Casein micelles are key structures in milk, influencing stability, nutritional properties, and functionality. Their hierarchical architecture, which is dynamic and responsive to environmental conditions, plays a crucial role in dairy processing. Understanding the structural and dynamic properties of casein micelles is essential for optimizing dairy products and processing techniques. This study presents a novel method for characterizing and evaluating casein micelles using a combination of Asymmetrical Flow Field-Flow Fractionation (AF4) and SmallAngle X-ray Scattering (SAXS) at synchrotron facilities. By coupling AF4 with SAXS, we can fractionate milk samples according to micelle size and gain insights into their structural organization. However, the highthroughput data generated in such experiments pose challenges for traditional data analysis. We introduce an automated data processing pipeline utilizing the McSAS software in combination with Indirect Fourier Transformation, allowing for efficient fitting of SAXS data and extraction of structural parameters such as radius of gyration (Rg) and maximum particle dimension (Dmax). This integrated approach provides a more detailed understanding of the heterogeneity and structural dynamics of casein micelles, revealing distinct features of their size distribution, internal cavities, and overall micelle structure across different fractions. The method offers a powerful tool for future investigations into the behavior of casein micelles under varying environmental conditions, with potential applications in optimizing dairy product formulations and studying casein micelle dynamics.

Effects of various heating temperatures (124, 129, 134, 140 or 143°C) and holding times (1 and 4 s) on milk quality using direct and indirect heating systems at pilot scale were investigated. Vitamin degradation was observed for both direct and indirect heating and the degradation increased with increasing temperature and holding time, however, no difference could be seen between heating systems. Slight increase in pH and decrease in ionic calcium for direct heating occurred. An increase in fat globule size and aggregate formation were noted for direct heating with more pronounced effects with increasing temperature. Physical stability of milk was unaffected on day of production; however, after 28 days of storage instability was more apparent for indirect heating. No effect of heating system was observed on colour. This suggests that heating may influence milk quality, however, no clear differences between direct and indirect heating were observed for the quality parameters investigated.

Kinetic thermal degradation models are vital components for optimization of food and bioproducts processing. Typically, models are fitted to laboratory scale experiments where vials are heated and held. However, these conditions are highly dissimilar from the thermal processes experienced in industrial production. Whereas fitting kinetic data to industrial scale production is often impossible due to cost and flexibility issues, this discrepancy between what is typically measured and what the models are intended for could be of concern. This study presents a case study where traditional laboratory experiments are fitted to vitamin degradation kinetic models in milk (vitamins B1, B2, and E). The best fitted kinetic models are then validated using five, carefully controlled industrial scale dairy processes. Results show that predictions are surprisingly close to the validation data (mean relative percentual deviation is -1.1%–+3% depending on vitamin), given the substantial difference between the laboratory and production scale setup. This offers empirical support for the conventional method of fitting kinetic parameters through simplified laboratory experiments to predict vitamin degradation during industrial scale processing of foods and bioproducts.

Micronutrients and physical stability of milk and cream were evaluated for different heat treatments at dairy production scale (HTST pasteurisation, high pasteurisation, ESL indirect and UHT direct and indirect). Changes caused due to heat treatments for macro and micro components (vitamins and minerals) as well as physical stability were investigated. Results show limited losses in macro components and total mineral concentrations for milk and cream after the studied heat treatments. Significant decrease in concentrations of vitamins B1, B2 and B12 and ionic calcium in milk was observed after high pasteurisation, whereas limited losses were observed after ESL and UHT processing. Casein micelle size in milk increased, however, no effect on physical stability was observed between heat treatments. This work shows that heating technologies at industrial dairy production scale investigated were mild and have low effects on micronutrients and physical stability of milk and cream.

