Genetic analysis of detailed milk protein composition and coagulation properties in Simmental cattle

The objective of this study was to estimate genetic parameters for milk protein fraction contents, milk protein composition, and milk coagulation properties (MCP). Contents of α_S1-, α_S2-, β-, γ-, and κ-casein (CN), β-lactoglobulin (β-LG), and α-lactalbumin (α-LA) were measured by reversed-phase HPLC in individual milk samples of 2,167 Simmental cows. Milk protein composition was measured as percentage of each CN fraction in CN (α_S1-CN%, α_S2-CN%, β-CN%, γ-CN%, and κ-CN%) and as percentage of β-LG in whey protein (β-LG%). Rennet clotting time (RCT) and curd firmness (a₃₀) were measured by a computerized renneting meter. Heritabilities for contents of milk proteins ranged from 0.11 (α-LA) to 0.52 (κ-CN). Heritabilities for α_S1-CN%, κ-CN%, and β-CN% were similar and ranged from 0.63 to 0.69, whereas heritability of α_S2-CN%, γ-CN%, and β-LG% were 0.28, 0.18, and 0.34, respectively. Effects of CSN2-CSN3 haplotype and BLG genotype accounted for more than 80% of the genetic variance of α_S1-CN%, β-CN%, and κ-CN% and 50% of the genetic variance of β-LG%. The genetic correlations among the contents of CN fractions and between CN and whey protein fractions contents were generally low. When the data were adjusted for milk protein gene effects, the magnitude of the genetic correlations among the contents of milk protein fractions markedly increased, indicating that they undergo a common regulation. The proportion of β-CN in CN correlated negatively with κ-CN% (r = −0.44). The genetic relationships between CN and whey protein composition were trivial. Low milk pH correlated with favorable MCP. Genetically, contents and proportions of α_S1- and α_S2-CN in CN were positively correlated with RCT. The relative proportion of β-CN in CN exhibited a genetic correlation with RCT of −0.26. Both the content and the relative proportion of κ-CN in CN did not correlate with RCT. Weak curds were genetically associated with increased proportions in CN of α_S1- and α_S2-CN, decreased contents of β-CN and κ-CN, and decreased proportion of κ-CN in CN. Negligible effects on the estimated correlations between a₃₀ and κ-CN contents or proportion in CN were observed when the model accounted for milk protein gene effects. Increasing β-CN and κ-CN contents and relative proportions in CN and decreasing the content and proportions of α_S1-CN and α_S2-CN and milk pH through selective breeding exert favorable effects on MCP.     

Identification of casein peptides in plasma of subjects after a cheese-enriched diet

Despite several studies support the functionality of casein peptides in vitro, a gap of knowledge still exist about their bioavailability.In this pilot study the bioavailability of phosphopeptides (CPPs) was investigated in four healthy subjects who consumed for one week 100 g/day of Parmigiano Reggiano cheese after one week of a dairy products-free diet. CPPs were detected in plasma samples from fasting subjects after the cheese-enriched diet and peptides were detected variously in almost all the 4 samples. Some α_s1- and α_s2-CN-derived CPPs, such as α_s1-CN (f43–52 and f43–50) and α_s2-CN (f8–12), (f7–12) and (f6–12) as well as four non-phosphorylated peptides belonging to the C-terminal end of β-CN (f193–209, f194–209, f200–209) were detected in plasma samples submitted to extraction and enrichment by hydroxyapatite (HA) chromatography followed by MALDI-TOF and nano LC-ESI/MS/MS analysis.Data indicated that casein oligopeptides are bioavailable after a continued intake of cheese. Future studies are warranted to ascertain this finding on a wider population and to clarify the mechanisms behind CPPs bioaccessibility and absorption.     

