Proteolytic activity and heat resistance of the protease AprX from Pseudomonas in relation to genotypic characteristics.

AprX is an alkaline metalloprotease produced by Pseudomonas spp. and encoded by its initial gene of the aprX-lipA operon. The intrinsic diversity among Pseudomonas spp. regarding their proteolytic activity is the main challenge for the development of accurate methods for spoilage prediction of ultra-high temperature (UHT) treated milk in the dairy industry. In the present study, 56 Pseudomonas strains were characterized by assessing their proteolytic activity in milk before and after lab-scale UHT treatment. From these, 24 strains were selected based on their proteolytic activity for whole genome sequencing (WGS) to identify common genotypic characteristics that correlated with the observed variations in proteolytic activity. Four groups (A1, A2, B and N) were determined based on operon aprX-lipA sequence similarities. These alignment groups were observed to significantly influence the proteolytic activity of the strains, with an average proteolytic activity of A1 > A2 > B > N. The lab-scale UHT treatment did not significantly influence their proteolytic activity, indicating a high thermal stability of proteases among strains. Amino acid sequence variation of biologically-relevant motifs in the AprX sequence, namely the Zn2+-binding motif at the catalytic domain and the C-terminal type I secretion signaling mechanism, were found to be highly conserved within alignment groups. These motifs could serve as future potential genetic biomarkers for determination of alignment groups and thereby strain spoilage potential.

Influence of Lactose on the Maillard Reaction and Dehydroalanine-Mediated Protein Cross-Linking in Casein and Whey.

A liquid chromatography-mass spectrometry method based on multiple reaction monitoring (MRM) was developed for the simultaneous quantification of markers representing two potentially competing pathways, the Maillard reaction and the dehydroalanine pathway. The two pathways involve the same residues in the proteins to some extent, namely, the essential amino acid lysine, as well as free-amino terminals available on proteins and polypeptides, competition between the two pathways in food systems may occur. The developed method comprises the following markers of the Maillard reaction: furosine, N-ε-(carboxyethyl)lysine (CEL) and N-ε-(carboxymethyl)lysine (CML), together with the dehydroalanine reaction pathway markers; lanthionine (LAN) and lysinoalanine (LAL), as well as lysine itself. The validated method was then used for the absolute quantification of heat-induced protein modifications in model systems of micellar casein and whey protein isolates (MCI and WPI, respectively) in the presence or absence of lactose. As expected, the Maillard reaction markers furosine, CEL and CML increased during the applied heat treatment in the presence of lactose, whereas the dehydroalanine markers, LAN and LAL increased with heating in both MCI and WPI, both in the presence and absence of lactose, although at lower levels in the presence of lactose, confirming the competing state of the two pathways.

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.

Phosphorylation and glycosylation isoforms of bovine κ-casein variant E in homozygous Swedish Red cow milk detected by liquid chromatography-electrospray ionization mass spectrometry.

Variations in the phosphorylation and glycosylation patterns of the common κ-casein (CN) variants A and B have been explored, whereas studies on variant E heterogeneity are scarce. This study reports for the first time the detailed phosphorylation and glycosylation pattern of the κ-CN variant E in comparison with variants A and B. Individual cow milk samples representing κ-CN genotype EE (n = 12) were obtained from Swedish Red cows, and the natural posttranslational modifications of its κ-CN were identified and quantified by liquid chromatography-electrospray mass spectrometry. In total, 12 unique isoform masses of κ-CN variant E were identified. In comparison, AA and BB milk consisted of 14 and 17 unique isoform masses, respectively. The most abundant κ-CN E isoform detected in the EE milk was the monophosphorylated, unglycosylated [1P 0G, ∼70%; where P indicates phosphorylation from single to triple phosphorylation (1-3P), and G indicates glycosylation from single to triple glycosylation (1-3G)] form, followed by diphosphorylated, unglycosylated (2P 0G, ∼12%) form, resembling known patterns from variants A and B. However, a clear distinction was the presence of the rare triphosphorylated, nonglycosylated (3P 0G, ∼0.05%) κ-CN isoform in the EE milk. All isoforms detected in variant E were phosphorylated, giving a phosphorylation degree of 100%. This is comparable with the phosphorylation degree of variants A and B, being also almost 100%, though with very small amounts of nonphosphorylated, glycosylated isoforms detected. The glycosylation degree of variant E was found to be around 17%, a bit higher than observed for variant B (around 14%), and higher than variant A (around 7%). Among glycosylation, the glycan e was the most common type identified for all 3 variants, followed by c/d (straight and branched chain trisaccharides, respectively), and b. In contrast to κ-CN variants A and B, no glycan of type a was found in variant E. Taken together, this study shows that the posttranslational modification pattern of variant E resembles that of known variants to a large extent, but with subtle differences.

