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Authenticity Assessment of Dairy ProductsMiguel Angel De La Fuente a & Manuela Jurez aa Instituto del Fro (CSIC), Jos Antonio Novais 10, Ciudad Universitaria s/n , Madrid, SpainPublished online: 18 Jan 2007.

To cite this article: Miguel Angel De La Fuente & Manuela Jurez (2005) Authenticity Assessment of Dairy Products, CriticalReviews 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)Copyright C Taylor and Francis Inc.ISSN: 1040-8398DOI: 10.1080/10408690490478127

Authenticity Assessmentof Dairy Products

MIGUEL ANGEL DE LA FUENTE and MANUELA JU AREZInstituto 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 substitutionof part of the fat or proteins, admixtures of milk of different species, additions of low-cost dairy products (mainly wheyderivatives), or mislabeling of products protected by denomination of origin. A range of analytical methods to detect fraudshave been developed, modified, and continually reassessed to be one step ahead of manufacturers who pursue these illegalactivities. Traditional procedures to assess the authenticity of dairy products include chromatographic, electrophoretic, andimmunoenzymatic methods. New approaches such as capillary electrophoresis, polymerase chain reaction, and isotope ratiomass spectrometry have also emerged alongside the latest developments in the former procedures. This work intends toprovide an updated and extensive overview since 1991 on the principal applications of all these techniques together withtheir advantages and disadvantages for detecting the authenticity of dairy products. The scope and limits of different toolsare also discussed.

Keywords cheese, fraud, milk, protected denomination of origin

INTRODUCTION

The authenticity of foods is currently of major concern for re-searchers, consumers, industries, and policymakers at all levelsof the production process. An authentic raw material or finishedproduct has to comply with labeling, principally in terms ofingredients, production technology, and genetic identity. Dairyproducts are of particular interest, because they are a group offoods that play an important role in feeding the population andare essential for certain groups of consumers (women, children,and the elderly). Milk is a fairly expensive raw material, andfrom an economic point of view it could, therefore, be attractiveto modify its composition and replace part of it with other dairyor non-dairy ingredients. European regulations are strict on thismatter: only skimming and some additions (minerals, vitamins,and milk proteins) are legally allowed in milk. For instance, re-placing milk fat or protein with another of different origin is apractice that is not allowed, even though this substitution couldenhance the nutritional value of the final product.

Ramos and Juarez1 and Juarez2 reviewed the main possiblefrauds in dairy products and the analytical procedures for detect-ing 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: [email protected]

has led to the manufacture of specialized milk products. Unfor-tunately, this progress has also provided opportunities for verysophisticated types of manipulations that are difficult to detect.One specific current challenge is to control the labeling claimson high-quality dairy products. Protected Denomination of Ori-gin (PDO) cheeses, for instance, have distinct characteristics andenhanced quality and are defined according to their geographi-cal area of production, as well as in terms of the materials andtechnology used in their manufacturing. The determination oforigin is a key component of PDO. Criteria and procedures forassessing the authenticity of these high-quality products need tobe developed.

Lees3 reported the legislation and analytical techniques cur-rently available for controlling the authenticity of milk productsin Europe. However, while strict standards and criteria for prod-uct definition exist, practical means for judging product authen-ticity are not always available. The main approach for solvingthese kinds of problems is to look for a specific marker or in-dicator in the product, which could be a component (complexmolecule, nucleic acid), or determine the ratio between somechemical constituents and assume that these ratios are a constantcomponent of the particular dairy product. From this perspec-tive, it seems to make sense that the addition of any substance tomilk products will modify the value of these ratios or highlightan anomaly in their chemical composition. In this area, manypattern classification procedures can be applied to the dataset to

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564 M. A. DE LA FUENTE AND M. JU AREZ

compare similarities or differences in the sample data with theoriginal data.

Analytical dairy science has advanced significantly over thelast decade, the main reason being the availability and improve-ments in advanced techniques, such as chromatography, im-munoenzymatic assays, and electrophoresis. In addition to newdevelopments in these techniques, the interdisciplinary and dy-namic nature of milk product analysis is being enhanced bythe application of disciplines already used to analyze otherfoodstuffs. Among them, capillary electrophoresis (CE), poly-merase chain reaction (PCR), and isotope ratio mass spectrome-try (IRMS) are just gaining popularity for solving dairy authen-ticity problems. The aim of this article is to provide an updatedreview (19912003) of the methodologies that have been iden-tified as showing potential for addressing the issues related tothe authenticity of dairy products and also to collate informa-tion on ongoing research into this matter. In line with Europeanregulations, we will focus primarily on the detection of foreignfat or protein in milk products, admixtures of the milk of differ-ent species, measurement of the presence of whey derivativesin milks, determination of geographical origin of high-qualitydairy products, and the study of the contents of heated/driedmilks added to milk.

FOREIGN FAT IN MILK FAT

Milk fat is a valuable fat for human nutrition and representsan expensive raw material. Of all milk fat products, butter is themost important one for economic reasons, and it must, there-fore, have high standards of quality. In this respect, the E.U. hasdeveloped strict standards of identity for butter and has estab-lished that this dairy product must be obtained exclusively frommilk or cream. In spite of this, butter has always been subjectedto adulteration by the addition of less expensive vegetable oranimal fats. As a result, the detection of non-milk fats in butteris far more important today than ever before, and a number ofinvestigations have been carried out by several research groupsto develop analytical methods for this purpose. In recent years,different reviews have also appeared with updated informationon the detection of non-milk fats in butter.49

Even though certain techniques of physical analysis such asscanning calorimetry have been successfully applied,1012 mostcurrent analytical approaches to authenticity issues in milk fatconstituents are essentially based on the Gas Chromatography(GC) separation of its components. Nowadays, the best way ofrevealing the presence of foreign fats in milk fat includes thestudy of the fatty acid composition, the triglyceride (TG) pro-file, and the different fractions of other minor lipid constituents,mainly from the unsaponifiable fraction. Other recent workshave considered the use of complementary techniques in theidentification of milk fat such as pyrogram fingerprints obtainedby pyrolysis GC with Flame Ionization Detector (FID), as well asthe determination of compounds contributing to milk fat aromaby headspace GC.13,14

Fatty Acid Composition

Analytical methods for assessing the authenticity of milk fatbased on fatty acid composition have a long tradition2,15 andwere adopted by the International Dairy Federation (IDF) as thebasis for the quantification of milk fat in fat mixtures.16 Thesemethods are supported by the fact that the presence of butyricacid is rather unique to milk fat. To increase the reliability ofthese methods, the content of other fatty acids was also includedin the calculation. For example, the amount of butyric acid orsome ratios involving butyric and other fatty acids (caproic, lau-ric or oleic acids) determined by analysis of fatty acid methylesters are still used as parameters to analyze admixtures of veg-etable fats to milk fat.17,18 However, low percentage (

AUTHENTICITY OF DAIRY PRODUCTS 565

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

Although the official E.U. method for the determination of thepurity of milk fat is founded on TG analysis by packed columnGLC, the original E.U. regulation31 and its revised versions34,35foresee capillary columns as an alternative technique, providedthe same results are obtained. Different works carried out to as-sess the authenticity of milk fat using short capillary (less than5 m) column GC have been described.22,3641 This abundanceof studies is due to the development of better columns with highthermal stability and selectivity of the stationary phase. In addi-tion, short capillary column analysis reduces the analysis time,consuming less carrier gas, and providing similar levels of accu-racy than packed columns. However, there has been some con-troversy about the quantitative aspects of TG profiling of milkfat by capillary column GC. Although capillary GC was equiv-alent to the packed column in terms of analytical precision andall the samples tested fulfilled the purity criteria (S-values), irre-spective of the chromatographic technique used, Ulberth et al.41observed that S-values obtained by capillary GC deviated tosome extent from those of the packed column, and differencesexceeded the value for reproducibility laid down in the Europeanrules. A later study39 was carried out by testing the purity of 50widely varying samples of milk fat in accordance with E.U.rules. For all the samples, the differences between S-values ob-tained from packed and capillary column data did not exceedthe reproducibility limits stipulated by E.U. rules. However, inorder to guarantee comparable results, these authors39 recom-mended rechecking by each laboratory wishing to use a capil-lary instead of a packed column to monitor the purity of milkfat.

