al.103 successfully applied

an ELISA based on PAB against bovine 3 -casein during an E.U.
collaborative study for the detection of cows milk in goats and
ewes milk and cheese.
The use of MAB in these studies is currently finding an increasing number of applications, replacing PAB (Table 1). MAB
have several advantages compared with PAB: their production
is less influenced by the purity of antigen; they give less crossreaction and provide more homogeneous affinity. Moreover, as
PAB production relies upon the immunization of laboratory animals, their use presents a problem in the long term when a consistent supply is needed for their large-scale production and commercialization. Hybridoma technology has provided the means
to overcome this problem, as it allows the continuous production
of MAB of consistent specificity. At the beginning of the nineties
some MAB directed towards milk proteins were isolated and
characterized.109111 However only more recent studies tested
the cross-reactivities of MAB against proteins from bovine or
caprine milks in dairy products. These MAB produced against
whey proteins112,113 or caseins114118 were used to detect the
presence of milk from different species in milk products.
Whey proteins may be denatured by thermal treatment modifying their immunoreactivity and consequently the reliability
of the method. On the contrary, immunoreactivity of bovine caseins is less affected by conventional heating and would have a
great potential for detecting and quantifying bovine milk in milk
mixtures and dairy products. Assays using two MAB raised in
mice against cows -lactoglobulin detected 1 part cows milk
per 100.000 parts goats or ewes milk.112 Preliminary immunological characterization119,120 of bovine caseins indicated that
-casein fraction bear the largest number of cow-specific epitopes and display the highest antigenicity. One of the MAB
obtained against -casein was applied in different ELISA to detect and quantify the presence of cows milk in goats and ewes
milks114,115 and cheeses.116
These procedures could be suitable for the commercial production of stable kits for caprine milk identification in unknown
milk mixtures on large- or small-scale inspection programs

Enzyme linked immunosorbent assay (ELISA) in dairy products authentication


Type of antigen

Type of

ELISA format

Goats milk in ewes milk

Goats milk in ewes milk and cheese
Cows milk in goats milk and cheese
Cows milk in ewes and goats cheese
Cows milk and caseinate in goats and ewes milk and cheese
Cows milk in goats milk
Cows milk in ewes and goats milk
Goats milk in ewes milk and cheese
Cows milk in goats and ewes milk
Cows milk in ewes milk and cheese
Cows milk in goats and ewes milk and cheese
Cows milk in goats milk

Whey proteins
s1 -casein
3 -casein
s2 -casein
Whey proteins


Indirect Competitive
Indirect Competitive
Indirect Competitive

Limit of detection





Table 2

Polymerase chain reaction in dairy products authentication


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Cows milk in goats and ewes milk and cheese

Cows milk in goats milk
Cows milk in goats milk
Cheeses from different species
Cows milk in goats, ewes, and buffalos milk and cheese
Cows milk in buffalos cheese

Limit of detection

Fragment of
amplified DNA

Cycles of

Use of restriction




Citochrome b mitochondrial
Citochrome b mitochondrial
Citochrome b mitochondrial
Citochrome b mitochondrial




and could also be applied to detect fraudulent substitution of

goats milk in traditional cheeses that must be made with pure
ewes milk (Manchego, Feta, Roquefort, Pecorino). S2 -casein
was characterized as the most immunoreactive caprine caseins
fraction.121 Immunization of mice with this purified fraction
meant that it was possible to detect eight MAB, and from these
it was the hybridoma cell line B2B that appeared to be species
monospecific and only reacted with the caprine S2 -casein.122
Thereafter, this antibody has been used in ELISA for the detection and quantification of the presence of goats milk in ewes
milk117 and cheese.118 It was confirmed that the antigenicity of
caseins was not affected by ripening, since no significant differences were noted between ewes cheese mixed with fresh,
semi-hard and hard goats cheese. Although experiments on the
use of the purified goat S2 -casein fraction subjected to limited
proteolysis by enzymes and determination by immunoblotting
of the location of the epitope recognized by the MAB B2B were
not performed, results presented from hard cheese118 suggested
that the epitope may be a short continuous peptide fragment.

Polymerase Chain Reaction (PCR)

The origin of dairy products from certain animal species can
also be determined by DNA studies. Milk from healthy mammary glands contains a large number of somatic cells (epithelial cells and leukocytes). These cells are concentrated during
processes, such as cheese making, and can be used to isolate
and amplify DNA and discriminate species. In comparison with
protein, DNA assays may have the advantage of increased sensitivity and rapid performance with high sample numbers being
automatically processed. Furthermore, these methods could be
advantageous for highly processed milk products treated with
high temperature and pressure or submitted to long periods of
storage, because they are methods based on more resistant material such as nucleic acids. As we mentioned above, protein-based
methods for species identification may fail after excessive proteolysis or heat-induced denaturation.
PCR makes it possible to amplify defined DNA-fragments
in a very short time by a factor up to some millions in three
steps (denaturation to obtain a single-stranded DNA, annealing
in which primers flank the molecular region of interest, and finally extension where the DNA polymerase synthesizes the new
strand). Subsequently, the amplified DNA can be analyzed by