Differential in vitro digestion rates in gastric phase of bovine milk with different κ-casein phenotypes.

Casein (CN) micelles will coagulate in the stomach after ingestion, which is similar to the cheesemaking process. Although genetic variants of bovine proteins, especially κ-CN, have been confirmed to influence the coagulation properties of the CN micelle, its influence on milk digestibility has not been revealed yet. This study aimed to investigate how genetic variants, glycosylation degree of κ-CN, and CN micelle size influence digestion rates during in vitro gastrointestinal digestion. Three milk pools, representing κ-CN phenotypes of either AA, BB, or AB composition were prepared from milk of individual Danish Holstein cows representing these different genotypes. In vitro digestion of the 3 milk pools, AA, BB, or AB, was investigated by sodium dodecyl sulfate-PAGE, liquid chromatography-mass spectrometry, and degree of hydrolysis. The results showed that κ-CN AA milk had faster digestion rate in the gastric phase compared with BB and AB milks, whereas only small differences were apparent in the intestinal digestion phase. The results further documented that the milk pools representing κ-CN phenotypes BB and AB had comparable overall glycosylation degrees (50.9% and 50.0%, respectively) and higher than that of the AA milk pool (46.9%). Further, the AA milk pool was associated with larger CN micelles. These differences in CN micelle sizes and glycosylation degrees can be part of underlying explanations for the differential in vitro digestion rates observed between the AA, BB, and AB κ-CN milk pools.

Factors influencing milk osteopontin concentration based on measurements from Danish Holstein cows.

Our objective was to determine the content of the bioactive protein osteopontin (OPN) in bovine milk and identify factors influencing its concentration. OPN is expressed in many tissues and body fluids, with by far the highest concentrations in milk. OPN plays a role in immunological and developmental processes and it has been associated with several milk production traits and lactation persistency in cows. In the present study, we report the development of an enzyme linked immunosorbent assay (ELISA) for measurement of OPN in bovine milk. The method was used to determine the concentration of OPN in milk from 661 individual Danish Holstein cows. The median OPN level was determined to 21.9 mg/l with a pronounced level of individual variation ranging from 0.4 mg/l to 67.8 mg/l. Breeding for increased OPN in cow's milk is of significant interest, however, the heritability of OPN in milk was found to be relatively low, with an estimated value of 0.19 in the current dataset. The variation explained by the herd was also found to be low suggesting that OPN levels are not affected by farm management or feeding. Interestingly, the concentration of OPN was found to increase with days in milk and to decrease with parity.

Quantitative LC-MS/MS analysis of high-value milk proteins in Danish Holstein cows.

High-value milk proteins, which can be obtained by optimized fractionation procedures, are ideal ingredients in many food applications. Thus, a simple and robust analytical method is required for the identification and quantification of these individual milk proteins. Here, we present a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using multiple reaction monitoring (MRM) to simultaneously detect and measure target peptides of two major milk proteins, α-lactalbumin (α-LA) and ß-casein (ß-CN), in raw milk samples from 662 Danish Holstein cows. The MRM quantification of α-LA and ß-CN was achieved with limit of detection (LOD) of 0.14 and 0.16 g/L, respectively and reproducibility of the assay <15%. By this newly established MRM-based method, the concentration of α-LA and ß-CN in an individual cow's milk ranged from 0.5 to 1.9 (average 1.1) g/L, and from 7.5 to 23.4 (average 15) g/L, respectively. There was no significant effect of parity, whereas significantly increasing concentrations of α-LA and ß-CN were observed through lactation (P < 0.001). This shows a considerable biological variation of these two ingredient milk proteins, providing potential varying outputs of fractionation in the dairy streams.

