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Milk and milk products Milk and milk products Presentation Transcript

  • Milk and Milk products Speaker: Manoj Solanki Dairy Chemistry NDRI, Karnal
  • Definition of Milk?Definition - Normal secretion of themammary gland of mammalsMore precisely it is a very complexproduct that contains more than100,000 chemicals that are either insolution, suspension or emulsion withwater.
  • WHY MILK IN DAILY DIET?• Milk contains more of the known essential nutrients required for health than any other single food.• Milk contains more utilizable calcium and good quality protein.• Casein major milk protein has unique property of influencing its own digestion as well as other proteins fed with it.• Conjugated linoleic acid of milk fat supresses cancer, atheroselerosis and fat deposition in the body.• Milk is an ideal food for elderly due to its high nutritive value.• Milk is both a fast and conveinent food no preparation, immediately ready for consumption.
  • A glass of LOW FAT MILK Regular Soft drink( 250 ml ) Rs. 10.00 ( 300 ml ) Rs.20. 00 Milk Soft drink
  • Main Ingredients Milk Total Milk SolidsWater 87.3 % 100-87.3= 12.7 % Fat 3.9 % Solids-not-fat 8.8 %
  • Composition of MilkWater 87.5%Lactose 4.9%Protein 3.2% Casein 2.9% Lactalbumin 0.52% Lactoglobulin 0.20%Fat 3.7%Minerals 0.72% Calcium 0.12% Phosphorus 0.11% Chlorine 0.11%
  • Other Compounds Normally Found in Milk• Acids – Citrate, Formate, Acetate, Lactate, Oxalate• Enzymes – Peroxidase, Catalase, Lipase, Phosphatase• Gases – Oxygen, Nitrogen, Carbon dioxide• Vitamins – A, C, D, B Complex
  • Composition of various milks (%) : Constituents Cow Milk Buffalo Milk Goat Milk Sheep Milk Human MilkMoisture 87.6 81 85.2 80.7 87.4Protein 3.3/3.4 4.3/3.9 3.7/3.5 4.8 1.6Fat 3.6/4.9 8.8/6.6 5.6/4.5 6 3.75Carbohydrate 4.5/4.1 5.0/5.2 4.7/4.6 4.9 6.98Mineral Matters (Ash) 0.7 .8/.7 0.8 0.8 0.21Calcium 0.12 0.21 0.17 - -Phosphorus 0.09 0.13 0.12 - -Iron (mg/100g) 0.2 0.2 0.3 - -Vitamin A (IU/100g) 180 162 182 189.7 189.9Vitamin B12 (mg/100g) 0.05 0.04 0.04 - -Riboflavin (mg/100g) 0.2 0.19 0.15 - -Vitamin C (mg/100g) 1.6 1.5 1.5 5.5 4Nicotinic acid (mg/100g) 0.1 0.1 0.1 - -Calories 67 117 72 - -Total solids 13.7 17.02 13.5 16.3 12.57Solids-not-fat 8.8 9.2 9 10.3 8.8
  • • Milk has well defined physical equilibrium - Three Phase• Emulsion• Colloids• Solution
  • Milk Composition• Solution - dissolve to the molecular level – Lactose – Minerals• Suspension - Colloid, particles so small they will not settle out – Proteins - Casein• Emulsion - compounds that dont mix with water – Butterfat
  • Milk lipids• oil-in water emulsion• Colloidal Phase • casein micelles • Calcium Phosphate • globular Proteins Whey Protein-colloidal solution Casein-colloidal suspension• True Solution-lactose, vitamins, acids, enzymes and some inorganic salts
  • Milk Fat Tri/Di/Monoglycerides 98-99% Phospholipids Sterols Vitamins A, D, E & K
  • Fat• Impart richness/smoothness• Triglycerides of fatty acids 95-99% milk fat,• DG 4.1% BM and 1.26-1.59% CM,• MG 0.7% BM and 0.016-0.038% CM• FFA 0.22% BM and 0.33%CM
  • Fat Globules
  • Fat Globule Fat Globule membrane Globule range in diameter ( 0.1 - 20µm); Av= 3.5µm. Size & size distribution may be measured by light microscopy, light scattering, electronic counting devices TGS (Coultercounter) 98-99% About 75% of globules are < 1µm dia. Thickness of FGM= 15nm (10-20nm)
