-
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'