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11.10.12 Milk Composition Dairy Proteins Cows Milk, a “3, 4, 5 Food” (approx.) 86% water Milk contains three phases: Milk Proteins (Cows’ Milk) Total Protein Total Caseins αs1 αs2 β κ (kappa) Total Whey Proteins α-lactalbumin β-lactoglobulin Serum Albumin Immunoglobulins Proteose peptone g/L 33 26 10 2.6 9.3 3.3 6.3 1.2 3.2 0.4 0.7 0.8 % of total protein 100 79.5 30.6 8.0 28.4 10.1 19.3 3.7 9.8 1.2 2.1 2.4 3% protein 4% fat 5% lactose Liquid, colloid & emulsion proteins are present in every phase Emulsion phase - the milk fat globule 95% of milk lipid is present in globules, mostly triglycerides Ensheathed in the membrane of the secreting cell, including its phospholipids and membrane proteins Serum proteins may bind, especially after homogenization (which decreases the size of the mfg, ∴increases surface area) € MFG Proteome ~120 proteins found in Cows’ milk, seem different than those of human milk. membrane/protein trafficking (23%) cell signaling (23%) for secretion? unknown functions (21%) fat transport/metabolism (11%) transport (9%) protein synthesis/folding (7%) immune proteins (4%) milk (serum) proteins (2%) Milk Protein Fractions-Traditional Skim Milk Filtrate pH 4.6 precipitate Whey or serum Casein 20% of protein 80% of protein 50%sat ammonium sulfate precipitate Lactoglobulins 12% of proteins Filtrate Lactalbumins + proteose peptone +NPN 8% of protein In blood: albumins are soluble in water & dilute salt; globulins precipitate in DI water 1 11.10.12 Milk Protein Commercial Fractions Skim Milk pH 4.6 Casein Curd Chymosin Heat, Ca++ &/or acid Casein Curd Co-precipitate Acid Casein Caseinate Heat aggregation Lactalbumin Aqueous Phase (Whey) Proteins β-Lactoglobulin α-Lactalbumin Bovine Serum Albumin (BSA) Immunoglobulins, esp in colostrum Proteose Peptones Rennet Casein Whey ultrafiltration Ion exchange Whey Prot Conc Whey Prot Isol Most abundant (absent in human milk) Ca++ binding Nutritional effects? Passive immunity transfer to calf Smallish glycosylated miniproteins Comparison - Whey Proteins β-Lactoglobulin Property α‑La‑B Molecular Weight 14,176 Amino acids 123 Proline 2 Cys 8 ‑S‑S‑ 4 Phospho‑ 0 Carbohydrate Hydrophobicity (kJ/res) 4.7 Charge mol % residues 28 Net charge/residue ‑0.02 Charge distribution even β-Lactoglobulin β‑LG‑B 18,363 162 8 5 2 0 5.1 30 ‑0.04 even BSA 66,267 582 34 35 17 0 0 4.3 34 ‑0.02 α-Lactalbumin • β-barrel, binding site inside • Hydrophobic on the inside • Small amount of helix • 5 Cys (1 -SH left over) Nearly half of total whey proteins Dominates T behavior β-barrel protein (“Lipocalin”) Exists as dimers (octamers @ pH ~4) Stable @ pH 2, where it is a monomer Binds lipids, retinol, but not in milk 8 genetic variants, mostly -A, -B Not present in human milk - immunogenic? Less functional than suggested by composition Very similar to lysozyme structure, except pI Two major genetic variants, -A and -B Binds Ca++ 8 Cys (4 S-S bonds), 4 Trp Relatively heat stable, releases Ca ion at pH <4 Modifier subunit of Lactose Synthase 2 11.10.12 α-Lactalbumin Very similar to lysozyme structure, except pI Two major genetic variants, -A and -B Binds Ca++ 8 Cys (4 S-S bonds), 4 Trp Proteose Peptones BSA & IG Bovine Serum Albumin binds lipids & metal ions In blood, may aid lipid transport Immunoglobulins - passive immunity Lease heat-stable proteins in milk IGG IGA IGM Properties of the αs-Caseins αS-casein ppts from micelle in 0.4M CaCl2, from solution in 10mM Ca++ αS1-casein - major component 2 hydrophobic, one polar region αS2-casein strongly negative N-terminus, positive Cterminus Acid-soluble, heat-stable, highly functional fraction Smallish peptides, some are fragments of βcasein, some glycosylated Component 3 (most hydrophobic) Component 5 (1-105) Component 8-fast (1-28) Component 8-slow (29-105) Caseins Property αSl‑B Molecular Weight 23,614 Amino acids/Molec 199 Proline 17 Cys 0 ‑S‑S‑ 0 Phospho‑ 8 Carbohydrate 0 Hydrophobicity (kJ/res) 4.9 Charge mol % residues 34 Net charge/residue ‑0.10 Charge distribution uneven αSl -A 25,230 207 10 2 ? 11 0 4.7 36 ‑0.07 uneven β‑A2 23,983 209 35 0 0 5 0 5.6 23 ‑0.06 very uneven k‑B 19,023 169 20 2 ? 1 + 5.1 21 ‑0.02 very‑uneven Properties of the Other Caseins β-casein Phosphoprotein Neg charge on N-term, neutral elsewhere 35 Pro residues k-casein - Micelle stabilizer γ-casein (& others) Glycoprotein, phosphoprotein, on C-terminal side C-terminal fragment of β-casein 3 11.10.12 Casein Micelle - Composition Component αs1-Casein αs2-Casein β-Casein k-Casein ϒ- & others Calcium Phosphate g/100g micelle 35.6 10 34 12 ~3 2.9 2.9 Casein Micelle - Structure The conc of Ca++ and HnPO4 exceeds the solubility product of several CaPO4 salts Micelle Cartoon Caseins lack rigid structure Associate with hydrophobic regions on the inside, phosphoserine residues on the outside (sub-micelles) Polar portion of k-casein holds -OPO3-2 apart Submicelles are crosslinked by Ca(PO4)6 chains bridging between -OPO3-2 groups on submicelles Digestible protein, available Ca++ & Phosphate Clotting of Milk core OPO3 Ser -casein Ca9(PO4)6 OPO3 -casein Ser OPO3 Ca9(PO4)6 OPO3 Ser Acid (HCl, HOAc, Lactic acid…) Ser Chymosin (works better at low pH) core Isoelectric precipitation of caseins Solubilization of Ca++ Clips k-casein between F105-M106 Disruption of micelle structure Precipitation of by Ca++ Coprecipitation with other caseins Further (slower) proteolysis in curd Enzymes Xanthine Oxidase Phosphatases Lactoferrin, Transferrin Lactoperoxidase Plasmin Catalase Lipases 4