rancid flavour when it is cleaved from glycerol by lipase action Saturated fatty acids(no double bonds ), such as myristic, palmitic, and stearic make up two thirds of milk fatty acids. Oleic acid is the most abundant unsaturated fatty acid in milk with one double bond While the cis form of geometric isomer is the most common found in nature, approximately 5% of all unsaturated bonds are in the trans position as a result of rumen hydrogenation Triglycerides account for 98.3% of milkfat. The distribution of fatty acids on the triglyceride chain, while there are hundreds of different combinations, is not random. The fatty acid pattern is important when determining the physical properties of the lipids In general, the Sni position binds mostly longer carbon length fatty acids, and the sN3 position binds mostly shorter carbon length and unsaturated fatty acids. For example C4-97 in sn3 C6-84% in Sn3 Cl8-58% in Sn1 The small amounts of mono-, diglycerides, and free fatty acids in fresh milk may be a product of early lipolysis or simply incomplete synthesis. Other classes of lipids include phospholipids (0.8%)which are mainly associated with the fat globule membrane, and cholesterol(0. 3%)which is mostly located in the fat globule core Milk Lipids- Physical Properties The physical properties of milkfat can be summerized as follows density at 20C is 915 kg m(-3* refractive index(589 nm) is 1.462 which decreases with increasing temperature olubility of water in fat is 0. 14%(w/w)at 20C and increases with increasing thermal conductivity is about 0. 17 J m(-1)s(-1)K(-1)at 20C specific heat at 40%C is about 2. lkJ kg(-1)K(-1) electrical conductivity is <10(-12)ohm(-1)cm(-1) dielectric constant is about 3.1 *the brackets around numbers denote superscript t room temperature, the lipids are solid, therefore, are correctly referred to as"fat" as opposed to"oil"which is liquid at room temperature. The melting points of individual triglycerides ranges from C for tributyric glycerol to 72C for tristearin. However, the final melting point of milkfat is at 37 C because higher melting triglycerides dissolve in the liquid fat. This temperature is significant because 37.C is the body temperature of the cow and the milk would need to be liquid at this temperature The melting curves of milkfat are complicated by the diverse lipid composition
16 rancid flavour when it is cleaved from glycerol by lipase action. Saturated fatty acids (no double bonds), such as myristic, palmitic, and stearic make up two thirds of milk fatty acids. Oleic acid is the most abundant unsaturated fatty acid in milk with one double bond. While the cis form of geometric isomer is the most common found in nature, approximately 5% of all unsaturated bonds are in the trans position as a result of rumen hydrogenation. Triglycerides account for 98.3% of milkfat. The distribution of fatty acids on the triglyceride chain, while there are hundreds of different combinations, is not random. The fatty acid pattern is important when determining the physical properties of the lipids. In general, the SN1 position binds mostly longer carbon length fatty acids, and the SN3 position binds mostly shorter carbon length and unsaturated fatty acids. For example: • C4 - 97% in SN3 • C6 - 84% in SN3 • C18 - 58% in SN1 The small amounts of mono- , diglycerides, and free fatty acids in fresh milk may be a product of early lipolysis or simply incomplete synthesis. Other classes of lipids include phospholipids (0.8%) which are mainly associated with the fat globule membrane, and cholesterol (0.3%) which is mostly located in the fat globule core. Milk Lipids - Physical Properties The physical properties of milkfat can be summerized as follows: • density at 20° C is 915 kg m(-3)* • refractive index (589 nm) is 1.462 which decreases with increasing temperature • solubility of water in fat is 0.14% (w/w) at 20° C and increases with increasing temperature • thermal conductivity is about 0.17 J m(-1) s(-1) K(-1) at 20° C • specific heat at 40° C is about 2.1kJ kg(-1) K(-1) • electrical conductivity is <10(-12) ohm(-1) cm(-1) • dielectric constant is about 3.1 *the brackets around numbers denote superscript At room temperature, the lipids are solid, therefore, are correctly referred to as "fat " as opposed to "oil" which is liquid at room temperature. The melting points of individual triglycerides ranges from -75° C for tributyric glycerol to 72° C for tristearin. However, the final melting point of milkfat is at 37° C because higher melting triglycerides dissolve in the liquid fat. This temperature is significant because 37° C is the body temperature of the cow and the milk would need to be liquid at this temperature. The melting curves of milkfat are complicated by the diverse lipid composition:
trans unsaturation increases melting points odd-numbered and branched chains decrease melting points Crystallization of milkfat largely determines the physical stability of the fat globule and the consistency of high-fat dairy products, but crystal behaviour is also complicated by the wide range of different triglycerides. There are four forms that milkfat crystals can occur in alpha, B, B1, and B 2, however, the alpha form is the least stable and is rarely observed in slowly cooled fat Milkfat Structure- Fat Globules More than 95% of the total milk lipid is in the form of a globule ranging in size from 0. 1 to 5 um in diameter. These liquid fat droplets are covered by a thin membrane, 8 to 10 nm in thickness, whose properties are completely different from both milkfat and plasma. The native fat globule membrane(fGm)is comprised of apical plasma membrane of the secretory cell which continually envelopes the lipid droplets as they pass into the lumen The major components of the native FGM, therefore, is protein and phospholipids. The phospholipids are involved in the oxidation of milk. There may be some rearrangement of the membrane after release into the lumen as amphiphil ic substances from the plasma sorb onto the fat globule and parts of the membrane dissolve into either the globule core or the serum. The FGM decreases the lipid-serum interface to very low values, 1 to 2.5 mN/m, preventing the globules from immediate flocculation and coalescence, as well as protecting them from enzymatic action It is well known that if raw milk or cream is left to stand, it will separate. Stokes'Law plasma phases of milk. However, in cold raw milk, creaming takes place faster than y and predicts that fat globules will cream due to the differences in densities between the fat predicted from this fact alone. IgM, an immunoglobulin in milk, forms a complex with lipoproteins. This complex, known as cryoglobulin precipitates onto the fat globules and causes flocculation. This is known as cold agglutination. As fat globules cluster, the speed of rising increases and sweeps up the smaller globules with them. The cream layer forms very rapidly, within 20 to 30 min, in cold milk Homogenization of milk prevents this creaming by decreasing the diameter and size distribution of the fat globules, causing the speed of rise to be similar for the majority of globules. As well, homogenization causes the formation of a recombined membrane which is much similar in density to the continuous phase Recombined membranes are very different than native FGM. Processing steps such as homogenization, decreases the average diameter of fat globule and significantly increases the surface area. Some of the native fgm will remain ad sorbed but there is no longer enough of it to cover all of the newly created surface area. Immediately after disruption of the fat globule, the surface tension raises to a high level of 15 mN/m and amphiphilic molecules in the plasma quickly adsorb to the lipid droplet to lower this value. The adsorbed layers consist mainly of serum proteins and casein micelles
17 • trans unsaturation increases melting points • odd-numbered and branched chains decrease melting points Crystallization of milkfat largely determines the physical stability of the fat globule and the consistency of high-fat dairy products, but crystal behaviour is also complicated by the wide range of different triglycerides. There are four forms that milkfat crystals can occur in; alpha, ß , ß ' 1, and ß ' 2, however, the alpha form is the least stable and is rarely observed in slowly cooled fat. Milkfat Structure - Fat Globules More than 95% of the total milk lipid is in the form of a globule ranging in size from 0.1 to 15 um in diameter. These liquid fat droplets are covered by a thin membrane, 8 to 10 nm in thickness, whose properties are completely different from both milkfat and plasma. The native fat globule membrane (FGM) is comprised of apical plasma membrane of the secretory cell which continually envelopes the lipid droplets as they pass into the lumen. The major components of the native FGM, therefore, is protein and phospholipids. The phospholipids are involved in the oxidation of milk. There may be some rearrangement of the membrane after release into the lumen as amphiphilic substances from the plasma adsorb onto the fat globule and parts of the membrane dissolve into either the globule core or the serum. The FGM decreases the lipid-serum interface to very low values, 1 to 2.5 mN/m, preventing the globules from immediate flocculation and coalescence, as well as protecting them from enzymatic action. It is well known that if raw milk or cream is left to stand, it will separate. Stokes' Law predicts that fat globules will cream due to the differences in densities between the fat and plasma phases of milk. However, in cold raw milk, creaming takes place faster than is predicted from this fact alone. IgM, an immunoglobulin in milk, forms a complex with lipoproteins. This complex, known as cryoglobulin precipitates onto the fat globules and causes flocculation. This is known as cold agglutination. As fat globules cluster, the speed of rising increases and sweeps up the smaller globules with them. The cream layer forms very rapidly, within 20 to 30 min., in cold milk. Homogenization of milk prevents this creaming by decreasing the diameter and size distribution of the fat globules, causing the speed of rise to be similar for the majority of globules. As well, homogenization causes the formation of a recombined membrane which is much similar in density to the continuous phase. Recombined membranes are very different than native FGM. Processing steps such as homogenization, decreases the average diameter of fat globule and significantly increases the surface area. Some of the native FGM will remain adsorbed but there is no longer enough of it to cover all of the newly created surface area. Immediately after disruption of the fat globule, the surface tension raises to a high level of 15 mN/m and amphiphilic molecules in the plasma quickly adsorb to the lipid droplet to lower this value. The adsorbed layers consist mainly of serum proteins and casein micelles
