FST 3325: Dairy Science and Technology

FST 3325: Dairy Science and Technology
Academic year: 2012 by Fabien Matsiko

INTRODUCTION Milk is a fluid secreted by the mammary glands of mammals
It provides good quality proteins, Ca, and vit A, D, B2 (riboflavin), niacin and folic acid High content of water (87% on average) Milk has shorter shelf life Shelf stable dairy products: yogurt, cheese and Kefir Overview of physical-chemical properties of milk Basic quality tests on raw milk Discussion of processing technologies of dairy products

Chap1. Chemical & Physical properties of Milk
1.1Milk Composition and Structure • Milk = polydisperse system of water with: – Emulsified fat globules enveloped by a membrane (Ø 0,1-10 μm) – Casein micelles (Ø nm) – Lipoprotein particles (Ø 10 nm) Milk plasma = Milk - fat globules ~ separated milk (skimmed milk) Milk serum = milk plasma - casein micelles = milk - fat, caseins and insoluble salts Dry matter (DM) = all dry milk components Fat-free DM = dry matter - fat

Milk viewed at different magnifications

Composition and Structure of milk (Approximate average quantities in 1kg of milk

Properties of the main structural elements of milk

1.2.Different kinds of milk
Some of the properties in comparison with cow’s milk: Goat's milk – relatively more proteins and fat – used for cheese manufacturing • Sheep's milk – rich in proteins and fats • Buffalo milk – composition comparable to cow's milk – high fat content (6-7%) • Horse's milk – low fat and protein content – used for yogurt – low nutritional value – used in Eastern countries, Russia, Mongolia

1.2.Different kinds of milk
Casein-rich milks: cow, sheep, goat, buffalo – usable for cheese manufacturing; compact coagulation – difficult digestion Albumin rich milks: humans, horse – no cheese manufacturing possible; fine coagulation -better digestion Unless specified otherwise, cow’s milk will be dicussed in this subject.

Composition of human milk with reference of cow’s milk
– specific composition – more natural defense components (immunoglobulins, oligosaccharides, Bifidus factors) – large amounts of lactose (= stimulates absorption of Ca, Mg, Mn, Cu and Zn) – Rich in vitamins and Poly-unsaturated fatty acids (n-6/n-3) – low in proteins (more whey proteins, fewer caseins) – Absence of allergens – Dangers: presence of (fat-soluble) pesticides (DDT,...), PCB’s, dioxins, Phthalates, Medicines, Nicotin, alcohol

1.3Factors influencing Composition of milk
Breed – a wide diversity – predominately the result of selection by people

Various breeds

Different breeds of cows and the gross composition of milk
Components Breeds Ayrshire Brown Swiss Guernsey Holstein Jersey Shorthorn Fat 3.95 3.98 4.72 3.54 5.13 3.5 Lactose 3.48 3.64 3.75 3.29 3.97 3.32 Ash 0.72 0.74 0.76 0.75 Total Solids 12.77 13.07 14.04 12.16 14.42 12.27 Different breeds of cows and the gross composition of milk

– milk composition is largely influenced by seasons
• Season/nutrition – milk composition is largely influenced by seasons – winter: silaged feed; summer: fresh grass - Differences in fat and protein content -Major Implications on processing

Stage of lactation -Time elapsed after calving influences milk composition -Colostrum milk = First milk produced after calving; more salts, proteins (mainly serum proteins) and less lactose; composition evolves towards its normal composition within 4 days - Further changes occur: decrement in protein content, casein, serum proteins, ashes, fat-free dry matter; at the end of the lactation, an increment in DM content occurs -Lactose content is almost constant during lactation; fat content is correlated to fat-free dry matter during lactation -pH changes: 6,6 6,7 6,9 (at the end)

Factors affecting the milk composition
• Age of cow – has a very small but consistent effect on milk composition fat and fat-free dry matter decrease slightly with each successive lactation • Mastitis – = inflammation of the udder – several species of pathogenic bacteria may cause mastitis – causes a decrease in milk yield and a change in milk composition – decrease in fat content, fat-free dry matter, lactose and casein and increase of serum proteins and chloride content – the number of somatic cells increases (> /ml) -catalase activity increases – Cl-lactose ratio increases

Correlations • Lactose and chlorides are negatively correlated
– the result of the activity of the udder which maintains milk isotonic – if lactose-production is restricted by an infected udder, more chlorides (and thus also sodium) are added to the milk by the udder – this phenomenon can be used to detect mastitis infection: the number of Koestler is a valuable parameter to differentiate normal from mastitis milk Normal milk=(100*% Clorides)/% Lactose= Mastititis milk=(100*% Clorides)/% Lactose> 3.0

1.3 Factors influencing the composition
• Method of milking – during milking the fat content of the milk leaving the udder increases (e.g. from 1-10%, marked differences among cows in this respect); fat-free dry matter remains constant – the interval between milking influences fat content, but not fat-free dry matter; longer intervals result in a higher fat content

1.3 Factors influencing the composition
• Individual – variations within breeds are the result of genetic and environmental factors - by selection high-productive animals are obtained • Quarters – differences in composition in the milk of different quarters of the udder of one cow mostly are negligible unless a quarter is or has been affected by mastitis • Other factors – contamination and processing

Chap.2 Chemical Constituents of Milk
2.1 Carbohydrates

2.1.1 Lactose Lactose: prominent carbohydrate of milks of most species virtually unique to milk having been found elsewhere only in fruits of certain members of Sapotacea The components and breakdown elements of lactose are found in minor quantities: glucose and galactose, lactic acid and butyric acid Other carbohydrates are found only in small quantities

Chemical and Biochemical Properties of Lactose
Lactose: Disaccharide of D-glucose and D-galactose joined in a ß-1,4glucosidic linkage – both moieties occur predominantly in the pyranose ring form – lactose = 1,4-β-D-galactopyranosyl- D-glucopyranose • Reducing sugar – property important in the analysis of milk – also causes non-enzymatic browning, called Maillard reaction • amino acids and reducing sugars react forming aroma products (HMF,aldehydes and pyrazines) and brown components; nutritional value of milk lowers during this reaction

Lactulose • Compound found in heated milk products
• Lobry de Bruyn-Alberda van Ekenstein transformation: mechanism by which lactulose is formed • Sweeter than lactose • Bifidus factor: promotes the growth of Bifidobacterium bifidum and thus beneficial in the diets of human infants • Concentrations up to about 1% may occur in commercial evaporated milk • A parameter to evaluate the thermal treatment of milk (sterilized versus UHT milk, limit of 600 ppm)

Lobry de Bruyn-Alberda van Ekenstein transformation

Lactose content • Relatively constant at 4,8 to 5,2 %
• Lower levels occur in colostrum and mastitis milk to • Lactose comprises about 52 % of milk solids-non-fat, about 70 % of whey solids and > 90 % of the solids in milk ultrafiltrate • Lactose intolerance – Caused by a shortage of the lactase-enzyme (β-galactosidase) – Results in non-hydrolysis of lactose into glucose and galactose – Undigested lactose may support growth of undesirable intestinal flora, as well as draw water into the intestine causing diarrhoea and abdominal cramps

Lactose sweetness Lactose is not as sweet as other common sugars:Sucrose, Fructose and glucose

• Relatively sweeter at higher concentration
• β-lactose is sweeter than α-lactose – this difference is not important since the small difference between freshly prepared solutions is eliminated quickly by equilibration of the anomers

Lactose mutarotation Lactose exists in both α and β forms (interchanging the OH and H on the reducing group) • Lactose is optically active because of its asymmetry – α-form can be distinguished from the β form by its greater rotation of polarized light α and β -forms of lactose exist in solution in a temperature dependent equilibrium [β]/[α] = 1,64 - 0,0027.T with T = temp. in °C • The equilibrium is reached after 24 hours – example: at 15 °C [β]/[α] = 1,60 or in this solution 38 % of the lactose is present in the α form, 62 % in the β form

Mutarotation in Lactose Solutions
A.Course of reaction (% finished ) as function of time B.Effect of pH on the mutarotation rate constant (K/h) C

Lactose solubility α and β lactose differ considerably in solubility and in the temperature dependence of solubility – this is influenced by the mutarotation • Lactose solutions can be supersaturated easily, i.e. nucleation does not occur easily – at concentrations over 2,1 times the final solubility, spontaneous crystallization occurs (homogeneous nucleation) – at a relative supersaturation below 1,6 seeding with lactose crystals usually is needed to induce crystallization (solution is meta-stable)

Lactose crystallisation
αLactose crystallises as a hydrate of equimolar amt of water and lactose – Crystallization is of great technological importance – because it may crystallize in some milk products, notably sweetened condensed milk and ice cream During evaporation processes, the solubility is exceeded – in condensed milk, a directed crystallization in the α-form is observed, so that crystals don't exceed 10 μm – crystals of 30 μm give the defect sandy mouthfeel

Lactose crystallization
Above 93 °C anhydrous β-lactose crystallizes – β-lactose dissolves much faster than α-lactose hydrate at room temperature, as its solubility is about 10 times higher and the crystals are usually smaller with a larger surface area

Amorphous Lactose • Is formed when a solution is dried rapidly, as in a spray drier, or frozen • Is often called “glass” • Is a very concentrated solution which dissolves on addition of water, but then α-lactose may start to crystallize • Is very hygroscopic, which is of importance in milk powder • Instant milk powder has a better solubility, caused by addition of moisture and a directed crystallization

Lactic acid fermentation
• Lactose can be fermented by bacteria that have a β- galactosidase (lactase)-system: LAB • Lactose glucose + galactose x lactic acid – glycolitic or Embden-Meyerhof pathway – dependent on conditions and culture, the following reaction products can be formed: D(-) lactic acid, L(+) lactic acid, racemic solution • When 1 % lactic acid is formed, the reaction process is slowed down due to the pH drop (at this moment 20% of the lactose is metabolized; yogurt: 40%)

Lactic acid fermentation
• Important flavor compounds are: – diacetyl (butter flavor) – acetaldehyde (yogurt flavor) – propionic acid can be formed out of lactic acid by propionic acid bacteria 3 CH3CHOHCOOH 2 CH3CH2COOH + CH3COOH + H2O + CO2

Alcohol fermentation • Lactose pyruvic acid acetaldehyde + CO2 ethanol
• This fermentation doesn't occur frequently • Mostly an undesired fermentation by yeasts • In some sour foaming milk drinks it is desired (kefir, koumiss)

Butyric acid fermentation
2 CH3CHOHCOOH C3H7COOH + 2 CO2 + 2 H2 This fermentation is highly undesirable in cheese because of the gas and off-flavor Characteristic for Clostridia bacteria

Other Carbohydrates Lactulose
– a derivative of lactose present in heated milk – often used as a parameter for heat-treatment of milk, so that sterilized and UHT milk can be distinguished • Free glucose and galactose – detected readily in fresh milk – lack of agreement on their concentrations • No carbohydrates as glycogen or starch

Other carbohydrates – present in small quantities
• Oligosaccharides – present in small quantities – composed out of glucose, galactose and fucose (6-deoxygalactose) – some are composed of hexosamines: N-acetylglucosamines • of great biological importance • bifidus factor in human milk • serological activity (anti-hemaglutination) – some are made of neuraminic derivatives as N-acetyl neuraminic acid (NANA) • bounded on k-casein • anti-bacterial activity

Lactose separation • Lactose is water-soluble • Follows the aqueous phase during separation processes • Present in skimmed milk, buttermilk, whey (often used as source for gaining of lactose)

2.2 Lipids • Milk fat content • Chemical properties • Physical properties • Deterioration of milk fat • Other lipid components • Fat globules

Milk Fat and Composition
Is variable • Dependent on a large number of factors – caused by differences between breed and individuals – influenced by the season

Chemical properties • Global composition of milk fat
– major fraction of neutral lipids, in particular triglycerides – small fraction of polar lipids with important structural functions • present in the fat globule, in the fat globule membrane and in the plasma

Chemical properties • Milk fat triglycerides
– a very great range of some 250 different fatty acid residues – a relatively high proportion of short-chain fatty acids (C4-C10) – butyric acid is specific for milk fat of ruminant species – a high proportion of saturated fatty acids is high (63% w/w) – oleic acid is the most abundant of the unsaturated fatty acids residues – other unsaturated fatty acids residues are present in a wide variety of chain length, unsaturation and isomers – several "odd" fatty acids (uneven, branched, keto, hydroxy) – fatty acids are stereospecifically distributed: asymmetric structure, which influences the texture of derived products

Chemical properties • Mono- and diglycerides and free fatty acids also occur in small quantities (hydrolytic products from lipolysis) • Fluctuations in the composition occur – dependent on feed, season and breed – are of major technological importance as they influence the physical properties of the milk fat (harder fat during winter periods)

Fatty Acid Composition of milk lipids

Physical Properties Melting Point Of major importance in the manufacturing of milk products and milk fat in particular • Is determined by the fatty acids – chain length and degree of saturation – place of the double bond – the trans-isomers – the place of the fatty acids within the triglycerides • Influenced by minor components – mono- and diglycerides – free fatty acids – phospholipds – water