various molecular biological procedures, mainly by size fragment length polymorphism, and visualized with ethidium bromide after electrophoresis in agarose gel. The decisive advantage
of PCR-analysis in comparison to protein chemical methods is
that, in contrast to specific proteins, the whole DNA is always
identically present in every organ of a specie. Because of this,
it is possible to determine molecular genetic differences very
clearly. Molecular biology techniques have been used to identify the species of origin in foods, especially in meat products.
The use of this methodology in dairy products has been limited
to detecting bacterial contaminants, and only in recent years has
it been applied to control authenticity and differentiate between
species relevant to the dairy industry (Table 2).
Although Lipkin et al.123 showed that it was possible to use
milk as a source of DNA and as a substrate for PCR, at that time
the quality of purified DNA was an unresolved problem, and
some difficulties emerged in recognizing the milks of closely
related species, (ewe, goat, cow, and buffalo) because of the
possibility of cross-reaction. It was also not possible to define
highly specific regions in genes, which could be used for an unequivocal determination by means of PCR. Furthermore, milk
would seem to be a very complex food system with an abundance
of potential PCR inhibitors. It has been reported124,125 that certain compounds in fungi-containing cheeses (Danish blue, vein
Brie, and Brie) may interact with the DNA or the enzymatic
PCR reaction and inhibit it.
Since then, new developments have improved the efficiency
of these techniques. Such advances include rapid and reliable
procedures for isolating genomic DNA from dairy samples based
on the use of resins126,127 or spin columns.125,128 The finding129
that free DNA is principally located in the cream fraction could
be of great relevance when very small amounts of target DNA
are present. Concerning the inhibitors of PCR reaction, it has
been tested that the heat treatment of milk128,130 and the drying
or freezing of the cheese or fungi used during cheesemaking128
did not seem to interfere with the detection method, whatever
the milk treatment or nature and ripening of the cheeses tested.
Material employed in amplification consists of nuclear or
mitochondrial DNA. Specific -casein gene PCR with universal primers encoding a partial sequence of gene was performed
to detect the corresponding DNA in dairy products.125,131 In the
PCR product from ovine or caprine -casein, DNA was shown
to contain a specific restriction enzyme site that is not present in
bovine -casein DNA. Accordingly, after selected restriction

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enzyme analysis and PAGE, the undigested bovine -casein

fragment can be detected as an additional band if cows milk
is present,125 or by more sensitive enzyme immunoassay using
primers labeled with biotin or digoxygenin.131 Branciari et al.127
described a procedure to determine the species of origin of milk
used in cheese manufacturing, based on isolating the mitochondrial DNA and the use of PCR-restriction fragment length polymorphism of cytochrome b gene sequences. Current knowledge
suggests that mitochondrial DNA may be more suitable than
nuclear DNA for this kind of analysis. This material is attractive
because of its variability (mitochondrial cytochrome b gene sequences differ by at least a few nucleotides, even in very closely
related species), the availability of mitochondrial sequence data
from many vertebrates and the high copy number relative to the
nuclear DNA.
The methods discussed above125,127,131 are based on PCR associated with restriction fragment length polymorphism, which
entails the amplification of a common fragment followed by digestion with one or more species-specific restriction enzymes.
In contrast, design of species-specific primers has meant that it
has been possible to simplify the technique avoiding the need for
subsequent restriction fragment analysis. Different works have
been reported in this direction.128,132,133
Another alternative approach includes duplex-PCR. It allows
co-amplification of separate regions of a single gene or specific fragments, each typical of a different animal species in
a single PCR reaction. The duplex-PCR technique has been
proposed134,135 to identify bovine and water buffalo DNA in a
single PCR assay in milk and mozzarella cheese originally made
from pure buffalo milk. Primers were designed within the cytochrome b gene region of the mitochondrial DNA. The results
of these experiments indicated the applicability of this method,
which showed an absolute specificity for the two species and
a high sensitivity even to low DNA concentrations. The minimum concentration of bovine tested in buffalo and buffalo in
bovine milk was 1%,134 just the percentage considered to be the
minimum legal limit.62
Molecular genetic techniques could also be used to identify the breeds involved in milk production. For instance, some
PDO cheeses have restrictions, because they should be made
only from specific breeds milk. Maudet and Taberlet136 developed a quick and easy DNA-based test to specifically detect
Holsteins breed milk in French PDO cheeses that are not allowed to contain milk from this breed. They found a molecular
marker for Holsteins milk detection in a gene affecting coat
color in cattle. A single nucleotide substitution in that gene allowed the differentiation of breeds. DNA extraction from cheese,
a pre-amplification of the gene and a competitive oligonucleotide
priming PCR were performed. 1% of Holstein milk was detectable in milk curd. The development and application of these
tests could be a powerful tool to authenticate PDO cheeses or
other high-quality dairy products made from milk from specific
breeds in the future.
Although the results of PCR applied to detect the authenticity of dairy products seem reliable, some parameters are still


difficult to control. The amount of DNA recoverable from milk

products is directly related to the somatic cell content of the raw
milk, and the somatic cell count varies according to individual
modifications, diseases, and number and stage of lactation. Cows
infected by mastitis have a very high number of somatic cells
compared with healthy individuals. Nevertheless, milk quality
control tests for quality payments currently carried out in many
countries, and the huge number of cows on dairy farms are factors that could help reduce the effects of mastitis. Similar problems can also arise with the habitual methods used for measuring
proteins, since protein levels vary between cows and breeds.
Despite the disadvantages that still exist, the PCR-method
for food analysis will become increasingly important in the future. Further developments could consist of extending the direct
identification to other milk-producing species in the same PCR
assay using the multiplex-PCR technique. This could allow all
the species present in a milk mixture to be detected at the same
time. Basic premises for this expected development are further
sequence data about the genomes of ruminants, the use of quantitative PCR to get a more accurate estimation of cows milk
proportion in other milks, development of new faster and costeffective DNA extraction and purification procedures prior to
amplification, and the use of real-time PCR135 together with fluorescence techniques to allow the quantitative determination of
the sequence of DNA that is amplified, thereby avoiding analysis
by gel electrophoresis.


