weak compared to the affinity in the presence of stabilizing anions (e.g., F ,SO42-).302

Heat induced changes on conformation also change binding behavior of proteins.Upon heat treatment at 75C for 10 and 20 min, the binding affinity of /3-lg for2-nonanone was reduced and the number of sites for binding was increased.303 Thiswas related to conformational changes and aggregation of /3-lg.

Measurement of Flavor BindingMethods for the measurement of flavor binding have recently been reviewed byWilson.304 Flavor binding is usually determined by equilibrium measurements usingheadspace analysis, membrane dialysis, and solvent extraction techniques.

4.4 Some Selected Processing Effects on the FunctionalProperties of Major Milk Proteins

The functional properties of milk proteins depend on the molecular structure, andconsequently on every factor which may modify the molecular structure, includingthe source of the milk, the type of protein (caseins and whey proteins), and theprocesses used for the preparation or isolation of the milk proteins.29'305"307 Chefteland Lorient,17 Kinsella,14 Harper,308 and especially Schmidt et al?09 have suggestedthat essentially every step in the processing of milk protein products is important,either directly or indirectly, in determining the final functional properties of milkproteins. Major processing steps that have been reported to affect the functionalproperties of major milk proteins are given in Table 4.11. However, in many in-stances, the mechanisms(s) by which a processing step changes functionality is notunderstood.

In this section, the effect on proteins and their functional properties of two proc-essing effects (heat treatments and filtration processes) are briefly discussed.

4.4.1 Effects of Heat TreatmentsHeat processing is generally considered to be one of the most important single factorinfluencing functionality, more particularly, whey protein functionality.64'308"314However, much of the effect of heat thermal treatment depends on the degree of thetreatment and on media conditions (pH, presence of ions such as Ca2 + ). Some ofthe contradictory results could possibly be explained by differences in heat treatmentparameters (Lorient et al 1991).29

4.4.1.1 Effects on CaseinsCaseins in micellar form, and especially sodium casemates, are exceptionally ther-mostable; typically, milk withstands heating at 1400C at pH 6.7 for 20 minutes beforecoagulation occurs and sodium caseinates withstands heating at 1400C for at least

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60 minutes.312 The remarkable stability of caseins at high temperatures is principallydue to the low levels of secondary and tertiary structures.

From a physicochemical point of view, heating or cooling milk above or belowphysiological temperature causes a shift in the calcium phosphate equilibrium whichaffects some properties of milk, especially rennet coagulability. On cooling, colloidalcalcium phosphate (CCP) dissolves, and some casein, especially /3-casein, disso-ciates from the micelles,315'316 contributing to the increase in the rennet coagulationtime (RCT) of milk observed during cold storage. Conversely, on heating, the soluble/3-casein reassociates with the micelles and the RCT is reduced.312 Furthermore, heattreatments in the range of 80-150C, such as preheating of milk, in-container ster-ilization, and UHT processes, induce changes in caseins such as (1) dephosphory-

Table 4.11 PROCESSING-RELATED VARIABLES THAT MAY AFFECTTHE FUNCTIONAL PROPERTIES OF CASEIN AND WHEYPROTEIN PRODUCTS

Processing Variables

Thermal treatmentForewarmingMilk pasteurizationMilk sterilizationEvaporation and concentrationDehydration

Pretreatment before fractionationLipid removalpH adjustment

Fractionation and isolationTechnique usedMiscellaneous factors (pumping, storage, etc.)

Cheese processingStarter usedCoagulant usedProcess modifications (cooking temperature,

calcium chloride, water washing, etc.)Storage factors

Casein or whey storage conditionsCasein or whey protein product storage

conditionsSanitation factors

Microbiological loadAntimicrobial agent added

a: Direct protein conformation or denaturation effect,b: Indirect protein effect or effect on compositional factors+ : Variable has an effect; : Variable has no effect.Adapted from Refs. 9, 64, 308, 309, 312.

Effect on FunctionalityCaseins Whey Proteins

Direct(a)

+

+

+

( + )

+++

( + )( + )

+

Indirect(b)

+

++

+

+

+++

( + )( + )

+

Direct(a)

+++++

+

( + )+

++

++

Indirect(b)

+++++

++

++

+++

+

+

lation, (2) proteolysis, (3) covalent bond formation, and (4) changes in casein mi-cellar structures, etc. (Table 4.12) which differ only in rate and not in nature.312

1. Casein is completely dephosphorylated in 5 h at 1200C and approximately50% dephosphorylation occurs within 1 h.317 Milk concentration increases the rateof dephosphorylation; preheating has no effect on the rate of dephosphorylation ofunconcentrated milk but reduces the rate for concentrated milk.318 Dephosphoryla-tion, which reduces protein charge, might be expected to affect the heat stability ofmilk but its specific contribution has not been quantified.313