Physical Properteis • Crystallization Nucleation • Crystal growth • Polymorphism

Nucleation and crystal growth
• Bulk milk fat – one catalytic impurity per mg suffices to ensure rapid crystallization – super cooling of 5 K suffices to induce nucleation – this concerns the highest melting triglycerides – as soon as these nuclei have been formed, they act in turn as catalytic impurities for the nucleation of other triglycerides – little hysteresis between solidification and melting curves • Emulsified milk fat – fat is finely divided: 1 mg of fat is divided among some 108 -globules, in homogenized milk among at least 1011 globules – in each globule at least one nucleus must be formed: the number of catalytic impurities may easily become limiting – super cooling can be more exquisite – hysteresis can be considerable

Polymorphism •Three polymorphic modifications α, β' and β – each modification is characterized by its crystal lattice type (mode of packing), not by its geometrical form (habit) – an important feature is the distance between the chains, the so called short spacing (X-ray diffraction patterns) – the melting points increase in the order a, b' and b, as do the melting heat and the density of the crystals; this implies that closeness and intricacy of fit of the molecules increase and their freedom of motion decreases – generally the a, b' modifications are not stable (meta stable) – Nucleation of a fat usually occurs in the a modification • mostly, after a little while, transition to a stabler polymorph occurs – In milk fat, a crystals can be very persistent – Conversely, in most fats the a modification has only a short -lifetime, while b' may persist longer

Deterioration of milk fat
Lipolysis Oxidation Fishy taste

Lipolysis • = Hydrolysis of fatty acid esters by the action of lipases
– results in the common flavor defect know as lipolytic or hydrolytic rancidity and is distinct from oxidative rancidity • By endogeneous lipases (mainly lipoprotein lipase) or by bacterial lipases • The properties of the fat globule membrane are of major importance – reduced contents of phospholipids or mastitis can increase the sensitivity of the fat globule for lipolysis – other factors that destabilize the fat globule membrane, especially agitation and foaming, also promote lipolysis – lipolysis is promoted in the manufacturing of cheese • Sensory perception of lipolytic rancidity is strongly affected by the pH of the product – at low pH they are more readily tasted – in fresh milk threshold values corresponded to acid degree values (ADV) of 4,1 to 4,5 mmol per 100 g of fat

Oxidation three stages initiation, propagation and termination
• Proceeds by the well-known auto oxidation reaction in three stages initiation, propagation and termination – unsaturated fatty acids are transformed to hydroperoxides, the primary reaction products – during propagation, antioxidant compounds such as tocopherols and ascorbic acid are depleted while peroxide derivatives of fatty acids accumulate – peroxides, which have little flavor, undergo further reactions to form a variety of carbonyl, the secondary reaction products, which are responsible for the rancid taste and odour

Factors affecting oxidation can be categorized into 2 groups
Intrinsic factors – metalloproteins such as milk peroxidase and xanthin oxidase – endogenous ascorbic acid, which acts as a cocatalyst with copper to promote oxidation – endogenous copper content – endogenous antioxidants, mainly tocopherols • Extrinsic factors – contamination with metals – temperature of storage – oxygen tension – heat treatment – agitation – light – acidity

Oxidation • Effect of heat treatment
– migration of copper from the plasma to the fat globule – denaturation of metalloproteins and increase the availability of metals for oxidation – high heat treatment stabilizes milk against oxidation • probably due to exposure of sulfhydryl groups of denaturated proteins and the releaseof hydrogen sulfide • Effect of homogenization – reduces the sensitivity of milkfat to both copper- and light-induced oxidation, probably because oxidation sensitive membrane lipids are displaced • Effect of light – photooxidation is accompanied by depletion of riboflavin, ascorbic acid and some amino acids – so-called sunlight-flavor is caused by oxidation of methionine to methional

Fishy taste Can be induced by transformation of Phosphatidylcholine to trimethylamine

Other lipids • Phospholipids • Sterols • Other lipids

Phospholipids

Sterols • Form the largest fraction of the unsaponifiable lipids
• Consist largely of cholesterol • A small fraction of the cholesterol is esterified (saponifiable sterol fraction) • Sterols can be found in: – The fat globule – The fat globule membrane – The serum phase

Other lipids • Pigments, mainly carotenoids (β-carotene = provitamin A) – concentration is season-dependent; in summer double quantities compared to winter – in sheep, goat and buffalo milk no β-carotene is present • Vitamins (mainly A, D and E) • Antioxidants (mainly tocopherols) • Polar fatty acids: keto acids en HO acids, important for lacton formation • Squalene

Fat globules • Milk fat present in milk as a globule that reduce interfacial tension of lipids-serum Fat globule distribution • Fat globule structure • Agglutination

Fat globules distribution
• Thermodynamically, no emulsion is stable – the stability is a kinetic time-dependent phenomenon – milk separates or creams spontaneously and rapidly and many processes involve manipulation of the creaming phenomenon – emulsion stability is largely dependent on the size distribution of the globules – in raw milk, fat globules range in size from 0,1 to 15 μm • The milk emulsion contains three distinct populations of fat globules – small globules (<1μm) that represent 80% of the total number of the globules, but only a small fraction of the total milk fat – large globules (>12μm) that represent 2-3% of the total milk fat – the medium group which represents 95% of the total milk fat

Fat globules distribution
• The distribution can be characterized by some parameters – the average diameter – the average volume – the average ratio volume/surface • A large activity at the level of the membrane: 70 m2 per liter of milk (3,5 % fat) • The fat globule size distribution can be influenced by many factors : – shift to larger globules during lactation – in fatty rich milk larger globules occur

Fat globule structure Fat globule membrane -8 to 10 nm in thickness
-reduces the lipid/serum interfacial tension -composition dependent on the procedure of isolation (difficult quantification)

Fat globules structure
Composition of natural fat globule membranes – phospholipids and cholesterol – proteins, especially glycoproteins with properties similar to the proteose-pepton-fraction of the serum – enzymes (xanthine oxidase and alkaline phosphatase) – high melting glycerides, which are probably crystallized on the membrane during cooling

Agglutination • In undisturbed milk, lipid globules rise and form a cream layer • The creaming effect can be described by the law of Stokes: v= 2r2 (dp - df) g 9ŋ V = speed of creaming g = gravity acceleration r = radius ŋ = viscosity dp, df = density of plasma and fat

Agglutination • = Flocculation of fat globules
– speed of spontaneous creaming is in reality much higher than predicted by the law of Stokes – adverse temperature effect is found; at lower temperatures creaming increases – doesn't often occur in heated milk: a consequence of denaturation of agglutinins – in homogenized milk no flocculation occurs, but clustering • Destabilization of the emulsion can be performed by – mechanical movement (free fat in cooled milk) – enzymes – churning (butter still contains fat globules; % of the fat

Destabilization processes in emulsions

Positive Nutritional Aspects of Milk Fat
• Conjugated linoleic acid (cis-9, trans-11 octadecanoic acid) • Produced in the rumen by Butyrivibrio fibrisolvens Up to 100 μmol/g milk fat • Potent anti-carcinogen (especially colon cancer, melanoma and breast cancer) • Antioxidant • Lowers blood LDL-cholesterol • Promotes muscle and fat growth, raises rate of metabolism • Positive influence on the immune system

Nutritional aspects of Milk Fat
• Sphengomyeline Structural function in membranes • Positive effects on cholesterol metabolism • Protection against fumonisin, bacterial toxins and pathogens • Anti-proliferative effect on cancer cells (colon cancer) by influencing cell differentiation, and programmed cell death (apoptosis)

Other positive nutritional aspects
• Butyric acid – Unique feature of milk fat (7-13 mol/100 mol milk fat) – Results from fermentation of non-absorbed carbohydrate by colonic microflora – Is utilized by colonocytes as an important energy source inducing colonic generation (importance of dietary fibers!) – Potent inhibitor of cell proliferation and inducer of differentiation and apoptosis in a number of cancer cell lines – Synergic anti carcinogenic effect with vit. A, D and E

Chemical & Physical Properties
Proteins Introduction Heterogeneity of milk proteins Caseins Casein micelles Whey proteins Milk fat globule membrane proteins Non-protein nitrogen Enzymatic coagulation of caseins Heat-induced coagulation Acid-induced coagulation Ethanol coagulation Age-gelation of sterilized milks Denaturation of whey proteins

Introduction • Milk proteins: of great importance
– human nutrition: nutritional functionality – behavior/properties of dairy products: technological functionality • Normal bovine milk: g protein/litre • Nitrogen content of milk – caseins – whey proteins – non-protein nitrogen (NPN) – minor proteins associated with the milk fat globule membrane

Amino acid composition of the milk proteins

Caseins • αs1-Caseins • αs2-Caseins • β-Caseins • k-Caseins

Casseins • Original definition:
“Those phosphoproteins which precipitate from raw skim” milk upon acidification to pH 4.6 at 20°C” Account for 76-86% of the total milk protein Essentially all occur in micelles Most of the caseins can be represented by four gene products: αs1-, αs2-, β- and k-caseins in the approximate ratio 40:10:35:12

Caseins • Nowadays: classification according to chemical structure, rather than on the basis of an operational definition – not all of the caseins contain phosphorus – some are found in acid whey • Nomenclature – a Greek letter with or without a numerical subscript to identify the family of proteins – an uppercase Latin letter to indicate the genetic variant – post-translational modifications such as number of phosphorylations or formation of sub-fractions are indicated after the genetic variant – e.g. as1-CN B-8P

Casseins • Caseins – contain high numbers of proline residues distributed relatively uniformly throughout the polypetide chains – proline inhibits formation of an ordered, stable a-helix lack of tertiary structure of caseins stability of caseins against heat denaturation considerable exposure of hydrophobic residues to water strong association of caseins insoluble in water • Milk protein genetic variants – Relationship between genetic polymorphism of milk proteins and technological properties – Milk composition is different for various phenotypes

Caseins • Contain negatively charged groups such as phosphates,side-chain carboxyls, terminal carboxyls, and sulfhydryls – binding of a number of different cations, such as calcium, barium, magnesium, potassium, sodium, etc. – highly charged segments facilitate electrostatic interactions • Calcium – calcium binding by phosphoseryl residues is of primary interest – involved in micelle formation

αsCasseins • Calcium-sensitive casein fraction
• Precipitated with 0.4 M CaCl2 at pH 7 and 4°C • αs1-caseins • αs2-caseins

αs1Cassein Five known genetic variants: A, B, C, D, E
– the polymorfs are breed specific – the B variant: predominant in Bos taurus (European cattle) • Predominant genetic B variant – consists of 199 amino acid residues – a calculated molecular weight of 23,614 D (dalton)

αs1Cassein • Polypeptide chain
– two predominantly hydrophobic regions (1 - 44, and ) – a highly charged polar zone ( ) – rather loose and flexible polypeptide • λ-caseins – may be present in milk in small amounts – fragments of αs1-casein as the result of plasmin proteolysis

αs2-Caseins Four recognized genetic variants: A, B, C, and D
– the same primary amino acid sequence – different degrees of posttranslational phosphorylation – A and D variants observed in European breeds (Bos taurus) • αs2-casein A-11P – primary structure of 207 amino acid residues – a calculated molecular weight of 25,230 D

αs2-Caseins • More sensitive to precipitation by Ca++ than αs1-casein
• Polypeptide chain – a remarkable dipolar structure – negative charges near the N-terminus – positive charges near the C-terminus

β-Caseins • 1 major component with at least seven genetic variant(A1, A2, A3, B, C, D, and E) 8 minor components: proteolytic fragments of the major component • A variants: predominant (nearly 100%) polymorphs in all species and strains of Bos

β -caseins β -casein A2-5P
– a single polypeptide chain of 209 residues – a calculated molecular weight of 23,983 D • Polypeptide chain – a strongly negatively charged N-terminal portion – rest of the molecule has virtually no net charge – the β-casein molecule is somewhat similar to an anionic detergent with a negatively charged head and an uncharged essentially hydrophobic tail

β -caseins • γ1-, γ2- and γ3-caseins – present in raw milk
– formed by the action of the plasmin on β-casein in milk – carboxyl terminal fragments of b-casein remaining associated with the casein micelle and recovered by pH 4.6 precipitation – corresponding N-terminal fragments • classified as proteose-peptones • hydrophilic and appear as heat-stable fractions in whey

ΚCaseins • A mixture of polymers held together by disulfide bonds
– monomers consist of a major carbohydrate-free component and at least six minor components • Minor k -casein components – same primary amino acid sequence as major component – contain various amounts and types of carbohydrate moieties attached to the polypeptide chain by post-translational glycosylation

Κcaseins • Two genetic variants are known: A and B
– the A variant tends to be the predominant variant in most breeds • k-casein B-1P – consists of 169 amino acid residues – a calculated molecular weight of 19,007 D • Possess considerable heterogeneity – genetic differences – variation in carbohydrate content and/or phosphate content – a possible variation in the para-k-portion

Κcaseins • Mostly contain only one phosphoseryl residue
• Do not bind Ca++ strongly and are soluble in the presence of high calcium concentrations • k -casein associates with as1- and/or b-casein stabilizes them against precipitation by Ca++ formation of stable colloidal particles similar to but less stable than native casein micelles

Casein Micelle • Casein micelle structure • Stability of micelles
• Forces

Casein Micelles • Micelles – = coarse colloidal particles
– ≈ 95% of the casein in normal un-cooled milk – comprised of some ,000 casein molecules – molecular weights of ≈108 Dalton – mean diameters of ≈100 nm (range nm)

