Identification of rare genetic variants of the αS-caseins in milk from native Norwegian dairy breeds and comparison of protein composition with milk from high-yielding Norwegian Red cows.

Several factors influence the composition of milk. Among these, genetic variation within and between cattle breeds influences milk protein composition, protein heterogeneity, and their posttranslational modifications. Such variations may further influence technological properties, which are of importance for the utilization of milk into dairy products. Furthermore, these potential variations may also facilitate the production of differentiated products (e.g., related to specific breeds or specific genetic variants). The objective of this study was to investigate the genetic variation and relative protein composition of the major proteins in milk from 6 native Norwegian dairy breeds representing heterogeneity in geographical origin, using the modern Norwegian breed, Norwegian Red, as reference. In total, milk samples from 144 individual cows were collected and subjected to liquid chromatography-electrospray ionization/mass spectrometry-based proteomics for identification of genetic and posttranslational modification isoforms of the 4 caseins (αS1-CN, αS2-CN, ß-CN, κ-CN) and the 2 most abundant whey proteins (α-lactalbumin and ß-lactoglobulin). Relative quantification of these proteins and their major isoforms, including phosphorylations of αS1-CN and glycosylation of κ-CN, were determined based on UV absorbance. The presence and frequency of genetic variants of the breeds were found to be very diverse and it was possible to identify rare variants of the CN, which, to our knowledge, have not been identified in these breeds before. Thus, αS1-CN variant D was identified in low frequency in 3 of the 6 native Norwegian breeds. In general, αS1-CN was found to be quite diverse between the native breeds, and the even less frequent A and C variants were furthermore detected in 1 and 5 of the native breeds, respectively. The αS1-CN variant C was also identified in samples from the Norwegian Red cattle. The variant E of κ-CN was identified in 2 of the native Norwegian breeds. Another interesting finding was the identification of αS2-CN variant D, which was found in relatively high frequencies in the native breeds. Diversity in more common protein genetic variants were furthermore observed in the protein profiles of the native breeds compared with milk from the high-yielding Norwegian Reds, probably reflecting the more diverse genetic background between the native breeds.

Degradation of ß-casomorphin-7 through in vitro gastrointestinal and jejunal brush border membrane digestion.

This work aimed to study the opioid peptide ß-casomorphin-7 (BCM7) degradation or stability during digestion using human gastrointestinal (GI) juices and porcine jejunal brush border membrane (BBM) peptidases. Synthetic BCM7 was subjected to in vitro digestion by GI fluids obtained from human volunteers for 180 min, and to downstream degradation with porcine BBM vesicles. The BCM7 was sampled at 4 time points over 24 h after BBM addition. The digests were profiled by HPLC-electrospray ionization mass spectrometry (ESI/MS) to monitor BCM7 during GI digestion, and intact BCM7 through BBM digestion was quantified by reverse-phase (RP)-HPLC. We found that BCM7 was partly digested with human GI enzymes, as 3 proteolytic fragments in addition to f(60-66) YPFPGPI were detected: f(62-66) FPGPI, f(60-65) YPFPGP and f(61-66) PFPGPI. The RP-HPLC analysis revealed that 42% of the initial peptide was degraded after only 2 h of BBM digestion, and as much as 79% was degraded after 4-h digestion with supplementation of BBM. In conclusion, this study showed that most of BCM7 was degraded during GI and BBM digestion, although a small amount (5%) was still detected after 24-h digestion. It remains to be studied whether the small amount of intact BCM7 detected after in vitro digestion is transported via active transceptors in the human intestinal epithelial cells and enters blood circulation.

Feeding a concentrate rich in rapeseed oil improves fatty acid composition and flavor in Norwegian goat milk.

