Commercial Milk Samples Research Articles

Electrochemical Detection of Melamine by Using Reduced Graphene Oxide-Copper Nanoflowers Modified Glassy Carbon Electrode.

In this work, a robust and reliable electrochemical sensor was developed for sensitive detection of non-electroactive melamine (MEL) using a modified glassy carbon electrode with ascorbic acid (AA) as the active recognition element. To increase the current signal of AA, the working electrode was successively modified with l-arginine (l-Arg) and reduced graphene oxide–copper nanoflower composite. The voltammetry measurements denoted that the hydrogen bonding was formed between AA and MEL. Using the optimum conditions, the proposed enhanced sensor can detect MEL concentrations ranging from 10 × 10–9 to 9.0 × 10–8 M with a detection limit of 5.0 × 10–9 M that is proportional to the decrease of AA in the anodic peak current. Finally, the proposed sensor was successfully applied for the determination of MEL in commercial infant milk samples and good recovery values were obtained.

Towards an optical multisensor system for dairy: Global calibration for fat analysis in homogenized milk

A new approach to constructing global calibration models for optical spectroscopic determination of fat content in normalized milk (homogenized milk with standardized nutrient content) in the region 400–1100 nm has been developed. Following the global modelling approach attempting to cover all possible variability of analyzed object, calibration models were built on a large set of commercial milk samples from many producers, collected in two different countries and varying from each other in nutrient composition and methods of technological processing. Suggested method employs spectral information related to the light scatter effect by colloidal particles of the milk constituents. Due to the application of advanced variable interval selection method, the root mean-square error of cross-validation (RMSECV) value of 0.12% in the range 1.5–6.0% of fat content has been achieved in the calibration by partial least-squares (PLS) regression method. Selected spectral intervals can be used for the development of an inexpensive and compact optical sensor for milk analysis based on light-emitting diodes (LEDs). Global calibration models developed in this study can be used for the practical analysis of fat in various normalized milk samples.

Analysis of antibiotic residues in raw and commercial milk in Punjab, India vis‐à‐vis human health risk assessment

AbstractThe present study quantitatively analyzed the occurrence of antibiotic residues in 524 milk samples (492 raw milk samples from dairy farms and 32 commercial milk samples) from Punjab, India using high‐performance liquid chromatography (HPLC). Tetracyclines, sulphonamides, fluoroquinolones (commonly used in veterinary practice in Punjab), and chloramphenicol (banned in food animals in India) were targeted and analyzed using multiresidue HPLC method. Out of 492 dairy farms milk samples, 78 (16%) were found to be positive for antibiotic residues with 20 (4%) exceeded the maximum residue limits (MRLs). Among 32 commercial milk samples, 4 (12.5%) were positive for antibiotic residues with one (3.1%) sample violated the MRL. Assessment of human health risks revealed that the current levels of antibiotic residues in milk pose no significant toxicological effects on the health of consumers. However, the results highlighted an immediate need of effective strategies for prevention of nonprudent use of antibiotics in veterinary practice for safeguarding consumer's health.

Chemical Composition of Commercial Cow's Milk.

Bovine milk is a nutritionally rich, chemically complex biofluid consisting of hundreds of different components. While the chemical composition of cow's milk has been studied for decades, much of this information is fragmentary and very dated. In an effort to consolidate and update this information, we have applied modern, quantitative metabolomics techniques along with computer-aided literature mining to obtain the most comprehensive and up-to-date characterization of the chemical constituents in commercial cow's milk. Using nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography-mass spectrometry (LC-MS), and inductively coupled plasma-mass spectrometry (ICP-MS), we were able to identify and quantify 296 bovine milk metabolites or metabolite species (corresponding to 1447 unique structures) from a variety of commercial milk samples. Through our literature analysis, we also found another 676 metabolites or metabolite species (corresponding to 908 unique structures). Detailed information regarding all 2355 of the identified chemicals in bovine milk have been made freely available through a Web-accessible database called the Milk Composition Database or MCDB ( http://www.mcdb.ca/ ).