In the quantitative analysis of milk fat TG, the introductiontechnique is critical, because the sample consists of a mixtureof TG with a wide range of volatilities, and relatively high tem-peratures are required for complete vaporization. Classic hotsplit injection was by far the least suitable technique since itcauses strong discrimination and decomposition. Although on-column injection has been the most frequently used approach toreduce these undesirable effects22,37,38,40,41 Programmed Tem-perature Vaporizer (PTV) Injection has also been assayed.40,42Banfi et al.42 demonstrated that PTV injection gives results com-parable to those obtained with the on-column injector. The re-peatability and reproducibility obtained with PTV widely fulfillthe requirements of the official E.U. method, demonstrating thatthis configuration can be applied as a valid alternative to theon-column injection system.

Owing to differences with cows milk fat TG profiles(Figure 1), the equations proposed by E.U. rules are not suit-able to monitor goats or ewes milk fat. To detect foreign fatin goats and ewes milk fat, new multiple regression equa-tions based on TG contents of the goats and ewes fats havebeen proposed.43,44 Fontecha et al.43 applied these mathemat-ical equations to provide a fast and highly sensitive means ofdetermining mixtures of non-milk fats in goats milk. Althoughthis procedure could also be very useful for specific detection

of mixtures of non-goat milk fat in goats milk, the limit ofdetection reached was not very low. The method for determina-tion of TG proposed by Precht25,26 was also applied for analyz-ing different types of cheeses to detect the presence of non-dairyfat.45 In cheeses with extensive lipolysis, the Precht formula er-roneously indicated the presence of non-dairy fat; this was at-tributed to the decrease in low-molecular weight TG producedby the lipases. In unlipolyzed samples, or those with only slightlipolysis, this equation, however, successfully highlighted thepresence of undeclared non-dairy fat. It was concluded that anew formula should be developed for cheeses with extensivelipolysis.

Analysis of Minor Components

Analysis of minor components, mainly constituents of theunsaponifiable matter, can be an indispensable tool for authenti-cation purposes. Traditionally, sterol analysis has been used forthe detection of admixtures of animal fats and vegetable oils bydetermination of cholesterol and phytosterols. This determina-tion is the most sensitive method for differentiating vegetableand animal fat. Animal fats, such as milk fat, primarily containcholesterol; phytosterols are not detectable or only present attrace levels. Among the different sterols present in vegetableoils, -sitosterol is usually the main constituent, therefore, asuitable marker for the detection of the addition of vegetable oilto milk fat.

The Provisional IDF method46 to determine sterols in milkfats involves saponification of the lipids, extraction of the un-saponifiable matter, pre-separation by Thin-Layer Chromatog-raphy (TLC), derivatization of the sterols, and subsequent GCanalysis (Figure 2). Presently, it is still published as a provisionalstandard, due to the absence of repeatability and reproducibility,even though several interlaboratory tests have been performedover the last few years. The high variability should be attributedto the steps required. Contarini et al.47 showed that the accuracyof determining cholesterol in milk fat by this method strictlydepended on evaluating the correction factor for the internalstandard; therefore, it should be done at the same time as thesample analysis. A detection limit of 45% for vegetable fatwas determined in milk fat.

Apart from its analytical disadvantages, the IDF method46 istedious and time-consuming and thus, not advisable as a rou-tine procedure. In order to carry out rapid and reliable deter-minations for sterols and simplify the analyses, different alter-natives have been developed over the last 10 yr (Figure 2). Theuse of fused silica columns GC4850 meant that the sterol frac-tion derivatization step could be eliminated, thereby reducingthe analysis time and improving sensitivity. The unsaponifiablematter was also directly analyzed by GC as free sterols51 ortrimethylsilyl derivatives.52 In this way, isolation of the sterolsby preparative TLC could be avoided. Alonso et al.53 analyzedthe sterol profile after sample alkali-catalyzed transesterificationwith 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(43)).

time and eliminates the need for saponification, extraction, andderivatization steps (Figure 2). It is, therefore, suitable for rou-tine use to detect possible additions of vegetable oils in milkfat.

Although most of the procedures used to assess the sterolscontents include a GC as a final step, determination by HPLChas also been reported.54,55 Kamm et al.56 developed a methodcombining HPLC and GC that involves transesterification of the

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Figure 2 Scheme of the analytical procedures to determine sterol fractioncomposition in milk fat by gas chromatography.

fat, pre-separation of the sterol fraction from other lipid con-stituents, and on-line transfer to the capillary GC system. Theon-line approach eliminated time-consuming sample prepara-tion steps prior to GC analysis, and meant that it was possibleto detect adulterations at low levels of cotton and rapeseed oilin milk fat using -sitosterol as a marker.

Other minor components have been reported to establish thecriteria for assessing the authenticity of milk fat and derivatives.Milk fat diglycerides C30-C36 showed characteristic finger-prints compared with other fat sources, especially with tallow.57The 3,5-cholestadiene, a derivative from partial dehydroxylationof cholesterol during refining treatment, was found to be an indexfor the addition of refined beef tallow to butter.58 These markerscould be used in addition to the most common ones described inthe previous paragraphs. Povolo et al.57 reported an interestingapproach based on the combination of data from the determi-nation of different compounds (diglycerides, 3,5-cholestadieneand TG) by GC to detect the presence of beef tallow in milkfat. When the results were evaluated separately, the methods didnot seem to be able to decrease the limit of detection below 5%.The application of multivariate statistical techniques increasedthe power of these analyses, and the statistical model calculatedwas able to detect the presence of 2% of beef tallow; it could beapplied when the results obtained by the Official Method35 areclose to their limit of detection.

ADMIXTURES OF THE MILKS OF DIFFERENTSPECIES

Identification of the species of origin for milk products isimportant from a theoretical standpoint and useful in practice,since it allows the detection of fraud by substituting a less costlytype of milk for one of a higher quality. In comparison with cowsdairy products, goats, ewes, and buffalos products are moreexpensive, and authentication is, therefore, of great economical

interest. Some dairy products should contain defined proportionsof milks from different ruminant species, whereas others, such asPDO cheeses, with a higher market value can only be made withpure milk from cow, goat, ewe, or buffalo, and milk additionfrom another species or even breed is not allowed. Adequatecontrol methods are required to check the presence of cheapermilk in PDO cheeses. Apart from economic loss, correct speciesidentification is important for the consumer for other reasons:medical requirements, food allergies, or religious practices. Thissituation has prompted research to find methods for detecting thespecies of origin of dairy products.

Although analysis of selected fatty acid by GC59,60 and TGby NMR61 to distinguish milks from different ruminant speciescan be found in the literature, most of the works on this issueinvolve protein fraction studies. The present European Com-munity reference method62 for the detection of cows milk inewes and goats milk products is based on isoelectric focus-ing (IEF) of -caseins, peptides originated from -casein afterenzymatic proteolysis by plasmin. A sample is judged as be-ing positive if both bovine 2- and 3-caseins are equal to orgreater than the level in the 1% standard (ovine or caprine). Fora more accurate analytical result, samples must be analyzed si-multaneously in an IEF gel with two reference standards of milkwith 0 and 1% cows milk, calculating the bovine milk presentby comparing the intensity of the bovine 2- or 3-caseins with1% bovine milk reference. Although the scope of the qualitativedetection of cows milk in milk and cheese of other species iscovered by this method, the quantitative determination of cows,ewes, and goats milk in ternary mixtures remains a subject forfurther study, because ewes and goats milk could not be dis-tinguished. Moreover, the IEF method is time-consuming andrequires special equipment, as well as specially trained technicalstaff. Several alternative methods (chromatographic, immuno-logical, and electrophoretical) have been published to authenti-cate the species of origin of milk products, and they might beapplicable to routine analysis. Identification procedures basedon DNA extraction and amplification are also now emerging.

Electrophoresis

Procedures based on different types of electrophoresisabound in the literature. Different studies6365 have tried to im-prove the sensitivity of the IEF reference method introducingdetection by immunoblotting with polyclonal antibodies (PAB)against bovine milk proteins. Mayer et al.66 complement IEFwith cation-exchange HPLC to determine para--caseins. Asthese molecules were not substantially affected by the degree ofripening, the method could be suitable for determination of thepercentages of cows, goats, and ewes milk in mixed cheese.However, quantitative results in cheeses had to be regarded asapproximate values. This is because the estimated percentageof cows milk in mixed cheese is greatly affected by the caseincontent of the milks used for cheese making and the differenttechnological procedures applied.