Disulfide bond formation is not crucial for the heat-induced interaction between ß-lactoglobulin and milk fat globule membrane proteins.

During heat treatment of milk, ß-lactoglobulin (ß-LG) associates with the milk fat globule membrane (MFGM). The objective of this study was to examine different binding types that could be involved in this process. First, we tested the thiol-disulfide bond interchange between ß-LG and MFGM by heating raw milk (87°C, 8 min) in the presence of different reagents capable of preventing this interaction, and then evaluated the presence of ß-LG in resulting MFGM preparations by sodium dodecyl sulfate-PAGE. Contrary to commonly accepted theory, ß-LG still associated with MFGM when milk was heated in the presence of 10 mM N-ethylmaleimide, dithiobis-nitrobenzoic acid, or dithioerythritol. This finding indicated that noncovalent binding could be involved in the interaction, and therefore these were studied next. Preventing noncovalent interactions by heating milk in the presence of 8 M urea (to inhibit formation of hydrogen bonds) or 2 M NaCl (to inhibit electrostatic and hydrophobic interactions) reduced the association of ß-LG and MFGM. Inhibiting both hydrogen and disulfide bond formation by addition of 8 M urea and 10 mM dithioerythritol or inhibiting hydrophobic interactions with 0.2% sodium dodecyl sulfate completely prevented the association. In contrast to the simple thiol-disulfide interaction model, the results suggest a more complex understanding of the interactions between ß-LG and MFGM during heating of milk. This indicates that disulfide formation between ß-LG and proteins in the MFGM is not required for the association, but that hydrophobic interactions and hydrogen bonding may be crucial. This novel insight into ß-LG and MFGM association is in contrast to the current literature and requires further study.

Liquid Chromatography Mass Spectrometry Quantification of α-solanine, α-chaconine, and Solanidine in Potato Protein Isolates.

For potato proteins to be used as a food ingredient, the level of natural potato defense substances, the glycoalkaloids (GAs), should be limited. In this work, a method is developed for quantification of the two major potato GAs, α-solanine and α-chaconine, as well as for their aglycon form, solanidine, using liquid chromatography-mass spectrometry single quadrupole in single ion monitoring mode. Standard solutions of GA and a food-grade potato protein powder was used to validate the method. A linear correlation between GA concentration and the ion intensity of >0.995 was obtained for all standard solutions. Recovery of GA in spiked samples was within the range 82%-106%. The method for GA quantification was applied to a variety of potato protein isolates. The results showed that total GA increased during the storage of the potatoes. Washing the potato protein isolates using water at a sufficient level was shown to be able to reduce the amount of GA below the threshold of 150 µg g-1, as needed for human consumption.

Influence of desialylation of caseinomacropeptide on the denaturation and aggregation of whey proteins.

The effect of the addition of caseinomacropeptide (CMP) or desialylated CMP on the heat-induced denaturation and aggregation of whey proteins was investigated in the pH range 3 to 7 after heating at 80°C for 30 min. The rate and temperature of denaturation, the extent of aggregation, and the changes in secondary structure of the whey proteins heated in presence of CMP or desialylated CMP were measured. The sialic acid bound to CMP favored the denaturation and aggregation of whey proteins when the whey proteins were oppositely charged to CMP at pH 4. A transition occurred at pH 6, below which the removal of sialic acid enhanced the stabilizing properties of CMP against the denaturation and aggregation of the whey proteins. At pH >6, the interactions between desialylated CMP and the whey proteins led to more extensive denaturation and aggregation. Sialic acid bound to CMP influenced the denaturation and aggregation behavior of whey proteins in a pH-dependent manner, and this should be considered in future studies on the heat stability of such systems containing CMP.

Impact of industrial cream heat treatments on the protein composition of the milk fat globule membrane.

