Qualitative and quantitative analysis of bovine milk adulteration in caprine and ovine milks using native-PAGE

Native-PAGE (polyacrylamide gel electrophoresis) was used for the simultaneous qualitative and quantitative analysis of bovine milk adulteration in caprine and ovine milk using whole milk samples as well as their whey protein fraction. Quantification was based on measuring band intensity of bovine β-lactoglobulins in all milk mixtures and bovine α-lactalbumin in caprine/bovine milk blends. Linear relationships were established between the band intensity of bovine β-lactoglobulins and α-lactalbumin vs. volume percentage of added bovine milk in all milk analysed, with the correlation coefficient from 0.9950 to 0.9998. These correlations enabling the quantification of bovine milk percentage within the wide range from 3% or 5% to 90% in caprine/bovine and ovine/bovine milk blends, respectively. The differences between the actual percentages of bovine milk present in the adulterated milk samples and those calculated using the regression lines were less than or equal to 5% for all samples. This method offers a rapid determination combined with unequivocal identification of the bovine whey proteins in almost every caprine/bovine or ovine/bovine milk mixtures.     

PCR detection of cows’ milk in water buffalo milk and mozzarella cheese

Polymerase chain reaction (PCR) has been applied for the specific detection of cows’ DNA in water buffalo milk and mozzarella cheese by using primers targeting the mitochondrial 12S ribosomal RNA gene. The use of specific primers for cow yielded a 346 bp fragment from cows’ milk DNA, whereas no amplification signal was obtained in sheep's, goats’ and water buffalo's milk DNA. Analysis of both buffalo milk and buffalo mozzarella cheese mixtures containing different percentages of cows’ milk or bovine mozzarella cheese, enabled the specific detection of cow's DNA with a sensitivity threshold of 0.1%. The proposed PCR assay represents a rapid and straightforward method for the detection of adulterations in water buffalo milk and mozzarella cheese.     

Detection of milk fat adulteration with admixture of foreign oils and fats using a fractionation technique and the apparent solidification time test

A fractionation technique followed by the apparent solidification time (AST) test was adopted for detecting the admixture of foreign oils and fats in milk fat. The AST values of the solid fraction obtained at 20°C, and solid and liquid fractions obtained at 18°C for pure cow milk fat, were 2 min 30 s, and 3 min 21 s and 3 min 31 s, while for buffalo milk fat they were 1 min 58 s, and 2 min 47 s and 3 min 10 s respectively. This new approach can detect some mixtures of foreign oils and fats in cow milk fat but not in buffalo milk fat.     

IDENTIFICATION OF MILK FROM GOAT, EWE AND COW IN THEIR MIXTURES BY POLYACRYLAMIDE GEL ELECTROPHORESIS

A rapid identification of milk from goat, ewe and cow was done in the raw milk mixtures by polyacrylamide gel electrophoresis method. Two kinds of milk were mixed in the following proportions: 75:25%, 50:50%, 25:75% (V/V). Raw goat milk was adulterated with increasing proportions of cow milk or ewe milk, and ewe milk was adulterated with cow milk. Ewe milk in goat milk was detected by the electrophoretic resolution of the casein fraction whereas cow milk in goat and in ewe milk was identified by the presence of two fast-moving whey proteins.     

Simultaneous detection of cow and buffalo species in milk from China, India, and Pakistan using multiplex real-time PCR

Asian countries are major producers of cow and buffalo milk. For quality and authenticity purposes, a multiplex real-time PCR assay was developed to specifically and simultaneously detect DNA from these 2 bovine species. Targeting the cytochrome b gene of mitochondrial DNA, common PCR primers amplified a 105-bp fragment, and 2 fluorescent probes specific to either cow or buffalo were designed for their identification. Specificity was successfully tested on 6 other species, including sheep and goat, and sensitivity reached 1% of cow DNA in buffalo DNA and vice versa. As an evaluation, the method was tested using 119 freeze-dried Asian milk samples from regional industrial milk facilities. Although these samples did not cover the entire Asian zone, the multiplex assay indicated that approximately 20% of the samples (mainly from India) showed high levels of cross-contamination of cow milk by buffalo milk, and vice versa. Fast, sensitive, and straightforward, this method is fit-for-purpose for the authenticity control of Asian milk.     