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Traditional methods such as polyacrylamide gel elec-trophoresis (PAGE) have been used to identify milk from goat,ewe, and cow in their mixtures67,68 to detect bovine milkin ovine or caprine cheeses,6971 or to ensure the authentic-ity of ovine yogurts and guarantee that they are accuratelylabeled.72 Another study65 detected denatured bovine whey pro-teins in cheeses from ewes and/or goats milk combining pro-tein separation by PAGE with western blotting detection usingcommercially available polyclonal anti-bovine -lactoglobulinantibodies.

In comparison with IEF, PAGE is less costly and requiresfewer technical skills. However, in a routine examination, itis still time-consuming and labor intensive. Furthermore, italso presents the disadvantage of poor visualization of smallmolecules. Presently, capillary electrophoresis (CE), with itshigh resolving power, rapid method development, easy sam-ple preparation, and low operation cost, has been shown tohave the greatest potential for different foods and also dairyproducts.73,74 Capillary zone electrophoresis (CZE) has beenused to detect adulteration of goats and ewes milk, with cowsmilk, on the basis of the different migration time of the S1-casein fractions of the different species. Cattaneo et al.75 demon-strated the capability of CZE to analyze milk mixtures and todetermine up to 8% of cows milk content with coated sil-ica capillary. The use of uncoated capillary CZE meant thatcosts were reduced, and that the addition of raw or reconsti-tuted cows milk in goats milk of as little as 1%76 could bedetected.

The differences between the CE profiles at low pH of the ca-sein fraction from 100% cows, ewes, or goats milk providedthe basis for the selection of different peaks, characteristic of thepresence of these milk types in ternary mixtures. Molina et al.77used the Principal Component Regression (PCR) and PartialLeast-Square regression (PLS) to predict the percentages of milkof each species in the mixture. Thereafter, they characterized78,79the casein fractions of cheeses made from goats, ewes, andcows milk to identify the major protein components of the ca-sein fraction and their breakdown products. Caprine para--casein and bovine -casein peaks were indicators of the pres-ence of milk of these species. These peaks could be seen incheeses made from mixtures and could be potential indicatorsfor identifying and quantifying these mixtures. Other authors80showed that a CZE electropherogram of the ethanol-water pro-tein fraction in isoelectric acidic buffers contains enough in-formation to classify cheeses according to both, genetic originand ripening time. On the basis of the electropherograms, andusing PLS multivariate regression, the contents of cows milkin presumably pure goat and ewe cheeses, as well as in binaryand ternary mixtures, was predicted with low relative standarddeviations.

The study of whey protein profile by CZE could also be usedas a tool to determine the content of cows milk in mixed dairyproducts. A method using a methyl silanized capillary column inborate buffer (pH 9.2) based on establishing different ratios be-tween the whey proteins present in the acid soluble fraction was

applied to detect bovine milk in buffalos,81 ewes,82 and goats83milk and cheese; amounts of between of 0.5% and 5% were de-tected. The qualitative analysis of ternary mixtures has also beenreported,83 but the loss of resolution between the -lactalbuminand -lactoglobulin caprine and the overlap between goat andewe -lactalbumins did not allow accurate quantitative analy-sis of triple mixtures and double mixtures of goat-ewe milk.Herrero-Martnez et al.84 developed a simple CE method forthe separation of bovine, caprine, and ovine whey proteins inbinary and ternary cheeses giving limits of detection as low as1% in very short times. They used acidic isolectric buffers sup-plemented with a surfactant (Tween 20) that allowed the sepa-ration of bovine -lactalbumin and -lactoglobulin from smallpeptides arising from casein degradation during cheese ripen-ing. An additional advantage was that the methodology protocoldid not require coated capillaries that are necessary in methodsadopting 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 along way to go before they are complete. We could safely say thatCE, accompanied by instrumental development (MS detection,new injection devices to enhance precision, microfabricated sys-tems for routine protein separation), could become much morepopular in the future.

Chromatographic Methods

High performance liquid chromatography (HPLC) is veryautomated and has proved to be a valuable quantitative tech-nique for protein analysis in dairy products. Reversed-phaseHPLC (RP-HPLC)8589 and ion-exchange chromatography90were used to separate whey proteins from milks of differ-ent species. Separation of caseins from bovine, ovine, andcaprine milk using various HPLC procedures has also beenreported.9193 However, separation time is usually within the3050 min range, which is rather long for some routine qual-ity control purposes. Moreover, some of these studies revealedcomplex chromatograms,87 while others did not detect the pres-ence of goats milk in triple mixtures of goat-ewe-cow milk andbinary combinations of goat-ewe milk.88

In a comparative study94 of caseins fraction of bovine, ovineand caprine milks, results obtained by HPLC matched the urea-PAGE method. However, although HPLC was more efficientfor quantitative results, electrophoresis was shown to be moresensitive to detecting adulterations.

In general terms, chromatographic and electrophoretic tech-niques based on physicochemical properties of mixed nativeproteins could become complicated after the proteolysis thatoccurs during ripening processes, since intermediate proteinand peptide products appear. Moroever, methods based onthe detection of heat-sensitive proteins such as whey pro-teins could become ineffective if heat-treated milk is used forprocessing.

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Immunological Methods

Immunological procedures based on antigen-antibody pre-cipitation reactions are suitable to authenticate dairy productsbecause of their sensitivity to differentiating milk proteins fromdifferent species. The main characteristic of these methods istheir high specificity derived from the antigen-antibody reac-tion. In addition, they have the advantage of being inexpensive,quick, sensitive and specific compared with conventional detec-tion methods. Immunoassay kits are easy to use in the laboratoryfor detecting the authenticity of dairy products and they can suc-cessfully be applied to the food sector. The most used immuno-logical technique for testing the authenticity of dairy productsis enzyme immunoassay using Enzyme Linked ImmunosorbentAssay (ELISA) format. The main advantages of ELISA, apartfrom the ones mentioned above, are that it provides quantitativeresults and can be widely used as a routine procedure.

Antibodies used in immunoassays, depending on the pro-duction method, can be polyclonal or monoclonal (MAB). Thegeneration of PAB is less expensive and time-consuming thanMAB, which could explain the higher number of studies on theformer in the literature. Numerous ELISA methods with PAB forthe detection of bovine proteins in goats and ewes milk,95104and goats milk in ovine milk products98,105108 based on differ-ent antigens (caseins and native and denatured whey proteins)and varying techniques have been reported. Details about themost recent approaches are shown in Table 1.

In the immunological detection of interespecific adulterationsof dairy products, the main problem lies in the production of an-tibodies exclusively specific for milk proteins from one species.A strategy to enhance specificity was based on the generationof antibodies against short specie-specific peptides located inthe primary sequence of caseins. PAB raised in rabbits againstthe bovine S1-casein sequence 140149100 appeared monospe-cific for bovine S1-casein, since no antibody-antigen complexwas formed with homologous ovine or caprine proteins. It wassuggested101 that only one amino acid, glutamic acid residuein position 148, was essential for the antigenic character of the

Table 1 Enzyme linked immunosorbent assay (ELISA) in dairy products authentication

Type of Limit of detectionMixture Type of antigen antibody ELISA format (%) ReferenceGoats milk in ewes milk Whey proteins Polyclonal Indirect 1 108Goats milk in ewes milk and cheese Caseins Polyclonal Sandwich 1 106Cows milk in goats milk and cheese s1-casein Polyclonal Competitive 101Cows milk in ewes and goats cheese -lactoglobulin Polyclonal Indirect Competitive 0.1 102Cows milk and caseinate in goats and ewes milk and cheese 3-casein Polyclonal Indirect Competitive 0.1 103Cows milk in goats milk Caseins Polyclonal Indirect


