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Acidified milk is normally divided into drinking (e.g. acidified milk drinks) and gelling (e.g. stirred/set yoghurt) types, and its texture is mainly determined by the state of casein micelles. Pol...

The present study examined the microstructure as well as the physicochemical properties of human milk during the second year of lactation in an attempt to explore its applicability for the formulation of food products. It was observed that human milk fat globules (MFG) droplet size increased within 3 days of milk extraction due to coalescence, as evidenced by confocal microscopy. Furthermore, a gradual decrease of the average MFG size was noted from the sixteenth (16th) to twenty-fifth (25th) month of lactation. It was also found that the size of casein micellar structures increased upon acidification to pH 4.3 (isoelectric point of human caseins). In addition, human milk proteins enhanced the stability of oil-in-water emulsions against coalescence compared to cow, sheep, and goat milk proteins employed as macromolecular emulsifying ingredients. The cold-acid-gels of human milk proteins showed a less elastic behavior than the other milk samples, possibly due to the different structure, composition and size of human casein micelles. Furthermore, the DSC thermograms showed that human whey proteins are denatured in the same temperature range as do the cow whey proteins, but exhibit different thermal transition profiles. Overall, the findings of this research confirm that both the structure and the physicochemical properties of human milk are affected by the stage of lactation. Moreover, the particular composition and structure of human milk proteins seem to be responsible for the special functional characteristics of human milk that may lead towards the formulation of innovative products., Graphical abstract Image 1, Highlights • The physicochemical properties of human milk in the 2 nd year were examined. • Enhanced stabilization of oil-in-water emulsions with human milk proteins (HMP). • Less elastic behavior of cold-acid HMP gels compared to other milk species proteins. • HMP exhibited different thermal transition profiles than cow milk proteins.

Acidification of milk destabilizes casein micelles (CMs) and results in network formation. This process is fundamental during the manufacturing of dairy gels such as cheese and yoghurts. Understanding the structural alterations of CMs during this process aids to predict the physicochemical and sensory properties of dairy gels. Herein we utilize direct Stochastic Optical Reconstruction Microscopy (dSTORM) to visualize individual hydrated CMs and characterize pH-dependent changes in CM size distributions. CMs were immobilized, fixed, labelled with caseins-specific primary antibodies, and imaged by dSTORM using Alexa Fluor 647 conjugated secondary antibodies. While antibodies specific to κ- and β-casein were used to stain CMs, only β-casein antibodies enabled reproducible imaging of micelles across the entire chosen pH window. Furthermore, CM’s structural evolution was studied at acidic pH values representing the conditions during acid milk gel formation and at three elevated pH values. dSTORM imaging of casein aggregates at pH 4.5, below the isoelectric point of caseins, showed that β-casein distribution throughout the protein network and resolved nano-sized pores within the structure. Moreover, automated and quantitative image analysis revealed that the average size of CMs increased upon alkalization to pH 7.5 and 8.3, whilst narrow size distributions were found upon acidification to pH 5.5.

Properties of casein micelles can be manipulated by changing environmental conditions, which leads to formation of particles of varying sizes with different physical properties. This study evaluated the impact of shear (100 and 1000 s−1) on the properties of casein micelles in milk protein concentrate in the pH range 2.0–7.5. Casein micelle size was not affected by shear at pH 6.7 or 7.5, whereas casein aggregates, which were formed at pH 4.6 and 2.0, had their size considerably reduced upon high shearing. The composition of the soluble phase was dependent on pH as increased concentrations of β-casein and αs-caseins were found at pH 4.6 and pH 2, respectively. Greatest dissociation of individual caseins from the micelle occurred at pH 2.0. These serum caseins appeared in a form of fairly small soluble aggregates mainly consisting of αS- and β-caseins. The composition of aggregates varied with applied shear, indicating orthokinetic aggregation and fragmentation.

Casein nanomicelles, a major fraction of milk protein, are emerging as a novel drug delivery system owing to their various structural and functional properties. Casein is further divided into α-, β- and κ-casein, and to date various models have been proposed to describe casein structure, but still no definite structure presenting a detailed assembly of the casein micelle has been found. Thus far, the submicellar model and Horne and Holt model are the most accepted models. This article presents a detailed review of casein micelles and their fractions, and the physicochemical properties that account for their numerous applications in nutraceutics, pharmaceutics and cosmetics. Due to their nanosize and self-assembling nature, casein nanomicelles are considered as excellent delivery carriers to provide better bioavailability and stability of various compounds such as vitamins, oils, polyphenols, fattyacids and minerals. Their amphiphilic nature also provides a great opportunity to deliver hydrophobic bioactives in various drug delivery systems such as nanoparticles, nanomicelles, nanogels and nanoemulsions to improve drug binding and targeting.