The impact of cream processing on milk fat globule membrane (MFGM) was assessed in an industrial setting for the first time. Three creams and their derived MFGM fractions from different stages of the pasteurization procedure at a butter dairy were investigated and compared to a native control as well as a commercial MFGM fraction. The extent of cross-linking of serum proteins to MFGM proteins increased progressively with each consecutive pasteurization step. Unresolved high molecular weight aggregates were found to consist of both indigenous MFGM proteins and ß-lactoglobulin as well as αs1- and ß-casein. With regards to fat globule stability and in terms of resistance towards coalescence and flocculation after cream washing, single-pasteurized cream exhibited reduced sensitivity to cream washing compared to non- and double-pasteurized creams. Inactivation of the agglutination mechanism and the increased presence of non-MFGM proteins may determine this balance between stable and non-stable fat globules.

A genome-wide association study reveals specific transferases as candidate loci for bovine milk oligosaccharides synthesis.

BACKGROUND: Human milk oligosaccharides (OS) play a key role in brain and gut microbiota development of the neonate, but the underlying biosynthetic steps of OS in the mammary gland are still largely unknown. As bovine milk contains OS with somewhat similar structures and functionalities there is increased interest in further understanding the genetic basis underlying the OS content of milk for eventual extraction and generation of value-added ingredients for infant formulas and nutraceuticals. The present study is the first to report on genetic parameter estimation as well as on a genome wide association study (GWAS) from the largest bovine milk OS dataset analyzed to date. RESULTS: In total 15 different bovine milk OS were monitored. Heritabilities ranged from 0 to 0.68 in Danish Holstein and from 0 to 0.92 in Danish Jersey. The GWAS identified in total 1770 SNPs (FDR < 0.10) for five different OS in Danish Holstein and 6913 SNPs (FDR < 0.10) for 11 OS in Danish Jersey. In Danish Holstein, a major overlapping QTL was identified on BTA1 for LNH and LNT explaining 24% of the variation in these OS. The most significant SNPs were associated with B3GNT5, a gene encoding a glycosyltransferase involved in glycan synthesis. In Danish Jersey, a very strong QTL was detected for the OS with composition 2 Hex 1 HexNAc (isomer 1) on BTA11. The most significant SNP had -log10(P-value) of 52.88 (BOVINEHD1100030300) and was assigned to ABO, a gene encoding ABO blood group glycosyltransferases. This SNP has been reported to be a missense mutation and explains 56% of the OS variation. Other candidate genes of interest identified for milk OS were ALG3, B3GALNT2, LOC520336, PIGV, MAN1C1, ST6GALNAC6, GLT6D1, GALNT14, GALNT17, COLGALT2, LFNG and SIGLEC. CONCLUSION: To our knowledge, this is the first study documenting a solid breeding potential for bovine milk OS and a strong indication of specific candidate genes related to OS synthesis underlying this genetic influence. This new information has the potential to guide breeding strategies to achieve production of milk with higher diversity and concentration of OS and ultimately facilitate large-scale extraction of bovine milk OS.

Effect of Casein Hydrolysates on Intestinal Cell Migration and Their Peptide Profiles by LC-ESI/MS/MS.

Potential beneficial effects of bioactive peptides derived from casein on epithelial cellular wound healing in the gastrointestinal tract were studied. Bovine casein was digested by a combination of pepsin and pancreatic proteases at different time intervals to represent ranges of duration of gastrointestinal digestion. Intestinal epithelial cells were used as an in vitro model of the small intestine. The effect of casein hydrolysates on cell migration was studied by scratch assay as a model of wound healing. Casein digested by pepsin and pancreatin for 10 to 30 min were found to have a significant stimulatory effect of >40% on cell migration relative to the control. A potential effect of casein gastrointestinal digests on gastro-intestinal wound healing has not previously been reported. The peptide profiles of active as well as inactive casein hydrolysates were characterised by liquid chromatography coupled to ion trap tandem mass spectrometry. By comparison of identified peptides in active and inactive casein hydrolysates, a pool of 11 peptides derived from casein were identified as potential candidates for effects on cell migration. Searching the milk bioactive peptide database (MBPDB) showed that 15 of the identified peptides had known biological functions such as antimicrobial, antioxidant, and immunomodulatory activity.