Table 2 Polymerase chain reaction in dairy products authentication

Limit of detection Fragment of Cycles of Use of restrictionMixture (%) amplified DNA amplification enzymes ReferenceCows milk in goats and ewes milk and cheese 0.5 -Casein 35 Yes 125Cows milk in goats milk 0.1 Citochrome b mitochondrial 3550 Not 128Cows milk in goats milk 0.1 Citochrome b mitochondrial 32 Not 132Cheeses from different species Citochrome b mitochondrial 35 Yes 127Cows milk in goats, ewes, and buffalos milk and cheese 5 -Casein 20 Not 131Cows milk in buffalos cheese 1 Citochrome b mitochondrial 35 Not 134135

and could also be applied to detect fraudulent substitution ofgoats milk in traditional cheeses that must be made with pureewes milk (Manchego, Feta, Roquefort, Pecorino). S2-caseinwas characterized as the most immunoreactive caprine caseinsfraction.121 Immunization of mice with this purified fractionmeant that it was possible to detect eight MAB, and from theseit was the hybridoma cell line B2B that appeared to be speciesmonospecific and only reacted with the caprine S2-casein.122Thereafter, this antibody has been used in ELISA for the detec-tion and quantification of the presence of goats milk in ewesmilk117 and cheese.118 It was confirmed that the antigenicity ofcaseins was not affected by ripening, since no significant dif-ferences were noted between ewes cheese mixed with fresh,semi-hard and hard goats cheese. Although experiments on theuse of the purified goat S2-casein fraction subjected to limitedproteolysis by enzymes and determination by immunoblottingof the location of the epitope recognized by the MAB B2B werenot performed, results presented from hard cheese118 suggestedthat the epitope may be a short continuous peptide fragment.

Polymerase Chain Reaction (PCR)

The origin of dairy products from certain animal species canalso be determined by DNA studies. Milk from healthy mam-mary glands contains a large number of somatic cells (epithe-lial cells and leukocytes). These cells are concentrated duringprocesses, such as cheese making, and can be used to isolateand amplify DNA and discriminate species. In comparison withprotein, DNA assays may have the advantage of increased sen-sitivity and rapid performance with high sample numbers beingautomatically processed. Furthermore, these methods could beadvantageous for highly processed milk products treated withhigh temperature and pressure or submitted to long periods ofstorage, because they are methods based on more resistant mate-rial such as nucleic acids. As we mentioned above, protein-basedmethods for species identification may fail after excessive pro-teolysis or heat-induced denaturation.

PCR makes it possible to amplify defined DNA-fragmentsin a very short time by a factor up to some millions in threesteps (denaturation to obtain a single-stranded DNA, annealingin which primers flank the molecular region of interest, and fi-nally extension where the DNA polymerase synthesizes the newstrand). Subsequently, the amplified DNA can be analyzed by

various molecular biological procedures, mainly by size frag-ment length polymorphism, and visualized with ethidium bro-mide after electrophoresis in agarose gel. The decisive advantageof PCR-analysis in comparison to protein chemical methods isthat, in contrast to specific proteins, the whole DNA is alwaysidentically present in every organ of a specie. Because of this,it is possible to determine molecular genetic differences veryclearly. Molecular biology techniques have been used to iden-tify the species of origin in foods, especially in meat products.The use of this methodology in dairy products has been limitedto detecting bacterial contaminants, and only in recent years hasit been applied to control authenticity and differentiate betweenspecies relevant to the dairy industry (Table 2).

Although Lipkin et al.123 showed that it was possible to usemilk as a source of DNA and as a substrate for PCR, at that timethe quality of purified DNA was an unresolved problem, andsome difficulties emerged in recognizing the milks of closelyrelated species, (ewe, goat, cow, and buffalo) because of thepossibility of cross-reaction. It was also not possible to definehighly specific regions in genes, which could be used for an un-equivocal determination by means of PCR. Furthermore, milkwould seem to be a very complex food system with an abundanceof potential PCR inhibitors. It has been reported124,125 that cer-tain compounds in fungi-containing cheeses (Danish blue, veinBrie, and Brie) may interact with the DNA or the enzymaticPCR reaction and inhibit it.

Since then, new developments have improved the efficiencyof these techniques. Such advances include rapid and reliableprocedures for isolating genomic DNA from dairy samples basedon the use of resins126,127 or spin columns.125,128 The finding129that free DNA is principally located in the cream fraction couldbe of great relevance when very small amounts of target DNAare present. Concerning the inhibitors of PCR reaction, it hasbeen tested that the heat treatment of milk128,130 and the dryingor freezing of the cheese or fungi used during cheesemaking128did not seem to interfere with the detection method, whateverthe milk treatment or nature and ripening of the cheeses tested.

Material employed in amplification consists of nuclear ormitochondrial DNA. Specific -casein gene PCR with univer-sal primers encoding a partial sequence of gene was performedto detect the corresponding DNA in dairy products.125,131 In thePCR product from ovine or caprine -casein, DNA was shownto contain a specific restriction enzyme site that is not present inbovine -casein DNA. Accordingly, after selected restriction

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enzyme analysis and PAGE, the undigested bovine -caseinfragment can be detected as an additional band if cows milkis present,125 or by more sensitive enzyme immunoassay usingprimers labeled with biotin or digoxygenin.131 Branciari et al.127described a procedure to determine the species of origin of milkused in cheese manufacturing, based on isolating the mitochon-drial DNA and the use of PCR-restriction fragment length poly-morphism of cytochrome b gene sequences. Current knowledgesuggests that mitochondrial DNA may be more suitable thannuclear DNA for this kind of analysis. This material is attractivebecause of its variability (mitochondrial cytochrome b gene se-quences differ by at least a few nucleotides, even in very closelyrelated species), the availability of mitochondrial sequence datafrom many vertebrates and the high copy number relative to thenuclear DNA.

The methods discussed above125,127,131 are based on PCR as-sociated with restriction fragment length polymorphism, whichentails the amplification of a common fragment followed by di-gestion with one or more species-specific restriction enzymes.In contrast, design of species-specific primers has meant that ithas been possible to simplify the technique avoiding the need forsubsequent restriction fragment analysis. Different works havebeen reported in this direction.128,132,133

Another alternative approach includes duplex-PCR. It allowsco-amplification of separate regions of a single gene or spe-cific fragments, each typical of a different animal species ina single PCR reaction. The duplex-PCR technique has beenproposed134,135 to identify bovine and water buffalo DNA in asingle PCR assay in milk and mozzarella cheese originally madefrom pure buffalo milk. Primers were designed within the cy-tochrome b gene region of the mitochondrial DNA. The resultsof these experiments indicated the applicability of this method,which showed an absolute specificity for the two species anda high sensitivity even to low DNA concentrations. The min-imum concentration of bovine tested in buffalo and buffalo inbovine milk was 1%,134 just the percentage considered to be theminimum legal limit.62

Molecular genetic techniques could also be used to iden-tify the breeds involved in milk production. For instance, somePDO cheeses have restrictions, because they should be madeonly from specific breeds milk. Maudet and Taberlet136 devel-oped a quick and easy DNA-based test to specifically detectHolsteins breed milk in French PDO cheeses that are not al-lowed to contain milk from this breed. They found a molecularmarker for Holsteins milk detection in a gene affecting coatcolor in cattle. A single nucleotide substitution in that gene al-lowed the differentiation of breeds. DNA extraction from cheese,a pre-amplification of the gene and a competitive oligonucleotidepriming PCR were performed. 1% of Holstein milk was de-tectable in milk curd. The development and application of thesetests could be a powerful tool to authenticate PDO cheeses orother high-quality dairy products made from milk from specificbreeds in the future.

Although the results of PCR applied to detect the authentic-ity of dairy products seem reliable, some parameters are still

difficult to control. The amount of DNA recoverable from milkproducts is directly related to the somatic cell content of the rawmilk, and the somatic cell count varies according to individualmodifications, diseases, and number and stage of lactation. Cowsinfected by mastitis have a very high number of somatic cellscompared with healthy individuals. Nevertheless, milk qualitycontrol tests for quality payments currently carried out in manycountries, and the huge number of cows on dairy farms are fac-tors that could help reduce the effects of mastitis. Similar prob-lems can also arise with the habitual methods used for measuringproteins, since protein levels vary between cows and breeds.