This project investigated the influence of the addition of high methoxyl pectin (HMP) and carboxymethylcellulose (CMC) with different combination ratios on the stability of acidified skim milk drinks (SAMD) and whole milk drinks (WAMD). The experimental results showed that with the addition of combination polysaccharides, the SAMDs were not stabilised after period of time, but the WAMDs showed a markedly reduced formation of serum phase. The presence of milk fat globule membranes (MFGM) aided the dispersion of casein micelles and prevented casein sedimentation and serum separation. The rheological properties of the unstable drinks in this study demonstrated severe shear-thinning behaviour and less frequency dependence, however, for the stable drinks, the opposite properties were observed. Drink stability improved when the amount of HMP increased in the polysaccharide ratio, highlighting the importance of the molecular properties of polysaccharides.

Agar is widely used in yogurt manufacture to improve the stability of milk gels. However, the formation and structural properties of acid-induced casein–agar gel have not been elucidated. In this study, different acidification temperatures were used to evaluate the evolution of acid-induced casein–agar double networks and their microstructure and penetration properties. Agar (0.25%, w/w) was mixed with a micellar casein dispersion (3%, w/w), acidified with glucono-δ-lactone at 30, 37, 42, or 45 °C, respectively, and then cooled to 10 °C. Compared with casein gel alone, the casein–agar gel had a higher firmness and water holding capacity (WHC). Moreover, these acid-induced casein–agar double networks formed different structures depending on the acidification temperature. When acidified below the agar transition temperature (at 30 or 37 °C), the agar formed a weak gel prior to casein network formation that hindered the movement of casein micelles. Therefore, the casein–agar mixtures formed double networks during the acidification stage with relatively low elasticity indexes (EIs). In contrast, casein–agar mixtures acidified above the agar transition temperature (at 42 or 45 °C) formed double networks during the cooling stage that exhibited higher EIs. Cryo-scanning electron microscopy showed denser networks in casein–agar gels acidified above the agar transition temperature. Furthermore, casein–agar gels acidified above the agar transition temperature showed a significantly higher firmness and WHC than those acidified below. Overall, acidification temperature significantly affected the gelation sequence and, thereby, the formation and structural properties of the acid-induced casein–agar double network.

Milk protein coagulation is not only important during the processing of milk into various dairy products, but also during digestion of milk. This review focusses on the gastric coagulation of milk proteins. During this process, coagulation of casein micelles and milk fat globules can occur due to pepsin-induced hydrolysis of the proteins that provide steric stabilisation. The gastric coagulation leads to delayed gastric emptying of casein and fat. Native whey proteins are not susceptible to gastric coagulation or delayed gastric emptying. Both heat treatment and homogenisation of milk lead to weaker gastric curds being formed, which are broken down more easily due to proteolysis and deformation. Incorporation of denatured whey proteins in gastric curds of heated milk delays their emptying. Understanding gastric coagulation and digestion behaviour allows tailoring of gastric transit via compositional differences or processing.

Food Hydrocolloids for Health 1, 100014 (2021). doi:10.1016/j.fhfh.2021.100014, Published by Elsevier, Amsterdam

Nanomedicine for cancer therapy is a successful tool to diminish the side effect of chemotherapeutics such as paclitaxel (PTX). In this regard, Abraxane®, a human serum albumin (HSA)-based nanomedicine system has shown lesser side effects than Taxol®. However, the large-scale production of HSA protein is limited and expensive, which is traduced in a high cost of the treatments in clinical applications. Thus, the use of easily-available alternative nanocarriers could increment the accessibility of patients to nanomedicine for cancer treatments. Casein is a low-cost protein able to self-assemble into micelles which could efficiently encapsulate PTX into their structure. In this work, the synthesis of chemically crosslinked casein micelles (CCM), used to prepare PTX-based nanoformulations, is presented. CCM@PTX nanoformulations showed promising results in vitro to be applied as nanomedicine for cancer therapy. Thus, the obtained nanoformulations are great candidates to be parenterally administered, accumulate in tumor by passive targeting without leakage of PTX in plasma, and release the drug within the tumor microenvironment, in response to overexpressed proteases such as trypsin. Fil: Cuggino, Julio César. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: Picchio, Matías Luis. Universidad Nacional de Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada; Argentina Fil: Gugliotta, Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Bürgi Fissolo, María de Los Milagros. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Ronco, Ludmila Irene. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: Calderón, Marcelo. Polymat; España Fil: Etcheverrigaray, Marina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina Fil: Alvarez Igarzabal, Cecilia Ines. Universidad Nacional de Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada; Argentina Fil: Minari, Roque Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: Gugliotta, Luis Marcelino. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina

