Protein-Based Animal Species Authentication in Dairy Products

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1		Joana Amaral	--	4060	2022-04-27 13:14:01	\|
2	format	Jason Zhu	-70 word(s)	3990	2022-04-28 03:52:28	\|

This entry is adapted from the peer-reviewed paper 10.3390/foods11081124

Milk is one of the most important nutritious foods, widely consumed worldwide, either in its natural form or via dairy products. Currently, several economic, health and ethical issues emphasize the need for a more frequent and rigorous quality control of dairy products and the importance of detecting adulterations in these products. For this reason, several conventional and advanced techniques have been proposed, aiming at detecting and quantifying eventual adulterations, preferentially in a rapid, cost-effective, easy to implement, sensitive and specific way. Protein-based techniques, including electrophoresis, chromatography and immunochemical assays, are considered current methodologies for assessing the authenticity of dairy products. They are generally considered fast, high throughput and cost-effective, being suitable approaches for the analysis of animal species in raw milk. However, when applied to processed foods, their reliability might be compromised due to protein denaturation and consequent epitope modification, disabling the immunorecognition of proteins. In recent years, the developments of mass spectrometry (MS) platforms for protein analysis, characterization and quantification have provided alternative approaches that rely on marker peptides instead of whole proteins, being suitable alternatives to analyze processed products.

Protein analysis Authenticity Dairy products

1. Electrophoretic Techniques

Different works using electrophoretic techniques have been reported so far for the detection of milk adulteration, including the use of polyacrylamide gel electrophoresis (PAGE) or, most frequently, the use of isoelectric focusing (IEF). Although PAGE is generally effective, its main limitation concerns the complex band pattern obtained, with frequent overlap of bands that can lead to an equivocal interpretation of results. Pesic et al. ^[1] suggested the use of a native PAGE electrophoresis for the qualitative and quantitative analysis of bovine adulteration in ovine or caprine milk based on bovine β-lactoglobulins (β-LG) and α-lactalbumins (α-LA). This method was considered a fast and convenient alternative for the detection and estimation of milk adulteration. However, its application is limited to fresh milk mixtures since heat processing and pH can cause the denaturation of whey proteins, with β-lactoglobulins being remarkably affected particularly by severe heat treatments including ultra-high temperature (UHT).

A similar approach consisting of isoelectric focusing (IEF) of γ-caseins, namely γ2-and γ3-caseins obtained by plasminolysis of β-casein, is currently the reference method in the EU for the determination of cow’s milk caseins in ovine, caprine, and water buffalo cheeses ^[2]^[3]^[4]^[5]^[6]. In this method, the samples should be analyzed together with reference standards containing 0% and 1% cows’ milk, being considered positive if both bovine γ2-and γ3-caseins, or the corresponding peak area ratios, are equal to or greater than the level of the 1% reference standard ^[6]. The method can be used for detecting either raw or heat-treated cow’s milk and caseinate in fresh or ripened cheeses made of ewes’, goats’ and buffalos’ milk or their mixtures, though not being suitable for the detection of milk and cheese adulteration by heat-treated bovine whey protein concentrates ^[6]. It is also not adequate for species quantification, especially in ternary mixtures due to the similarities between some species, such as ovine and caprine ^[3]^[4]^[7]. In fact, the reference method fails in detecting goat’s milk in sheep’s cheese and milk. Additionally, other works demonstrated that the evaluation of cow’s milk casein in water buffalo cheese by IEF is sometimes uncertain due to the presence of interfering co-migrating bands that can result in false positives ^[4]^[7]^[8]. Recently, Caira et al. ^[8] used a proteomic approach to demonstrate that this false positive result was due to the water buffalo fragment β-casein(f100-209), which was also formed after plasminolysis of buffalo’s milk or dairy products and co-migrates in IEF with bovine γ2-casein. To avoid false positives due to a water buffalo casein band with an isoelectric point similar to that of bovine γ2-casein, Addeo et al. ^[7] proposed the use of IEF coupled to immunoblotting to detect the presence of cow’s milk in water buffalo cheeses. The methodology proved to be successful in evaluating the authenticity of pure water buffalo milk and cheeses, with a limit of detection up to 0.25% bovine milk (v/v), which was lower than that described by the EU reference method (1%).

