Factors influencing casein micelle size in milk of individual cows: Genetic variants and glycosylation of κ-casein

The average casein micelle size varies widely between milk samples of individual cows. The factors that cause this variation in size are not known but could provide more insight into casein micelle structure and into the physiology of casein micelle formation. The objective of this research was therefore to determine factors that influence average casein micelle size in milk from individual cows. Average casein micelle size of milk samples was associated with the A and B genetic variants of κ-casein, and differences in concentration of glycosylated κ-casein as a fraction of total milk protein. Milk samples with a low average casein micelle size were associated with the B variant of κ-casein and a higher relative concentration of glycosylated κ-casein, compared with milk samples with a high average casein micelle size. Differences observed may be attributed to the effect of glycosylated κ-casein groups on casein micelle formation in the mammary gland.     

Effect of κ-casein B relative content in bulk milk κ-casein on Montasio, Asiago, and Caciotta cheese yield using milk of similar protein composition

The aim of this study was to investigate the effect exerted by the relative content of κ-casein (κ-CN) B in bulk milk κ-CN on coagulation properties and cheese yield of 3 Italian cheese varieties (Montasio, Asiago, and Caciotta). Twenty-four cheese-making experiments were carried out in 2 industrial and 1 small-scale dairy plant. Detailed protein composition of bulk milk of 380 herds providing milk to these dairies was analyzed by reversed-phase HPLC. To obtain 2 experimental milks differing in the relative content of κ-CN B in κ-CN, herds were selected on the basis of bulk milk protein composition and relative content of κ-CN genetic variants. Milk was collected and processed separately for the 2 groups of selected herds. A difference of 20% in the relative content of κ-CN B in κ-CN was obtained for the 2 experimental milks for Montasio and a difference of 15% for Asiago and Caciotta. The 2 experimental milks were of similar protein and CN content, casein number, pH, CN composition, and β-CN genetic composition. For each cheese-making trial, amounts of milk, ranging from 2,000 to 6,000 kg, were manufactured. Each vat contained milk collected at least from 4 dairy herds. Cheese yield after brining and at the end of the aging was recorded. Milk with a greater proportion of κ-CN B in κ-CN (HIGHB) exhibited similar coagulation properties and greater cheese yield compared with milk with a lower proportion of κ-CN B in κ-CN (LOWB). The increased cheese yield observed for HIGHB when manufacturing Montasio cheese was ascribed to a greater fat content compared with LOWB. The probability of HIGHB giving a cheese yield 5% greater than that of LOWB ranged from 51 to 67% for Montasio cheese, but was less than 21% for Asiago and Caciotta cheeses. Variation in relative content of κ-CN B in κ-CN content did not relevantly affect industrial cheese yield when milks of similar CN composition were processed. An indirect effect due to the increased κ-CN content of κ-CN B milk is thought to explain the favorable effects of κ-CN B on cheese yield reported in the literature.     

Effects of diet on casein and fatty acid profiles of milk from goats differing in genotype for αS1-casein synthesis

This study investigated the interactions between nutrition and the genotype at α_S1-CN loci (CSN1S1) in goats, evaluating the impact of fresh forage-based diets and an energy supplement on the casein and fatty acid (FA) profiles of milk from Girgentana goats. Twelve goats were selected for having the same genotype at the α_S2-CN, β-CN, and κ-CN loci and differing in the CSN1S1 genotype: homozygous for strong alleles (AA) or heterozygous for strong and weak alleles (AF). Goats of each genotype were divided into three groups and, according to a 3 × 3 Latin square design, fed ad libitum three diets: sulla fresh forage (SFF), SFF plus 800 g/day of barley (SFB), and mixed hay plus 800 g/day of barley (MHB). The SFB diet led to higher-energy intake and milk yield. The energy-supplemented diets (SFB, MHB) reduced milk fat and urea and increased coagulation time. The fresh forage diets (SFF, SFB) increased dry matter (DM) and crude protein (CP) intake and milk β-CN. Diet had a more pronounced effect than CSN1S1 genotype on milk FA profile, which was healthier from goats fed the SFF diet, due to the higher content of rumenic acid, polyunsaturated, and omega-3 FAs. The AA milk had longer coagulation time and higher curd firmness, higher short- and medium-chain FAs (SMFA), and lower oleic acid than AF milk. Significant diet by genotype interactions indicated the higher milk yield of AA goats than AF goats with the higher-energy SFB diet and the lower synthesis of SMFA in AF than in AA goats with the SFF diet.     