International audience; Heat treatments induce structural modifications of bovine milk proteins. In this study, we aimed to investigate how these structural modifications are affected by the whey protein profile of infant milk formulas (IMFs) and heat treatment parameters. Three model IMFs (1.3% of proteins; caseinwhey protein ratio 4060) differing by the whey protein profile (13.37 α-lactalbumin (α-LA)β-lactoglobulin (β-LG), 13.261.80 α-LAβ-LGlactoferrin (LF) or 10.130.40 α-LAβ-LGLF), were heated at 67.5 °C or 80 °C to reach an akin whey protein denaturation extent of 65%. Protein structures were analyzed by asymmetrical flow field-flow fractionation coupled with multiangle light scattering and differential refractometer, transmission electron microscopy and electrophoresis. The unheated IMFs were used as reference. The results showed that LF addition in IMFs induced partial casein micelle disintegration before heating. In the absence of added LF, the heat-denatured whey proteins either formed soluble whey protein aggregates or casein micelle-bound whey protein aggregates. The latter were favored at the expense of soluble aggregates in the heated IMFs with the LF content increase and the concomitant β-LG content decrease. Consequently, the casein micelle structure was strongly dependent on the β-LG and LF amounts in IMFs and on the heating temperature. In the IMFs containing β-LG and LF and at temperature greater than the β-LG denaturation temperature, the casein micelles exhibited filamentous appendages on its surface after heating. Below the denaturation temperature of β-LG and in IMFs containing trace β-LG amount, the heated casein micelles were perfectly spherical with a smooth surface. © 2020 Elsevier Ltd

The influence of different casein-based ingredients on the physicochemical properties of native casein micelles (CMs) was studied. Sodium caseinate (SCN) and β-casein concentrate (BCC) were mixed with milk protein concentrate (MPC) at different ratios (1:3, 1:1, 3:1), at a total protein concentration of 20 g/L. Tea polyphenols (TPP) were added to select samples to investigate their effect on CM-SCN and CM-BCC interactions. The results demonstrated decreasing structural integrity of CMs and increased levels of non-micellar casein, due to addition of SCN to MPC, which led to a decreasing turbidity over time and an increase in surface hydrophobicity. Addition of BCC to MPC resulted in an increase in turbidity over time and a decrease in surface hydrophobicity of the system. TEM images suggested that small micelles from BCC interacted with the surface of CMs. The SDS-PAGE results show that CMs in samples with TPP dissociated less, indicating that TPP stabilized the CMs structure. In conclusion, the two casein ingredients, SCN and BCC, have a different effect on the colloidal and physicochemical properties of CMs in MPC suspensions. This study will contribute to a greater understanding of the effect of mixed protein formulation strategies on the functional properties of beverage systems.

Over the past 60 years considerable progress has been made on understanding the nature of casein interactions. Although there is ongoing debate, most researchers now view that casein micelles are assembled due to the concerted action of two major types of interactions, namely hydrophobic interactions, and formation of calcium phosphate nanoclusters across phosphoserine clusters. This paper provides the authors’ perspectives on these interactions, reviews evidence supporting the involvement of these two types of interactions, critiques some of the proposed models, and gives several examples where these interactions are manipulated to modify the functionality of dairy products.

In the present work, the influence of agar oligosaccharide (AO) on the stability of directly acidified milk drinks (DAMDs) was investigated. The experimental results showed that the DAMDs stabilized by AO in combination with anionic polysaccharide had smaller particle diameter values and lower sedimentation fractions than that stabilized by the commercial reference under the same pH conditions. Cryo-SEM images showed a remarkable density porous network organized by agar oligosaccharides based polysaccharide mixtures. It revealed that the stabilizing mechanism of polysaccharide mixtures containing agar oligosaccharides was dominated not only by electrostatic interactions between casein particles and anionic polysaccharides, but also by the three-dimensional network structure provided by agar oligosaccharides molecules, which could separate casein micelles and hold particles in suspension for a long storage time.

The spontaneous self-assembly of biological macromolecules is of great scientific interest and of significant importance in biological as well as industrial processes. Interestingly, proteins have ...