Optimisation of microwave-assisted acid hydrolysis for the determination of seleno-amino acids bound to proteins in powdered milk, lyophilized milk and infant formula

Selenium (Se) is an essential element present in milk as seleno-amino acids and seleno-proteins. To perform an accurate seleno-amino acid analysis, a complete hydrolysis of proteins is necessary. Microwave-assisted acid hydrolysis (MAAH) can be used; however, some variables must be optimised to avoid incomplete hydrolysis and oxidation. Multivariate calibration showed that the volume of the reducing agent, the concentration of the acid used, and the microwave power were statistically significant (95% confidence), and since these are the variables controlling the system, a final optimisation was performed to increase the method efficiency. This was performed using a 2(4−1) central composite design. A robustness study was performed employing the Taguchi design, which demonstrated that the optimised MAAH technique was not dependent on the protein concentration of the milk samples. Following MAAH optimisation, a 30-fold recovery improvement was obtained for seleno-amino acids, reaching a limit of detection of 0.053 μg 100g−1 for selenocysteine. Selenocysteine, selenomethionine, and seleno-methyl-selenocysteine were determined at concentration ranges of 5.06 ± 0.20–22.5 ± 0.90, 0.75 ± 0.03–19.7 ± 1.2 and 1.25 ± 0.05–16.3 ± 0.85 respectively, in commercial powder milk samples and infant formula, lyophilized selenium-enriched ewe milk, lyophilized human milk and reference material ERM-BD150 skimmed milk powder.

New Poly(Ionic Liquid) Based Fiber for Determination of Oxytetracycline in Milk Samples by Application of SPME-CE Technique.

In this work, a procedure using solid phase microextraction in combination with capillary electrophoresis was developed for the determination of oxytetracycline in milk samples. The method involves the synthesis of poly(1-allyl-3-methyl imidazolium) chloride film on a stainless-steel bar via electropolymerization and its use as an adsorbent for oxytetracycline (OT) by an ionic exchange mechanism. The coated fiber is then immersed in milk samples for retention of oxytetracycline residues, followed by elution, drying, and reconstitution before analysis with capillary electrophoresis. The proposed method achieves a limit of detection of 70 µg L−1 with adequate precision and uncertainty, making this methodology appropriate for the determination of OT in milk samples. The method was applied to the pre-concentration and quantification of oxytetracycline in ten commercial milk samples. Two tested samples were positive for the presence of oxytetracycline but the concentration was below the maximum residue limit according to the international normative standard. The proposed methodology was evaluated according to the Eco-Scale approach, and the total score of 51 indicated that the methodology proposed is both green and acceptable despite the multi-stage character. SPME-CE methodology allows us to perform the sample pre-treatment and determination of OT in an effective and greener way, decreasing the number of steps during the analysis and the generation of waste.

Paper-based miniaturized immunosensor for naked eye ALP detection based on digital image colorimetry integrated with smartphone

Alkaline phosphatase (ALP) is a metalloprotein found naturally in raw milk samples and is considered as an important biomarker in quality control of milk. Its easy, personalized, as well as instrument-less detection is important to ensure the pasteurization and its differentiation form raw milk. In view of such importance of APL, we have developed an office punching machine crafted paper biosensor for naked eye detection of ALP in milk samples. The quantitative estimation is done by digital image colorimetry (DIC) based technique integrated with smartphone. The sensor-probe was developed by the covalent immobilization of ALP antibody (anti-ALP) onto the functionalized paper surface. The fabrication of the biosensing probe was characterized using DIC, Fourier transform infrared spectroscopy (FTIR), and atomic force microscopy (AFM). The detection was based on immunocomplexation between the sensor-probe and ALP, which generates blue-green precipitate as an analytical signal by exploiting the catalytic activity of ALP towards 5-bromo-4-chloro 3-indolyl phosphate (BCIP). The dose dependent appearance of the blue-green complex was captured using smartphone camera and DIC was employed using Red, Green, and Blue (RGB) profiling system, where the maximum sensitivity was obtained for the red color channel. Based on the DIC analysis, a wide dynamic range for the ALP detection is obtained from 10 to 1000 U/mL with the detection limit of 0.87 (± 0.07) U/mL. The designed paper-based biosensor is successfully applied to detect ALP in commercial and raw milk samples. Interferences due to components present in the milk samples was evaluated and the long-term stability of the designed biosensor was examined. Based on the detection principle, a miniaturized kit [20.0 mm (L) × 20.0 mm (W) × 2.15 mm (H)] was developed and applied for the ALP detection to demonstrate the instrument-free direct in-kitchen applicability.