Impaired quality due to a high content of free fatty acids (FFA) and off-flavors has caused challenges in the development of Norwegian goat milk products. The present study aimed to examine the effect of lipid-supplemented concentrates on milk fat content, fatty acid composition, FFA, lipoprotein lipase activity, sensory properties, and size of milk fat globules of goat milk. Thirty goats assigned to 3 experimental groups were fed different concentrates from 60 d in milk (DIM) until late lactation (230 DIM). The diets were (1) control concentrate (no added fat); (2) control concentrate with 8% (added on air-dry basis) hydrogenated palm oil enriched with palmitic acid (POFA); and (3) control concentrate with 8% (added on air-dry basis) rapeseed oil (RSO). The POFA group produced milk with the highest fat content, and fat content was positively correlated with the mean size of milk fat globules. Goats in the RSO group had a higher content of long-chain and unsaturated fatty acids, whereas milk from goats in the POFA group had a higher content of palmitic and palmitoleic acids (C16:0 and C16:1 cis). The control group produced milk with a higher content of short-, medium-, odd-, and branched-chain fatty acids compared with the 2 other groups. The content of FFA in milk was low in early and late lactation and peaked in mid lactation (90 DIM). A high content of FFA was correlated with poor sensory properties (tart/rancid flavor). The RSO group produced milk with lower content of FFA and off-flavors in mid lactation and a higher proportion of unsaturated fatty acids. Therefore, replacement of palm oil with rapeseed oil as a lipid source in dairy goat feed would be favorable.

Grazing season and forage type influence goat milk composition and rennet coagulation properties.

Two different types of pasture (cultivated and rangeland) and 2 different hay qualities (high and low quality) were examined for their effects on goat milk composition and rennet coagulation properties. Furthermore, the effect of dietary treatments in both the early and late grazing season was studied. As lactation stage is known to influence milk composition, the goats in the early and late grazing season were in the same lactation stage at the start of the experiment. The milk composition was influenced both by dietary treatment and season. Milk from goats on pasture was superior to those on hay by containing a higher content of protein and casein, and the goats on cultivated pasture had the highest milk yield. Casein composition was significantly influenced by forage treatment. Goats grazing on cultivated pasture had higher contents of αs1-casein and also of κ-casein compared with the other treatments, whereas goats grazing on rangeland had the highest content of ß-casein. Factors such as milk yield, casein micelle size, αs2-casein, and calcium content were reduced in late compared with early season. More favorable rennet coagulation properties were achieved in milk from the early grazing season, with shorter firming time and higher curd firmness compared with milk from the late grazing season, but the firming time and curd firmness were not prominently influenced by forage treatment. The content of αs2-casein and calcium in the milk affected the firming time and the curd firmness positively. The influence of season and forage treatment on especially milk yield, casein content, and rennet coagulation properties is of economic importance for both the dairy industry and goat milk farmers.

Newly identified mutations at the CSN1S1 gene in Ethiopian goats affect casein content and coagulation properties of their milk.

Very high casein content and good coagulation properties previously observed in some Ethiopian goat breeds led to investigating the αs1-casein (CSN1S1) gene in these breeds. Selected regions of the CSN1S1 gene were sequenced in 115 goats from 5 breeds (2 indigenous: Arsi-Bale and Somali, 1 exotic: Boer, and 2 crossbreeds: Boer × Arsi-Bale and Boer × Somali). The DNA analysis resulted in 35 new mutations: 3 in exons, 3 in the 5' untranslated region (UTR), and 29 in the introns. The mutations in exons that resulted in an amino acid shift were then picked to evaluate their influence on individual casein content (αs1-, αs2-, ß-, and κ-CN), micellar size, and coagulation properties in the milk from the 5 goat breeds. A mutation at nucleotide 10657 (exon 10) involved a transversion: CAG→CCG, resulting in an amino acid exchange Gln77→Pro77. This mutation was associated with the indigenous breeds only. Two new mutations, at nucleotide 6072 (exon 4) and 12165 (exon 12), revealed synonymous transitions: GTC→GTT in Val15 and AGA→AGG in Arg100 of the mature protein. Transitions G→A and C→T at nucleotides 1374 and 1866, respectively, occurred in the 5' UTR, whereas the third mutation involved a transversion T→G at nucleotide location 1592. The goats were grouped into homozygote new (CC), homozygote reference (AA), and heterozygote (CA) based on the nucleotide that involved the transversion. The content of αs1-CN (15.32g/kg) in milk samples of goats homozygous (CC) for this newly identified mutation, Gln77→Pro77 was significantly higher than in milks of heterozygous (CA; 9.05g/kg) and reference (AA; 7.61g/kg) genotype animals. The αs2-, ß-, and κ-CN contents showed a similar pattern. Milk from goats with a homozygous new mutation had significantly lower micellar size. Milk from both homozygote and heterozygote new-mutation goats had significantly shorter coagulation rate and stronger gel than the reference genotype. Except the transversion, the sequence corresponded to allele A and presumably derived from it. Therefore, this allele is denoted by A3. All goats from the reference genotype (AA) were homozygous for the allele at nucleotide position 1374 and 1866, whereas all mutations in the 5' UTR existed in a heterozygous form in both heterozygous (CA) and the new mutation (CC) genotype. The newly identified mutation (CC) detected in some of the goat breeds is, therefore, important in selection for genetic improvement and high-quality milk for the emerging goat cheese-producing industries. The finding will also benefit farmers raising these goat breeds due to the increased selling price of goats. Further studies should investigate the effect of this amino acid exchange on the secondary and tertiary structure of the αs1-CN molecule and on the susceptibility of peptide hydrolysis by digestive enzymes.