Casein micelles are present in bovine milk as colloidal particles with diameters of 20-600 nm, which are complex macromolecular assemblies composed of four distinct types of casein and colloidal calcium phosphate (CCP). Multiple structural models of casein micelles have been proposed based on their biochemical or physical properties and observed using electron microscopy. However, the CCP distribution and crosslinking structure between CCP and casein remain unclear. Therefore, the internal structure of casein micelles in raw milk was observed using cryo-electron microscopy of vitreous sections (CEMOVIS) with high precision at high resolution. The results confirmed that the average casein micelle diameter was about 140 nm, and that the CCP diameter in casein micelles was about 2-3 nm, with an average diameter of 2.3 nm. The distribution of CCP in casein micelles was not uniform, with an average interval between CCPs of about 5.4 nm. Areas containing no black particles (attributed to CCP) were present, with an average size of about 19.1 nm. Considering previous reports, these areas possibly correspond to pores or cavities filled with water. Based on differences in the density of structures in casein micelles, we estimated that some of the casein aggregates were able to connect with CCP in a string.

Variation in temperature causes changes in the composition of the casein micelles. Milk samples with 10, 20, 30, 40, and 50% reduction of total β-CN from casein micelles were prepared and analysed. The micelle structure was maintained with minor changes in z-average diameters, zeta potentials, and micellar calcium. During acid gelation decreases in maximum storage modulus, fracture force and firmness were observed with gradual removal of β-CN, but with no significant differences regarding the gelation time among the samples. Decreases in gelation time, maximum G′, fracture force and firmness occurred with gradual removal of β-CN during rennet-induced gelation. Thus, the changes in composition of casein micelle during low temperature treatments had an influence on the gelation properties of bovine milk.

The objectives of this study were to apply response surface methodology to optimize fat-soluble vitamin loading in re-assembled casein micelles, and to evaluate vitamin D stability of dry formulations during ambient or accelerated storage and in fortified fluid skim milk stored under refrigeration. Optimal loading of vitamin A (1.46-1.48mg/100mgcasein) was found at 9.7mM phosphate, 5.5mM citrate and 30.0mM calcium, while optimal loading of vitamin D (1.38-1.46mg/100mg casein) was found at 4.9mM phosphate, 4.0mM citrate and 26.1mM calcium. In general, more vitamin D was retained in vitamin D-re-assembled casein micelles than control powders during storage, while vitamin D loss was not different for vitamin D-re-assembled casein micelles and control fortified milks after 21days of refrigerated storage with light exposure. In conclusion, re-assembled casein micelles with high loading efficiency show promise for improving vitamin D stability during dry storage.

We have reported a simple method for separating lactic acid bacteria (LAB) from yogurt. This method is based on the process of destructions and denaturation of casein micelle aggregates by vortexing, and can supply samples containing only LAB. Recovered LAB were clearly observable by microscopy, meaning that morphological changes could be directly detected at the single-cell level. This method will be a helpful tool for the analyzing various LAB, including their enzyme activity and protein expression.

The effects of heat on the casein proteins and the casein micelles, including their interactions with denatured whey proteins are reviewed. Aspects from chemical changes to amino acid functional groups, hydrolysis of peptide bonds, iso-peptide bonds and protein cross-link formation, reactions of thiol groups/disulphide bonds, fibril formation, interaction of denatured whey proteins with caseins/casein micelles and the dissociation of casein micelles on heating is covered. Where appropriate, the relevance of these heat-induced changes in commercial processing is discussed.

In our preceding study (Part I), the thermal denaturation and aggregation of enriched pea protein fractions, namely vicilin/convicilin 7S (Vic) and legumin 11S (Leg), were investigated in the absence or in presence of casein micelles (CM) at pH ≈ 7.1. The present report (Part II) focuses on the glucono-δ-lactone (GDL) acid-induced gelation of either co-heated proteins in admixture (namely route 1) or thermally-aggregated pea proteins mixed with unheated CM (route 2), while applying a pea protein (either non fractionated PP or Vic or Leg) - to - CM weight ratio of 1: 1 and total protein concentration of 3.6 wt%. The pea protein thermal aggregates obtained by route 1 were of lower size and less soluble than those yielded in isolation. For route 1, gelation of the heated CM – Vic mixture was triggered at a higher pH value and led to higher final storage modulus G′ than corresponding protein samples in isolation. In contrast, the presence of large and sedimentable aggregates in the case of the CM – PP and CM – Leg mixtures impaired gelation. Concerning route 2, either the PP or Vic aggregates mixed with unheated CM resulted in rapid gelation and higher final G’ values than those measured for their single-protein sample counterparts. Viscoelastic properties of the mixed gel depended on the pea protein fraction used, thermal aggregation route, extent of physical interactions between pea proteins during acidification and further involvement of CM. Hence, route 2 would be more reliable than route 1 to produce a “mixed” dairy-like gelled product containing pea proteins with improved texture properties.

