Milk Proteins PDF

Milk proteins Milk proteins are natural vehicles for probiotics cells and, owing to their structural and physico-chemical properties, they can be used as a delivery system. There are 3 or 4 caseins in the milk of most species; the different caseins are distinct molecules but are similar in structur...

Milk proteins Milk proteins are natural vehicles for probiotics cells and, owing to their structural and physico-chemical properties, they can be used as a delivery system. There are 3 or 4 caseins in the milk of most species; the different caseins are distinct molecules but are similar in structure. All other proteins found in milk are grouped together under the name of whey proteins. The major whey proteins in cow milk are beta-lactoglobulin and alpha-lactalbumin. For example, the proteins have excellent gelation properties and this specificity has been recently exploited to encapsulate probiotic cells. The results of these studies are promising and using milk proteins is an interesting method because of their biocompatibility. María Encarnación Morales, María Adolfina Ruiz, in Nutraceuticals, 2016  Introduction Normal bovine milk contains about 3.5% protein The concentration changes significantly during lactation Especially during the first few days post-partum (Figure 4.1); the greatest change occurs in the whey protein fraction (Figure 4.2)  The properties of many dairy products: – depend on the properties of milk proteins (also the fat, lactose and the salts). Casein [kéisi:n] products are almost exclusively milk protein The high heat treatments are possible – because of the high heat stability of the principal milk proteins, the caseins Thermization is used to extend the keeping quality of raw milk (the length of time that milk is suitable for consumption) when it cannot be immediately used in other products, such as cheese.  Heterogeneity of milk proteins On acidification to pH 4.6 (at around 30°C) – about 80% of the total protein in bovine milk precipitates out of solution; this fraction is casein – The protein which remains soluble under these conditions: whey protein or non-casein nitrogen.  Heterogeneity of milk proteins There are several major differences between the caseins and whey proteins: *Rennet is a complex of enzymes – In contrast to the caseins, the whey proteins do not precipitate from produced in any mammalian solution when the pH of milk is adjusted to 4.6 stomach. Rennet contains many – Only the casein fraction of milk protein is normally incorporated into enzymes, including a protease that coagulates the milk, causing cheese products it to separate into solids (curds) – Chymosin(rennin) and some other proteinases (enzymes in rennets) and liquid (whey) produce a very slight, specific change in casein, resulting in its coagulation in the presence of Ca2+ – Whey proteins undergo no such alteration – The substrate of chymosin is K-casein which is specifically cleaved at the peptide bond between amino acid residues 105 and 106, phenylalanine and methionine – The coagulability(응집성) of casein through the action of rennets is utilized in the manufacture of most cheese products Rennet-induced casein micelle aggregation model  Heterogeneity of milk proteins – Casein is very stable to high temperatures; Whey protein is heat sensitive – Caseins are phospho-proteins, containing (~0.85% phosphorus); Whey proteins contain no phosphorus – The phosphate groups of casein can bind to calcium which is nutritionally valuable – Casein is low in sulphur (0.8%); whey proteins are relatively rich (1.7%) – The sulphur of casein is present mainly in methionine (Sulphur-containing essential amino acids) – Whey proteins contain significant amounts of sulphur in cysteine, cystine (cysteine-S-S-cysteine), and methionine  Heterogeneity of milk proteins – Casein is synthesized in the mammary gland and is found nowhere else in nature – Whey proteins are molecularly dispersed in solution – The caseins have a complicated quaternary structure and exist in milk as large colloidal aggregates – Both the casein and whey protein groups are heterogeneous, each containing several different proteins.  