Lotte Bach Larsen

Bovine milk procathepsin D and cathepsin D: coagulation and milk protein degradation.

Cathepsin D is an indigenous aspartic proteinase in bovine milk. By competitive enzyme-linked immunosorbent assay the amount of immunoreactive cathepsin D and procathepsin D in bovine skim milk was estimated to be 0.4 microgram/ml. Immunoreactive cathepsin D purified from whey consisted of a small fraction of mature cathepsin D, but the major form was the proenzyme procathepsin D. A preparation of bovine milk procathepsin D was, like mature cathepsin D, able to degrade purified alpha s1-, alpha s2-, beta- and kappa-casein and alpha-lactalbumin, while beta-lactoglobulin was resistant to cleavage. The cleavage sites in these proteins were determined and compared with those of chymosin. Cathepsin D was capable of generating the alpha s1-I, beta-I, beta-II and beta-III fragments originally described from the action of chymosin on the respective caseins, and these fragments were subjected to further proteolysis. Cathepsin D was also able to liberate the caseinomacropeptide from purified kappa-casein, and to coagulate bovine skim milk. This demonstrated that milk contains an indigenous coagulation enzyme present mainly in the whey fraction.

The milk acid proteinase cathepsin D: a review

Cathepsin D is a lysosomal aspartic proteinase with a low pH optimum, found in many tissues and in bovine milk. It is synthesised as its inactive zymogen, procathepsin D. Procathepsin D can convert itself, by an autoproteolytic pathway, into an active intermediate, pseudocathepsin D, while other proteases are involved in the processing to mature cathepsin D. Cathepsin D is known to participate in a range of physiological processes. Pro-, pseudo- and mature cathepsin D have been purified from bovine milk, but procathepsin D is the major form present in milk. Cathepsin D activity in milk appears correlated with somatic cell count. The active forms of the enzyme readily hydrolyse αS1-, αS2- and β-caseins, in the case of αS1-casein and β-casein with a specificity similar to that of chymosin. Cathepsin D can also produce para-κ-casein from κ-casein and, at high concentrations, can coagulate milk. Cathepsin D appears to be able to at least partially survive commercial pasteurisation processes. In recent years, increasing evidence has been documented indicating a role for this enzyme in proteolysis in cheese during ripening, most clearly in cheese where rennet activity is low, such as Swiss cheese, Quarg and Feta. Further research is required to evaluate the significance of this enzyme in more detail.

Isolation and characterization of plasminogen and plasmin from bovine milk

Abstract A procedure was developed for the isolation of plasminogen and plasmin from bovine milk. Pure undegraded plasminogen with N-terminal amino acid sequence and mobility in SDS-PAGE similar to plasminogen isolated from bovine blood was obtained. Additionally, the preparation contained the proteolytically modified midi-plasmin, consisting of kringle 4,5 and the light chain with an apparent molecular weight of 50 k Da in unreduced SDS-PAGE, and resulting in two bands of 30 and 26 k Da after reduction. Some 15% of the amino acid sequence of plasminogen/midi-plasmin isolated from milk was determined. The partial amino acid sequence was identical to that previously reported for plasminogen isolated from bovine blood (J. Schaller et al., (1985). Eur. J. Biochem., 149, 267–278), and to the bovine liver plasminogen cDNA sequence (L. Berglund et al., (1995). Int. Dairy J., 5, 593–603). Enzyme-linked immunosorbent assay (ELISA) and Western blotting experiments revealed that immunoreactive plasminogen was associated with acid-precipitated casein, rennet-coagulated casein and casein micelles. Some was found in acid whey and to a lesser extent in rennet whey. The amount of plasminogen associated with the milk fat globule membrane was very low and reflected the presence of casein. By Western blotting, immunoreactive plasminogen was found in zones with apparent molecular weights of 85, 80 and 50 k Da in unreduced gels. These hands may represent two plasminogen forms (85 and 80 k Da) and midi-plasmin/midi-plasminogen (50 k Da). The total concentration of plasminogen in bovine milk was estimated to 1.5 μg/mL.

Procathepsin D cannot autoactivate to cathepsin D at acid pH

The amino acid sequence of the propart of bovine procathepsin D was determined at the protein level. Incubation of the isolated procathepsin D at pH 3.5–5.0 for 30–120 min leads to a 2 kDa reduction in its molecular mass, as seen by SDS‐PAGE. The activation product is pseudocathepsin D and is the result of a proteolytic cleavage between LeuP26 and IleP27 in the propart. Incubation at pH 5.0 for 20 h of either procathepsin D or pseudocathepsin D results in both cases in approximately equal amounts of pseudocathepsin D and a further processed intermediate, nine amino acids shorter than pseudocathepsin D. No reaction products corresponding to cathepsin D with a mature amino terminus were observed, showing that autoproteolysis alone cannot generate the mature form found in the lysosomes.