Capillary electrophoresis (CE) has been suggested as an alternative to gel electrophoresis-based methods for the authenticity assessment of dairy products because of its higher resolution power, low operation cost and high throughput ^[9]^[10]. Somma et al. ^[10] compared the efficiency of ultra-thin-layer IEF with capillary isoelectric focusing (cIEF) applied to the separation and identification of the main peptides arising from the hydrolysis of water buffalo and bovine β-caseins. Additionally, cIEF was used in combination with mass spectrometry for structural confirmation of the separated peptides. cIEF proved to be faster and more convenient because it does not require gel staining, though the cow-specific markers were only detectable at 5% cow’s milk addition in water buffalo’s milk, a value well above the sensitivity of the IEF method (0.5%). Nevertheless, both methods could be useful for detecting the fraudulent addition of cow’s milk to buffalo’s milk, which is very important in the production of Mozarella di Bufala cheese. More recently, Trimboli et al. ^[11] proposed the use of a routine CE method for human blood and urine protein analysis as a tool to authenticate ewe’s skimmed milk. The method was based on the separation of skimmed milk proteins and the use of a characteristic peak for ewe’s milk quantification in ovine/bovine skimmed milk mixtures, allowing people to detect a minimum amount of 5% of added cow’s milk with good linearity, precision and accuracy. A similar approach, using a routine CE method for blood analysis, was also attempted for detecting as low as 1% cow’s milk in buffalo’s milk and predicting the amount of fraudulently added milk by exploiting cow’s α-lactalbumin as a marker of adulteration ^[12]. Although most works dealing with the application of CE have been applied to milk mixtures, its use for the successful identification of animal species in cheese samples has also been demonstrated ^[13]^[14].

2. Immunochemical Techniques

Immunochemical methods are often used in the food industry for the qualitative and quantitative detection of food components and/or contaminants, being applied since the early 1980s to answer to the analytical demands in the dairy industry ^[3]^[5]^[15]. Essentially, an immunochemical assay consists of the reaction of an antigen with a specific antibody ^[5]. Therefore, immunochemical techniques provide highly specific and sensitive methods, being applied to a variety of complex food products. Compared with electrophoretic and chromatographic techniques, they are considered generally simpler, of lower cost, more sensitive and specific ^[3]^[5].

Enzyme-linked immunosorbent assay (ELISA) is the immunochemical technique most frequently used in dairy product analysis with diverse formats, including direct, indirect, sandwich and competitive, being applied to detect whey proteins and caseins. ELISA are frequently used in the analysis of milk and dairy products because of their easy application in routine analysis, low-cost, speed and sensitivity. However, the selected antisera influences the specificity and sensitivity of the method, thus requiring specific antibodies capable of differentiating species, without providing false positives due to cross-reactivity with non-target species or other food ingredients ^[15]^[16]^[17]. This could be achieved by the use of novel immunoreagents obtained by antipeptide antibody technology, suitable for milk species identification ^[18]. The characteristics, advantages and limitations of antibody-based techniques for the assessment of dairy products authenticity have been reviewed by Pizzano et al. ^[5].

ELISA has been used for species authentication in milk and dairy products since the late 1980s ^[5]. Hurley et al. ^[19] described the development of an indirect competitive ELISA, using bovine immunoglobin G (IgG) as a target, due to its high immunogenicity, to detect the presence of cow’s milk in other types of milk. The sensitivity of this technique was assayed using raw, pasteurized and previously frozen cow’s milk, concluding that high temperatures caused specific epitope modification. The detection limit in this method was 1 µg/mL of bovine IgG (0.1%), highlighting its high sensitivity without cross-reactivity with other species. Another study aiming at detecting cheese adulterations also targeting bovine IgG, but applying a sandwich ELISA, was performed by the researchers ^[16]. This methodology allowed further lowering the sensitivity to 0.001% of bovine milk in goat soft cheese and 0.01% of bovine milk in sheep and buffalo soft cheese.

ELISA targeting fairly thermostable proteins, such as caseins, has been proposed as a feasible alternative to detect adulterations in heat-treated milk and dairy products. Among caseins, bovine β-caseins present a high specific antigenicity, not being affected by heat treatment and having a concentration more or less stable and independent of season, climatic and feeding conditions ^[20]^[21]^[22]. Therefore, different ELISA have become available in the format of commercial kits for routine surveillance tests. The performance of such kits has been evaluated in different studies showing their usefulness for qualitative purposes but exhibiting inconsistencies in quantitative determinations of cheese adulteration. In 2008, Costa et al. ^[23] evaluated two specific commercial ELISA kits to quantify the amount of cow’s and goat’s milk added to sheep’s milk and cheese and concluded that they were more successful in detecting the adulteration in milk than in cheeses. More recently, Zeleňáková et al. ^[24] tested the reliability of a commercial ELISA (RC-bovino from Zeu-Inmunotec, Spain), concluding that the quantification of cow’s milk in sheep’s cheese was not exact, possibly due to modifications in the cheese matrix that take place during the manufacturing process. The same commercial ELISA kit was also used by Stanciuc et al. ^[25] to qualitatively detect the presence of cow’s milk in goat’s and sheep’s cheeses for confirmation of positive results obtained with a immunochromatographic method. From 73 tested samples from Romania, 67.3% of sheep’s cheeses and 79.7% of goat’s cheeses were adulterated by the addition of cow’s milk, suggesting the need to improve the quality control in the cheese industry. Another commercial kit (Casein ELISA set, SEDIUM R&D) was used by Zeleňáková et al. ^[26] to detect and quantify cow’s milk caseins in sheep’s milk and cheese, obtaining a calibration curve in the range of 0.5–50% using different mixtures of heat-treated milks. When applied to cheeses, the kit did not provide any relation between the presence of caseins and the increase in the cow’s milk proportion in the mixture, either using raw or pasteurized milk, concluding its inadequacy for cheese analysis. By contrary, the use of a sandwich ELISA kit (β-Lactoglobulin ELISA Set, SEDIUM R&D) targeting bovine β-lactoglobulin to detect adulterations in sheep’s milk and cheese was able to provide a quantitative analysis within 0.2–20 mg/kg ^[27].

