Research ArticleChanges of pH in 120573-Lactoglobulin and 120573-Casein Solutionsduring High Pressure Treatment
Karsten Olsen Bo B Jespersen and Vibeke Orlien
Food Chemistry Department of Food Science Faculty of Science University of Copenhagen Rolighedsvej 301958 Frederiksberg Denmark
Correspondence should be addressed to Vibeke Orlien vorfoodkudk
Received 29 October 2014 Accepted 9 February 2015
Academic Editor Guang Zhu
Copyright copy 2015 Karsten Olsen et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
The pH changes in the milk systems 120573-lactoglobulin B 120573-casein and mixture of 120573-lactoglobulin and 120573-casein (pH 7 and ionicstrength 008M) were measured in situ during increasing pressure up to 500MPa An initial decrease to pH 67 was observed from01 to 150MPa for 120573-lactoglobulin followed by an increase to pH 73 at 500MPa The initial decrease is suggested to be causedby the deprotonation of histidine while the increase is suggested to result from an increase of hydroxide ions due to the loss oftertiary structure The change in pH of the 120573-casein solution displayed an almost linear increasing pressure dependency up toa pH of 77 at 500MPa The limited tertiary structure of 120573-casein could allow exposure of all amino acids thus the increase ofpH can be caused by binding of water protons resulting in an increase of hydroxide ions Addition of 120573-casein to 120573-lactoglobulin(11) was found to suppress the initial pH decrease found for the 120573-lactoglobulin solution The pH change of the mixture did notsuggest any intermolecular interaction and a simple additive model is proposed to calculate the pH change of the mixture fromthe corresponding individual samples
1 Introduction
Controlling the pH during the various steps during produc-tion of dairy products is crucial for the product quality Asreviewed by Salaun et al [1] the buffering capacity of theproduct plays a major role in the variations in pH Regardingcowrsquos milk the caseins whey proteins soluble minerals andcolloidal calcium phosphate (CCP) contribute 35 5 40and 20 respectively to milkrsquos buffering capacity [1] ThepH decrease during heat treatment of milk above 90∘C isthus explained by lactose degradation casein dephosphory-lation and calcium phosphate precipitationThe opposite pHincrease during cheese ripening is explained by lactic aciddegradation and ammonia production by microorganismsWhen milk has been high pressure (HP) treated between 250and 600MPa the buffering capacity is maximal at pH 52ndash54compared to pH of 48ndash50 for untreated milk The quantita-tive importance of this is that micellar calcium solubilisationin pressurized milk was promoted at this higher pH andmore calcium ions were found in milk treated at 600MPafor 30min upon acidification to 52 compared to untreated
milk [2] Most likely the different buffering capacities are aresult of different states of colloidal calciumphosphate duringthe pressurizing process Indeed Orlien et al [3] showed themajor influence of the pH of the milk on the barostability ofthe casein micelles under pressure and explained this by thegreater importance of the state of calcium and the associatedHP effect on the CCP balance in the micelle-serum systemFurthermore the pH of the milk has major impact on theextent of 120573-lactoglobulin (120573-Lg) denaturation and it has beenshowed that HP-induced denaturation of 120573-Lg decreased atacidic pH but increased at alkaline pH [4 5]
The pH and HP influences on the physicochemicalchanges in milk are thus important for the technologicalaspects of dairy products However variations in pH of milkor milk systems during HP-treatment have not yet beenelucidated
In recent years it has been shown that the pH ofweak acids changes under pressure due to effect on theionization equilibrium [6ndash11] Depending on the nature ofthe solution pH may decrease increase or remain constantwhen subjected to high pressureThe pressure dependence of
Hindawi Publishing CorporationJournal of SpectroscopyVolume 2015 Article ID 935901 6 pageshttpdxdoiorg1011552015935901
2 Journal of Spectroscopy
aqueous solutions is governed by the partial molar volumesunder the actual conditions in the equilibrium solutionsDissociation of acids increases upon pressurizing if theion-pair formation is accompanied by a substantial volumereduction Consequently the buffering capacity of certainbuffers decreases with pressure with significant changes inpH upon pressurizing To our knowledge only one study onpH changes in protein solution under pressure exists the pHincreased up to 15 units in a 120573-Lg solution at pressure upto 300MPa followed by a decrease to the initial pH of 40for further pressurization up to 500MPa [7] This study wastherefore undertaken to monitor the effect on changes in pHof a 120573-Lg solution at its natural pH and in combination with120573-casein (120573-CN) during HP-treatment and offers additionalinformation to the understanding of themolecular propertiesof 120573-Lg under pressure
2 Materials and Methods
21 Chemicals 120573-lactoglobulin from bovine milk geneticvariant B was isolated from acidic whey of fresh skim milkof homozygotic cows and purified according to the methoddescribed by [12] 120573-casein-A1015840A1015840 (120573-CN) was a gift fromDairy Technology Department of Food Science Universityof Copenhagen 31015840310158401015840-Dibromothymolsulfonphthalein (bro-mothymol blue) was obtained from Merck (Germany) Allother chemicals were of analytical grade and solutions werebased on highly purified water (Milli-Q Plus Millipore CorpBedford MA USA)
22 High Pressure Spectrophotometer The intensity of lighttransmitted through the solutions under investigation ofvarying pressure was measured in situ in a thermostated highpressure optical cell (Type 7402006 from SITEC Sieber Engi-neering AG Switzerland) equipped with a hand-operatedpressure generating system (Type 7501700 from SITECSieber Engineering AG Switzerland) as described by [7]Thepressure generating system and the optical cell were filledwith the relevant solution and the intensity spectrum from350 to 700 nmwas recorded at each step of pressures between01 and 500MPa The intensity spectra were converted toabsorption spectra using 119860
120582= minus log(119868
120582119868ref120582
) where 119868120582and
119868ref120582
are intensities at wavelength 120582 for the solution and forwater as reference respectively The intensity spectrum ofwater was recorded prior to measurement of the indicatorsolution
23 In Situ Spectrophotometry A stock solution of bromoth-ymol blue (36 sdot 10minus4M) in water was prepared Solutionswith bromothymol blue in its acidic alkaline or partiallytransformed form were prepared by adjusting aliquots topH 30 100 and 70 respectively with appropriate amountsof HCl or NaOH and to an ionic strength at 008M withNaCl and a final bromothymol blue concentration of 36 sdot10minus5M Prior to the optical measurements under pressurethe pH of all solutions was measured as a reference withglass electrode (713 pHMeterMetrohm Switzerland) againstinternational activity pH standards 120573-Lg was dissolved in
water as a stock solution and stored at 5∘C overnight forequilibration The 120573-Lg solutions for measurement underpressure were made by adding an appropriate aliquot ofbromothymol blue (final concentrations of 36sdot10minus5M) toaliquots of the stock solution giving final120573-Lg concentrationsof 100 and 300mgmL The pH of the 120573-Lg solutions wasadjusted with HCl or NaOH to pH 30 pH 100 and pH70 and to an ionic strength at 008M with NaCl 120573-CN wasdissolved in water (with 2mgmL NaCl) as a stock solutionand stored at 5∘C overnight for equilibration The 120573-CNsolutions for measurement under pressure were made byadding an appropriate aliquot of bromothymol blue (finalconcentrations of 36sdot10minus5M) to aliquots of the stock solutiongiving final 120573-CN concentrations of 100mgmL The pH ofthe 120573-CN solution was adjusted to pH 70 and to an ionicstrength at 008M with NaCl For the mixed 120573-Lg and 120573-CNsolution an appropriate volume of 120573-Lg stock solution wasadded to the 120573-CN solution to obtain a concentration of100mgmL for both proteins The solutions were filled inthe optical cell of the high pressure spectrophotometer anda series of intensity spectra were recorded and converted toabsorption spectra as described above
The method developed by Orlien et al [7] was used tomonitor in situ pH changes in solutions of 120573-Lg and 120573-CNandmixture thereof under pressure bymeasuring the absorp-tion spectra of the respective proteins in solution underacid and basic conditions and the partially transformed andcalculating the change in pH in accordance with
ΔpH (119875) = Δ log( 120572 (119875)1 minus 120572 (119875)
) (1)
where ΔpH(119875) = pH(01MPa) minus pH(119875) The degree ofdissociation 120572(119875) of the indicator is calculated by
120572 (119875) =119860119909(119875) minus 119860
119886(119875)
119860119887(119875) minus 119860
119886(119875) (2)
