Casein Micelle Size Research Articles

Linking variation in the casein fraction and salt composition to casein micelle size in milk of Dutch dairy goats

The casein composition, salt composition, and micelle size varies substantially between milk samples of individual animals. In goats, the links between those casein characteristics are unknown and could provide useful insights into goat casein micelle structure. In this study, the casein and salt composition of 42 individual Dutch goats from 17 farms was studied and linked to casein micelle size. Micelle size, the proportions of individual caseins, and protein content were associated with one another. Milk with smaller casein micelles was higher in protein content, salt content, and proportion of αs1-CN, but lower in αs2-CN and β-CN. The higher salt content in milk with small casein micelles was mainly attributed to a higher protein content, but changes in casein composition might additionally contribute to differences in mineralization. The nonsedimentable casein content in goat milk correlated with nonsedimentable fractions of β-CN and κ-CN and was independent of micelle size. Between large and small casein micelles, goat casein micelles showed more differences in casein and salt composition than bovine micelles, indicating differences in internal structure. Nevertheless, the casein mineralization in goat milk was similar to casein mineralization in bovine milk, indicating that mineralization of casein micelles follows a general principle. These results can help to better understand how composition and micelle structure in goat milk are related to each other, which may be useful to improve processing and product properties of goat milk in the future.

Milk protein polymorphisms of Aosta Valley cattle breeds

Abstract Cow’s milk proteins are encoded by highly polymorphic genes characterized by several mutations which result in different allelic variants. Each allelic variant has different possible effects on cheese-making properties and on human health. β-casein A1-A3-I-B, k-casein B and β-lactoglobulin B are supposed to influence milk cheese-making properties by increasing cheese or milk yield, by varying chemical parameters, by having small casein micelle size, and by influencing rennet coagulation time (RCT) and curd-firming rate (CFR). In addition, β-casein A1-B are also considered to be a risk factor for different health diseases such as ischemic heart disease (IHD), type 1 diabetes (T1D), decreased glutathione (GSH) concentration, and milk intolerance. An LC-MS method was applied to profile, for the first time, the main milk proteins genetic variants from Aosta Valley autochtonous cattle breeds. Analyses performed on milk collected from bovines of three cattle breeds (Red Pied – VRP, Black Pied – VNP and Chestnut – CAS), either from IAR experimental farm or from herds of dairy-producers in Aosta Valley region, showed quite high frequencies of β-casein A2 and A3/I, and low frequencies of β-casein A1. Moreover, low frequencies of β-casein B in VRP breed and high frequencies of the same variant in CAS, and VNP breeds have been found. As far as k-casein is concerned allelic variant B is the most diffused in VRP while allelic variant A in VNP and CAS breeds. Finally, β-lactoglobulin most diffused allelic variant for all breeds is B. Results suggest that Aosta Valley milk has good cheese-making properties and good frequencies of β-casein A2 which may be related to beneficial effects on human health. In light of these results, it is important to develop breeding programs which take into consideration milk proteins polymorphisms to further increase the milk suitability for cheese-making process and to decrease the presence of β-casein A1 and B in drinking milk which can be a risk factor for human health.

Structural Properties of Casein Micelles with Adjusted Micellar Calcium Phosphate Content.

Micellar calcium phosphate (MCP) content of skim milk was modified by pH adjustment followed by dialysis. Turbidity, casein micelle size and partitioning of Ca and caseins between the colloidal and soluble phases of milk were determined. Protein structure was characterised by Fourier transform infrared (FTIR) spectroscopy and proton nuclear magnetic resonance (1H NMR), whereas organic and inorganic phosphorus were studied by phosphorus-31 nuclear magnetic resonance (31P NMR). The sample with the lowest MCP content (MCP7) exhibited the smallest particle size and turbidity, measuring 83 ± 8 nm and 0.08 ± 0.01 cm-1, respectively. Concentrations of soluble caseins increased with decreasing MCP levels. At ~60% MCP removal, FTIR analysis indicated a critical stage of structural rearrangement and 31P NMR analysis showed an increase in signal intensity for Ca-free Ser-P, which further increased as MCP concentration was further reduced. In conclusion, this study highlighted the importance of MCP in maintaining micellar structure and its impact on the integrity of casein micelle.