Increasing cheese and caseinate industrial production gives
rise to larger volumes of whey. The overall production of whey
powder exceeds the demand for this product. Whey solids may
be fraudulently added to dairy products. This practice, in which
a portion of casein from the milk is replaced by whey proteins,
soluble peptides, and lactose, has attracted great interest; a variety of analytical procedures have been developed to detect their
presence qualitatively and quantitatively.
Special attention has been given to detecting the addition of
rennet whey solids to liquid milk and milk powder. Determination of rennet whey is based on the detection and quantification
of caseinomacropeptide (CMP), the hydrophilic fragment of casein released by chymosin during milk clotting. In this process,
chymosin cleaves the Phe105 -Met106 peptide bond in -casein,
which destabilizes the casein micelles. Para--casein (1105)
remains in the precipitated caseins, whereas CMP (106169) is
recovered in the rennet whey. On rare occasions, -casein is split
at position 106107, resulting in the formation of pseudo-CMP,
lacking the N-terminal Met. CMP should be absent from milk
and has been used traditionally as an index or marker of rennet
whey solids.
The E.U. official method138 for detecting rennet whey in
milk powder is based on CMP analyses by HPLC on wide pore
reversed-phase and UV detector after sample treatment with
8% TCA. This procedure employs an extremely flat gradient

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of acetonitrile, which generates just enough resolution between

CMP and pseudo-CMP. However, under certain circumstances
false positive results could not be excluded, mainly due to
the co-elution with CMP of proteolytic products arising from
the action of psychrotrophic proteinases on -casein. These
proteinases (mainly from enzymes of Pseudomonas), which
progressively split -casein in milk, can give rise to degradation products similar to CMP, depending on the storage conditions and composition. Alternatives based on RP-HPLC to
improve the E.U. method138 include reducing the TCA concentration during extraction139141 and applying selective detection
with pulsed electrochemicals.142 Procedures employing cationexchange chromatography without the use of TCA143,144 have
also been proposed, but it is unlikely that pseudo-CMP can be
separated from CMP, due to a lack of charge difference. In general terms, chromatographic procedures do not have enough resolving power to separate the peptides arising from the action
of residual or reactivated proteolytic enzymes during storage
of long-life products, such as UHT milk. These degradation
products cannot be separated by HPLC and can complicate the
detection of CMP and give false positive results.
Although other methods, such as IEF145 and ELISA with
PAB against the 130152 fragment of bovine -casein,146 have
also been assayed, CE has already become the most powerful
tool for detecting CMP in dairy products. CE has the potential
to give rapid separations with high plate numbers, provided that
adsorption from the proteins to the capillary wall is suppressed.
Furthermore, it allows a very high resolution of proteins and
peptides that differ in just one amino acid residue, and unlike
conventional electrophoresis, there is no limitation in the size of
the components to be separated. Otte et al.147 tested that CMP
could be separated from whey protein using untreated capillary
column at low pH (2.5). Van Riel and Olieman148 published a
very selective CE approach that separates CMP from pseudoCMP and could prevent false positive results in dairy products.
They used a hydrophilic coated capillary at low pH, affording
a limit of detection of 0.4% of rennet whey solids in milk and
buttermilk powder. Thereafter, it was applied for the determination of rennet whey in UHT milks,149 and it also permitted the
detection of 1% ovine and caprine cheese whey in bovine milk
when a western blotting detection system was incorporated.150
The investigation of the suitability of this CE method to detect rennet whey added to UHT milk revealed that, except for the
case of freshly processed milk manufactured from raw materials
of good microbiological quality, the formation on storage of proteolytic breakdown products closely related to CMP hampered
accurate determination.149 In fact, it was later demonstrated151
that thermostable proteinases from psychrotrophic bacteria, although less specific than chymosin, can also split -casein at
position 105106, leading to the formation of CMP, as well as
other very similar peptides (Figure 3). To solve this problem,
linear discriminant functions, using ratios for CE peak areas
of CMP and two other CMP-like degradation products were
defined.152 These functions permitted the detection of rennet
whey solids added to UHT milk, except in the case of very severe