  • Milk fat Globules• Covered by a thin membrane from secretory cells (Protein and Phospholipids)• Helps prevent coalescence and flocculation• Protects fat from enzymatic action• Properties enable the production of butter, whipped cream and ice cream• Homogenization – Decreases diameter making them more stable in suspension
  • Milk fat composition:S.No Constituents Range Location1 Triacylglycerol 98 – 99% Fat Globule core2 Phospholipids 0.2 – 1.0% FGM & Serum3 Sterols 0.25 – 0.40% Fat globule, FGM & Serum4 FFA Traces Fat globule & Serum5 Waxes Traces Fat globule6 Squaline Traces Fat globule Fat soluble Vitamins Traces Fat globule Vit - A 7 – 8.5 µg / g fat7 Carotenoids 8 – 10 µg / g fat Vit- E 2 – 50 µg/g fat Fat globule Vit- D Traces Vit-K Traces
  • Major Fatty Acids of Bovine Milk• Milk Fat consist of >200 different FA , only 15 fatty acid occur in proportion greater than 1% of milk fat• Saturated fatty acids (65%) – No double bonds – Related to some health concerns• Monounsaturated fatty acids (25%) – One double bond – Thought to be generally healthy• Polyunsaturated fatty acids (10%) – Greater than one double bond
  • Fatty Acid Profile of milk fat• High conc. of short chain fatty acids (C4:0–C8:0 carbon atoms) Help to maintain a degree of liquidation in the relatively saturated milk fat at body temp, and this may be important for efficient secretion• Appreciable amount of medium chain fatty acids. (C10:0– C14:0 carbon atoms) Absorbed directly via portal blood stream rather than through the lymphatic system, so direct contribution to energy metabolism ( especially brain) of new born.)• Difference in long chain fatty acids (14 – 26 carbon atoms) ability to oxidize long chain fatty acids is not developed fully at birth
  • Importance of Lipids1. Economics: Price of milk, because it is the costliest component of milk.2.Nutritional Function: Rich source of energy. Highest caloric values per weight (9Kcal/gm) Carrier of fat soluble Vitamins – A,D,E,K Source of essential fatty acids – linoleic & Arachidonic acid. Source of prostaglandins – Earlier sexual maturity, increased work capacity.
  • • Milk fat contain lactones which contribute to flavour of milk 70-120mg/kg milk fat• Milk fat is regarded as the most digestible of the various dietary fats and oils• Digestibility of milk fat is 99% whereas palm oil show 91%
  • Why digestibility of milk fat is good• Most of the FA present in milk are liquid• The digestibility of fat whose melting point is below 450C is 95% or better• Positioning of the fatty acid in TG• Smaller fat globules are absorbed directly
  • Benefits of milk Fat• Antimicrobial activity- by reducing surface tension & by decreasing pH• SCFA- butyric acid promote the growth of Bifidobacterium in colon• Anticarcinogenic role-CLA CLA-antioxidant, boost immune system, reduce risk of heart disease by reducing levels of prostaglandins, CLA also improve bone density increase insulin sensitivity• Sphingolipids- ceramides-regulate cell function, protective effect against colon cancer
  • Milk Fat and CVD• Milk contain relatively low cholesterol Cow-2.8mg/g Buffalo-1.9 mg/g• Exp conducted on animals can not be extrapolated to humans• Exp animals have low cholesterol levels and absorption is more Rat- 50-80% Monkey & dog- 40-75% Rabbits –upto 90%• Lipid Hypothesis Stearic & palmitic-36%-hypercholesterolemic
  • Non – nutritional Functions:Improve the physical properties in:- Baked goods – Bread, Cakes,Biscuits Cookies, Pasteries, crackers by trapping air in dough. Lubricating action provides characteristic form and texture. Frying Medium. Flavour & Palatability.