Preparation of casein and whey proteins Skim milk is used as the starting material for the preparation of casein and whey proteins – Acid precipitation Acidification of milk to about pH 4.6 induces coagulation of the casein For laboratory-scale production of casein, HCl is usually used for acidification; acetic or lactic acids are used less frequently The whey proteins may be recovered from the whey by salting out(염석), dialysis(투석) or ultrafiltration (한외여과) – Centrifugation Most (90-95%) of the casein in milk is sedimented by centrifugation at 100,000 g for 1 h – Centrifugation of calcium-supplemented milk Addition of CaCl2 (0.2 M) causes aggregation of the casein; it can be readily removed by low-speed centrifugation – Salting-out methods Addition of (NH4)2SO4, MgSO4, NaCl to milk  Preparation of casein and whey proteins – Ultrafiltration The casein micelles are retained by fine-pore filters – Gel fltration Filtration through cross-linked dextrans (a complex, branched glucan (polysaccharide made of many glucose molecules)) – Precipitation with ethanol 40% ethanol – Cryoprecipitation Casein, in a mainly micellar form, is destabilized and precipitated by freezing milk at about - 10°C – Rennet coagulation – Other methods for the preparation of whey proteins Ion exchange chromatography: isolates (containing 90-95% protein), industrially used Dialysis Gel-filtration Ultracentrifugation Ion-exchange chromatography  Heterogeneity and fractionation of casein β-, γ-, κ-, αs- (αs1 -, αs2-) casein αs1-, αs2-, β-, κ-caseins which represent 37, 10, 35, and 12% of whole casein, respectively Fractionation of the casein: ion-exchange chromatography (DEAE-cellulose). High performance ion-exchange gives excellent results for small amounts of sample Resolution of caseins by electrophoresis. Casein can resolve into about 20 bands. Electrophoresis in polyacrylamide gels (PAGE) Electrophoresis (SDS-PAGE)  Microheterogeneity of the caseins Variability in the degree of phosphorylation Disulphide (S-S) bonding – αs1-, β-casein: no cystein or cystine – αs2-, κ-casein: two cysteins per mole exist as disulphide bonds Hydrolysis of primary caseins by plasmin (an important enzyme present in blood and milk that degrades many blood plasma proteins) – γ-Caseins are produced from β-casein by proteolysis by plasmin (an indigenous proteinase in milk) – Isolated αs2-casein in solution is also very susceptible to plasmin Variations in the degree of glycosylation (a carbohydrate is attached to a hydroxyl or other functional group of another molecule) – κ-Casein is the only one of the principal milk proteins which is normally glycosylated Genetic polymorphism (다형성) – Whey protein, β-lactoglobulin (β-lg), exists in two forms, A and B, which differ from each other by only a few amino acids  Some important properties of the caseins Amino acid composition – All the caseins have a high content (35-45%) of non-polar amino acids (Val, Leu, Ile, Phe, Try, Pro) → poorly soluble in aqueous systems – The high content of phosphate groups, low level of sulphur- Sodium caseinate powder can be used containing amino acids and high carbohydrate content in the case in a variety of foods, including: of K-casein offset the influence of non-polar amino acids → protein powder coffee creamer soluble in aqueous systems cheese ice cream cheese-flavored snacks margarine – The high viscosity is due to the high water binding capacity (WBC) cereal bars of casein (about 2.5 g H2O/g of protein) processed meats chocolate → Such high WBC gives casein very desirable functional properties for bread incorporation into various foods What Is Sodium Caseinate And How Its Made? -Milk is curdled by adding enzymes or an acidic substance such as lemon juice or vinegar. -The solid curds are separated from the whey, which is the liquid part of the milk. -Once the curd has been separated, they are treated with an alkali called sodium hydroxide, and are then dried and formed into a powder. -This product is called sodium caseinate, which is extracted from casein and contains 90% protein. -Casein and sodium caseinate are almost the same products and can be used in the same manner, but they vary on a chemical level. Uses Of Sodium Caseinate Protein supplement - This sodium caseinate powder can be used as a protein powder because it provides a rich source of high quality protein. It contains 90% protein. This essential nutrient is