proteolysis, occurring after very long storage periods or at high

Whey other than rennet cannot be tested by detecting and
quantifying CMP. For example, there are no specific indicators
for the addition of acid whey to milk products. In these cases,
the approach for detecting whey additions consists of determining the whey protein in total protein ratio (WPTPR). Although
about 80% of milk protein are caseins and 20% are whey, there
is a wide variation in the range of naturally occurring WPTPR.
Protein denaturation, chemical reactions that take place during
milk processing, and proteolysis may alter protein characteristics, and therefore, influence the accurate determination of the
different protein fractions in dairy products.
Traditional procedures to calculate WPTPR include the determination of casein-bound phosphorus, direct measurement of
casein and whey proteins by SDS-PAGE and polarographic evaluation of cysteine/cystine, which are indicative of whey protein.
However, these classic methods are tedious, time-consuming
and are not suitable for proteolyzed products, such as cheese
and high protein denaturation material, because of the thermal processing. In the last few years, new and more rapid analytical methods have been proposed as alternatives. Schmidt
et al.153 presented a fast (less than 2 min) and versatile approach for discrimination of pure milk samples from milk samples with added whey proteins based on pyrolisis-mass spectrometry. Non-invasive techniques include Fourier Transform
Infrared (FTIR) spectroscopy154 photoacoustic spectroscopy,155
CE,156 and principally UV spectroscopy.156161
Meisel157 proposed a method to determine WPTPR using
fourth derivative spectroscopy based on differences in the UV
spectrum between tryptophan and tyrosine, as well as on differences in the tryptophan to tyrosine ratio, which is 0.19 for
casein and 0.59 for whey proteins. A similar procedure based
on the zero or first-order derivative UV spectroscopy in alkali
was applied for the determination of whey powder to milk powder ratio.158,159 Peng and Puhan160 and Miralles et al.161 used
the fourth derivative UV spectroscopy method for assessing the
quality of different types of milks. With this method it was possible to detect adulterations of UHT milk with whey over 5%,
determining the WPTPR in UHT milk irrespective of the bacterial count of raw milk and storage time.161 Sample preparation
only included skimming to avoid interferences in the measurement and skim milk dilution with guanidine HCl buffer.
With CE, WPTPR is determined as the ratio between the area
of the capillary electrophoretic peaks corresponding to whey
proteins and the area of these peaks corresponding to whey proteins plus caseins. Miralles et al.156 compared CE, SDS-CE, and
UV fourth-derivative absorption spectroscopy to accurately determine the WPTPR in raw, pasteurized, and UHT milks. The results confirmed that the heat treatment applied to milk, although
severe enough to produce lactosylation of the proteins, does
not influence the WPTPR determined by these three methods
(Table 3). However, CE and SDS-CE still need expensive equipment and qualified operators, whereas UV fourth-derivative
absorption spectroscopy is a technique available in most


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Figure 3 Capillary electrophoresis patterns of a stored UHT milk sample after 130 h incubation at 6 C (a) and a sample of skim milk powder containing 5%
rennet whey powder. (Reproduced with permission (149)).



Table 3 Whey protein percentage to total protein ratio in raw, pasteurized,

and UHT milks using capillary electrophoresis (CE), sodium dodecyl sulphate
capillary electrophoresis (SDS-CE), and UV-Fourth derivative absorption
spectroscopy (UV-4th derivative) (Data taken from reference 156)
Whey protein/Total protein (%)
Type of milk

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UV-4th derivative

17.1 2.1
16.6 2.2
16.8 2.1

18.5 2.4
17.7 2.2
17.0 2.0

17.2 1.6
18.8 2.2
17.2 1.8

laboratories, which makes it more suitable for routine analysis. In order to introduce an E.U. reference method to determine
this ratio, these authors156 recommended more studies in milk
samples from different countries and in milks with different proteolysis degrees caused by storage.


Vegetable proteins can be added to milk to make the cost of the
product lower or healthier because of the increased fiber content.
These proteins often have good hydration properties, producing
a higher moisture content in cheese. Despite the good nutritional
and functional properties of these products, their use as supplements and substitutes for bovine milk protein is forbidden. Their
addition is not legal, since by definition, a dairy product can only
be obtained by processing milk or components of milk origin.
Therefore, the determination of the presence and amount of these
vegetable proteins in milk or dairy products is necessary.
Although wheat gluten, pea, rice, potato, bean, or soluble
cereal hydrolysates proteins can also be used as vegetable substitutes, soy protein, due to its low price and high availability in
the market is likely to be the major adulterant. Several preparations of soy proteins are commercially available, such as soy
flour (4252% of protein), concentrates (6269% of protein),
isolates (8287% of protein), and hydrolysates (around 20% of
protein). For their detection, several analytical techniques can
be applied. In the early nineties information available on methods for detecting the possible addition of such protein to milk
was still limited to immunological and electrophoretic procedures that were able to detect around 5% soybean proteins in
milk.2 Recently, during the last ten years, new developments in
these techniques and new approaches, such as CE, HPLC, and
biosensors, have become available for use.
RP-HPLC has been shown to be useful to analyze soy bean
proteins and achieve the simultaneous separation of soya bean
and bovine whey proteins.162,163 Espeja et al.164 by RP-HPLC
perfusion achieved the simultaneous separation of soy and milk
proteins in cows, goats, and ewes milk spiked with these proteins in less than 2 min using a linear acetonitrile-water-0.1%
trifluoracetic acid binary gradient. The standard addition method
was, however, required to quantify cows milk samples due to
the existence of matrix interferences. It is well known that quan-