Peptides inhibitory to endopeptidase and aminopeptidase fromLactococcus lactis ssp.lactis MG1363, released from bovineβ-casein by chymosin,trypsin or chymotrypsin

Peptides inhibitory to the 70-kDa endopeptidase (PepO) from the cytoplasm ofLactococcus lactis ssp.lactis MG1363 were isolated from the supernatant (pH 4.6) of chymosin, tryptic and α-chymotryptic hydrolysates ofβ-casein (β-CN) by reversed-phase HPLC and identified by sequencing and mass spectrometry. Chymosin releasedβ-CN f193–209, kinetic constant (K _i) of which for inhibition of PepO was 60 μM. This peptide also inhibited (K _i=1700 μM) the 95-kDa aminopeptidase (PepN) fromL. lactis ssp.lactis MG 1363. Trypsin released two PepO-inhibitory peptides: one,β-CN f69–97, was not degradable by PepO (K _i=4.7 μM), while the other,β-CN f141–163, was degradable by PepO but competitively inhibited hydrolysis of methionine enkephalin by PepO. A peptide,β-CN f69–84, which inhibited PepO with aK _i of 8.1 μM, was isolated from the α-chymotryptic hydrolysate. Peptides released fromβ-CN by trypsin or chymotrypsin had very little inhibitory activity against PepN. PepO degradedβ-CN f193–209 very slowly compared with the hydrolysis of methionine enkephalin. All four inhibitory peptides (β-CN f193–209, f69–97, f69–84, f141–163) were readily degraded by thermolysin.     

Cloning and characterization of debittering peptidases,PepE, PepO,PepO2, PepO3, and PepN,of Lactobacillus helveticus WSU19

Peptidases of Lactobacillus helveticus WSU19 are important for debittering aged Cheddar–type cheese. Our objective was to determine specificities of aminopeptidase N (PepN) and endopeptidases E, O, O2, and O3 (PepE, PepO, PepO2, and PepO3) of Lb. helveticus WSU19 on the bitter peptide, β–CN f193–209. Aminopeptidase and endopeptidase genes of Lb. helveticus WSU19 were cloned in Escherichia coli DH5α. The β–CN f193–209 peptide was digested by cell–free extracts from peptidase–positive clones under cheese ripening conditions. The degradation pattern was analyzed qualitatively using matrix–assisted laser desorption/ionization time–of–flight mass spectrometry. Proline residues precluded PepN activity on β–CN f193–209. Complete degradation of β–CN f193–209 by PepN required post–proline endopeptidases, particularly PepO and PepO3. PepO–like endopeptidase activities on Pro₂₀₆–Ile₂₀₇ prevented formation of bitter peptides from the C–terminus of β–CN f193–209. PepE cleaved β–CN f193–209 only when combined with PepN or PepO–like endopeptidases. Aminopeptidase and post–proline endopeptidase activities contributed to the initial degradation of β–CN f193–209.     

Effect of ripening time on peptide dynamics and bioactive peptide composition in Tulum cheese

Skin bag Tulum cheeses traditionally produced in the Central Taurus region of Turkey were studied to identify peptide profiles by liquid chromatography–tandem mass spectrometry over 180 d of ripening. After mass spectrometry analysis, 203 peptides were identified: 59 from α_S1-casein (CN), 11 from α_S2-CN, 129 from β-CN, and 4 from κ-CN. Numbers of α_S1- and β-CN-derived peptides increased with increasing number of ripening days due to the dependence of newly formed peptides on proteolysis. However, similar increases were not observed for α_S2- and κ-CN-derived peptides. Most identified peptides consisted of β-CN-derived peptides, followed by α_S1-, α_S2-, and κ-CN-derived peptides. Among these, bioactive peptides were found, including antihypertensive, antibacterial, antioxidant, dipeptidyl peptidase-4 inhibitory, metal chelating, skin regenerating, glucagon-like peptide-1 secretion enhancing, opioid, cathepsin B inhibitory, prolyl endopeptidase inhibitory, immunomodulatory, brain function improving, antiamnesic, antihypercholesterolemic, anti-inflammatory, and anticarcinogenic peptides.     