Composition of casein micelles
– consist on dry weight base of » 94% protein and » 6% of small ions principally calcium, phosphate, magnesium and citrate, referred to collectively as colloidal calcium phosphate (CCP) – milk micelles are highly hydrated, typically ≈ 2 g H2O/g protein

• Occurring in all milks that have been examined in detail
Casein Micelle • Important and characteristic macromolecular assembly of mammalian biology • Occurring in all milks that have been examined in detail • Biological functions – to form a coagulum in the stomach of the nursling, allowing the slow release of nutrients down the digestive tract – to act as a means of transporting calcium and phosphate in a readily assimilable form from mother to young – to provide a source of amino acids – to produce biologically active peptides by enzymatic cleavage of casein polypeptide chains such as the casomorphins

Models of Casein micelle structure
• Detailed structure is still not known! • Submicelles – basic units that compose a micelle – nm in diameter – a porous structure – varying composition of the four principal caseins linked together in the micelles by colloidal calcium phosphate (cementing agent) and hydrophobic bonds Walstra , 1999 structure

Models of casein micelle structure
David Horne, 2003 Double binding model

Models of casein micelle structure
• Dalgleish (2004) – No spherical sub-micelles – Caseins organized as tubules Calcium phosphate nanoclusters Gaps big enough which allow proteins to pass trough or bind Κ-casein on surface available for reaction with denaturated whey proteins Κ-casein on surface available for reaction with denaturated whey proteins

Casein micelle structure
• k-casein – the principal micelle-stabilizing factor – located predominantly on the surface – very hydrophilic C-terminal part = flexible "hairs” – essential in providing stability against flocculation of the micelles – hydrodynamic thickness of the hairy layer ≈ 7 nm

Stability of casein micelle
• Heat stability – limited heat treatment has no effect on the micelles – sterilization can disintegrate micelles into a fibrillar structure – stable at the following temperature-time combinations: 10 minutes at 130 °C and 50 minutes at 115 °C • Lowering temperature – lower temperatures cause a disintegration of micelles in particular β-casein is sensitive – important as milk is frequently cooled to below 5°C – effect is reversible when increasing the temperature – low temperatures give a finer, more voluminous precipitate and a weaker gel by acidification

Stability of casein micelle
• Change in mineral composition – removal of calcium ions from the micelle causes reversible dissociation of β- and k-casein from the micelles without real micelle disintegration – addition of excess Ca++ favors micellar component aggregation – aggregation of casein micelles in concentrated and frozen milk products seems to be caused by salting out (a decrease steric repulsion) • Dehydration – dehydration leads to micelle-aggregation – dehydration of k-casein causes presumably a decrease in voluminosity and steric repulsion

Whey Proteins

Whey Proteins • β-Lactoglobulin • α-Lactalbumin • Bovine serum albumin
• Immunoglobulins • Lactotransferrin • Enzymes

Whey Proteins Whey proteins are the major nitrogen compounds remaining in milk after precipitation of the caseins by acid (pH 4.6) or by rennet (pH ≈6.7) and represent ≈ 20% of the nitrogen in bovine milk • Include a characteristic group of globular proteins • Are synthesized in the mammary gland of the cow – e.g. β-lactoglobulin and α-lactalbumin or • Are derived from the blood – e.g. bovine serum albumin and immunoglobulins

Whey Proteins • Some minor whey proteins have antimicrobial properties, e.g. lactotransferrin, lactoperoxidase, and lysozyme • Some identified polypeptides include the proteose pepton components which occur in the acid and rennet whey and the glycomacropeptides present only in rennet whey • Milk contains ≈ 30 enzymes, derived mainly from blood and secretory cell membranes. Some of these enzymes, especially lipoprotein lipase and proteinase, are technologically important in milk and dairy products

Whey proteins Is a major milk protein – ≈ 50% of the whey proteins
•β lactoglobulins Is a major milk protein – ≈ 50% of the whey proteins – ≈12% of total milk proteins • Monomeric molecular weight is about 18,300 D • Has a high affinity for retinol – speculations that its biological function is related to vitamin A transport • 8 genetic variants: A, B, C, D, Dr, E, F, G – A and B, have been found in all breeds of Bos taurus and B. indicus examined – some breeds of B. taurus also secrete variants C and D – milk of the Draughtmaster breed contains a fifth variant Dr – other variants are found in the milk of B. grunniens (Yak) and B. javanicus (Bali cattle)

Primary structure of Bos β- lactoglobulin &α Lactalbumine

β -Lactoglobulin – depends on the pH
• Conformation – depends on the pH – between pH 6.7 and 5.2 (its isoelectric point) at room temperature: dimerization (MW of 36,700 D) – between pH 5.2 and 3.5: octamerization of especially the A variant (MW of 147,000 D); maximal octamerization from pH 4.4 to 4.7 at 0°C – below pH 3.5: monomers due to strong electrostatic repulsive forces – the B variant (predominant one in Western cattle) octamerizes to a much smaller extend, possibly due to increased electro-static repulsion

β -Lactoglobulin • Has a free thiol group at Cys119 or Cys121
– of great importance for changes occuring in milk during heating – is involved in reactions with other proteins, notably k-casein and α-lactalbumin • Binds a variety of hydrophobic molecules

α-Lactalbumin • Represents » 20% of the proteins of bovine whey
• Is a small molecule with a molecular weight of about 14 kD • Iso-electric point is pH 4.8 • 2 to (3) genetic variants, A, B, (C) – only variant B is found in the milks of western breeds – milks of African Fulani and Indian Zebu cattle contain both A and B variants (differing by only one amino acid replacement) • Several minor components – the same amino acid composition as the major component – glycosylated forms that contain mannose, galactose, fucose, Nacetylglucosamine,N-acetylgalactosamine, and N-acetylneuraminic acid

α-Lactalbumin • Amino acid sequence is similar to that of lysozyme
– α-lactalbumin arose in evolution by gene duplication of an ancestral gene coding for lysozyme • Is necessary for the synthesis of lactose – interacts with galactosyltransferase (an enzyme which catalyzes the transfer of galactose) – without α-lactalbumin, glucose is an extremely poor substrate for galactosyl-transferase

α-Lactalbumin • Contains eight cysteine groups, all of which are involved in disulfide bonds • Has a highly ordered secondary structure • Has a compact, spherical tertiary structure • Undergoes a conformational change at pH < 4.0 – is accompanied by the release of bound calcium – results in temperature and concentration dependent aggregation • Thermal denaturation of a-lactalbumin – is quite reversible – is also accompanied by a release of bound calcium • Is stabilized against heat denaturation and aggregation in the presence of calcium

α-Lactalbumin • Has rather good emulsifying and foaming properties, although the native molecule reveals low surface hydrophobicity • The susceptibility of α-lactalbumin to surface denaturation at oil-water or air-water interfaces is involved in its emulsifying and foaming properties

Bovine serum albumin • Is identical to the blood bovine serum albumine (BSA), the major protein of blood plasma – milk ≈ 0.4 g BSA/litre – blood contains g BSA/litre • BSA contains 582 amino acids and has a calculated molecular weight of 66,267 D • Has no specific function in milk? – Is presumably a leakage protein • Is a well-known transport protein for insoluble fatty acids in the blood circulatory system – binding of fatty acids stabilizes the protein molecule against heat denaturation – this ability to bind fatty acids may promote lipolysis

Bovine serum albumin • Has a rather complex tertiary structure
– three about equal-sized major globular domains dissimilar in hydrophobicity, net charge, ligand binding sites – one free thiol – 17 disulfide linkages

Bovine serum albumin • Behavior of BSA in milk and milk products
– little is known – possible influence on properties of milk and milk products? • Heat-induced gelation behaviour at pH 6.5 – initiated by an intermolecular thiol disulfide interchange • BSA is highly soluble up to 35% (w/w) in pure water at 3°C • BSA undergoes extensive precipitation in the temperature range 40-45°C – this changed solubility above 40°C parallels the reversible (partial) unfolding of BSA • On acidification to pH 4.0, the BSA undergoes acid denaturation

Immunoglobulin(lg) • Are antibodies synthesized in response to stimulation by macromolecular antigens foreign to the animal • Bovine colostrum contains up to 100 g Ig/litre – concentration decreases to < 1 g/litre within a week of parturition • Their primary function in milk is to provide passive immunity for the neonate

Immunoglobulin • Are a very complex, heterogeneous group of proteins
• Five principal classes of Ig are recognized – IgG (sub-classes IgG1, IgG2, IgG3, IgG4) – IgA (sub-classes IgA1, IgA2) – IgM – IgD – IgE • Up to 80% of the immunoglobulin in milk or whey is Ig-G • The basic sub-unit in each class consists of four polypeptide chains linked by disulfide bridges – two heavy (H, MW ≈ ,000) chains – two light (L, MW = 22,400) chains

Four-peptide chain structure of immunoglobulins

Transferrins • = a group of evolutionary related iron-binding proteins
• Best characterized members – sero-transferrin (present in blood plasma and other extracellular fluids, e.g., spinal fluid, semen) – lacto-transferrin (milk, pancreatic juice, tears, leucocytes)

Lactotransferrin • A monomeric glycoprotein with a molecular weight of
about 80 kD • Has the ability to bind reversibly two Fe+++ per molecule (concomitantly ie together with two HCO3-) • Suppresses bacterial activity by removing the iron that is required for bacterial growth • Concentration – in bovine colostrums: ≈1 mg/ml – in milk: mg/ml • Several improved methods for their isolation have been published – because of the apparent physiological and nutritional significance of lacto-transferrins

Milk fat globule membrane proteins
• MFGM – a thin membrane that surrounds the fat globules in milk – contains approximately 50% protein and accounts for about 1% of the total protein of the milk • Total MFGM protein content – as observed is dependent upon the past history of the membrane from its formation to its analysis – both the temperature and the time of storage before analysis can alter the membrane composition and physical state • SDS-PAGE / IEF Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis IEF IsoElectroFluoroscence – most universally accepted method for characterization of membrane proteins – milk fat globule membrane proteins are classified based on their electrophoretic mobilitie

Estimated composition of natural MFGM

Non-protein nitrogen • = a large number of N-containing compounds of low molecular weight occur in milk • mg of N per litre milk • Wide variations in concentrations – probably arise from the fact that many of them are metabolites of amino acids and nucleic acids and from the fact that their concentrations in milk depend on the amounts of those substances consumed by the cow

Principal NPN components in milk

Enzymatic coagulation of caseins
• Introduction • Primary phase of rennet action • Secondary (non-enzymatic) phase of coagulation • Curd formation • Final stage • Rennets

• = clotting of milk by proteolytic enzymes • Represents one of the oldest operations in food technology – is used since antiquity for the manufacture of cheese • Is a complex process

• A primary enzymic phase – k-casein is altered and loses its ability to stabilize the remainder of the caseinate complex • A secondary non-enzymic phase – aggregation of the altered casein micelles takes place • A third step: liquid to solid – the aggregate of casein micelles forms a firm gel structure • A possibly separate fourth step – the curd structure tightens and syneresis (the expulsion of water by the curd arising from structural rearrangements after formation) occurs. In cheese making, this syneresis enhanced by cutting • The enzymic reaction and the subsequent aggregation can occur simultaneously during the later stages • Without primary enzyme action, no clotting occurs

Primary phase of rennet action
• Involves the enzymatic cleavage of the Phe105-Met106 linkage of k-casein • This results in the formation of – soluble glycomacropeptide (GMP) which diffuses away from the micelle and is washed out ( ) – para-k-casein = a distinctly hydrophobic peptide that remains on the micelle (1-105) • A relatively quick reaction – turnover: 100 s-1 in milk, pH 6.7, 30°C – seems independent of micelle size – decreasing temperature by 10°C reduces the rate by a factor 2

Properties of k-casein and the polypeptides formed by chymosin action

Secondary phase of coagulation
• A non-enzymatic phase = aggregation of the renneted micelles • Hydrolysis of k-casein reduces the intermicellar repulsive forces (electrostatic and steric) and the micelle stability • No aggregation until % of k-casein has been destroyed • During the last 20% of the proteolytic reaction – concentration of micelles capable of aggregating increases – the aggregation rate increases rapidly • Aggregation rate – is unaffected by the concentration of rennet – is unaffected by the size of the casein micelles – is very sensitive to the concentration of calcium ions – is very sensitive to the temperature

Secondary phase of coagulation
• Milk will not clot at less than about 15°C – this is a direct consequence of the slowness of the aggregation reaction at low temperatures • Low temperatures (less than 8°C) – allow proteolysis of k-casein in the absence of coagulation – coagulation occurs on subsequent warming • Temperatures above 45°C – aggregation is very efficient – approaches the theoretical maximum particle collision rate • Addition of CaCl2 – decreases the coagulation time – at high calcium chloride concentrations (0.4 M), the coagulation time is retarded severely and only weak curd is obtained

Curd formation casein micelles
• Is characterized by a steady aggregation of the rennet treated casein micelles – Chains of micelles are formed at first – By the rennet clotting time (RCT), the chains have begun to link into a loose network – The network extends and becomes more differentiated, with the chains of micelles aligning together • The visually-observed rennet clotting time (RCT) – when the curd starts to form – the resultant of two reactions: the enzymatic reaction is largely determinant, as flocculation time is insignificant to splitting time – at 4°C, the enzymatic reaction may be complete in about 3 h, while clotting takes a week

