Glycosylation of κ-casein: Genetic and nongenetic variation and effects on rennet coagulation properties of milk

The aims of this study were to investigate genetic and nongenetic variation in the degree of glycosylation of κ-casein (κ-CN) and to estimate the effects of glycosylated (G-κCN) and unglycosylated (U-κCN) κ-CN contents on milk coagulation properties of Simmental cows. Measures of contents of the main casein fractions, G-κCN, and U-κCN, and assessment of genotypes at CSN2, CSN3, and BLG were obtained by reversed-phase HPLC analysis of 2,015 individual milk samples. Content of total κ-CN (κ-CNtot, g/L) was the sum of G-κCN and U-κCN, and the glycosylation degree of κ-CN (GD) was measured as the ratio of G-κCN to κ-CNtot. Rennet coagulation time (RCT) and curd firmness were measured by using a computerized renneting meter. Measures of curd firmness were adjusted for RCT before statistical analysis. Variance components of κ-CNtot, G-κCN, U-κCN, and GD were estimated by Bayesian procedures and univariate linear models that included the class effects of the herd-test-day, parity, days in milk, genotypes at milk protein genes, and animal. These class effects, those of G-κCN, U-κCN, and content of other caseins, and the linear effect of milk pH were accounted for by models investigating the influence of κ-CN glycosylation on coagulation properties. The GD ranged from 22 to 76%, indicating that variation in G-κCN depends on the variation both in κ-CNtot and in the efficiency of κ-CN glycosylation. Genotype CSN3 BB exhibited high G-κCN and U-κCN relative to that of CSN3 AA. Heritability of G-κCN, U-κCN, and GD was high and ranged from 0.46 to 0.56. A large proportion of the additive genetic variation in G-κCN and U-κCN was attributable to influence of CSN and BLG, but these genes did not affect variation in GD, and across-genotypes differences in the trait were small or trivial. Average RCT of the milk class having the highest G-κCN was, on average, 2 min (standard deviation 0.5) shorter than that of the lowest class. Conversely, U-κCN and content of other caseins were not associated with any effect on RCT, except for a slight delay in coagulation when U-κCN was very high. Curd firmness increased when the contents of both κ-CN fractions and other caseins increased. This study provides evidence that the positive association between RCT and κ-CN content is exclusively attributable to the glycosylated fraction of the protein. Because exploitable additive genetic variation in G-κCN exists, improvement of κ-CN composition through selective breeding might be an effective way to enhance milk coagulation properties.     

Influence of chemical and biochemical characteristics on the texture of Appenzeller® cheese

Texture properties are very important quality parameters of Appenzeller^® cheese. The Appenzeller^® Cheese Makers Association defines optimal texture quality as being characterised by a softness that lasts at least three months. During winter production, texture-related problems are sporadically encountered. In the present study, ten Appenzeller^® cheese loaves with optimal texture properties aged for 90 days and ten loaves with dry and firm texture of the same age were selected and characterised through a uniaxial compression test and with physicochemical and biochemical methods. All cheeses had been produced in the same week during winter (January) in different cheese factories and ripened for three months. The aim of the study was to determine the main factors responsible for classification into the two groups based on univariate and multivariate statistical analysis tools. The three main parameters influencing the force at 33% deformation in this study were calcium, water, and free amino acids.     

Immunoreactive properties of α-casein and κ-casein: Ex vivo and in vivo studies

The aim of this study was to evaluate the ex vivo and in vivo studies immune potential of α- and κ-casein. Ex vivo, naïve mouse splenocytes were stimulated with α- or κ-casein. After 120 h of culture, the proliferation index (PI), determined by 3-(4,5 dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and carboxyfluorescein diacetate N-succinimidyl ester (CFSE) staining, did not vary for either antigen, suggesting similar ex vivo immunogenic potential of both casein fractions. In vivo, BALB/ccmdb mice were sensitized with α- or κ-casein and then gavaged with primary antigen. Mice immunized with α-casein had higher levels of IgG (2^16.33) and IgA (2^10.22) in serum at the end of the experiment compared with mice immunized with κ-casein (2¹⁵ and 2^9.3 for IgG and IgA, respectively). The use of α-casein for mouse immunization and ex vivo lymphocyte stimulation resulted in higher concentrations of secreted cytokines (IL-4, IL-10) compared with κ-casein stimulation. This is consistent with increasing regulatory T cell (Treg) lymphocyte populations, independent of the antigen used for stimulation. In summary, the immunogenic potential of α- and κ-casein was similar. Humoral and cellular immune responses confirmed their strong, independent potential to induce B and T cells. We propose that the lymphocyte proliferation index be used as an initial screening for protein immunogenicity.     