Impaired rennet coagulation properties in milk could lead to prolonged processing times and production losses. Heritability for milk coagulation has previously been estimated to be 0.28 to 0.45, indicating that genetic selection can be used to manipulate this trait. The CN proteins are expressed by the genes CSN1S1, , CSN2, , CSN1S2, , and CSN3 and are located on bovine chromosome 6. To better understand the effect of genetic variation in the CN genes on milk coagulation, blood and milk samples from 30 Swedish Red Dairy Cattle (RDC) with divergent coagulation properties were investigated. DNA from the 30 cows was sequenced for the CN genes to determine the theoretical AA sequence and to look for genetic variation in the untranslated regions. The aim is to confirm the protein genetic variants previously reported, while searching for additional genetic variation in the CN genes of 30 RDC. We observed genetic variation in 116 SNPs in the known CN genes where 10% of the SNPs are exon variants and the remaining 90% are intron variants. A total of 2.5% of the SNPs are found in the 5 '- '- or 3 '-untranslated '-untranslated region (UTR) regions of the exons; 2% are synonymous variants and 6% are missense variants that concurred with the known protein variants for CSN1S1, , CSN2, , and CSN3. . Furthermore, 6% of the SNPs are splice polypyrimidine tract intron variants. The 2 genetic variants in the 5 '- '- and 3 '-UTR '-UTR in CSN1S1 and CSN3 are found with protein variants CSN1S1C and CSN3B. . Because both UTR variants are associated with gain and loss of micro RNA and transcription factors, this could explain differences in expression of the genetic protein variants. Preliminary chi-squared analysis and comparison with previous GWAS studies showed potential connections between the identified SNPs and coagulation properties of milk. By advancing the knowledge of the connection between the DNA sequence and the functional properties of the CN proteins, we hope to learn more about the cheese coagulation properties of milk from RDC.

Changes in nutritional and technological properties during storage of milk and cream subjected to different heat treatments at dairy production scale (high-temperature-short-time pasteurisation, extended shelf-life treatment and ultra-high temperature treatment) were investigated. Results indicate a decrease in pH with longer storage times, whereas no changes in ionic calcium concentration occurred during shorter storage. Vitamin losses ranged from 1-22% for different heat treatments and more pronounced effects of vitamin degradation was observed with more intense heat treatment and longer storage times. Vitamin B12 concentration tended to decrease more over time than vitamins B1, B2 and E and more losses in vitamin concentration were found after storage than after heat treatment. Significant effects on physical stability and colour changes in milk and cream were observed during prolonged storage. This suggests that larger changes in nutritional and technological quality of heat-treated milk and cream at dairy production scale occur during longer storage times.

Individual milk samples were used to investigate how much of the variation in rennet gelation properties that can be explained by variation in milk composition by linear regression using the ordinary least squares method so as to optimise cheese milk. Natural variation in casein (CN) content was shown to have a large effect on gel strength but did not affect gel formation kinetics. Ionic calcium concentration was more important for initial gel formation whereas total calcium content is a better predictor for the developed gel structure. Of the individual milk proteins, κ-CN had the largest effect on gelation explaining up to 20% of the variation in gel forming kinetics and gel strength, while post-translational modifications of κ-CN did not explain more variation than total κ-CN. However, the relative concentration of glycosylated κ-CN seems to be more important for gel-build-up kinetics and un-glycosylated κ-CN for the gel-structure.

In the present study, digestion pattern of purified bovine κ-casein (κ-CN) variants A, B, E as well as desialylated variant B, using INFOGEST 2.0 in vitro gastrointestinal digestion were investigated using peptidomics. Peptide profiles of the digests were identified and quantified using ion abundancies by liquid chromatography electrospray quadropole time of flight mass spectrometry (LC-ESI/Q-TOF MS/MS). Results showed that the κ-CN variants A and E had comparable digestion patterns at most digestion time points. In the in vitro gastric and in the initial intestinal phases fewer peptides and with lower total abundances were identified for variant B compared to variants A and E, indicating a slower digestion rate for κ-CN B. By desialylation, the digestion rate of desialylated variant B in both gastric and initial intestinal phases increased compared to the natural sialylated counterpart. Bioinformatics search revealed nine potential bioactive peptides released from all three variants A, B and E by the in vitro intestinal digestion, with four additional potential bioactive peptides being released after desialylation of κ-CN B.

Nutritional and technological properties of milk and cream were evaluated for different heat treatments at dairy production scale (HTST pasteurisation, high pasteurisation, ESL indirect and UHT direct and indirect). Changes caused due to heat treatments for macro and micro components (vitamins and minerals) of the nutritional attributes as well as technological properties were investigated. Results show limited losses in macro components and total mineral concentrations for milk and cream after the studied heat treatments. Significant decrease in concentrations of vitamins B1, B2 and B12 and ionic calcium in milk was observed after high pasteurisation, whereas limited losses were observed after ESL and UHT processing. Casein micelle size in milk increased, however, no effect on physical stability was observed between heat treatments. This suggests that heating technologies at dairy production scale today are mild and have low effects on nutritional and technological properties of milk and cream.

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.