Comparison of three methods to determine the whey protein to total protein ratio in milk

Three methods for quantification of the ratio of whey protein in total protein [capillary electrophoresis (CE), sodium dodecyl sulfate capillary electrophoresis (SDS-CE), and UV fourth derivative absorption spectroscopy (UV-4th Der.)] were applied to raw (n = 21), pasteurized (n = 5) and UHT (n = 18) milk samples. All methods effectively measured the whey protein to total protein ratio independently of the heat treatment applied to the milk. Mean values obtained by CE, SDS-CE and UV-4th Der. were respectively, 17.1, 18.5, and 17.2% for raw milks, 16.6, 17.7, and 18.8% for pasteurized milks, and 16.8, 17.0, and 17.2% for UHT milks. (Key words: whey protein to total milk protein ratio, capillary electrophoresis, sodium dodecyl sulfate capillary electrophoresis, fourth derivative ultraviolet spectroscopy)     

The binding of orally dosed hydrophobic active pharmaceutical ingredients to casein micelles in milk

Casein proteins (α_S1-, α_S2-, β- and κ-casein) account for 80% of the total protein content in bovine milk and form casein micelles (average diameter = 130 nm, approximately 10¹⁵ micelles/mL). The affinity of native casein micelles with the 3 hydrophobic active pharmaceutical ingredients (API), meloxicam [351.4 g/mol; log P = 3.43; acid dissociation constant (pK_a) = 4.08], flunixin (296.2 g/mol; log P = 4.1; pK_a = 5.82), and thiabendazole (201.2 g/mol; log P = 2.92; pK_a = 4.64), was evaluated in bovine milk collected from dosed Holstein cows. Native casein micelles were separated from raw bovine milk by mild techniques such as ultracentrifugation, diafiltration, isoelectric point precipitation (pH 4.6), and size exclusion chromatography. Acetonitrile extraction of hydrophobic API was then done, followed by quantification using HPLC-UV. For the API or metabolites meloxicam, 5-hyroxy flunixin and 5-hydroxy thiabendazole, 31 ± 3.90, 31 ± 1.3, and 28 ± 0.5% of the content in milk was associated with casein micelles, respectively. Less than ～5.0% of the recovered hydrophobic API were found in the milk fat fraction, and the remaining ～65% were associated with the whey/serum fraction. A separate in vitro study showed that 66 ± 6.4% of meloxicam, 29 ± 0.58% of flunixin, 34 ± 0.21% of the metabolite 5-hyroxy flunixin, 50 ± 4.5% of thiabendazole, and 33 ± 3.8% of metabolite 5-hydroxy thiabendazole was found partitioned into casein micelles. Our study supports the hypothesis that casein micelles are native carriers for hydrophobic compounds in bovine milk.     

Short communication: Space allocation in intensive Mediterranean buffalo production influences the profile of functional biomolecules in milk and dairy products