Final stage • Is not well defined and includes
– syneresis and firming of the curd – a loss of paracasein micelle identity – non-specific proteolysis of caseins in the coagulum • The paracasein micelles fuse into larger units as CCP rearranges throughout the micellar region – this may be analogous to binding between submicelles in a micelle • Syneresis is enhanced by applying external pressure to the curd or deforming it (stirring, cheddaring)

Renneting of milk S= degree of splitting of k casein
A= Agregation of casein micelle F= Firmness of the coagulum(gel)

Rennets • Traditional rennet
= a crude enzyme extract prepared from calves stomachs used in the milk-clotting-process • Shortages of this material ! – use of pepsins and acid proteases produced by other animals, fungi and microorganisms • Nowadays any milk-clotting enzyme preparation yielding a relatively stable curd is designated as rennet

Animal rennets • Calf rennet
– generally refers to an enzyme extract obtained from the fourth stomach (abomasum) of 10- to 30-day-old un-weaned calves – used to coagulate milk for cheese production • Chymosin – the purified milk-clotting enzyme present in crude rennet – a molecular weight of 35,600 D – belongs to the group of aspartic proteases – the standard against which all other types of milk-clotting enzymes are compared – coagulates milk rapidly at its natural pH with little further degradation of the milk proteins

Animal rennet • As the calf ages, chymosin is replaced by pepsin
– in cattle, the chymosin secretion never comes to a complete stop – pepsin can also clot milk • Pepsin’s use as a 100% replacement for calf rennet in cheese making is limited due to shortcomings (i) set time for curd formation is extented (ii) the curd is softer than that obtained with calf rennet (iii) bitter peptides are formed (iv) fat loss is excessive (v) the cheese flavor is generally bland (vi) above pH 6.5 pepsin activity falls of so rapidly that its use is limited to the production of only some sweet (e.g. swiss cheese) and some Italian varieties of cheeses

Animal rennet • A worldwide shortage of calf rennet has developed
– largely due to an increase in cheese production – a greater tendency to slaughter "mature" calves • Stomachs of other young mammals (lambs) – obvious sources of rennet substitutes – they produce acid proteinases that may be expected to have properties in common with calf rennet – pepsins and chymosins have also been isolated from the adult cow, sheep, buffalo, chicken, rabbit, swine, and goat • Apparently, humans, dogs, and possibly young pigs do not secrete chymosin

Plant Rennet • Many proteolytic enzymes of vegetable origin are milk coagulants – papain from papaya – ficin from Ficus spp. – bromelain from pineapple • Most plant coagulants – proved to be non-specific in their proteolytic activity – the excessive proteolysis results in bitter products • At present there is no commercially available rennet derived from a higher plant

Plant rennets

Bacterial rennets • A number of bacteria are reported to produce milk-clotting enzymes • Possible advantages – high titer of the active preparation – short generation times • Commercial production of rennets with bacteria has not been successful – invariably strong and nonspecific proteolytic action – loss of fat and nitrogen in the whey – reduced yield – poor quality of the aged cheese

Bacteria reported to produce milk coagulants

Fungal rennet Many species of filamentous fungi produce a chymosin like enzyme • Most fungal coagulants are much too proteolytic for use as rennets • The coagulants from three fungal species have proved suitable for large-scale commercialization – Endothia parasitica (Cryphonectria parasitica) – Mucor miehei (Rhizomucor miehei) – Mucor pusillus (Rhizomucor pusillus)

Recombinant calf chymosin
• Differences still exist between microbial and calf chymosin (i) the ratio of proteolytic to milk-clotting activity is still a little higher or microbial rennets, resulting in greater hydrolysis of cheese protein during ripening and leading to soft body and texture (ii) microbial rennets are more thermostable, cannot be inactivated at normal pasteurization temperature, and therefore present problems in processing of cheese whey (iii) change in milk-clotting activity from calf to microbial rennet will require adjustment of the process parameters such as temperature, pH, calcium concentration etc.

Recombinant calf chymosin
• Calf chymosin has been cloned in suitable microbes • Commercial calf chymosin produced by recombinant DNA technology – Aspergillus niger var. awamori : Chymogen® (Chr. Hansen) – Escherichia coli K12 : Chy-Max® (Pfizer) – Kluyveromyces lactis : Maxiren® (Gist-Brocades) • A variety of cheeses have been produced using recombinant chymosin – no significant differences could be detected among cheeses made by rennet and recombinant chymosin

Heat-induced coagulation
• Bovine milk withstands high processing temperatures – is essential for many modern milk-processing operations The heat stability of milk is normally far in excess of that required to withstand all the commercial processes to which it is subjected. • Non-casein proteins of milk are relatively heat-labile • Caseins are remarkably heat-stable – sodium caseinate in water can withstand heating at 140°C for >60 min at pH 6.7 and typical bulk bovine milk is stable for »20 min at 140°C

• Most protein denaturation reactions are very pH dependent – the pH change caused by heating is primarily responsible for heat coagulation – the decrease in pH upon heating is partially due to changes in the buffer capacity of milk salts and the release of carbon dioxide • Heating milk at a pH < 6.5 for 20 to 30 min at 100°C – it coagulates to form a gel – casein micelles have denatured whey proteins attached to the micelle surfaces Heating milk at a pH > 6.7 – very intensive heat treatment is required for precipitation – a gel does not form

• High heat stability of milk casein micelles – reflects the open, random-coil structure of the caseins – treatment at elevated temperatures has little effect on its (absent?) secondary and tertiary structures – hydrophobic bonds that play a major role in the association of caseins at low or ambient temperature are non-existent at temperatures > 100 °C and hence there is little tendency to aggregate at high temperatures – negatively charged phosphate groups of casein may contribute to its remarkably high heat stability

Acid induced coagulation
• Caseins are insoluble at their iso-electric points (≈ pH 4.6) at temperatures above 20 °C • Caseins remain more or less soluble if milk is acidified to pH 4.6 at temperatures lower than 6°C – the precipitate formed becomes progressively coarser and more rubbery as the temperature at precipitation is increased – raising the temperature gives an immediate precipitation • Although the iso-electric point of the γ-caseins is about 6, they also precipitate at pH 4.6 • Proteose peptons are soluble at pH 4.6

Ethanol coagulation • Milk coagulates on addition of ethanol (usually to ≈ 40%) • Ethanol stability = the ability of milk to withstand treatment with ethanol without coagulation – has been used as a not-very-effective selection test for milk for the manufacture of sterilized concentrated milk – is strongly influenced by the Ca++-concentration and the pH

Age-gelation of sterilized milk
• In-container sterilized evaporated milk occasionally gels during storage • UHT-sterilized (concentrated) milks – are very prone to gelation during storage – limits the usefulness of such products • The cause (s) and mechanism of age-gelation – have not yet been definitively established – age-gelation of unconcentrated UHT milk • plasmin and extracellular proteinases secreted by psychotrophic bacteria are major causative factors – age-gelation of UHT-sterilized concentrated milk • physicochemical changes in the casein micelles appear to be principally responsible for milk sterilized by a direct UHT process

Age-gelation of sterilized milks
• UHT sterile milk concentrate – contains casein micelle-denatured whey protein complexes that are about double the size of native casein micelles – these complexes undergo extensive aggregation during storage – this eventually causes age thickening • Age thickening is promoted by – high milk solids content – addition of alkali to raise pH – addition of citrate, phosphate, and other anions that lower Ca ion activity • Addition of Ca++ improves stability against age thickening

Age thickening of concentrated milk
Dotted lines show milk with added polyphosphate Solid lines indicate milk without added polyphosphate

Denaturation of whey proteins
– have typical globular conformations – are relatively heat labile – denaturation occurs upon heating • Susceptibility to heat denaturation is influenced by – pH – Ca++-concentration – protein concentration – presence of sugars, polyhydric alcohols and protein modifying agents • The ranking order of the heat sensitivity α-lactalbumin > β-lactoglobulin > BSA > immunoglobulins

• Heat-induced interaction between b-lactoglobulin and k-casein – plays a major role in determing the heat stability and rennet clotting behaviour of milk – β-Lactoglobulin precipitates on casein micelles upon heating – β-Lactoglobulin is also precipitated by destabilization of the micelles (β-lactoglobulin - k-casein interaction)

Enzymes

Enzymes • Milk contains both indigenous and exogenous enzymes,
the latter being mainly bacterial • Most significant bacterial enzymes occurring in milk are heat-stable lipases and proteinases elaborated by psychotrophic bacteria • Cause several defects in milk but also can be responsible for some beneficial effects – desired transformations, e.g. ripening of cheese (natural proteases) – undesired degradation processes, e.g. lipolysis of raw milk; – indicator enzymes for heating of milk – antimicrobial effects of certain enzymes – quality parameter

Endogenous Enzymes of bovine milk

Enzymes • Lactoperoxidase • Xanthine oxidase • Catalase • Lipase
• Phosphatase • Protease • Reductase • Other enzymes

Lactoperoxidase • Catalyzes oxidation by H2O2 of a long list of electron-donor compounds, including – aromatic amines – phenols – aromatic acids – leuko dyes – tryosine and tryptophan – ascorbate – iodide – nitrite – thiocyanate • Transfer oxygen in its atomic form from hydrogen peroxide to its substrates

Lactoperoxidase • May amount to as much as 1% of the total serum proteins of milk (i.e. 60 mg.kg-1) • Its properties are well-known (pH-optimum 6,8; molecular weight ) • Is an indicator enzyme for high pasteurization • Catalyzes oxidation of thiocyanate (SCN-) to a product (OSCN- ) that inhibits certain bacteria • Thiocyanate is a natural constituent of milk and hydrogen peroxide is produced by some bacteria themselves (self-inhibition) • In some countries this effect is enhanced by further addition of hydrogen peroxide

Xanthine oxidase • Is very prominent in milk
• Most of it is associated with the fat globule membrane • Milk is a source of choice for isolating this enzyme for investigation • Different names are used for xanthine oxidase; e.g – Schardinger enzyme – adenine oxidase – nitrate reducing enzyme – aldehyde oxidase

Xanthine oxidase • Catalyzes the reaction of purine bases (xanthine and hypoxanthine) to ureic acid & hydrogen peroxide • Can reduce NO3 (which occurs in milk only in trace quantities) to NO2 which is a powerful inhibitor of some bacteria – put to use in the manufacture of certain types of cheese, where a little nitrate is added to the milk to prevent late blowing of the cheese by Clostridia • Is especially activated by pasteurization and homogenization

Catalase • Catalyzes the decomposition of H2O2 to H20 and O2
• In milk its activity parallels leukocyte count and is higher in mastitic milk and colostrum than in normal milk • Its activity increases with the multiplication of bacteria • Is inactivated at 65°C after 30 minutes and is used as a test to control heat treatment of milk • The catalase procedure – H2O2 is added to milk to inactivate spore forming bacteria – excess of H2O2 is decomposed by the catalase

Lipases • Catalyzes the hydrolysis of triglycerides to glycerol and fatty acids • Membrane lipase – is originally present on the casein micelles, but adsorbs irreversible to the fat gobule membrane during cooling of milk – is often called lipoprotein lipase – is responsible for the rancid flavor of raw milk • Plasma lipase – stays in the plasma during the cooling of milk – can be activated by mechanical movement • During cool conservation of milk, lipolysis can occur as a consequence of the combination temperature - mechanical movement • Thermo-labile enzymes

Phosphatase • Also called phosphomonoesterases
• Of natural origin and responsible for hydrolysis of phosphoesters • Alkaline phosphatase – pH-optimum is about 9,6 – is destroyed by pasteurization and an index for the efficiency of pasteurization – two major isoenzymes have been identified, α and β phosphatase,mainly located in the milk plasma and fat globule membrane – reactivation can occur • Acid phosphatase – pH optimum of 4,5 – extremely heat stable, but is sensitive to light and UV – is present in low concentrations, in particular in skimmed milk

Protease • Plasmin – the principal milk protease
– belongs to the alkaline serine proteinase class – is probably identical to the plasmin of blood – is present on the casein micelles – has alkaline properties – survives pasteurization – is relatively resistant to UHT-treatment; can cause a bitter flavor (hydrophobic peptides of low molecular weight) and changes in viscosity and appearance – is also of importance during ripening of cheese • A second protease – with maximal activity at pH 4,0 – is very heat sensitive and hydrolyses preferably α-caseins – classification not yet clear; an aspartate proteinase?