  • Milk SNF Protein 3.25% (3/4 casein) Lactose 4.6% Minerals 0.65% Ca, P, citrate, Mg, K, Na, Zn, Cl, Fe, Cu, sulfate, bicarbonate, many others Acids 0.18% citrate, formate, acetate, lactate, oxalate Enzymes peroxidase, catalase, phosphatase, lipase Gases oxygen, nitrogen Vitamins C, thiamine, riboflavin, others
  • Protein – Wide Range of Functions in Living Organisms Cells, Tissues & MusclesP BloodROT Enzymes & HormonesEIN Antibodies Recovery of Wear & Tear
  • Protein – In Milk PricingPrice of Raw Milk to Producers – Traditional basis • Fat and / or Solids not fatDemand of Milk Fat Preference for Milk Protein• Component Pricing based on Protein – Already Popular in Western Countries
  • 30
  • Protein Composition of Milk Approx. % of skim milk Protein protein Isoelectric point Molecular weight alpha-Casein 45-55 4.1 23,000 kappa-Casein 8-15 4.1 19,000 B-Casein 25-35 4.5 24,000 gamma-Casein 3-7 5.8-6.0 30,650 alpha-Lactalbumin 2-5 5.1 14,437 B-Lactoglobulin 7-12 5.3 18,000 Blood serum albumin 0.7-1.3 4.7 68,000 Lactoferrin 0.2-0.8 87,000 Immunoglobulins IgG1 1-2 160,000 IgG2 0.2-0.5 160,000 IgM 0.1-0.2 ~1,000,000 IgA 0.05-0.10 ~400,000Proteose peptone fraction 2-6 3.3-3.7 4,100-200,000
  • Various proteins in milk• s1-Casein,s2-Casein,-Casein, -Casein and K casein• -lactoglobulin• -lactalbumin• Immunoglobulin• Proteose peptone• Serum albumin• Lactoferrin
  • • Casein micelles- 70-110 nm CM 110-160nm BM• Soluble casein• Casein-Acid, Rennet• Caseinates• Whey protein concentrates, Whey protein Isolates, Whey powders
  • Preparation of casein and whey proteins Acid (isoelectric) precipitationCentrifugationCentrifugation of calcium-supplemented milkSalting-out methodsUltrafiltrationGel filtration (gel permeation chromatography)Precipitation with ethanolCryoprecipitationRennet coagulation
  • Microheterogeneity of the caseins Each of the four caseins, αs1 -, αs2 -, β- and κ –caseins, exhibits variability,which refers to as microheterogeneity, arising from five causes :-1. Variability in the degree of phosphorylation Each of the 4 caseins is phosphorylated to a characteristic but variable level Casein Number of phosphate residues αs1 8, occasionally 9 αs2 10, 11, 12 or 13 β 5, occasionally 4 κ 1, occasionally 2 or perhaps 3The number of phosphate groups in the molecule is indicated as αs1-CN 8P or αs1-CN9P, etc. (CN = casein)2. Disulphide bonding αs1 & β no cysteine or cystine αs2 & κ each contains two cysteines per mole It normally exist as intermolecular disulphide bonds
  • 3. Hydrolysis of primary caseins by plasmin γ-Caseins are produced from β-casein by proteolysis by plasmin, an indigenous proteinase in milk. Corresponding N-terminal fragments are the principal components of the proteose- peptone (PP) fraction, i.e. PP5 (β-CN fl-105/107), PP8 slow (β-CN f29-105/107) and PP8 fast (β-CN fl-28). Normally, the γ-caseins represent only about 3% of whole casein but levels may be very much higher (up to 10%) in late lactation and mastitic milks
  • 4. Variations in the degree of glycosylation κ -Casein is normally glycosylated containing about 5% of carbohydrate (NANA or sialic acid, galactose and N-acetyl galactosamine Carbohydrates are located towards C-terminal of the molecule, attached through an 0- threonyl linkage, mainly to Thr131 of κ –casein Number of oligosaccharides per κ -casein molecule varies from 0 to 4 κ -casein in colostrum is even more highly glycosylated; more sugars are present & the structures are more complex and uncertain. Carbohydrate bestows on κ -casein quite high solubility and hydrophilicity.5. Genetic polymorphism Aschaffenburg & Drewry (1956) discovered that the whey protein, β-lactoglobulin (β- lg), exists in two forms, A & B - differ from each other by only a few amino acids. Milk of any individual animal may contain β-lg A or B or both, & the milk is indicated as AA, BB or AB with respect to β -lg. This phenomenon was referred to as genetic polymorphism Occurs in all milk proteins; a total of ~30 variants have been demonstrated by PAGE