Enhanced reversible redox activity of hemin on cellulose microfiber integrated reduced graphene oxide for H2O2 biosensor applications

In recent years, the carbohydrate polymers incorporated composite materials have shown significant interest in the bioanalytical chemistry due to their enhanced catalytic performances of various enzymes or mimics. This paper reports the fabrication of novel H2O2 biosensor using a hemin immobilized reduced graphene oxide-cellulose microfiber composite (hemin/RGO-CMF). The RGO-CMF composite was prepared by the reduction of GO-CMF composite using vitamin C as a reducing agent. Various physio-chemical methods have applied for the characterization of RGO-CMF composite. Cyclic voltammetry results revealed that the hemin/RGO-CMF composite shows a better redox electrochemical behavior than hemin/RGO and hemin/GO-CMF. Under optimized conditions, the hemin/RGO-CMF composite exhibit a linear response to H2O2 in the concentration range from 0.06 to 540.6 μM with the lower detection limit of 16 nM. The sensor also can able to detect the H2O2 in commercial contact lens solution and milk samples with functional recovery, which authenticates the potential ability in practical sensors.

A sample stacking-capillary electrophoresis method for simultaneous determination of nitrate and thiocyanate ions of ultra-heat-treated milk samples

Ultra-heat-treated (UHT) cow milk is mostly consumed in Turkey. Heating milk can cause some chemical and biochemical changes that affect different components of milk. However, studies on the constituents of UHT milk are scare. In the present study, nitrate and thiocyanate contents of commercial UHT~whole cow milk were determined simultaneously by sample stacking-capillary electrophoresis. The best separation of nitrate and thiocyanate ions was performed by reducing the electroosmotic flow at pH 4.0 using 30 mM formic acid and 30 mM sodium sulfate containing separation electrolyte. Analysis was completed in less than 3 min. The limits of detection obtained at a signal-to-noise ratio of 3 for nitrate and thiocyanate were 0.03 mg/L (0.48 μ role=presentation> μ μ \mu mol/L) and 0.06 mg/L (1.03 μ role=presentation> μ μ \mu mol/L), respectively. The nitrate concentration in commercial UHT milk samples ranged between 0.20 and 1.50 mg/L (3.23-24.20 μ role=presentation> μ μ \mu mol/L) and the thiocyanate ion concentration ranged between 0.41 and 0.67 mg/L (7.06-11.55 μ role=presentation> μ μ \mu mol/L).

Rapid detection and characterization of postpasteurization contaminants in pasteurized fluid milk