Reductase • Related to the reducing properties of microorganisms and leukocytes • Can be used as quality-parameter • Causes discoloration of methylene blue and other dyes • Has lost its importance as a result of better cooling conditions, which has changed the bacterial flora in milk

Other enzymes • Amylases
– catalyze the hydrolysis of starch to dextrin and maltose, dependent on its nature (α- or β-amylases) – the α-amylase is inactivated by heating 55°C during 30 minutes, – the β-amylase keeps its activity after 30 minutes at 65°C. • Lysozyme – is quantitatively an important fraction of the whey proteins of human milk (400 mg.l-1) – less important in cow's milk – is a powerful bactericide as it attacks polysaccharides of the bacterial cell wall, causing lysis of the bacteria – it releases bifidus factors • Lactase – normally doesn't occur, and if so only in small quantities

Chemical and Physical Properties of Milk
Minerals and Vitamins

Minerals and ionic equilibra
• Ash content • Composition of mineral fraction • Equilibra • Trace elements

Ash content • ≈ 0,70 - 0,85 % < actual present salt content
• Comprises – oxides of Na, K, Ca, Mg, Fe, P and S – some chlorides – S and parts of P and Fe of organic origin

Some minor components in milk

The composition of mineral fraction
• Three families of salt constituents 1. Na, K and Ca, which exist almost entirely as free ions in milk and are readily diffusible (present in milk ultrafiltrate); the concentrations of these 3 ions are negatively correlated to lactose, as required to maintain osmotic equilibrium of milk with blood 2. colloidal Ca, Mg, inorganic P and citrate; total concentrations of Ca, Mg, P and citrate in milk plasma are 30,3; 5,2; 21,4 and 9,5 mM. 3. salts, whose concentrations are affected by the natural pH of milk, namely, diffusible Ca, diffusible Mg, diffusible citrate, Ca2+ and HPO4-2

Equilibra • = the form in which the mineral occurs, whether as ions, whether in colloidal form • Of importance for physicochemical properties of milk -heat stability -gelling -clotting • Ca/P-equilibrum • Factors influencing equilibra

Ca/P-equilibrum • Forms of calcium 1. 20 % as organic Ca: Ca-caseinate
2. 80 % as inorganic Ca 50 % as inorganic colloidal tricalciumphosphate 20 % as not ionized Ca salt : Ca citrate and phosphate 10 % ionic Ca • Forms of phosphorus 1. 30 % as organic P 10 % in phopsholipids and other esters 20 % in caseins as phophoserine 2. 70 % as inorganic P 35 % colloidal tricalciumphosphate 35 % soluble phosphate

Ca/P-equilibrium • Dialysis of milk doesn't result in removal of all Ca and P, which means that these elements are partly bound • A part of the calciumphosphate is present in an insoluble form, the colloidal calciumphosphate • Milk is supersaturated in calcium phosphate; this is precipitated on the micelles • On the casein, cations are bound as counter-ion for the negative charge of the protein

Factors influencing equilibrium
• Acidification – During acidification more Ca will be present in its ionic form; the colloidal calcium phosphate will be more soluble – By acidification micelles are destabilized; the negative charge of the micelles decreases, which causes a higher concentration of ionic Ca • Heating – During heating a part of the calcium phosphate will become insoluble and precipitates on the micelles; this can be accompanied by a slight pH-drop – Heating above 100°C causes splitting of phosphate from the caseins and creates acids out of lactose; the result is a pH-drop that influences the equilibrium

Factors influencing equilibra
• Water removal – During evaporation processes, the concentration of solubilized components increases, which can affect the equilibria • Additions – Addition of CaCl2 causes an increase of Ca-activity and a decrease in the stability of the micelles – Addition of sodium citrate and certain phosphates enhances the stability of the micelles

Trace elements • Content of trace elements • Role of Cu and Fe

Content of trace elements
• Some of these trace elements have an important role in the activity of enzymes • Manganese is known to play an important role in the development of lactic acid bacteria

Trace elements

Role of Cu and Fe • Cu and Fe have a technological significance as catalyst of oxidation reactions • Cu is present in fat globule, where it can catalyze oxidation of phospholipids •Cu can enter the milk via dust, apparatus and water; this Cu is distributed between membrane and serum. • Technological processes influence the distribution of Cu ions: – acidification causes a migration of Cu to the fat globule membrane – during cooling Cu migrates from the membrane to the plasma – during heating Cu migrates to the membrane, but high pasteurization hinders this transport; the distribution of Cu, during decreaming, depends on the history of milk (heating, cooling)

Chemical and Physical Properties of milk
Vitamins

Vitamins • Milk is a good source of diverse vitamins
• There is often a difference upon their solubility in fat or water

Fat Soluble Vitamins • Vitamin A • Vitamin D • Vitamin E • Vitamin K

Vitamin A • Vitamin A and its analogous are important for eye functioning • β-carotene – also called pro-vitamin A – is also present in milk – is responsible for the yellow color of butter • Content – whole milk : 0,4 mg per kg – skimmed milk : 20 μg per kg – butter: 7,5 mg per kg. • During summer, milk contains 50% more vitamin A than during winter months • Pasteurization, sterilization and drying have nearly no effect on vitamin A or β-carotene content

Vitamin D • Is important for the skeleton
• Content of milk: about 2 μg per kg – is very dependent on seasonal variations – summer milk contains 2 to 3 times more vitamin D than winter milk • Is very stable and its content isn't affected by pasteurization or sterilization

Vitamin E • Vitamin E (tocopherol) deficiency causes sterility in male
and female animals • Content of human milk is about ten times that of cow's milk • The need for vitamin E increases as the amount of unsaturated fatty acids in food increases • Content – milk: 1 mg per liter – butter: 23 mg per kg – principal content is higher during summer than during winter, caused by the higher tocopherol content of fresh grass • Heat or other processes don't cause a significant decrease in vitamin E content

Vitamin K • Only in traces in milk, if at all
• Human needs for this vitamin are supplied from consumption of plant materials containing it and by microbial synthesis in the digestive tract

Water soluble Vitamins in Milk
• Vitamin B1 • Vitamin B2 • Niacin • Vitamin B6 • Pantothenic acid • Biotin • Folic acid • Vitamin B12 • Vitamin C

Vitamin B1 • = Thiamine • Is essential for growth and metabolism of all animals but also of many plants and microorganisms • In milk not only present in its free form, but also phosphorylated (18-45%) and as a protein complex (5- 17%) • Mean concentration in milk: about 0,43 mg per liter • Daily requirement of an adult person: about 1,0 to 1,4 mg • More or less destroyed by heat, dependent on time – temperature combination – loss in pasteurized milk and UHT milk: about 3-4% – loss in sterilized milk: can mount up to 45%

Vitamin B2 • = Riboflavin • Very important in – hydrogen transport
– carbohydrate and protein metabolism – transformations of sugar and proteins into fatty acids – eye-functioning • Mostly present in its free form in cow's milk • Content of whole, skimmed and butter milk : about 1,7 mg per liter • Daily requirement of an adult person: 1,5 to 1,7 mg • In the absence of oxygen very persistent during heat treatment. – the effect of pasteurization and sterilization is small

Niacin • Is a component of co-enzymes that are involved in the citric acid cycle, metabolism of fatty acids and the glycol acid cycle • Content of milk: about 0,9 mg per liter • In skimmed and butter milk, this content decreases • Daily requirement of a grown person: up to 13 to 18 mg

Vitamin B6 • Is very important in protein metabolism
• Main content of milk: about 0,6 mg per liter • Daily requirement: about 1 to 4 mg per day • Pasteurization or homogenization give no significant losses of vitamin B6 content • Great losses are caused by sterilization (up to 50%) • Vitamin B6 is also very sensitive to light

Pantothenic acid • Is involved in the metabolism of carbohydrates, fats and amino acids • Mean content of milk: about 3,4 mg per liter • Daily requirement of a grown person: about 10 to 15 mg • Very stable during a number of processes (e.g. manufacture of evaporated milk, sterilized milk and milk powder)

Biotin • Is involved in carbohydrate and fatty acids metabolism
• Milk is a good source of biotin • Mean content – whole milk: 0,030 mg per liter – skimmed milk: 0,016 mg per liter – buttermilk: 0,011 mg per liter • Daily requirement of an adult person is only 30 μg • Light and heat have only a small effect – loss in pasteurized and UHT: less than 10 % – loss in sterilized milk: up to 10 to 15 %

Folic acid • Is necessary for the normal development of red blood cells • Few data about the contents in milk are available – in raw milk about 0,06 mg per kg – this value is also reached in cheese • Is very sensitive to heat, oxygen and light – during sterilization, losses of 50 % can occur

Vitamin B12 • Is the only vitamin that contains a metal (cobalt)
• Deficiencies of this vitamin can cause anemia • Is only present in animal products and vegetarians may have these deficiencies • Mean content of milk: about 4,2 μg per liter • Daily requirement of a grown person: only 1 μg • Is sensitive to heat and oxygen – in strongly heated products, the losses can reach 100 %

Vitamin C • Is involved in the formation of intercellular material
(collagen) • Content – fresh raw milk: nearly 20 mg per liter – skimmed milk: 16 mg per liter – butter milk: 12 mg per liter • Daily requirement of an adult person 55 to 60 mg • Is very sensitive to light, heat and metals (Cu, Fe). – during pasteurization 20 % is destroyed – during evaporation 75 % is destroyed – during sterilization 50 to 100 % is destroyed

Chemical & Physical Properties of milk
Other Compounds

Other compounds • Organic acids • Gasses • Biological factors

Organic acid • Citric acid
– is very important for the stability of milk – is also a substrate for microorganisms (used as starter culture to make dairy products) and is transformed to aromas Besides citric acid, also lactic acid, hippuric acid (benzoic acid + glycocol) and orotinic acid (precursor of nucleotides) are found

Gases • After milking 8 % (volume) gases, especially CO2 (6%),are present • During processing (heating) and storage, CO2 content decreases, while O2 and N2 content increase • Raw commercial milk contains at room temperature about 1,3 % N2 and 0,5 % O2 by volume, or about 15 and 6 mg.kg-1

Biological Factors • Bifidus factors • Streptogenines
– peptides formed out of casein – growth factors for lactic acid bacteria • Lactenines – active against lactic acid bacteria – L1, L3 agglutinines – L2 lactoperoxidase

Physical and Chemical Properties of Milk
Physical Properties

Physical Properties • Density • Dry matter • Freezing point • Acidity
• Redox potential

Density • Can be expressed as mass density or volumetric mass and is designated as ρ • Is temperature-dependent • Recknagel effect – with a change in temperature, a slow stabilization of the density has to be considered – density is dependent on the physical properties of fat and the hydration of proteins can take time • The density of milk – at 20°C is on average about 1030 kg.m-3 – normally varies within the range of 1027 to 1033 kg.m-3

Density • The density of milk – is dependent on composition
– can be calculated from the density and mass fraction of individual components – at 20°C the densities of water, milk fat, protein, lactose and other components are 998.2, 918, 1400, 1780 and 1850 respectively • Fat content decreases the density of milk, while solubilized components increase its density – as a consequence, the density of skimmed milk is higher than that of whole milk. – addition of water causes a decrease of the milk density

Dry matter • Is in essence a chemical property
• Has a major influence on physical properties • Its mean value in milk is 12,50 % • Dry matter is determining weight loss during heating • The dry matter can also be calculated from some characteristics by the formula: DM=1.23 F +2.6 *100 (ρ )/ ρ20 with ρ20 = volume mass at 20°C D.M. = dry matter (%) F = fat content (%) Fat-free dry matter (F.F.D.M.) is defined as F.F.D.M. = D.M. - F

Freezing point • Is a colligative property that is determined by the molarity of solutes rather than by the percentage by weight or volume • Is a constant that is used to determine addition of water, which has a warmer freezing point as a result • Is determined by some components in solution: lactose and salts

Freezing point • Freezing point determinations may be done by the Horvet procedure • For scaling, solutions of sugar or NaCl are used – 7 % sucrose = -0,422°H (6,879 g NaCl diluted to 1000 ml with water) – 10 % sucrose = -0,621 °H (10,175 g NaCl diluted to 1000 ml with water) – Conversion formulas are given m°H = 1,0356 m°C m°C = 0,9656 m°H

Freezing point • The freezing point of milk is usually in the range of -0,512 to -0,550°C with an average of -0,522°C • If the freezing point of unwatered milk is known, the relationship between added water and freezing point depression is given by W= (C-D)(100-S)/C with W = % (w/w) extraneous water in suspect milk C = actual or reference freezing point of genuine milk D = freezing point of suspect milk S = the percentage (w/w) of total solids in the suspected milk

Freezing point • Soured or fermented milk is not suitable for added water testing – because the freezing point is lowered by lactic acid and increased concentrations of soluble minerals • Several reports suggest that heat treatment of milk, including UHT and retort sterilization causes little permanent effect on freezing points • It has also been suggested that freezing points are not a reliable index for added water in processed milk

Acidity • An important quality parameter, but is also a characteristic
for fermentation processes • Fresh milk normally has a pH of 6,6 to 6,8. – is not the result of lactic acid, but a result of dry matter • The acidity can be expressed in: – D°(Dornic): number of 0,1 ml NaOH N/9 needed to neutralize 10 ml of milk – N°(Normal grades): number of 0,1 ml NaOH 0,1N to neutralize 10 ml of milk – S.H. (Soxhlet-Henkel): number of 0,1 ml NaOH 0,25N to neutralize 10 ml of milk – % lactic acid • Earlier, triation of milk at reception was based on the acidity: alizarol test, bromocresol test, alcohol test, ...

