Detection Of Milk Adulteration Research Articles

Metabolomic biomarkers identify differences in milk produced by Holstein cows and other minor dairy animals

Several milk metabolites are associated with breeds or species of dairy animals. A better understanding of milk metabolites from different dairy animals would advance their use in evaluating milk traits and detecting milk adulteration. The objective of this study was to characterize the milk metabolite profiles of Chinese Holstein, Jersey, yak, buffalo, goat, camel, and horse and identify any differences using non-targeted metabolomic approaches. Milk samples were tested using nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography–tandem mass spectrometry (LC-MS). Data were analyzed using a multivariate analysis of variance and differences in milk metabolites between Holstein and the other dairy animals were assessed using orthogonal partial least-squares discriminant analysis. Differential metabolites were identified and some metabolites, such as choline and succinic acid, were used to distinguish Holstein milk from that of the other studied animals. Metabolic pathway analysis of different metabolites revealed that glycerophospholipid metabolism as well as valine, leucine, and isoleucine biosynthesis were shared in the other ruminant animals (Jersey, buffalo, yak, and goat), and biosynthesis of unsaturated fatty acids was shared in the non-ruminant animals (camel and horse). These results can be useful for gaining a better understanding of the differences in milk synthesis between Holstein and the other dairy animals.

Development of QCM Biosensor with Specific Cow Milk Protein Antibody for Candidate Milk Adulteration Detection

Adulteration of goat milk is usually done using cow’s milk product. Cow milk is used as it is widely available and its price is cheaper compared to goat milk. This paper shows a development of candidate tools for milk adulteration using cow milk. A quartz crystal microbalance immunosensor was developed using commercial crystal resonator and polyclonal antibody specific to cow milk protein. A specific protein at 208 KDa is found only in cow milk and does not exist in goat milk. The existence of this protein can be used as an indicator of cow milk content in a target solution. To detect the PSS 208 kDa protein, antibody specific to the PSS 208 was developed. The purified antibody was immobilized on top of the sensor surface on a polystyrene layer. The fraction of the immobilized antibody on the sensor was found at 1.5% of the given antibody. Using a static reaction cell, the developed immunosensor could detect the specific cow milk protein in buffer solution. The detection limit is 1 ppm. A linear relationship between frequency change and specific protein of cow milk concentration is found from a concentration of 1 ppm to 120 ppm.

Preservatives and neutralizing substances in milk: analytical sensitivity of official specific and nonspecific tests, microbial inhibition effect, and residue persistence in milk

<p>Milk fraud has been a recurring problem in Brazil; thus, it is important to know the effect of most frequently used preservatives and neutralizing substances as well as the detection capability of official tests. The objective of this study was to evaluate the analytical sensitivity of legislation-described tests and nonspecific microbial inhibition tests, and to investigate the effect of such substances on microbial growth inhibition and the persistence of detectable residues after 24/48h of refrigeration. Batches of raw milk, free from any contaminant, were divided into aliquots and mixed with different concentrations of formaldehyde, hydrogen peroxide, sodium hypochlorite, chlorine, chlorinated alkaline detergent, or sodium hydroxide. The analytical sensitivity of the official tests was 0.005%, 0.003%, and 0.013% for formaldehyde, hydrogen peroxide, and hypochlorite, respectively. Chlorine and chlorinated alkaline detergent were not detected by regulatory tests. In the tests for neutralizing substances, sodium hydroxide could not be detected when acidity was accurately neutralized. The yogurt culture test gave results similar to those obtained by official tests for the detection of specific substances. Concentrations of 0.05% of formaldehyde, 0.003% of hydrogen peroxide and 0.013% of sodium hypochlorite significantly reduced (P<0.05) the microbial counts in milk after 24 and 48h refrigeration. Formaldehyde and sodium hypochlorite remained detectable in milk after 48 and 24h of refrigeration, respectively; while hydrogen peroxide could not be detected after 24h. Official tests for the detection of milk fraud by the addition of preservatives and neutralizing substances present limitations and may be ineffective in detecting milk adulteration</p>

A Quick Detection of Melamine Adulteration in Milk and Dairy Products Using First-Order Multivariate Calibration