  • Nomenclature of the caseins Heterogeneity of bovine casein
  • Casein• Phosphoproteins – about 80% of all milk proteins• Lack Disulfide bonds and has little secondary and tertiary structure• Form a micelle (140nm) - hydrophobic – Calcium and phosphates stabilize structure• Several forms – Alpha - phosphorylated form S1 and S2 variants (39%) – Beta - major (28%) – Kappa - glycoprotein (10%) • Stabilizes the micelle – Gamma - fragments of beta (3%)• Precipitated by – pH below 4.6 – Low solubility – Clots in stomach – Ultracentrifugation – Ultrafiltration - reduce trucking costs of milk used for cheese. – Rennin - cheese • Cleaves Kappa casein
  • Whey proteins About 20% of the total protein of bovine milk belongs to a group of proteinsgenerally referred to as whey or serum proteins or non-casein nitrogen Whey proteins, as a group, are – 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: Ultrafiltration/diafiltration of acid or rennet whey Ion-exchange chromatography Demineralization by electrodialysis and/or ion exchange, thermal evaporation of water and crystallization of lactose Thermal denaturation, recovery of precipitated protein by filtration/ centrifugation and spray-drying, to yield lactalbumin
  • β- lactoglobulin OccuranceMajor whey protein (WP)-50% of total WP & 12% of total protein of milkPrincipal WP in bovine, ovine caprine & buffalo milksDoes not occur in human, rat, mouse or guinea-pig milksContains 4 genetic variants – A, B, C & D in bovine milk Amino acid composition Amino acid sequence of bovine β-lg consists of 162 residues per monomer Physiological function Acts as a carrier for retinol (vit. A) Binds retinol in a hydrophobic pocket, protect it from oxidation Transport it through stomach to small intestine where it is transferred to a retinol binding protein, which has similar structure to β-lg β-lg also binds FFA & thus it stimulates lipolysis
  • Whey Protein: -LactoglobulinMost abundant whey protein (approximately half of thetotal protein in bovine whey) (not in humans) Structure 162 amino acids, 9 beta strands, alpha-helix, 2 disulfides, 1 free sulphhydryl group (-SH) β-barrel domain hydrophobic pocket Stability pH temperature surface activity 46
  • • source of essential and branched chain amino acids. A retinol- binding protein also exists within the β-lactoglobulin structure having potential to modulate lymphatic response.• Various peptides derived from proteolytic digestion of β-Lg by enzymes have shown inhibitory activity against Angiotensin- Converting Enzyme (ACE) which plays a major role in the regulation of blood pressure.• The active peptides were usually short (< 8 amino acids) and could be enriched from a mixture of proteins and other peptides using UF with low molecular mass cut-off membranes.• β-Lg exert an antimicrobial function in vivo after its partial digestion by endopeptidases of the pancreas, and small targeted modification in the sequence of these peptides could be useful in expanding their anti-microbial function. 47
  • α -lactalbumin OccuranceRepresents about 20% of bovine whey protein & 3.5% of total milk proteinPrincipal protein in human milkSmall protein of molecular mass 14 kDa Amino acid composition Contain 123 residues per monomer & is rich in tryptophan & sulphur Principal α-la contains no phosphorus or carbohydrate Metal binding and heat stabilityα-la is a metallo-protein & binds Ca2+ per mole in a pocket containing 4 Asp residuesCa2+ containing protein is quite heat stable & hence imparts high heat stability to α-la (the protein renatures after heat denaturation) Metal free protein is denatured at quite low temperatures & does not renature on cooling
  • -Lactalbumin• The component of the lactose synthetase enzymeUDP-galactose + glucose Lactose synthetase lactose + UDP• Structure – 123 amino acids – 4 disulfides – 4 α-helices – 2 β-strands – 1 calcium 49