where 119860(119875) is the absorbance of the partially transformedindicator (index 119909) and absorbance of the indicator in itsacidic form (index 119886) and in its basic form (index 119887)respectively at the respective pressure
24 Statistical Analysis The statistical analysis is carried outin R (R version 1121) with RKWard (Version 054 KDEversion 451) as graphical user interface used for scriptmarkup and piping to the Rterminal The following add-on packages were also used MASS (Version 73-7) nlme(Version 31-97) and gmodels (Version 2150)
3 Results and Discussion
120573-Lg is a compactly folded globular protein and consists of162 amino acid where 53 residues have titratable side groups[13] 120573-Lg is a dimer at pH 7 and each monomer has 2ionisable histidine side groups However the two histidineresidues differ considerably in solvent accessibility due tothe conformation of the protein molecule As the solvent-accessible area of His146 is around 126 A2 at pH 7 it is
Journal of Spectroscopy 3
available to titration while in contrast with the solvent-accessible area of His161 around 12 A2 it becomes buriedin the interior of the native protein [14] The effective p119870
119886
of ionisable side groups in a protein depends on differentmolecular microenvironments and may have either higheror lower value than the respective free amino acid thusthe p119870
119886of His146 is reported to vary from 62 to 77
and the p119870119886of His161 is reported to vary from 58 to 85
[13]Spectrophotometric measurement of pH with acid-base
indicators is based on differences in absorption spectrabetween the acidic form and the basic form of the indi-cator molecule and the useful range depends on p119870
119894[7]
Previously we have developed a self-consistent method formeasurement of changes in pH with pressure based on thefact that the indicator p119870
119894is insensitive to pressure and
that the indicator molecule does not bind to the protein[7] A number of possible indicators (neutral red phenolred and bromothymol blue) were investigated in detail overthe spectral range 350ndash700 nm in solutions of their acidicand basic forms and of a mixture around p119870
119894value of
each indicator since these indicators were considered goodcandidates for pressure insensitive indicators Bromothymolblue was chosen for the investigation of pH changes of the120573-Lg solutions under pressure due to the sufficient differencein the absorption spectra for the acid base and partiallytransformed forms Figure 1 shows the absorption spectra forbromothymol blue in aqueous solutions of 120573-Lg at differentpH as a function of pressure The increase in molar absorp-tivity upon pressurizing reflects the concentration increasewith pressure as the system is compressedThe small variationin absorption spectra of the acid form with pressure is mostlikely a combined effect of compressibility of the solventof deformation of windows of the high pressure cell andto a much lesser degree of conformational changes of theindicator As seen in Figure 1 the clear distinction betweenthe acid and basic form ensures an accurate calculation of thedegree of dissociation at each pressure [7] This distinctionis emphasized since the changes of the absorption spectraduring pressurization reflect the shifted equilibrium HIn +
H2O119870119894
999448999471 Inminus + H3O+ according to the spectral changes
of bromothymol blue in water at the relevant pHs (datanot shown) and thus function as a sensor to probe theresult of a diffusion controlled transfer of protons from thesolvent to the acidic and basic side groups of the protein incontact with the indicator At initial pH of 7 and pressure(01MPa) the two absorption bands at 431 and 619 nmconfirm that bromothymol blue is in a partially transformedform yet mostly in its acidic form (Figure 1) reflecting thatthe two histidine residues are between being protonated anddeprotonated according to the rather large span of theirrespective p119870
119886 Upon pressurizing the absorption spectra
of bromothymol blue change corresponding to changes inthe acidbase equilibrium of the indicator and as seenthe intensity of both absorption bands increased when thepressure increased to 500MPa120573-Lg is the most abundant whey protein and is used
in many applications for its various functional properties
000408121620
350 400 450 500 550 600 650 700 750 800
Abs pH = 3
pH = 10
120582 (nm)
350 400 450 500 550 600 650 700 750 80000
02
04
06
08
0150100150200250
300350400450500
pH = 7
120582 (nm)
Abs
Figure 1 Pressure-induced changes in the visible absorption spectraof bromothymol blue in an aqueous solution of120573-Lg B (100mgmL)at acidic (pH 30) and basic (pH 100) conditions compared to thepartially transformed form at pH 70 Pressure level in MPa is givenby the legend The abrupt decrease in absorbance at 650 nm is dueto the deuterium lamp in the spectrophotometer
which depend on the physiochemical state and pH Anothergroup of milk proteins that contributes to the milk bufferingcapacity is the caseins and it is reported that the purecaseins have maximal buffering capacity around pH 5ndash55due to phosphoserine and histidine residues [1] Moreoverthe different genetic variants have different physicochemicalproperties and different interacting behaviour with othermilk constituents depending on the solution conditions120573-CN is the most hydrophobic but also highest chargedcasein (minus13 at pH 66) due to an unevenly distribution ofhydrophobic (a long C-terminal without any charged sidegroups) and hydrophilic (a short N-terminal with all chargedside groups) residues resulting in a distinct amphipathic char-acter This unique characteristic makes 120573-CN very useful forfoodmanufacturers and is one of the most abundant proteinsin various food products The caseins have little tertiarystructure and can barely be denatured but it has been foundthat at the 120573-Lg 120573-CNmolar ratio of 1 013 or greater 120573-CNis able to suppress the heat-induced aggregation of 120573-Lg [15]Figure 2 shows the absorption spectra for bromothymol bluein aqueous solutions of 120573-CN as a function of pressure andthe absorption spectra change considerably with a markedincrease in the basic absorption band upon pressurizationWith 120573-Lg in the solution the increase in the basic absorptionband was less marked (Figure 2)
From the spectral data and using spectra of the acidic andbasic 120573-Lg solutions (Figure 1) as the acidic and basic formof bromothymol blue respectively in the calculation of 120572(119875)
Figure 2 Pressure induced changes in the visible absorption spectraof bromothymol blue in an aqueous solution of120573-Lg B (100mgmL)and of120573-LgB+120573-CN (1 1mgmL) at the partially transformed format pH 70 Pressure level in MPa is given by the legend The abruptdecrease in absorbance at 650 nm is due to the deuterium lamp inthe spectrophotometer
(2) the change in pH was calculated according to (1) and theresults are presented in Figure 3
The major factors that control the impact of HP on themolecular structure is the electrostriction of charged andpolar groups elimination of packing defects and the solva-tion of hydrophobic groups Pressure treatment was foundto induce changes in the pH of the 120573-Lg aqueous solutionsdependent on the working pressure and the minimum in pHwas found to be around 150MPa (Figure 3) The pH profileobserved for 120573-Lg with an initial pH 70 (Figure 3) is oppositeto the observed pressure dependency for 120573-Lg with an initialpH of 40 [7] and reflects the difference in the titratable sitesat the respective pH (histidine versus aspartic and glutamicacid) and in the titration behaviour during pressurizationdue to conformational changes (extent of residues beingexposed to solvent versus buried) From Figure 3 it is seenthat high pressure of an unbuffered aqueous solution of 120573-Lgwith an initial pH of 7 induces an immediate decrease inpH up to 150MPa followed by an abrupt increase in pHbeyond the initial pH following a gradual increase to a pHaround 73 at 500MPa Several investigations of high pressureeffects on 120573-Lg have been carried out and several modelsfor the resulting conformational changes have accordinglybeen suggested The study of denaturation of 120573-Lg in skimmilk leads to the overall reaction scheme dissociation ofthe dimer to monomers unfolding of the monomeric (still)native structure and irreversible aggregation with 120573-Lg orcaseins [16] The pressure denaturation of purified 120573-Lgwas earlier described as a three-step process including an
0 50 100 150 200 250 300 350 400 450 500
minus06
minus04
minus02
00
02
04
ΔpH
120573-Lg 1mgmL120573-Lg 3mgmL120573-CN 1mgmL
120573-Lg + 120573-CN (1 1)Calculated mix
P (MPa)
Figure 3 Pressure dependence of pH of aqueous solutions of 120573-LgB at 100mgmL (◼) and 300mgmL (e) 120573-CN 100mgmL (998787)and 120573-Lg B + 120573-CN (1 1mgmL) (995333) all with an initial pH of 70The change in pH ΔpH(119875) = pH(01MPa) minus pH(119875) is calculatedfrom spectral data according to (1) The dashed line represents theΔpH calculated from the changes of 120573-Lg (100mgmL) and 120573-CN(100mgmL) according to (3)