Lateral flow immunoassays (LFIA) are alternative tools very easy to handle by non-expert workers. Thus, they can be applied in-field for screening purposes and are appropriate to be used by the cheese industry to quickly check and control the genuineness of the milk used along its production chain. Recently, Galan-Malo et al. ^[28] developed and validated a rapid test based on LFIA able to detect down to 0.5% of cow’s milk in goat’s, sheep’s or buffalo’s milk without identifying any false-positives among over 146 negative assayed samples.

Although most available immunochemical assays concern the authentication of sheep’s, goat’s and buffalo’s milk and/or cheeses, some studies have addressed other animal species. Pizanno et al. ^[18] developed an ELISA based on the use of antipeptide antibodies raised against the 1–18 sequence stretch of cow’s β-casein to successfully detect the presence of low levels (0.5%, v/v) of cow’s milk fraudulently blended with high-valued donkey’s milk. An indirect competitive ELISA to detect cow’s milk in yak’s milk using a specific monoclonal antibody for bovine β-casein (mAb 1-9B) was developed by Ren et al. ^[29]. The method allowed detecting 10 µg/mL of bovine milk in yak’s milk and was not affected by any external factors such as temperature and milk treatment.

3. Chromatographic and Mass Spectrometry Techniques

Up until now, different chromatographic techniques, including either gas or liquid chromatography, have been applied to authenticate dairy products because of their relative simplicity and speed, as well as possibility of automation ^[3]^[15]. High-performance liquid chromatography (HPLC) with ultraviolet (UV) detection was firstly used for the separation of the different casein fractions, relying on both normal (NP) or reverse-phase (RP) columns to identify cow’s milk in goat’s and sheep’s milk ^[30]^[31]^[32]^[33]. However, UV detection has drawbacks related to low specificity in the presence of co-eluting peaks or interferents. Thus, during the past decade, the technological advances, mainly in the area of mass spectrometry (MS) detection, have steadily replaced UV detectors, whenever the detection of food frauds is concerned. Soft-ionization techniques, such as electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI), have made possible to accurately analyze proteins and peptides, therefore allowing their use as reliable biomarkers for dairy product authentication. Peptides as biomarkers present advantages over proteins, which are affected by thermal processing ^[34]. Owing to the specificity, fastness, sensitivity and high reproducibility of the mass spectra, several methodologies based on MALDI time-of-flight mass spectrometry (TOF MS) have been developed, so far, to obtain informative fingerprints of milk proteins towards dairy product authentication ^[35].

Based on MALDI-TOF MS analysis of intact proteins of different milk species, Cozzolino et al. ^[36] suggested α-lactalbumin and β-lactoglobulin as markers for detecting cow’s milk added to sheep’s and buffalo’s milk or cheese. Researchers also demonstrated the usefulness of the method in detecting the addition of powdered to fresh milk based on the presence of lactosylated forms originated by heat processing. The analysis of entire proteins by direct MALDI-TOF MS coupled to unsupervised statistical analysis was also successfully proposed for milk authentication by Di Girolamo et al. ^[35] and Nicolaou and Goodacre ^[37]. Identical results were obtained by Kuckova et al. ^[38] regarding the identification of the species of origin in milk, though the same was not verified when the method was applied to analyze commercial cheeses, which could be attributed either to protein profile modifications or to adulteration of ovine and caprine cheeses. Recently, Rau et al. ^[39] demonstrated the feasibility of MALDI-TOF MS combined with a small in-house validated database, containing more than 150 reference spectra of milk and cheese, as a rapid, easy and robust method to identify the species of origin in mozzarella and white brined cheeses. The direct protein extraction without applying a tryptic digestion step allowed performing the analysis in less than 30 min with reduced analytical costs.