Thermal effects on IgM-milk fat globule-mediated agglutination.

The process of agglutination causes firm cream layers in bovine milk, and a functioning agglutination mechanism is paramount to the quality of non-homogenized milks. The phenomenon is not well-described, but it is believed to occur due to interactions between immunoglobulins (Ig) and milk fat globules. For the first time, this paper demonstrates how the process of agglutination can be visualized using confocal laser scanning microscopy, rhodamine red and a fluoresceinisothiocynat-conjugated immunoglobulin M antibody. The method was used to illustrate the effect on agglutination of storage temperature and pasteurization temperature. Storage at 5 °C resulted in clearly visible agglutination which, however, was markedly reduced at 15 °C. Increasing storage temperature to 20 or 37 °C cancelled any detectable interaction between IgM and milk fat globules, whereby the occurrence of cold agglutination was documented. Increasing 20 s pasteurization temperatures from 69 °C to 71 °C and further to 73 °C lead to progressively higher inactivation of IgM and, hence, reduction of agglutination. Furthermore, 2-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that changes in storage temperature caused a redistribution of Ig-related proteins in milk fat globule membrane isolates. Poly-immunoglobulin G receptor was present in milk fat globule preparations stored at cold (4 °C) conditions, but absent at storage at higher temperature (25 °C). The findings provide valuable knowledge to dairy producers of non-homogenized milk in deciding the right pasteurization temperature to retain the crucial agglutination mechanism.

Digestibility of glycated milk proteins and the peptidomics of their in vitro digests.

BACKGROUND: Milk proteins are widely used in food production and are often glycated by reducing sugar. Although many studies have reported the digestibility of glycated milk protein, most have focused on measuring degree of hydrolysis (DH), showing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) image of digests. Detailed information on the changes in peptide composition of digests has seldom been revealed. Therefore, in addition to measuring the DH and showing the SGS-PAGE images of digests, we also analyzed the peptidomics in digests using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) and Mascot database in this work to further reveal the influence of glycation on protein nutrition. RESULTS: Compared with ß-lactoglobulin and bovine serum albumin (BSA), DH of ß-casein was suppressed to a lesser extent by glycation in both gastric and intestinal stages. Aggregates of glycated BSA were less sensitive to the action of digestive enzymes throughout gastrointestinal digestion according to SDS-PAGE images. Changes in the peptide composition of digests induced by glycation were distinctly displayed, showing both absence of peptides and occurrence of new peptides, based on the results obtained from LC-ESI-MS/MS. CONCLUSIONS: Glycation can greatly change the peptide composition in digests of milk protein. The nutritional impact of the change in the peptide composition requires further investigation, and the impact of MRPs in unabsorbed digests on the gut flora should be an interesting field for further studies. © 2018 Society of Chemical Industry.

Comparison of milk protein composition and rennet coagulation properties in native Swedish dairy cow breeds and high-yielding Swedish Red cows.