initial pressure-melted state for pressure up to 50MPa areversible denaturation up to 200MPa and an irreversibledenaturation above 200MPa [17] Likewise a three-stagedenaturation model for purified 120573-Lg was suggested withthree discernible structural stages stage I (up to 150MPa)is the native stable structure in stage II (200ndash450MPa) thenative monomers are reversibly interchanged with nonnativemonomers and disulfide-bonded dimer and in stage III (over500MPa) unfolded monomers and dimers interact to formaggregates [18] The pressure-induced structural changes areas emphasized by Anema [16] a complex series of moreconsecutive andor concurrent pathways than the three men-tioned general steps dependent on conditions like solventpressure duration and temperature Thus the HP effect onthe structure of 120573-Lg could be brought together dissociationinto monomers various molten globule structures denatu-ration and aggregation It was shown that upon these HP-induced conformational changes charged residues undergoa change from buried to exposed leading to an unexpectedpH variation of a 120573-Lg solution under pressure [7] The pHprofiles in Figure 3 show that the initial step where dimers aredissociated into monomers at low pressure of 50MPa resultsin no (1mgmL) or minor (3mgmL) changes in pH of thesolution The following transformation into molten globulesis identical to the changes in the 120573-Lg structure in aqueoussolution with initial pH of 40 under increasing pressure [7]despite beingwith a different effect on solution pHThe initialdissociation increases the accessible surface area resulting inan increase in hydration of the protein molecules resultingin the contraction of solvent water (due to electrostriction)and leading to volume decrease and disruption of ion pairs
Journal of Spectroscopy 5
in the protein molecule The p119870119886for histidine residue 146
is 75 and 68 for the dimer and monomer respectivelywhile p119870
119886for histidine residue 161 is 65 and 58 for the
dimer and monomer respectively [13] Hence the pH ofthe 120573-Lg solution under pressure depends on whether theimidazole rings are protonated or deprotonated Accordingto the equilibrium HisH+ + H2O 999448999471 His + H3O
+ at theinitial pH before pressurisation most of the His146 wasprotonated and most of the His161 was deprotonated 120573-Lgdissociated from dimer to monomer at the initial increaseof pressure and the His146 became available for solventand shifted to the deprotonated state (due to lower p119870
119886
for the monomer) resulting in a decrease in pH Duringpressurisation up to 150MPa the dimers are dissociated andprovide pathways for water to penetrate into the interiorof the monomers leading to the molten globule states Inparticular the dissociation at the dimerization area resultedin an increased accessibility of His161 When the remainingprotonated His161 experiences the solvent pH (still higherthan the p119870
119886for the monomer) it became deprotonated
resulting in further decrease of pH The pressure and solventpH effects are optimally balanced at 150MPa where themaximum decrease in pH occurs (Figure 3) At furtherincrease in pressure the tertiary structure of 120573-Lg is disruptedleading to a denatured proteinmaking both histidine residuesavailable for the solvent water As a result of gradual waterpenetration and the accompanying electrostriction of waterthe deprotonated His residues will reassociate the protonfrom water which will lead to an excess amount of OHminus inthe solution corresponding to an increase in pH as seen inFigure 3 As seen in Figure 3 the extent of the pH change isdependent on the concentration of 120573-Lg and increasing con-centration from 1 to 3mgmLbrings about a higher amount ofhistidine residues in effect resulting in a greater pH changeA pH reduction of the observed magnitude (034 units for120573-Lg at 3mgmL at 500MPa) can be of major importancefrom a technological point of view Adjusting the pH ofmilk by 05ndash07 units prior to HP-treatment at 250ndash600MPaat 20∘C was found to reduce the extent of denaturation of120573-Lg considerably compared to milk at normal pH [4 5]Interestingly after reaching a minimum in pH at 150MPa thepH increased abruptly for both concentrations of 120573-Lg andincreased gradually at further pressurizing
The nonglobular 120573-CN is therefore insensitive forpressure-induced conformational changes At the same timeit is highly charged and may therefore give electrostrictiona significant role in the pH behaviour under pressure Theamphiphilic nature of120573-CNusually results in self-associationinto large oligomersmicelle upon dissolving in aqueousmedia but the concentration used and the preparation of thesolution in this study ensure a solution of 120573-CN monomersAt 01MPa and pH 70 both the 5 histidine residues (p119870
119886asymp
65 [1]) and the 5 phosphoserine residues (p1198701198862asymp 63ndash
68 for phosphoserine residues in 120573-CN [19]) are shiftedto the deprotonated states Hence no specific structuralrearrangement of the protein will affect solvent pH onlythe three ldquosimplerdquo ionisation equilibria related to histidinephosphoserine and water self-ionisation will govern the pHchange of the 120573-CN solution under pressure The underlying
electrostrictive effect is from the water self-ionisation whichis increasingly promoted at increasing pressure thereby gen-erating charged hydronium and hydroxide ions It is acceptedthat exposure andor generation of polar and charged groupswill lead to a decrease in volume due to electrostrictivepacking effects Thus the H
3O+ ions are reassociated with
the deprotonated His and Ser-PO3
2minus in effect shifting thecorresponding equilibria to the protonated states This willpromote a further dissociation of water which will lead to anexcess amount of OHminus in the solution corresponding to anincrease in pH as seen in Figure 3
Mixing equal amounts of 120573-Lg and 120573-CN resulted in adepressing effect of the individual pressure effects on bothproteins as seen in Figure 3The initial pHdecrease caused bythe dimer dissociation and deprotonation of His146 in 120573-Lgwas counterbalanced by the increasing concentration of OHminusdue to the electrostrictive effect of the 120573-CN-water systemThis finding indicates that the stability of120573-Lg under pressuredepends on the treatment media and is in agreement withother reports [20 21] It was found that 120572
119904-casein suppressed
the pressure-induced aggregation of 120573-Lg because of thechaperone property of casein [20] Whether 120573-CN acted in asimilar chaperone manner thereby reducing the dissociationand denaturation effect or it was a simple balancing of the pHdue to release of H
3O+ and OHminus from the 120573-Lg and 120573-CN
respectively cannot be deduced from this study Howeverthe subsequent pH increase in the solution of the mixedproteins seemed to be an equal contribution of the effect onpH by the individual protein-water systems Moreover thepressure course of the pH changes of the mixed solution canbe modulated by a simple equation based on the individualHP-pH progress (Figure 3)
The pressure-induced changes in pH of a 120573-Lg solutionat its natural pH was determined by the pressure-induceddissociation and unfolding of120573-Lg and the concurrent degreeof accessibility of titratable side groups in this case the twohistidine side chains The importance of the HP effect on thestructural changes of the protein and the rearrangement ofthe protein-water system on solvent pH was supported bythe pressure effect on pH in a 120573-CN solution 120573-CN lacks athree-dimensional structure hence the pressure-induced pHchanges were explained purely by the shifts in the equilibriaof the histidine and phosphoserine residues as affected bypressure and waterrsquos self-ionisation The HP-induced pHchanges in a mixed 120573-Lg and 120573-CN solution were found tobe a simple mix of the effects from the individual pH profilesunder pressure
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
6 Journal of Spectroscopy
References
[1] F Salaun B Mietton and F Gaucheron ldquoBuffering capacity ofdairy productsrdquo International Dairy Journal vol 15 no 2 pp95ndash109 2005
[2] M H Famelart F Gaucheron F Mariette Y Le Graet KRaulot and E Boyaval ldquoAcidification of pressure-treated milkrdquoInternational Dairy Journal vol 7 no 5 pp 325ndash330 1997
[3] V Orlien L Boserup and K Olsen ldquoCasein micelle dissoci-ation in skim milk during high-pressure treatment effects ofpressure pH and temperaturerdquo Journal of Dairy Science vol93 no 1 pp 12ndash18 2010
[4] M Arias R Lopez-Fandino and A Olano ldquoInfluence of pH onthe effects of high pressure onmilk proteinsrdquoMilchwissenschaftvol 55 no 4 pp 191ndash194 2000
[5] T Huppertz P F Fox and A L Kelly ldquoHigh pressure treatmentof bovine milk effects on casein micelles and whey proteinsrdquoJournal of Dairy Research vol 71 no 1 pp 97ndash106 2004