Identification of lactoferrin peptides generated by digestion with human gastrointestinal enzymes.

Lactoferrin (LF) is a protein present in milk and other body fluids that plays important biological roles. As part of a diet, LF must survive gastrointestinal conditions or create bioactive fragments to exert its effects. The degradation of LF and formation of bioactive peptides is highly dependent on individual variation in intraluminal composition. The present study was designed to compare the degradation and peptide formation of bovine LF (bLF) following in vitro digestion under different simulated intraluminal conditions. Human gastrointestinal (GI) juices were used in a 2-step model digestion to mimic degradation in the stomach and duodenum. To account for variation in the buffering capacity of different lactoferrin-containing foods, gastric pH was adjusted either slowly or rapidly to 2.5 or 4.0. The results were compared with in vivo digestion of bLF performed in 2 volunteers. High concentration of GI juices and fast pH reduction to 2.5 resulted in complete degradation in the gastric step. More LF resisted gastric digestion when pH was slowly reduced to 2.5 or 4.0. Several peptides were identified; however, few matched with previously reported peptides from studies using nonhuman enzymes. Surprisingly, no bovine lactoferricin, f(17-41), was identified during in vitro or in vivo digestion under the intraluminal conditions used. The diversity of enzymes in human GI juices seems to affect the hydrolysis of bLF, generating different peptide fragments compared with those obtained when using only one or a few proteases of animal origin. Multiple sequence analysis of the identified peptides indicated a motif consisting of proline and neighboring hydrophobic residues that could restrict proteolytic processing. Further structure analysis showed that almost all proteolytic cutting sites were located on the surface and mainly on the nonglycosylated half of lactoferrin. Digestion of bLF by human enzymes may generate different peptides from those found when lactoferrin is digested by nonhuman enzymes. The degradation of LF in the GI tract should be taken into consideration when health effects are proposed, because LF has now been approved by the European Food Safety Authority as a dietary supplement in food products.

Importance of casein micelle size and milk composition for milk gelation.

The economic output of the dairy industry is to a great extent dependent on the processing of milk into other milk-based products such as cheese. The yield and quality of cheese are dependent on both the composition and technological properties of milk. The objective of this study was to evaluate the importance and effects of casein (CN) micelle size and milk composition on milk gelation characteristics in order to evaluate the possibilities for enhancing gelation properties through breeding. Milk was collected on 4 sampling occasions at the farm level in winter and summer from dairy cows with high genetic merit, classified as elite dairy cows, of the Swedish Red and Swedish Holstein breeds. Comparisons were made with milk from a Swedish Red herd, a Swedish Holstein herd, and a Swedish dairy processor. Properties of CN micelles, such as their native and rennet-induced CN micelle size and their zeta-potential, were analyzed by photon correlation spectroscopy, and rennet-induced gelation characteristics, including gel strength, gelation time, and frequency sweeps, were determined. Milk parameters of the protein, lipid, and carbohydrate profiles as well as minerals were used to obtain correlations with native CN micelle size and gelation characteristics. Milk pH and protein, CN, and lactose contents were found to affect milk gelation. Smaller native CN micelles were shown to form stronger gels when poorly coagulating milk was excluded from the correlation analysis. In addition, milk pH correlated positively, whereas Mg and K correlated negatively with native CN micellar size. The milk from the elite dairy cows was shown to have good gelation characteristics. Furthermore, genetic progress in relation to CN micelle size was found for these cows as a correlated response to selection for the Swedish breeding objective if optimizing for milk gelation characteristics. The results indicate that selection for smaller native CN micelles and lower milk pH through breeding would enhance gelation properties and may thus improve the initial step in the processing of cheese.