2. Although the nature of the proteolysis products formed on heating has notbeen studied in detail,312 some authors have reported the appearance of glycopeptidesin milk heated at temperatures >50C,319 and of peptides similar to the glyco-macropeptide after a treatment at 1200C for 20 minutes.320 Furthermore, formationof nonprotein nitrogen from milk proteins at temperatures >100C is almost linearwith time; 10 to 20% of total nitrogen is solubilized after 5 h at 1200C317 or 60minutes at 135C.321

3. During heat treatment of proteins, reactions can occur between reactive sidechains of some amino acids, such as Iysine and cysteine, and other amino acidresidues, carbohydrates, or lipids. The browning that occurs when milk is heated attemperatures > 1000C is a consequence of the Maillard reaction between the carbonylgroup of lactose and the e-amino group of lysine.

4. Heating milk causes a number of changes in casein micelles such as the ag-gregation of casein micelles during UHT sterilization.322""324 This increase in caseinmicelle size probably results from the combined effects of the heat denaturation ofwhey proteins and their deposition onto micellar surfaces and from the increase inmicellar calcium which may lead to calcium bridges between micelles.324 The in-crease in micelle size during heating is also accompanied by a large increase in thenumber of very small particles.325'326 These particles may be formed by the breakingup of casein micelles327"329 due to the removal of colloidal calcium by soluble citrate.The citrate is normally neutralized by soluble calcium but calcium phosphate pre-cipitates when the milk is heated. Finally, at normal pH, milk coagulation occurs at14O0C after about 20 minutes. The heat stability of milk, which is considerable

Table 4.12 SOME HEAT-INDUCED CHANGES INMILK PROTEINS

Protein Type or Structure

Caseins

Micellar structure

Whey proteins

Modifications

DephosphorylationProteolysisCovalent bond formation

Zeta-potentialHydration changesAssociation-dissociation

Unfolding-aggregationDisulfide interchange

economic importance, is influenced by many compositional factors as well as proc-essing effects.313-330-331 In the case of pH, Rose332'333 showed that the heat coagu-lation time-pH profile of most milks (type A) showed a maximum at approximately6.7 and a minimum at 6.9. The pH effect in milk coagulation is a function ofK-casein concentration on micelle surfaces and the /3-lg concentration in the milkserum. The minimum appears to be due to the dissociation of K-casein from thecasein micelles at pH >6.9 while the maximum is related to the presence of /3-lg.Some milk samples from individual cows fail to show minimum and maximumpoints on the curve, but instead coagulation time increases as the pH increases from6.2 to 7.4: such milk is referred to as "type B " . Tessier and Rose334 eliminated theminimum in the curve of type A milk by adding K-casein, thus converting it to typeB. They also converted type B milk to type A by salting out some K-casein or byadding /3-lg.

4.4.1.2 Effects on Whey ProteinsHeating globular proteins causes them to unfold and this unfolding is accompaniedby an endothermal heat effect (heat uptake). This effect may be observed by differ-ential scanning calorimetry as a function of temperature or time.335 Table 4.12presents the denaturation characteristics of some whey proteins.

1. /3-lactoglobulin. With a denaturation temperature of 78C, /3-lg is the moststable of the serum proteins. A second thermal change appears near 14O0C causedby the breakdown of disulphide bonds and additional unfolding of the molecule.335The heat denaturation of /3-lg is pH dependent. After an acidic heat treatment (pH2.5,900C, 10 to 15 minutes), /3-lg is still soluble. Two molecular species are present:one (60%) is soluble at pH 4.5 and is identical to native protein; the other (40%),insoluble at pH 4.5, has been irreversibly denatured but without aggregation, prob-ably due to the electrostatic repulsions at this pH.336-337 Heating at pH 4.5 (70 to85C, 15 to 30 minutes) resulted in a denatured /3-lg insoluble throughout the pHrange. Proteins are aggregated due to the formation of intermolecular disulphidebonds. Heat treatments at neutral pH have also been examined. At 800C, pH 6.8 to7.5, /3-lg is partially denatured without aggregation and loss of solubility. It seemsthat thiol groups, unmasked and activated at pH >6.8, initiate intramolecular disul-fide rearrangements that stabilize the molecule.335