Microbial spoilage of pasteurized fluid milk is typically due to either (1) postpasteurization contamination (PPC) with psychrotolerant gram-negative bacteria (predominantly Pseudomonas) or (2) growth of psychrotolerant sporeformers (e.g., Paenibacillus) that have the ability to survive pasteurization when present as spores in raw milk, and to subsequently grow at refrigeration temperatures. While fluid milk quality has improved over the last several decades, continued reduction of PPC is hampered by the lack of rapid, sensitive, and specific methods that allow for detection of PPC in fluid milk, with fluid milk processors still often using time-consuming methods (e.g., Moseley keeping quality test). The goal of this project was to utilize a set of commercial fluid milk samples that are characterized by a mixture of samples with PPC due to psychrotolerant gram-negative bacteria and samples with presence and growth of psychrotolerant sporeforming bacteria to evaluate different approaches for rapid detection of PPC. Comprehensive microbiological shelf-life characterization of 105 pasteurized fluid milk samples obtained from 20 dairy processing plants showed that 60/105 samples reached bacterial counts >20,000 cfu/mL over the shelf-life due to PPC with gram-negative bacteria. Among these 60 samples with evidence of gram-negative PPC spoilage over the shelf-life, 100% (60/60) showed evidence of contamination with noncoliform, non-Enterobacteriaceae (EB) gram-negative bacteria (e.g., Pseudomonas), 20% (12/60) showed evidence of contamination with coliforms, and 7% (4/60) showed evidence of contamination with noncoliform EB. Among the remaining 45 samples, 28 showed levels of gram-positive bacteria above 20,000 cfu/mL and the remaining 17 samples did not exceed 20,000 cfu/mL over the shelf-life. Evaluation of the same set of 105 samples using 6 different approaches {all possible combinations of 2 different enrichment protocols (13°C or 21°C for 18 h) and 3 different plating media [crystal violet tetrazolium agar, EB Petrifilm (3M, St. Paul, MN), and Coliform Petrifilm]} showed that enrichment at 21°C for 18 h, followed by plating on crystal violet tetrazolium agar provided for the most sensitive, accelerated detection of samples that reached >20,000 cfu/mL due to PPC with psychrotolerant gram-negatives (70% sensitivity). These results show that tests still required and traditionally used in the dairy industry (e.g., coliform testing) are not suitable for monitoring for PPC. Rather, approaches that allow for detection of all gram-negative bacteria are essential for improved detection of PPC in fluid milk.

Fast quantification of bovine milk proteins employing external cavity-quantum cascade laser spectroscopy

Analysis of proteins in bovine milk is usually tackled by time-consuming analytical approaches involving wet-chemical, multi-step sample clean-up procedures. The use of external cavity-quantum cascade laser (EC-QCL) based IR spectroscopy was evaluated as an alternative screening tool for direct and simultaneous quantification of individual proteins (i.e. casein and β-lactoglobulin) and total protein content in commercial bovine milk samples. Mid-IR spectra of protein standard mixtures were used for building partial least squares (PLS) regression models. A sample set comprising different milk types (pasteurized; differently processed extended shelf life, ESL; ultra-high temperature, UHT) was analysed and results were compared to reference methods. Concentration values of the QCL-IR spectroscopy approach obtained within several minutes are in good agreement with reference methods involving multiple sample preparation steps. The potential application as a fast screening method for estimating the heat load applied to liquid milk is demonstrated.

Investigation on Ruthenium(II) Bipyridine/AgIII Complexes Chemiluminescence System and Its Application for Sensitive Norfloxacin and Ofloxacin Detection

The reaction mechanism of AgIII complex ([Ag(HIO6)2]5–) with ruthenium(II) bipyridine (Ru(bpy)32+) in acid medium was investigated. A novel flow injection chemiluminescence (CL) analysis method was developed for the detection of norfloxacin (NFLX) and ofloxacin (OFLX) in commercial drug, milk and human urine samples based on AgIII-ruthenium(II) bipyridine in acidic solution. Under optimal conditions, CL intensities were proportional to drug concentrations in the range of 1.6 × 10-9 to 8 × 10-6 g mL-1 for NFLX and 1.0 × 10-9 to 4 × 10-6 g mL-1 for OFLX. The detection limit was 1.0 × 10-9 g mL-1 for NFLX and 0.8 × 10-9 g mL-1 for OFLX. On the basis of the CL, UV and fluorescence spectra, possible reaction mechanisms were proposed.