required for the body for building and repairing muscle tissues, improving bone health and boosting metabolism. As sodium caseinate is high in protein, it would make an excellent protein supplement choice among athletes and people involved in strength training. Sodium Caseinate Food additive - In the food industry, sodium caseinate is used as a food additive. It can be used to change the texture and stabilise many kinds of food products such as ice cream, cheese, coffee creamer, cereal bars, chocolate, bread, margarine, cheese-flavored snacks and processed meats – All the caseins have a very high proline content → very low content of α-helix or β-sheet structures in the caseins (due to inhibiting hydrogen bond for amino- and carboxyl- groups of the backbone) → readily susceptible to proteolysis → this is an important characteristic in neonatal nutrition. – As a group, the caseins are deficient in sulphur amino acids which limits their biological value. (the principal sulphydryl-containing protein in milk is the whey protein β-lactoglobulin). – Sulphur: in metabolic reactions, serve as electron donors and electron acceptors. Present in vitamins. Important part in enzymes and in antioxidant molecules(glutathione). – The caseins, especially αs2-casein, are rich in lysine, an essential amino acid in which many plant proteins are deficient. Proline  Primary structure of casein – All caseins (polar and non-polar residues) are not uniformly distributed but occur in clusters, giving hydrophobic region and hydrophilic regions. – This feature makes the caseins good emulsifiers. (Emulsion is a mixture of two or more liquids that are normally non-mixable)  Casein phosphorus – Milk contains about 900 mg phosphorus/L – Casein contains about 0.85% phosphorus inorganic: soluble and colloidal phosphates organic: phospholipids, casein and sugar phosphates, nucleotides – Phosphorus is important: 1) nutritionally because it can bind large amounts of Ca2+, Zn2+ and other metals 2) increases the solubility of caseins 3) high heat stability of casein 4) important in the coagulation of rennet-altered casein – The phosphorus is covalently bound to the protein – Phosphate is bound mainly to serine – Phosphorylation occurs in the Golgi membrane  Casein carbohydrate – αs1-, αs2, β-caseins contain no carbohydrate – κ-casein contains about 5% carbohydrate (N-acetylneuraminic acid (sialic acid), galactose and N-acetylgalactosamine), Glycosylation – The carbohydrate provide on κ-casein quite high solubility and hydrophilicity  Secondary and tertiary structures of casein Caseins have relatively little secondary or tertiary structure, probably due to the presence of high levels of proline residues, especially in β-casein, which disrupt α-helix and β-sheet. The lack of secondary and tertiary structures is probably significant for the following reasons: – 1) The caseins are readily susceptible to proteolysis This has obvious advantages for the digestibility of the caseins, nutritionally. The caseins are also readily hydrolysed in cheese, which is important for the development of cheese flavor and texture – 2) The caseins adsorb readily at air-water and oil-water interfaces due to a) their open structure b) relatively high content of non-polar amino acid residues c) the uneven distribution of amino acids. This gives the caseins very good emulsifying and foaming properties, which are widely exploited in the food industry. – 3) The lack of higher structures probably explains the high stability of the caseins to denaturing agents, including heat  Molecular size of casein – All the caseins are relatively small molecules, ranging in molecular weight from about 20 to 25 kDa Hydrophobicity – The caseins are often considered to be rather hydrophobic molecules Influence of Ca2+ on caseins – αs1- and αs2-caseins are insoluble in calcium-containing solutions – β-casein is soluble at high concentrations of Ca2+ (0.4M) at temperatures below 18°C – κ-casein is soluble in Ca2+ at all concentrations.  