Fat Destabilization While homogenization is the principal method for acheiving stabil ization of the fat emulsion in milk, fat destabiliza tion is necessary for structure formation in butter, whipping cream and ice cream. Fat destabilization refers to the process of clustering and clumping (partial coalescence) of the fat globules which leads to the development of a continuous internal fat network or matrix structure in the product. Fat destabilization (sometimes "fat agglomeration")is a general term that describes the summation of several different phenomena These include Coalescence an irreversible increase in the size of fat globules and a loss of identity of the coalescing globules Flocculation a reversible(with minor energy input)agg lomeration/clustering of fat globules with no loss of identity of the globules in the floc; the fat globules that flocculate; they can be easily redispersed if they are held together by weak forces, or they might be harder to red isperse to they share part of their interfacial layers Partial coalescence ible agglomeration/clustering of fat globules, held together by combination of fat crystals and liquid fat, and a retention of ident ity of individual globules as long as the crystal structure is maintained (i.e, temperature dependent, once the crystals melt, the cluster coalesces). They usually come together in a shear field, as in whipping, and it is env isioned that the crystals at the surface of the droplets are responsible for causing colliding globules to stick together, while the iquid fat partially flows between they and acts as the"cement". Partial coalescence dominates structure formation in whipped, aerated dairy emulsions, and it should be emphasized that crystals within the emulsion droplets are responsible for it a good reference for more information on fat globules can be found in Mulder and Walstra Milk Lipids- Functional Properties Like all fats, milkfat provides lubrication. They impart a creamy mouth feel as opposed to a dry texture. Butter flavour is unique and is derived from low levels of short chain fatty acids If too many short chain fatty acids are hydrolyzed(separated) from the triglycerides, however, the product will taste rancid. Butter fat also acts as a reservoir for other flavours especially in aged cheese. Fat globules produce ashortening effect in cheese by keeping the protein matrix extended to give a soft texture. Fat substitutes are designed to mimic the globular property of milk fat. The spreadable range of butter fat is 16-24 C. Unfortunately butter is not spreadable at refrigeration temperatures. Milk fat provides energy (1g =9 cal. ) and nutrients(essential fatty acids, fat soluble vitamins) Milk proteins: Introduction and review 18
18 Fat Destabilization While homogenization is the principal method for acheiving stabilization of the fat emulsion in milk, fat destabilization is necessary for structure formation in butter， whipping cream and ice cream. Fat destabilization refers to the process of clustering and clumping (partial coalescence) of the fat globules which leads to the development of a continuous internal fat network or matrix structure in the product. Fat destabilization (sometimes "fat agglomeration") is a general term that describes the summation of several different phenomena. These include: Coalescence: an irreversible increase in the size of fat globules and a loss of identity of the coalescing globules; Flocculation: a reversible (with minor energy input) agglomeration/clustering of fat globules with no loss of identity of the globules in the floc; the fat globules that flocculate ; they can be easily redispersed if they are held together by weak forces, or they might be harder to redisperse to they share part of their interfacial layers; Partial coalescence: an irreversible agglomeration/clustering of fat globules, held together by a combination of fat crystals and liquid fat, and a retention of identity of individual globules as long as the crystal structure is maintained (i.e., temperature dependent, once the crystals melt, the cluster coalesces). They usually come together in a shear field, as in whipping, and it is envisioned that the crystals at the surface of the droplets are responsible for causing colliding globules to stick together, while the liquid fat partially flows between they and acts as the "cement". Partial coalescence dominates structure formation in whipped, aerated dairy emulsions, and it should be emphasized that crystals within the emulsion droplets are responsible for its occurrence. A good reference for more information on fat globules can be found in Mulder and Walstra. Milk Lipids - Functional Properties Like all fats, milkfat provides lubrication. They impart a creamy mouth feel as opposed to a dry texture. Butter flavour is unique and is derived from low levels of short chain fatty acids. If too many short chain fatty acids are hydrolyzed (separated) from the triglycerides, however, the product will taste rancid. Butter fat also acts as a reservoir for other flavours, especially in aged cheese. Fat globules produce a 'shortening' effect in cheese by keeping the protein matrix extended to give a soft texture. Fat substitutes are designed to mimic the globular property of milk fat. The spreadable range of butter fat is 16-24° C. Unfortunately butter is not spreadable at refrigeration temperatures. Milk fat provides energy (1g = 9 cal.), and nutrients (essential fatty acids, fat soluble vitamins). Milk Proteins: Introduction and Review