In this work, a reasonable substitute with laborious and expensive liquid chromatography for quick melamine detection in milk and dairy products is outlined. To accomplish this aim, first-order multivariate calibration methods including partial least squares (PLS) and net analyte preprocessing-partial least squares (NAP/PLS) are adopted for melamine quantification in complex food extracts down to 0.07 mg/kg without the need for efficient sample clean up. Solution pH has a significant influence on the performance of the proposed method and poor prediction was observed at pH 2.0 and 4.0. NAP/PLS outperformed PLS for melamine quantification, where analysis was carried out over the spectral domain 215–260 nm and pH 2–8. By using NAP/PLS, melamine is accurately quantified in spiked milk and dairy products (0.5–2.0 mg/kg) containing varying levels of fat (0–29%) and protein (3–23%), which proves the workability of the proposed method for handling wide spectrum of foodstuffs. At spiking level of 0.5 mg/kg, the average recoveries were acceptable (91.2–105.3%). However, modest recoveries were observed for food items containing high levels of protein and fat. Statistical tests confirmed that (with few exceptions) the NAP/PLS is of comparable analytical performance for highly advanced liquid chromatography which gives more chance for the current method to be applied for quick melamine detection in milk and dairy products. The proposed methods would be applied in food laboratories, especially when advanced instruments are not available. Practical Applications A new application of multivariate calibration for melamine detection (down to 0.07 mg/kg) in different types of milk and two dairy products is reported. The proposed analytical method would be of great importance from practical point of view considering the fact that chromatographic separation was avoided. The proposed methods would be applied in food laboratories, especially when advanced instruments are not available.

Intellectual property and technology management in dairy sciences.

Intellectual Property (IP) in dairy sciences at the Indian Council of Agricultural Research (ICAR) such as processes of animal nutrition and dairy food, milk adulteration detection kits, food processing machinery, and other know-how were managed by protecting them as patents at IPO, and transferred to their end-users. A total of 75 patent applications was filed in nine subject specific areas of dairy sciences, out of that 12% were granted for a period of 20 years. As per IPC these applications were fallen under its six classes viz. A, B, C, F, G and H and spread in 22 sub-classes. These IP assets were transferred to 26 public and private organization by signing agreements for 45 partnerships. These transferred technologies belong to six different subject specific areas of dairy science, where transfer of IP protected assets were on its higher side with 34%, which is a success indicator for a research organization. To speed up this process ICAR has taken the initiative with NAIP and established BPD Unit at NDRI, Karnal, which is a leading dairy science institute and also having a functional unit of TBI with the support of DST. With these new business dimensions 20 EDPs were organized, where 300 researchers, entrepreneurs, business organizations and dairy farmers were interacted to use the untapped IPs for the development dairy sector as a profitable business.

Performance improvement and single laboratory validation of classical qualitative methods for the detection of adulterants in milk: starch, chlorides and sucrose

Simple modifications in classical methods for detection of milk adulteration were validated with significant performance improvement.

A new comprehensive index for discriminating adulteration in bovine raw milk

This paper proposes a new comprehensive index, called Q, which can effectively discriminate artificial adulterated milk from unadulterated milk. Both normal and adulterated samples of bovine raw milk were analysed by Fourier transform infrared spectroscopic instrument to measure the traditional indices of quality, including fat (FAT), protein (PRO), lactose (LAC), total solids (TS), non-fat solid (NFS), freezing point (FP) and somatic cell counts (SCC). From these traditional indices, this paper elaborates a method to build the index Q. First, correlated analysis and principle component analysis were used to select parameter pairs TS-FAT and FP-LAC as predominant variables. Second, linear-regression analysis and residual analysis are applied to determine the index Q and its discriminating ranges. The verification and two-blind trial results suggested that index Q could accurately detect milk adulteration with maltodextrin and water (as low as 1.0% of adulteration proportions), and with other nine kinds of synthetic adulterants (as low as 0.5% of adulteration proportions).

Detection of Urea Adulteration in Milk Using Near-Infrared Raman Spectroscopy

We report results of a spectroscopic study to assess the applicability of near-infrared Raman spectroscopy for quantitative determination of urea adulteration in milk without any preprocessing requirement. Different batches of milk adulteration were prepared with each batch containing milk samples added with urea at various concentrations ranging from 10 to 1,000 mg/dl. The Raman spectra were measured from these urea-mixed milk samples with a Raman spectroscopy setup that incorporates a 785-nm diode laser for Raman excitation. An algorithm based on partial least square (PLS) regression was developed to quantitatively predict the concentration of urea based on the measured sets of Raman spectra of the urea-mixed milk samples. The results showed that Raman spectroscopy in combination with the PLS-based chemometric algorithm could detect urea mixed in milk samples with an accuracy of >90 %.