Action of rennets on casein – κ-casein is the only major casein hydrolysed by rennets during the primary phase of milk coagulation (in cheese making) Casein association – κ-casein is present largely as disulphide-linked polymers – At 4 °C, β-casein exists in solution as monomers; at 8.5°C, 20 monomers chains – αs1-casein polymerizes to form tetramers of molecular mass, 113 kDa – The major caseins interact with each other and, in the presence of Ca2+ these associations lead to the formation of casein micelles. Casein Micelle Structure - Composition and general features – About 95% of the casein exists in milk as large colloidal particles, known as micelles (colloid is a substance dispersed throughout another substance) – On a dry matter basis, casein micelles contain 94% protein and 6% low molecular weight species (calcium, magnesium, phosphate and citrate) – The micelles are highly hydrated, binding about 2.0g of H2O/g protein – Casein micelles are generally spherical in shape, with diameters ranging from 50 to 500nm (average 120nm) – There are 1014-1016 micelles/ml milk; they are roughly two micelle diameters (240nm) apart. – The surface area of the micelles is very large; the surface properties of the micelles are critical to their behavior. – The white color of milk is due largely to light scattering by the casein micelles 2) Stability of micelles – stable to the normal processes (very stable at high temperatures) – can be sedimented by ultracentrifugation and re-dispersed readily by mild agitation. – stable to commercial homogenization – stable to high [Ca2+], up to at least 200mM at temperatures up to 50°C – aggregate and precipitate from solution when the pH is adjusted to the isoelectric point of caseins (pH4.6) – Many proteinases catalyse the hydrolysis of a specific bond in κ-casein – destabilized by 40% ethanol at pH 6.7 and by lower concentrations if the pH is reduced – destabilized by freezing due to a decrease in pH and an increase in the [Ca2+] in the unfrozen phase of milk 3) Principal micelle characteristics – Knowledge of micelle structure is important because the stability and behavior of the micelles are central to many dairy processing operations, (e.g., cheese manufacture, stability of sterilized sweetened-condensed and reconstituted milks and frozen products) – κ-Casein, which represents about 15% of total casein, is a critical feature of micelle structure and stability. Stabilize calcium-sensitive αs1, αs2 and β-caseins (85% of total casein) – Micelle has a porous structure in which the protein occupies about 25% of the total volume – Chymosin and similar proteinases specifically hydrolyse most of the micellar κ- casein – When heated in the presence of whey proteins (as in normal milk), κ-casein and β- lactoglobulin interact to form a disulphide-linked complex → modify rennet coagulability and heat stability – The micelles can be destabilized by alcohols and acetone-electrostatic interactions in micelle structure – As the temperature is lowered, β-casein dissociate from the micelles 4) Micelle structure- subunit (submicelles) Schmidt, Walstra, Ono: The κ-casein content of the submicelles varies and that the κ-casein deficient submicelles are located in the interior of the micelles with the κ-casein-rich submicelles concentrated at the surface Some αs1, αs2 and β-caseins are also exposed on the surface  Because the micelles are closely packed, inter-micellar collisions are frequent; however, the micelles do not normally remain together after collisions The micelles are stabilized by two principal factors: – (1) a surface (zeta) potential of -20mV at pH 6.7, which is probably too small for colloidal stability (0에 가 까울수록 안정성 떨어짐) – (2) steric stabilization (입체적 안전성) due to the protruding κ-casein hairs Zeta potential is the potential difference between the dispersion medium and the stationary layer of fluid attached to the dispersed particle Liquid system에서 분산되어 있는 파티클 (콜로이드 상태)은 전기적인 charge를 가짐 수용액에서 이온을 가져와서 생김 전기적으로 음성 또는 양성을 띰 Casein micelles in milk Casein micelles form Casein micelles chains in yogurt form clusters in cheese curd  Whey proteins About 20% of the total protein of bovine milk: whey or serum proteins or non-casein nitrogen Preparation – 1. the proteins remaining soluble at pH 4.6 – 2. soluble in saturated NaCl – 3. soluble after rennet coagulation of the caseins – 4. by gel permeation chromatography – 5. by ultracentrifugation, with or without added Ca2+  On a commercial scale, whey protein-rich products are prepared by: 1. Ultrafiltration after acid or