Effect of κ-casein B relative content in bulk milk κ-casein on Montasio, Asiago, and Caciotta cheese yield using milk of similar protein composition

The aim of this study was to investigate the effect exerted by the relative content of κ-casein (κ-CN) B in bulk milk κ-CN on coagulation properties and cheese yield of 3 Italian cheese varieties (Montasio, Asiago, and Caciotta). Twenty-four cheese-making experiments were carried out in 2 industrial and 1 small-scale dairy plant. Detailed protein composition of bulk milk of 380 herds providing milk to these dairies was analyzed by reversed-phase HPLC. To obtain 2 experimental milks differing in the relative content of κ-CN B in κ-CN, herds were selected on the basis of bulk milk protein composition and relative content of κ-CN genetic variants. Milk was collected and processed separately for the 2 groups of selected herds. A difference of 20% in the relative content of κ-CN B in κ-CN was obtained for the 2 experimental milks for Montasio and a difference of 15% for Asiago and Caciotta. The 2 experimental milks were of similar protein and CN content, casein number, pH, CN composition, and β-CN genetic composition. For each cheese-making trial, amounts of milk, ranging from 2,000 to 6,000 kg, were manufactured. Each vat contained milk collected at least from 4 dairy herds. Cheese yield after brining and at the end of the aging was recorded. Milk with a greater proportion of κ-CN B in κ-CN (HIGHB) exhibited similar coagulation properties and greater cheese yield compared with milk with a lower proportion of κ-CN B in κ-CN (LOWB). The increased cheese yield observed for HIGHB when manufacturing Montasio cheese was ascribed to a greater fat content compared with LOWB. The probability of HIGHB giving a cheese yield 5% greater than that of LOWB ranged from 51 to 67% for Montasio cheese, but was less than 21% for Asiago and Caciotta cheeses. Variation in relative content of κ-CN B in κ-CN content did not relevantly affect industrial cheese yield when milks of similar CN composition were processed. An indirect effect due to the increased κ-CN content of κ-CN B milk is thought to explain the favorable effects of κ-CN B on cheese yield reported in the literature.     

Effect of κ-carrageenan on rheological properties, microstructure, texture and oxidative stability of water-in-oil spreads

The effect of κ-carrageenan concentration (0-7.5 g kg⁻¹) on the rheology, microstructure, texture and oxidative stability of water-in-oil (W/O) spreads (600 g fat kg⁻¹ emulsion) was examined over 60 days storage time. Results showed that increasing the κ-carrageenan concentration to 7.5 g kg⁻¹ significantly increased the viscosity of the aqueous phase (to 42.7 mPa s at 60 °C) resulting in gelation of the aqueous phase on cooling. The microstructure of the spreads was disrupted by higher levels of κ-carrageenan, resulting in a less homogeneous distribution of the aqueous phase. Melt temperature (where tan δ > 1) decreased significantly from 62 to 56.2 °C with increasing κ-carrageenan concentration from 0 to 7.5 g kg⁻¹. The firmness and the G′ at 6 °C for all samples were significantly increased after 60 days storage with only small effects due to κ-carrageenan levels. Oxidation of the fat phase was evident by the significant increases in peroxide values of all spreads on storage, with κ-carrageenan exhibiting no antioxidant behaviour. While increased κ-carrageenan levels modified the microstructure of W/O spreads in terms of the droplet size of the aqueous phase and its distribution few changes were evident in the continuous fat phase.     

Effects of genetic variants and sialylation on in vitro digestibility of purified κ-casein