Application of proteomics to the areas of milk production, processing and quality control – A review

Milk contains a complex mixture of proteins that undergo several qualitative and quantitative changes during processing and storage. Elucidation of these changes is important to optimise milk processing and storage parameters. Proteomics has become widely used in the field of research on milk proteins due to improvements in the tools and diversified ways in which the methodologies of protein analysis have been developed. This review describes the use of proteomics in studies on milk proteins in relation to species–species differences, post‐translational modification, effect of lactation, mastitis, changes in heated milk and during cheese ripening, and the detection of milk adulteration.

Determination of whey proteins in different types of milk

Protein analysis is very important both in terms of milk protein allergy, and of milk and dairy product adulteration (β-lactoglobulin may be an important marker in the detection of milk adulteration). The aim of this study was to detect major whey proteins α-lactalbumin and β-lactoglobulin and their genetic variants by reversed-phase high-performance liquid chromatography. Milk samples from cows (n = 40), goats (n = 40) and sheep (n = 40) were collected at two farms and milk bars in the Czech Republic from April to June 2010. The concentration of α-lactalbumin was higher in goat’s milk (1.27 ± 0.05 g·l-1, P < 0.001) and cow’s milk (1.16 ± 0.02 g·l-1, P = 0.0037) compared to sheep’s milk (0.95 ± 0.06 g·l-1); however, concentration of α-lactalbumin in goat’s milk and cow’s milk did not differ significantly (P < 0.05). Goat’s milk contained less β-lactoglobulin (3.07 ± 0.08 g·l-1) compared to cow’s milk (4.10 ± 0.04 g·l-1, P < 0.001) or sheep’s milk (5.97 ± 0.24 g·l-1, P < 0.001). A highly significant positive correlation (r = 0.8686; P < 0.001) was found between fraction A and B of β-lactoglobulin in sheep’s milk, whereas in cow’s milk there was a negative correlation (r = -0.3010; P = 0.0296). This study summarizes actual information of the whey protein content in different types of milk which may be relevant in assessing their allergenic potential.

Chemical and functional properties of glycomacropeptide (GMP) and its role in the detection of cheese whey adulteration in milk: a review

Glycomacropeptide (GMP) is a C-terminal part (f 106–169) of kappa-casein which is released in whey during cheese making by the action of chymosin. GMP being a biologically active component has gained much attention in the past decade. It also has unique chemical and functional properties. Many of the biological properties have been ascribed to the carbohydrate moieties attached to the peptide. The unique set of amino acids in GMP makes it a sought-after ingredient with nutraceutical properties. Besides its biological activity, GMP has several interesting techno-functional properties such as wide pH range solubility, emulsifying properties as well as foaming abilities which are shown to be promising for applications in food and nutrition industry. These properties of GMP have given new dimension for the profitable utilization of cheese whey to the dairy industry. A number of protocols for isolation of GMP from cheese whey have been reported. Moreover, its role in detection of sweet/rennet whey adulteration in milk and milk products has also attracted attention of various researchers, and many GMP-specific analytical methods have been proposed. This review discusses the chemico-functional properties of GMP and its role in the detection methods for checking cheese or sweet whey adulteration in milk. Recent concepts used in the isolation of GMP from cheese whey have also been discussed.

MALDI-TOF mass spectrometric determination of intact phospholipids as markers of illegal bovine milk adulteration of high-quality milk

In the dairy industry one of the most common frauds is mixing high-value milk (sheep's and goats') with milk of lower value (cows'). This illegal practice has commercial, ethical, and serious sanitary consequences because consumers can be exposed to hidden allergens contained in the undeclared cows' milk. Here, we investigated the possibility of using matrix-assisted laser-desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry (MS) as a rapid, sensitive, and accurate technique for detection of milk adulteration by analysis of phospholipid profiles. Lipid extracts of pure raw milk, commercial milk, and binary mixtures of cows' and goats' milk and cows' and sheep's milk (the concentrations of each milk varied from 0 % to 50 %) were analyzed with α-cyano-4-chlorocinnamic acid as matrix. The abundance ratio of the ions at m/z 703 and m/z 706 was found to be species-correlated and was used as marker of cows' milk in sheep's and goats' milk. Furthermore, the procedure could potentially be applied to cheese samples, because peaks at m/z 703 and 706 were also found in several commercial cheese samples. This approach proved to be an efficient, rapid, and inexpensive method of detecting milk fraud.