  • • α-Lactalbumin (α-La) is one of the main proteins found in human and bovine milk comprising approximately 20-25% of whey proteins.• The peptide with the amino acids sequence Tyr-Gly-Leu-Phe (f 50- 53), released from α-La by pepsin treatment is shown to inhibit ACE.• Recently, a folding variant of α-La named HAMLET/ BAMLET for human/bovine α-La made lethal to tumour cells has been discovered, which selectively enters tumour cells and induces an apoptosis like mechanism.• α-La was observed to improve cognitive performances in stress- vulnerable individuals by increased brain tryptophan and serotonin activity.• Other clinical trials suggested that α-La could be used to improve sleep in adults suffering from nutritional disturbances. 50
  • • Purified α-La is most readily used in infant formula manufacturing, as it has the most structurally similar protein profile compared to human.• α-La is rich in essential amino acids, this protein is ideally suited to fortifying infant formulae. Clinical trials with α-La enriched infant formula have also been shown to exhibit antimicrobial activity.• At a standard pasteurization temperature of 72oC for 15 s, the bioactive whey proteins retain most of their activity. 51
  • Biological function α-la plays imp. role in lactose synthesisUDP-D –Galactose + D-glucose Lactose - Lactose + UDP synthetaseLactose synthetase consists of dissimilar A (UDP-galactosyl transferase) protein subunits B (α-la) In the absence of B protein, the A protein acts as a non-specific galactosyltransferase but in the presence of B protein, it becomes very specific & transfersgalactose only to glucose to form lactose α-la is therefore a ‘specifier protein’ Concentration of lactose in milk is directly related to the concentration of α-la inmilk
  • Bovine Serum AlbuminNormal bovine milk – 0.1 – 0.4 gl-1; 0.3 – 1.0 % of total NLarge molecule of molecular mass 66 kDa; 582 amino acidsContains 17 disulphides & 1 sulphydrylBSA in milk is a result of leakage from bloodBinds metals and fatty acids and hence stimulate lipase activity.Immunoglobulins Mature milk – 0.6 – 1.0 g Igl-1 (3.0 % of total N), Colostrum -100 gl-1 Complex protein consisting of 5 classes – IgG, IgA, IgD, IgM & IgE IgG, IgA, & IgM are present in milk & IgG occurs as IgG1 & IgG2 IgG consists of two long (heavy) & two shorter (light) polypeptide chains linked by disulphides Principal Ig in bovine milk is IgG1 while in human milk it is IgA Physiological function – provides various types of immunity in the body
  • Lactoferrin• Lactoferrin is an iron-binding glycoprotein of the transferrin family which was first fractionated as an unknown “red fraction” from cows’ milk by Sørensen and Sørensen (1939).• Bovine and human lactoferrins has a sequence identity of 69%.• Human milk has the highest LF concentrations (1.5 to 2.0 mgmL-1) of the mammalian milks compared with the average LF concentration in bovine milk of 0.1 mgmL-1.• Lactoferrin can bind the free ferric iron with high affinity, and thus function as a local antioxidant, protecting the immune cells against the free radicals produced by them. 54
  • Immunoglobulins• An immunoglobulin (Ig) is an antibody or gamma-globulin. There are five classes of antibodies-IgA, IgD, IgE, IgG, and IgM. The IgG class antibodies comprise 80-90% of total Igs (about 50 g L-1) in the early bovine colostrum from the milking of the first day after parturition.• The biological function of immunoglobulins (Igs) in bovine milk and colostrums is to protect mammary gland against pathogens and to provide the calf with an immunological protection against surrounding pathogens.• Immunoglobulins, together with lactoferrin, lacto-peroxidase and lysozyme form the very important anti-microbial system of bovine lacteal secretions. 55
