minor ions, lipase and plasmin enzymes, and entrapped milk serum. These micelles a rather porous structures, occupy ing about 4 ml/g and 6-12% of the total volume fraction of milk The"casein sub-micelle"model has been prominent for the last several years, and model, and mounting research evidence to suggest that there is not a defined sub-micellar illustrated and described with the following link, but there is not universal acceptance of this structure to the micelle at all. Another model of a more open structure is also defined with the following link In the submicelle model, it is thought that there are small aggregates of whole casein, containing 10 to 100 casein molecules, called submicelles. It is thought that there are two different kinds of submicelle; with and without kappa-casein. These submicelles contain a hydrophobic core and are covered by a hydrophilic coat which is at least partly comprised of the polar moieties of kappa-casein. The hydrophilic CMP of the kappa-casein exists as a flexible hair The open model also suggests there are more dense and less dense regions within the midelle, but there is less of a well-defined structure. In this model, calcium phosphate nanoclusters bind caseins and provide for the differences in density within the casein Colloidal calcium phosphate( CCP)acts as a cement between the hundreds or even thousands of submicelles that form the casein micelle. Binding may be covalent or electrostatic Submicelles rich in kappa-casein occupy a surface position, whereas those with less are buried in the interior. The resulting hairy layer, at least 7 nm thick, acts to prohibit further aggregation of submicelles by steric repulsion. The casein micelles are not static; there are three dynamic equilibria between the micelle and its surround ings the free casein molecules and submicelles the free submicelles and micelles the dissoved colloidal calcium and phosphate The following factors must be considered when assessing the stability of the casein Role of catt More than 90% of the calcium content of skim milk is associated in some way or another with the casein micelle. The removal of ca++ leads to reversible dissociation of B -casein without micellular d isintegration. The add ition of ca++ leads to aggregation H Bonding: Some occurs between the individual caseins in the micelle but not much becaus there is no secondary structure in casein protel Disulphide bonds alpha(sl)and B-caseins do not have any cysteine residues. If any S-s bonds occur
21 minor ions, lipase and plasmin enzymes, and entrapped milk serum. These micelles are rather porous structures, occupying about 4 ml/g and 6-12% of the total volume fraction of milk. The "casein sub-micelle" model has been prominent for the last several years, and is illustrated and described with the following link, but there is not universal acceptance of this model, and mounting research evidence to suggest that there is not a defined sub-micellar structure to the micelle at all. Another model of a more open structure is also defined with the following link. In the submicelle model, it is thought that there are small aggregates of whole casein, containing 10 to 100 casein molecules, called submicelles. It is thought that there are two different kinds of submicelle; with and without kappa-casein. These submicelles contain a hydrophobic core and are covered by a hydrophilic coat which is at least partly comprised of the polar moieties of kappa-casein. The hydrophilic CMP of the kappa-casein exists as a flexible hair. The open model also suggests there are more dense and less dense regions within the midelle, but there is less of a well-defined structure. In this model, calcium phosphate nanoclusters bind caseins and provide for the differences in density within the casein micelle. Colloidal calcium phosphate (CCP) acts as a cement between the hundreds or even thousands of submicelles that form the casein micelle. Binding may be covalent or electrostatic. Submicelles rich in kappa-casein occupy a surface position, whereas those with less are buried in the interior. The resulting hairy layer, at least 7 nm thick, acts to prohibit further aggregation of submicelles by steric repulsion. The casein micelles are not static; there are three dynamic equilibria between the micelle and its surroundings: • the free casein molecules and submicelles • the free submicelles and micelles • the dissoved colloidal calcium and phosphate The following factors must be considered when assessing the stability of the casein micelle: Role of Ca++: More than 90% of the calcium content of skim milk is associated in some way or another with the casein micelle. The removal of Ca++ leads to reversible dissociation of ß -casein without micellular disintegration. The addition of Ca++ leads to aggregation. H Bonding: Some occurs between the individual caseins in the micelle but not much because there is no secondary structure in casein proteins. Disulphide Bonds: alpha(s1) and ß-caseins do not have any cysteine residues. If any S-S bonds occur