Simple membrane filtration method for estimating numbers of Paenibacillus spp. spores in raw milk,using β-galactosidase activity as a selection criterion

Paenibacillus spp. are spore-forming bacteria that adversely affect the quality of dairy products. There is currently no appropriate method for enumerating Paenibacillus spp. spores. We developed a simple membrane filtration method to enumerate Paenibacillus spp. spores in raw milk, using β-galactosidase activity as a selection criterion. Although Paenibacillus spp. spores are relatively small, use of a membrane filter with 0.65-μm pore size allowed us to easily filter raw milk with sufficient recovery. The membrane was put on plates containing X-gal, and detection of β-galactosidase-positive colonies enabled selective enumeration of Paenibacillus spp. spores. We investigated Paenibacillus spp. spore levels in raw milk from six different areas in the Tokachi region, Hokkaido, Japan over 1 year. There were ≤10 spores 100 mL⁻¹ throughout the year, with no significant differences between areas or seasons. Paenibacillus amylolyticus and Paenibacillus odorifer were the predominant species, accounting for 50.6% and 27.4% of the total spores, respectively.     

Size of native and heated casein micelles,content of protein and minerals in milk from Norwegian Red Cattle—effect of milk protein polymorphism and different feeding regimes

Milk samples of 59 cows of the Norwegian Red Cattle breed receiving three different supplementary concentrates, were analysed for genotypes of caseins and whey proteins, the content of different milk salts (Ca²⁺, Ca, Mg and citrate), the content of total protein, casein and whey protein and the mean micellar size of native and heated casein micelles. The genotype of α_s1-casein had a statistically significant effect on the content of protein and casein, and the content of whey protein and the casein number were significantly influenced by different feeding regimes, and the content of citrate. The mean size of native and heated casein micelles was significantly influenced by the feeding regimes, genotype of α_s1-casein (native mean size only) and κ-casein, pH and the content of casein, whey protein and casein number. The heat-induced changes in mean micellar size were significantly affected by the calcium ion activity which accounted for approximately 40% of the total variation.     

Double-antibody based immunoassay for the detection of β-casein in bovine milk samples

The concentration of casein (CN) is one of the most important parameters for measuring the quality of bovine milk. Traditional approach to CN concentration determination is Kjeldahl, which is an indirect method for determination of total nitrogen content. Here, we described a double-antibody based direct immunoassay for the detection of β-CN in bovine milk samples. Monoclonal antibody (McAb) was used as capture antibody and polyclonal antibody (PcAb) labelled with horseradish peroxidase (HRP) as detection antibody. With the direct immunoassay format, the linear range of the detection was 0.1–10.0 μg mL⁻¹. The detection limit was 0.04 μg mL⁻¹. In addition, the concentration of β-CN in real bovine milk samples has been detected by the developed immunoassay. There was a good correlation between the results obtained by the developed technique and Kjeldahl method from commercial samples. Compared to the traditional approach, the advantage of the assay is no need of time-consuming sample pretreatment.     

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.     

Apparent voluminosity of casein micelles in the temperature range 35–70 °C

Casein micelles are basic units of dairy matrices that can be aggregated by reducing their electrostatic repulsion or by surface modifications. Studying casein micelles in terms of a colloidal suspension demands of a preferably complete physico-chemical characterisation. Process control in dairy technology as well as mechanistic models that explain the formation and structure of dairy products build on the specific hydration of the casein micelles. The effect of the temperature on the voluminosity of native casein micelles at pH 6.6–6.7 was investigated using rheometry covering a wide range of temperatures and mass fractions. Increasing temperature resulted in a constant decrease of the voluminosity up to 70 °C. A quadratic polynomial was tested to fit the temperature dependency the best. The apparent voluminosity of casein micelle suspensions was determined with 4.0 mL g⁻¹ at 35 °C, 3.6 mL g⁻¹ at 50 °C, and 3.5 mL g⁻¹ at 70 °C.     