Other approaches have relied on a bottom-up proteomic strategy, based on MS analysis of peptides obtained after enzymatic digestion ^[39]^[40]^[41]^[42]. Calvano et al. ^[40] reported several bovine-specific peptide markers in milk tryptic digests that can be useful for detecting adulterations by cow’s milk addition to goat’s or sheep’s milk. Since the detection of sheep’s milk adulterated with goat’s milk is a difficult task because of their similar protein profiles, two goat-specific peptide markers assigned to κ-casein were identified ^[40]. Caira et al. ^[41] used a MALDI-TOF MS method to simultaneously determine the presence of water buffalo’s and cow’s milk in Italian water buffalo’s mozzarella cheese. Since crossbreeding with other water buffalo breeds has been avoided in indigenous Mediterranean Italian buffalo, these animals generally exhibit reduced milk protein polymorphisms when compared to other international breeds. Therefore, hundreds of milk samples (Italian and from several other countries) were analyzed, aiming at identifying signature peptides associated with water buffalo origin for the authentication of PDO products ^[41]. Caseins were the target proteins owing to the identified differences between indigenous and international breeds, namely the unique presence of a β-CN A variant and an internally deleted αs1-CN (f35-42) variant in international water buffalo milk samples. The peptidomic approach allowed the identification of several tryptic signature peptides as molecular marker candidates to detect the addition of imported water buffalo’s milk in Italian PDO products, as well as adulterations with cow’s milk blending. The proposed methodology enabled the specific detection of international water buffalo and bovine caseins down to 2% and 0.78%, respectively. MALDI-TOF MS has also been proposed to detect the adulteration of water buffalo’s ricotta with bovine milk based on a specific peptide marker, corresponding to the region 149–162 of β-lactoglobulin, enabling its detection down to 5% ^[42]. Nardiello et al. ^[43] proposed the use of a nano LC−ESI-ion-trap tandem mass spectrometry (nano LC-ESI-IT-MS/MS) methodology combined with a database post-processing to validate peptide sequence assignments and determine the species of origin in milk samples. Bovine species-specific peptides originated from αS1-casein and β-lactoglobulin were identified as suitable authenticity markers with detection levels as low as 1%.

MALDI-TOF MS has also been referred to as a tool for selecting the most suitable peptide makers in further analysis by liquid chromatography coupled to mass spectrometry (LC-MS) ^[44]^[45]^[46]. In fact, LC-MS has been increasingly applied in food analysis owing to its powerful capacity in detecting and quantifying specific analytes in complex mixtures, offering particularly enhanced selectivity and sensitivity when multiple reaction monitoring (MRM) scanning is applied ^[47]^[48]. Cuollo et al. ^[44] used two techniques, namely MALDI-TOF MS and LC-ESI/MS, to detect specific signature peptides to differentiate cow’s, sheep’s, goat’s and water buffalo’s milks, with both approaches providing similar sensitivities (1% for caprine and 0.5% for the other species). αs1-CN (f8-22) peptide was selected as a convenient marker for cow’s, sheep’s and water buffalo’s milk, while αs1-CN (f8-22) was for goat’s milk. MALDI-TOF MS data were tentatively used to perform quantitative analysis based on synthetically modified proteotypic peptides as internal standards, but accurate evaluation of caprine milk in quaternary mixtures was only achieved by LC-ESI-MS.

Sforza et al. ^[49] described an LC-MS method to evaluate the presence of cow’s milk in fresh sheep’s milk cheese targeting short marker peptides, namely αs1-CN (f1-23) and αs1-CN (f1-14), generated from proteolytic activities of the rennet enzyme chymosin and starter lactic acid bacteria, respectively. While the first peptide was degraded over time, thus being undetectable after long ageing periods, the second was frequently observed in cow’s milk cheeses. Despite this occurrence, the researchers referred to its detection in hard cheeses aged for more than 30 months. Moreover, the degradation of αs1-CN (f1-23) peptide also led to other fragments that could be detected. The method allowed the detection of cows’ milk down to 1% in all the analyzed cheeses, demonstrating the usefulness of these two candidate biomarkers to assess the addition of cow’s milk in fresh sheep’s cheese ^[49]. Czerwenka et al. ^[50] developed an LC-MS method to detect the adulteration of cow’s milk in water buffalo’s milk and mozzarella cheese, targeting the whey β-lactoglobulin as an adulteration marker. Since this water-soluble protein is mainly present in the whey fraction and not in the cheese, the analyzed parts were the brine in which this type of cheese is usually sold, or in the exudate obtained after cheese centrifugation. Researchers showed that sufficient amounts of β-lactoglobulin were present either in the brine or exudate, allowing the detection of adulterations with cow’s milk. The application of this method to assess 18 commercial samples of water buffalo mozzarella cheese allowed detecting three adulterated products. However, quantitative determination presented several pitfalls because of the variability the target the analyte between and within the two blended milks and the lack of an internal standard. Quantification of the fraudulent addition of bovine milk in the production of buffalo mozzarella PDO cheese was claimed by Russo et al. ^[47], based on UPLC-MS/MS exploiting the MRM mode, though the protein level in the studied cheeses was not taken into consideration. The use of MRM allowed a highly selective and sensitive detection and quantification of the chosen proteotypic marker, even in complex matrices, by simultaneously monitoring both their parent and one or more product ions. The selection of the species-specific proteotypic marker—phosphorylated β-CN (f33-48) tryptic peptide—was performed by an untargeted LC-MS/MS analysis by means of a quadrupole TOF MS equipped with an ESI source (ESI-Q-q-TOF). Additionally, to select the best conditions for trypsin digestion, a preliminary study was conducted by MALDI-TOF MS. Overall, the method allowed targeting the marker peptides with high specificity, thus being adequate for the authentication of complex matrices such as dairy products ^[47].