Recent studies have reported a very high frequency of noncoagulating milk in Swedish Red cows. The underlying factors are not fully understood. In this study, we explored rennet-induced coagulation properties and relative protein profiles in milk from native Swedish Mountain and Swedish Red Polled cows and compared them with a subset of noncoagulating (NC) and well-coagulating (WC) milk samples from modern Swedish Red cows. The native breeds displayed a very low prevalence of NC milk and superior milk coagulation properties compared with Swedish Red cows. The predominant variants in both native breeds were αS1-casein (αS1-CN) B, ß-CN A2 and ß-lactoglobulin (ß-LG) B. For κ-CN, the B variant was predominant in the Swedish Mountain cows, whereas the A variant was the most frequent in the Swedish Red Polled. The native breeds displayed similar protein composition, but varied in content of αS1-CN with 9 phosphorylated serines (9P) form. Within the Swedish Mountain cows, we observed a strong inverse correlation between the relative concentration of κ-CN and micelle size and a positive correlation between ionic calcium and gel firmness. For comparison, we investigated a subset of 29 NC and 28 WC milk samples, representing the extremes with regard to coagulation properties based on an initial screening of 395 Swedish Red cows. In Swedish Red, NC milk properties were found to be related to higher frequencies of ß-CN A2, κ-CN E and A variants, as well as ß-LG B, and the predominant composite genotype of ß- and κ-CN in the NC group was A2A2/AA. Generally, the A2A2/AA composite genotype was related to lower relative concentrations of κ-CN isoforms and higher relative concentrations of αS1-, αS2-, and ß-CN. Compared with the group of WC milk samples, NC milk contained a higher fraction of αS2-CN and α-lactalbumin (α-LA) but a lower fraction of αS1-CN 9P. In conclusion, milk from native Swedish breeds has good characteristics for cheese milk, which could be exploited in niche dairy products. In milk from Swedish Mountain cows, levels of ionic calcium seemed to be more important for rennet-induced gel firmness than variation in the relative protein profile. In Swedish Red, lower protein content as well as higher fraction of αS2-CN and lower fraction of αS1-CN 9P were related to NC milk. Further, a decrease in the frequency of the composite ß-κ-CN genotype A2A2/AA through selective breeding could have a positive effect on milk coagulation properties.

Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography.

Studies have suggested that nanoscale extracellular vesicles (EV) in human and bovine milk carry immune modulatory properties which could provide beneficial health effects to infants. In order to assess the possible health effects of milk EV, it is essential to use isolates of high purity from other more abundant milk structures with well-documented bioactive properties. Furthermore, gentle isolation procedures are important for reducing the risk of generating vesicle artefacts, particularly when EV subpopulations are investigated. In this study, we present two isolation approaches accomplished in three steps based on size-exclusion chromatography (SEC) resulting in effective and reproducible EV isolation from raw milk. The approaches do not require any EV pelleting and can be applied to both human and bovine milk. We show that SEC effectively separates phospholipid membrane vesicles from the primary casein and whey protein components in two differently obtained casein reduced milk fractions, with one of the fractions obtained without the use of ultracentrifugation. Milk EV isolates were enriched in lactadherin, CD9, CD63 and CD81 compared to minimal levels of the EV-marker proteins in other relevant milk fractions such as milk fat globules. Nanoparticle tracking analysis and electron microscopy reveals the presence of heterogeneous sized vesicle structures in milk EV isolates. Lipid analysis by thin layer chromatography shows that EV isolates are devoid of triacylglycerides and presents a phospholipid profile differing from milk fat globules surrounded by epithelial cell plasma membrane. Moreover, the milk EV fractions are enriched in RNA with distinct and diverging profiles from milk fat globules. Collectively, our data supports that successful milk EV isolation can be accomplished in few steps without the use of ultracentrifugation, as the presented isolation approaches based on SEC effectively isolates EV in both human and bovine milk.

Short communication: Inhibition of angiotensin 1-converting enzyme by peptides derived from variants of bovine ß-casein upon apical exposure to a Caco-2 cell monolayer.

This study investigated the consequence of genetically contingent amino acid substitutions in bovine ß-casein (CN) genetic variants A1, A2, B, and I on the structure and bioactive potential of peptides following in vitro digestion. The ß-CN variants were digested in vitro using pepsin and pancreatin, and a peptide profile was obtained by liquid chromatography tandem mass spectrometry, revealing among others, the ß-casomorphin precursor peptides VYPFPGPIHN and VYPFPGPIPN, derived from variant A1/B and from A2/I, respectively. These 2 peptides were synthesized and assessed for angiotensin 1-converting enzyme (ACE) inhibitory capacity before and after incubation with a monolayer of Caco-2 intestinal cells. The VYPFPGPIHN was a stronger ACE inhibitor than VYPFPGPIPN, with the concentration needed to reach half-maximal inhibition (IC50) of 123 ± 14.2 µM versus 656 ± 7.6 µM. Exposure to a Caco-2 intestinal cell monolayer did not affect ACE inhibition by VYPFPGPIHN, but resulted in an almost 2-fold increase in inhibition by VYPFPGPIPN after incubation. Subsequent tandem mass spectrometric analysis identified the truncated peptide VYPFPGPIP, suggesting hydrolysis by a cell membrane associated peptidase. Thus, genetic variation in bovine ß-CN results in the generation of peptides that differ in bioactivity, and are differently affected by intestinal brush border peptidases.