Species identification of ruminant milk by genotyping of the κ-casein gene

The development of molecular genetic and bioinformatic systems for identifying the species of milk and the raw material composition of dairy products is of great scientific and practical importance with the purpose of introducing developments in the system for controlling the turnover of falsified products. The aim of the research is to develop a method of PCR-RFLP analysis for species identification of milk and dairy products from agricultural ruminant animals by the κ-casein gene (CSN3) with the possibility of qualitative and relative quantitative assessment of species-specific DNA of the tested biomaterial. The objects of research were samples of raw milk and milk powder, pasteurized cream, and hard and semi-hard cheeses. The developed method of species identification of milk and dairy products includes sample preparation of the studied samples, nucleic acid extraction, combined PCR-RFLP technique, detection of obtained results by the method of horizontal electrophoresis in agarose gel and their analysis, including using the developed mathematical algorithms and software. The synergistic effect established in combined operation of 2 restriction enzymes ensured their application in a mix with increased performance in an ergonomic way in the context of DNA authentication of cow, goat, and sheep milk and dairy products based on them. The specificity and sensitivity of the proposed method is potentially suitable for implementing the development of a system to control the turnover of falsified and counterfeit goods.     

Measurement of casein in milk by Kjeldahl and sodium dodecyl sulfate–polyacrylamide gel electrophoresis

Our objectives were to determine if milk casein as a percentage of true protein (CN%TP) estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) is equivalent to CN%TP estimated by Kjeldahl, and to determine the proportion of casein (CN), casein proteolysis products (CNPP), and serum protein (SP) from milk true protein (TP) that goes into the Kjeldahl noncasein nitrogen (NCN) filtrate and the proportion that stays in the NCN precipitate using SDS-PAGE. Raw milk samples were collected from 16 mid-lactation Holstein cows twice a week for 2 wk. These milks were analyzed for Kjeldahl total nitrogen, nonprotein nitrogen, and NCN content in duplicate, and by SDS-PAGE. The CN%TP determined by Kjeldahl was compared with the CN%TP estimated by SDS-PAGE calculated in 2 ways: as a percentage of only intact caseins divided by TP and as a percentage of both intact caseins and CNPP divided by TP. Three milks varying in fat, lactose, TP, CN, and SP content were formulated. These milks were analyzed in duplicate for Kjeldahl total nitrogen, nonprotein nitrogen, and NCN content, and each of the NCN filtrate and NCN precipitate were analyzed in duplicate by SDS-PAGE for relative quantity (%) of CN, CNPP, and SP. We found that the estimate of CN%TP by Kjeldahl was higher than the estimate of CN%TP by SDS-PAGE that was calculated as only intact CN divided by the total of all protein bands. However, no difference was detected in the estimate of CN%TP by Kjeldahl compared with CN%TP by SDS-PAGE when CNPP were included as CN in the calculation of SDS-PAGE results. Based on SDS-PAGE results, we found that a majority (89%) of the CNPP from the milk (approximately 10.13 out of 11.41% TP) were retained in the Kjeldahl NCN precipitate. Thus, CN%TP measured by Kjeldahl underestimates the amount of proteolytic damage that has been done to CN in milk. It is important for the dairy industry to correctly and rapidly measure the extent of proteolytic damage to milk protein to correctly value milk from a product quality and yield point of view. A rapid and quantitative measure of proteolytic damage to milk protein is needed.     