Milk with different κ-casein (CN) phenotypes has previously been found to influence its gastric digestion rate. Therefore, the aim of the present study is to disentangle contributions of genetic variation and its related sialylation on the in vitro digestion process of κ-CN. Accordingly, κ-CN was purified from milk representing homozygous cows with κ-CN phenotypes AA, BB, or EE and used as substrate molecules in model studies using the INFOGEST 2.0 in vitro static digestion model. Furthermore, the effect of removal of the terminal sialic acids present on the O-linked oligosaccharides of the purified κ-CN A, B, and E protein variants were studied by desialylation enzymatic assays. The κ-CN proteins were purified by reducing anion exchange chromatography with purities of variants A, B, and E of 93.0, 97.1, and 90.0%, respectively. Protein degradations of native and desialylated κ-CN isolates in gastric and intestinal phases were investigated by sodium dodecyl sulfate-PAGE, degree of hydrolysis (DH), and liquid chromatography electrospray ionization mass spectrometry. It was shown that after purification, the κ-CN molecules reassembled into multimer states, which then constituted the basis for the digestion studies. As assessed by DH, purified variants A and E were found to exhibit faster in vitro digestion rates in both gastric and intestinal phases compared with variant B. Desialylation increased both gastric and intestinal digestion rates for all variants, as measured by DH. In the gastric phase, desialylation promoted digestion of variant B at a rate comparable with native variants A and E, whereas in the intestinal phase, desialylation of variant B promoted better digestion than native A or E. Taken together, the results confirm that low glycosylation degree of purified κ-CN promotes faster in vitro digestion rates, and that desialylation of the O-linked oligosaccharides further promotes digestion. This finding could be applied to produce dairy products with enhanced digestibility.     

Influence of processing and κ-carrageenan on properties of whipping cream

The aim of this work was to characterise the influence of ultra-high-temperature (UHT) treatment and high-pressure homogenisation on functional properties of whipping cream (30% fat content) in relation to adding κ-carrageenan (0%, 0.02% and 0.04% in milk plasma). The determination of the particle size distribution, which was measured by laser diffraction and verified by microscopic observation, indicated that the diameter of fat globules decreased significantly by homogenisation but clusters of small fat globules were produced before the carrageenan–casein micelles aggregates. The viscosity of cream was increased and the thixotropic behaviour was observed both by adding carrageenan and by homogenisation. The homogenisation significantly increased colloidal stability during storage and milk plasmas’ release was minimised in combination with carrageenan addition. The most influenced functional properties were: the whippability, which was significantly impaired by homogenisation, and the stability of whipped foam, which was significantly improved with the increase of the carrageenan concentration.     

Genomic architecture of bovine κ-casein and β-lactoglobulin

The objective of this study was to characterize the genetic architecture underlying the absolute concentrations of 2 important milk proteins, κ-casein (κ-CN) and β-lactoglobulin (β-LG), in a backcross population of (Holstein × Jersey) × Holstein cattle. A genome-wide association analysis was performed using a selective DNA pooling strategy and the Illumina BovineHD BeadChip assay [777,000 (777K) SNP markers; Illumina Inc., San Diego, CA]. After correction for multiple testing, 25 single nucleotide polymorphisms were found to be associated with κ-CN and 36 single nucleotide polymorphisms were associated with β-LG. A pathway association analysis revealed 15 Gene Ontology (GO) terms associated with the κ-CN trait and 28 GO terms associated with β-LG. In addition, several GO terms were associated with both milk proteins. Further analysis revealed that κ-CN and β-LG production is regulated by both kinase and phosphatase activity, including mechanisms regulating the extracellular matrix. These results are in concordance with the complex multihormonal process controlling the expression of milk proteins and interactions between mammary epithelial cells and extracellular matrix components. Although κ-CN and β-LG milk proteins are expressed by single genes, the results from this study showed that many loci are involved in the regulation of the concentration of these 2 proteins.     

Preparation of and studies on the functional properties and bactericidal activity of the lysozyme–xanthan gum conjugate

The purpose of this research was to produce a xanthan gum–lysozyme conjugate (Lyz–XM) and to investigate the effect of conjugation on functional properties and antimicrobial properties of the conjugated products. The antimicrobial activity of Lyz is limited to Gram positive bacteria, but its antibacterial spectrum can be extended towards Gram negative bacteria by conjugation with carbohydrate through the Maillard reaction. Lyz was allowed to react with XG under mild Maillard reaction condition (pH 8.5 and 60 °C for 10 days) and conjugation was confirmed by SDS-PAGE, enzyme activity and determination of sugar content of the product. Results showed that under optimal conditions approximately 1.9 mmol XG was attached to one mol Lyz. The Lyz–XG conjugate showed higher solubility at acidic pHs and at different temperatures, increased heat stability with improved emulsion and foaming properties. Additionally, Lyz–XG conjugate showed antioxidant properties and significantly inhibited the growth of Staphylococcus aureus and Escherichia coli in a dose dependent manner. These findings may broaden food applications of Lyz as a functional ingredient with high quality emulsifier, foam producer or natural antibacterial agent in food or pharmaceutical industries.     