The ability of milk to coagulate is crucial for cheese production. Poorly or non-coagulating milk will affect industrial cheese production with lower cheese yield and longer processing time. We have previously shown that up to 19% of milk from Swedish Red Dairy Cattle is poorly coagulating and 18% is noncoagulating. We have also identified specific milk proteins and genetic variants as potential biomarkers for selective breeding. However, these variants are not fully characterized and show conflicting results between presumed genetic variants and actual protein masses. We re-sequenced the casein genes in 30 cows with varying milk coagulation properties and detailed analyses of their milk proteins. The analyses of three genes confirmed existing variation in the casein gene, and identified 77 single nucleotide polymorphisms in introns as well as at least ten insertions or deletions. No new coding variants were detected so far.

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.

Summary During whey powder production, the feed is subjected to several heat treatments which can cause lactosylation of proteins. In this study, lactosylation of whey proteins was evaluated in spray‐dried powders before and after storage by varying the native protein fraction as well as the serum protein/lactose ratio in the powders. The lactosylation of native α‐lactalbumin and β‐lactoglobulin in the powders before storage was not affected to a large extent by the protein denaturation or if the feed had been heat treated in a high or low lactose environment. After storage (relative humidity of 23.5%, 30 °C, 25 days), the kinetic of lactosylation tended to increase with increasing native protein fraction and bulk protein content in the powders. An explanation could be that proteins dissolved in the lactose glassy structure might have a lower reactivity, while proteins present in the protein glassy structure with dissolved lactose may display higher lactosylation reactivity.

To keep their functional properties, it is crucial that protein aggregates maintain their structure after spray drying and that the powders can be fully rehydrated. In this study, microgels and fractal aggregates were prepared by heating a mixture of milk serum protein concentrate and lactose (40/60; %, w/w) at 85 degrees C for 15 min by varying the pH. Various fractions of native proteins were added to the systems prior to spray drying. This study showed that microgels and fractal aggregates kept their structure after spray drying and reconstitution. The particle morphology could be correlated to the stiffness of the interface of the feed droplet. The forced imbibition rate showed a negative correlation with increasing amount of aggregated proteins in the powders that seems to be a result of denatured/aggregated proteins present at the surface. These findings are of importance for the formulation of spray-dried powders with improved rehydration characteristics. (C) 2020 The Author(s). Published by Elsevier Ltd.

Experimentally determined vitamin degradation kinetic parameters are vital input for process optimization, e.g., of nutrient rich beverages such as milk and fruit juices. The standard method of obtaining these parameters is to subject a test-tube of the nutrient (in its food matrix) to a water bath at elevated temperatures and measure remaining concentration as a function of time. It is well known that accurate kinetic fitting requires measuring the temperature inside the test tube as a function of time (due to lag it is not identical to the water-bath temperature), and this is included in contemporary studies. However, the importance of axial temperature gradients for valid kinetic parameter estimations has not been discussed to the same extent. This contribution combines experiments and physical modelling to show how an axial temperature gradient evolves in a test tube (R = 13 mm, Z = 75 mm), due to free convection currents (T-infinity - T-0 = 57-71 degrees C). Consequently, the test tube temperature is not uniform in such an experiment. Thus, the positioning of the thermocouple in a test-tube-in-water bath experiment is essential for ensuring a valid estimation of the kinetic parameters. Simulations suggest that positioning it half-way between top and bottom is a suitable best practice. (C) 2021 The Author(s). Published by Elsevier B.V. on behalf of Institution of Chemical Engineers.

Milk oligosaccharides are of high interest due to their bioactive properties. This study is the first to characterise milk oligosaccharides from native North European cattle breeds, as represented by 80 milk samples collected from eight native breeds originated from Norway (Norwegian Doela cattle and Norwegian Telemark cattle), Sweden (Swedish Mountain cattle), Denmark (Danish Red anno 1970), Iceland (Icelandic cattle), Lithuania (native Lithuanian Black and White) and Finland (Western Finncattle and Eastern Finncattle). Using high-performance liquid-chromatography chip/quadrupole time-of-flight mass-spectrometry, 18 unique monosaccharide compositions and a multitude of isomers were identified. No N-glycolylneuraminic acid was identified among these breeds. Western Finncattle milk was most abundant in neutral, acidic and fucosylated oligosaccharides. Further, Eastern Finncattle milk was significantly higher in acidic oligosaccharides and Icelandic cattle milk significantly higher in fucosylated oligosaccharides, compared to the mean. This study highlights specific native breeds of particular interest for future exploitation of milk oligosaccharides and breeding strategies.