Influence of milk pre-heating conditions on casein–whey protein interactions and skim milk concentrate viscosity

The viscosity of concentrates (50–55% total solids) prepared from skim milk heated (5 min at 80 or 90 °C) at pH 6.5 and 6.7 was examined. The extent of heat-induced whey protein denaturation increased with increasing temperature and pH. More denatured whey protein and κ-casein were found in the serum phase of milk heated at higher pH. The viscosity of milk concentrates increased considerably with increasing pH at concentration and increasing heating temperature, whereas the distribution of denatured whey proteins and κ-casein between the serum and micellar phase only marginally influenced concentrate viscosity. Skim milk concentrate viscosity thus appears to be governed primarily by volume fraction and interactions of particles, which are governed primarily by concentration factor, the extent of whey protein denaturation and pH. Control and optimization of these factors can facilitate control over skim milk concentrate viscosity and energy efficiency in spray-drying.     

Isolation and characterisation of κ-casein/whey protein particles from heated milk protein concentrate and role of κ-casein in whey protein aggregation

Milk protein concentrate (79% protein) reconstituted at 13.5% (w/v) protein was heated (90 °C, 25 min, pH 7.2) with or without added calcium chloride. After fractionation of the casein and whey protein aggregates by fast protein liquid chromatography, the heat stability (90 °C, up to 1 h) of the fractions (0.25%, w/v, protein) was assessed. The heat-induced aggregates were composed of whey protein and casein, in whey protein:casein ratios ranging from 1:0.5 to 1:9. The heat stability was positively correlated with the casein concentration in the samples. The samples containing the highest proportion of caseins were the most heat-stable, and close to 100% (w/w) of the aggregates were recovered post-heat treatment in the supernatant of such samples (centrifugation for 30 min at 10,000 × g). κ-Casein appeared to act as a chaperone controlling the aggregation of whey proteins, and this effect was stronger in the presence of α_S- and β-casein.     

Heat-induced interactions of β-lactoglobulin and α-lactalbumin with the casein micelle in pH-adjusted skim milk

Skim milks, adjusted to pH 6.48, 6.60 or 6.83, were heated for various temperature–time combinations in a pilot-scale ultra-high temperature (UHT) plant. Heat-treated samples were ultracentrifuged and their supernatants analysed by quantitative polyacrylamide gel electrophoresis in order to measure the extent of β-lactoglobulin (β-lg) and α-lactalbumin (α-la) denaturation and their subsequent association with the casein micelle. The activation energy of β-lg denaturation decreased as the pH increased. In contrast, there was no apparent trend for α-la. The extent of β-lg and α-la association with the micelle increased with heating time and temperature. The association of both proteins with the micelle was markedly affected by the milk pH. The rate and extent of association were greatest at pH 6.48, and least at pH 6.83. α-La continued to associate with the micelle although most of the β-lg had already associated. It was possible that α-la interacted at the micelle surface with β-lg that had previously associated with the micelle. A pseudo-first-order mathematical model was used to calculate the apparent rate constant for β-lg association with the micelle.     

Pilot-scale β-casein depletion from micellar casein via cold microfiltration in the diafiltration mode

The aim of this study was to produce pilot scale batches of β-casein concentrate and micellar casein concentrate with reduced β-casein level. The isolation of β-casein was done using membrane filtration at cold temperatures (≤5 °C). A micellar casein concentrate was obtained from skim milk by means of warm microfiltration (MF) at 50 °C (0.1 μm pore size, ceramic membranes). The concentrate was stored at 2–3 °C for approximately 40 h to induce temperature-dependent dissociation of β-casein from casein micelles. β-casein was separated from the cold-stored concentrate using MF (0.3 μm pore size, organic membranes) at ≤5 °C. β-Casein permeate was warmed up to 50 °C to lead self-association of β-casein micelles before ultrafiltration at 50 °C (10 kDa cut-off, organic membranes). Two streams, a β-casein-depleted and a β-casein concentrate, were generated. A purity of 92.64% and a yield of up to 18.07% were achieved for β-casein.     