Redox potential • The redox potential of milk is in the range of +0.2 to +0.3 V and is mainly determined by dissolved oxygen • Milk is essentially oxygen free when secreted but about 0,3 mM O2 is present after equilibrium with air is established • Removal of oxygen by nitrogen lowers the Eh of milk to about -0,12 V • Decreased oxygen tension by bacterial respiration is the basis of the methylene blue reduction test for milk bacterial quality • The other redox systems of significance in milk are ascorbate and riboflavin

Redox potential • Ascorbate in freshly drawn milk is all in the reduced form, but oxidizes during refrigerated storage; this process also produces singlet oxygen which is involved in lipid oxidation • Riboflavin is important for photooxidation reactions in milk; methionine is transformed into methional, which is the principal component of "sunlight" flavor in milk • Heat treatment is well known to increase the reducing capacity, mainly due to activation of protein thiol groups and products of Maillard browning reactions – activated thiol groups cause cooked flavor which decreases as cysteine bonds reform on standing

Technology of Milk and Milk Products
Primary Treatments

Primary treatments • Reception • Centrifugation • Standardization
• Homogenization

Raw milk quality • Fresh raw milk collected aseptically from the udder: bacteria / cm3 • Milk produced under normal hygienic conditions: < bacteria / cm3 • High counts are indicative of a breakdown in hygiene – contamination of the cow's udder – contamination from inadequately cleaned and sterilized equipment – the result of mastitic infection • Raw milk should be maintained at less than 4 °C – between milking and processing – to prevent spoilage and pathogenic organisms

Reception • At delivery, a sample is taken per tanker
• After a quick control, the tanker is unloaded • The milk is cooled over a plate heat exchanger and pumped into storage tanks • After each collection, the tanker is cleaned and disinfected by a CIP (clean-in place) system In some cases the milk is collected via a collecting station and transferred to the processing industry • For a temporary stabilization, a thermisation (60-65°C, 10- 20s) can be applied • Some regular tests that are performed include freezing point depression, antibiotics, fat content

Centrifugation • Aim (1) to obtain skimmed milk and cream
these phases can afterwards be mixed together to obtain the desired fat content (2) to remove the visible dirt (3) to remove bacterial spores (bactofugation)

Spontaneous creaming • Because of the difference in density between
– fat globules (df = 920 kg.m-3 at room temperature) – plasma (dp = 1030 kg.m-3 at room temperature) • Phenomenon we often want to prevent • Is much enhanced when the globules have been aggregated into floccules, clusters or granules • Stokes Law: V= 2r2 (dp- df )g/9η v = creaming velocity (m/s); r = radius of fat globules (m) dp = density plasma (kg/m3); df = density fat (kg/m3) g = gravity acceleration (m/s2); η = viscosity (Pa.s)

Spontaneous decreaming
• Example r = 1mm η = 1, Pa.s at 20 °C dp-df = 103,4 kg/m3 at 20 °C v = 0,45 mm/h

Centrifugal decreaming
• A considerable increase in the velocity of rising or settling can be obtained in a centrifugal field • The following formula can be used: 2r2 (dp- df )g*Z/9η Z = centrifugal constant R = radius of the orbital path (m) n = number of revolutions per minute (rpm) • Same example of spontaneous creaming R = 0,12 m n = 6000 rpm v = 0,60 mm/s Z=Rn2/894

Operation of Centrifuge

Operation of Centrifuge
• Cream separator – bowl base and lid – separating discs (conical) – feed inlet and outlet – sludge chamber • Separators have up to 120 discs – one above another – angle of inclination is 45°to 60° – outer diameter 200 to 300 mm – made of stainless steel with a wall thickness of 0,4 mm – space between the discs varies between 0,4 and 2 mm – laminar flow through the narrow gaps ensuring that separation is not adversely affected by turbulence – larger gaps are necessary if there is danger of clogging – holes forming a channel for the ascending liquids

Types of Centrifuge • Tubular bowl centrifuge
– long, narrow, cylindrical bowl rotating at high speed in an outer stationary casing. – The feed is introduced through a stationary pipe to the bottom of the bowl and quickly accelerated to bowl speed by means of vanes – The two liquids are removed from the annular layers formed through a circular weir system and discharged into stationary covers.

Types of Centrifuge • Disc-bowl centrifuge
– a relatively shallow, wide cylindrical bowl rotates at moderate speed in a stationary casing – the bowl is usually bottom driven – the feed is normally introduced to the bottom of the bowl through a centrally located feed pipe from above – the bowl contains a number of closely spaced metal cones, called discs, which rotate with the bowl and are located one above the other with a fixed clearance between them – the discs have one or more sets of matching holes which form channels through which the feed material flows – the separated liquids are removed by means of a weir system

Types of Centrifuge • Semi open design
– The centrifugal force throws the milk outwards to form a ring with a cylindrical inner surface. – This is in contact with air at atmospheric pressure, which means that the pressure of the milk at the surface is also atmospheric. – The pressure increases progressively with increasing distance from the axis of rotation to a maximum at the periphery of the bowl. – Cream moves inwards towards the axis of rotation and passes through channels to the cream paring chamber (paring disks) – The skim milk leaves the disc stack at the outer edge and passes between the top disc and the bowl hood to the skim milk paring chamber. – The rims of the stationary paring discs dip into the rotating columns of liquid, continuously paring out a certain amount. The kinetic energy of the rotating liquid is converted into pressure in the paring disc.

Types of Centrifuge Semi open design Paring Discks

Types of Centrifuge • Hermetic separator – The bowl of a hermetic
separator is completely filled with milk during operation. There is no air in the centre. – The pressure generated by the external product pump is sufficient to overcome the flow resistance through the separator to the discharge pump at the outlets for cream and skimmilk. – With this type of separator it is possible to obtain a high concentrated cream (>75%), which is not the case with a semi open separator.

Types of Centrifuge • Nozzle-discharge (self-cleaning) centrifuge
– = a self-opening centrifuge. – is of the disc-bowl type but the bowl is usually biconal in shape – a number of ports are spaced around the bowl usually at its largest diameter – these centrifuges are used to clarify milk from visible dust

Process Parameters • De-creaming of milk is largely dependent on temperature • Usually de-creaming is carried out at elevated temperatures – viscosity decreases – difference dp-df increases • The optimal decreaming temperature is 45 to 55°C • Elevated temperature has also defects – e.g. an increased risk of precipitation on the apparatus

Performance of a Separator
• Is judged by the residual fat content of the separated milk or by the degree of cream separation from the whole milk • The degree of cream separation is defined as: E= total fat in cream/total fat in the whole milk should be as high as possible Furthermore: mw = mc + ms and: mw.fw = mc.fc + ms.fs mw = mass of the whole milk fw = fat content of the whole milk mc = mass of the cream fc = fat content of the cream ms = mass of the separated milk fs = fat content of the sep. milk

Performance of a separator
• E therefore becomes: E=mc.fc / mw.fw= fc(fw-fs)/fw(fc-fs) • As fs << fc, and fc ≈ fc-fs : E=1-fs/fw • E is determined by different factors: – functioning of the separator – temperature – presence of gasses – the season – quality of the raw milk, especially the amount of free fatty acids

Standardization • Continue standardization can be achieved by means of
pipe and valve connections • A part of the cream flow is led into the separated milk, so that the desired fat content is obtained • Mass balance equation: mC.fC + mS.fS = (mC + mS).fM mC : mass flow cream fC : fat content cream mS : mass flow skimmed milk fS : fat content skimmed milk mM : mass flow stand. milk fM : fat content stand. milk • After some transformations: mc/ms= fM-fs/fc-fM

Standardization • Direct automated standardization of milk
– The pressure control system at the skim milk outlet maintains a constant pressure, regardless of fluctuations in the pressure drop over downstream equipment. – The cream regulating system maintains a constant fat content in the cream discharged from the separator by adjusting the flow of cream discharged. This adjustment is independent of variations in the throughput or in the fat content of the incoming whole milk. – Finally, the ratio controller mixes cream of constant fat content with skim milk in the necessary proportions to give standardized milk of a specified fat content. – The standard deviation, based on repeatability, should be less than 0.03% for milk and 0.2 – 0.3% for cream.

Homogenization • Is used principally to prevent or delay the formation of a cream layer in full cream milk • A reduction in the size of the fat globules • As a result the stability of the emulsion is improved • Raw and homogenized milk at 350 atm

Principle • Homogenization divides globules into smaller ones with
diameters down to < 1 µm, depending on the pressure • Done by forcing all of the milk at high pressures through a narrow slit, which is only slightly larger than the diameter of the globules themselves • The velocity in the narrowest slit can be 100 to 250 m/s • This causes high shearing stress and microturbulence • Fat globules become deformed, then become wavy and then break up

Principle of Homogenization
Scheme of Homogenizer

Homogenization Parameters
• Pressure is the most important parameter • Pressure influence on homogenization degree : log dva = const - 0,6 log P dva = average diameter P = homogenization pressure • The temperature has different effects – by lowering the viscosity, turbulence increases – the fluidity of the fat plays an important role • Normally, homogenization is executed at 50 to 70°C.

Homogenization parameters
• The ratio fat to surface active material determines also the degree of homogenization – for the homogenization of whole milk, there is normally enough protein present – for fat-rich products, the amount of proteins is a limiting factor; as a result, the fat globules can aggregate to clusters

Influence of Homogenization
• Homogenization causes a change in following properties : – fat globule diameter – clustering is directly proportional to pressure and fat content, and inversely proportional to fat-free dry matter – creaming is strongly reduced – the thickness of the membrane increases with an increasing homogenization pressure (± 15 nm in homogenized milk) – heat stability decreases with increasing homogenization

Effect on the product The fat globules are surrounded by a membrane
-5 to 10 nm thickness -Is approximately composed of 1/3 phosphatides (Phospholipids) and 2/3 proteins -has emulsifying properties and keeps emulsion, milk stable -Phosphatides mainly lecithins form the inner part of membrane -The non polar groups of this monomolecular layer are oriented inside

Effect on product During homogenization, the original membrane is destroyed The first result is rise in interfacial tension Surface active components form a new membrane by adsorption and interfacial tension soon falls again The new emulsion therefore remains stable again after homogenization The new membrane mainly consists of casein, the proportion of phosphatides having decreased

Heat Treatment

Introduction • Heat treatments: on a large scale in the dairy industry
• Intermediate products and end products • Destruction of all or part of the microorganisms in milk – pasteurization: destroys pathogenic microorganisms and partly spoilage microorganisms, inactivates some enzymes – sterilization: destroys all microorganisms and inactivates the majority of enzymes • Optimized heating methods (e.g. UHT): to retain as much as possible the good organoleptic and nutritive properties

Pasteurization • Batch heating – 62 to 65°C for 30 minutes
– rarely applied (in developed countries) • HTST – short time heating: 72 to 76°C for 15 to 40 seconds – continuously in plate heat exchangers • Flash heating – high temperature heating: 85 to 90°C for 1 to 4 seco nds – used for cream and yogurt milk – peroxidase enzyme inactivated • Ultra pasteurisation – °C for 2s – Extended shelf life products (ESL) – days shelf life – Refrigeration remains necessary

Time/Temperature Combination Innactivation Curves

Sterilization • Destruction of the whole microflora including spores
(except heat resistant spores, e.g. B. stearothermophilus) • Complete enzyme inactivation (except some heat resistant lipases and proteases derived from Pseudomonas strains) • 109 to 115°C for 40 to 20 minutes • Side effects – non-enzymatic browning (Maillard) – cooked or caramelized flavour due to the decomposition of lactose

Ultra High Temperature Treatment
• Less discoloration and flavor changes compared to classical sterilization • °C for seconds • Direct UHT – steam injection followed by vacuum cooling – less defects • Indirect UHT – heat exchangers – more defects

Apparatus and process line
Plate Heat Exchanger

Tubular Heat Exchanger

Scraped Heat Exchanger

Direct UHT with steam injection

Indirect UHT

Effect on product • Endogenous milk enzymes • Proteins
• Bacterial enzymes • Rennet coagulation • Acid coagulation • Nutritional quality • Sensory quality and flavor • Proteins – whey proteins – caseins • Lactose • Milkfat globules • Milk salts

Whey Proteins • β-lactoglobulin, α-lactalbumin, bovine serum albumine (BSA) and immuno-globulins • Well developed secondary and tertiary structures • Susceptible to thermal denaturation: unfolding – tends to enhance intermolecular interactions – frequently leads to a loss of protein solubility • α-lactalbumine: – lowest denaturation temperature Td – able to renature after moderate heating: β-lac more affected

Whey proteins • Mildest heat treatments: thermisation and pasteurization – no significant effect on the whey proteins • More severe pasteurization (> 72°C and/or > 15 sec) – may partly denature whey protein – effect on further processing operations (e.g. increased rennet coagulation time), or on sensory qualities of the finished product (e.g. cooked flavour development in highly pasteurized milk) • More severe heating conditions – complex formation of β-lactoglobulin with k-casein – reduced rennetability of heated milk – applied in yogurt manufacturing process (90 °C, 5 minutes) – incorporation of whey proteins in fresh cheeses

Caseins • Heterogeneous group phospho-proteins (80 % of proteins)
• Four types: αs1-, αs2-, β-, k-cas (38%, 10%, 36%, 13%) • Random coil proteins • Casein in micellar form – exceptionally heat stable – withstands 20 minutes heating at 140 °C, pH 6,7 – heat stability of milk decreases abruptly at pH < 6,4 • Interaction between β-lactoglobulin and k-casein – reaction between thiol groups

Lactose • Milk products: very sensitive to thermally induced non-enzymatic sugar degradation reactions • Maillard browning reactions – involves lactose and lysine rich milk proteins – may significantly compromise the nutritional value, especially in the case of sterilized milk: • destruction of essential amino acids • destruction of vitamins • limitation of the bioavailability of other amino acids