Despite the claimed advantage of quantitative analysis by LC-MS methods, it must be referred that it mainly gives an estimation of the fraud extent since the protein content of milk is known to vary with different factors, with the breed and season being of most relevance ^[45]^[46]^[47]^[48]^[49]^[50]^[51]. Trying to overcome this aspect, Gunning et al. ^[48] proposed the use of MRM MS-targeting αS1-casein to detect the addition of cow’s milk to buffalo mozzarella cheese. The relative amounts of each species in binary mixtures were determined based on corresponding peptides arising from a corresponding protein strategy and the ratios of transition peak areas. Moreover, identical peptides with the same sequences in both species were used to establish the relative levels of both species of αS1-casein in the component mixtures. The method was applied in a survey of 28 products sold in UK retail and restaurants, enabling researchers to verify that almost 2/3 were suspicious of being adulterated with cow’s milk. An UHPLC-MS/MS method also exploiting MRM mode, using at least two transitions for each compound, has recently been reported by Ke et al. ^[52] to quantify cow’s whey and whole milk powder in goat’s and sheep’s milk products, including infant formula. This method allowed the simultaneous quantification of four caseins (β-CN, αs1-CN, αs2-CN, and κ-CN) and two whey proteins (α-lactalbumin, β-lactoglobulin) based on the detection of their signature peptides. Isotopic labeled signature peptides were used as internal standards to compensate the matrix effect. The method was successfully validated regarding several parameters. Calibration curves for the tryptic signature peptides presented good linearity, the limits of quantification were between 0.01–0.05 g/100 g for the target proteins and the method showed high precision, reproducibility and recovery rates. The analysis of 11 commercial samples of goat infant formula milk powder revealed some adulterations among the evaluated products ^[52].

Although proteomic approaches developed so far mostly rely on the target identification of marker peptides, recently an untargeted UHPLC−MS/MS high resolution MS (HRMS) combined with chemometrics, was proposed to discriminate among cow’s, goat’s and buffalo’s milk samples ^[53]. The approach allowed the identification of different marker compounds, suggesting β-carotene and ergocalciferol for cow’s and water buffalo’s milk identification, respectively. Moreover, the levels of octanoic, nonanoic and decanoic acids were found to be higher in goat’s than in cow’s and buffalo’s milk ^[53].

Recently, the development of ambient ionization techniques, such as direct analysis in real time (DART), enabled a high-throughput and easy analysis of food. The potential of this ionization technique coupled to HRMS and chemometrics was exploited for dairy product authentication, including the discrimination of cow’s, goat’s and sheep’s milk. Results showed that DART-HRMS analysis of the non-polar fraction of milk had a limited discrimination potential, probably due to the high variability in triacylglycerols (TAG) among each group of samples ^[54].

Although the application of both chromatographic and mass spectrometry techniques to dairy product authentication mainly relies on protein analysis, other compounds such as fatty acids and TAG have also been addressed for this purpose ^[55]^[56]^[57]. Bratu et al. ^[58] used GC-MS analysis of fatty acid methyl esters coupled to principal component analysis (PCA) to differentiate 25 different cheeses (including cow, goat and sheep). Although sample discrimination in 3 groups was achieved using 12 components, more studies should be performed comprising a higher number of samples, also including model cheeses made with mixtures of milk besides pure milk cheeses. Vieitez et al. ^[59] showed that the addition of cow’s milk to pure goat’s milk influences the TAG profile by determining the partition number (PN), which characterizes the molecular structure of TAG. The analysis of blends containing 10, 20 and 50% of cow’s milk showed that the addition of cow’s milk to goat’s milk affects the TAG profile by decreasing TAG with PN between 38 and 42, while increasing it with PN between 46 and 50. Of the 15 commercial samples evaluated, 3 presented a different TAG profile, suggesting their possible adulteration with cow’s milk.