titative determination of soy proteins presents problems, particularly when mixed in low proportions with other products
and further heat treated. Cattaneo et al.165 tested gel permeation
chromatography (GPC), SDS-PAGE and IEF for detection of
soy proteins in melted cheeses, after selective sample treatment
with a tetraborate-EDTA buffer. The use of this buffer was revealed to be of vital importance for removing interfering peaks
or bands from milk proteins both in GPC and electrophoresis.
Soya proteins are insoluble in a tetraborate-EDTA buffer, which
thus allows the removal of soluble caseins from samples. SDSPAGE took advantage of the size exclusion capacity of the gel in
order to separate and detect the presence of soy proteins with the
most sensitivity (0.06% soy protein in total protein). This technique also had the advantage that it was not affected by heat
treatment. Results were, however, difficult to quantify, and the
technique was rather time-consuming.
The first application of CE to the analysis of soy and milk
proteins on the basis of their different CE pattern was developed
by Kanning et al.166 using a hydrophilic coated capillary and
a low pH buffer. Later, these studies were accomplished by an
SDS-CE after a tetraborate-EDTA sample treatment.167 This approach allowed the separation of the basic subunits of glycinin
and the and  subunits of -conglycinin from the main milk
protein peaks (Figure 4) reducing the limit of detection of soy
protein in milk powder to 1%. SDS-CE afforded less resolution
than SDS-PAGE for the separation of soy and milk proteins,
but presented the advantage of providing shorter analysis times,
offering the possibility of screening more samples in a similar
period of time. Unfortunately, the addition of soy-protein hydrolysates could not be determined.167
A promising methodology for the detection of plant proteins
in milk products consists of the direct biosensor immunoassay format, whereby antibodies are immobilized on the sensor
surface, and the binding of plant proteins of the immobilized
antibodies is detected directly. Haasnoot et al.168 designed an
optical biosensor (Biacore 3000) for the simultaneous detection
of soy, pea, and soluble wheat protein in milk powders. Purified
PAB raised against the three protein sources were immobilized
in different flow channels on the biosensor chip. The total run
time was 5 min, and the limits of detection in milk powder were
below 0.1%. These kinds of instruments can be future routine
procedure alternatives.
A collaborative study169171 involving 8 international laboratories was conducted to evaluate 2 electrophoretic procedures (SDS-PAGE and SDS-CE combined with a tetraborateEDTA sample pre-treatment) and an indirect competitive ELISA
method using PAB for the determination of the fraudulent addition of vegetable proteins. Levels of soy, pea, and wheat proteins
in different dairy products (milk powders, cheeses, and yogurts)
subjected to low and high heat treatments were studied. In-house
pre-validation tests showed that SDS-CE was more suitable than
SDS-PAGE for the detection of soy and pea proteins in milk
powder. Quantification of band volumes by SDS-PAGE was
difficult due to variations in the background among gels and
the irregularity of bands, thus, results showed poor run-to-run


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Figure 4 SDS-CE electropherograms of samples of milk powder containing 0% (a), 1% (b), 2% (c), and 5% (d) of soya protein in total protein, once the
milk proteins were removed by treatment with a tetraborate-EDTA buffer. Peaks: 1 = Basic subunits of glycinin, 2 = acid subunits of glycinin, 3 = chain of
-conglycinin 4 = and  chains of -conglycinin.) (Reproduced with permission (167)).

repeatability. SDS-CE and ELISA were selected for validation

through collaborative trials. SDS-CE provided a slightly better
accuracy, but ELISA presented the advantage of being suitable
for samples containing wheat proteins, which could not be detected by SDS-CE. Precision parameters showed that, in general
terms, repeatability was similar for both techniques. The highest
percentage of reproducibility for SDS-CE was attributed to the
dependence of the results on the equipment and conditions used
in each laboratory (detector sensitivity, sample loading, voltage,
temperature control). ELISA overestimated the percentages of
adulteration in the analysis of low-heat-treated milk samples,

probably due to non-specific binding of antibodies with certain

milk constituents, whereas values obtained for high-heat-treated
samples were lower than the real values. This was attributed
to protein denaturation that could have caused aggregation and
consequently decreased immunoreactivity.


Regional origin assignment of highly valuable dairy products is of considerable importance for legal, fiscal, and trade

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controls. It is also of value for ensuring fair competition and

protecting consumers against fraud due to mislabeling. Milk
and milk products of defined regional origin are highly valued
by consumers and command a premium price. Most of the PDO
cheeses traded in the E.U. are classified according to their region
of production. The PDO system legislates for the monitoring of
geographical origin of raw materials in PDO products. As the
popularity of PDO status labeling has grown, not only to protect
the interests of the regional producer, but also as an important
marketing strategy, the need for analytical methods capable of
verifying geographical origin claims at the retail level has become increasingly important. Moreover, the confirmation of the
geographical origin also has wide implications for dairy products, such as butters, which receive preferential taxation rates
for importation into the E.U.
The ability to differentiate dairy samples from different countries or regions to assess authenticity is being finely explored.
Conventional authentication relies upon the availability of sitespecific microbiological and physic-chemical parameters. The
analysis of metabolic profiles of microbial isolates used in conjugation with artificial neural networks has meant that it has been
possible to infer the geographical origin of Portugese cheeses
and it has been suggested as a tool for PDO certification,172
whereas the flavor capabilities (based on the identification by
GC-MS of volatile odor-conferring molecules) and the microbial diversity have been proved to be closely linked and related to the geographical origin of natural whey cultures used
for water buffalo mozzarella cheese manufacture.173 Grappin
et al.174 were able to correctly discriminate the origin of 20
Comte cheeses made in 5 different cheese plants according to
some physic-chemical variables, microbial counts, and sensory
characteristics. Other works175,176 have found significant differences in the amount of terpenes and conjugated linoleic acid
(CLA) in Gruy`ere cheese between a lowland and a highland production zone. Collomb et al.177 confirmed that CLA and some
trans fatty acids could also be interesting potential indicators for
the origin of cream and PDO cheeses. Fat content and pH value,
as well as biochemical parameters, such as L- and D-lactate
and piruvate, meant that it was possible to partially discriminate
between the regions when these indicators were combined by
principal component analysis in Emmentaler cheese.178 More
research carried out by GC/MS and MS-based electronic nose
have showed the potential of volatile compounds to discriminate
Emmentaler cheese samples of different geographical origin.179
The non-protein nitrogen and the water-soluble nitrogen fractions of these cheeses also showed significant interregional differences due mostly to the different ripening times.180 However,
conventional analytical methodology allowing the unequivocal
determination of the origin of these products is not available yet.
Thus, no precise and well established conclusions can be drawn.
FTIR techniques in combination with multivariate chemometrics have also been investigated for their potential for discriminating cheese from several geographical regions.181,182 Pillonel et al.182 correctly classified 20 Emmentaler cheese samples from 6 different European regions by near and mid-FTIR.