Effect of composition, casein genetic variants and glycosylation degree on bovine milk whipping properties

This study investigated the individual and combined effects of ĸ-Casein (ĸ-CN; AA, AB, BB), β-Casein (β-CN; A1A1, A1A2, A2A2) and high and low ratios of glycosylated ĸ-CN to total ĸ-CN, referred to as the glycosylation degree (GD), on bovine cream whipping properties. The genetic variants of individual cows were identified using reversed-phase high-performance liquid chromatography (RP-HPLC) and verified through liquid chromatography–mass spectrometry (LC-MS). A previously discovered relationship between days-in-milk and GD was validated and used to obtain high and low GD milk. Whipped creams were created through the mechanical agitation of fat standardised cream from milk of different ĸ-CN, β-CN, and GD combinations, and whipping properties (the ability to whip, overrun, whipping time and firmness) were evaluated. No significant correlation was measured in whipping properties for cream samples from milks with different ĸ-CN and β-CN genetic variants. However, 80 % of samples exhibiting good whipping properties (i.e., the production of a stiffened peak) were from milk with low GD suggesting a correlation between whipping properties and levels of glycosylation. Moreover, cream separated from skim milk of larger casein micelle size showed superior whipping properties with shorter whipping times (<5 min), and higher firmness and overrun. Milk fat globule (MFG) size, on the other hand, did not affect whipping properties. Results indicate that the GD of κ-CN and casein micelle size may play a role in MFG adsorption at the protein and air interface of air bubbles formed during whipping; hence, they govern the dynamics of fat network formation and influencing whipping properties.

Digestive properties and peptide profiles exhibited significant differences between skim camel milk and bovine milk powder after static in vitro simulated infant gastrointestinal digestion

This study aims to analyze the differences in digestion properties and peptide profiles between the skim camel and bovine milk powder after static in vitro simulated infant gastrointestinal digestion. The hydrolysis degree of camel milk proteins exceeded by 13.18% that of bovine milk. The concentration and release rate of free amino groups in the camel milk digesta was higher than that of bovine milk powder, which was likely due to the higher β-/αs-casein ratio and larger casein micelle size in camel milk. Camel milk powder presented higher β-CN coverage and comparatively shorter bioactive peptides compared to bovine milk powder. The anti-inflammatory peptide KVLPVPQ displayed the highest abundance in camel milk powder. Outcomes of this study showed that camel milk proteins possessed superior digestibility and unique peptides, which outlined the potential nutritional implications of camel milk for infants.

Effect of CSN3 Gene Polymorphism on the Formation of Milk Gels Induced by Physical, Chemical, and Biotechnological Factors.

During the last decade, research into genetic markers in the casein gene cluster has been actively introduced in cattle breeding programs. A special interest has been paid to the polymorphism of the CSN3 gene, responsible for the expression of the k-casein, playing a key role in protein coagulation, interaction with whey proteins, stabilization, and aggregation of casein micelles. This paper aimed to determine the effect of CSN3 genetic polymorphism on acid; rennet; acid-rennet; heat- and acid-induced as well as heat- and calcium-induced coagulation in skimmed milk; and protein-standardized milk systems (UF, NF, RO, VE). The influence of polymorphic variants of the CSN3 gene on the coagulation ability of milk proteins was assessed by the particle size of casein micelles, protein retention factor in the clot, and coagulation ability (duration of induction period, mass coagulation period, dynamic viscosity in gel point). The correlation between CSN3 gene polymorphism and protein coagulation was revealed. Milk systems obtained from CSN3 BB milk were found to have the shortest duration of coagulation, formation of better gel strength values, and increased yield compared to CSN3 AA. This study will improve the efficiency of milk processing and optimize the technology of dairy product production.