References

Pesic, M.; Barac, M.; Vrvic, M.; Ristic, N.; Macej, O.; Stanojevic, S. Qualitative and quantitative analysis of bovine milk adulteration in caprine and ovine milks using native-PAGE. Food Chem. 2011, 125, 1443–1449.
Amaral, J.S.M.I.; Pissard, A.; Pierna, J.A.F.; Baeten, V. Milk and milk products. In Foodintegrity Handbook; Morin, J.-F., Lees, M., Eds.; Eurofins Analytics France: Nantes, France, 2018; pp. 3–26.
Poonia, A.; Jha, A.; Sharma, R.; Singh, H.B.; Rai, A.K.; Sharma, N. Detection of adulteration in milk: A review. Int. J. Dairy Technol. 2017, 70, 23–42.
Spoljaric, J.; Mikulec, N.; Plavljanic, D.; Radeljevic, B.; Havranek, J.; Antunac, N. Proving the adulteration of ewe and goat cheeses with cow milk using the reference method of isoelectric focusing of gamma-casein. Mljekarstvo 2013, 63, 115–121.
Pizzano, R.; Nicolai, M.A.; Manzo, C.; Addeo, F. Authentication of dairy products by immunochemical methods: A review. Dairy Sci. Technol. 2011, 91, 77–95.
European Commission. Commission Implementing Regulation (EU) 2018/150 of 30 January 2018 amending Implementing Regulation (EU) 2016/1240 as regards methods for the analysis and quality evaluation of milk and milk products eligible for public inter-vention and aid for private storage. Off. J. Eur. Union 2018, L26, 14–47.
Addeo, F.; Pizzano, R.; Nicolai, M.A.; Caira, S.; Chianese, L. Fast Isoelectric Focusing and Antipeptide Antibodies for Detecting Bovine Casein in Adulterated Water Buffalo Milk and Derived Mozzarella Cheese. J. Agric. Food Chem. 2009, 57, 10063–10066.
Caira, S.; Nicolai, M.A.; Lilla, S.; Calabrese, M.G.; Pinto, G.; Scaloni, A.; Chianese, L.; Addeo, F. Eventual limits of the current EU official method for evaluating milk adulteration of water buffalo dairy products and potential proteomic solutions. Food Chem. 2017, 230, 482–490.
De la Fuente, M.A.; Juarez, M. Authenticity assessment of dairy products. Crit. Rev. Food Sci. 2005, 45, 563–585.
Somma, A.; Ferranti, P.; Addeo, F.; Mauriello, R.; Chianese, L. Peptidomic approach based on combined capillary isoelectric focusing and mass spectrometry for the characterization of the plasmin primary products from bovine and water buffalo beta-casein. J. Chromatogr. A 2008, 1192, 294–300.
Trimboli, F.; Morittu, V.M.; Cicino, C.; Palmieri, C.; Britti, D. Rapid capillary electrophoresis approach for the quantification of ewe milk adulteration with cow milk. J. Chromatogr. A 2017, 1519, 131–136.
Trimboli, F.; Costanzo, N.; Lopreiato, V.; Ceniti, C.; Morittu, V.M.; Spina, A.; Britt, D. Detection of buffalo milk adulteration with cow milk by capillary electrophoresis analysis. J. Dairy Sci. 2019, 102, 5962–5970.
Molina, E.; Ramos, M.; Amigo, L. Characterisation of the casein fraction of Iberico cheese by electrophoretic techniques. J. Sci. Food Agric. 2002, 82, 1240–1245.
Herrero-Martinez, J.M.; Simo-Alfonso, E.F.; Ramis-Ramos, G.; Gelfi, C.; Righetti, P.G. Determination of cow’s milk and ripening time in nonbovine cheese by capillary electrophoresis of the ethanol-water protein fraction. Electrophoresis 2000, 21, 633–640.
Reid, L.M.; O’Donnell, C.P.; Downey, G. Recent technological advances for the determination of food authenticity. Trends Food Sci. Technol. 2006, 17, 344–353.
Hurley, I.P.; Coleman, R.C.; Ireland, H.E.; Williams, J.H.H. Use of sandwich IgG ELISA for the detection and quantification of adulteration of milk and soft cheese. Int. Dairy J. 2006, 16, 805–812.
Asensio, L.; Gonzalez, I.; Garcia, T.; Martin, R. Determination of food authenticity by enzyme-linked immunosorbent assay (ELISA). Food Control 2008, 19, 1–8.
Pizzano, R.; Salimei, E. Isoelectric Focusing and ELISA for Detecting Adulteration of Donkey Milk with Cow Milk. J. Agric. Food Chem. 2014, 62, 5853–5858.
Hurley, I.P.; Coleman, R.C.; Ireland, H.E.; Williams, J.H.H. Measurement of bovine IgG by indirect competitive ELISA as a means of detecting milk adulteration. J. Dairy Sci. 2004, 87, 543–549.
Hurley, I.P.; Ireland, H.E.; Coleman, R.C.; Williams, J.H.H. Application of immunological methods for the detection of species adulteration in dairy products. Int. J. Food Sci. Technol. 2004, 39, 873–878.
Zelenakova, L.; Golian, J.; Zaiac, P. Application of ELISA tests for the detection of goat milk in sheep milk. Milchwissenschaft 2008, 63, 137–141.
Song, H.X.; Xue, H.Y.; Han, Y. Detection of cow’s milk in Shaanxi goat’s milk with an ELISA assay. Food Control 2011, 22, 883–887.