[6] S KMin P S Chaminda and SK Sastry ldquoIn situmeasurementof reaction volume and calculation of pH of weak acid buffersolutions under high pressurerdquo Journal of Physical Chemistry Bvol 115 no 20 pp 6564ndash6571 2011
[7] VOrlien KOlsen and LH Skibsted ldquoIn situmeasurements ofpH changes in 120573-lactoglobulin solutions under high hydrostaticpressurerdquo Journal of Agricultural and Food Chemistry vol 55no 11 pp 4422ndash4428 2007
[8] V M Stippl A Delgado and T M Becker ldquoDevelopment of amethod for the optical in-situ determination of pH value duringhigh-pressure treatment of fluid foodrdquo Innovative Food Scienceamp Emerging Technologies vol 5 no 3 pp 285ndash292 2004
[9] M Hayert J-M Perrier-Cornet and P Gervais ldquoA simplemethod for measuring the pH of acid solutions under highpressurerdquo The Journal of Physical Chemistry A vol 103 no 12pp 1785ndash1789 1999
[10] T K Hitchens and R G Bryant ldquoPressure dependence of weakacid ionization in deuterium oxide solutionsrdquo The Journal ofPhysical Chemistry B vol 102 no 6 pp 1002ndash1004 1998
[11] R C Neuman Jr W Kauzmann and A Zipp ldquoPressuredependence of weak acid lonization in aqueous buffersrdquo TheJournal of Physical Chemistry vol 77 no 22 pp 2687ndash26911973
[12] K R Kristiansen J Otte R Ipsen and K B Qvist ldquoLarge-scalepreparation of 120573-lactoglobulin A and B by ultrafiltration andion-exchange chromatographyrdquo International Dairy Journalvol 8 no 2 pp 113ndash118 1998
[13] F Fogolari L Ragona S Licciardi et al ldquoElectrostatic prop-erties of bovine 120573-lactoglobulinrdquo Proteins Structure Functionand Genetics vol 39 no 4 pp 317ndash330 2000
[14] B Y QinM C Bewley L K Creamer HM Baker E N Bakerand G B Jameson ldquoStructural basis of the tanford transition ofbovine 120573-lactoglobulinrdquo Biochemistry vol 37 no 40 pp 14014ndash14023 1998
[15] Y H Yong and E A Foegeding ldquoEffects of caseins on thermalstability of bovine 120573-lactoglobulinrdquo Journal of Agricultural andFood Chemistry vol 56 no 21 pp 10352ndash10358 2008
[16] S G Anema R Stockmann and E K Lowe ldquoDenaturationof 120573-lactoglobulin in pressure-treated skim milkrdquo Journal ofAgricultural and Food Chemistry vol 53 no 20 pp 7783ndash77912005
[17] H Stapelfeldt and L H Skibsted ldquoPressure denaturation andaggregation of 120573-lactoglobulin studied by intrinsic fluorescence
depolarization Rayleigh scattering radiationless energy trans-fer and hydrophobic fluoroprobingrdquo Journal of Dairy Researchvol 66 no 4 pp 545ndash558 1999
[18] T Considine H Singh H A Patel and L K CreamerldquoInfluence of binding of sodium dodecyl sulfate all-trans-retinol and 8-anilino-1-naphthalenesulfonate on the high-pressure-induced unfolding and aggregation of 120573-lactoglobulinBrdquo Journal of Agricultural and Food Chemistry vol 53 no 20pp 8010ndash8018 2005
[19] J-J Baumy P Guenot S Sinbandhit and G Brule ldquoStudyof calcium binding to phosphoserine residues of beta-caseinand its phosphopeptide (1ndash25) by 13P NMRrdquo Journal of DairyResearch vol 56 no 3 pp 403ndash409 1989
[20] J-S He S Zhu T-H Mu Y Yu J Li and N Azumaldquo120572s-casein inhibits the pressure-induced aggregation of 120573-lactoglobulin through its molecular chaperone-like propertiesrdquoFood Hydrocolloids vol 25 no 6 pp 1581ndash1586 2011
[21] C Mazri L Sanchez S J Ramos M Calvo and M D PerezldquoEffect of high-pressure treatment on denaturation of bovine 120573-lactoglobulin and 120572-lactalbuminrdquo European Food Research andTechnology vol 234 no 5 pp 813ndash819 2012
aqueous solutions is governed by the partial molar volumesunder the actual conditions in the equilibrium solutionsDissociation of acids increases upon pressurizing if theion-pair formation is accompanied by a substantial volumereduction Consequently the buffering capacity of certainbuffers decreases with pressure with significant changes inpH upon pressurizing To our knowledge only one study onpH changes in protein solution under pressure exists the pHincreased up to 15 units in a 120573-Lg solution at pressure upto 300MPa followed by a decrease to the initial pH of 40for further pressurization up to 500MPa [7] This study wastherefore undertaken to monitor the effect on changes in pHof a 120573-Lg solution at its natural pH and in combination with120573-casein (120573-CN) during HP-treatment and offers additionalinformation to the understanding of themolecular propertiesof 120573-Lg under pressure
2 Materials and Methods
21 Chemicals 120573-lactoglobulin from bovine milk geneticvariant B was isolated from acidic whey of fresh skim milkof homozygotic cows and purified according to the methoddescribed by [12] 120573-casein-A1015840A1015840 (120573-CN) was a gift fromDairy Technology Department of Food Science Universityof Copenhagen 31015840310158401015840-Dibromothymolsulfonphthalein (bro-mothymol blue) was obtained from Merck (Germany) Allother chemicals were of analytical grade and solutions werebased on highly purified water (Milli-Q Plus Millipore CorpBedford MA USA)
22 High Pressure Spectrophotometer The intensity of lighttransmitted through the solutions under investigation ofvarying pressure was measured in situ in a thermostated highpressure optical cell (Type 7402006 from SITEC Sieber Engi-neering AG Switzerland) equipped with a hand-operatedpressure generating system (Type 7501700 from SITECSieber Engineering AG Switzerland) as described by [7]Thepressure generating system and the optical cell were filledwith the relevant solution and the intensity spectrum from350 to 700 nmwas recorded at each step of pressures between01 and 500MPa The intensity spectra were converted toabsorption spectra using 119860
120582= minus log(119868
120582119868ref120582
) where 119868120582and
119868ref120582
are intensities at wavelength 120582 for the solution and forwater as reference respectively The intensity spectrum ofwater was recorded prior to measurement of the indicatorsolution
23 In Situ Spectrophotometry A stock solution of bromoth-ymol blue (36 sdot 10minus4M) in water was prepared Solutionswith bromothymol blue in its acidic alkaline or partiallytransformed form were prepared by adjusting aliquots topH 30 100 and 70 respectively with appropriate amountsof HCl or NaOH and to an ionic strength at 008M withNaCl and a final bromothymol blue concentration of 36 sdot10minus5M Prior to the optical measurements under pressurethe pH of all solutions was measured as a reference withglass electrode (713 pHMeterMetrohm Switzerland) againstinternational activity pH standards 120573-Lg was dissolved in
water as a stock solution and stored at 5∘C overnight forequilibration The 120573-Lg solutions for measurement underpressure were made by adding an appropriate aliquot ofbromothymol blue (final concentrations of 36sdot10minus5M) toaliquots of the stock solution giving final120573-Lg concentrationsof 100 and 300mgmL The pH of the 120573-Lg solutions wasadjusted with HCl or NaOH to pH 30 pH 100 and pH70 and to an ionic strength at 008M with NaCl 120573-CN wasdissolved in water (with 2mgmL NaCl) as a stock solutionand stored at 5∘C overnight for equilibration The 120573-CNsolutions for measurement under pressure were made byadding an appropriate aliquot of bromothymol blue (finalconcentrations of 36sdot10minus5M) to aliquots of the stock solutiongiving final 120573-CN concentrations of 100mgmL The pH ofthe 120573-CN solution was adjusted to pH 70 and to an ionicstrength at 008M with NaCl For the mixed 120573-Lg and 120573-CNsolution an appropriate volume of 120573-Lg stock solution wasadded to the 120573-CN solution to obtain a concentration of100mgmL for both proteins The solutions were filled inthe optical cell of the high pressure spectrophotometer anda series of intensity spectra were recorded and converted toabsorption spectra as described above
The method developed by Orlien et al [7] was used tomonitor in situ pH changes in solutions of 120573-Lg and 120573-CNandmixture thereof under pressure bymeasuring the absorp-tion spectra of the respective proteins in solution underacid and basic conditions and the partially transformed andcalculating the change in pH in accordance with
ΔpH (119875) = Δ log( 120572 (119875)1 minus 120572 (119875)
) (1)
where ΔpH(119875) = pH(01MPa) minus pH(119875) The degree ofdissociation 120572(119875) of the indicator is calculated by
120572 (119875) =119860119909(119875) minus 119860
119886(119875)
119860119887(119875) minus 119860
119886(119875) (2)
where 119860(119875) is the absorbance of the partially transformedindicator (index 119909) and absorbance of the indicator in itsacidic form (index 119886) and in its basic form (index 119887)respectively at the respective pressure
24 Statistical Analysis The statistical analysis is carried outin R (R version 1121) with RKWard (Version 054 KDEversion 451) as graphical user interface used for scriptmarkup and piping to the Rterminal The following add-on packages were also used MASS (Version 73-7) nlme(Version 31-97) and gmodels (Version 2150)
3 Results and Discussion