within the micelle, they are not the driving force for stabilization Hydrophobic Interactions: Caseins are among the most hydrophobic proteins and there is some evidence to suggest they play a role in the stability of the micelle. It must be remembered that / drophobic interactions are very temperature sensitive Electrostatic Interactions Some of the subunit interactions may be the result of ionic bonding, but the overall micellar structure is very loose and open van der waals forces: No sucess in relating these forces to micellular stability Steric stabilization: As already noted, the hairy layer interferes with interparticle approach There are several factors that will affect the stability of the casein micelle system Salt content: affects the calcium activity in the serum and calcium phosphate content of the micelles p lowering the pH leads to dissolution of calc ium phosphate until, at the isoelectr point(pH 4.6), all phosphate is dissolved and the caseins precipitate Temperature at 4C, beta-casein begins to dissociate from the micelle, at 0 C, there is no micellar aggregation; freezing produces a precipitate called cryo-casein Heat Treatment: whey proteins become adsorbed, altering the behaviour of the micelle Dehydration: by ethanol, for example, leads to aggregation of the micelles When two or more of these factors are applied together, the effect can also be additive Casein micelle aggregation Caseins are able to aggregate if the surface of the micelle is reactive. The Schmidt model further illustrates this Although the casein micelle is fairly stable, there are four major ways in which aggregation can be induced 1. chymosin-rennet or other proteolytic enzymes as in Cheese manufacturing 2. acid 3. heat 4. age gelation Enzyme Coagulation Chymosin, or rennet, is most often used for enzyme coagulation. During the primary stage rennet cleaves the Phe(105)-Met(106) linkage of kappa-casein resulting in the formation of
22 within the micelle, they are not the driving force for stabilization. Hydrophobic Interactions: Caseins are among the most hydrophobic proteins and there is some evidence to suggest they play a role in the stability of the micelle. It must be remembered that hydrophobic interactions are very temperature sensitive. Electrostatic Interactions: Some of the subunit interactions may be the result of ionic bonding, but the overall micellar structure is very loose and open. van der Waals Forces: No sucess in relating these forces to micellular stability. Steric stabilization: As already noted, the hairy layer interferes with interparticle approach. There are several factors that will affect the stability of the casein micelle system: Salt content: affects the calcium activity in the serum and calcium phosphate content of the micelles. pH: lowering the pH leads to dissolution of calcium phosphate until, at the isoelectric point (pH 4.6), all phosphate is dissolved and the caseins precipitate. Temperature: at 4° C, beta-casein begins to dissociate from the micelle, at 0° C, there is no micellar aggregation; freezing produces a precipitate called cryo-casein. Heat Treatment: whey proteins become adsorbed, altering the behaviour of the micelle. Dehydration: by ethanol, for example, leads to aggregation of the micelles. When two or more of these factors are applied together, the effect can also be additive. Casein micelle aggregation Caseins are able to aggregate if the surface of the micelle is reactive. The Schmidt model further illustrates this. Although the casein micelle is fairly stable, there are four major ways in which aggregation can be induced: 1. chymosin - rennet or other proteolytic enzymes as in Cheese manufacturing 2. acid 3. heat 4. age gelation Enzyme Coagulation Chymosin, or rennet, is most often used for enzyme coagulation. During the primary stage, rennet cleaves the Phe(105)-Met(106) linkage of kappa-casein resulting in the formation of