Temperature-dependent dynamics of bovine casein micelles in the range 10–40 °C

Milk is a complex colloidal system that responds to changes in temperature imposed during processing. Whilst much has been learned about the effects of temperature on milk, little is known about the dynamic response of casein micelles to changes in temperature. In this study, a comprehensive physico-chemical study of casein micelles in skim milk was performed between 10 and 40 °C. When fully equilibrated, the amount of soluble casein, soluble calcium and the pH of skim milk all decreased as a function of increasing temperature, whilst the hydration and volume fraction of the casein micelles decreased. The effect of temperature on casein micelle size, as determined by dynamic light scattering and differential centrifugation, was less straightforward. Real-time measurements of turbidity and pH were used to investigate the dynamics of the system during warming and cooling of milk in the range 10–40 °C. Changes in pH are indicative of changes to the mineral system and the turbidity is a measure of alterations to the casein micelles. The pH and turbidity showed that alterations to both the casein micelles and the mineral system occurred very rapidly on warming. However, whilst mineral re-equilibration occurred very rapidly on cooling, changes to the casein micelle structure continued after 40 min of measurement, returning to equilibrium after 16 h equilibration. Casein micelle structure and the mineral system of milk were both dependent on temperature in the range 10–40 °C. The dynamic response of the mineral system to changes in temperature appeared almost instantaneous whereas equilibration of casein was considerably slower, particularly upon cooling.     

Nutritional utilization in Malagueña dairy goats differing in genotypes for the content of alphaS1-casein in milk

A study was carried out with 20 goats of the Malagueña breed, half with a high (HG) and half with a low (LG) genetic capability for α_S1-casein (AS1-CN) synthesis, to determine whether the 2 different genotypes (that cause differences in goat milk composition) are related to differences in nutritional feed utilization. Among the 10 HG goats, 7 had BB and 3 had AB genotypes for AS1-CN, whereas there were 7 EF and 3 FF genotypes in the 10 LG goats. The goats were fed diets differing in crude protein content (13.6 vs. 17.7% dry matter for diets 1 and 2, respectively). For each genotype group, a balance trial was conducted with each of the 2 diets in a 2-period balanced changeover designed with half the animals consuming diet 1 and the other half diet 2, determining individual feed intake and the utilization of N and energy in the diets. Greater voluntary feed intake on a metabolic body weight basis among the HG goats was identified as the first possible cause of their milk production. The HG goats also had a greater level of feed utilization, on a metabolic body weight basis, for N and energy intake. Greater ratios of N balance/ digestible N, milk protein N/digestible N, milk energy/ digestible energy, and milk energy/ME were found for HG goats compared with LG. These effects appear to be dependent on the level of protein in the diet, indicating interactive effects. The greater N and energy utilization of HG versus LG goats may explain the differences in milk composition between the 2 genotype groups.     

Effect of heating on the distribution of transforming growth factor-��2 in bovine milk

The objective of this study was to characterize the impact of heat treatments on the distribution of transforming growth factor-beta (TGF-??2) between cream and skim milk and between the casein and whey fractions of skim milk. Skimming removed 45% and 62% of the TGF-??2 from raw and pasteurized milks and only 8% of the total TGF-??2 in skimmed pasteurized milk was found in whey, compared to 37% in whey from raw skimmed milk. The TGF-??2 content of whey decreased as the heat treatment of the milk increased in intensity (thermization > pasteurization > UHT sterilization). Using milk held for 1 or 2 min at temperatures ranging from 57 to 84 °C, it was shown that TGF-??2 in the whey portion decreases at temperatures above 66 °C and becomes undetectable at temperatures higher than 76 °C. Altogether, these data on the heat-induced changes in TGF-??2 content of cream, skim milk, casein and whey reveal a potentially negative impact of certain heat treatments in developing TGF-??2-enriched fractions from milk.     