The aim of the present study was to determine if space allocation influenced the concentration of biomolecules in buffalo milk and dairy products. Intensively housed buffaloes (n = 96) were randomly assigned to 2 groups according to days in milk, parity, and milk yield: group S10 had a space allocation of 10 m² per buffalo and group S15 had a space allocation of 15 m² per buffalo. Individual milk yield was recorded daily. Twice a month, a bulk milk sample was collected for each group, as well as whey, ricotta, and mozzarella cheese, to assess cheese yield and to conduct HPLC-electrospray ionization-tandem mass spectrometry, milk antioxidant activity, and cell viability analyses. We tested milk extracts from the 2 groups in vitro to evaluate their efficacy in counteracting endothelial oxidative damage induced by high glucose. We evaluated reproductive function in 28 buffaloes from each group using the Ovsynch-timed artificial insemination program. We observed no differences in milk quantity or quality in terms of fat, protein, or lactose, and reproductive function did not differ between the 2 groups. Compared with group S10, group S15 had higher concentrations of carnitine (56.7 ± 1.1 vs. 39.8 ± 0.7 mg/L in milk and 40.9 ± 0.8 vs. 31.7 ± 0.7 mg/L in whey), acetyl-l-carnitine (51.9 ± 0.3 vs. 39.7 ± 0.7 mg/L in milk and 41.1 ± 1.7 vs. 28.7 ± 2.6 mg/L in whey), propionyl-l-carnitine (34.8 ± 1.0 vs. 21.0 ± 0.9 mg/L in milk and 26.9 ± 0.8 vs. 17.6 ± 1.2 mg/L in whey), glycine betaine (23.1 ± 1.9 vs. 13.5 ± 1.6 mg/L in milk and 10.7 ± 0.4 vs. 7.9 ± 0.5 mg/L in whey), and δ-valerobetaine (24.2 ± 0.5 vs. 16.7 ± 0.5 mg/L in milk and 22.0 ± 0.9 vs. 15.5 ± 0.7 mg/L in whey). Group S15 also had higher total antioxidant activity than group S10 (56.7 ± 1.9 vs. 46.4 ± 1.13 mM Trolox equivalents). Co-incubation of high-glucose-treated endothelial cells with milk extracts from group S15 improved cell viability compared with cells treated with high glucose only; it also reduced intracellular lipid peroxidation (144.3 ± 0.4 vs. 177.5 ± 1.9%), reactive oxygen species (141.3 ± 0.9 vs. 189.3 ± 4.7 optical density units), and cytokine release (tumor necrosis factor-α, IL-1β, IL-6). Greater space allocation was associated with higher levels of biomolecules in buffalo milk. This could have been the result of improved welfare in buffaloes that were allocated more space.     

Detection of milk mixtures in Halloumi cheese

A capillary electrophoresis method has been applied to the detection of illegal addition of milk from goat and/ or cow in Halloumi cheese, traditionally made with sheep milk. The electrophoretic profiles of the casein from Halloumi cheeses have revealed that caprine para-kappa-casein and bovine alphas1-casein peaks point to the presence of low percentages of goat's and/or cow's milk added to Halloumi cheese. Stepwise multiple linear regression has been used to predict these percentages with a standard error of the estimation of 2.14%. The analytical method combined with the statistical application is valid for the prediction of percentages higher than 2% of goat's and percentages of 5% of cow's milk added to the cheese either in fresh or ripened cheese. The standard error of estimation was higher for the prediction of cow's milk than for goat's milk.     

Proteins recovered from acid whey/skim milk mixtures heated at alkaline pH values

Skim milk and mixtures prepared by combining acid whey with skim milk at volume ratios of 2:1, 1:1, 1:2, 1:3 and 1:4 were adjusted to pH 7.5 and heated at 90°C × 15 min. Protein was isolated from these heated samples by precipitation at pH 4.6 and it was found that 65% of the whey protein was recovered in each case. Non-recovered proteins included the proteose peptones and small quantities of β-lactoglobulin, α-lactalbumin and bovine serum albumin. The solubility of these isolates, which contained from 10–25% whey protein, decreased to > 95% when the whey protein exceeded ˜16%. Further characterization of the isolate, prepared from the 1:1 volume ratio of acid whey and skim milk, showed that ˜50% of the whey protein was insoluble, bound to casein and non-functional while the other ˜50% was complexed with casein and was soluble. The addition of a reducing agent suggests that sulphydryl bonding alone is not responsible for complex formation.     

Buffalo vs. cow milk fat globules: Size distribution,zeta-potential,compositions in total fatty acids and in polar lipids from the milk fat globule membrane

Although buffalo milk is the second most produced milk in the world, and of primary nutritional importance in various parts of the world, few studies have focused on the physicochemical properties of buffalo milk fat globules. This study is a comparative analysis of buffalo and cow milk fat globules. The larger size of buffalo fat globules, 5 vs. 3.5 μm, was related to the higher amount of fat in the buffalo milks: 73.4 ± 9.9 vs. 41.3 ± 3.7 g/kg for cow milk. Buffalo milks contained significantly lower amount of polar lipids expressed per gram of lipids (0.26% vs. 0.36%), but significantly higher amount of polar lipids per litre of milk (+26%). Buffalo and cow milk fat globule membranes contain the same classes of polar lipids; phosphatidylethanolamine, sphingomyelin (SM) and phosphatidylcholine (PC) being the main constituents. A significant higher percentage of PC and lower percentage of SM were found for buffalo milks. The fatty acid analysis revealed that saturated fatty acids, mainly palmitic acid, trans fatty acids, linolenic acid (ω3) and conjugated linolenic acid were higher in buffalo milk than in cow milk. Such results will contribute to the improvement of the quality of buffalo milk-based dairy products.     