120573-Lg is a compactly folded globular protein and consists of162 amino acid where 53 residues have titratable side groups[13] 120573-Lg is a dimer at pH 7 and each monomer has 2ionisable histidine side groups However the two histidineresidues differ considerably in solvent accessibility due tothe conformation of the protein molecule As the solvent-accessible area of His146 is around 126 A2 at pH 7 it is
Journal of Spectroscopy 3
available to titration while in contrast with the solvent-accessible area of His161 around 12 A2 it becomes buriedin the interior of the native protein [14] The effective p119870
119886
of ionisable side groups in a protein depends on differentmolecular microenvironments and may have either higheror lower value than the respective free amino acid thusthe p119870
119886of His146 is reported to vary from 62 to 77
and the p119870119886of His161 is reported to vary from 58 to 85
[13]Spectrophotometric measurement of pH with acid-base
indicators is based on differences in absorption spectrabetween the acidic form and the basic form of the indi-cator molecule and the useful range depends on p119870
119894[7]
Previously we have developed a self-consistent method formeasurement of changes in pH with pressure based on thefact that the indicator p119870
119894is insensitive to pressure and
that the indicator molecule does not bind to the protein[7] A number of possible indicators (neutral red phenolred and bromothymol blue) were investigated in detail overthe spectral range 350ndash700 nm in solutions of their acidicand basic forms and of a mixture around p119870
119894value of
each indicator since these indicators were considered goodcandidates for pressure insensitive indicators Bromothymolblue was chosen for the investigation of pH changes of the120573-Lg solutions under pressure due to the sufficient differencein the absorption spectra for the acid base and partiallytransformed forms Figure 1 shows the absorption spectra forbromothymol blue in aqueous solutions of 120573-Lg at differentpH as a function of pressure The increase in molar absorp-tivity upon pressurizing reflects the concentration increasewith pressure as the system is compressedThe small variationin absorption spectra of the acid form with pressure is mostlikely a combined effect of compressibility of the solventof deformation of windows of the high pressure cell andto a much lesser degree of conformational changes of theindicator As seen in Figure 1 the clear distinction betweenthe acid and basic form ensures an accurate calculation of thedegree of dissociation at each pressure [7] This distinctionis emphasized since the changes of the absorption spectraduring pressurization reflect the shifted equilibrium HIn +
H2O119870119894
999448999471 Inminus + H3O+ according to the spectral changes
of bromothymol blue in water at the relevant pHs (datanot shown) and thus function as a sensor to probe theresult of a diffusion controlled transfer of protons from thesolvent to the acidic and basic side groups of the protein incontact with the indicator At initial pH of 7 and pressure(01MPa) the two absorption bands at 431 and 619 nmconfirm that bromothymol blue is in a partially transformedform yet mostly in its acidic form (Figure 1) reflecting thatthe two histidine residues are between being protonated anddeprotonated according to the rather large span of theirrespective p119870
119886 Upon pressurizing the absorption spectra
of bromothymol blue change corresponding to changes inthe acidbase equilibrium of the indicator and as seenthe intensity of both absorption bands increased when thepressure increased to 500MPa120573-Lg is the most abundant whey protein and is used
in many applications for its various functional properties
000408121620
350 400 450 500 550 600 650 700 750 800
Abs pH = 3
pH = 10
120582 (nm)
350 400 450 500 550 600 650 700 750 80000
02
04
06
08
0150100150200250
300350400450500
pH = 7
120582 (nm)
Abs
Figure 1 Pressure-induced changes in the visible absorption spectraof bromothymol blue in an aqueous solution of120573-Lg B (100mgmL)at acidic (pH 30) and basic (pH 100) conditions compared to thepartially transformed form at pH 70 Pressure level in MPa is givenby the legend The abrupt decrease in absorbance at 650 nm is dueto the deuterium lamp in the spectrophotometer
which depend on the physiochemical state and pH Anothergroup of milk proteins that contributes to the milk bufferingcapacity is the caseins and it is reported that the purecaseins have maximal buffering capacity around pH 5ndash55due to phosphoserine and histidine residues [1] Moreoverthe different genetic variants have different physicochemicalproperties and different interacting behaviour with othermilk constituents depending on the solution conditions120573-CN is the most hydrophobic but also highest chargedcasein (minus13 at pH 66) due to an unevenly distribution ofhydrophobic (a long C-terminal without any charged sidegroups) and hydrophilic (a short N-terminal with all chargedside groups) residues resulting in a distinct amphipathic char-acter This unique characteristic makes 120573-CN very useful forfoodmanufacturers and is one of the most abundant proteinsin various food products The caseins have little tertiarystructure and can barely be denatured but it has been foundthat at the 120573-Lg 120573-CNmolar ratio of 1 013 or greater 120573-CNis able to suppress the heat-induced aggregation of 120573-Lg [15]Figure 2 shows the absorption spectra for bromothymol bluein aqueous solutions of 120573-CN as a function of pressure andthe absorption spectra change considerably with a markedincrease in the basic absorption band upon pressurizationWith 120573-Lg in the solution the increase in the basic absorptionband was less marked (Figure 2)
From the spectral data and using spectra of the acidic andbasic 120573-Lg solutions (Figure 1) as the acidic and basic formof bromothymol blue respectively in the calculation of 120572(119875)
Figure 2 Pressure induced changes in the visible absorption spectraof bromothymol blue in an aqueous solution of120573-Lg B (100mgmL)and of120573-LgB+120573-CN (1 1mgmL) at the partially transformed format pH 70 Pressure level in MPa is given by the legend The abruptdecrease in absorbance at 650 nm is due to the deuterium lamp inthe spectrophotometer
(2) the change in pH was calculated according to (1) and theresults are presented in Figure 3
The major factors that control the impact of HP on themolecular structure is the electrostriction of charged andpolar groups elimination of packing defects and the solva-tion of hydrophobic groups Pressure treatment was foundto induce changes in the pH of the 120573-Lg aqueous solutionsdependent on the working pressure and the minimum in pHwas found to be around 150MPa (Figure 3) The pH profileobserved for 120573-Lg with an initial pH 70 (Figure 3) is oppositeto the observed pressure dependency for 120573-Lg with an initialpH of 40 [7] and reflects the difference in the titratable sitesat the respective pH (histidine versus aspartic and glutamicacid) and in the titration behaviour during pressurizationdue to conformational changes (extent of residues beingexposed to solvent versus buried) From Figure 3 it is seenthat high pressure of an unbuffered aqueous solution of 120573-Lgwith an initial pH of 7 induces an immediate decrease inpH up to 150MPa followed by an abrupt increase in pHbeyond the initial pH following a gradual increase to a pHaround 73 at 500MPa Several investigations of high pressureeffects on 120573-Lg have been carried out and several modelsfor the resulting conformational changes have accordinglybeen suggested The study of denaturation of 120573-Lg in skimmilk leads to the overall reaction scheme dissociation ofthe dimer to monomers unfolding of the monomeric (still)native structure and irreversible aggregation with 120573-Lg orcaseins [16] The pressure denaturation of purified 120573-Lgwas earlier described as a three-step process including an
0 50 100 150 200 250 300 350 400 450 500
minus06
minus04
minus02
00
02
04
ΔpH
120573-Lg 1mgmL120573-Lg 3mgmL120573-CN 1mgmL
120573-Lg + 120573-CN (1 1)Calculated mix
P (MPa)
Figure 3 Pressure dependence of pH of aqueous solutions of 120573-LgB at 100mgmL (◼) and 300mgmL (e) 120573-CN 100mgmL (998787)and 120573-Lg B + 120573-CN (1 1mgmL) (995333) all with an initial pH of 70The change in pH ΔpH(119875) = pH(01MPa) minus pH(119875) is calculatedfrom spectral data according to (1) The dashed line represents theΔpH calculated from the changes of 120573-Lg (100mgmL) and 120573-CN(100mgmL) according to (3)