Effects of β-κ-casein (CSN2-CSN3) haplotypes and β-lactoglobulin (BLG) genotypes on milk production traits and detailed protein composition of individual milk of Simmental cows

The aim of this study was to investigate the effects of CSN2-CSN3 (β-κ-casein) haplotypes and BLG (β-lactoglobulin) genotypes on milk production traits, content of protein fractions, and detailed protein composition of individual milk of Simmental cows. Content of the major protein fractions was measured by reversed-phase HPLC in individual milk samples of 2,167 cows. Protein composition was measured as percentage of each casein (CN) fraction to total CN and as percentage of β-lactoglobulin (β-LG) to total whey protein. Genotypes at CSN2, CSN3, and BLG were ascertained by reversed-phase HPLC, and CSN2-CSN3 haplotype probabilities were estimated for each cow. Traits were analyzed by using a linear model including the fixed effects of herd-test-day, parity, days in milk, and somatic cell score class, linear regressions on haplotype probabilities, class of BLG genotype, and the random effect of the sire of the cow. Effects of haplotypes and BLG genotypes on yields were weak or trivial. Genotype BB at BLG and haplotypes carrying CSN2 B and CSN3 B were associated with increased CN content and CN number. Haplotypes including CSN3 B were associated with increased κ-CN content and percentage of κ-CN to total CN and with decreased percentages of α_S1- and γ-CN to total CN. Allele CSN2 B had the effect of increasing β-CN content and decreasing content of α_S1-CN. Haplotypes including allele CSN2 A¹ exhibited decreased β-, α_S2-, and γ-CN concentrations and increased α_S1- and κ-CN contents, whereas CSN2 I had positive effects on β-CN concentration and trivial effects on content of other protein fractions. Effects of haplotypes on CN composition were similar to those exerted on content of CN fractions. Allele BLG A was associated with increased β-LG concentration and percentage of β-LG to total whey protein and with decreased content of other milk proteins, namely β-CN and α_S1-CN. Estimated additive genetic variance for investigated traits ranged from 14 to 39% of total variance. Increasing the frequency of specific genotypes or haplotypes by selective breeding might be an effective way to change milk protein composition.     

Comprehensive analysis of proteolysis during 8 months of ripening of high-cooked Old Saare cheese

We applied capillary electrophoresis, liquid chromatography coupled with tandem mass-spectrometry (MS/MS), and ultra-performance liquid chromatography to determine the composition of water-insoluble and water-soluble proteinaceous fractions of the cheese and to study in detail the degradation of caseins during 8 mo of ripening of Estonian high-temperature cooked hard cheese Old Saare. The application of high-resolution and high-accuracy MS/MS enabled identification of more than 3,000 small peptides, representing a fairly full casein peptidome containing peptides of 4 to 25 AA in length: 1,049 from β-casein (CN), 944 from α_S1-CN, 813 from α_S2-CN, and 234 from κ-CN. The majority of β-CN- and α_S1-CN-derived peptides originated from the N-terminal parts of the molecule, f6-93 and f1-124, respectively; peptides from α_S2-CN arose predominantly from the C-terminal end f100-162. At the beginning of ripening, we found a relatively high amount of peptides originating from the glycomacropeptide part of κ-CN, whereas peptides from para-κ-CN prevailed during the later stages of ripening of the cheese. The cleavage patterns of β-CN, α_S2-CN, as well as α_S1-CN, showed that primary proteolysis was started mainly by plasmin, although a low proteolytic activity of chymosin was also evident. Based on the analysis of cleavage sites, we observed a significant participation of proteolytic enzymes, including amino- and carboxypeptidases, of both mesophilic and thermophilic starter bacteria in further hydrolysis of oligopeptides during the ripening. Several new phosphopeptides were detected in the result of MS/MS data analysis. The profiles of the estimated concentrations of phosphopeptides revealed that those originating from β-CN and α_S1-CN accumulated during cheese maturation. In contrast, we did not notice any generation of phosphopeptides from the highly phosphorylated part of α_S2-CN, f25-80, presumably due to the inaccessibility of this region to the action of plasmin and chymosin. The analysis of cleavage sites and the combination of principal component and clustering analyses provided a characterization of the complex dynamics of formation and degradation of peptides during cheese maturation. We made an attempt to obtain a comprehensive picture of proteolysis during Old Saare cheese ripening on the basis of the detailed peptidomic data, including also the less abundant peptides determined by MS/MS, and complemented by the data on intact caseins and free AA and reported the results in the paper.     