NMR-Based Milk Metabolomics

Milk is a key component in infant nutrition worldwide and, in the Western parts of the world, also in adult nutrition. Milk of bovine origin is both consumed fresh and processed into a variety of dairy products including cheese, fermented milk products, and infant formula. The nutritional quality and processing capabilities of bovine milk is closely associated to milk composition. Metabolomics is ideal in the study of the low-molecular-weight compounds in milk, and this review focuses on the recent nuclear magnetic resonance (NMR)-based metabolomics trends in milk research, including applications linking the milk metabolite profiling with nutritional aspects, and applications which aim to link the milk metabolite profile to various technological qualities of milk. The metabolite profiling studies encompass the identification of novel metabolites, which potentially can be used as biomarkers or as bioactive compounds. Furthermore, metabolomics applications elucidating how the differential regulated genes affects milk composition are also reported. This review will highlight the recent advances in NMR-based metabolomics on milk, as well as give a brief summary of when NMR spectroscopy can be useful for gaining a better understanding of how milk composition is linked to nutritional or quality traits.

Relationship between the metabolite profile and technological properties of bovine milk from two dairy breeds elucidated by NMR-based metabolomics.

The aim of the present study was to investigate the relationship between the metabolite profile of milk and important technological properties by using nuclear magnetic resonance (NMR)-based metabolomics. The metabolomics approach was introduced for the metabolic profiling of a set of milk samples from two dairy breeds representing a wide span in coagulation properties. The milk metabolite profiles obtained by proton and carbon NMR spectroscopy could be correlated to breed and, more interestingly, also with the coagulation profile, as established by traditional methods by using principal component analysis (PCA). The metabolites responsible for the separation into breed could mainly be ascribed to carnitine and lactose, whereas the metabolites varying in the samples with respect to coagulation properties included citrate, choline, carnitine, and lactose. The results found in the present study demonstrated a promising potential of NMR-based metabolomics for a rapid analysis and classification of milk samples, both of which are useful for the dairy industry.

The occurrence of noncoagulating milk and the association of bovine milk coagulation properties with genetic variants of the caseins in 3 Scandinavian dairy breeds

Substantial variation in milk coagulation properties has been observed among dairy cows. Consequently, raw milk from individual cows and breeds exhibits distinct coagulation capacities that potentially affect the technological properties and milk processing into cheese. This variation is largely influenced by protein composition, which is in turn affected by underlying genetic polymorphisms in the major milk proteins. In this study, we conducted a large screening on 3 major Scandinavian breeds to resolve the variation in milk coagulation traits and the frequency of milk with impaired coagulation properties (noncoagulation). In total, individual coagulation properties were measured on morning milk collected from 1,299 Danish Holstein (DH), Danish Jersey (DJ), and Swedish Red (SR) cows. The 3 breeds demonstrated notable interbreed differences in coagulation properties, with DJ cows exhibiting superior coagulation compared with the other 2 breeds. In addition, milk samples from 2% of DH and 16% of SR cows were classified as noncoagulating. Furthermore, the cows were genotyped for major genetic variants in the αS1- (CSN1S1), β- (CSN2), and κ-casein (CSN3) genes, revealing distinct differences in variant frequencies among breeds. Allele I of CSN2, which had not formerly been screened in such a high number of cows in these Scandinavian breeds, showed a frequency around 7% in DH and DJ, but was not detected in SR. Genetic polymorphisms were significantly associated with curd firming rate and rennet coagulation time. Thus, CSN1S1 C, CSN2 B, and CSN3 B positively affected milk coagulation, whereas CSN2 A(2), in particular, had a negative effect. In addition to the influence of individual casein genes, the effects of CSN1S1-CSN2-CSN3 composite genotypes were also examined, and revealed strong associations in all breeds, which more or less reflected the single gene results. Overall, milk coagulation is under the influence of additive genetic variation. Optimal milk for future cheese production can be ensured by monitoring the frequency of unfavorable variants and thus preventing an increase in the number of cows producing milk with impaired coagulation. Selective breeding for variants associated with superior milk coagulation can potentially increase raw milk quality and cheese yield in all 3 Scandinavian breeds.