2. a-lactalbumin. With a denaturation temperature of 62C, a-la is the least stablewhey protein, but requires the most heat per gram for unfolding. It has long beenassumed that a-la. was the most stable serum protein due to the reversibility of theheat denaturation at pH 6. Recent studies have clearly shown that the reversibledenaturation of a-la is due to calcium ion dissociation and reassociation from theprotein338 which is a calcium metalloprotein. Solubility studies on purified wheyproteins as a function of pH and temperature showed that a-la is insoluble from pH3.5 to 5. A solubility minimum is attained at pH 4.2 which corresponds to theisoelectric point of a-la.339 The partial, reversible thermal denaturation of a-la andits effect on the solubility of the protein at reduced pH values has been exploited inthe development of a process for whey protein fractionation.340-341

A large variety of heat treatments have been studied to increase the utilization ofwhey proteins17'23'26'342"344 as well as the impact of heat treatments inherent to theprocessing of milk such as pasteurization. Indeed, even mild heat treatments suchas standard pasteurization have been shown to affect the functionality of whey pro-tein concentrates.345-346 Morr345 reported that pasteurization (72C for 15 seconds)of cheese whey increased the foaming of a cheese whey concentrate at both pH 4.5and pH 9.0, whereas the pasteurization of acid whey decreased the foaming of anacid whey protein concentrate. Mangino et ai346 studying these same products,found that the binding of alkanes by whey protein concentrates was increased by thepasteurization of both types of whey.

Lorient et al.29 have studied the emulsifying and foaming properties of purifieda-la and /3-lg as a function of heat treatment and pH. The two proteins show im-proved emulsifying activity when heated at 700C for 30 minutes at acid or neutralpH; the activity of /3-lg is always higher. When heated at 900C for 60 minutes,emulsifying activity is only improved at acid pH. As for foaming properties, thecombined effects of pH and heat treatment appear to be different for the two proteins;a positive effect when heated at basic, neutral or isoelectric pH for /3-lg, and annegative effect a-la (especially at pH 2). Conversely, the foaming properties ofa-la are improved at pH 2-5.

4.4.2 Membrane Separation ProcessesNew developments in membrane separation processes and their application in thedairy industry have opened up new possibilities both for the production and utili-zation of milk protein ingredients. The use of classical isolation methods such asprecipitation with acid, heat or chemicals, and isoelectric coagulation, affect thenative state of milk proteins and thus their functional properties. Conversely, the useof membrane processes for separation or concentration is based on differences in thephysical characteristics of milk components such as their molecular weight. As aconsequence, the native state of the proteins is not altered.347'348

Membrane separation processes are generally divided into four categories ac-cording to the molecular size of the retained solutes. Fig. 4.10348 shows schematicallythe spectrum of particle sizes encountered in various dairy systems in relation toalternate filtration-based separation processes available to the dairy industry. Infor-mation on recent engineering advances involving these processes may be foundelsewhere.349"352 For the purpose of this monograph, the following names and mean-ings as defined by Jelen348 are used.

Microfiltration (MF) being more specifically used to remove large particles suchas casein fines, microorganisms, or microbial spores, fat globules, somatic cells,phopholipoprotein particles, etc. (Fig. 4.10) from whey or milk is not treated in thefollowing section. However, recent information on the influence of operating param-eters, and applications of MF in the dairy industry may be found in Olesen andJensen,353 Pedersen,354 and Pearce et al.355

Figure 4.10 Spectrum of application of membrane separation processes in the dairy industry.(Adapted from Ref. 348.)

4.4.2.1 Reverse Osmosis (RO)In reverse osmosis (RO), a purified liquid is separated from the feed solution, whichcontains solutes (usually low molecular weight salts) or other liquids. The use ofRO is increasing in the dairy industry for many reasons. First, the concentration offood process356 streams to 10 to 25% total solids can, in some cases, be accomplishedat lower cost with RO than with evaporation. Second, low temperature concentrationby RO minimizes loss of volatile flavor components and adverse changes in heat-sensitive food components. RO can also be used to treat effluent streams to producereusable water.

Fouling is a major problem for the RO of whey. Calcium salts, especially calciumphosphate, are primary foulants.357'358 Whey pretreatments (acidification, heat treat-ment) to remove or reduce the effects of calcium salts have been studied to improveperformance. However, as explained in a preceding section, the effect of these treat-ments on whey functionality must be considered.

4.4.2.2 NanoMtration (NF)The main emerging applications for the dairy industry of NF is for the partial de-mineralization of whey-like materials.359"360 Since NF is used mainly for the re-moval of mineral ions that contribute to the osmotic pressure in dairy systems,361*362the operating pressure reported for some of the experimental uses is lower than thepressures used in RO.

Particle Size(Hin)

Approx. MolecularWeight (D)

ParticleCharacteristics

Approx. Flux(L/m2h)

Approx. OperatingPressure (Bar)

Relative size ofmilk systemscomponents

Process forSeparation

io'