The primary structure of proteins consists of a polypeptide chain of amino acids residues joined together by peptide linkages, which may also be cross-linked by disulphide bridges Amino acids contain both a weakly basic amino group, and a weakly acid carboxyl group both connected to a hydrocarbon chain, which is unique to different amino acids. The three-dimensional organization of proteins, or conformation, also involves secondary tertiary, and quaternary structures. The secondary structure refers to the spatial arrangement of amino acid residues that are near one another in the linear sequence. The alpha-helix and B-pleated sheat are examples of secondary structures arising from regular and periodic steric relationships. The tertiary structure refers to the spatial arrangement of amino acid residues that are far apart in the linear sequence, giving rise to further coiling and folding. If the protein is tightly coiled and folded into a somewhat spherical shape, it is called a globular protein. If the protein consists of long polypeptide chains which are intermolecularly linked, they are called fibrous proteins. Quaternary structure occurs when proteins with two or more polypeptide chain subunits are associated Milk Protein fractionation The nitrogen content of milk is distributed among caseins(76%), whey proteins(18%), and non-protein nitrogen(NPN)(6%). This does not include the minor proteins that are associated with the FGM. This nitrogen distribution can be determined by the row land fractionation method 1. Precipitation at pH 4.6-separates caseins from whey nitrogen 2. Precipitation with sodium acetate and acetic acid(pH 5.0)-separates total protein from whey NPn The concentration of proteins in milk is as follows: grams/ litre of total protein Total Protein 100 Total Caseins 79.5 30.6 alpha s2 2.6 8.0 9.3 28.4 3.3 Total Whey proteins alpha lactalbumin 1.2 3.7 beta lactoglobulin 3.2 9.8 Immunoglobulins 0.7 Proteose peptone 0.8 Caseins, as well as their structural form-casein micelles, whey proteins, and milk enzymes will now be examined in further detail
19 The primary structure of proteins consists of a polypeptide chain of amino acids residues joined together by peptide linkages, which may also be cross-linked by disulphide bridges. Amino acids contain both a weakly basic amino group, and a weakly acid carboxyl group both connected to a hydrocarbon chain, which is unique to different amino acids. The three-dimensional organization of proteins, or conformation, also involves secondary, tertiary, and quaternary structures. The secondary structure refers to the spatial arrangement of amino acid residues that are near one another in the linear sequence. The alpha-helix and ß -pleated sheat are examples of secondary structures arising from regular and periodic steric relationships. The tertiary structure refers to the spatial arrangement of amino acid residues that are far apart in the linear sequence, giving rise to further coiling and folding. If the protein is tightly coiled and folded into a somewhat spherical shape, it is called a globular protein. If the protein consists of long polypeptide chains which are intermolecularly linked, they are called fibrous proteins. Quaternary structure occurs when proteins with two or more polypeptide chain subunits are associated. Milk Protein Fractionation The nitrogen content of milk is distributed among caseins (76%), whey proteins (18%), and non-protein nitrogen (NPN) (6%). This does not include the minor proteins that are associated with the FGM. This nitrogen distribution can be determined by the Rowland fractionation method: 1. Precipitation at pH 4.6 - separates caseins from whey nitrogen 2. Precipitation with sodium acetate and acetic acid (pH 5.0) - separates total proteins from whey NPN The concentration of proteins in milk is as follows: grams/ litre % of total protein __________________________________________________________________________ Total Protein 33 100 Total Caseins 26 79.5 alpha s1 10 30.6 alpha s2 2.6 8.0 beta 9.3 28.4 kappa 3.3 10.1 Total Whey Proteins 6.3 19.3 alpha lactalbumin 1.2 3.7 beta lactoglobulin 3.2 9.8 BSA 0.4 1.2 Immunoglobulins 0.7 2.1 Proteose peptone 0.8 2.4 __________________________________________________________________________ Caseins, as well as their structural form - casein micelles, whey proteins, and milk enzymes will now be examined in further detail