initial pressure-melted state for pressure up to 50MPa areversible denaturation up to 200MPa and an irreversibledenaturation above 200MPa [17] Likewise a three-stagedenaturation model for purified 120573-Lg was suggested withthree discernible structural stages stage I (up to 150MPa)is the native stable structure in stage II (200ndash450MPa) thenative monomers are reversibly interchanged with nonnativemonomers and disulfide-bonded dimer and in stage III (over500MPa) unfolded monomers and dimers interact to formaggregates [18] The pressure-induced structural changes areas emphasized by Anema [16] a complex series of moreconsecutive andor concurrent pathways than the three men-tioned general steps dependent on conditions like solventpressure duration and temperature Thus the HP effect onthe structure of 120573-Lg could be brought together dissociationinto monomers various molten globule structures denatu-ration and aggregation It was shown that upon these HP-induced conformational changes charged residues undergoa change from buried to exposed leading to an unexpectedpH variation of a 120573-Lg solution under pressure [7] The pHprofiles in Figure 3 show that the initial step where dimers aredissociated into monomers at low pressure of 50MPa resultsin no (1mgmL) or minor (3mgmL) changes in pH of thesolution The following transformation into molten globulesis identical to the changes in the 120573-Lg structure in aqueoussolution with initial pH of 40 under increasing pressure [7]despite beingwith a different effect on solution pHThe initialdissociation increases the accessible surface area resulting inan increase in hydration of the protein molecules resultingin the contraction of solvent water (due to electrostriction)and leading to volume decrease and disruption of ion pairs
Journal of Spectroscopy 5
in the protein molecule The p119870119886for histidine residue 146
is 75 and 68 for the dimer and monomer respectivelywhile p119870
119886for histidine residue 161 is 65 and 58 for the
dimer and monomer respectively [13] Hence the pH ofthe 120573-Lg solution under pressure depends on whether theimidazole rings are protonated or deprotonated Accordingto the equilibrium HisH+ + H2O 999448999471 His + H3O
+ at theinitial pH before pressurisation most of the His146 wasprotonated and most of the His161 was deprotonated 120573-Lgdissociated from dimer to monomer at the initial increaseof pressure and the His146 became available for solventand shifted to the deprotonated state (due to lower p119870
119886
for the monomer) resulting in a decrease in pH Duringpressurisation up to 150MPa the dimers are dissociated andprovide pathways for water to penetrate into the interiorof the monomers leading to the molten globule states Inparticular the dissociation at the dimerization area resultedin an increased accessibility of His161 When the remainingprotonated His161 experiences the solvent pH (still higherthan the p119870
119886for the monomer) it became deprotonated
resulting in further decrease of pH The pressure and solventpH effects are optimally balanced at 150MPa where themaximum decrease in pH occurs (Figure 3) At furtherincrease in pressure the tertiary structure of 120573-Lg is disruptedleading to a denatured proteinmaking both histidine residuesavailable for the solvent water As a result of gradual waterpenetration and the accompanying electrostriction of waterthe deprotonated His residues will reassociate the protonfrom water which will lead to an excess amount of OHminus inthe solution corresponding to an increase in pH as seen inFigure 3 As seen in Figure 3 the extent of the pH change isdependent on the concentration of 120573-Lg and increasing con-centration from 1 to 3mgmLbrings about a higher amount ofhistidine residues in effect resulting in a greater pH changeA pH reduction of the observed magnitude (034 units for120573-Lg at 3mgmL at 500MPa) can be of major importancefrom a technological point of view Adjusting the pH ofmilk by 05ndash07 units prior to HP-treatment at 250ndash600MPaat 20∘C was found to reduce the extent of denaturation of120573-Lg considerably compared to milk at normal pH [4 5]Interestingly after reaching a minimum in pH at 150MPa thepH increased abruptly for both concentrations of 120573-Lg andincreased gradually at further pressurizing
The nonglobular 120573-CN is therefore insensitive forpressure-induced conformational changes At the same timeit is highly charged and may therefore give electrostrictiona significant role in the pH behaviour under pressure Theamphiphilic nature of120573-CNusually results in self-associationinto large oligomersmicelle upon dissolving in aqueousmedia but the concentration used and the preparation of thesolution in this study ensure a solution of 120573-CN monomersAt 01MPa and pH 70 both the 5 histidine residues (p119870
119886asymp
65 [1]) and the 5 phosphoserine residues (p1198701198862asymp 63ndash
68 for phosphoserine residues in 120573-CN [19]) are shiftedto the deprotonated states Hence no specific structuralrearrangement of the protein will affect solvent pH onlythe three ldquosimplerdquo ionisation equilibria related to histidinephosphoserine and water self-ionisation will govern the pHchange of the 120573-CN solution under pressure The underlying
electrostrictive effect is from the water self-ionisation whichis increasingly promoted at increasing pressure thereby gen-erating charged hydronium and hydroxide ions It is acceptedthat exposure andor generation of polar and charged groupswill lead to a decrease in volume due to electrostrictivepacking effects Thus the H
3O+ ions are reassociated with
the deprotonated His and Ser-PO3
2minus in effect shifting thecorresponding equilibria to the protonated states This willpromote a further dissociation of water which will lead to anexcess amount of OHminus in the solution corresponding to anincrease in pH as seen in Figure 3
Mixing equal amounts of 120573-Lg and 120573-CN resulted in adepressing effect of the individual pressure effects on bothproteins as seen in Figure 3The initial pHdecrease caused bythe dimer dissociation and deprotonation of His146 in 120573-Lgwas counterbalanced by the increasing concentration of OHminusdue to the electrostrictive effect of the 120573-CN-water systemThis finding indicates that the stability of120573-Lg under pressuredepends on the treatment media and is in agreement withother reports [20 21] It was found that 120572
119904-casein suppressed
the pressure-induced aggregation of 120573-Lg because of thechaperone property of casein [20] Whether 120573-CN acted in asimilar chaperone manner thereby reducing the dissociationand denaturation effect or it was a simple balancing of the pHdue to release of H
3O+ and OHminus from the 120573-Lg and 120573-CN
respectively cannot be deduced from this study Howeverthe subsequent pH increase in the solution of the mixedproteins seemed to be an equal contribution of the effect onpH by the individual protein-water systems Moreover thepressure course of the pH changes of the mixed solution canbe modulated by a simple equation based on the individualHP-pH progress (Figure 3)
The pressure-induced changes in pH of a 120573-Lg solutionat its natural pH was determined by the pressure-induceddissociation and unfolding of120573-Lg and the concurrent degreeof accessibility of titratable side groups in this case the twohistidine side chains The importance of the HP effect on thestructural changes of the protein and the rearrangement ofthe protein-water system on solvent pH was supported bythe pressure effect on pH in a 120573-CN solution 120573-CN lacks athree-dimensional structure hence the pressure-induced pHchanges were explained purely by the shifts in the equilibriaof the histidine and phosphoserine residues as affected bypressure and waterrsquos self-ionisation The HP-induced pHchanges in a mixed 120573-Lg and 120573-CN solution were found tobe a simple mix of the effects from the individual pH profilesunder pressure
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
6 Journal of Spectroscopy
References
[1] F Salaun B Mietton and F Gaucheron ldquoBuffering capacity ofdairy productsrdquo International Dairy Journal vol 15 no 2 pp95ndash109 2005
[2] M H Famelart F Gaucheron F Mariette Y Le Graet KRaulot and E Boyaval ldquoAcidification of pressure-treated milkrdquoInternational Dairy Journal vol 7 no 5 pp 325ndash330 1997
[3] V Orlien L Boserup and K Olsen ldquoCasein micelle dissoci-ation in skim milk during high-pressure treatment effects ofpressure pH and temperaturerdquo Journal of Dairy Science vol93 no 1 pp 12ndash18 2010
[4] M Arias R Lopez-Fandino and A Olano ldquoInfluence of pH onthe effects of high pressure onmilk proteinsrdquoMilchwissenschaftvol 55 no 4 pp 191ndash194 2000
[5] T Huppertz P F Fox and A L Kelly ldquoHigh pressure treatmentof bovine milk effects on casein micelles and whey proteinsrdquoJournal of Dairy Research vol 71 no 1 pp 97ndash106 2004
[6] S KMin P S Chaminda and SK Sastry ldquoIn situmeasurementof reaction volume and calculation of pH of weak acid buffersolutions under high pressurerdquo Journal of Physical Chemistry Bvol 115 no 20 pp 6564ndash6571 2011
[7] VOrlien KOlsen and LH Skibsted ldquoIn situmeasurements ofpH changes in 120573-lactoglobulin solutions under high hydrostaticpressurerdquo Journal of Agricultural and Food Chemistry vol 55no 11 pp 4422ndash4428 2007
[8] V M Stippl A Delgado and T M Becker ldquoDevelopment of amethod for the optical in-situ determination of pH value duringhigh-pressure treatment of fluid foodrdquo Innovative Food Scienceamp Emerging Technologies vol 5 no 3 pp 285ndash292 2004