By studying the hydration of casein micelles using a variety of techniques, a distinction could be made between water that appeared bound by the protein (∼0.5 g g−1 protein), water associated with the κ-casein brush (∼1.0 g g−1 protein) and water entrapped in the casein micelles (∼1.8 g g−1 protein), yielding a total micellar hydration of ∼3.3 g g−1 protein, in line with casein micelle voluminosity derived from intrinsic viscosity measurements. For caseinate particles, however, the main contributor to intrinsic viscosity was not protein hydration but the non-spherical particle shape. These non-spherical particles in caseinate are likely to be naturally present as primary casein particles (PCP) in casein micelles. PCP could be used to build casein micelles by controlled introduction of micellar salts. Based on the findings of this study, casein micelles could be described as a porous network of non-spherical PCP linked by calcium phosphate nanoclusters.

In this study, skim milk powder (SMP) aqueous dispersions were subjected to a preliminary wet heat incubation at various temperatures (70?90 ?C) to improve the heat stability of recombined filled evaporated milk (RFEM) emulsions. The determination of the free amino group and free lactose content and SDS-PAGE revealed that both whey proteins and caseins were involved in the conjugation with lactose upon preliminary wet heating at different temperatures. The results of the non-sedimentable serum protein content, the particle size and relative viscosity of the SMP dispersion indicated that both association of whey proteins with casein micelles and casein dissociation from the micelles occurred during the incubation. The heat stability of the subsequently produced RFEM emulsions was greatly enhanced: RFEM emulsions could withstand a typical in-container sterilization procedure (i.e. 30 min of heating at 120 ?C) without coagulation (D4,3

Bioactive food components have potential health benefits but are highly susceptible for degradation under adverse conditions such as light, pH, temperature and oxygen. Furthermore, they are known to have poor solubilities, low stabilities and low bioavailabilities in the gastrointestinal tract. Hence, technologies that can retain, protect and enable their targeted delivery are significant to the food industry. Amongst these, microencapsulation of bioactives has emerged as a promising technology. The present review evaluates the potential use of casein micelles (CMs) as a bioactive delivery system. The review discusses in depth how physicochemical and techno-functional properties of CMs can be modified by secondary processing parameters in making them a choice for the delivery of food bioactives in functional foods. CMs are an assembly of four types of caseins, (αs1, αs2, β and κ casein) with calcium phosphate. They possess hydrophobic and hydrophilic properties that make them ideal for encapsulation of food bioactives. In addition, CMs have a self-assembling nature to incorporate bioactives, remarkable surface activity to stabilise emulsions and the ability to bind hydrophobic components when heated. Moreover, CMs can act as natural hydrogels to encapsulate minerals, bind with polymers to form nano capsules and possess pH swelling behaviour for targeted and controlled release of bioactives in the GI tract. Although numerous novel advancements of employing CMs as an effective delivery have been reported in recent years, more comprehensive studies are required to increase the understanding of how variation in structural properties of CMs be utilised to deliver bioactives with different physical, chemical and structural properties.

Properties of yoghurt extremely depend on the application of heat treatment to milk prior to manufacture. After heating the milk, denatured whey protein and casein makes complexes which dramatically improve the yoghurt texture such as firmness and water holding capacity etc. The formation of complexes, micelle bound or soluble whey protein/k-casein complexes, and their localization between casein micelles and serum depends on the physical properties (heat treatment, pH, compositions) of milk. We review the formation of these complexes and their effect on the physical, rheological and microstructural properties of acid milk gels. In order to investigate the interactions of denatured whey protein-casein, the formation of covalent and non-covalent bond namely thiol/disulphide, hydrophobic and electrostatic etc. are reviewed, with the emphasis on acid gelation and final yoghurt texture. This review summarizes the different factors and interactions on the formation of the micelle bound complexes or soluble denatured whey protein/k-casein complexes in heated milk and how these complexes affect the yoghurt properties. This information could be fundamental knowledge for developing a desirable yoghurt texture, may have potential interest for future research.