Despite the disadvantages that still exist, the PCR-methodfor food analysis will become increasingly important in the fu-ture. Further developments could consist of extending the directidentification to other milk-producing species in the same PCRassay using the multiplex-PCR technique. This could allow allthe species present in a milk mixture to be detected at the sametime. Basic premises for this expected development are furthersequence data about the genomes of ruminants, the use of quan-titative PCR to get a more accurate estimation of cows milkproportion in other milks, development of new faster and cost-effective DNA extraction and purification procedures prior toamplification, and the use of real-time PCR135 together with flu-orescence techniques to allow the quantitative determination ofthe sequence of DNA that is amplified, thereby avoiding analysisby gel electrophoresis.

ADDITION OF WHEY PROTEINS TO DAIRY PRODUCTS

Increasing cheese and caseinate industrial production givesrise to larger volumes of whey. The overall production of wheypowder exceeds the demand for this product. Whey solids maybe fraudulently added to dairy products. This practice, in whicha portion of casein from the milk is replaced by whey proteins,soluble peptides, and lactose, has attracted great interest; a vari-ety of analytical procedures have been developed to detect theirpresence qualitatively and quantitatively.

Special attention has been given to detecting the addition ofrennet whey solids to liquid milk and milk powder. Determina-tion of rennet whey is based on the detection and quantificationof 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) isrecovered in the rennet whey. On rare occasions, -casein is splitat position 106107, resulting in the formation of pseudo-CMP,lacking the N-terminal Met. CMP should be absent from milkand has been used traditionally as an index or marker of rennetwhey solids.

The E.U. official method138 for detecting rennet whey inmilk powder is based on CMP analyses by HPLC on wide porereversed-phase and UV detector after sample treatment with8% TCA. This procedure employs an extremely flat gradient

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of acetonitrile, which generates just enough resolution betweenCMP and pseudo-CMP. However, under certain circumstancesfalse positive results could not be excluded, mainly due tothe co-elution with CMP of proteolytic products arising fromthe action of psychrotrophic proteinases on -casein. Theseproteinases (mainly from enzymes of Pseudomonas), whichprogressively split -casein in milk, can give rise to degrada-tion products similar to CMP, depending on the storage con-ditions and composition. Alternatives based on RP-HPLC toimprove the E.U. method138 include reducing the TCA concen-tration during extraction139141 and applying selective detectionwith pulsed electrochemicals.142 Procedures employing cation-exchange chromatography without the use of TCA143,144 havealso been proposed, but it is unlikely that pseudo-CMP can beseparated from CMP, due to a lack of charge difference. In gen-eral terms, chromatographic procedures do not have enough re-solving power to separate the peptides arising from the actionof residual or reactivated proteolytic enzymes during storageof long-life products, such as UHT milk. These degradationproducts cannot be separated by HPLC and can complicate thedetection of CMP and give false positive results.

Although other methods, such as IEF145 and ELISA withPAB against the 130152 fragment of bovine -casein,146 havealso been assayed, CE has already become the most powerfultool for detecting CMP in dairy products. CE has the potentialto give rapid separations with high plate numbers, provided thatadsorption from the proteins to the capillary wall is suppressed.Furthermore, it allows a very high resolution of proteins andpeptides that differ in just one amino acid residue, and unlikeconventional electrophoresis, there is no limitation in the size ofthe components to be separated. Otte et al.147 tested that CMPcould be separated from whey protein using untreated capillarycolumn at low pH (2.5). Van Riel and Olieman148 published avery selective CE approach that separates CMP from pseudo-CMP and could prevent false positive results in dairy products.They used a hydrophilic coated capillary at low pH, affordinga limit of detection of 0.4% of rennet whey solids in milk andbuttermilk powder. Thereafter, it was applied for the determina-tion of rennet whey in UHT milks,149 and it also permitted thedetection of 1% ovine and caprine cheese whey in bovine milkwhen a western blotting detection system was incorporated.150

The investigation of the suitability of this CE method to de-tect rennet whey added to UHT milk revealed that, except for thecase of freshly processed milk manufactured from raw materialsof good microbiological quality, the formation on storage of pro-teolytic breakdown products closely related to CMP hamperedaccurate determination.149 In fact, it was later demonstrated151that thermostable proteinases from psychrotrophic bacteria, al-though less specific than chymosin, can also split -casein atposition 105106, leading to the formation of CMP, as well asother very similar peptides (Figure 3). To solve this problem,linear discriminant functions, using ratios for CE peak areasof CMP and two other CMP-like degradation products weredefined.152 These functions permitted the detection of rennetwhey solids added to UHT milk, except in the case of very severe

proteolysis, occurring after very long storage periods or at hightemperatures.

Whey other than rennet cannot be tested by detecting andquantifying CMP. For example, there are no specific indicatorsfor the addition of acid whey to milk products. In these cases,the approach for detecting whey additions consists of determin-ing the whey protein in total protein ratio (WPTPR). Althoughabout 80% of milk protein are caseins and 20% are whey, thereis a wide variation in the range of naturally occurring WPTPR.Protein denaturation, chemical reactions that take place duringmilk processing, and proteolysis may alter protein characteris-tics, and therefore, influence the accurate determination of thedifferent protein fractions in dairy products.

Traditional procedures to calculate WPTPR include the de-termination of casein-bound phosphorus, direct measurement ofcasein and whey proteins by SDS-PAGE and polarographic eval-uation of cysteine/cystine, which are indicative of whey protein.However, these classic methods are tedious, time-consumingand are not suitable for proteolyzed products, such as cheeseand high protein denaturation material, because of the ther-mal processing. In the last few years, new and more rapid an-alytical methods have been proposed as alternatives. Schmidtet al.153 presented a fast (less than 2 min) and versatile ap-proach for discrimination of pure milk samples from milk sam-ples with added whey proteins based on pyrolisis-mass spec-trometry. Non-invasive techniques include Fourier TransformInfrared (FTIR) spectroscopy154 photoacoustic spectroscopy,155CE,156 and principally UV spectroscopy.156161

Meisel157 proposed a method to determine WPTPR usingfourth derivative spectroscopy based on differences in the UVspectrum between tryptophan and tyrosine, as well as on dif-ferences in the tryptophan to tyrosine ratio, which is 0.19 forcasein and 0.59 for whey proteins. A similar procedure basedon the zero or first-order derivative UV spectroscopy in alkaliwas applied for the determination of whey powder to milk pow-der ratio.158,159 Peng and Puhan160 and Miralles et al.161 usedthe fourth derivative UV spectroscopy method for assessing thequality of different types of milks. With this method it was pos-sible to detect adulterations of UHT milk with whey over 5%,determining the WPTPR in UHT milk irrespective of the bacte-rial count of raw milk and storage time.161 Sample preparationonly included skimming to avoid interferences in the measure-ment and skim milk dilution with guanidine HCl buffer.

With CE, WPTPR is determined as the ratio between the areaof the capillary electrophoretic peaks corresponding to wheyproteins and the area of these peaks corresponding to whey pro-teins plus caseins. Miralles et al.156 compared CE, SDS-CE, andUV fourth-derivative absorption spectroscopy to accurately de-termine the WPTPR in raw, pasteurized, and UHT milks. The re-sults confirmed that the heat treatment applied to milk, althoughsevere enough to produce lactosylation of the proteins, doesnot influence the WPTPR determined by these three methods(Table 3). However, CE and SDS-CE still need expensive equip-ment and qualified operators, whereas UV fourth-derivativeabsorption 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 6C (a) and a sample of skim milk powder containing 5%rennet whey powder. (Reproduced with permission (149)).

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Table 3 Whey protein percentage to total protein ratio in raw, pasteurized,and UHT milks using capillary electrophoresis (CE), sodium dodecyl sulphatecapillary electrophoresis (SDS-CE), and UV-Fourth derivative absorptionspectroscopy (UV-4th derivative) (Data taken from reference 156)

Whey protein/Total protein (%)Type of milk CE SDS-CE UV-4th derivative

Raw 17.1 2.1 18.5 2.4 17.2 1.6Pasteurized 16.6 2.2 17.7 2.2 18.8 2.2UHT 16.8 2.1 17.0 2.0 17.2 1.8

laboratories, which makes it more suitable for routine analy-sis. In order to introduce an E.U. reference method to determinethis ratio, these authors156 recommended more studies in milksamples from different countries and in milks with different pro-teolysis degrees caused by storage.