Casein The case in content of milk represents about 80% of milk proteins. The principal casein fractions are alpha(sI)and alpha(s2 )-caseins, B-casein, and kappa-casein. The distinguishing property of all caseins is their low solubility at pH 4.6. The common compositional factor is that caseins are conjugated proteins, most with phosphate group(s) esterified to serine residues. These phosphate groups are important to the structure of the casein micelle. Calcium binding by the individual caseins is proportional to the phosphate content The conformation of caseins is much like that of denatured globular proteins. The high number of prolnein of close-packed, ordered secondary structures. Caseins contain ne residues in caseins causes particular bending of the protein chain and inhibits the form disulfide bonds. As well, the lack of tertiary structure accounts for the stability of caseins against heat denaturation because there is very little structure to unfold. Without a tertiary structure there is considerable exposure of hydrophobic residues. This results in strong association reactions of the caseins and renders them insoluble in water Within the group of caseins, there are several distinguishing features based on their charge distribution and sensitivity to calcium precipitation alpha(sl)-casein:(molecular weight 23,000; 199 residues, 17 proline residues) region, which contains all but one of eight phosphate groups. It can be precipitate ar Two hydrophobic regions, containing all the proline residues, separated by a po calcium alpha(s2)-casein:(molecular weight 25,000; 207 residues, 10 prolines) Concentrated negative charges near N-terminus and positive charges near C-terminus. It can also be precipitated at very low levels of calcium. B-casein:(molecular weight 24,000; 209 residues, 35 prolines) Highly charged N-terminal region and a hydrophobic C-terminal region. Very amphiphilic protein acts like a detergent molecule. Self association is temperature dependant; will form a large polymer at 20C but not at 4 C. Less sensitive to calcium precipItation kappa-casein:(molecular weight 19,000: 169 residues, 20 prolines) Very resistant to calcium precipitation, stabilizing other caseins. Rennet cleavage at the Phe105-Met106 bond eliminates the stabilizing ability, leaving a hydrophobic ppa-casein, and a hydrophilic portion called kappa-casein glycomacropeptide(GMP), or more accurately, caseinomacropeptide(CMP) Structure: The Casein Micelle Most, but not all, of the casein proteins exist in a colloidal particle known as the casein micelle. Its biological function is to carry large amounts of highly insoluble Cap to mammalian young in liquid form and to form a clot in the stomach for more efficient nutrition. Besides casein protein, calcium and phosphate, the micelle also contains citrate
20 Caseins The casein content of milk represents about 80% of milk proteins. The principal casein fractions are alpha(s1) and alpha(s2)-caseins, ß -casein, and kappa-casein. The distinguishing property of all caseins is their low solubility at pH 4.6. The common compositional factor is that caseins are conjugated proteins, most with phosphate group(s) esterified to serine residues. These phosphate groups are important to the structure of the casein micelle. Calcium binding by the individual caseins is proportional to the phosphate content. The conformation of caseins is much like that of denatured globular proteins. The high number of proline residues in caseins causes particular bending of the protein chain and inhibits the formation of close-packed, ordered secondary structures. Caseins contain no disulfide bonds. As well, the lack of tertiary structure accounts for the stability of caseins against heat denaturation because there is very little structure to unfold. Without a tertiary structure there is considerable exposure of hydrophobic residues. This results in strong association reactions of the caseins and renders them insoluble in water. Within the group of caseins, there are several distinguishing features based on their charge distribution and sensitivity to calcium precipitation: alpha(s1)-casein: (molecular weight 23,000; 199 residues, 17 proline residues) Two hydrophobic regions, containing all the proline residues, separated by a polar region, which contains all but one of eight phosphate groups. It can be precipitated at very low levels of calcium. alpha(s2)-casein: (molecular weight 25,000; 207 residues, 10 prolines) Concentrated negative charges near N-terminus and positive charges near C-terminus. It can also be precipitated at very low levels of calcium. ß -casein: (molecular weight 24,000; 209 residues, 35 prolines) Highly charged N-terminal region and a hydrophobic C-terminal region. Very amphiphilic protein acts like a detergent molecule. Self association is temperature dependant; will form a large polymer at 20° C but not at 4° C. Less sensitive to calcium precipitation. kappa-casein: (molecular weight 19,000; 169 residues, 20 prolines) Very resistant to calcium precipitation, stabilizing other caseins. Rennet cleavage at the Phe105-Met106 bond eliminates the stabilizing ability, leaving a hydrophobic portion, para-kappa-casein, and a hydrophilic portion called kappa-casein glycomacropeptide (GMP), or more accurately, caseinomacropeptide (CMP). Structure: The Casein Micelle Most, but not all, of the casein proteins exist in a colloidal particle known as the casein micelle. Its biological function is to carry large amounts of highly insoluble CaP to mammalian young in liquid form and to form a clot in the stomach for more efficient nutrition. Besides casein protein, calcium and phosphate, the micelle also contains citrate