[9] M Hayert J-M Perrier-Cornet and P Gervais ldquoA simplemethod for measuring the pH of acid solutions under highpressurerdquo The Journal of Physical Chemistry A vol 103 no 12pp 1785ndash1789 1999
[10] T K Hitchens and R G Bryant ldquoPressure dependence of weakacid ionization in deuterium oxide solutionsrdquo The Journal ofPhysical Chemistry B vol 102 no 6 pp 1002ndash1004 1998
[11] R C Neuman Jr W Kauzmann and A Zipp ldquoPressuredependence of weak acid lonization in aqueous buffersrdquo TheJournal of Physical Chemistry vol 77 no 22 pp 2687ndash26911973
[12] K R Kristiansen J Otte R Ipsen and K B Qvist ldquoLarge-scalepreparation of 120573-lactoglobulin A and B by ultrafiltration andion-exchange chromatographyrdquo International Dairy Journalvol 8 no 2 pp 113ndash118 1998
[13] F Fogolari L Ragona S Licciardi et al ldquoElectrostatic prop-erties of bovine 120573-lactoglobulinrdquo Proteins Structure Functionand Genetics vol 39 no 4 pp 317ndash330 2000
[14] B Y QinM C Bewley L K Creamer HM Baker E N Bakerand G B Jameson ldquoStructural basis of the tanford transition ofbovine 120573-lactoglobulinrdquo Biochemistry vol 37 no 40 pp 14014ndash14023 1998
[15] Y H Yong and E A Foegeding ldquoEffects of caseins on thermalstability of bovine 120573-lactoglobulinrdquo Journal of Agricultural andFood Chemistry vol 56 no 21 pp 10352ndash10358 2008
[16] S G Anema R Stockmann and E K Lowe ldquoDenaturationof 120573-lactoglobulin in pressure-treated skim milkrdquo Journal ofAgricultural and Food Chemistry vol 53 no 20 pp 7783ndash77912005
[17] H Stapelfeldt and L H Skibsted ldquoPressure denaturation andaggregation of 120573-lactoglobulin studied by intrinsic fluorescence
depolarization Rayleigh scattering radiationless energy trans-fer and hydrophobic fluoroprobingrdquo Journal of Dairy Researchvol 66 no 4 pp 545ndash558 1999
[18] T Considine H Singh H A Patel and L K CreamerldquoInfluence of binding of sodium dodecyl sulfate all-trans-retinol and 8-anilino-1-naphthalenesulfonate on the high-pressure-induced unfolding and aggregation of 120573-lactoglobulinBrdquo Journal of Agricultural and Food Chemistry vol 53 no 20pp 8010ndash8018 2005
[19] J-J Baumy P Guenot S Sinbandhit and G Brule ldquoStudyof calcium binding to phosphoserine residues of beta-caseinand its phosphopeptide (1ndash25) by 13P NMRrdquo Journal of DairyResearch vol 56 no 3 pp 403ndash409 1989
[20] J-S He S Zhu T-H Mu Y Yu J Li and N Azumaldquo120572s-casein inhibits the pressure-induced aggregation of 120573-lactoglobulin through its molecular chaperone-like propertiesrdquoFood Hydrocolloids vol 25 no 6 pp 1581ndash1586 2011
[21] C Mazri L Sanchez S J Ramos M Calvo and M D PerezldquoEffect of high-pressure treatment on denaturation of bovine 120573-lactoglobulin and 120572-lactalbuminrdquo European Food Research andTechnology vol 234 no 5 pp 813ndash819 2012
available to titration while in contrast with the solvent-accessible area of His161 around 12 A2 it becomes buriedin the interior of the native protein [14] The effective p119870
119886
of ionisable side groups in a protein depends on differentmolecular microenvironments and may have either higheror lower value than the respective free amino acid thusthe p119870
119886of His146 is reported to vary from 62 to 77
and the p119870119886of His161 is reported to vary from 58 to 85
[13]Spectrophotometric measurement of pH with acid-base
indicators is based on differences in absorption spectrabetween the acidic form and the basic form of the indi-cator molecule and the useful range depends on p119870
119894[7]
Previously we have developed a self-consistent method formeasurement of changes in pH with pressure based on thefact that the indicator p119870
119894is insensitive to pressure and
that the indicator molecule does not bind to the protein[7] A number of possible indicators (neutral red phenolred and bromothymol blue) were investigated in detail overthe spectral range 350ndash700 nm in solutions of their acidicand basic forms and of a mixture around p119870
119894value of
each indicator since these indicators were considered goodcandidates for pressure insensitive indicators Bromothymolblue was chosen for the investigation of pH changes of the120573-Lg solutions under pressure due to the sufficient differencein the absorption spectra for the acid base and partiallytransformed forms Figure 1 shows the absorption spectra forbromothymol blue in aqueous solutions of 120573-Lg at differentpH as a function of pressure The increase in molar absorp-tivity upon pressurizing reflects the concentration increasewith pressure as the system is compressedThe small variationin absorption spectra of the acid form with pressure is mostlikely a combined effect of compressibility of the solventof deformation of windows of the high pressure cell andto a much lesser degree of conformational changes of theindicator As seen in Figure 1 the clear distinction betweenthe acid and basic form ensures an accurate calculation of thedegree of dissociation at each pressure [7] This distinctionis emphasized since the changes of the absorption spectraduring pressurization reflect the shifted equilibrium HIn +
H2O119870119894
999448999471 Inminus + H3O+ according to the spectral changes
of bromothymol blue in water at the relevant pHs (datanot shown) and thus function as a sensor to probe theresult of a diffusion controlled transfer of protons from thesolvent to the acidic and basic side groups of the protein incontact with the indicator At initial pH of 7 and pressure(01MPa) the two absorption bands at 431 and 619 nmconfirm that bromothymol blue is in a partially transformedform yet mostly in its acidic form (Figure 1) reflecting thatthe two histidine residues are between being protonated anddeprotonated according to the rather large span of theirrespective p119870
119886 Upon pressurizing the absorption spectra
of bromothymol blue change corresponding to changes inthe acidbase equilibrium of the indicator and as seenthe intensity of both absorption bands increased when thepressure increased to 500MPa120573-Lg is the most abundant whey protein and is used
in many applications for its various functional properties
000408121620
350 400 450 500 550 600 650 700 750 800
Abs pH = 3
pH = 10
120582 (nm)
350 400 450 500 550 600 650 700 750 80000
02
04
06
08
0150100150200250
300350400450500
pH = 7
120582 (nm)
Abs
Figure 1 Pressure-induced changes in the visible absorption spectraof bromothymol blue in an aqueous solution of120573-Lg B (100mgmL)at acidic (pH 30) and basic (pH 100) conditions compared to thepartially transformed form at pH 70 Pressure level in MPa is givenby the legend The abrupt decrease in absorbance at 650 nm is dueto the deuterium lamp in the spectrophotometer
which depend on the physiochemical state and pH Anothergroup of milk proteins that contributes to the milk bufferingcapacity is the caseins and it is reported that the purecaseins have maximal buffering capacity around pH 5ndash55due to phosphoserine and histidine residues [1] Moreoverthe different genetic variants have different physicochemicalproperties and different interacting behaviour with othermilk constituents depending on the solution conditions120573-CN is the most hydrophobic but also highest chargedcasein (minus13 at pH 66) due to an unevenly distribution ofhydrophobic (a long C-terminal without any charged sidegroups) and hydrophilic (a short N-terminal with all chargedside groups) residues resulting in a distinct amphipathic char-acter This unique characteristic makes 120573-CN very useful forfoodmanufacturers and is one of the most abundant proteinsin various food products The caseins have little tertiarystructure and can barely be denatured but it has been foundthat at the 120573-Lg 120573-CNmolar ratio of 1 013 or greater 120573-CNis able to suppress the heat-induced aggregation of 120573-Lg [15]Figure 2 shows the absorption spectra for bromothymol bluein aqueous solutions of 120573-CN as a function of pressure andthe absorption spectra change considerably with a markedincrease in the basic absorption band upon pressurizationWith 120573-Lg in the solution the increase in the basic absorptionband was less marked (Figure 2)
From the spectral data and using spectra of the acidic andbasic 120573-Lg solutions (Figure 1) as the acidic and basic formof bromothymol blue respectively in the calculation of 120572(119875)
Figure 2 Pressure induced changes in the visible absorption spectraof bromothymol blue in an aqueous solution of120573-Lg B (100mgmL)and of120573-LgB+120573-CN (1 1mgmL) at the partially transformed format pH 70 Pressure level in MPa is given by the legend The abruptdecrease in absorbance at 650 nm is due to the deuterium lamp inthe spectrophotometer
(2) the change in pH was calculated according to (1) and theresults are presented in Figure 3