Direct nanoHPLC-ESI-QTOF MS/MS analysis of tryptic caseinophosphopeptides

Caseinophosphopeptides (CPPs) were generated following tryptic hydrolysis of sodium caseinate. Hydrolysate peptides were separated and identified using nano-HPLC ESI-QTOF MS/MS. Sequence coverage in the 3 h hydrolysate was 79.4%, 55.6%, 80.9% and 68.1% for α_s1-, α_s2-, β- and κ-casein (CN), respectively. Variable levels of serine phosphorylation in β-CN f1–25 were observed in the 3 h hydrolysate. Analysis of β-CN f1–25 4P demonstrated that this peptide was stable during the course of hydrolysis. The effect of heat treatment (75 °C, 45 min) at pH 6.0, 7.0 and 8.0 on the peptide profile of the 3 h hydrolysate was studied. Compared to pH 6.0 and 8.0, least modification in phosphopeptide profiles was observed for the hydrolysate sample heated at pH 7.0. Different dephosphorylation and oxidation patterns were also observed following heat treatment at the three pH values. These results demonstrate that heat treatment, in addition to pH, has a major effect on both the phosphorylated and non-phosphorylated peptide profiles of CN hydrolysates.     

Occurrence of β-casein fragments in cold-stored and curdled river buffalo (Bubalus bubalis L.) milk

The safeguard of river buffalo Mozzarella cheese, a Protected Designation of Origin dairy product, has prompted an analytical study to trace the milk and curd used as raw material in cheesemaking. This is to prevent the illegal use of milk or curd from different geographical areas outside of those indicated in the official production protocol. For this purpose, we studied primary proteolysis occurring in fresh and frozen milk and curd to identify a molecular marker that could indicate the raw material used. Whole casein from frozen river buffalo milk was separated using cation-exchange chromatography and sodium dodecyl sulfate-PAGE, and a protein component with an estimated molecular weight of 15.3 kDa was detected. This protein component was revealed in fresh river buffalo milk as a faint electrophoresis band, which drastically increased in intensity in refrigerated and frozen milk as well as in curd and was found to be associated with β-CN through hydrophobic interaction. By using matrix-assisted laser desorption/ionization-time of flight peptide mass mapping, this component was identified as the C-terminal fragment f(69-209) of β-CN (expected molecular weight of 15,748.8 Da). β-Casein f(69-209), originating from the early hydrolysis of Lys₆₈-Ser₆₉ by plasmin, has no counterpart in bovine milk. The increased rate of hydrolysis by plasmin toward the cleavage site Lys₆₈-Ser₆₉ has to be ascribed to the elevated proline content of the peptide 61-73. The favored production of β-CN f(69-209) has also drawn attention to the complementary proteose peptone β-CN f(1-68) that is presumed to play a physiological role in inducing milk secretion similar to that of β-CN f(1-29). The higher in vivo and in vitro production rate, compared with γ₁-CN formation, indicates that β-CN f(69-209) and its complementary fragment are candidate molecular markers to evaluate milk and curd freshness. We used indirect ELISA analysis based on the determination of remaining nonhydrolyzed β-CN to perform a quantitative evaluation of proteolysis.     

Qualitative and Quantitative Determination of Caseins with Reverse-Phase and Anion-Exchange HPLC

Whole native caseinate (WNC) and casein (CN) fractions from preparative DE-52 cellulose urea columns were chromatographed using C-8 reverse-phase (RP) and DEAE-type anion-exchange (AEx) HPLC systems. With RP, α_S2-CN and κ-CN eluted first as several small peaks; α_S1-CN eluted later as two peaks, followed by β-CN peaks. With AEx, κ-CN eluted early as a group of peaks, β-CN eluted next, and α_S1-CN and α_S2-CN coeluted last. Standard curves were prepared for α_S1-CN and β-CN using RP-PHLC and showed correlation coefficients of 0.99 and 0.98, respectively. The caseins in WNC, nonfat dry milk casein, commercial casein(ates) and caseins from milks of individual cows were determined.     