Naked eye colorimetric quantification of protein content in milk using starch-stabilized gold nanoparticles

Abstract An easy-to-perform colorimetric analysis of protein content in milk based on aggregation-induced color change of starch-stabilized gold nanoparticles (AuNPs) has been developed. The synthesized AuNPs were employed without further surface modification. The assay relies on the additional stabilization power of proteins that inhibits the aggregation of AuNPs induced by hydrochloric acid. In the presence of milk proteins, the assay shows red to blue color shift as the total protein concentration decreases. Since the localized surface plasmon resonance (LSPR) expresses correlation with protein concentration, the protein content can be quantified. With an optimized condition, the assay is so sensitive that it can accurately determine protein content from 2.93 × 10 −1 to 2.93 × 10 −3 mg/mL via naked eye observation based on color change. Moreover, an extra accuracy can be achieved via the corresponding UV–visible spectra. Adulterating substance study showed that starch, lactose, table sugar, melamine, and sodium chloride cannot deceive the assay. The developed assay is very simple, requires only 6 min of analysis time without any extraction process. This technique provides a great potential for on-site milk analysis, milk adulteration detection as well as in-house use.

Direct estimation of sialic acid in milk and milk products by fluorimetry and its application in detection of sweet whey adulteration in milk

Sialic acid, being a biologically active compound, is recognised as an important component of milk and milk products. Almost all the sialic acid estimation protocols in milk require prior hydrolysis step to release the bound sialic acid followed by its estimation. The objective of this work was to estimate sialic acid in milk and milk products by fluorimetric assay which does not require a prior hydrolysis step thus decreasing the estimation time. The recovery of added sialic acid in milk was 91·6 to 95·8%. Sialic acid in milk was found to be dependent on cattle breed and was in the range of 1·68-3·93g/kg (dry matter basis). The assay was further extended to detect adulteration of milk with sweet whey which is based on the detection of glycomacropeptide (GMP) bound sialic acid in adulterated milk. GMP is the C-terminal part of κ-casein which is released into the whey during cheese making. For detection of adulteration, selective precipitation of GMP was done using trichloroacetic acid (TCA). TCA concentration in milk was first raised to 5% to precipitate milk proteins, especially κ-casein, followed by raising the TCA concentration to 14% to precipitate out GMP. In the precipitates GMP bound sialic acid was estimated using fluorimetric method and the fluorescence intensity was found to be directly proportional to the level of sweet whey in adulterated milk samples. The method was found to detect the presence of 5% sweet whey in milk.

Detection of sheep and goat milk adulterations by direct MALDI–TOF MS analysis of milk tryptic digests

In dairy field, one of the most common frauds is the adulteration of higher value types of milk (sheep's and goat's) with milk of lower value (cow's milk). This illegal practice has an economic advantage for milk producers and poses a threat for consumers' health because of the presence of hidden allergens as, for example, cow milk proteins, in particular, α(s1)-casein and β-lactoglobulin. The urgent need of sensitive techniques to detect this kind of fraud brought to the development of chromatographic, immunoenzymatic, electrophoretic and mass spectrometric assays. In the current work, we present a fast, reproducible and sensitive method based on the direct matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) MS analysis of milk tryptic digests for the detection of milk adulteration by evaluating specie-specific markers in the peptide profiles. Several pure raw and commercial milk samples and binary mixtures containing cows' and goats', cows' and sheep's and goats' and sheep's milk (concentrations of each milk varied from 0% to 100%) were prepared, and tryptic digests were analyzed by MALDI-TOF MS. The use of the new MALDI matrix α-cyano-4-chlorocinnamic acid allowed to detect cow and goat milk peptide markers up to 5% level of adulteration. Finally, from preliminary data, it seems that the strategy could be successfully applied also to detect similar adulterations in cheese samples.

Performance study of a ‘constant phase angle based’ impedance sensor to detect milk adulteration

In this paper, a new type of impedance sensor is proposed to detect milk adulteration. The measurement is based on the change in constant value of the phase angle of the impedance sensor due to the change of ionic property of the adulterated milk. The performance of the sensor is tested for three different types of adulterated milk (milk adulterated with tap-water, urea and liquid-whey). It has been observed that the sensor can identify 5% of liquid-whey adulteration, 10% tap-water adulteration, and 0.6 mg of urea per ml of milk. The sensor is also tested to distinguish between synthetic milk and original milk and the result shows that it can differentiate these two when the synthetic milk is reconstructed by adding minimum15% of adulteration.