FOREIGN VEGETABLE PROTEINSIN MILK PRODUCTS

Vegetable proteins can be added to milk to make the cost of theproduct lower or healthier because of the increased fiber content.These proteins often have good hydration properties, producinga higher moisture content in cheese. Despite the good nutritionaland functional properties of these products, their use as supple-ments and substitutes for bovine milk protein is forbidden. Theiraddition is not legal, since by definition, a dairy product can onlybe obtained by processing milk or components of milk origin.Therefore, the determination of the presence and amount of thesevegetable proteins in milk or dairy products is necessary.

Although wheat gluten, pea, rice, potato, bean, or solublecereal hydrolysates proteins can also be used as vegetable sub-stitutes, soy protein, due to its low price and high availability inthe market is likely to be the major adulterant. Several prepa-rations of soy proteins are commercially available, such as soyflour (4252% of protein), concentrates (6269% of protein),isolates (8287% of protein), and hydrolysates (around 20% ofprotein). For their detection, several analytical techniques canbe applied. In the early nineties information available on meth-ods for detecting the possible addition of such protein to milkwas still limited to immunological and electrophoretic proce-dures that were able to detect around 5% soybean proteins inmilk.2 Recently, during the last ten years, new developments inthese techniques and new approaches, such as CE, HPLC, andbiosensors, have become available for use.

RP-HPLC has been shown to be useful to analyze soy beanproteins and achieve the simultaneous separation of soya beanand bovine whey proteins.162,163 Espeja et al.164 by RP-HPLCperfusion achieved the simultaneous separation of soy and milkproteins in cows, goats, and ewes milk spiked with these pro-teins in less than 2 min using a linear acetonitrile-water-0.1%trifluoracetic acid binary gradient. The standard addition methodwas, however, required to quantify cows milk samples due tothe existence of matrix interferences. It is well known that quan-

titative determination of soy proteins presents problems, par-ticularly when mixed in low proportions with other productsand further heat treated. Cattaneo et al.165 tested gel permeationchromatography (GPC), SDS-PAGE and IEF for detection ofsoy proteins in melted cheeses, after selective sample treatmentwith a tetraborate-EDTA buffer. The use of this buffer was re-vealed to be of vital importance for removing interfering peaksor bands from milk proteins both in GPC and electrophoresis.Soya proteins are insoluble in a tetraborate-EDTA buffer, whichthus allows the removal of soluble caseins from samples. SDS-PAGE took advantage of the size exclusion capacity of the gel inorder to separate and detect the presence of soy proteins with themost sensitivity (0.06% soy protein in total protein). This tech-nique also had the advantage that it was not affected by heattreatment. Results were, however, difficult to quantify, and thetechnique was rather time-consuming.

The first application of CE to the analysis of soy and milkproteins on the basis of their different CE pattern was developedby Kanning et al.166 using a hydrophilic coated capillary anda low pH buffer. Later, these studies were accomplished by anSDS-CE after a tetraborate-EDTA sample treatment.167 This ap-proach allowed the separation of the basic subunits of glycininand the and subunits of -conglycinin from the main milkprotein peaks (Figure 4) reducing the limit of detection of soyprotein in milk powder to 1%. SDS-CE afforded less resolutionthan 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 similarperiod of time. Unfortunately, the addition of soy-protein hy-drolysates could not be determined.167

A promising methodology for the detection of plant proteinsin milk products consists of the direct biosensor immunoas-say format, whereby antibodies are immobilized on the sensorsurface, and the binding of plant proteins of the immobilizedantibodies is detected directly. Haasnoot et al.168 designed anoptical biosensor (Biacore 3000) for the simultaneous detectionof soy, pea, and soluble wheat protein in milk powders. PurifiedPAB raised against the three protein sources were immobilizedin different flow channels on the biosensor chip. The total runtime was 5 min, and the limits of detection in milk powder werebelow 0.1%. These kinds of instruments can be future routineprocedure alternatives.

A collaborative study169171 involving 8 international lab-oratories was conducted to evaluate 2 electrophoretic proce-dures (SDS-PAGE and SDS-CE combined with a tetraborate-EDTA sample pre-treatment) and an indirect competitive ELISAmethod using PAB for the determination of the fraudulent addi-tion of vegetable proteins. Levels of soy, pea, and wheat proteinsin different dairy products (milk powders, cheeses, and yogurts)subjected to low and high heat treatments were studied. In-housepre-validation tests showed that SDS-CE was more suitable thanSDS-PAGE for the detection of soy and pea proteins in milkpowder. Quantification of band volumes by SDS-PAGE wasdifficult due to variations in the background among gels andthe 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 themilk 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 validationthrough collaborative trials. SDS-CE provided a slightly betteraccuracy, but ELISA presented the advantage of being suitablefor samples containing wheat proteins, which could not be de-tected by SDS-CE. Precision parameters showed that, in generalterms, repeatability was similar for both techniques. The highestpercentage of reproducibility for SDS-CE was attributed to thedependence of the results on the equipment and conditions usedin each laboratory (detector sensitivity, sample loading, voltage,temperature control). ELISA overestimated the percentages ofadulteration in the analysis of low-heat-treated milk samples,

probably due to non-specific binding of antibodies with certainmilk constituents, whereas values obtained for high-heat-treatedsamples were lower than the real values. This was attributedto protein denaturation that could have caused aggregation andconsequently decreased immunoreactivity.

GEOGRAPHICAL ORIGIN OF DAIRY PRODUCTS

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

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controls. It is also of value for ensuring fair competition andprotecting consumers against fraud due to mislabeling. Milkand milk products of defined regional origin are highly valuedby consumers and command a premium price. Most of the PDOcheeses traded in the E.U. are classified according to their regionof production. The PDO system legislates for the monitoring ofgeographical origin of raw materials in PDO products. As thepopularity of PDO status labeling has grown, not only to protectthe interests of the regional producer, but also as an importantmarketing strategy, the need for analytical methods capable ofverifying geographical origin claims at the retail level has be-come increasingly important. Moreover, the confirmation of thegeographical origin also has wide implications for dairy prod-ucts, such as butters, which receive preferential taxation ratesfor importation into the E.U.

The ability to differentiate dairy samples from different coun-tries or regions to assess authenticity is being finely explored.Conventional authentication relies upon the availability of site-specific microbiological and physic-chemical parameters. Theanalysis of metabolic profiles of microbial isolates used in con-jugation with artificial neural networks has meant that it has beenpossible to infer the geographical origin of Portugese cheesesand it has been suggested as a tool for PDO certification,172whereas the flavor capabilities (based on the identification byGC-MS of volatile odor-conferring molecules) and the micro-bial diversity have been proved to be closely linked and re-lated to the geographical origin of natural whey cultures usedfor water buffalo mozzarella cheese manufacture.173 Grappinet al.174 were able to correctly discriminate the origin of 20Comte cheeses made in 5 different cheese plants according tosome physic-chemical variables, microbial counts, and sensorycharacteristics. Other works175,176 have found significant dif-ferences in the amount of terpenes and conjugated linoleic acid(CLA) in Gruye`re cheese between a lowland and a highland pro-duction zone. Collomb et al.177 confirmed that CLA and sometrans fatty acids could also be interesting potential indicators forthe origin of cream and PDO cheeses. Fat content and pH value,as well as biochemical parameters, such as L- and D-lactateand piruvate, meant that it was possible to partially discriminatebetween the regions when these indicators were combined byprincipal component analysis in Emmentaler cheese.178 Moreresearch carried out by GC/MS and MS-based electronic nosehave showed the potential of volatile compounds to discriminateEmmentaler cheese samples of different geographical origin.179The non-protein nitrogen and the water-soluble nitrogen frac-tions of these cheeses also showed significant interregional dif-ferences due mostly to the different ripening times.180 However,conventional analytical methodology allowing the unequivocaldetermination 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 chemo-metrics have also been investigated for their potential for dis-criminating cheese from several geographical regions.181,182 Pil-lonel et al.182 correctly classified 20 Emmentaler cheese sam-ples from 6 different European regions by near and mid-FTIR.

Despite the few samples, clear trends were observed. Neverthe-less, further analysis with more samples will be necessary toconfirm these results and build a validated prediction model.