Structural equation modeling for unraveling the multivariate genomic architecture of milk proteins in dairy cattle

The aims of this study were to investigate potential functional relationships among milk protein fractions in dairy cattle and to carry out a structural equation model (SEM) GWAS to provide a decomposition of total SNP effects into direct effects and effects mediated by traits that are upstream in a phenotypic network. To achieve these aims, we first fitted a mixed Bayesian multitrait genomic model to infer the genomic correlations among 6 milk nitrogen fractions [4 caseins (CN), namely κ-, β-, α_S1-, and α_S2-CN, and 2 whey proteins, namely β-lactoglobulin (β-LG) and α-lactalbumin (α-LA)], in a population of 989 Italian Brown Swiss cows. Animals were genotyped with the Illumina BovineSNP50 Bead Chip v.2 (Illumina Inc.). A Bayesian network approach using the max-min hill-climbing (MMHC) algorithm was implemented to model the dependencies or independence among traits. Strong and negative genomic correlations were found between β-CN and α_S1-CN (?0.706) and between β-CN and κ-CN (?0.735). The application of the MMHC algorithm revealed that κ-CN and β-CN seemed to directly or indirectly influence all other milk protein fractions. By integrating multitrait model GWAS and SEM-GWAS, we identified a total of 127 significant SNP for κ-CN, 89 SNP for β-CN, 30 SNP for α_S1-CN, and 14 SNP for α_S2-CN (mostly shared among CN and located on Bos taurus autosome 6) and 15 SNP for β-LG (mostly located on Bos taurus autosome 11), whereas no SNP passed the significance threshold for α-LA. For the significant SNP, we assessed and quantified the contribution of direct and indirect paths to total marker effect. Pathway analyses confirmed that common regulatory mechanisms (e.g., energy metabolism and hormonal and neural signals) are involved in the control of milk protein synthesis and metabolism. The information acquired might be leveraged for setting up optimal management and selection strategies aimed at improving milk quality and technological characteristics in dairy cattle.     

Immunoglobulin E epitope mapping by microarray immunoassay reveals differences in immune response to genetic variants of caseins from different ruminant species

The allergenicity of the caseins (CN), one of the major allergens in cow milk, is well characterized and their immunoglobulin E (IgE)-binding epitopes have been identified. However, investigations about the allergenic potential of the genetic variants occurring in the caseins are lacking. Therefore, this study determined the influence of the genetic polymorphism on IgE binding to epitopes of bovine casein variants. Furthermore, differences in IgE binding between epitopes of goats and water buffaloes were analyzed. A set of 187 peptides, covering the previously identified sequential IgE-binding epitopes of α_S1-, α_S2-, β-, and κ-CN variants from cows and the corresponding homologous peptides of water buffaloes and goats, were synthesized and tested by means of peptide microarray for IgE binding, using sera from 16 cow milk-sensitized individuals. Seven of the 16 sera samples showed positive signals on microarrays and were included in this study. In 5 α_S1-CN variants (A, B, C, E, and I), the AA substitution or deletion affected the immunoreactivity of epitopes AA 4 to 23, AA 17 to 36, AA 83 to 102, AA 173 to 192, and AA 175 to 194, as well as of the variant-specific peptides AA 184 to 196, AA 187 to 199, AA 174 to 193, and AA 179 to 198, which were found to resist gastrointestinal digestion. Variation in IgE binding was further detected for peptides AA 103 to 123 and AA 108 to 129 of 3 β-CN variants (A¹, A², and B). The majority of sera showed IgE binding to α_S1-CN peptides of cows and the homologous counterpart of goats and water buffaloes. However, α_S1- and β-CN epitopes from goats and water buffaloes had lower immunoreactivity than those of cows, but, in some cases, higher or exclusive IgE binding was observed. The results of this study indicate that genetic variants of the caseins differ in their allergenicity. This might be useful in the search for a suitable protein source for cow milk-allergic patients. In addition, milk from water buffaloes and goats harbor an allergenic potential due to cross-reactivity of IgE antibodies with cow milk caseins and are, therefore, not an acceptable alternative in the nutrition of cow milk-allergic patients.     