Detection of rennet whey solids in UHT milk by capillary electrophoresis

The fraudulent presence of rennet whey solids in UHT milk was studied by capillary electrophoresis (CE). Commercial UHT samples of different origins, genuine milk samples and milk samples adulterated with rennet whey were analysed. Linear discriminant functions using ratios of peak areas of caseinmacropeptide (CMP) and two other CMP-like degradation products were defined. The interference of proteolysis in the detection was estimated in samples adulterated on purpose with rennet whey, UHT treated in a pilot plant, and stored at 10, 20 and 30°C for up to 150 days. The application of the classification functions obtained allowed the detection of rennet whey solids added to milk. Only interferences due to very severe proteolysis, occurring after very long storage periods and/or at storage temperatures above room temperature, were observed.     

Short communication: Persistent contamination by Listeria monocytogenes of bovine raw milk investigated by whole-genome sequencing

Following the persistent detection of Listeria monocytogenes in raw bovine milk sold through a vending machine, the 120 lactating cows of the herd producing the milk were subjected to bacteriological investigation. A single cow with subclinical mastitis (1.2–1.3 × 10⁵ somatic cells/mL) and persistent L. monocytogenes excretion was detected. The cow was subjected to antimicrobial therapy, but L. monocytogenes excretion remained high (>3.0 × 10² cfu/mL). Following culling of the infected cow, L. monocytogenes disappeared from the tank milk, and further isolates were recovered from the mammary parenchyma and lymph nodes of the infected cow. To investigate the clonal nature of the contamination, all isolates recovered in the study (n = 13) were analyzed by serogroup PCR, pulsed-field gel electrophoresis, and whole-genome sequencing. Our results demonstrated the clonal nature of the contamination. All isolates belonged to lineage II, serogroup IIa, sequence type 37, clonal complex 37 and harbored some virulence determinants. This case showed that, although relatively rare, prolonged milk contamination by L. monocytogenes can originate from subclinical and persistently infected cows, posing a health risk to consumers.     

双向电泳技术鉴别牛羊乳品真实性

采用双向电泳技术对牛羊乳品的蛋白质指纹进行分析。首先通过全乳图谱分析牛羊乳的差异蛋白,然后针对婴幼儿配方粉、乳清粉等开展方法特异性研究,通过婴幼儿配方粉添加实验开展方法灵敏度研究,最后对市售配方粉、液态乳、酸乳等进行检测。总体上,双向电泳技术信息丰度高,信息直观,重复性良好,能准确分辨牛羊乳及乳清,灵敏度达到5%。通过检测,8 份市售样品中有1 份酸羊乳实际为牛乳,图谱蛋白指纹信息清晰,直观反映产品真实性。     

Effect of different time intervals on the viscosity of batter and on the sensory qualities of puras (pancakes) prepared using chhana and paneer whey

The viscosity of pura (pancakes) batter samples prepared with bovine, buffalo and mixed wheys (80 : 20 whey to water) was measured at different time intervals at a temperature of 25  ±  2°C using a Brookfield viscometer (LVT 98534). The viscosity of sweet pura batter samples increased after 1 h, remained constant for another 2 h and then decreased, whereas the viscosity of salty pura batter samples increased from freshly prepared batter to 1 h and then remained constant. Sensory evaluation of both sweet and salty puras at different time intervals from batter preparation was also carried out. The overall acceptability scores for sweet puras prepared using bovine milk chhana whey, buffalo milk paneer whey and mixed milk chhana whey and stored for up to 4 h at 25  ±  2°C and for 24 h at 4–5°C were not significantly different, and the same conclusion applied to salty puras made from bovine milk chhana whey. It was concluded that sweet and salty pura batters can be prepared from any chhana or paneer whey.     

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.     