Natural isotope fractionation could possibly be the best an-swer to the question of the geographical origin of foods. Thisapproach is based on the small but significant different ratios ofthe stable isotopes of bioelements, mainly nitrogen (15N/14N),carbon (13C/12C), oxygen (18O/16O), sulfur (34S/32S), and hy-drogen (2H/1H) present in certain organic molecules, due to thekinetic-chemical and physical factors that can be correlated withthe metabolic and/or geographical origin of a product. The rel-ative abundance of the heavy and light stable isotopes of theseelements varies in the environment around us as a result of bio-chemical and physical processes. These processes are said tofractionate the isotopes of an element. This fractionation leadsto characteristic isotopic signatures or fingerprint for a particulargeographical location.

For many years, Isotope Ratio Mass Spectrometry (IRMS)has been the official technique for detecting adulteration inhoney and has been used in other foods, such as wines andfruit juices.183,184 More recently, studies published in the liter-ature have shown that determining the ratios of stable isotopesof bioelements by mass spectrometry can also be applied tomilk products. The principle of IRMS consists of measuring theisotope ratio of an analyte converted into a simple gas, isotopi-cally representative of the original sample before entering theion source of an IRMS system.

The first condition for a stable isotope method to be used as aroutine is that there is a scientific explanation (physical, chemi-cal, or biochemical) for variations of the isotope ratios in naturalsubstances. Based on this knowledge, the establishment of a rel-evant database for statistical evaluation is required. The secondrequisite is a sufficient number of laboratories able to applythe method with good reproducibility and repeatability. This isusually tested by interlaboratory comparison. Furthermore, theability of laboratories to apply such standard methods properlyin routine work should be tested in regularly repeated intercom-parison exercises, where the result of the single laboratory incomparison to the mean value of all other laboratories is mostrelevant. In addition, it would be advisable to complement IRMSwith conventional methods of analysis: GC,185 HPLC,186,187 orICP.188 Stable isotope procedures are not supposed to replaceclassic analytical methodologies, rather they are to serve as ad-ditional indispensable tools for authentication.

The results of the multi-element stable isotope ratio analysisindicate the possibility of performing a regional origin assign-ment for dairy products. The measurement of carbon, oxygen,and nitrogen stable isotope abundances in milk reflected the iso-topic composition of the diet fed to the dairy cows. This diet andits isotopic ratios depend on geographical and climatic factors.Kornexl et al.189 and Rossmann et al.190 classified milks fromdifferent Bavarian regions and Alpine areas by IRMS. Milksfrom zones dominated by grassland typically show relativelynegative 13C-values, while in regions dominated by crop culti-vation 13C-values are more positive: extensive production based

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on greenland feed, or more intensive farming with a higher de-gree of corn. Differences in plants, and primarily in soil, arecaused by nitrogen sources and complex processes. Fertilizersare nitrogen sources that particularly influence the 15N contentsof plants and subsequent fodder. Specific environments can alsobe more or less suitable for growing different plant species, someof which exhibit very low 15N contents (legume, grass), whileothers preferably grow in soils with higher 15N-values (rape,maize). The difference in the 18O-value of milk relative to thatof the source of water is an additional assignment criterion andis a good indicator of temperature and climate (continental ormaritime) of the region. Concerning sulphur fractionation, ithas been reported183,185 that the geology of a region (igneous orsedimentary, acidic or basic) could considerably influence 34S-values of soils. Possible effects of industrial emissions on thesevalues of the soil via wet and dry deposition also have to be con-sidered. Lamprecht et al.191 and Lamprecht and Haberhauer186developed procedures for isolation by cation-exchange chro-matography and enrichment of methionine-bound sulphur incasein for stable isotope ratio analysis. These techniques wereused to create a database for determining the country of originof the milk samples.

Multi-element stable isotope analysis, together with discrim-inant analysis of the results and evaluation by comparison withdata for certified authentic samples, can be a powerful tool forsolving the problem of butter origin assignment.188 Rossmannet al.192 carried out determinations of the light elements (C, N,O, and S) and a heavy trace element (Sr) for butter from sev-eral European countries and others outside the E.U. 87Sr/86Srin butter provided the means whereby regions with similar oridentical climatic conditions could be further subdivided ac-cording to their respective geological conditions. Low 87Sr/86Srvalues in soil are encountered in mafic rock terrain (volcanicor ultrabasic, basaltic) and in sedimentary carbonate-rich rocks,whereas higher values are found in old acidic (igneous or meta-morphic, granitoidic) areas. The results of this study192 indicatedthat IRMS can reliably detect the regional origin of butter.

15N/14N and 13C/12C of caseins and the ratios between somefree amino acids, such as Thr/Pro, Ile/Pro, Met/Pro, and His/Pro,were successfully used to identify the place of origin of ewesmilk in PDO Italian cheeses produced in different regions.187Wietzerbin et al.193 also confirmed the usefulness of isotopictechniques to control the compliance of PDO cheeses with therestricted rules associated with their production, including theauthentication of the geographical origin by comparison with

Table 4 Overview of methods (19912003) for milk and dairy products authenticity testing

Mixture or addition Method Marker/Parameter Reference

Buttermilk powder in skim dry milk SDS-PAGE Proteins of fat globule membrane 204Added water to milk Cryoscopy and Titration Freezing point and titrable acidity 205Distinguish natural from imitation Mozzarella cheese RP-HPLC Lysinoalanine 206Artificial flavoring in cheeses GC-IRMS 2H/1H of benzaldehyde 207Acid casein and caseinates in processed cheeses CE Intact -casein 208Rennet casein in processed cheeses CE Para--casein 209Cheaper clotting enzymes during PDO cheese making CE Peptides 210

authentic samples. This study also detected fraudulent practices,such as the use of corn silage in the cattle diet (when only theuse of grass is claimed) or the addition of foreign components.

ADDITION OF HEATED AND DRIED MILKTO DAIRY PRODUCTS

Although the addition of milk powders is a practice that is al-lowed in yogurts to enhance their functional properties, in otherdairy products it is considered fraudulent. Raw milk has a betterflavor than its heat-treated equivalent and the nutritional qualityof reconstituted dried milk powder is not the same as fresh wholemilk because of certain constituents. Additionally, the develop-ment of off-flavor and reduction in protein digestibility of driedmilk powder under adverse conditions of storage also reduces thequality of liquid milk when it is adulterated with this powder.Moreover, for some consumers, raw milk cheeses are consid-ered to have a better flavor than equivalent products made fromheat-treated milk. Thus, although some manufacturing practicesinclude the addition of dried milk to cheese milk, thereby in-creasing the cheese yield and reducing the production cost, thecheese quality can be negatively affected. All these problemspoint to the need to develop methods to detect the adulterationof raw milk with cheaper powdered reconstituted milk.

Although techniques, such as the determination of milkRNase activity,194 the study by CE of the ratio of -casein to-lactalbumin,195 and the use of Near Infrared Spectrometry,196have been reported to assess the adulteration of fresh or pasteur-ized milk with dry milk, most of the procedures to detect thisaddition are based on measuring the content of products formedas a result of the Maillard reaction or milk heat treatment. Othersindicators reported in the literature are the hydroxymethylfur-fural determined by visible spectrometry197,198 and mainly thefurosine, generated from acid hydrolysis of the lactulose lysinecomplex formed during heating.199201 Furosine measured byHPLC was successfully conducted as an index to detect theadulteration of raw, pasteurized milks and cheese with reconsti-tuted dried milk. However, this determination had an importantlimitation in that there was a wide range in the amount of furo-sine formed with the varying time and temperature limits in theUHT process.

In order to circumvent this disadvantage, other works202,203suggested determining the ratio of lactulose to furosine as anindicator. Milk drying promotes intensive Maillard reaction and

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Table 5 Overview of the main instrumental methods more frequently used for authenticity testing of dairy products

Marker Analytical method Usefulness Advantages Disadvantages ReferencesFatty Acids GC

Long capillarycolumns

Detection of non-dairy(vegetable and animal)fats in milk fat

Well standardizedIt is adopted by the International

Dairy Federation as the officialmethod.