Effects of milk protein polymorphism and composition,casein micelle size and salt distribution on the milk coagulation properties in Norwegian Red cattle

Effects of milk protein polymorphism and composition, casein micelle size and salts distribution on the coagulation properties of milk from 99 Norwegian Red cattle (NRF) were studied. Genetic variants of α_S1-casein (CN), β-CN, κ-CN and β-lactoglobulin (LG) affected rennet coagulation properties of milk. Significant effects of κ-CN and the composite genotype α_S1-β-κ-CN were observed on acid coagulation properties. Relative concentrations of milk proteins were significantly affected by individual casein genotypes and the composite genotype of α_S1-β-κ-CN while, the relative concentration of β-LG was only affected by β-LG genotypes. The salts distribution in milk and the concentration of milk proteins affected both rennet and acid coagulation properties. Milk protein genotypes associated with better rennet coagulation, impaired the acid coagulation properties. However, α_S1-β-κ-CN BB-A¹A²-BE and BB-A²A²-BB were associated with poor rennet and acid coagulation properties. Breeding programs should focus on decreasing these genotypes in NRF cattle.     

Differentiation of intracellular peptidases of starter and adjunct cultures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Selection of starter and adjunct cultures is important to minimize bitterness of Cheddar and Gouda cheeses. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry may be useful for rapid screening of cheese cultures for propensity to produce bitter cheese. The objective of this study was to demonstrate the application of MALDI-TOF for differentiating intracellular peptidase activities of starter and adjunct cultures on β-CN f193-209 under simulated cheese condition. Bovine β-casein was incubated with chymosin in 9.55 g/l citrate buffer (pH 5.4, 40 g/l sodium chloride) at 30°C for 24 h, followed by incubation with cell-free extract (CFE) of starter or adjunct culture. Mixed strains of Lactococcus lactis ssp. lactis and L. lactis ssp. cremoris designated as 56 and 105 were the sources of nonbitter and bitter starter cultures, respectively. Lactobacillus helveticus WSU-19 and W900R represented adjunct cultures having high and low debittering activities, respectively. The degradation pattern of β-CN f193-209 by CFE of WSU-19 indicates general aminopeptidase and endopeptidase activities, while degradation of the peptide by CFE of W900R, 56, and 105 are mainly from endopeptidase activity. The rates of β-CN f193-209 hydrolysis by CFE of WSU-19, W900R, 56, and 105 are 6.90, 0.38, 0.39, and 0.23 mg/l per h, respectively.     

Angiotensin-converting enzyme inhibitory activity in Mexican Fresco cheese

The objective of this study was to evaluate if Mexican Fresco cheese manufactured with specific lactic acid bacteria (LAB) presented angiotensin I-converting enzyme inhibitory (ACEI) activity. Water-soluble extracts (3 kDa) obtained from Mexican Fresco cheese prepared with specific LAB (Lactococcus, Lactobacillus, Enterococcus, and mixtures: Lactococcus-Lactobacillus and Lactococcus-Enterococcus) were evaluated for ACEI activity. Specific peptide fractions with high ACEI were analyzed using reverse phase-HPLC coupled to mass spectrometry for determination of amino acid sequence. Cheese containing Enterococcus faecium or a Lactococcus lactis ssp. lactis-Enterococcus faecium mixture showed the largest number of fractions with ACEI activity and the lowest half-maximal inhibitory concentration (IC₅₀; <10 μg/mL). Various ACEI peptides derived from β-casein [(f(193-205), f(193-207), and f(193-209)] and α_S1-casein [f(1-15), f(1-22), f(14-23), and f(24-34)] were found. The Mexican Fresco cheese manufactured with specific LAB strains produced peptides with potential antihypertensive activity.     

Milk nutrition and childhood epilepsy: An ex vivo study on cytokines and oxidative stress in response to milk protein fractions