Despite the few samples, clear trends were observed. Nevertheless, further analysis with more samples will be necessary to
confirm these results and build a validated prediction model.
Natural isotope fractionation could possibly be the best answer to the question of the geographical origin of foods. This
approach is based on the small but significant different ratios of
the stable isotopes of bioelements, mainly nitrogen (15 N/14 N),
carbon (13 C/12 C), oxygen (18 O/16 O), sulfur (34 S/32 S), and hydrogen (2 H/1 H) present in certain organic molecules, due to the
kinetic-chemical and physical factors that can be correlated with
the metabolic and/or geographical origin of a product. The relative abundance of the heavy and light stable isotopes of these
elements varies in the environment around us as a result of biochemical and physical processes. These processes are said to
fractionate the isotopes of an element. This fractionation leads
to characteristic isotopic signatures or fingerprint for a particular
geographical location.
For many years, Isotope Ratio Mass Spectrometry (IRMS)
has been the official technique for detecting adulteration in
honey and has been used in other foods, such as wines and
fruit juices.183,184 More recently, studies published in the literature have shown that determining the ratios of stable isotopes
of bioelements by mass spectrometry can also be applied to
milk products. The principle of IRMS consists of measuring the
isotope ratio of an analyte converted into a simple gas, isotopically representative of the original sample before entering the
ion source of an IRMS system.
The first condition for a stable isotope method to be used as a
routine is that there is a scientific explanation (physical, chemical, or biochemical) for variations of the isotope ratios in natural
substances. Based on this knowledge, the establishment of a relevant database for statistical evaluation is required. The second
requisite is a sufficient number of laboratories able to apply
the method with good reproducibility and repeatability. This is
usually tested by interlaboratory comparison. Furthermore, the
ability of laboratories to apply such standard methods properly
in routine work should be tested in regularly repeated intercomparison exercises, where the result of the single laboratory in
comparison to the mean value of all other laboratories is most
relevant. In addition, it would be advisable to complement IRMS
with conventional methods of analysis: GC,185 HPLC,186,187 or
ICP.188 Stable isotope procedures are not supposed to replace
classic analytical methodologies, rather they are to serve as additional indispensable tools for authentication.
The results of the multi-element stable isotope ratio analysis
indicate the possibility of performing a regional origin assignment for dairy products. The measurement of carbon, oxygen,
and nitrogen stable isotope abundances in milk reflected the isotopic composition of the diet fed to the dairy cows. This diet and
its isotopic ratios depend on geographical and climatic factors.
Kornexl et al.189 and Rossmann et al.190 classified milks from
different Bavarian regions and Alpine areas by IRMS. Milks
from zones dominated by grassland typically show relatively
negative 13 C-values, while in regions dominated by crop cultivation 13 C-values are more positive: extensive production based


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on greenland feed, or more intensive farming with a higher degree of corn. Differences in plants, and primarily in soil, are
caused by nitrogen sources and complex processes. Fertilizers
are nitrogen sources that particularly influence the 15 N contents
of plants and subsequent fodder. Specific environments can also
be more or less suitable for growing different plant species, some
of which exhibit very low 15 N contents (legume, grass), while
others preferably grow in soils with higher 15 N-values (rape,
maize). The difference in the 18 O-value of milk relative to that
of the source of water is an additional assignment criterion and
is a good indicator of temperature and climate (continental or
maritime) of the region. Concerning sulphur fractionation, it
has been reported183,185 that the geology of a region (igneous or
sedimentary, acidic or basic) could considerably influence 34 Svalues of soils. Possible effects of industrial emissions on these
values of the soil via wet and dry deposition also have to be considered. Lamprecht et al.191 and Lamprecht and Haberhauer186
developed procedures for isolation by cation-exchange chromatography and enrichment of methionine-bound sulphur in
casein for stable isotope ratio analysis. These techniques were
used to create a database for determining the country of origin
of the milk samples.
Multi-element stable isotope analysis, together with discriminant analysis of the results and evaluation by comparison with
data for certified authentic samples, can be a powerful tool for
solving the problem of butter origin assignment.188 Rossmann
et al.192 carried out determinations of the light elements (C, N,
O, and S) and a heavy trace element (Sr) for butter from several European countries and others outside the E.U. 87 Sr/86 Sr
in butter provided the means whereby regions with similar or
identical climatic conditions could be further subdivided according to their respective geological conditions. Low 87 Sr/86 Sr
values in soil are encountered in mafic rock terrain (volcanic
or ultrabasic, basaltic) and in sedimentary carbonate-rich rocks,
whereas higher values are found in old acidic (igneous or metamorphic, granitoidic) areas. The results of this study192 indicated
that IRMS can reliably detect the regional origin of butter.
N/14 N and 13 C/12 C of caseins and the ratios between some
free amino acids, such as Thr/Pro, Ile/Pro, Met/Pro, and His/Pro,
were successfully used to identify the place of origin of ewes
milk in PDO Italian cheeses produced in different regions.187
Wietzerbin et al.193 also confirmed the usefulness of isotopic
techniques to control the compliance of PDO cheeses with the
restricted rules associated with their production, including the
authentication of the geographical origin by comparison with
Table 4

authentic samples. This study also detected fraudulent practices,

such as the use of corn silage in the cattle diet (when only the
use of grass is claimed) or the addition of foreign components.