Estimation of genetic parameters and detection of chromosomal regions affecting the major milk proteins and their post translational modifications in Danish Holstein and Danish Jersey cattle.

BACKGROUND: In the Western world bovine milk products are an important protein source in human diet. The major proteins in bovine milk are the four caseins (CN), αS1-, αS2-, ß-, and k-CN and the two whey proteins, ß-LG and α-LA. It has been shown that both the amount of specific CN and their isoforms including post-translational modifications (PTM) influence technological properties of milk. Therefore, the aim of this study was to 1) estimate genetic parameters for individual proteins in Danish Holstein (DH) (n = 371) and Danish Jersey (DJ) (n = 321) milk, and 2) detect genomic regions associated with specific milk protein and their different PTM forms using a genome-wide association study (GWAS) approach. RESULTS: For DH, high heritability estimates were found for protein percentage (0.47), casein percentage (0.43), k-CN (0.77), ß-LG (0.58), and α-LA (0.40). For DJ, high heritability estimates were found for protein percentage (0.70), casein percentage (0.52), and α-LA (0.44). The heritability for G-k-CN, U-k-CN and GD was higher in the DH compared to the DJ, whereas the heritability for the PD of αS1-CN was lower in DH compared to DJ, whereas the PD for αS2-CN was higher in DH compared to DJ. The GWAS results for the main milk proteins were in line what has been earlier published. However, we showed that there were SNPs specifically regulating G-k-CN in DH. Some of these SNPs were assigned to casein protein kinase genes (CSNK1G3, PRKCQ). CONCLUSION: The genetic analysis of the major milk proteins and their PTM forms revealed that these were heritable in both DH and DJ. In DH, genomic regions specific for glycosylation of k-CN were detected. Furthermore, genomic regions for the major milk proteins confirmed the regions on BTA6 (casein cluster), BTA11 (PEAP), and BTA14 (DGAT1) as important regions influencing protein composition in milk. The results from this study provide confidence that it is possible to breed for specific milk protein including the different PTM forms.

Estimation of genetic parameters and detection of quantitative trait loci for minerals in Danish Holstein and Danish Jersey milk.

BACKGROUND: Bovine milk provides important minerals, essential for human nutrition and dairy product quality. For changing the mineral composition of the milk to improve dietary needs in human nutrition and technological properties of milk, a thorough understanding of the genetics underlying milk mineral contents is important. Therefore the aim of this study was to 1) estimate the genetic parameters for individual minerals in Danish Holstein (DH) (n=371) and Danish Jersey (DJ) (n=321) milk, and 2) detect genomic regions associated with mineral content in the milk using a genome-wide association study (GWAS) approach. RESULTS: For DH, high heritabilities were found for Ca (0.72), Zn (0.49), and P (0.46), while for DJ, high heritabilities were found for Ca (0.63), Zn (0.57), and Mg (0.57). Furthermore, intermediate heritabilities were found for Cu in DH, and for K, Na, P and Se in the DJ. The GWAS revealed a total of 649 significant SNP markers detected for Ca (24), Cu (90), Fe (111), Mn (3), Na (1), P (4), Se (12) and Zn (404) in DH, while for DJ, a total of 787 significant SNP markers were detected for Ca (44), Fe (43), K (498), Na (4), Mg (1), P (94) and Zn (3). Comparing the list of significant markers between DH and DJ revealed that the SNP ARS-BFGL-NGS-4939 was common in both breeds for Zn. This SNP marker is closely linked to the DGAT1 gene. Even though we found significant SNP markers on BTA14 in both DH and DJ for Ca, and Fe these significant SNPs did not overlap. CONCLUSION: The results show that Ca, Zn, P and Mg show high heritabilities. In combination with the GWAS results this opens up possibilities to select for specific minerals in bovine milk.