We present a pilot study on the effects of milk protein fractions [α_S1-casein (CN), α_S2-CN, κ-CN, β-CN, and a mix of α-lactalbumin (α-LA) and β-lactoglobulin (β-LG)] from different animal species (bovine, ovine, and caprine) on pro- and anti-inflammatory cytokines and oxidative status in cultured peripheral blood mononuclear cells from children with generalized epilepsy. Peripheral blood mononuclear cells (PBMC) were obtained by density gradient from blood of 10 children with generalized epilepsy (5 males; mean age 33.6 ± 5.4 mo) and 10 controls (5 males; mean age 35.6 ± 6.8 mo). Children with epilepsy were grouped according to cytokine levels as follows: children with epilepsy having low levels of cytokines not different from those of control children (LL-EC); children with epilepsy having cytokine levels at least 5-fold higher (medium levels) than those of control children (ML-EC); and children with epilepsy having cytokine levels at least 10-fold higher (high levels) than those of control children (HL-EC). The production of tumor necrosis factor-α (TNF-α), IL-10, IL-6, and IL-1β was studied in cultured PBMC incubated with α_S1-CN, α_S2-CN, κ-CN, β-CN, and a mix of α-LA and β-LG from bovine, caprine, and ovine milks. The levels of reactive oxygen and nitrogen species (ROS/RNS) and catalase activity were assessed in cultured supernatant. In the HL-EC group, β-CN from small ruminant species (ovine and caprine) induced the highest levels of TNF-α, whereas PBMC incubated with α_S2-CN from ovine milk and the mix of β-LG and α-LA from all tested milk species had the lowest levels of TNF-α. Within the HL-EC group, production of IL-1β was higher for bovine and ovine α_S2-CN fractions and lower for caprine and ovine β-CN and κ-CN. In the HL-EC group, IL-6 was higher in cultured PBMC incubated with α_S2-CN from bovine and ovine milk than from caprine milk. The cytokine IL-10 did not differ among milking species. The highest levels of ROS/RNS were found after incubation of PBMC with the β-CN fraction in bovine milk. Catalase activity was higher in PBMC cultured with β-CN isolated from bovine and caprine milk and with α_S1-CN from ovine milk.     

Genome-wide association study for αS1- and αS2-casein phosphorylation in Dutch Holstein Friesian

Phosphorylation of caseins (CN) is a crucial post-translational modification that allows caseins to form colloid particles known as casein micelles. Both α_S1- and α_S2-CN show varying degrees of phosphorylation (isoforms) in cow milk and were suggested to be more relevant for stabilizing internal micellar structure than β- and κ-CN. However, little is known about the genetic background of individual α_S2-CN phosphorylation isoforms and the phosphorylation degrees of α_S1- and α_S2-CN (α_S1-CN PD and α_S2-CN PD), defined as the proportion of isoforms with higher degrees of phosphorylation in total α_S1- and α_S2-CN, respectively. We aimed to identify genomic regions associated with these traits using 50K single nucleotide polymorphisms for 1,857 Dutch Holstein Friesian cows. A total of 10 quantitative trait loci (QTL) regions were identified for all studied traits on 10 Bos taurus autosomes (BTA1, 2, 6, 9, 11, 14, 15, 18, 24, and 28). Regions associated with multiple traits were found on BTA1, 6, 11, and 14. We showed 2 QTL regions on BTA1, one affecting α_S2-CN production and the other harboring the SLC37A1 gene, which encodes a phosphorus antiporter and affects α_S1- and α_S2-CN PD. The QTL on BTA6 harbors the casein gene cluster and affects individual α_S2-CN phosphorylation isoforms. The QTL on BTA11 harbors the PAEP gene that encodes for β-lactoglobulin and affects relative concentrations of α_S2-CN-10P and α_S2-CN-11P as well as α_S1-CN PD and α_S2-CN PD. The QTL on BTA14 harbors the DGAT1 gene and affects relative concentrations of α_S2-CN-10P and α_S2-CN-11P as well as α_S1-CN PD and α_S2-CN PD. Our results suggest that effects of identified genomic regions on phosphorylation of α_S1- and α_S2-CN are related to changes in milk synthesis and phosphorus secretion in milk. The actual roles of SLC37A1, PAEP, and DGAT1 in α_S1- and α_S2-CN phosphorylation in Dutch Holstein Friesian require further investigation.     