Milk Fat globules • Heat-induced changes
– chemical (reactions of fatty acid residues) – physical (creaming, flocculation, coalescence, disruption) • Unheated natural milk fat globules – quite stable against flocculation and coalescence – cold agglutination (precipitation of immunoglobulins) • The effect of heating – inactivation of immunoglobulines (no cold agglutination) – homogenization: heat stability¯ – no changes in size distribution (except direct UHT-heating) – no coagulation of the fat globules

Milk Salts • Dynamic equilibrium shifts
– concentrations of Ca and P in the serum phase decrease with increasing heating temperature – lesser effects appear on Mg and citrate – reversible when moderate heating temperature ( °C) • Irreversible changes – irreversible breakdown of lactose to lactic and formic acid: permanent decrease in pH after cooling – dephosphorylation of casein: progressively more important as heating temperature and time increase

Endogenous Milk Enzymes
• The activity and stability of endogenous milk enzymes – over 60 in raw milk – influenced by temperature, pH, thermal conductivity and the availability of substrates, activators or inhibitors • Thermal inactivation of some milk enzymes is taken as a control of heat treatments – example: the universal alkaline phosphatase test as an index of adequate pasteurization

Bacterial enzymes • Heat stable extracellular enzymes of many psychrotrophic bacteria (Pseudomonas spp.), stable in UHT-range – proteinases: • may cause gelation of UHT-sterilized milks • responsible for taste and textural changes – lipases: • influence taste: hydrolytic rancidity – phospholipases: • are responsible for bitterness of milk or cream • Heat-stable enzyme secretion in cold-stored raw milk is dependent on the initial microbial quality of the raw milk (thermisation)

Rennet coagulation temperatures) impairs its renneting properties:
• Severe heating of cheese milk (> pasteurization temperatures) impairs its renneting properties: – β-lactoglobulin - k-casein complex: -S-S- bridge – sterically hinders the aggregation of rennet-altered micelles – reversion (if not too severe conditions of heating): by reducing the pH, adding CaCl2, or by acidification (to pH 4,6) followed by re-neutralization to the original pH of the milk • The main reason for severely heating cheese milk: – incorporation denatured whey proteins into the cheese: yield increases – flavour defects, too high moisture content, does not melt – not for ripened cheese

Acid coagulability • Milk acidification
– colloidal calcium phosphate (CCP) progressively solubilizes – aggregation of the casein occurs near the iso-electric point (pH 4,6) – acid casein production (syneresis) – formation of a gel network with good water-holding capacity

Acid coagulation • Milk heating prior to acidification:
– production of fresh cheeses, fermented milk products and yogurt – incorporation of whey proteins: influences the rheological properties and mechanism of structure formation – destruction of pathogenic organisms and phages – reduction total number of microorganisms: increased shelf life – improves the culture medium for the starter bacteria: • formation of growth-promoting substances (such as formic acid) • lowering the redox potential • inactivating antimicrobial substance – improved nutritional status: better digestible denatured proteins – possible destruction of some heat-labile vitamins

Nutritional Quality • Pasteurization:
– virtually no change in nutritional quality – during storage: possible loss of light-sensitive vit A and riboflavin – nutritional quality of proteins and amino acids unaltered • UHT treatment: – possible loss of folacin, ascorbic acid, vitamin B12 and thiamine – during storage: folacin, vit B6, vit A and some vit B12 can be lost • Sterilization: – Maillard reactions (destruction of essential amino acids) – considerable destruction of vitamins

Sensorial quality • Two major flavors characterize heat treated milk:
– cooked, caramelized flavors: during heat treatment – stale and oxidized flavors: during storage • Flavor of heat treated milk affected by: – sterilized > indirect UHT > direct (injection) UHT > direct (infusion) UHT ≈ pasteurized > raw milk – milk quality – packaging material – storage time and temperature – oxygen content during heat treatment and subsequent storage

Fermented dairy products
Milk and Milk Products Fermented dairy products

Fermented milk products
• Definition - description • Mechanism of growth • The manufacture of yogurt – Ingredients – Processing of yogurt – Structural and chemical changes • Other fermented milks

Definition- description
• Fermented milk is the non-leaked product obtained by coagulating skimmed milk, semi-skimmed milk or full fat milk by inoculation with lactic acid bacteria, with or without the association of yeasts • Yogurt is a fermented milk, obtained by a simultaneous operation of L. bulgaricus and S. thermophilus and in which the two specific cultures remains active (= living) until consumption o Dry matter on fat free product > 8.2% o Yoghurt: min 3% fat o Partially skimmed yoghurt: between 1 and 3% fat o Skimmed yoghurt: <1% fat - By fermentation a characteristic mild clean lactic flavour and typical aroma is produced

Definition-description
• Thermal treated fermented milk is fermented milk that is submitted to heat treatment after fermentation to enhance shelf life, but by which the specific flora is destroyed • Thermal treated yogurt doesn't exist • Among fermented milk also churned (butter-) milk is sold • Other examples of fermented milks – bioyogurt – acidophilus milk – bifidus milk – kefir – koumiss – sour cream

Mechanism of growth • The growth of yogurt bacteria shows some specific aspects • Both organisms can grow separately in milk, but stimulate each others growth when brought together (synergism) • Symbiosis of yogurt bacteria – S. thermophilus produces formic acid and CO2 that stimulates L. bulgaricus – L. bulgaricus produces low-molecular peptides and amino acids that stimulate S. thermophilus – The oxygen content is very critical, if it is higher than 4 mg/kg growth is delayed – The formed lactic acid is important as inhibition factor; the ratio bars to cocs is stabilized

The manufacture of yogurt
• Ingredients – Milk ingredients – Yogurt sweeteners – Yogurt fruit preparations – Stabilizers and thickeners – Flavours and colours – Preservatives – Vitamin and mineral fortifications – Yogurt cultures

Milk ingredients • The main ingredient of yogurt is milk or ingredients derived from milk • By combining milk ingredients with different composition with respect to milk solids, fat and protein and even protein type and functionality, it is possible to tailor products with specific desired properties and composition • The most commonly used milk based ingredients are – fresh whole milk – fresh skimmed milk – fresh cream (30-48% butterfat) – skimmed milk concentrate (evaporated or UF) – skimmed milk powder – milk protein concentrates and isolates

Yogurt sweetners • The sweetness source is commonly sucrose, either in powder or in liquid form • Raw cane sugar or honey may be used as well as de-colorized,de-flavorized, de-acidified and deodorized fruit concentrates • Low-calorific sweeteners are used in low-calorie formulations and the favored one is aspartame

Yogurt fruit preparations
• Yogurt fruit is generally added to the cooled yogurt after fermentation • It is usually supplied as a pasteurized fruit preparation to which, in addition to fruit, sugar, stabilizers and thickeners, flavouring and colourings have been added • Traditionally a high brix 'jam', with a total sugar content of around 60%, was used • Nowadays a lower brix fruit preparation (30-50°) is used • Yogurt fruit preparation is typically added at a dosage of 8-25% of the final recipe, with 10-15% being most common

Stabilizers & Thickners
• The most used stabilizers and thickeners are – modified starches – native starches – gelatin – pectin – locust bean gum • Sometimes used – agar – carrageenan – guar gum

Stabilizers & thickners
• Main functions – they provide viscosity and body in the final product and influence processing viscosity; – they influence structure and texture; – they control the degree of gelation required in stirred yogurt; – they help prevent serum release; – they influence creaminess and mouthfeel; – they enable calorific reduction without loss of quality; – they replace milk solids and fat and enable formulations to be more cost effective. • Typical inclusion rates of 0,05-1% have dramatic effects on the properties of the final product

Flavors and colors • Common natural colours are
– betamin from beetroot – anthocyanin from grape skin – carmine from cochineal – turmeric from the turmeric plant rhizome

Preservatives • When used, the common preservative in yogurt is sorbic acid added as its potassium salt. • It has a very selective action against yeasts and moulds • Another preservative used in fruit preparations is sulphur dioxide

Vitamins and mineral fortification
• These are generally added to milk before fermentation but some could be incorporated via the fruit preparation

Yogurt culture • The culture organisms used to manufacture yogurt are usually controlled by national legislation, but are most commonly mixed cultures of Lactobacillus bulgaricus and Streptococcus thermophilus • As with cheese cultures phage attack can be a problem if cultures are not rotated • The main functions of the yogurt culture are generally – to produce lactic acid to sour the milk from pH 6,5-6,7 to below pH4,6 – to produce the characteristic flavour of yogurt – to produce a firm yogurt curd that will provide stability and viscosity in the final product

Yogurt culture • Between 0,9-1,2% lactic acid is produced by the
breakdown of lactose; sucrose added to sweeten the final yogurt is not metabolized • The characteristic flavour of yogurt is due largely to acetaldehyde produced mainly by L. bulgaricus • Slime-producing or filant cultures, e.g. Streptococcus filant: produce exopolysacharide – are commonly used to give extra body and glossy appearance to yogurt, particularly low fat formulations – do however also give the product a somewhat long or ropy texture often considered undesirable. – main advantage is cost saving, enabling a lower milk solids level to be used without loss of viscosity

Yogurt culture • In recent years bio-active cultures have a an increasing popularity and are being used as a marketing element • These cultures contain species of L. acidophilus or Bifido-bacterium to provide organisms that can survive and grow in human intestines and contribute to general health and well-being • Several products claim to have a positive influence on human's health, although nothing scientifically has been proven yet

Processing of yogurt • Phases important during yogurt manufacturing
– increasing dry matter: to increase waterbinding properties, 1,5 to 2,5% milk powder is added – heat treatment: after homogenization, a heat treatment of 5-10 min.at 85°C is applied, aim is to better the structure of the product and to destroy natural inhibition factors, sufficient de-aeration is also needed, especially with continuous heating – fermentation is carried out in tanks and afterwards filled for stirred yogurt, or fermentation is carried out in the package for set yogurt • for set yogurt the yogurt milk is after pretreatment cooled to 40-45°C, ented with 2-3% culture and incubated in the recipient, herefore incubation rooms or tunnels are used, after fermentation to pH 4,5 the products are cooled • for stirred yogurt a short acidification of 3 h at 45°C or a long acidification of --15h at 31°C is applied, to enhance slime formation tw o cultures are usually used,after incubation to pH 4,5 there is stirred, cooled and filled, the mechanical treatments have an important influence on the structure of the yogurt

Flow diagram: Yogurt standardized milk Homogenize 55°C & 20 MPa
High Pasteurization 85°C for 5 min Cool to 45°C Cool to 30-32°C Innoculate 2.5 % Starter 0.025% Incubate 16-20 Hours Stirring Cool to 6 °C Packaging Cool to 6°C Stirred yogurt Incubation 2.5 hours set yogurt Flow diagram: Yogurt

Structural & chemical changes
• Yogurt shows a gel structure • In the protein matrix the components are included, the bacteria are bound to the protein by slime filaments • During fermentation the following conversions take place – Lactose is transformed for 20-30%; lactose is split into glucose and galactose by the b-galactosidase of S. thermophilus and L. bulgaricus – Glucose is mainly homofermentatively transformed to pyruvic acid. – Pyruvic acid is further transformed to lactic acid; L. bulgaricus forms D-lactic acid and S. thermophilus forms L+lactic acid – Pyruvic acid is further converted to small quantities of diacetyl and formic acid, acetaldehyde is the main aroma component – Proteins are coagulated and proteolysis is caused by L. bulgaricus. Fats are not changed – Vitamins are partly metabolized, partly synthesized

Structural & chemical changes
• Yogurt is said to have an influence on intestinal flora • It can inhibit tumors and gives a better resorption of lactose • Yogurt has a limited shelf life (storage temperature < 7°C) especially when infection with yeasts occurs; acidification continue • To improve shelf life, sometimes a heat treatment is applied – In function of the intensity apart from the yeasts and moulds, also bacteria and enzymes are inactivated

Other fermented milk • Among the other fermented milks, churned milk is the most important one • Churned milk is obtained as a by-product during churning of cream, during churning of milk, during acidification of separated milk with a butter culture

Other fermented milk • Fromage frais (fresh cheese) are cultured
products obtained by fermenting milk to pH with mixed mild cheese cultures, usually comprising Streptococcus cremoris and Streptococcus lactis, and sometimes Leuconostoc citrovorum and Streptococcus diacetylactis • This is followed by removal of whey and concentration of the solids (by using a cheese cloth, centrifugation or ultra filtration) to obtain a stiff, white to off-white, spreadable paste that should be homogenous, short textured and free from serum or graininess • The products should have a clean lactic flavour and be mildly acidic

Fat rich dairy products

Fat rich dairy products
• Cream • Butter • Butter oil and special products

Cream • Cream = fraction that rises to the top of whole raw milk when left to stand = o/w emulsion prepared out of milk by enrichment of the fat content • According to food legislation – cream is the product with at least 20% fat – whipping cream is the product with at least 40% fat – thinned cream is the product with at least 4% at maximum 20% fat – Double cream contains at least 48% fat – Clotted cream contains at least 55% fat • An intermediate product for the manufacturing of butter • Also for direct consumption after heat treatment (pasteurization, sterilization or UHT) • Further distinction: coffee cream, double cream,