Background The use of biopolymers for the delivery of functional food ingredients and pharmaceuticals has increased dramatically. In this context a great attention has been paid to caseins and the casein micelle over the years, because of their superior functional qualities and nutritional properties. Scope and approach This review summarizes the unique properties of caseins and the casein micelle and how these properties enhance the successful delivery of sensitive food ingredients and bioactives. This information could be used as a baseline to enhance the capacity of casein and casein micelles in delivering biomolecules through better utilization of their unique properties. Key findings and conclusions Stability of caseins and the casein micelle during heating, freezing, and drying make them valuable in delivering food ingredients and bioactives. However, environmental modifications such as changes in pH perturb the stability of the casein micelle structure. Biological and physical functions of caseins are governed by their composition and flexible conformation. These unique carrying properties of caseins and the casein micelle and their physical and physicochemical changes in response to various environmental conditions such as heat, pH and high pressure can be successfully adapted in delivering functional food ingredients and bioactives in forms of micro-and nanocapsule, emulsions, hydrogels and edible films/coatings.

In this Research Communication we present a study of the effect of Ca-binding salts on the recovery of milk fat globule membrane (MFGM) from buttermilk. Sodium phosphate buffer was used for the purpose of MFGM recovery from buttermilk for the first time and we showed that 0.1 M buffer at pH 7.2 was the most effective for the recovery of MFGM. The fact of high efficacy of sodium phosphate buffer in recovery of MFGM from buttermilk allowed us to suggest that MFGM in buttermilk is present in association with casein through Ca- bridges formed between phospholipids of MFGM and phosphate groups of casein, primarily with k-casein as the peripheral protein of casein micelles.

This research paper addresses the hypothesis that comparative genomics can give a new insight into the functionality of casein genes with respect to the casein micelle. Comparative genomics is a rapidly emerging field in computational biology whereby two or more genomes are compared in order to obtain a global view on genomes as well as assigning previously unknown functions for genes. Casein genes are among the most rapidly evolving mammalian genes, with the gene products mainly grouped into four types (αs1-, αs2-, β- and κ-casein). Functionally, casein genes are central to the casein micelle, the exact structure of which is still a subject of intense debate. Moreover, and adding to this complexity, some mammals lack some of the casein genes, although casein micelles have been observed in their milk. This observation has prompted an investigation into the distribution of casein genes across a host of mammalian species. It was apparent from this study that casein gene sequences are very diverse from each other and we confirmed that many mammalian species lack one or more of the casein genes. The genes encoding β- and κ-caseins are present in most mammals whereas α-casein encoding genes are less represented. This suggests different mechanisms for casein micelle formation in different species as well as the functions that are assigned to each individual casein.

In bovine milk, caseins are considered as the dominant proteins, which in total accounts for 80% of the total protein content. With balanced hydrophobic and hydrophilic amino acid chains, caseins can self-assemble into micellar forms at nanoscale and can be used as an effective carrier for delivery of hydrophobic bioactive compounds. Due to their random coil structures, caseins possess excellent stability during thermal processing, enabling their applications in food and beverages. In addition, the recent studies and understanding of the physicochemical properties of individual casein proteins have expanded their potential for other applications. This chapter reviews the physicochemical properties of caseins and casein micelles, available technologies to manipulate nanostructures of caseins, and the utilization of caseins for delivery of bioactive compounds as functional food ingredients.

This study aimed to determine the interactive effects of casein, whey proteins and calcium ions on the rheological properties of calcium-induced skim milk gels. Skim milk blends with different casein to whey protein ratios (95:5, 93:7, 90:10, 85:15 and 80:20) were either not preheated or preheated at 90 °C for 10 min, and calcium chloride was added at 0–40 mmol L−1. Results suggested that the final gel strength (G′) was dependent on the concentration of added calcium chloride, ratio of casein to whey proteins, and whether the whey proteins were denatured by preheat treatment. Denatured whey proteins in preheated skim milk blends appeared to lead to two competing effects on the gel strength: reduced gel strength due to competition with casein micelles for calcium ion binding; and enhanced gel strength by participating in the gel structure through aggregation via calcium-bridging, hydrophobic interactions, and disulphide bonding.

Protein-based hydrogels have received attention for biomedical applications and tissue engineering because they are biocompatible and abundant. However, the poor mechanical properties of these hydrogels remain a hurdle for practical use. We have developed a highly stretchable and notch-insensitive hydrogel by integrating casein micelles into polyacrylamide (PAAm) networks. In the casein-PAAm hybrid gels, casein micelles and polyacrylamide chains synergistically enhance the mechanical properties. Casein-PAAm hybrid gels are highly stretchable, stretching to more than 35 times their initial length under uniaxial tension. The hybrid gels are notch-insensitive and tough with a fracture energy of approximately 3000 J/m2. A new mechanism of energy dissipation that includes friction between casein micelles and plastic deformation of casein micelles was suggested.