In vitro gastrointestinal digestion of bovine αS1- and αS2-casein variants gives rise to different IgE-binding epitopes

The occurrence and differences of resistant regions containing IgE-binding epitopes of α_S1-casein (α_S1-CN) variants B and C, as well as α_S2-CN A and B, after in vitro gastrointestinal digestion was investigated using mass spectrometry. The amino acid substitutions characterising the genetic variants affected the peptide pattern arising from the caseins and thus modifications in their allergenic epitopes occurred. Peptides f174–193 in α_S1-CN B and f179–198 in α_S1-CN C correspond to the IgE-binding epitope f173–194, which has been reported as one of the major epitopes in α_S1-CN B. Within α_S2-CN, the two variant-specific peptides, f7–29 from variant A and f1–22 from variant B, contain the previously identified IgE-binding epitope f1–20. These peptides, and in consequence the protein variants, may exhibit different immunoreactions, which could be significant in the production of milk with improved nutritional properties, such as hypoallergenic quality, by selection and breeding of cows with particular milk protein genotypes.     

Effects of the detailed protein composition of milk on curd yield and composition measured by model micro-cheese curd making of individual milk samples

The effect of the contents of casein (CN) and whey protein fractions on curd yield (CY) and composition was estimated using 964 individual milk samples. Contents of α_S1-CN, α_S2-CN, β-CN, γ-CN, glycosylated κ-CN (Gκ-CN), unglycosylated κ-CN, β-LG, and α-LA of individual milk samples were measured using reversed-phase HPLC. Curd yield and curd composition were measured by model micro-cheese curd making using 25 mL of milk. Dry matter CY (DMCY) was positively associated with all casein fractions but especially with α_S1-CN and β-CN. Curd moisture decreased at increasing β-CN content and increased at increasing γ-CN and Gκ-CN content. Due to their associations with moisture, Gκ-CN and β-CN were the fractions with the greatest effect on raw CY, which decreased by 0.66% per 1-standard deviation (SD) increase in the content of β-CN and increased by 0.62% per 1-SD increase in the content of Gκ-CN. The effects due to variation in percentages of the casein fractions in total casein were less marked than those exerted by contents. A 1-SD increase in β-CN percentage in casein (+3.8% in casein) exerted a slightly negative effect on DMCY (β = ?0.05%). Conversely, increasing amounts of α_S1-CN percentage were associated with a small increase in DMCY. Hence, results suggest that, at constant casein and whey protein contents in milk, the DMCY depends to a limited extent on the variation in the α_S1-CN:β-CN ratio. κ-Casein percentage did not affect DMCY, indicating that the positive relationship detected between the content of κ-CN and DMCY can be attributed to the increase in total casein resulting from the increased amount of κ-CN and not to variation in κ-CN relative content. However, milk with increased Gκ-CN percentage in κ-CN also shows increased raw CY and produces curds with increased moisture content. Curd yield increased at increasing content and relative proportion of β-LG in whey protein, but this is attributable to an improved capacity of the curd to retain water. Results obtained in this study support the hypothesis that, besides variation in total casein and whey protein contents, variation in protein composition might affect the cheese-making ability of milk, but this requires further studies.     

Effects of milk protein variants on the protein composition of bovine milk

The effects of β-lactoglobulin (β-LG), β-casein (β-CN), and κ-CN variants and β-κ-CN haplotypes on the relative concentrations of the major milk proteins α-lactalbumin (α-LA), β-LG, α_S1-CN, α_S2-CN, β-CN, and κ-CN and milk production traits were estimated in the milk of 1,912 Dutch Holstein-Friesian cows. We show that in the Dutch Holstein-Friesian population, the allele frequencies have changed in the past 16 years. In addition, genetic variants and casein haplotypes have a major impact on the protein composition of milk and explain a considerable part of the genetic variation in milk protein composition. The β-LG genotype was associated with the relative concentrations of β-LG (A » B) and of α-LA, α_S1-CN, α_S2-CN, β-CN, and κ-CN (B > A) but not with any milk production trait. The β-CN genotype was associated with the relative concentrations of β-CN and α_S2-CN (A² > A¹) and of α_S1-CN and κ-CN (A¹ > A²) and with protein yield (A² > A¹). The κ-CN genotype was associated with the relative concentrations of κ-CN (B > E > A), α_S2-CN (B > A), α-LA, and α_S1-CN (A > B) and with protein percentage (B > A). Comparing the effects of casein haplotypes with the effects of single casein variants can provide better insight into what really underlies the effect of a variant on protein composition. We conclude that selection for both the β-LG genotype B and the β-κ-CN haplotype A²B will result in cows that produce milk that is more suitable for cheese production.