Costa, N.; Ravasco, F.; Miranda, R.; Duthoit, M.; Roseiro, L.B. Evaluation of a commercial ELISA method for the quantitative detection of goat and cow milk in ewe milk and cheese. Small Rumin. Res 2008, 79, 73–79.
Zeleňáková, L.; Židek, R.; Čanigová, M.; Ziarovska, J.; Zajác, P.; Marsalkova, L.; Fikselová, M.; Golian, J. Research And Practice: Quantification Of Raw And Heat-Treated Cow Milk in Sheep Milk, Cheese And Bryndza By ELISA Method. Potravinarstvo 2016, 10, 14–22.
Stanciuc, N.; Rapeanu, G. Identification of adulterated sheep and goat cheeses marketed in Romania by immunocromatographic assay. Food Agric. Immunol. 2010, 21, 157–164.
Zeleňáková, L.; Žıdek, R.; Čanigová, M.; Ziarovska, J.; Zajác, P.; Marsalkova, L.; Fikselová, M.; Golian, J. Reliability of cow casein quantitation in sheep milk and cheese by ELISA method. J. Food Phys. 2010, 23, 22–26.
Zelenakova, L.; Zidek, R.; Canigova, M. Optimization of ELISA method for detection of bovine beta-lactoglobulin in sheep milk and sheep milk products. Milchwissenschaft 2011, 66, 278–281.
Galan-Malo, P.; Mendiara, I.; Razquin, P.; Mata, L. Validation of a rapid lateral flow method for the detection of cows’ milk in water buffalo, sheep or goat milk. Food Addit. Contam. Part A 2018, 35, 599–604.
Ren, Q.R.; Zhang, H.; Guo, H.Y.; Jiang, L.; Tian, M.; Ren, F.Z. Detection of cow milk adulteration in yak milk by ELISA. J. Dairy Sci. 2014, 97, 6000–6006.
Veloso, A.C.A.; Teixeira, N.; Ferreira, I.M.P.L.V.O. Separation and quantification of the major casein fractions by reverse-phase high-performance liquid chromatography and urea-polyacrylamide gel electrophoresis—Detection of milk adulterations. J. Chromatogr. A 2002, 967, 209–218.
Enne, G.; Elez, D.; Fondrini, F.; Bonizzi, I.; Feligini, M.; Aleandri, R. High-performance liquid chromatography of governing liquid to detect illegal bovine milk’s addition in water buffalo Mozzarella: Comparison with results from raw milk and cheese matrix. J. Chromatogr. A 2005, 1094, 169–174.
Rodriguez, N.; Ortiz, M.C.; Sarabia, L.; Gredilla, E. Analysis of protein chromatographic profiles joint to partial least squares to detect adulterations in milk mixtures and cheeses. Talanta 2010, 81, 255–264.
Manzo, N.; Pizzolongo, F.; Montefusco, I.; Romano, A.; Masi, P.; Romano, R. Using whey proteins to detect the addition of bovine milk fat in buffalo cream destined for the butter-making process. Food Control 2017, 81, 164–167.
Buckley, M.; Melton, N.D.; Montgomery, J. Proteomics analysis of ancient food vessel stitching reveals > 4000-year-old milk protein. Rapid Commun. Mass Spectrom. 2013, 27, 531–538.
Di Girolamo, F.; Masotti, A.; Salvatori, G.; Scapaticci, M.; Muraca, M.; Putignani, L. A Sensitive and Effective Proteomic Approach to Identify She-Donkey’s and Goat’s Milk Adulterations by MALDI-TOF MS Fingerprinting. Int. J. Mol. Sci. 2014, 15, 13697–13719.
Cozzolino, R.; Passalacqua, S.; Salemi, S.; Garozzo, D. Identification of adulteration in water buffalo mozzarella and in ewe cheese by using whey proteins as biomarkers and matrix-assisted laser desorption/ionization mass spectrometry. J. Mass Spectrom. 2002, 37, 985–991.
Nicolaou, N.; Xu, Y.; Goodacre, R. MALDI-MS and multivariate analysis for the detection and quantification of different milk species. Anal. Bioanal. Chem. 2011, 399, 3491–3502.
Kuckova, S.; Zitkova, K.; Novotny, O.; Smirnova, T. Verification of cheeses authenticity by mass spectrometry. J. Sep. Sci. 2019, 42, 3487–3496.
Rau, J.; Korte, N.; Dyk, M.; Wenninger, O.; Schreiter, P.; Hiller, E. Rapid animal species identification of feta and mozzarella cheese using MALDI-TOF mass-spectrometry. Food Control 2020, 117, 107349.
Calvano, C.D.; De Ceglie, C.; Monopoli, A.; Zambonin, C.G. Detection of sheep and goat milk adulterations by direct MALDI-TOF MS analysis of milk tryptic digests. J. Mass Spectrom. 2012, 47, 1141–1149.
Caira, S.; Pinto, G.; Nicolai, M.A.; Chianese, L.; Addeo, F. Simultaneously tracing the geographical origin and presence of bovine milk in Italian water buffalo Mozzarella cheese using MALDI-TOF data of casein signature peptides. Anal. Bioanal. Chem. 2016, 408, 5609–5621.
Russo, R.; Rega, C.; Chambery, A. Rapid detection of water buffalo ricotta adulteration or contamination by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 2016, 30, 497–503.
Nardiello, D.; Natale, A.; Palermo, C.; Quinto, M.; Centonze, D. Milk authenticity by ion-trap proteomics following multi-enzyme digestion. Food Chem. 2018, 244, 317–323.