Although the addition of milk powders is a practice that is allowed in yogurts to enhance their functional properties, in other
dairy products it is considered fraudulent. Raw milk has a better
flavor than its heat-treated equivalent and the nutritional quality
of reconstituted dried milk powder is not the same as fresh whole
milk because of certain constituents. Additionally, the development of off-flavor and reduction in protein digestibility of dried
milk powder under adverse conditions of storage also reduces the
quality of liquid milk when it is adulterated with this powder.
Moreover, for some consumers, raw milk cheeses are considered to have a better flavor than equivalent products made from
heat-treated milk. Thus, although some manufacturing practices
include the addition of dried milk to cheese milk, thereby increasing the cheese yield and reducing the production cost, the
cheese quality can be negatively affected. All these problems
point to the need to develop methods to detect the adulteration
of raw milk with cheaper powdered reconstituted milk.
Although techniques, such as the determination of milk
RNase activity,194 the study by CE of the ratio of -casein to
-lactalbumin,195 and the use of Near Infrared Spectrometry,196
have been reported to assess the adulteration of fresh or pasteurized milk with dry milk, most of the procedures to detect this
addition are based on measuring the content of products formed
as a result of the Maillard reaction or milk heat treatment. Others
indicators reported in the literature are the hydroxymethylfurfural determined by visible spectrometry197,198 and mainly the
furosine, generated from acid hydrolysis of the lactulose lysine
complex formed during heating.199201 Furosine measured by
HPLC was successfully conducted as an index to detect the
adulteration of raw, pasteurized milks and cheese with reconstituted dried milk. However, this determination had an important
limitation in that there was a wide range in the amount of furosine formed with the varying time and temperature limits in the
UHT process.
In order to circumvent this disadvantage, other works202,203
suggested determining the ratio of lactulose to furosine as an
indicator. Milk drying promotes intensive Maillard reaction and

Overview of methods (19912003) for milk and dairy products authenticity testing

Mixture or addition
Buttermilk powder in skim dry milk
Added water to milk
Distinguish natural from imitation Mozzarella cheese
Artificial flavoring in cheeses
Acid casein and caseinates in processed cheeses
Rennet casein in processed cheeses
Cheaper clotting enzymes during PDO cheese making




Cryoscopy and Titration

Proteins of fat globule membrane

Freezing point and titrable acidity
2 H/1 H of benzaldehyde
Intact -casein




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Table 5

Overview of the main instrumental methods more frequently used for authenticity testing of dairy products


Analytical method

Fatty Acids

Long capillary


Packed and short



2 and 3


Identification of the
specie of origin (cow,
ewe, goat and buffalo)

Well standardized
EU reference method
Limit of detection low (1%)

Whey proteins
and caseins


Identification of the
specie of origin (cow,
ewe, goat and buffalo)

Low cost and technical skills

Whey proteins
and caseins


Identification of the
specie of origin (cow,
ewe, goat and buffalo)

High resolving power

Little or no sample preparation
It permits the identification of ternary
samples (cow + goat + ewe)

Whey proteins
and caseins


Identification of the
specie of origin (cow,
ewe, goat and buffalo)

High repeatability
Easy collection to re-analyze

Fragments of
dairy proteins

Identification of the
specie of origin (cow,
ewe, goat and buffalo)


Identification of the
specie of origin (cow,
ewe, goat and buffalo)
Breed identification.

High specificity and sensitivity

Low cost per analysis
Useful as routine method
It permits the identification of ternary
High specificity and sensitivity
Technique not affected by ripening or
sample heat treatments
It could be quantitative.




Detection of non-dairy
Well standardized
(vegetable and animal) It is adopted by the International
fats in milk fat
Dairy Federation as the official
Detection of non-dairy
Cheap, fast and well standardized
fats in milk fat
Applicability confirmed (method
Detection of cows milk
adopted by the EU)
fat in fat from goats or Limit of detection below 5%
Detection of vegetable
Very selective and sensitive.
fats in milk fat

Limited by the natural variability of
fatty acids
High detection limits (>15%) Large
datasets for statistic are required
Accuracy is affected in lipolyzed
It requires long previous studies to
determine the standard triglyceride
Tedious and time consuming.
High variability depending mainly on
the several steps required.
It is time-consuming.
It requires special equipment and
enzyme treatment
It is not possible to distinguish ewes
from goats dairy products.
Time consuming
Poor visualization of small molecules
and low resolving power
It is affected by heat treatment (whey
protein denaturation) and ripening
(casein proteolysis).
Low precision
No quantitative method
It is affected by heat treatment (whey
protein denaturation) and ripening
(casein proteolysis)
Long analysis times
Complex chromatograms
Difficulties in detecting triple mixtures
It is affected by heat treatment (whey
protein denaturation) and ripening
(casein proteolyis).
Generation of antibodies is very
MAB production can take long periods
of time