the soluble CMP which diffuses away from the micelle and para-kappa-casein, a distinctly hydrophobic peptide that remains on the micelle. The patch or reactive site, as illustrated in the above image, that is left on the micelles after enzymatic cleavage is necessary before aggregation of the paracasein micelles can begin During the secondary stage, the micelles aggregate. This is due to the loss of steric repulsion of the kappa-casein as well as the loss of electrostatic repulsion due to the decrease in pH. As the pH approaches its isoelectric point(pH 4.6), the caseins aggregate cy to aggr interactions. Calcium assists coagulation by creating isoelectric cond itions and by acting as a brid ge bet elles. The ten of coagulation is very important both the primary and secondary stages. With an increase in temperature up to 40% C, the rate of the rennet reaction increases. During the secondary stage, increased temperatures increase the hydrophobic reaction. The tertiary stage of coagulation involves the rearrangement of micelles after a gel has formed. There is a loss of paracasein identity as the milk curd firms and syneresis begins Acid Coagulation Acidification causes the casein micelles to destabilize or aggregate by decreasing their electric charge to that of the isoelectric point. At the same time, the acidity of the medium increases the solubility of minerals so that organic calcium and phosphorus contained in the micelle gradually become soluble in the aqueous phase. Casein micelles disintegrate and casein precipitates. Aggregation occurs as a result of entropically driven hydrophobic Interactions Heat At temperatures above the boiling point casein micelles will irreversibly aggregate On heating, the buffer capacity of milk salts change, carbon dioxide is released, organic acids are produced, and tricalcium phophate and casein phosphate may be precipitated with the release of hydrogen ions Age gelation Age gelation is an aggregation ph enomenon that affects shelf-stable, sterilized dairy products, such as concentrated milk and UHT milk products. After weeks to months storage of these products, there is a sudden sharp increase in viscosity accompanied by visible gelation and irreversible aggregation of the micelles into long chains forming a three-dimensional network. The actual cause and mechanism is not yet clear, however, some theories exist 1. Proteolytic breakdown of the casein: bacterial or native plasmin enzymes that are asistant to heat treatment may lead to the formation of a gel 2. Chemical reactions: poly merization of casein and whey proteins due to Maillard type
23 the soluble CMP which diffuses away from the micelle and para-kappa-casein, a distinctly hydrophobic peptide that remains on the micelle. The patch or reactive site, as illustrated in the above image, that is left on the micelles after enzymatic cleavage is necessary before aggregation of the paracasein micelles can begin. During the secondary stage, the micelles aggregate. This is due to the loss of steric repulsion of the kappa-casein as well as the loss of electrostatic repulsion due to the decrease in pH. As the pH approaches its isoelectric point (pH 4.6), the caseins aggregate. The casein micelles also have a strong tendency to aggregate because of hydrophobic interactions. Calcium assists coagulation by creating isoelctric conditions and by acting as a bridge between micelles. The temperature at the time of coagulation is very important to both the primary and secondary stages. With an increase in temperature up to 40° C, the rate of the rennet reaction increases. During the secondary stage, increased temperatures increase the hydrophobic reaction. The tertiary stage of coagulation involves the rearrangement of micelles after a gel has formed. There is a loss of paracasein identity as the milk curd firms and syneresis begins. Acid Coagulation Acidification causes the casein micelles to destabilize or aggregate by decreasing their electric charge to that of the isoelectric point. At the same time, the acidity of the medium increases the solubility of minerals so that organic calcium and phosphorus contained in the micelle gradually become soluble in the aqueous phase. Casein micelles disintegrate and casein precipitates. Aggregation occurs as a result of entropically driven hydrophobic interactions. Heat At temperatures above the boiling point casein micelles will irreversibly aggregate. On heating, the buffer capacity of milk salts change, carbon dioxide is released, organic acids are produced, and tricalcium phophate and casein phosphate may be precipitated with the release of hydrogen ions. Age Gelation Age gelation is an aggregation phenomenon that affects shelf-stable, sterilized dairy products, such as concentrated milk and UHT milk products. After weeks to months storage of these products, there is a sudden sharp increase in viscosity accompanied by visible gelation and irreversible aggregation of the micelles into long chains forming a three-dimensional network. The actual cause and mechanism is not yet clear, however, some theories exist: 1. Proteolytic breakdown of the casein: bacterial or native plasmin enzymes that are resistant to heat treatment may lead to the formation of a gel 2. Chemical reactions: polymerization of casein and whey proteins due to Maillard type