Effect of high-pressure treatment and a bacteriocin-producing lactic culture on the proteolysis, texture, and taste of Hispánico cheese

The effects of high-pressure treatment, by itself or in combination with a bacteriocin-producing culture added to milk, on the proteolysis, texture, and taste of Hispánico cheese were investigated. Two vats of cheese were manufactured from a mixture of cow and ewe milk. Milk in one vat was inoculated with 0.5% Lactococcus lactis ssp. lactis INIA 415, a nisin Z and lacticin 481 producer; 0.5% L. lactis ssp. lactis INIA 415-2, a bacteriocin-nonproducing mutant; and 2% of a commercial Streptococcus thermophilus culture. Milk in the other vat was inoculated with 1% L. lactis ssp. lactis INIA 415-2 and 2% S. thermophilus culture. After ripening for 15 d at 12°C, half of the cheeses from each vat were treated at 400 MPa for 5 min at 10°C. Ripening of high-pressure-treated and untreated cheeses continued at 12°C until d 50. High-pressure treatment of cheese made from milk without the bacteriocin producer accelerated casein degradation and increased the free AA content, but it did not significantly influence the taste quality or taste intensity of the cheese. Addition of the bacteriocin producer to milk lowered the ratio of hydrophobic peptides to hydrophilic peptides, increased the free AA content, and enhanced the taste intensity. The combination of milk inoculation with the bacteriocin producer and high-pressure treatment of the cheese resulted in higher levels of both hydrophobic and hydrophilic peptides but had no significant effect on the free AA content, taste quality, or taste intensity.     

Ultrasonic effect on physicochemical and functional properties of α-lactalbumin

Ultrasound is the sound whose frequency is too high for humans to hear which is within the frequency range of 20 Hz-20 kHz, and the frequency of ultrasound is above 20 kHz. The aim of this study was to observe the effect of ultrasound and sonication on α-lactalbumin (α-LA) with a view to improving its physicochemical and functional properties. In this work both low-intensity ultrasound (500 kHz bath) and the high-intensity ultrasound (20 kHz probe and 40 kHz bath) were used. Ten per cent wt (g g⁻¹ dry matter) protein model suspensions of α-lactalbumin (α-LA) were treated with ultrasound probe (20 kHz for 15 and 30 min) and ultrasound baths (40 kHz and 500 kHz for 15 and 30 min).Changes in pH values, electrical conductivity, solubility measurements, foaming properties, as well as rheological and freezing-thawing properties have been examined. The protein fractions of α-lactalbumin were analyzed before and after ultrasound treatment by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis).The result showed that pH did not change significantly upon ultrasound however conductivities increased significantly after 20 kHz sonication. Electrical conductivity decreased significantly for ultrasound treatments in baths at 40 kHz and 500 kHz for all samples. Solubility increased significantly for all samples at 20 kHz. Foam capacities and foam stabilities were improved after ultrasound treatments for both 20 kHz and 40 kHz treatments. Foaming properties were not improved for protein model suspensions for 500 kHz treatments. The molecular weight of the protein decreased significantly after ultrasound treatments both using a 20 kHz probe and 40 kHz bath. The flow behaviour of α-lactalbumin was observed to be shear-thickening after all treatments. Apparent viscosity data calculated with power law equation (R² = 0.983-0.999) have not been changed significantly after all treatments. A remarkable decrease of initial freezing point was obtained after 20 kHz treatments.     

The effects of drying parameters and conditioning on mechanical properties of latex‐backed carpet

In this study, the effects of drying parameters and conditioning on the mechanical properties of latex‐backed carpet were investigated. Mechanical properties clearly correlated with the lowest moisture content (MC) experienced by samples in either drying or conditioning. Lowering MC was necessary to develop mechanical properties; however, the highest mechanical properties were achieved at the highest latex temperature, indicating that raising latex temperature can improve mechanical properties. The effect of conditioning on mechanical properties depended on the MC of the latex backing at the end of drying.     

Synergistic effects between κ-carrageenan and locust bean gum on physicochemical properties of edible films made thereof

The development of mixed systems, formed by locust bean gum (LBG), and κ-carrageenan (κ-car) can offer new interesting applications such as the development of edible films with particular properties. κ-car/LBG blend films with different ratios were developed, and their effects on films’ physical properties were assessed. Thermogravimetric analysis (TGA), X-ray diffraction (XRD) patterns, dynamic mechanical analysis (DMA) and Fourier-transform infrared (FTIR) spectroscopy techniques were used to highlight the interactions between the two polysaccharides. The addition of κ-car to LBG improved the barrier properties of the films leading to a decrease of water vapor permeability (WVP). Improved values of elongation-at-break (EB) were registered when the ratio of κ-car/LBG was 80/20 or 40/60 (% w/w). Moreover, the κ-car/LBG blend films enhance the tensile strength (TS) compared to κ-car and LBG films. FTIR results suggested that hydrogen bonds interactions between κ-car and LBG have a great influence in films’ properties e.g. moisture content, WVP. Therefore, different κ-car/LBG ratios can be used to tailor edible films with enhanced barrier and mechanical properties.     