Biochemical and molecular characterization of polymorphisms of αs1-casein in Sudanese camel (Camelus dromedarius) milk

This work was designed to detect occurrence of biochemical polymorphism of α_s1-casein in two ecotypes of Sudanese camel (Camelus dromedarius) and to characterize these variants on molecular level. Milk samples were screened for α_s1-casein variability by isoelectric focusing, using skimmed milk, as well as isolated α_s1-casein. Two protein patterns, named α_s1-casein A and C, were identified, whereas the major allele A revealed frequencies of 0.8214 and 0.8615 in the two ecotypes. CSN1S1*A and CSN1S1*C are both characterized by missing of exon 16 on mRNA-level compared with the previously described CSN1S1*B. However, the sequence of exon 16 occurs on DNA-level in both. Therefore, this exon seems to be skipped out during mRNA-processing. Furthermore, CSN1S1*C shows a single G > T nucleotide substitution in exon 5, leading to a non-synonymous amino acid exchange (p.Glu30 > Asp30; GenBank ID: JF429138). A polymerase chain reaction-restriction fragment length polymorphism-method was established as a DNA-based test for this nucleotide substitution.     

Allergic responses induced by goat milk αS1-casein in a murine model of gastrointestinal atopy

Up to 3% of young children develop milk allergy and this may influence the development of immune-mediated diseases in later life. One protein that has been associated with allergic reactions to ruminant milk is α(S1)-casein (CN). Studies suggest that goat milk with low levels of α(S1)-CN may reduce allergenicity of milk, but the dose response to α(S1)-CN has not been confirmed. In this study, we examined the immune response to varying levels of goat α(S1)-CN in a mouse model of gastrointestinal allergy. BALB/c mice (aged 5 wk) were given intraperitoneal injections with α(S1)-CN and aluminum as adjuvant at 1 and 3 wk to sensitize mice to the antigen. In wk 5, groups of fasting mice (n=8/group) were challenged 4 times on alternate days by intragastric gavage with saline or 2, 10, or 20mg of α(S1)-CN. Serum levels of specific IgE, IgG(1), and IgG(2a) antibodies and mouse mast cell protease-I were determined. Interleukin-4, IL-10, and IFN-γ responses to 48-h activation with antigen were measured in cultured splenocytes. We determined that mice sensitized with α(S1)-CN had higher titers of specific IgG(1) and IgE antibodies compared with controls; however, groups challenged with differing doses of α(S1)-CN did not differ. The group challenged with the highest dose of α(S1)-CN had a 10-fold increase in mouse mast cell protease-I compared with the group challenged with saline. Both IL-4 and IL-10 were produced in a dose-dependent manner by cultured splenocytes incubated with α(S1)-CN. Overall, α(S1)-CN stimulated the production of cytokines associated with allergic disease in a dose-dependent manner. Thus, milk with lower levels of α(S1)-CN should contribute to a lesser antigenic burden.     

Optimization of β-casein stabilized nanoemulsions using experimental mixture design

The objective of this study was to determine the effect of changing viscosity and glass transition temperature in the continuous phase of nanoemulsion systems on subsequent stability. Formulations comprising of β-casein (2.5%, 5%, 7.5%, and 10% w/w), lactose (0% to 20% w/w), and trehalose (0% to 20% w/w) were generated from Design of Experiments (DOE) software and tested for glass transition temperature and onset of ice-melting temperature in maximally freeze-concentrated state (T(g) ' & T(m) '), and viscosity (μ). Increasing β-casein content resulted in significant (P < 0.0001) increases in viscosity and T(m) ' (P= 0.0003), and significant (P < 0.0001) decreases in T(g) '. A mixture design was used to predict the optimum levels of lactose and trehalose required to attain the minimum and maximum T(g) ' and viscosity in solution at fixed protein contents. These mixtures were used to form the continuous phase of β-casein stabilized nanoemulsions (10% w/w sunflower oil) prepared by microfluidization at 70 MPa. Nanoemulsions were analyzed for T(g) ' & T(m) ', as well as viscosity, mean particle size, and stability. Increasing levels of β-casein (2.5% to 10% w/w) resulted in a significant (P < 0.0001) increase in viscosity (5 to 156 mPa.s), significant increase in particle size (P= 0.0115) from 186 to 199 nm, and significant decrease (P= 0.0001) in T(g) ' (-45 to -50 °C). Increasing the protein content resulted in a significant (P < 0.0001) increase in nanoemulsion stability. A mixture DOE was successfully used to predict glass transition and rheological properties for development of a continuous phase for use in nanoemulsions.