Investigating the effect of diluents and fat globules on the size measurement of casein micelles by dynamic light scattering

The hydrodynamic size of casein micelles was examined using dynamic light scattering (DLS). Four types of diluents [ultrafiltration (UF) permeate, lactose-free simulated milk ultrafiltrate (SMUF), calcium imidazole buffer, and deionised water] were compared in their effect on the stability of casein micelles. Results of dilution series and kinetic size measurements revealed that both UF permeate and lactose-free SMUF enable size measurements that are hardly affected by the required dilution step, whereas a calcium imidazole buffer containing 10 mm CaCl2 only could provide accurate sizing results within a short time after dilution. The contribution of residual fat globules to the particle size distribution was negligible in skim milk. For semi-skimmed milk, the size of the casein micelles could be estimated by subtracting the contribution of the fat droplets (obtained upon dilution in a casein dissolving solution) from the raw DLS data (as obtained upon dilution in milk ultrafiltrate).

Effect of sodium alginate on the yogurt stability was dependent on the thickening effect and interaction between casein micelles and sodium alginate

The effect of sodium alginate (SA) on the yogurt stability and the related mechanisms were investigated. It was found that low-concentration SA (≤0.2 %) increased the yogurt stability, while high-concentration SA (≥0.3 %) decreased the yogurt stability. Sodium alginate increased the viscosity and viscoelasticity of yogurt and this effect was positively correlated with its concentration, suggesting that SA worked as the thickening agent in yogurt. However, addition of ≥0.3 % SA damaged the yogurt gel. These results suggested that interaction between milk protein and SA might play an important role in the yogurt stability besides the thickening effect. Addition of ≤0.2 % SA did not change the particle size of casein micelles. However, addition of ≥0.3 % SA induced aggregation of casein micelles and increased the size. And the aggregated casein micelles precipitated after 3 h storage. Isothermal titration calorimetry analysis showed that casein micelles and SA were thermodynamically incompatible. These results suggested that the interaction between casein micelles and SA induced aggregation and precipitation of casein micelles, which was critical in the destabilization of yogurt. In conclusion, the effect of SA on the yogurt stability was dependent on the thickening effect and the interaction between casein micelles and SA.

Microscopical Evaluation of the Effects of High-Pressure Processing on Milk Casein Micelles.

The effect of different high-pressure processing (HPP) treatments on casein micelles was analyzed through scanning electron microscopy (SEM) and a particle size distribution analysis. Raw whole and skim milk samples were subjected to HPP treatments at 400, 500 and 600 MPa for Come-Up Times (CUT) up to 15 min at ambient temperature. Three different phenomena were observed in the casein micelles: fragmentation, alterations to shape and agglomeration. The particle size distribution analysis determined that, as pressure and time treatment increased, the three phenomena intensified. First, the size of the casein micelles began to decrease as their fragmentation occurred. Subsequently, the casein micelles lost roundness, and their shape deformed. Finally, in the most intense treatments (higher pressures and/or longer times), the micelles fragments began to agglomerate, which resulted in an increase in their average diameter. Homogenization and defatting had no significant effect on the casein micelles; however, the presence of fat in whole milk samples was bioprotective, as the effects of the three phenomena appeared faster in treated skim milk samples. Through this study, it was concluded that the size and structure of casein micelles are greatly altered during high-pressure treatment. These results provide information that broadens the understanding of the changes induced on casein micelles by high-pressure treatments at room temperature.

Structural changes induced by pasteurisation and/or high-pressure treatment of skim caprine milk

The effects of pasteurisation, high-pressure (HP), and a combination of pasteurisation and high-pressure (PHP) on the physicochemical properties and protein structure of caprine skim milk was investigated. Samples treated by PHP generally had a higher pH, whey protein denaturation, surface hydrophobicity, and intrinsic fluorescence than those treated only with heat or pressure. In contrast, the size of skim milk casein micelles decreased significantly with an increase in pressure level and time; however, the effect was less marked when heat and pressure treatments were combined. For the PHP and HP samples, as the level and time of pressure increased, the α-helix and β-turn content reduced, whereas β-sheet and random coil were induced. Thus, PHP treatment could be used as a good alternative technology in the dairy industry to promote the functional properties of skim caprine milk.