Milk protein genetic variants and isoforms identified in bovine milk representing extremes in coagulation properties

A gel-based proteomic approach consisting of 2-dimensional gel electrophoresis coupled with mass spectrometry was applied for detailed protein characterization of a subset of individual milk samples with extreme rennet coagulation properties. A milk subset with either good or poor coagulation abilities was selected from 892 Danish Holstein-Friesian and Jersey cows. Screening of genetic variants of the major milk proteins resulted in the identification of common genetic variants of β-casein (CN; A(1), A(2), B), κ-CN (A, B), and β-lactoglobulin (LG; A, B), as well as a low frequency variant, κ-CN variant E, and variants not previously reported in Danish breeds (i.e., β-CN variant I and β-LG variant C). Clear differences in the frequencies of the identified genetic variants were evident between breeds and, to some extent, between coagulation groups within breeds, indicating that an underlying genetic variation of the major milk proteins affects the overall milk coagulation ability. In milk with good coagulation ability, a high prevalence of the B variants of all 3 analyzed proteins were identified, whereas poorly coagulating milk was associated with the β-CN variant A(2), κ-CN variant A or E, and β-LG variant A or C. The β-CN variant I was identified in milk with both good and poor coagulation ability, a variant that has not usually been discriminated from β-CN variant A(2) in other studied cow populations. Additionally, a detailed characterization of κ-CN isoforms was conducted. Six κ-CN isoforms varying in phosphorylation and glycosylation levels from each of the genetic variants of κ-CN were separated and identified, along with an unmodified κ-CN form at low abundance. Relative quantification showed that around 95% of total κ-CN was phosphorylated with 1 or 2 phosphates attached, whereas approximately 35% of the identified κ-CN was glycosylated with 1 to 3 tetrasaccharides. Comparing isoforms from individual samples, we found a very consistent κ-CN isoform pattern, with only minor differences in relation to breed, κ-CN genetic variant, and milk coagulation ability.

Nuclear magnetic resonance metabonomics reveals strong association between milk metabolites and somatic cell count in bovine milk

Somatic cell count (SCC) is associated with changes in milk composition, including changes in proteins, lipids, and milk metabolites. Somatic cell count is normally used as an indicator of mastitis infection. The compositional changes in protein and fat affect milk coagulation properties, and also the metabolite composition is thought to contribute to differential milk properties. Milk somatic cells comprise different cell types, which may contribute to differential milk metabolite fingerprints. In this study, milk from a relatively large number of individual cows, representing significant differences in SCC, were analyzed by nuclear magnetic resonance (NMR)-based metabonomics, and the milk metabolite profiles were analyzed for differences related to SCC. Global principal component analysis performed on 876 samples from 2 Danish dairy breeds and orthogonal projection of latent structures discriminant analysis performed on a smaller subset (n=70) representing high (SCC >7.2×10(5) cells/mL) and low (SCC <1.4×10(4) cells/mL) milk SCC identified latent variables, which could be attributed to milk with elevated SCC. In addition, partial least squares regression between the NMR milk metabolite profiles and SCC revealed a strong correlation. The orthogonal projection of latent structures discriminant analysis and partial least squares regressions pinpointed specific NMR spectral regions and thereby identification of milk metabolites that differed according to SCC. Relative quantification of the identified metabolites revealed that lactate, butyrate, isoleucine, acetate, and β-hydroxybutyrate were increased, whereas hippurate and fumarate were decreased in milk with high levels of somatic cells.

The influence of feed and herd on fatty acid composition in 3 dairy breeds (Danish Holstein, Danish Jersey, and Swedish Red)

The composition of milk fat from dairy cows is related to both genetic and environmental factors. Here, the effect of feed and herd was examined in 3 Scandinavian breeds, namely Danish Holstein-Friesian (DH), Danish Jersey (DJ), and Swedish Red (SR). In total, milk samples from 1,298 cows kept in indoor housing systems were collected from 61 conventional dairy herds in Denmark and Sweden. The fatty acid (FA) composition of milk was determined by gas chromatography and the content of α-tocopherol by HPLC. Based on the 17 individual FA determined, distinct FA profiles were observed for all breeds using univariate and multivariate statistics. The DJ cows were characterized by higher levels of saturated short-chain FA; in contrast, DH cows had higher content of unsaturated C18 FA, whereas higher levels of primarily C14:0, C14:1, C18:1 cis-9, and C18:3n-3 were evident in SR cows. This variation in milk fat composition across breeds was further reflected in different desaturase indices, which were generally higher in SR cows. In addition, α-tocopherol differed significantly among breeds, with DJ cows having the highest content. Herd-specific feeding plans were collected, and different feed items were separated into 4 broad feed categories, including grass products, maize silage, grain, and concentrate. The pronounced differences in overall feed composition among breeds were, to a large extent, due to regional differences between countries, with SR receiving higher levels of grain and grass silage compared with the Danish breeds. Within breeds, differences in feeding regimens among herds were furthermore higher in SR. Significant correlations between feed category and individual FA were observed in all breeds. Furthermore, variance components were estimated and used to determine the proportion of phenotypic variation that could be explained by herd. The herd effect for individual FA was generally lower for DH compared with the 2 other breeds. In addition, very low herd effects were shown for C14:1 and C16:1 in all breeds, suggesting that the content of these FA is mainly genetically regulated.