Little is known about the extent of variation and activity of naturally occurring milk glycosidases and their potential to degrade milk glycans. A multi-omics approach was used to investigate the relationship between glycosidases and important bioactive compounds such as free oligosaccharides and O-linked glycans in bovine milk. Using 4-methylumbelliferone (4-MU) assays activities of eight indigenous glycosidases were determined, and by mass spectrometry and 1H NMR spectroscopy various substrates and metabolite products were quantified in a subset of milk samples from eight native North European cattle breeds. The results showed a clear variation in glycosidase activities among the native breeds. Interestingly, negative correlations between some glycosidases including β-galactosidase, N-acetyl-β-d-glucosaminidase, certain oligosaccharide isomers as well as O-linked glycans of κ-casein were revealed. Further, a positive correlation was found for free fucose content and α-fucosidase activity (r = 0.37, p-value < 0.001) indicating cleavage of fucosylated glycans in milk at room temperature. The results obtained suggest that milk glycosidases might partially degrade valuable glycans, which would result in lower recovery of glycans and thus represent a loss for the dairy ingredients industry if these activities are pronounced.

Milk that does not coagulate after rennet addition, also called noncoagulating (NC) milk, is unwanted in cheese production due to prolonged processing time. Amounts of whey and casein proteins, genetic variants, as well as posttranslational modifications (PTM) of proteins are all contributing factors in rennet-induced coagulation of milk. In this study, we conducted a wide-ranging investigation of milk proteins in milk samples from 616 Swedish Red dairy cattle using liquid chromatography-high resolution mass spectrometry. Relative concentration of proteins, genetic variants, and PTM were compared between NC milk and coagulating milk. The PTM investigated were phosphorylation of caseins and glycosylation of κ-casein. Several genetic variants and PTM were found, including rare phosphorylation variants of the αS-caseins. Genetic variants were found to effect the expressed amount of different proteins. Further, the effect of protein amounts and PTM on a binary NC milk trait was modeled using a generalized linear model. The model showed that NC milk significantly correlated with higher relative concentrations of α-lactalbumin and β-casein and lower relative concentrations of β-lactoglobulin and κ-casein. Regarding PTM of caseins, an effect on NC milk from a lower relative concentration of αS1-casein with 8 phosphate groups were found, even though an effect from total relative concentration of αS1-casein was not found. This study has provided insights into protein variants and PTM important for NC milk to improve this undesirable property.

Non-coagulating milk is unwanted in cheese production because it impairs chymosin-induced coagulation properties in bulk milk. Milk samples were analysed to investigate the coagulation process in non-coagulating milk for comparison with milk samples that have a high coagulation ability. This was done by measuring two peptides after chymosin cleavage, namely para-κ-casein and caseinomacropeptide, in all the milk samples using capillary electrophoresis. The results showed that cleavage of κ-casein into para-κ-casein and caseinomacropeptide occurred in both coagulation groups, suggesting that the aggregation stage is the cause of non-coagulating milk. Non-coagulating milk had lower content of para-κ-casein after chymosin addition compared with coagulating milk, which might be the result of a lower content of available κ-casein. Furthermore, this study indicates that there are different mechanisms that explain good coagulation ability and lack of coagulation ability of milk. The results can be useful in understanding the potential chemical mechanisms behind non-coagulating milk.

The rennet-induced coagulation ability of milk is important in cheese production. For Swedish Red Dairy Cattle (RDC), this ability is reduced because of a high prevalence of noncoagulating (NC) milk. In this study, we simultaneously combined genetic parameters for NC milk, milk coagulation properties, milk composition, physical traits, and milk protein composition. Our aim was to estimate heritability and genetic and phenotypic correlations for NC milk and 24 traits (milk coagulation properties, milk composition, physical traits, and milk protein composition). Phenotypes and ∼7,000 SNP genotypes were available for all 600 Swedish RDC. The genotypes were imputed from ∼7,000 SNP to 50,000 SNP. Variance components and genetic parameters were estimated with an animal model. In Swedish RDC, a moderate heritability estimate of 0.28 was found for NC milk. For the other 24 traits, heritability estimates ranged from 0.12 to 0.77 (standard errors from 0.08 to 0.18). A total of 300 phenotypic and genetic correlations were estimated. For phenotypic and genetic correlations, 172 and 95 were significant, respectively. In general, most traits showing significant genetic correlations also showed significant phenotypic correlations. In this study, phenotypic and genetic correlations with NC milk suggest that many correlations between traits exist, making it difficult to predict the real consequences on the composition of milk, if selective breeding is applied on NC milk. We speculate that some of these consequences may lead to changes in the composition of milk, most likely affecting its physical and organoleptic properties. However, our results suggest that κ-casein could be used as an indicator trait to predict the occurrence of NC milk at the herd level.