Modification of Lipid Profile in Commercial Cow Milk Samples before and after Their Expiration Date: Evaluation of Storage Crucial Parameters and Possible Environmentally Friendly Disposal Alternatives

Milk waste is considered a highly polluting material and its disposal is an economic and environmental problem for the dairy sector. Despite this, it can be turned into a source of nutraceutical products and biodiesel substrate. The objective of this study was to determine the qualitative and quantitative variation of single fatty acids in cow milk samples before expiration date and within 28 days after expiration date in order to monitor how the profile of the lipid fraction is influenced by different physicochemical parameters. It was shown that lipolysis in milk is a process independent of time but dependent on the values of pH and total titratable acidity, while crucial parameters for the lipid oxidation are temperature and time of exposure to atmospheric oxygen. All of these factors are at the basis of the efficacy of milk storage conditions. Moreover, our data demonstrate that milk, several weeks after its expiry date, is a rich source of fatty acids that may be recovered as potential substrates for the formulation of economically viable products and eco-friendly diesel-like fuels.

One-pot synthesis of a multi-template molecularly imprinted polymer for the extraction of six sulfonamide residues from milk before high-performance liquid chromatography with diode array detection.

A highly selective molecularly imprinted polymer sorbent was synthesized and employed for the simultaneous determination of six sulfonamide antibiotic residues (sulfanilamide, sulfacetamide, sulfadiazine, sulfathiazole, sulfamerazine, and sulfamethizole) in milk samples. Multi-analyte imprinted particles were used as a sorbent in solid-phase extraction. Sulfonamides were separated on a high-performance liquid chromatography column (Merck-Lichrospher RP18e, 5 μm 250 × 4mm) and further identified and quantified by diode array detection. Several parameters including required loading of the molecularly imprinted polymer sorbent, mass of milk, volume, and type of elution solvent, as well as time for absorption and elution were investigated to obtain optimal experimental conditions. For comparison purpose, a non-imprinted polymer was applied under the optimum conditions. The validation study according to the European Union Decision 2002/657/EC was based on the investigation of linearity, selectivity, stability, limits of detection and quantitation, decision limit, detection capability, trueness, precision, and ruggedness according to Youden's approach. The decision limit and detection capability values in the milk were achieved from 101.9 to 113.5μg/kg and from 114.4 to 135.4μg/kg, respectively, depending on the target sulfonamide drug. Finally, the optimized protocol was successfully applied to commercial milk samples and human breast milk.

Identification of the Peptide PyroQ-βCasein194-209 as a Highly Specific and Sensitive Marker to Differentiate between Ultrahigh-Temperature Processed (UHT) Milk and Mildly Heated Milk.

In this study, a new approach was introduced to identify marker peptides that reflect the thermal treatment of commercial milk samples and differentiate ultrahigh-temperature processed (UHT) milk from mildly heated milk. Peptide profiles of training set samples, pasteurized (n = 20), extended shelf life (n = 29), and UHT (n = 29) milk, were recorded by MALDI-TOF-MS after StageTip microextraction. As marker candidates, 13 peptides were selected, and their cutoff levels were defined. The quality of the cutoff levels was then tested with a blind test set. Thus, the peptide m/z 1701.0, which was identified as pyroQ-βcasein194-209, could ideally differentiate UHT milk from mildly heated milk with an accuracy of 100%. Due to its high reliability and sensitivity, this peptide may be applied in routine analysis to monitor thermal processing of milk. An additional heating experiment showed that the marker peptide candidates are formed during milk processing by endogenous enzymes and selective thermal cleavage.

Direct duplex real-time loop mediated isothermal amplification assay for the simultaneous detection of cow and goat species origin of milk and yogurt products for field use

A multiple loop-mediated isothermal amplification (LAMP) method was developed to detect cow and goat milk in the field using a portable fluorescence device. For rapid on-site detection, this duplex LAMP assay was used in combination with direct amplification, without DNA extraction. The cow- and goat-specific LAMP primer sets were designed based on the mitochondrial cytochrome b gene, and showed specificity against 13 other animal species in the reactions. The sensitivity of the duplex LAMP assay for cow and goat was 0.1 and 1 pg, respectively. The detection limit for both target species in milk mixtures was 2%. This assay successfully amplified and identified the two target species in 24 samples of commercial milk and yogurt products, with 30 min sampling-to-result analysis time. Therefore, this direct duplex real-time LAMP assay is useful for on-site simultaneous detection of cow and goat milk in commercial products, a capability needed to confirm accurate labeling.