Ultrasound-Assisted Encapsulation of Anthraquinones Extracted from Aloe-Vera Plant into Casein Micelles.

Aloe-vera extracted anthraquinones (aloin, aloe-emodin, rhein) possess a wide range of biological activities, have poor solubility and are sensitive to processing conditions. This work investigated the ultrasound-assisted encapsulation of these extracted anthraquinones (AQ) into casein micelles (CM). The particle size and zeta potential of casein micelles loaded with aloin (CMA), aloe-emodin (CMAE), rhein (CMR) and anthraquinone powder (CMAQ) ranged between 171–179 nm and −23 to −17 mV. The AQ powder had the maximum encapsulation efficiency (EE%) (aloin 99%, aloe-emodin 98% and rhein 100%) and encapsulation yield, while the whole leaf Aloe vera gel (WLAG) had the least encapsulation efficiency. Spray-dried powder (SDP) and freeze-dried powder (FDP) of Aloe vera showed a significant increase in size and zeta potential related to superficial coating instead of encapsulation. The significant variability in size, zeta potential and EE% were related to anthraquinone type, its binding affinity, and its ratio to CM. FTIR spectra confirmed that the structure of the casein micelle remained unchanged with the binding of anthraquinones except in casein micelles loaded with whole-leaf aloe vera gel (CMWLAG), where the structure was deformed. Based on our findings, Aloe vera extracted anthraquinones powder (AQ) possessed the best encapsulation efficiency within casein micelles without affecting its structure. Overall, this study provides new insights into developing new product formulations through better utilization of exceptional properties of casein micelles.

O137 Casein micelle size and size distribution in milk from ewes fed a diet including broccoli or cauliflower

O137 Casein micelle size and size distribution in milk from ewes fed a diet including broccoli or cauliflower

Seasonal Variations in the Composition and Physicochemical Characteristics of Sheep and Goat Milks

There has been growing consumer interest in sheep and goat milk products as alternatives to cow milk products. The physicochemical characteristics of milk vary not only between ruminant species, but also during different seasons; they determine the nutritional quality and processing properties of the milk. In this study, we characterized sheep and goat milks from New Zealand over the seasons for their composition (macronutrients, macro- and micro-minerals, fatty acids, and proteins) and physicochemical properties (e.g., ionic calcium, fat globule size, casein micelle size, viscosity, and melting behavior of milk fat). Heat-induced (95 °C for 5 min) protein interactions and changes in the physical properties of the milks were also investigated. The compositional and structural features of sheep and goat milks were identified and compared with those reported for cow milk. Seasonal variations in the milk characteristics were more pronounced for sheep milk than goat milk and were probably affected by the production systems. Sheep milk, particularly in the late season, had the largest heat-induced increases in casein micelle size and viscosity, probably arising from the greater casein–whey protein and casein–casein interactions during heat treatment. This study provides comprehensive information on the properties of sheep and goat milks and highlights the interaction effects between species, season, and processing.

Development in Maillard Reaction and Dehydroalanine Pathway Markers during Storage of UHT Milk Representing Differences in Casein Micelle Size and Sedimentation.