or other chemical reactions 3. Formation of kappa-casein-B-lactoglobulin complexes An excellent source of information on casein micelle stability can be found in Walstra Whey proteins The proteins appearing in the supernatant of milk after precipitation at pH 4.6are collectively called whey proteins. These globular proteins are more water soluble than caseins and are subject to heat denaturation. Native whey proteins have good gelling and whipping properties. Denaturation increases their water holding capacity. The principle fractions are B-lactoglobulin, alpha-lactalbumin, bovine serum albumin(BSA), and immunoglobulins (g) B-Lactoglobulins:(MW-18000: 162 residues) This group, includ ing eight genetic variants, comprises approximately half the total whey proteins. B-Lactoglobulin has two internal disulfide bonds and one free thiol group The conformat ion includes considerable secondary structure and exists naturally as a noncovalent linked dimer. At the isoelectric point(pH 3.5 to 5.2 ), the dimers are further associated to octamers but at ph below 3. 4, the are dissociated to monomers Ipha-Lactalbumins:(MW-14,000; 123 residues)These proteins contain eight cysteine groups, all involved in internal disulfide bonds, and four tryptophan residues alpha-Lactalbumin has a highly ordered secondary structure, and a compact, spherical tertiary structure. Thermal denaturation and pH <4.0 results in the release of bound calcium Enzymes Enzymes are a group of proteins that have the ability to catalyze chemical reactions and the speed of such reactions. The action of enzymes is very specific. Milk contains both indigenous and exogenous enzymes. Exogenous enzymes mainly consist of heat-stable enzymes produced by psychrotrophic bacteria: lipases, and proteinases. There are many indigenous enzymes that have been isolated from milk. The most significant group are the hydrolases ipoprotein lipase alkaline phosphatase Lipoprotein lipase(LPL): A lipase enzyme splits fats into glycerol and free fatty acids This enzy me is found mainly in the plasma in association with casein micelles. The milkfat is protected from its action by the FGM. If the FGM has been damaged, or if certain cofactors(blood serum lipoproteins )are present, the LPL is able to attack the lipoproteins of the fgm. lipolysis may be caused in this way
24 or other chemical reactions 3. Formation of kappa-casein-ß -lactoglobulin complexes An excellent source of information on casein micelle stability can be found in Walstra. Whey Proteins The proteins appearing in the supernatant of milk after precipitation at pH 4.6 are collectively called whey proteins. These globular proteins are more water soluble than caseins and are subject to heat denaturation. Native whey proteins have good gelling and whipping properties. Denaturation increases their water holding capacity. The principle fractions are ß -lactoglobulin, alpha-lactalbumin, bovine serum albumin (BSA), and immunoglobulins (Ig). ß -Lactoglobulins: (MW - 18,000; 162 residues) This group, including eight genetic variants, comprises approximately half the total whey proteins. ß -Lactoglobulin has two internal disulfide bonds and one free thiol group. The conformation includes considerable secondary structure and exists naturally as a noncovalent linked dimer. At the isoelectric point (pH 3.5 to 5.2), the dimers are further associated to octamers but at pH below 3.4, they are dissociated to monomers. alpha-Lactalbumins: (MW - 14,000; 123 residues) These proteins contain eight cysteine groups, all involved in internal disulfide bonds, and four tryptophan residues. alpha-Lactalbumin has a highly ordered secondary structure, and a compact, spherical tertiary structure. Thermal denaturation and pH <4.0 results in the release of bound calcium. Enzymes Enzymes are a group of proteins that have the ability to catalyze chemical reactions and the speed of such reactions. The action of enzymes is very specific. Milk contains both indigenous and exogenous enzymes. Exogenous enzymes mainly consist of heat-stable enzymes produced by psychrotrophic bacteria: lipases, and proteinases. There are many indigenous enzymes that have been isolated from milk. The most significant group are the hydrolases: • lipoprotein lipase • plasmin • alkaline phosphatase Lipoprotein lipase (LPL): A lipase enzyme splits fats into glycerol and free fatty acids. This enzyme is found mainly in the plasma in association with casein micelles. The milkfat is protected from its action by the FGM. If the FGM has been damaged, or if certain cofactors (blood serum lipoproteins) are present, the LPL is able to attack the lipoproteins of the FGM. Lipolysis may be caused in this way