Manufacturing cream • Milk maintained < 5 °C until required for separation • Milk heated for separation to °C for reasons of efficiency and product quality – separation at temperatures < 40 °C will yield cream with a high viscosity, but may result in the development of off-flavors due to lipolytic activity – separation at temperatures > 55 °C will cause the cream to thicken rapidly and excessively in storage • Decreaming is very important for the profitability of a factory – fat losses in skimmed milk may maximally reach 0,05% – decreaming is coupled to pasteurization

Manufacturing of cream
• Usually a separate pasteurization is applied – the milk is pre-warmed and centrifuged – afterwards the two phases, skimmed milk and cream, are separately pasteurized – skimmed milk serves as pre-warming flow for the incoming milk – pasteurization of full milk followed by centrifugation is rarely applied • Desired fat content of the cream for further butter making is 38 to 45% • Cream is used for direct consumption after – standardization – thermal treatment – mild homogenization (low pressure)

Butter • Definition • Treatment of cream for butter making
– Pre-treatment of cream – Cream ripening • Butter production – Churning process – Structure of butter – Production of butter – Other processes

Butter • = a w/o emulsion mainly produced by churning of cream
• According to food legislation – butter must contain at least 82% fat and at most 2% FFDM – the salt content is limited to 1% • Can be classified according to the type of processing 1. dairy butter out of unpasteurized cream farm butter out of cream after spontaneous acidification 2. churned butter or sour butter (pH of serum < 5,5) continuous butter or sweet butter (pH of serum > 5,5) • Main consumer perceived benefits – colour and appearance – flavour – texture and mouthfeel – spreadability – keeping quality

Treatment of cream for butter making
• Pre-treatment of cream • Cream ripening

Pre-treatment of cream
• Of great importance because the quality of the butter depends almost entirely on the treatment of the cream • The fat content aimed at depends on the further use of the cream % for churning in the butter churn % for butter making by the continuous process % for ripened cream % for sweet cream 82 % for butter making by the Alfa process

Pretreatment ofcream • Cream heat treatment
– high pasteurization: minimum claim is a negative phosphatase test – usually a higher heat treatment is applied: 95°C during 30-40 seconds or even °C during 15 seconds formation of free SH-groups Antioxidative effect inactivation of lipases and proteases • Facultative treatments – deaeration or degassing before pasteurization to remove gasses and undesired flavours the cream is brought at 75-85°C under vacuum – desodorization applied after pasteurization carried out under vacuum at 95°C

Cream ripening • Covers a physical and a biological phase
• Physical cream ripening • Biological cream ripening

Physical cream ripening
• Aim = to obtain a directed crystallization • Can as such influence the structure of the butter • Is needed because fluid fat is difficult to churn and too many fat losses can occur • Soft milk fat (unsaturated - in summer) soft and greasy butter • Hard milk fat (saturated – in winter) hard and stiff butter. • Different temperature schemes can be followed • Earlier the Swedish or the Alnaro procedure was followed the temperature is adapted to the iodine value T1 T2 T3 Sumer Winter T1: crystallization temperature T2: temperature of primary ripening T3: temperature of second ripening

Soft Winter butter • When cooling quickly
– rapid formation of crystals. – Triglycerides with low melting points are “trapped” in the same crystals mixed crystals are formed. – Low ratio of liquid to solid fat – Hard butter • This can be avoided – The cream is heated carefully to a higher temperature – The low-melting triglycerides are melted out of the crystals – The melted fat is then re-crystallized at a slightly lower temperature, resulting in a higher proportion of “pure” crystals and a lower proportion of mixed crystals. A higher liquid-to-solids ratio and a softer fat.

Biological cream ripening
• Was previously generally applied and is now limited (sweet butter) or not applied at all • Aim = an acidification and aroma formation • Actual processes – 3-5% starter culture is added – during the warm period • After the desired acidification, e.g. sour butter pH » 5,2, cooling is applied to churning temperature – T = 12°C during winter – T = 9°C during summer – or cooling is carried out to below 7°C to inhibit further acidification • The main aroma component diacetyl is only formed at pH < 5,2 and so only occurs in aromatic butter

Butter production • There are four basic processes involved in the
manufacture of butter 1. concentration of the fat phase of milk 2. Crystallisation of the fat phase of milk 3. phase separation of the oil-in-water (o/w) emulsion 4. formation of a plastical water-in-oil (w/o) emulsion • Churning process • Structure of butter • Production of butter • Other processes

Churning process • Churning = intense mechanical movement + air inclusion rapid motion of the fat globules in relation to each other collisions with surfaces and high turbulence fat crystals penetrate and disrupt the membranes destruction of the 5 to 10 nm thick fat globule membrane the emulsion is destabilized liquid fat leaves the fat globules coalescence to form fat agglomerates or butter granules o/w emulsion of cream is transformed to w/o emulsion of butter • Agglomeration is difficult – at too low temperature when the proportion of liquid fat is too low – at too high temperature when all the fat is in the liquid form

Concentrated and dried products
Milk and Milk Products Concentrated and dried products

Outlines Definitions • Unsweetened condensed milk
• Other concentrated products • Dried dairy products • Dried dairy ingredients

Definitions • Concentrated and dried dairy products are milk products
with an extended shelf life • Concentrated products: partial water removal – no organisms survive due to • Sterilization • increased osmotic pressure • Concentrated and dried milk products have several advantages: – Storage: requires small space under regular storage conditions and retains high quality at the same time – Economy: transport costs are reduced – Use in emergencies: wars, epidemics, earthquakes – Formulations: tailored food products, e.g. for sportsmen,

Unsweetened condensed milk Milk Clarification Sediments Cooling 4°C
Storage 4°C First Standardization Preheating ( °C for 1-6min) Fat Evaporation (45-70°C) Homogenization (P1: MPa; P2: 5-10 MPa Water Second standardization Stabilizers Packaging Packages Sterilization ( °C for 15-20min or 140 °C for 3 Seconds) Unsweetened concentrated milk 10°C Unsweetened condensed milk

Unsweetened condensed milk
• Based on evaporation – partial removal of water from milk – extends the shelf life by suppressing the microorganisms present in the milk • Milk quality for unsweetened condensed milk is very important – milk solids are concentrated – product is planned for long storage

Preheating – necessary for the stability of the condensed milk during sterilization – shortens the staying period of the milk in the evaporator – Produces a stabilizing effect caused by a decrease of calcium and phosphorus during heating • The preheating is carried out in continual heat exchangers of plate or tubular type – 93 to 100°C for 10 to 25 min. – 115 to 128°C for 1 to 6 min.

Evaporation • Evaporation is carried out under partial vacuum – Reduction of the evaporation temperature – Temperatures used are never below 45°C, in order to eliminate the growth of staphylococci – Tubular and plate evaporators – Single-effect or multiple-effect of two or more units up to eight – The falling film tubular evaporator • The leading evaporator in dairy industry • Tubes are about 3 to 5 cm in diameter and 15 m long • The tubes are heated with steam.

• Homogenization – Improvement of the stability of milk fat emulsion – Decrease of the average diameter of milk fat globules • Second standardization – Adjustment of the ratio of milk fat to nonfat milk solids (if the first standardization was not conducted) – Standardization of the total dry matter is standardized (if the first standardization has been carried out). • Heat stability depends on its salt balance – Addition of stabilizing salts • calcium, potassium or sodium carbonates and bicarbonates • potassium or sodium citrates • phosphates and other salts

• Packaging – Usually packaged in cans of various sizes depending on use • Sterilization – Sterilized ( °C for min.) – Continuous flow sterilization of evaporated milk is common ( °C), followed by packaging under aseptic conditions. • Evaporated milk can successfully be stored up to a year without any significant quality change at temperatures of 6-8°C.

Sweetened condensed milk
• Part of the water from fresh milk is evaporated • Sugar is added to the concentrated milk in order to extend shelf life • By increasing the osmotic pressure, the growth of microorganisms is prevented

Sweetened condensed milk
• hydrolysis of lactose – Milk is cooled to 5-10°C after pasteurization – Lactose is hydrolyzed by β-galactosidase, obtained from Saccharomyces fragilis. – The sweetness of the final product is approximately the same – In order to prevent lactose crystallization, acid-hydrolyzed sugar syrup may be added

Heat treatment, evaporation, sugar addition and standardization
• Main goal of heat treatment: – Total inactivation of osmophilic microorganisms – Inactivation of enzymes, particularly lipase and proteases – Decrease of fat separation – Inhibition of oxidative changes – Influence on the viscosity of the final product – The most frequently applied temperatures are 100 to 120°C. • Age thickening – A consequence of physicochemical changes in casein • Sugar addition is the way to prolong shelf life of this product – Sucrose, glucose, dextrose and others could be applied – Addition of sucrose before heat treatment increases the thermo-resistance of bacteria and their enzymes and significantly intensifies age thickening during storage. • During second standardization, total solids, sugar and fat contents are controlled

Cooling with crystallization
During cooling of the product after evaporation and sugar addition, lactose crystallization is induced • This is caused by: – Temperature decrease – High lactose concentration – Presence of high concentrations of added sugar – Relatively small amount of water • To avoid formation of crystals larger than 15 mm (sandiness), inoculation with powdered lactose crystals are used and the process is completed with rapid cooling and simultaneous agitation

Milk and Milk Products Dried dairy products

Milk powder • Milk powder
– =Dairy products from which the water has been removed to the greatest extent possible which prevents the growth of microorganisms • Skimmed Milk Powder – SMP produced by a 'low heat method' is simply pasteurized – SMP produced by a 'high heat method' requires heating at 85 to 88°C for 15 to 30 min. in addition to pasteurization. – Intensive heat treatment is applied to powder to be used in the bakery industry, where a degree of milk protein denaturation is desirable. • Standardization is needed to adjust the ratio of milk fat to total solids as required in the final product.

Flow diagram of processing milk powder
Clarification Cooling (4°C) Sediments Standardization Fat Heat treatment 88-90°C for 3-5min or 130°C for several seconds Evaporation until 30-35% TS; 40-50% TS Homogenization (5-15 MPa) Water Drying °C; °C Packaging Packages Milk powder 20°C Flow diagram of processing milk powder

Heat treatment – Carried out at temperatures higher than those required for pasteurization – Destroys all pathogenic and most of the non-pathogenic microorganisms – Inactivates enzymes – Activates the reducing SH groups of β-lactoglobulin, increasing the resistance of the powder to oxidative changes during storage • Evaporation – For roller drying evaporation is performed to 33-35% total solids – For spray drying is performed to 40-50% total solids • Homogenization – not obligatory – usually applied in order to decrease the free fat content – Fat globules depleted of protective membranes reduce milk powder solubility and increase their susceptibility to oxidative rancidity

Drying • Milk is most commonly dried by roller drying or spray
drying in a stream of hot air: • Roller drying – Roller drying at atmospheric pressure – Vacuum roller drying • Spray drying – Centrifugal atomization – Pressure atomization – Foam spray drying – Steam swept wheel atomization – Venturi spraying

Roller drying • Roller drying
– Commonly used in the production of skim milk powder and whole milk powder which find applications in other food industries (confectionery, feed blends) because of low product solubility – Direct contact of a layer of concentrated milk with the hot surface of rotating rollers adversely affects the milk components and causes irreversible changes. – Caramelization, Maillard's reaction and lactose degradation occurs • Vacuum roller drying – Operating temperatures below 100°C – Eliminates an oxygen effect

Spray drying Spray drying
– Mainly used for drying milk and milk products – Evaporated milk is atomized into fine droplets and exposed to a hot air flow in a spray-drying chamber, which may be in horizontal or vertical position – Air is filtered and heated up to °C – Air is usually indirect heated with steam or oil • Relation of milk flow and air stream – Concurrent flow – Countercurrent flow – Mixed flow • Concurrent flow • In spite of the inferior heat economy, is preferred in the dairy industry, as it improves product quality

Atomizers • Basic function
– To provide a high surface-to-mass ratio, enabling quick heat transfer with a high evaporation rate – The two atomizing designs: • centrifugal (rotary) atomizer • pressure (nozzle) atomizer • Powder particles gain spherical shapes during drying, with trapped air, thus gaining a low bulk density • Atomization parameters affect the properties of the product: – Bulk density – Size and size distribution of powder particles – Incorporated air content – Moisture content – Others

Atomizers A: Atomizing disc with curved channels B. Single
component nozzle C. Dual component nozzle

Standardization process

Standardization 100 kg of milk with 4% fat are centrifuged into 90.1 skimmed milk with 0.05 % fat and 9.9 kg cream with 40% fat. How much of this cream will be added to the skimmed milk to get the standardized milk with 3% fat. How much standardized milk will be obtained ? How much will the excess/surplus cream be?

Solution Mass balance: Skimmed milk + cream= Standardized milk
Lets X and Y be cream and standardized milk, respectively 90.1+ X=Y (1) Fat balance: 0.0005* X= 0.03Y(2) Combining (1) and (2)

Solution 0.0005*90.1+ 0.4X= 0.03 (90.1+ X) 0.04505+ 0.4X=2.703+0.03X
X= 7.18= 7.2 kg Standardized milk= 90.1 kg+ 7.2 kg= 97.3 kg with 3 % fat Cream in surplus= 9.9 kg kg= 2.7 kg

Question 2 How much skim milk containing 0.1 % fat is needed to reduce the fat of 200 kg of cream from 34% to 30% ? How much standardized cream will be obtained ?

FST 3325: Dairy Science and Technology

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