  • • A treatment with specific colostral Igs reduced the degree of gastric inflammation and Helicobacter colonization in mice and the degree of inflammation in children too.• When used as a mouth rinse for 3 days a immune preparation resulted in a higher resting pH in dental plaque of adults as compared with the control groups.• Infants receiving the formula containing the immune preparation (0.5 g per kg body weight for 7 days) had a significantly lower incidence of diarrhoea than those given a formula containing the control preparation during the 6 months follow-up period• There is evidence suggesting that shigellosis could be prevented by using colostral Ig preparations from cows immunized against certain Sh. flexneri antigens 56
  • • In clinical trials, the Ig preparations have seldom caused any undesired symptoms and the health risks are mainly due to allergenic properties of whey proteins.• Bovine Ig for human application is only in its infancy. There are uncertainties w.r.t. dosage and timing of administration.• Immunoglobulins are heat-sensitive molecules subject to human digestive action and may present difficulties with respect to palatability or shelf life of food products due to contaminating enzymes of a proteolytic nature. 57
  • Major differences between the caseins and whey proteins1. Caseins precipitate from the solution when the pH of milk is adjusted to 4.6 where as the whey proteins do not precipitate and remains in solution2. Chymosin and some other proteinases (known as rennets) produce a very slight, specific change in casein, resulting in its coagulation in the presence of Ca2+, whereas the whey proteins undergo no such alteration3. Casein is very stable to high temperatures; milk may be heated at its natural pH 6.7 at 100°C/24h without coagulation and it withstand heating at 140°C/20min. The whey proteins, on the other hand, are relatively heat labile, being completely denatured by heating at 90°C for 10min4. Caseins are phosphoproteins, containing, on average, 0.85% phosphorus, while the whey proteins contain no phosphorus
  • 5. Casein is low in sulphur (0.8%) while the whey proteins are relatively rich (1.7%)6. Casein is synthesized in the mammary gland while some of the whey proteins (β-lg and α-la) are also synthesized in the mammary gland, while others (e.g. bovine serum albumin and the immunoglobulins) are derived from the blood7. The whey proteins are molecularly dispersed in solution or have simple quaternary structures, whereas the caseins have a complicated quaternary structure and exist in milk as large colloidal aggregates, referred to as micelles, with particle masses of 106-109 DaOther protein fractions Milk contains two other groups of proteins or protein-like material,  proteose-peptone fraction and  non-protein nitrogen (NPN) fraction
  • Major bioactive protein components in bovine colostrum and milk Values expressed as weight of Colostrum Milk protein L-1 Immunoglobulin (Ig) A 3.2-6.2 g 0.1 g IgG1 48-87 g 0.4 g IgG2 1.6-2.9 g 0.05 g IgM 3.7-6.1 g 0.05 g Insulin like growth factor (IGF) -I 0.1-2 mg 25 µg IGF-II 0.1-2 mg 2 µgTransforming growth factor (TGF) -β 20-40 mg 1-2 µg EGF 4-8 mg 2 µg Lactoferrin 1.5-2 mg 0.1µg Lysozyme 0.1-0.7 mg 0.1-0.3 mg Lactoperoxidase 30 mg 20 mg 60
  • Lysozyme• Lysozyme is an important antimicrobial agent in milk, which kills bacteria by cleaving the β-1,4-glycosidic bond between N-acetylglucoseamine residues of the peptidoglycan in the bacterialcell wall.• LZ is a major component of the whey fraction in human milk (0.4 g L-1) although its concentration in bovine milk is several orders of magnitude lower (0.13 mg L-1).• Lysozyme in both cow and buffalo milk exhibited maximum activity at pH 7.4. 61
  • Lactoperoxidase (LP)• This enzyme system catalyzes peroxidation of thiocyanate and some halides (such as iodine and bromium), which ultimately generates products that inhibit and/or kill a range of bacterial species.• The important biological function of this enzyme is the bactericidal affect against gram –ve as well as gram +ve bacteria in the presence of hydrogen peroxide and SCN or halogens.• Lactoperoxidase is able to form oxidized halides and pseudohalides, which are potent biocidal small molecules.• Many attempts have been made to use LP as an ingredient for extending the shelf life of milk and milk products. 62