Ultra-high temperature (UHT) processing of milk can result in protein changes during storage; however, the progress of dehydroalanine (DHA) mediated protein cross-linking and Maillard reactions in relation to the sediment formation have not been investigated previously. Liquid chromatography–mass spectrometry, based on multiple reaction monitoring (MRM), was used to absolutely quantify concentrations of furosine, N-ε-(carboxyethyl)lysine (CEL), N-ε-(carboxymethyl)lysine (CML), lanthionine (LAN) and lysinoalanine (LAL) in skim milk and sediment of UHT milk produced from raw milk with either small or large casein micelles. The results showed a higher molar proportion of the advanced stage Maillard reaction products CEL and CML in the sediment, compared to early stage Maillard reaction product furosine, whereas furosine was predominant in the skim milk. Both LAL and LAN increased during storage in the skim milk phase, however only LAL was identified in the sediment. The milk pool with large native casein micelles, known to have a higher percentage of sedimentation, contained higher proportions of furosine, CEL, CML and LAL in the sediment compared to milk with smaller native casein micelles. The study demonstrates the potential contribution of processing-induced protein-protein interactions to sedimentation in UHT milk during storage.

Kinetics of heat-induced interactions among whey proteins and casein micelles in sheep skim milk and aggregation of the casein micelles

The interactions among the proteins in sheep skim milk (SSM) during heat treatments (67.5-90°C for 0.5-30 min) were characterized by the kinetics of the denaturation of the whey proteins and of the association of the denatured whey proteins with casein micelles, and changes in the size and structure of casein micelles. The relationship between the size of the casein micelles and the association of whey proteins with the casein micelles is discussed. The level of denaturation and association with the casein micelles for β-lactoglobulin (β-LG) and α-lactalbumin (α-LA) increased with increasing heating temperature and time; the rates of denaturation and association with the casein micelles were markedly higher for β-LG than for α-LA in the temperature range 80 to 90°C; the Arrhenius critical temperature was 80°C for the denaturation of both β-LG and α-LA. The casein micelle size increased by 7 to 120 nm, depending on the heating temperature and the holding time. For instance, the micelle size (about 293 nm) of SSM heated at 90°C for 30 min increased by about 70% compared with that (about 174.6 nm) of unheated SSM. The casein micelle size increased slowly by a maximum of about 65 nm until the level of association of the denatured whey proteins with casein micelles reached 95%, and then increased markedly by a maximum of about 120 nm when the association level was greater than about 95%. The marked increases in casein micelle size in heated SSM were due to aggregation of the casein micelles. Aggregation of the casein micelles and association of whey protein with the micelles occurred simultaneously in SSM during heating.

Kinetic aspects of casein micelle cross-linking by transglutaminase at different volume fractions

Milk protein concentrates (MPC) are widespread food ingredients, due to their high protein-to-solids ratio, nutritional value, and technological properties, which can be tailored to various end uses. Interest has been given to modifying such ingredients through enzymatic cross-linking by microbial transglutaminase. However, no systematic studies on the cross-linking kinetics of casein micelles at different concentration factors are currently available, although the enzyme might act in a considerably different fashion at decreased inter-particle distances. In this study, cross-linking of casein micelles was studied in a 4x MPC obtained by ultrafiltration, and compared to that of the original skim milk. The cross-linking kinetics were evaluated by following the degree of casein polymerisation using size exclusion chromatography. The extent of polymerisation for both skim milk and 4x MPC could be scaled to a master curve by normalising the incubation time to concentration factor and enzyme level, indicating similar cross-linking kinetics. Likewise, the cross-linking of individual casein types followed the same trend regardless of treatment. However, the casein micelle size increased with cross-linking at 4x but decreased at 1x concentration, suggesting enhanced cross-linking on the surface of the casein micelles in MPC suspensions. These results are relevant for the design of novel milk protein ingredients.

Characterization of Structural Changes of Casein Micelles at Different PH Using Microscopy and Spectroscopy Techniques.

This study aimed to evaluate the influence of pH changes on morphometric parameters of casein micelles and a general overview of their conformational structure through microscopy techniques, Raman spectroscopy and multivariate analysis. It was found that casein micelles morphology and protein secondary structure depend strongly upon pH. The changes of arithmetic average roughness (Ra), size, and shape of casein micelles at different pH are properly characterized by atomic force and cryo-transmission electron microscopy. Morphometric changes of casein micelles were correlated correctly with folding and unfolding of casein molecules as evaluated by Raman spectroscopy when the pH was varied. The novelty of this contribution consists in demonstrating that there is a close structure-functionality relationship between the morphometric parameters of proteins and their secondary structure. Knowledge about casein micelles can help improve their use of its diverse applications.