It is affected by the number of somatic

cells (therefore it is influenced by
physiological factors).
It requires previous identification of
specific DNA fragments.
It does not have enough resolving power
Detection of rennet whey It is a well standardized method
(official E.U.)
Degradation products can affect CMP
Quantitative procedure
analysis (in long-life products)
Detection of rennet whey High resolution of peptides that differ in Low precision and non-quantitative
only one amino acid.
Proteolytic breakdown products do not It would need standardization.
interfere in the determination.
Suitable for routine analysis
It requires tedious calibration studies
Whey protein in UV Spectroscopy Detection of acid whey
with different types of milk samples.
total protein
Easy sample preparation
Selection of suitable antigens still
Addition of vegetable
Large sample throughput
remains the major problem
High sensitivity
It permits the detection of wheat proteins Semi-quantitative
and adulteration of high heat milk
Addition of vegetable
Rapid and automated analysis
It is not independent of milk-processing
High resolution
Wheat proteins are not detected.
Low reproducibility
The quality of the results has to be
Identification of place of It is the only method able to determine
Isotope ratios
unambiguously the regional origin of
continuously controlled by regular
(C, N, O, S
milk and dairy products.
blind analysis.
and Sr)
Detects fraudulent
It requires databases and a sufficient
practices such as the
number of laboratories able to apply
use of supplements in
the method.
the cattle diet or
addition of foreign











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low lactose formation, due to the low water activity and relatively mild thermal conditions. As a consequence, the lactulose/furosine ratio in UHT milks is approximately 16 times
higher than in commercial milk powder samples. Ratios lower
than 6.0 in processed UHT milk may indicate the presence of reconstituted milk. Linear correlations between these two parameters have been reported for UHT milk and an equation allowed
accurate detection of milk powder in UHT milk.203 In spite of
these advances, the different processing practices employed in
the industry, the protein concentration of milk, the poor quality of the raw milk, severe preheating or recycling of the UHT
and prolonged storage at high ambient temperature can give rise
to different ratios. Therefore more research and studies will be
needed to develop a reference method to detect this practice.
The issues described above are the problems that have been
more extensively studied. Other procedures described in the
literature in the last thirteen years to solve and detect practices
that are not allowed are listed in Table 4. Among them, the addition of caseinates or rennet casein in different dairy products has
aroused great interest and will require more studies in the future.
Table 5 summarizes the main characteristics of the most cited
methods for assessing the authenticity of dairy products. Owing to the great amount of interest in this subject, which has
given rise to a large number of publications in recent years on
the use of high-resolution techniques, positive results can be expected in the near future in the field of authenticity controls of
dairy products. Nevertheless, this assessment will be a difficult
task, and in most cases will require the measurement of several markers. It will also have to take into account natural and
technology-induced variations. It should be borne in mind that
the availability on the market of new dairy products can create
new and potential areas of deception. Furthermore, fraudulent
practices tend to be quite innovative and manufacturers are well
aware of the weaknesses in food inspection systems. Therefore, within the food quality framework, authorities will need
to develop new methods and will have to be constantly adapting existing methods to detect frauds and protect consumers
fundamental rights.
The main developments in the future can be expected to center not only on enhancements to existing analytical methods, but
also on the prior stages of sample preparation, which tend to be
difficult to automate. The future of milk product authentication
techniques embraces the field of mixture analyses avoiding complex protocols for sample preparation, using automated methods
or coupling with separation techniques. This is applicable to the
PCR technique, which needs further development of rapid and
general sample preparation protocols before it can be used as an
ordinary detection method in milk products. Another promising
alternative is CE, which offers a number of advantages includ-


ing simplicity, efficiency, and low cost of the determinations.

The transfer of these analytical determinations from research to
the routine laboratory will be useful for the dairy industry and
consumers, since they will help improve the optimization of processing technologies in the industry and ensure that the product
reaching the consumer complies with labeling. The future perspectives of CE will also lie in improved instrumentation, like
injection and detection, which would provide enhanced precision, and could provide reliable quantitative data, similar to the
data supplied by HPLC. More work will also be required on
long capillary column GC and complementary techniques, such
as MS, to identify and quantify individual TG. These methods
could help monitor new value-added fractions of milk fat with
enhanced nutritional and functional properties generally produced by interesterification of fractionation.
It also seems evident that the control of authenticity of dairy
products may not only be attainable with a purely chemical vision. Physical analysis, supported by IR and UV spectroscopy or
IRMS, for instance, is appropriate both for global characterization of the sample and for a compound-by-compound characterization. In this field, the future seems to be linked to the increasing
development of analytical solutions that combine powerful analytical devices and data processing software.
Purity criteria have to be empirically determined by analyzing
a wide array of genuine products, as well as creating and regularly updating a database with information on the concentration
ranges of certain indicative components of the milk product concerned, mainly PDO. In order to solve difficult cases, more than
one analyte has to be considered for detecting fraud. Likewise,
a combination of different analytical techniques to determine
dissimilar characteristics of a commodity could be more useful than relying on one single method. Given the complexity
of some problems, univariate statistics have to be substituted
by intricate statistical algorithms to aid pattern recognition and
classification of genuine and fraudulent products.
Finally, more effort is still needed for the validation of recent
analytical methods. This validation of new procedures, as well
as the development and assessment of new reference materials
via collaborative trials, will continue to be an important issue
in the authenticity of dairy products. In the future, it is also
expected that all official methods for milk product testing will
evolve and be based on modern instrumental techniques, thereby
substituting the most laborious procedures.
The authors acknowledge financial support to the research
projects AGL2002-00887 and AGL2005-04760-C02-01 from
the Spanish Ministerio de Ciencia y Tecnologa.
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