Effects of γ-irradiation on Listeria monocytogenes population,colour, texture and sensory properties of Feta cheese during cold storage

Feta, a white brine cheese, was produced and contaminated with Listeria monocytogenes. Contamination occurred either at the beginning (pre-process contamination) or at the end of Feta manufacturing (post-process contamination). In the first case the milk was contaminated with 10³ cfu/ml, and 2 months later, in the final product, the L. monocytogenes population was approximately 10⁵ cfu/g. In the second case, the brine (NaCl, 7% w/v), in which the Feta was packaged, was contaminated with 10³ cfu/ml. Contaminated Feta samples were vacuum-packaged and exposed to irradiation doses of 1.0, 2.5 and 4.7 kGy and stored at 4 °C for a month. In the pre-process contaminated samples none of the irradiation doses eliminated L. monocytogenes; however the highest dose reduced the viable population to a level which is in compliance with EC regulations. In the post-process contamination, the 2.5 kGy and 4.7 kGy doses reduced L. monocytogenes counts below the detection limit. Irradiation had no effect on the texture of Feta. Irradiation at 4.7 kGy increased Feta's redness and decreased its yellowness and lightness. Sensorial analyses showed that at the 4.7 kGy dose, the aroma profile of Feta was temporarily affected, since it was restored after 30 days of cold storage.     

Isolation and characterisation of κ-casein/whey protein particles from heated milk protein concentrate and role of κ-casein in whey protein aggregation

Milk protein concentrate (79% protein) reconstituted at 13.5% (w/v) protein was heated (90 °C, 25 min, pH 7.2) with or without added calcium chloride. After fractionation of the casein and whey protein aggregates by fast protein liquid chromatography, the heat stability (90 °C, up to 1 h) of the fractions (0.25%, w/v, protein) was assessed. The heat-induced aggregates were composed of whey protein and casein, in whey protein:casein ratios ranging from 1:0.5 to 1:9. The heat stability was positively correlated with the casein concentration in the samples. The samples containing the highest proportion of caseins were the most heat-stable, and close to 100% (w/w) of the aggregates were recovered post-heat treatment in the supernatant of such samples (centrifugation for 30 min at 10,000 × g). κ-Casein appeared to act as a chaperone controlling the aggregation of whey proteins, and this effect was stronger in the presence of α_S- and β-casein.     

Species identification of ruminant milk by genotyping of the κ-casein gene

The development of molecular genetic and bioinformatic systems for identifying the species of milk and the raw material composition of dairy products is of great scientific and practical importance with the purpose of introducing developments in the system for controlling the turnover of falsified products. The aim of the research is to develop a method of PCR-RFLP analysis for species identification of milk and dairy products from agricultural ruminant animals by the κ-casein gene (CSN3) with the possibility of qualitative and relative quantitative assessment of species-specific DNA of the tested biomaterial. The objects of research were samples of raw milk and milk powder, pasteurized cream, and hard and semi-hard cheeses. The developed method of species identification of milk and dairy products includes sample preparation of the studied samples, nucleic acid extraction, combined PCR-RFLP technique, detection of obtained results by the method of horizontal electrophoresis in agarose gel and their analysis, including using the developed mathematical algorithms and software. The synergistic effect established in combined operation of 2 restriction enzymes ensured their application in a mix with increased performance in an ergonomic way in the context of DNA authentication of cow, goat, and sheep milk and dairy products based on them. The specificity and sensitivity of the proposed method is potentially suitable for implementing the development of a system to control the turnover of falsified and counterfeit goods.     