Plasmin: Plasmin is a proteolytic enzyme; it splits proteins. Plasmin attacks both B-casein and alpha(s2)-casein. It is very heat stable and responsible for the development of bitterness in pasteurized milk and UhT processed milk. It may also play a role in the ripening and flavour development of certain cheeses, such as Swiss cheese Alkaline phosphatase: Phosphatase enzymes are able to split specific phosporic acid esters into phosphoric acid and the related alcohols Unlike most milk enzymes, it has a ph and temperature optima differing from physiological values; pH of 9.8. The enzyme is destroye by minimum pasteurization temperatures, therefore, a phosphatase test can be done to ensure proper pasteurization Lactose: Lactose is a disaccharide(2 sugars) made up of glucose and galactose(which are oth monosaccharides It comprises 4.8 to 5.2% of milk, 52%of milk SNF, and 70% of whey solids. It is not as sweet as sucrose. When lactose is hydrolyzed by B-D-galactosidase (lactase), an enzyme that splits these monosaccharides, the result is increased sweetness, and depressed freezing One of its most important functions is its utilization as a fermentation substrate Lactic acid bacteria produce lactic acid from lactose, which is the beginning of many fermented dairy products. Because of their ability to metabolize lactose, they have a competitive advantage over many pathogenic and spoilage organisms Some people suffer from lactose intolerance; they lack the lactase enzyme, hence they cannot digest lactose, or dairy products containing lactose. Crystallization of lactose occurs in an alpha form which commonly takes a tomahawk shape This results in the defect called cream, sweetened condensed milk. In addition to lactose, fresh milk contains other ( s, ice sandiness. Lactose is relatively insoluble which is a problem in many dairy product carbohydrates in small amounts, includ ing glucose, galactose, and oligosaccharides Vitamins: V itamins are organic substances essential for many life processes. Milk includes fat soluble vitamins A. D.E. and K. vitamin A is derived from retinol and B-carotene Because milk is an important source of dietary vitamin A, fat reduced products which have lost vitamin A with the fat, are required to supplement the product with vitamin a Milk is also an important source of dietary water soluble vitamins Bl-thiamine B2-riboflavin B6-pyridoxine B12-cyanocobalamin niacin pantothenic acid
25 Plasmin: Plasmin is a proteolytic enzyme; it splits proteins. Plasmin attacks both ß -casein and alpha(s2)-casein. It is very heat stable and responsible for the development of bitterness in pasteurized milk and UHT processed milk. It may also play a role in the ripening and flavour development of certain cheeses, such as Swiss cheese. Alkaline phosphatase: Phosphatase enzymes are able to split specific phosporic acid esters into phosphoric acid and the related alcohols. Unlike most milk enzymes, it has a pH and temperature optima differing from physiological values; pH of 9.8. The enzyme is destroyed by minimum pasteurization temperatures, therefore, a phosphatase test can be done to ensure proper pasteurization. Lactose: Lactose is a disaccharide (2 sugars) made up of glucose and galactose (which are both monosaccharides). It comprises 4.8 to 5.2% of milk, 52% of milk SNF, and 70% of whey solids. It is not as sweet as sucrose. When lactose is hydrolyzed by ß -D-galactosidase (lactase), an enzyme that splits these monosaccharides, the result is increased sweetness, and depressed freezing point. One of its most important functions is its utilization as a fermentation substrate. Lactic acid bacteria produce lactic acid from lactose, which is the beginning of many fermented dairy products. Because of their ability to metabolize lactose, they have a competitive advantage over many pathogenic and spoilage organisms. Some people suffer from lactose intolerance; they lack the lactase enzyme, hence they cannot digest lactose, or dairy products containing lactose. Crystallization of lactose occurs in an alpha form which commonly takes a tomahawk shape. This results in the defect called sandiness . Lactose is relatively insoluble which is a problem in many dairy products, ice cream, sweetened condensed milk. In addition to lactose, fresh milk contains other carbohydrates in small amounts, including glucose, galactose, and oligosaccharides. Vitamins:Vitamins are organic substances essential for many life processes. Milk includes fat soluble vitamins A , D, E, and K. Vitamin A is derived from retinol and ß -carotene. Because milk is an important source of dietary vitamin A, fat reduced products which have lost vitamin A with the fat, are required to supplement the product with vitamin A. Milk is also an important source of dietary water soluble vitamins: • B1 - thiamine • B2 - riboflavin • B6 - pyridoxine • B12 - cyanocobalamin • niacin • pantothenic acid