Limited by the natural variability offatty acids

High detection limits (>15%) Largedatasets for statistic are required

1620

Triglycerides GCPacked and short

capillarycolumns

Detection of non-dairyfats in milk fat

Detection of cows milkfat in fat from goats orewes

Cheap, fast and well standardizedApplicability confirmed (method

adopted by the EU)Limit of detection below 5%

Accuracy is affected in lipolyzedsamples

It requires long previous studies todetermine the standard triglyceridecomposition.

2145

Phytosterols GC Detection of vegetablefats in milk fat

Very selective and sensitive. Tedious and time consuming.High variability depending mainly on

the several steps required.

465356

2 and 3caseins

IEF Identification of thespecie of origin (cow,ewe, goat and buffalo)

Well standardizedEU reference methodLimit of detection low (1%)

It is time-consuming.It requires special equipment and

enzyme treatmentIt is not possible to distinguish ewes

from goats dairy products.

626570

Whey proteinsand caseins

PAGE Identification of thespecie of origin (cow,ewe, goat and buffalo)

Low cost and technical skills Time consumingPoor visualization of small molecules

and low resolving powerIt is affected by heat treatment (whey

protein denaturation) and ripening(casein proteolysis).

677294


CE Identification of thespecie of origin (cow,ewe, goat and buffalo)

High resolving powerLittle or no sample preparationIt permits the identification of ternary

samples (cow + goat + ewe)

Low precisionNo quantitative methodIt is affected by heat treatment (whey

protein denaturation) and ripening(casein proteolysis)

7784


HPLC Identification of thespecie of origin (cow,ewe, goat and buffalo)

QuantitativeHigh repeatabilityEasy collection to re-analyze

Long analysis timesComplex chromatogramsDifficulties in detecting triple mixturesIt is affected by heat treatment (whey

protein denaturation) and ripening(casein proteolyis).

85899194

Fragments ofdairy proteins

ELISA (MAB) Identification of thespecie of origin (cow,ewe, goat and buffalo)

High specificity and sensitivityLow cost per analysisUseful as routine methodIt permits the identification of ternary

samples.

Generation of antibodies is veryexpensive.

MAB production can take long periodsof time

112122

DNA PCR Identification of thespecie of origin (cow,ewe, goat and buffalo)

Breed identification.

High specificity and sensitivityTechnique not affected by ripening or

sample heat treatmentsIt could be quantitative.

It is affected by the number of somaticcells (therefore it is influenced byphysiological factors).

It requires previous identification ofspecific DNA fragments.

125127128130137

CMP HPLC Detection of rennet whey It is a well standardized method(official E.U.)

Quantitative procedure

It does not have enough resolving powerDegradation products can affect CMP

analysis (in long-life products)

138144

CMP CE Detection of rennet whey High resolution of peptides that differ inonly one amino acid.

Proteolytic breakdown products do notinterfere in the determination.

Low precision and non-quantitativemethod

It would need standardization.

147152

Whey protein intotal proteinratio

UV Spectroscopy Detection of acid whey Suitable for routine analysisCheapEasy sample preparation

It requires tedious calibration studieswith different types of milk samples.

156161

Vegetableproteins

ELISA (PAB) Addition of vegetableproteins

Large sample throughputHigh sensitivityIt permits the detection of wheat proteins

and adulteration of high heat milkpowders.

Selection of suitable antigens stillremains the major problem

Semi-quantitative

169171

Vegetableproteins

CE Addition of vegetableproteins

Rapid and automated analysisHigh resolution

It is not independent of milk-processingconditions.

Wheat proteins are not detected.Low reproducibility

167169170

Isotope ratios(C, N, O, Sand Sr)

IRMS Identification of place oforigin

Detects fraudulentpractices such as theuse of supplements inthe cattle diet oraddition of foreigncomponents

It is the only method able to determineunambiguously the regional origin ofmilk and dairy products.

The quality of the results has to becontinuously controlled by regularblind analysis.

It requires databases and a sufficientnumber of laboratories able to applythe method.

186187189193

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low lactose formation, due to the low water activity and rel-atively mild thermal conditions. As a consequence, the lac-tulose/furosine ratio in UHT milks is approximately 16 timeshigher than in commercial milk powder samples. Ratios lowerthan 6.0 in processed UHT milk may indicate the presence of re-constituted milk. Linear correlations between these two param-eters have been reported for UHT milk and an equation allowedaccurate detection of milk powder in UHT milk.203 In spite ofthese advances, the different processing practices employed inthe industry, the protein concentration of milk, the poor qual-ity of the raw milk, severe preheating or recycling of the UHTand prolonged storage at high ambient temperature can give riseto different ratios. Therefore more research and studies will beneeded to develop a reference method to detect this practice.

OTHER AUTHENTICITY ISSUES

The issues described above are the problems that have beenmore extensively studied. Other procedures described in theliterature in the last thirteen years to solve and detect practicesthat are not allowed are listed in Table 4. Among them, the addi-tion of caseinates or rennet casein in different dairy products hasaroused great interest and will require more studies in the future.

FUTURE AND CONCLUDING REMARKS

Table 5 summarizes the main characteristics of the most citedmethods for assessing the authenticity of dairy products. Ow-ing to the great amount of interest in this subject, which hasgiven rise to a large number of publications in recent years onthe use of high-resolution techniques, positive results can be ex-pected in the near future in the field of authenticity controls ofdairy products. Nevertheless, this assessment will be a difficulttask, and in most cases will require the measurement of sev-eral markers. It will also have to take into account natural andtechnology-induced variations. It should be borne in mind thatthe availability on the market of new dairy products can createnew and potential areas of deception. Furthermore, fraudulentpractices tend to be quite innovative and manufacturers are wellaware of the weaknesses in food inspection systems. There-fore, within the food quality framework, authorities will needto develop new methods and will have to be constantly adapt-ing existing methods to detect frauds and protect consumersfundamental rights.

The main developments in the future can be expected to cen-ter not only on enhancements to existing analytical methods, butalso on the prior stages of sample preparation, which tend to bedifficult to automate. The future of milk product authenticationtechniques embraces the field of mixture analyses avoiding com-plex protocols for sample preparation, using automated methodsor coupling with separation techniques. This is applicable to thePCR technique, which needs further development of rapid andgeneral sample preparation protocols before it can be used as anordinary detection method in milk products. Another promisingalternative 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 tothe routine laboratory will be useful for the dairy industry andconsumers, since they will help improve the optimization of pro-cessing technologies in the industry and ensure that the productreaching the consumer complies with labeling. The future per-spectives of CE will also lie in improved instrumentation, likeinjection and detection, which would provide enhanced preci-sion, and could provide reliable quantitative data, similar to thedata supplied by HPLC. More work will also be required onlong capillary column GC and complementary techniques, suchas MS, to identify and quantify individual TG. These methodscould help monitor new value-added fractions of milk fat withenhanced nutritional and functional properties generally pro-duced by interesterification of fractionation.

It also seems evident that the control of authenticity of dairyproducts may not only be attainable with a purely chemical vi-sion. Physical analysis, supported by IR and UV spectroscopy orIRMS, for instance, is appropriate both for global characteriza-tion of the sample and for a compound-by-compound characteri-zation. In this field, the future seems to be linked to the increasingdevelopment of analytical solutions that combine powerful an-alytical devices and data processing software.

Purity criteria have to be empirically determined by analyzinga wide array of genuine products, as well as creating and regu-larly updating a database with information on the concentrationranges of certain indicative components of the milk product con-cerned, mainly PDO. In order to solve difficult cases, more thanone analyte has to be considered for detecting fraud. Likewise,a combination of different analytical techniques to determinedissimilar characteristics of a commodity could be more use-ful than relying on one single method. Given the complexityof some problems, univariate statistics have to be substitutedby intricate statistical algorithms to aid pattern recognition andclassification of genuine and fraudulent products.

Finally, more effort is still needed for the validation of recentanalytical methods. This validation of new procedures, as wellas the development and assessment of new reference materialsvia collaborative trials, will continue to be an important issuein the authenticity of dairy products. In the future, it is alsoexpected that all official methods for milk product testing willevolve and be based on modern instrumental techniques, therebysubstituting the most laborious procedures.

ACKNOWLEDGEMENTS

The authors acknowledge financial support to the researchprojects AGL2002-00887 and AGL2005-04760-C02-01 fromthe Spanish Ministerio de Ciencia y Tecnologa.

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