Fourier transform infrared spectroscopy and multivariate analysis for the detection and quantification of different milk species

The authenticity of milk and milk products is important and has extended health, cultural, and financial implications. Current analytical methods for the detection of milk adulteration are slow, laborious, and therefore impractical for use in routine milk screening by the dairy industry. Fourier transform infrared (FT-IR) spectroscopy is a rapid biochemical fingerprinting technique that could be used to reduce this sample analysis period significantly. To test this hypothesis we investigated 3 types of milk: cow, goat, and sheep milk. From these, 4 mixtures were prepared. The first 3 were binary mixtures of sheep and cow milk, goat and cow milk, or sheep and goat milk; in all mixtures the mixtures contained between 0 and 100% of each milk in increments of 5%. The fourth combination was a tertiary mixture containing sheep, cow, and goat milk also in increments of 5%. Analysis by FT-IR spectroscopy in combination with multivariate statistical methods, including partial least squares (PLS) regression and nonlinear kernel partial least squares (KPLS) regression, were used for multivariate calibration to quantify the different levels of adulterated milk. The FT-IR spectra showed a reasonably good predictive value for the binary mixtures, with an error level of 6.5 to 8% when analyzed using PLS. The results improved and excellent predictions were achieved (only 4–6% error) when KPLS was employed. Excellent predictions were achieved by both PLS and KPLS with errors of 3.4 to 4.9% and 3.9 to 6.4%, respectively, when the tertiary mixtures were analyzed. We believe that these results show that FT-IR spectroscopy has excellent potential for use in the dairy industry as a rapid method of detection and quantification in milk adulteration.

Application of immunological methods for the detection of species adulteration in dairy products

SummaryA number of enzyme‐linked immunosorbent assays (ELISAs) have been developed for the detection of milk adulteration in dairy products. Target antigens have been caseins, lactoglobulins, immunoglobulins and other whey proteins. Polyclonal and monoclonal antibodies have been used in a variety of formats including direct, indirect, competitive and sandwich ELISAs. ELISAs have been successfully applied to the detection of cows' milk adulteration of sheep, goat and buffalo milk. Goat milk adulteration of sheep milk has also been detected. A number of ELISAs have also been applied to cheese. It is recommended that ELISA should be used in combination with PCR to ensure compliance with current legislation.

Measurement of bovine IgG by indirect competitive ELISA as a means of detecting milk adulteration.

The aim of this work was to develop an assay capable of detecting adulteration of high premium milk with milk from cheaper sources. An indirect, competitive ELISA was developed for the rapid detection of cows' milk in the milk of goat, sheep, and buffalo. The assay uses a monoclonal antibody produced against bovine IgG. This antibody recognizes a species-specific epitope on the heavy chain of both bovine IgG1 and IgG2. A peroxidase-conjugated anti-mouse IgG antibody was used to detect bound monoclonal antibody and subsequent enzymatic conversion of substrate resulted in clear differences in absorbance when assaying different mixtures of milks adulterated with cows' milk. Once optimized, the ELISA was found to be highly specific. Detection limits of the assay are 1.0 microg/mL of bovine IgG, or 0.1% (vol/vol) adulteration with cows' milk. The assay was highly reproducible (CV < 10%) and performed equally well when used to detect bovine IgG in mixtures with the 3 types of milk tested. The ELISA performance makes it suitable for development as a kit, for use in the field as a high throughput screening ELISA.

Evaluation of cheese authenticity and proteolysis by HPLC and urea–polyacrylamide gel electrophoresis

Chromatographic and electrophoretic methods have been established as useful tools in characterising cheese ripening and in the detection of milk adulteration. The purpose of this work was to evaluate casein proteolysis of cheeses made from bovine, ovine or mixtures of bovine and ovine milks, as well as ovine cheese authenticity, for 30 days of ripening by HPLC and urea–polyacrylamide gel electrophoresis. Complementary information was obtained by both techniques when applied to the study of casein proteolysis during 30 days of ripening of ovine milk cheeses, ovine milk cheeses with 10% and 20% of bovine milk and bovine milk cheeses, manufactured according to the traditional Terrincho technology. For ovine cheeses, α-casein was the fraction that showed the higher degradation during cheese ripening. A similar behaviour was observed for ovine milk cheese with 10% of bovine milk. The profile for ovine milk cheese with 20% of bovine milk was more similar to that obtained for bovine cheese. Concerning bovine milk cheeses, electrophoresis was the most sensitive technique for the evaluation of proteolysis in these cheeses. Ten and 20% of bovine milk could be detected in ovine milk cheeses by urea–polyacrylamide gel electrophoresis and HPLC, respectively, even after 30 days of ripening.