External cavity-quantum cascade laser (EC-QCL) spectroscopy for protein analysis in bovine milk

The analytical determination of bovine milk proteins is important in food and non-food industrial applications and yet, rather labour-intensive wet-chemical, low-throughput methods have been employed since decades. This work proposes the use of external cavity-quantum cascade laser (EC-QCL) spectroscopy for the simultaneous quantification of the most abundant bovine milk proteins and the total protein content based on the chemical information contained in mid-infrared (IR) spectral features of the amide I band. Mid-IR spectra of protein standard mixtures were used for building partial least squares (PLS) regression models. Protein concentrations in commercial bovine milk samples were calculated after chemometric compensation of the matrix contribution employing science-based calibration (SBC) without sample pre-processing. The use of EC-QCL spectroscopy together with advanced multivariate data analysis allowed the determination of casein, α-lactalbumin, β-lactoglobulin and total protein content within several minutes.

A new UHPLC method for the quantitation of furosine as heat load indicator in commercial liquid milk

The concentration of the artificial compound furosine in liquid milk can be used as an indicator to assess the heat load applied onto the product, which allows discrimination between differently processed milk (pasteurized, Extended Shelf Life (ESL) milk, Ultra-High Temperature (UHT) treated milk). In this work, a new UHPLC method for the rapid determination of furosine in 2min only (total runtime 6min) is presented, using PDA detection at 280nm. The method was validated for the analysis of liquid milk samples regarding linearity, instrumentation precision, limits of detection and quantification, repeatability of sample work-up and recovery. Moreover, the method was compared to an existing RP-HPLC method. Selected commercial liquid milk samples (n=36) were analyzed, showing lowest furosine amounts for pasteurized milk, whereas highest amounts were found in UHT milk. For ESL milk, quite diverse concentrations were analyzed ranging from 11.70 up to 115.16mg furosine 100g−1 protein, illustrating the different processing technologies of this category with lowest amounts for filtered ESL milk, followed by directly heated, and indirectly heated ESL milk samples, respectively.

Use of Carbon Nanotubes as a Solid Support To Establish Quantitative (Centrifugation) and Qualitative (Filtration) Immunoassays To Detect Gentamicin Contamination in Commercial Milk.

Current methods to detect gentamicin (GEN), a broad-spectrum antibiotic that causes ototoxicity and nephrotoxicity when present in excess, have several limitations. Hence, we have developed two methods using multi-walled carbon nanotubes (MWCNTs) as a solid support to detect GEN. Hybridoma cells (2D12) producing high-sensitivity antibodies against GEN were established. The goat anti-mouse antibody was immobilized on MWCNTs directly or using bifunctional polyethylene glycol as a linker. On the basis of the physical characteristics of MWCNTs, a quantitative method involving centrifugation separation and a qualitative method involving filtration separation were established. Various experimental parameters were optimized for GEN detection, and recovery tests were performed. For the quantitative method, the limit of detection (LOD) was 0.048 ng/mL, whereas for the qualitative method, a LOD of 0.1 ng/mL was observed by the naked eye. The proposed immunoassays were applied to commercial milk samples. Thus, these methods show potential application for the detection of GEN.

Fluorescent sensor assay for β-lactamase in milk based on a combination of aptamer and graphene oxide

Fluorescent sensor assay (FSA) was developed by using a fluorescein-labeled aptamer assembled onto Graphene oxide (GO) in order to determine β-lactamase in milk. Under optimal conditions, FSA indicated a detection range from 1 to 46 U/mL with a limit of detection (LOD) of 0.5 U/mL (R2 = 0.999, n = 3). In addition, commercial milk samples tainted with β-lactamase were detected by the established FSA with a recovery rate between 96.04 and 119.67%. Additionally, the reliability and sensitivity of FSA was validated by enzyme-linked immunoassay (ELISA) with a high correlation of 0.993. Thus, these data, combined with the ease and speed of the assay, suggest that the developed FSA may represent a promising method for monitoring β-lactamase contamination in milk.