rennet whey to remove varying amounts of lactose, and spray-drying to produce whey protein concentrates (30-80% protein) 2. Ion-exchange chromatography: proteins are adsorbed on an ion exchanger, washed free of lactose and salts and then eluted by pH adjustment. The elute is free of salts by ultrafiltration and spray-dried to yield whey protein isolate, containing about 95% protein 3. Demineralization by electrodialysis and/or ion exchange, thermal evaporation of water and crystallization of lactose 4. Thermal denaturation, recovery of precipitated protein by filtration/centrifugation and spray-drying, to yield lactalbumin which has very low solubility and limited functionality - Biological value (BV) is a measure of the proportion of absorbed protein from a food which becomes incorporated into the proteins of the organism's body. - It captures how readily the digested protein can be used in protein synthesis in the cells of the organism. Heterogeneity of whey proteins – Whey protein contained two groups of proteins which could be fractionated by saturated MgSO4, or half saturated (NH4)2SO4 → the precipitate (roughly 20% of total N) was referred to as lactoglobulin and the soluble protein as lactalbumin – The lactoglobulin fraction consists mainly of immunoglobulins (Ig) – The lactalbumin fraction of bovine milk contains three main proteins, β- lactoglobulin (β-lg, 50%), α-lactalbumin (α-la, 20%) and blood serum albumin (BSA, 10%) – And trace amount of other proteins (lactotransferrin, serotransferrin and several enzymes)  β-Lactoglobulin Occurrence and microheterogeneity – β-Lactoglobulin (globular protein) is a major protein in bovine milk, representing about 50% of total whey protein and 12% of the total protein of milk – β-lg is the principal whey protein (WP) in bovine: four genetic variants of bovine β-lg, designated A, B, C and D Amino acid composition – Rich in sulphur amino acids which give it a high biological value – The cysteine is especially important since it reacts with the disulphide of κ-casein and significantly affects rennet coagulation following heat denaturation and the heat stability properties of milk; it is also responsible for the cooked flavor of heated milk – Heat denaturing the whey protein triggers hydrophobic interactions with other proteins, and the formation of a protein gel  Primary structure – Bovine β-lg: 162 residues per monomer. Secondary structure – Highly structured: 10-15% α-helix, 43% β-sheet, – 47% unordered structure Tertiary structure – X-ray crystallography: compact globular structure Quaternary structure  Physiological function – β-Lactoglobulin exhibits a number of biological activities, including antiviral, prevention of pathogen adhesion, anticarcinogenic and hypocholesterolemic effects. – β-lg has the ability to bind hydrophobic components, for example retinol and long-chain fatty acids. – Bind ligands and transport small hydrophobic ligands: β-lg has multiple ligand binding sites, β-lg is capable of binding to vitamins A and D, palmitic acid, and other hydrophobic compounds. – β-lg exhibits strong binding affinities for fatty acids, phospholipids, and aromatics compounds. – Binding of these molecules to β-lg can alter their biological activities. – Examples of this include angiotensin-converting enzyme (ACE) inhibition, alterations in antimicrobial and anticarcinogenic activity, hypocholesterolemic and metabolic effects, and modulation of other physiological functions. – Due to its ability to bind hydrophobic molecules, β-lg was used to improve the encapsulation properties of liposomes and to serve as a stable system for vitamin E delivery.  