The effect of addition of low and high acyl gellan (LAG/HAG) at three different concentrations on the microstructure and texture of acidified milk systems with different casein to whey protein ratios was investigated. The systems with added LAG exhibited a continuous gel network whereas micro-phase separation occurred when HAG was added. The continuous gel network was indicative of complexation between the polysaccharides and casein, which in turn led to enhanced textural properties and reduced syneresis. Those properties were reduced by decreasing the casein to whey protein ratio as fewer complexes were formed. HAG appeared to be incompatible with casein micelles, which resulted in a micro-phase separated system. This caused decreased textural properties but a higher physical stability due to HAG present in the serum phase available to bind water. By decreasing the casein to whey protein ratio the textural properties were improved at expense of the physical stability, indicating a preferential binding between HAG and whey proteins.

This study examined the milk quality of the autochthonous cattle breed Modenese (MO) in comparison with Italian Friesian (IF), at peak and mid lactation, with the aim to support the interest in sustainability of the local cattle breeds in Europe. Forty-eight individual milk samples were collected from 11 MO and 14 IF pluriparous cows, housed in a free stall barn under similar conditions of feeding and management, at 8 and 21 weeks post-calving; daily milk yield (MY) and body condition score (BCS) were individually recorded. Breed differences were observed, being MY lower in MO cows, but BCS, titratable acidity, freezing point, casein (MC), total calcium (Ca), total phosphorus (P) and colloidal P contents were higher compared to IF cows. Time affected BCS, MY, milk protein (MP), MC, urea, casein P and the micellar content of colloidal Ca and colloidal inorganic P. A factorial analysis was performed and four common factors were obtained with a cumulative explained variance of 77.7% of the total original. MO milk showed a nutritionally interesting mineral profile and processing properties suitable for the production of typical cheese that could be interesting for improving the interest in the conservation of animal genetic resources.

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.

Caseins display the major protein fraction in milk, and thus, significantly impact on milk's techno-functional properties such as foaming. As the micellar structure is mainly stabilized by hydrophobic as well as electrostatic forces, a gradual increase in pH value from 6.0 to 11.0 induced pronounced structural modifications. In consequence of alkalinisation, micelle size and composition changed. This had an impact on the solution properties such as turbidity. Furthermore, interfacial characteristics, e.g. surface pressure evolution and surface dilatational properties were affected. Different situations occurred regarding interfacial covering, which was also reflected in the resulting foam structures. The highest foam stability was obtained for pH 9.0. At this pH, highly voluminous casein micelles combined with a considerable amount of serum casein formed maximal viscoelastic surface layers. Micelle dissociation reduced viscoelasticity as well as foam stability, whilst drainage increased.

Cryoconcentration combined with a cascade effect was used to concentrate skim milk up to 25.12% total dry matter. Size, shape, and inter-micellar distance of casein micelles were characterized by ZetasizerNano-ZS, transmission electron microscopy, and ImageJ analyses. Flow properties of the cryoconcentrated skim milk were evaluated during 5 weeks of storage under refrigerated condition at 4 °C. Milk color was also evaluated according to the L *, a *, and b * system. The cryoconcentrated skim milk obtained after three cryoconcentration cycles was characterized by a monomodal distribution of its micelles with a tendency to smaller casein micelles. Approximately 60% of the total micellar volume was occupied by the casein micelles with a size of 100–200 nm, less than 18% of the volume with a size of 50–100 nm and only less than 1% was occupied by micelles with a size > 350 nm. This result shows that cryoconcentration changed the distribution of the mean size of the casein micelles to smaller units. No significant difference was observed on the inter-micellar distance. Cryoconcentration significantly improved the color of skim milk by increasing the L * value up to 67 which was similar to that of whole milk. Transition from a Newtonian to a non-Newtonian behavior was observed from the fourth week storage with a slight increase of casein micelle size. Industrial relevance A concentration procedure of skim milk based on a complete block cryoconcentration technique was proposed. Application of this sub-zero technology permitted the concentration of skim milk total dry matter up to 25%. The casein micelle size was positively affected by moving the major part of the micelles toward the smaller size, whereas the inter-micellar distance was not affected. This new knowledge can be exploited in milk-based products to enhance the product stability. The cryoconcentrated skim milk color was positively affected since its L * value, which represents the milk whiteness, was significantly improved. The flow behavior of the cryoconcentrated milk was of Newtonian type up to 4 weeks of storage at 4 °C. The generated knowledge in this study can be easily used by the milk processing industry in order to make stable milk product with high dry matter content without adding milk powder, which negatively affects the product sensory properties (floury consistency).

