Professional Documents
Culture Documents
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
To link to this article: http://dx.doi.org/10.1080/10408690490478127
Authenticity Assessment
of Dairy Products
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.
Keywords
INTRODUCTION
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.
E-mail: ifraf91@if.csic.es
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
563
564
Analysis of Triglycerides
FOREIGN FAT IN MILK FAT
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
565
M. A. DE LA FUENTE AND M. JUAREZ
566
Figure 1
(43)).
Gas chromatographic profiles of triglycerides of goat (A) and cow (B) milk fat samples using short capillary column. (Reproduced with permission
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.
567
Electrophoresis
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.
568
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
antibodies.
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
detected.
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
deviations.
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
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
processing.
569
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
Mixture
Type of antigen
Type of
antibody
ELISA format
Whey proteins
Caseins
s1 -casein
-lactoglobulin
3 -casein
Caseins
-lactoglobulin
s2 -casein
-casein
-casein
-casein
Whey proteins
Polyclonal
Polyclonal
Polyclonal
Polyclonal
Polyclonal
Polyclonal
Monoclonal
Monoclonal
Monoclonal
Monoclonal
Monoclonal
Monoclonal
Indirect
Sandwich
Competitive
Indirect Competitive
Indirect Competitive
Indirect
Sandwich
Indirect Competitive
Indirect
Immunostick
Competitive
Limit of detection
(%)
1
1
0.1
0.1
<0.1
<0.1
0.1
0.5
0.5
0.2
Reference
108
106
101
102
103
104
112
117118
114
115
116
113
M. A. DE LA FUENTE AND M. JUAREZ
570
Table 2
Mixture
Limit of detection
(%)
Fragment of
amplified DNA
Cycles of
amplification
Use of restriction
enzymes
Reference
0.5
0.1
0.1
5
1
-Casein
Citochrome b mitochondrial
Citochrome b mitochondrial
Citochrome b mitochondrial
-Casein
Citochrome b mitochondrial
35
3550
32
35
20
35
Yes
Not
Not
Yes
Not
Not
125
128
132
127
131
134135
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
571
572
573
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)).
M. A. DE LA FUENTE AND M. JUAREZ
574
Raw
Pasteurized
UHT
CE
SDS-CE
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.
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
575
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)).
576
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
577
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.
15
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
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
Method
Marker/Parameter
Reference
SDS-PAGE
Cryoscopy and Titration
RP-HPLC
GC-IRMS
CE
CE
CE
204
205
206
207
208
209
210
M. A. DE LA FUENTE AND M. JUAREZ
578
Table 5
Overview of the main instrumental methods more frequently used for authenticity testing of dairy products
Marker
Analytical method
Fatty Acids
GC
Long capillary
columns
Triglycerides
GC
Packed and short
capillary
columns
Phytosterols
GC
2 and 3
caseins
IEF
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
PAGE
Identification of the
specie of origin (cow,
ewe, goat and buffalo)
Whey proteins
and caseins
CE
Identification of the
specie of origin (cow,
ewe, goat and buffalo)
Whey proteins
and caseins
HPLC
Identification of the
specie of origin (cow,
ewe, goat and buffalo)
Quantitative
High repeatability
Easy collection to re-analyze
Fragments of
ELISA (MAB)
dairy proteins
Identification of the
specie of origin (cow,
ewe, goat and buffalo)
DNA
Identification of the
specie of origin (cow,
ewe, goat and buffalo)
Breed identification.
PCR
Usefulness
Advantages
Detection of non-dairy
Well standardized
(vegetable and animal) It is adopted by the International
fats in milk fat
Dairy Federation as the official
method.
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%
ewes
Detection of vegetable
Very selective and sensitive.
fats in milk fat
Disadvantages
Limited by the natural variability of
fatty acids
High detection limits (>15%) Large
datasets for statistic are required
Accuracy is affected in lipolyzed
samples
It requires long previous studies to
determine the standard triglyceride
composition.
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
expensive.
MAB production can take long periods
of time
References
1620
2145
4653
56
6265
70
6772
94
7784
8589
9194
112122
125
127128
130137
138144
147152
156161
169
171
167
169170
186187
189193
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.
OTHER AUTHENTICITY ISSUES
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.
FUTURE AND CONCLUDING REMARKS
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-
579
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