Antibacterial effect and hydrophobicity of yak κ-casein hydrolysate and its fractions

The antibacterial effects of yak κ-casein hydrolysate and its fractions on Escherichia coli were determined. Their surface hydrophobicity and amino acid profiles were also evaluated. Compared with intact yak κ-casein, yak κ-casein hydrolysate obtained with trypsin (YCHT) over 4 h of hydrolysis, permeate of YCHT obtained with 10 kDa cut-off membrane and permeate-F2 obtained from Sephadex G-25 gel filtration of permeate, exhibited a significantly higher antibacterial effect. Compared with YCHT and its permeate, permeate-F2 showed higher surface hydrophobicity when using PRODAN as a probe. Compared with native κ-casein, YCHT and permeate-F2 have a higher content and ratio of Lys, Pro, Phe and Leu residues. In contrast to its antibacterial effect, YCHT showed no cytotoxicity to mouse leukaemic monocyte macrophage RAW 264.7 cells at the concentrations used from 0 mg mL⁻¹ to 4 mg mL⁻¹. Therefore, yak κ-casein hydrolysate and its fractions may be used as potential inhibitors of E. coli.     

Occurrence,origin and fate of pyroglutamyl-γ3-casein in cheese

During plasmin-catalysed proteolysis of β-casein (β-CN) in cheese, pyroglutamyl-γ₃-casein (pγ₃-CN), originates from the cyclisation of the N-terminal Glu of γ₃-CN (β-CN f108–209). This peptide progressively increased in content during ripening of Grana Padano and Parmigiano-Reggiano Protected Designation of Origin cheeses. A preliminary survey revealed pγ₃-CN in both hard and extra-hard cheese varieties; in semi-hard cheeses it was present only in mature samples. To understand the mechanisms of pγ₃-CN formation/degradation, in vitro trials (real cheese, model cheeses and buffered γ₃-CN solutions) were performed. Both the effect of cheese pH and the capacity of certain microorganisms to cyclise the N-terminal Glu residue of γ₃-CN were studied. The results suggested that pγ₃-CN formed spontaneously during cheese ripening, mainly at the acid pH characteristic of hard and extra-hard cheeses; the role of cyclase enzymes was negligible. Differences in pγ₃-CN degradation were observed due to the proteolytic activity of bacteria involved in the cheese ripening.     

Effects of oxidative treatment on the physicochemical,rheological and functional properties of oat β-glucan

The objective of this work was promote oxidation of β-glucan from oat bran with hydrogen peroxide at different concentration levels (0.3%, 0.6% and 0.9% H₂O₂) and reaction times (30 and 60 min), and evaluate the physicochemical and functional properties of isedoxidised β-glucan with in-vitro tests. An increase in carbonyl and carboxyl groups and alterations in swelling power were verified in the oxidised β-glucan. The cholic acid binding capacity increased in the oxidised β-glucan; however, the fat binding capacity was not affected. After chemical digestion, the available glucose of the oxidised β-glucan was increased. Oxidation with hydrogen peroxide decreased the viscosity, hardness, adhesiveness and gumminess of the β-glucan gels. More studies are necessary to determine the effect of the oxidative treatment of β-glucan on its technological properties in food products, and biological properties should be examined with in-vivo studies.     

Caprine and ovine Greek dairy products: The official German method generates false-positive results due to κ-casein gene polymorphism

Caseins are widely used for species identification of dairy products. Isoelectric focusing (IEF) of para-κ-casein peptide is used as the official German method for the differentiation between caprine (isoform A) and ovine (isoform B) dairy products, based on their different isoelectric points. The discrimination between Greek goat and ewe dairy products using IEF has, however, been shown to be problematic because of the existence of the ewe isoform in milk from Greek indigenous dairy goats. This could be due to nucleotide polymorphisms within the goat κ-casein gene of Greek indigenous breeds, which alter the isoelectric point of the para-κ-casein peptide and lead to false positive results. Previous DNA analysis of the goat κ-casein gene has shown high levels of polymorphism; however, no such information is available for Greek indigenous dairy goats. Therefore, 87 indigenous dairy goats were sequenced at exon IV of κ-casein gene. In total, 9 polymorphic sites were detected. Three nonsynonymous point mutations were identified, which change the isoelectric point of the goat para-κ-casein peptide so that it appears identical to that of the ewe peptide. Ten composite genotypes were reconstructed and 6 of them included the problematic point mutations. For the verification of genetic results, IEF was carried out. Both goat and ewe patterns appeared in the problematic genotypes. The frequency of these genotypes could be characterized as moderate (0.23) to high (0.60) within Greek indigenous breeds. However, this is not an issue restricted to Greece, as such genotypes have been detected in various non-Greek goat breeds. In conclusion, IEF based on the official German method is certainly inappropriate for ovine and caprine discrimination concerning Greek dairy goat products, and consequently a new method should be established.