Structural and rheological properties of the clots formed by ruminant milks during dynamic in vitro gastric digestion: Effects of processing and species

Ruminant milks coagulate during gastric digestion, which plays an important role in controlling digestion kinetics. Previous studies on cow milk showed that the structural and rheological properties of the clots formed in the gastric environment affect their breakdown and consequently their emptying rate into the intestine. This study investigated the impacts of homogenisation and heat treatment on the structural and rheological properties of the clots formed by sheep, goat and cow milks during simulated dynamic gastric digestion, with empasis on the non-bovine milks. Overall, with increasing intensity of processing, the gastric clots formed by ruminant milk became more fragmented, contained more moisture and had lower consistency. The consistency of the clots appeared to be associated with the presence of colloidal calcium phosphate in the casein micelles and the moisture content of the clot. A structural rearrangement of the clots may occur during the early stages of digestion because the forces maintaining the clot network change as the pH decreased. Between different species, we observed different impacts of individual processing treatments on the macrostructure and the consistency of the clots. Homogenisation had greater impacts on goat and sheep milk clots than cow milk clots whereas heat treatment (95 °C for 5 min) had the most effect on homogenised cow milk clots. Differences in the physicochemical properties of the ruminant milks, e.g. casein micelle size and ionic calcium concentration, may have been responsible for the variations observed between species. This study furthered our understanding of the gastric coagulation of ruminant milk and highlighted the species-specific processing impacts (processing × species interactions) on the structural and rheological properties of gastric milk clots.

Effect of Different Temperature-Controlled Ultrasound on the Physical and Functional Properties of Micellar Casein Concentrate.

Micellar casein concentrate (MCC) is a novel dairy ingredient with high protein content. However, its poor functional properties impair its potential for further application, highlighting the importance of using innovative processing methods to produce modified MCC, such as ultrasound (US). This work investigated the impact of US on the physical and functional properties of MCC under temperature-controlled and -uncontrolled conditions for different time intervals. Under temperature-controlled ultrasound (TC-US) treatment, a reduction was found in the supernatant particle size of casein micelles. Soluble calcium content and hydrophobicity increased following ultrasound treatment at 20 °C, resulting in a remarkable improvement in emulsification. However, long-time ultrasonication led to an unstable state, causing the MCC solutions to show shear thinning behavior (pseudoplastic fluid). Compared with 50 °C temperature-controlled ultrasonication, ultrasonication at 20 °C had a greater influence on particle size, viscosity and hydrophobicity. These findings indicate that 20 °C TC-US could be a promising technology for the modification of MCC.

Processing sheep milk by cold plasma technology: Impacts on the microbial inactivation, physicochemical characteristics, and protein structure

This study aimed to evaluate the impacts of cold plasma processing time of 30 s, 180 s, and 300 s (CSM30, CSM180, and CSM300) on the microbial inactivation, physicochemical characteristics, and protein structure of raw sheep milk. Pasteurized sheep milk was used as the positive control. The microbial inactivation rate of CSM300 sample was similar to the pasteurized sample, and the former had a significantly smaller casein micelle size and polydispersity index (P < 0.05). The pH, and a* value decreased from 6.79 to 6.65, and −1.74 to −1.98, respectively (P < 0.05) with increasing processing time. Also, the moisture content of the freeze-dried milk decreased from 43.4 g/kg to 28.9 g/kg. The Fourier-transform infrared spectra indicated that the CSM180 and CSM300 samples had higher proportions of β-turn structures and a diminished-random coil conformation, the latter could induce an appropriate conformational transformation from α-helix to β-sheet. Scanning electron microscopy images confirmed that the CSM300 sample promoted a more uniform microstructure and a better dispersion state of proteins. These results might provide practical information regarding cold plasma–treated sheep milk, which can be considered as a potential alternative to conventional pasteurization.