Milk protein solutions [10% protein, 40/60 whey protein/casein ratio containing whey protein concentrate (WPC) and low-heat or high-heat milk protein concentrate (MPC)] containing fat (4% or 14%) and 70–80% water, form gels with interesting textural and functional properties if heated at high temperatures (90 °C, 15 min; 110 °C, 20 min) without stirring. Adjustment of pH before heating (HCl or glucono-δ-lactone) produces soft, spoonable gels at pH 6.25–6.6, but very firm, cuttable gels at pH 5.25–6.0. Gels made with low-heat MPC, WPC and low fat gave some syneresis; high-fat gels were slightly firmer than low-fat gels. Citrate markedly reduced gel firmness; adding calcium had little effect on firmness, but increased syneresis of low-heat MPC/WPC gels. The gels showed resistance to melting, and could be boiled or fried without flowing. Microstructural analysis indicated a network structure of casein micelles and fat globules interlinked by denatured whey proteins.

Kappa-casein (κCN) and beta-casein (βCN) are disordered proteins present in mammalian milk. In vitro, βCN self-assembles into core-shell micelles. κCN self assembles into similar micelles, as well as into amyloid-like fibrils. Recent studies indicate that fibrillization can be suppressed by mixing βCN and κCN, but the mechanism of fibril inhibition has not been identified. Examining the interactions of native and reduced kappa-caseins (N-κCN and R-κCN) with βCN, we expose a competition between two different self-assembly processes: micellization and fibrillization. Quite surprisingly, however, we find significant qualitative and quantitative differences in the self-assembly between the native and reduced κCN forms. Specifically, thermodynamic analysis reveals exothermic demicellization for βCN and its mixtures with R-κCN, as opposed to endothermic demicellization of N-κCN and its mixtures with βCN at the same temperature. Furthermore, with time, R-κCN/βCN mixtures undergo phase separation into pure βCN micelles and R-κCN fibrils, while in the N-κCN/βCN mixtures fibril formation is considerably delayed and mixed micelles persist for longer periods of time. Fibrils formed in N-κCN/βCN mixtures are shorter and more flexible than those formed in R-κCN/βCN systems. Interestingly, in the N-κCN/βCN mixtures, the sugar moieties of N-κCN oligomers seem to organize on the mixed micelles surface in a manner similar to the organization of κCN in milk casein micelles.

Skim milk powders with various levels of sodium hexametaphosphate (NaHMP) were prepared. Reconstituted skim milk samples were prepared from these powders. NaHMP slightly reduced the pH, markedly reduced the serum and ionic calcium and markedly increased the serum phase orthophosphate levels of the milks. This shift in the mineral equilibrium resulted in a drastic reduction in casein micelle integrity, with a marked dissociation of casein from the micelles. κ-Casein was the predominant casein dissociated, although significant levels of α S -casein and β-casein were also transferred to the serum phase. This dissociation of the casein micelles caused a marked decrease in size and scattering properties of the casein micelles. In addition, a small decrease in the zeta potential of the casein micelles in the milk was observed. Heat treatment of the milks with added NaHMP induced further dissociation of κ-casein, although much of the α S -casein and β-casein re-associated with the micelles.

Casein micelle hydrogels were developed using transglutaminase (Tgase) as a crosslink agent in order to encapsulate anthocyanins from jaboticaba fruit (Myrciaria cauliflora). Spray dried casein micelles (CMs) powder was rehydrated in ultrapure water at a concentration of 4.5% w/w, and Tgase was added at 3 units/g of casein. The suspensions were incubated at 45 °C for 1 h, followed by enzyme deactivation at 85 °C for 5 min. Jaboticaba extract (JE), obtained from jaboticaba peel, was added to the suspensions at a concentration of 2% (w/w) at 25 °C. In the suspensions, Tgase promoted a reduction in CMs size and an increase in the degree of casein polymerization. The presence of JE did not affect CMs size or charge. The hydrogel samples were produced by acidification of the suspensions using 2% w/w of glucono – δ – lactone until pH 4.5 at 30 °C. The hydrogels were analyzed using small deformation rheology and confocal laser scanning microscopy. Tgase treatment promoted the formation of a more compact protein matrix compared to the samples without the enzyme. The presence of JE decreased hydrogel elasticity and increased hydrogel pore size. The JE release profile was evaluated by immersing the hydrogels in three buffer solutions at pH 2.0, 4.5 and 7.0. The release rates for the hydrogels with Tgase were lower for all pH values. Solutions with higher pH values induced faster release rates. These findings can be applied to specific delivery systems, such as the transport of JE in an intestinal environment.