Cuollo, M.; Caira, S.; Fierro, O.; Pinto, G.; Picariello, G.; Addeo, F. Toward milk speciation through the monitoring of casein proteotypic peptides. Rapid Commun. Mass Spectrom. 2010, 24, 1687–1696.
Camerini, S.; Montepeloso, E.; Casella, M.; Crescenzi, M.; Marianella, R.M.; Fuselli, F. Mass spectrometry detection of fraudulent use of cow whey in water buffalo, sheep, or goat Italian ricotta cheese. Food Chem. 2016, 197, 1240–1248.
Galliano, F.; Saletti, R.; Cunsolo, V.; Foti, S.; Marletta, D.; Bordonaro, S.; D’Urso, G. Identification and characterization of a new beta-casein variant in goat milk by high-performance liquid chromatography with electrospray ionization mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun. Mass Spectrom. 2004, 18, 1972–1982.
Russo, R.; Severino, V.; Mendez, A.; Lliberia, J.; Parente, A.; Chambery, A. Detection of buffalo mozzarella adulteration by an ultra-high performance liquid chromatography tandem mass spectrometry methodology. J. Mass Spectrom. 2012, 47, 1407–1414.
Gunning, Y.; Fong, L.K.W.; Watson, A.D.; Philo, M.; Kemsley, E.K. Quantitative authenticity testing of buffalo mozzarella via alpha(s1)-Casein using multiple reaction monitoring mass spectrometry. Food Control 2019, 101, 189–197.
Sforza, S.; Aquino, G.; Cavatorta, V.; Galaverna, G.; Mucchetti, G.; Dossena, A.; Marchelli, R. Proteolytic oligopeptides as molecular markers for the presence of cows’ milk in fresh cheeses derived from sheep milk. Int. Dairy J. 2008, 18, 1072–1076.
Czerwenka, C.; Muller, L.; Lindner, W. Detection of the adulteration of water buffalo milk and mozzarella with cow’s milk by liquid chromatography-mass spectrometry analysis of beta-lactoglobulin variants. Food Chem. 2010, 122, 901–908.
Fuselli, F.; Tidona, F. Foreign milk in sheep’s, goat’s and water buffalo milk cheeses. In Handbook of Cheese in Health: Production, Nutrition and Medical Science; Preedy, V.R., Watson, R.R., Patel, V.B., Eds.; Wageningen Academic Publishers: Wageningen, The Netherlands, 2013; pp. 397–411.
Ke, X.; Zhang, J.S.; Lai, S.Y.; Chen, Q.; Zhang, Y.; Jiang, Y.R.; Mo, W.M.; Ren, Y.P. Quantitative analysis of cow whole milk and whey powder adulteration percentage in goat and sheep milk products by isotopic dilution-ultra-high performance liquid chromatography-tandem mass spectrometry. Anal. Bioanal. Chem. 2017, 409, 213–224.
Jia, W.; Dong, X.Y.; Shi, L.; Chu, X.G. Discrimination of Milk from Different Animal Species by a Foodomics Approach Based on High-Resolution Mass Spectrometry. J. Agric. Food Chem. 2020, 68, 6638–6645.
Hrbek, V.; Vaclavik, L.; Elich, O.; Hajslova, J. Authentication of milk and milk-based foods by direct analysis in real time ionization-high resolution mass spectrometry (DART-HRMS) technique: A critical assessment. Food Control 2014, 36, 138–145.
Blasi, F.; Lombardi, G.; Damiani, P.; Simonetti, M.S.; Giua, L.; Cossignani, L. Triacylglycerol stereospecific analysis and linear discriminant analysis for milk speciation. J. Dairy Res. 2013, 80, 144–151.
Chmilenko, F.A.; Minaeva, N.P.; Sidorova, L.P. Complex chromatographic determination of the adulteration of dairy products: A new approach. J. Anal. Chem. 2011, 66, 572–581.
Cossignani, L.; Pollini, L.; Blasi, F. Invited review: Authentication of milk by direct and indirect analysis of triacylglycerol molecular species. J. Dairy Sci. 2019, 102, 5871–5882.
Bratu, A.; Mihalache, M.; Hanganu, A.; Chira, N.A.; Todasca, M.C.; Rosca, S. Gas Chromatography Coupled with Chemometric Method for Authentication of Romanian Cheese. Rev. Chim.-Buchar. 2012, 63, 1099–1102.
Vieitez, I.; Irigaray, B.; Callejas, N.; Gonzalez, V.; Gimenez, S.; Arechavaleta, A.; Grompone, M.; Gambaro, A. Composition of fatty acids and triglycerides in goat cheeses and study of the triglyceride composition of goat milk and cow milk blends. J. Food Compos. Anal. 2016, 48, 95–101.

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Subjects: Agriculture, Dairy & Animal Science; Food Science & Technology

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Joana Amaral

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Mónica Honrado

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1. Electrophoretic Techniques

2. Immunochemical Techniques

3. Chromatographic and Mass Spectrometry Techniques

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