α-Lactalbumin α-Lactalbumim represents about 20% of the proteins of bovine whey (3.5% of total milk protein) Amino acid composition – Rich in tryptophan, rich in sulphur (1.9%, cysteine) Primary structure – There is considerable homology between the sequence of α-la and lysozymes (gycoside hydrolases, damage bacterial cell walls) from many sources Secondary and tertiary structure – A compact globular protein – The tertiary structure of a-la is very similar to that of lysozyme. Quarternary structure – α-la associates under a variety of environmental conditions but the association process has not been well studied. α-Lactalbumin  Biological function – Role in lactose synthesis: (Uridine diphosphate galactose) – Lactose synthetase consists subunit A (UDP-galactosyl transferase) and B (α-la). – Subunit B specifically transfers galactose to glucose to form lactose – The concentration of lactose in milk is directly related to the concentration of α-la – ALA has the ability to interact with hydrophobic substances such as retinol, vitamin D3, hydrophobic peptides, and fatty acids – ALA based nanoparticles or nanotubes could find applications in foods and pharmaceuticals for delivery of bioactive substances – Immunomodulatory Properties: α-Lactalbumin can have immune-boosting effects. It can bind to and help kill harmful bacteria, supporting the immune system and promoting gut health, especially in newborns. – A complex of α-lactalbumin and oleic acid can selectively induce cell death in tumor cells, suggesting potential as an anticancer agent. – Antioxidant Properties: α-Lactalbumin contains sulfur-containing amino acids like cysteine, which can help boost the body's antioxidant defenses by increasing levels of glutathione, a powerful antioxidant.  Metal binding and heat stability – α-la is a metallo-protein – it binds one Ca2+ per mole in a pocket containing four Aspartic acid residues – the Ca-containing protein is quite heat stable (the protein renatures following heat denaturation) ASP ASP LYS ASP ASP  Blood serum albumin Normal bovine milk contains a low level of blood serum albumin (BSA); presumably as a result of leakage from blood BSA is quite a large molecule (molecular mass 66 kDa; 582 amino acids) It binds metals and fatty acids (stimulate lipase activity)  Immunoglobulins (Ig) Mature milk contains 0.6-1 g/L (3% of total N) Colostrum contains up to 100 g/L; the levels of which decreases rapidly postpartum. There are five classes of Ig: IgA, IgG, IgD, IgE and IgM IgA, IgG and IgM are present in milk. The physiological function of Ig is to provide various types of immunity in the body  Minor milk proteins Milk contains numerous minor proteins, including perhaps 60 enzymes, some of which, lipase, proteinase, phosphatases and lactoperoxidase Comparison of human and bovine milks Milk is species-specific, designed to meet the nutritional and physiological requirements of the young of that species (4,300 species of mammals)  Synthesis and secretion of milk proteins Sources of amino acids – Amino acids for milk protein synthesis are obtained ultimately from blood plasma Amino acid transport into the mammary cell – Since the cell membranes are composed predominantly of lipids, amino acids (which are hydrophilic) cannot enter by diffusion and are transported by special carrier systems (not yet identified). Synthesis of milk proteins – Synthesis of the major milk proteins occurs in the mammary gland – DNA, RNA (mRNA, tRNA, rRNA) – Protein synthesis actually takes place in the ribosomes of the rough endoplasmic reticulum (RER) which contain rRNA. – Serum albumin and some of the immunoglobulins are transferred from the blood  Messenger RNA (mRNA) carries information about a protein sequence to the ribosomes Transfer RNA (tRNA) is a small RNA chain of about 80 nucleotides that transfers a specific amino acid to a growing polypeptide chain at the ribosomal site of protein synthesis Ribosomal RNA (rRNA) is the catalytic component of the ribosomes; rRNA and protein combine to form a nucleoprotein, called a ribosome  Secretion of milk-specific proteins – Synthesis in ribosome→ER lumen→Golgi lumen (cis cisternae→trans cisternae) – Proteins appear to enter the complex at the cis face and progress, undergoing post-translational modification, towards the trans face (achieved by budding and fusion of vesicles) – In the apical cytosol there are numerous protein-containing secretory vesicles – Move to the apical plasma membrane and fuse with it, releasing their contents by exocytosis. Secretory vesicles seem to become attached to the cytoplasmic face of the apical plasma membrane. Secretory vesicle membrane becomes incorporated into the apical membrane as a consequence of exocytosis.  The synthesis and secretion of milk proteins involves eight steps: 1) transcription, 2) translation, 3) segregation, 4) modification, 5) concentration, 6) packaging, 7) storage and 8) exocytosis