Real-time PCR和双向电泳联合鉴别婴幼儿配方羊乳粉的乳源成分

为实现对婴幼儿配方羊乳粉中乳源成分的准确判别,将基因检测作为初筛方法,以蛋白质双向电泳作为确证方法,研究该技术路线下对乳源成分检测的灵敏度、准确性等指标。优化建立的基于嗜热蛋白酶的一步式DNA提取法,仅需通过温控反应在17 min内完成DNA步骤,极大缩短了样品前处理时间;建立的快速实时聚合酶链式反应（real-time polymerase chain reaction,real-time PCR）程序仅需28 min 26 s即可完成检测。通过牛（家牛、水牛、牦牛）、羊（山羊和绵羊）、山羊、绵羊单一乳源成分,以及哺乳动物内参照检测体系,可以实现对低至10～100 pg/μL DNA的检测,对混合样品中牛源性成分检测的灵敏度可达1%（m/m）。然后结合双向电泳技术进一步确证产品的蛋白成分来源,可以判定牛基因检测阳性的样品是否含有来源于牛乳酪蛋白或乳清蛋白,进而对样品的真实性进行判定。real-time PCR法和双向电泳技术联合检测解决了以往婴幼儿配方乳粉检测中因配料复杂乳源判别困难的瓶颈问题。     

Real-time TaqMan PCR for quantitative detection of cows’ milk in ewes’ milk mixtures

A real-time PCR based on the amplification of a fragment of the mitochondrial 12S ribosomal RNA gene was developed and evaluated for the detection and quantification of cows’ milk in raw and heat-treated cow/sheep milk mixtures. The method combines the use of cow-specific primers that amplify a 252 bp fragment from cow DNA, and mammalian-specific primers amplifying a 428 bp fragment from mammalian species DNA, which is used as an endogenous control. The method measures PCR product accumulation through a 6-carboxyfluorescein-labeled fluorogenic probe (TaqMan). A comparison of the cycle number at which mammalian and cow-specific PCR products were first detected, in combination with the use of reference standards of known bovine content, allowed the determination of the percentage of cows’ milk in mixtures. Experimental raw and heat-treated binary mixtures were analyzed, demonstrating the specificity and sensitivity of the assay for detection and quantification of cows’ milk in the range 0.5–10%.     

Detection of cow milk adulteration in yak milk by ELISA

In the current study, a simple, sensitive, and specific ELISA assay using a high-affinity anti-bovine β-casein monoclonal antibody was developed for the rapid detection of cow milk in adulterated yak milk. The developed ELISA was highly specific and could be applied to detect bovine β-casein (10–8,000 μg/mL) and cow milk (1:1,300 to 1:2 dilution) in yak milk. Cross-reactivity was <1% when tested against yak milk. The linear range of adulterant concentration was 1 to 80% (vol/vol) and the minimum detection limit was 1% (vol/vol) cow milk in yak milk. Different treatments, including heating, acidification, and rennet addition, did not interfere with the assay. Moreover, the results were highly reproducible (coefficient of variation <10%) and we detected no significant differences between known and estimated values. Therefore, this assay is appropriate for the routine analysis of yak milk adulterated with cow milk.     

Use of duplex polymerase chain reaction (duplex-PCR) technique to identify bovine and water buffalo milk used in making mozzarella cheese

Molecular biology techniques have been used for species identification in food of animal origin in relatively recent years. A polymerase chain reaction (PCR) based method, the multiplex PCR, was recently applied to species identification in meat and meat products. It allows co-amplification of separate regions of a single gene or specific fragments, each typical of a different animal species in a single PCR reaction, using different pairs of primers in the same reaction mix. In the present paper, the duplex-PCR technique is proposed to identify bovine and water buffalo DNA in a single PCR assay in milk and mozzarella cheese (a typical Italian cheese, originally made from pure water buffalo milk). Because of its lower cost, undeclared bovine milk is added to water buffalo milk for making different kinds of mozzarella cheese. The results of this experiment indicate the applicability of this method, which showed an absolute specificity for the two species and a high sensitivity even down to low DNA concentrations (1 pg). In bovine and water buffalo mixtures of both milk and mozzarella cheese, the minimum concentration tested was 1% of bovine in water buffalo milk and water buffalo in bovine milk. The importance of the somatic cell content in raw milk is also discussed with special reference to the evaluation of mixtures (milk or cheese) of the two species.