The major factors that control the impact of HP on themolecular structure is the electrostriction of charged andpolar groups elimination of packing defects and the solva-tion of hydrophobic groups Pressure treatment was foundto induce changes in the pH of the 120573-Lg aqueous solutionsdependent on the working pressure and the minimum in pHwas found to be around 150MPa (Figure 3) The pH profileobserved for 120573-Lg with an initial pH 70 (Figure 3) is oppositeto the observed pressure dependency for 120573-Lg with an initialpH of 40 [7] and reflects the difference in the titratable sitesat the respective pH (histidine versus aspartic and glutamicacid) and in the titration behaviour during pressurizationdue to conformational changes (extent of residues beingexposed to solvent versus buried) From Figure 3 it is seenthat high pressure of an unbuffered aqueous solution of 120573-Lgwith an initial pH of 7 induces an immediate decrease inpH up to 150MPa followed by an abrupt increase in pHbeyond the initial pH following a gradual increase to a pHaround 73 at 500MPa Several investigations of high pressureeffects on 120573-Lg have been carried out and several modelsfor the resulting conformational changes have accordinglybeen suggested The study of denaturation of 120573-Lg in skimmilk leads to the overall reaction scheme dissociation ofthe dimer to monomers unfolding of the monomeric (still)native structure and irreversible aggregation with 120573-Lg orcaseins [16] The pressure denaturation of purified 120573-Lgwas earlier described as a three-step process including an
0 50 100 150 200 250 300 350 400 450 500
minus06
minus04
minus02
00
02
04
ΔpH
120573-Lg 1mgmL120573-Lg 3mgmL120573-CN 1mgmL
120573-Lg + 120573-CN (1 1)Calculated mix
P (MPa)
Figure 3 Pressure dependence of pH of aqueous solutions of 120573-LgB at 100mgmL (◼) and 300mgmL (e) 120573-CN 100mgmL (998787)and 120573-Lg B + 120573-CN (1 1mgmL) (995333) all with an initial pH of 70The change in pH ΔpH(119875) = pH(01MPa) minus pH(119875) is calculatedfrom spectral data according to (1) The dashed line represents theΔpH calculated from the changes of 120573-Lg (100mgmL) and 120573-CN(100mgmL) according to (3)
initial pressure-melted state for pressure up to 50MPa areversible denaturation up to 200MPa and an irreversibledenaturation above 200MPa [17] Likewise a three-stagedenaturation model for purified 120573-Lg was suggested withthree discernible structural stages stage I (up to 150MPa)is the native stable structure in stage II (200ndash450MPa) thenative monomers are reversibly interchanged with nonnativemonomers and disulfide-bonded dimer and in stage III (over500MPa) unfolded monomers and dimers interact to formaggregates [18] The pressure-induced structural changes areas emphasized by Anema [16] a complex series of moreconsecutive andor concurrent pathways than the three men-tioned general steps dependent on conditions like solventpressure duration and temperature Thus the HP effect onthe structure of 120573-Lg could be brought together dissociationinto monomers various molten globule structures denatu-ration and aggregation It was shown that upon these HP-induced conformational changes charged residues undergoa change from buried to exposed leading to an unexpectedpH variation of a 120573-Lg solution under pressure [7] The pHprofiles in Figure 3 show that the initial step where dimers aredissociated into monomers at low pressure of 50MPa resultsin no (1mgmL) or minor (3mgmL) changes in pH of thesolution The following transformation into molten globulesis identical to the changes in the 120573-Lg structure in aqueoussolution with initial pH of 40 under increasing pressure [7]despite beingwith a different effect on solution pHThe initialdissociation increases the accessible surface area resulting inan increase in hydration of the protein molecules resultingin the contraction of solvent water (due to electrostriction)and leading to volume decrease and disruption of ion pairs
Journal of Spectroscopy 5
in the protein molecule The p119870119886for histidine residue 146
is 75 and 68 for the dimer and monomer respectivelywhile p119870
119886for histidine residue 161 is 65 and 58 for the
dimer and monomer respectively [13] Hence the pH ofthe 120573-Lg solution under pressure depends on whether theimidazole rings are protonated or deprotonated Accordingto the equilibrium HisH+ + H2O 999448999471 His + H3O
+ at theinitial pH before pressurisation most of the His146 wasprotonated and most of the His161 was deprotonated 120573-Lgdissociated from dimer to monomer at the initial increaseof pressure and the His146 became available for solventand shifted to the deprotonated state (due to lower p119870
119886
for the monomer) resulting in a decrease in pH Duringpressurisation up to 150MPa the dimers are dissociated andprovide pathways for water to penetrate into the interiorof the monomers leading to the molten globule states Inparticular the dissociation at the dimerization area resultedin an increased accessibility of His161 When the remainingprotonated His161 experiences the solvent pH (still higherthan the p119870
119886for the monomer) it became deprotonated
resulting in further decrease of pH The pressure and solventpH effects are optimally balanced at 150MPa where themaximum decrease in pH occurs (Figure 3) At furtherincrease in pressure the tertiary structure of 120573-Lg is disruptedleading to a denatured proteinmaking both histidine residuesavailable for the solvent water As a result of gradual waterpenetration and the accompanying electrostriction of waterthe deprotonated His residues will reassociate the protonfrom water which will lead to an excess amount of OHminus inthe solution corresponding to an increase in pH as seen inFigure 3 As seen in Figure 3 the extent of the pH change isdependent on the concentration of 120573-Lg and increasing con-centration from 1 to 3mgmLbrings about a higher amount ofhistidine residues in effect resulting in a greater pH changeA pH reduction of the observed magnitude (034 units for120573-Lg at 3mgmL at 500MPa) can be of major importancefrom a technological point of view Adjusting the pH ofmilk by 05ndash07 units prior to HP-treatment at 250ndash600MPaat 20∘C was found to reduce the extent of denaturation of120573-Lg considerably compared to milk at normal pH [4 5]Interestingly after reaching a minimum in pH at 150MPa thepH increased abruptly for both concentrations of 120573-Lg andincreased gradually at further pressurizing
The nonglobular 120573-CN is therefore insensitive forpressure-induced conformational changes At the same timeit is highly charged and may therefore give electrostrictiona significant role in the pH behaviour under pressure Theamphiphilic nature of120573-CNusually results in self-associationinto large oligomersmicelle upon dissolving in aqueousmedia but the concentration used and the preparation of thesolution in this study ensure a solution of 120573-CN monomersAt 01MPa and pH 70 both the 5 histidine residues (p119870
119886asymp
65 [1]) and the 5 phosphoserine residues (p1198701198862asymp 63ndash
68 for phosphoserine residues in 120573-CN [19]) are shiftedto the deprotonated states Hence no specific structuralrearrangement of the protein will affect solvent pH onlythe three ldquosimplerdquo ionisation equilibria related to histidinephosphoserine and water self-ionisation will govern the pHchange of the 120573-CN solution under pressure The underlying
electrostrictive effect is from the water self-ionisation whichis increasingly promoted at increasing pressure thereby gen-erating charged hydronium and hydroxide ions It is acceptedthat exposure andor generation of polar and charged groupswill lead to a decrease in volume due to electrostrictivepacking effects Thus the H
3O+ ions are reassociated with
the deprotonated His and Ser-PO3
2minus in effect shifting thecorresponding equilibria to the protonated states This willpromote a further dissociation of water which will lead to anexcess amount of OHminus in the solution corresponding to anincrease in pH as seen in Figure 3
Mixing equal amounts of 120573-Lg and 120573-CN resulted in adepressing effect of the individual pressure effects on bothproteins as seen in Figure 3The initial pHdecrease caused bythe dimer dissociation and deprotonation of His146 in 120573-Lgwas counterbalanced by the increasing concentration of OHminusdue to the electrostrictive effect of the 120573-CN-water systemThis finding indicates that the stability of120573-Lg under pressuredepends on the treatment media and is in agreement withother reports [20 21] It was found that 120572
119904-casein suppressed
the pressure-induced aggregation of 120573-Lg because of thechaperone property of casein [20] Whether 120573-CN acted in asimilar chaperone manner thereby reducing the dissociationand denaturation effect or it was a simple balancing of the pHdue to release of H
3O+ and OHminus from the 120573-Lg and 120573-CN
respectively cannot be deduced from this study Howeverthe subsequent pH increase in the solution of the mixedproteins seemed to be an equal contribution of the effect onpH by the individual protein-water systems Moreover thepressure course of the pH changes of the mixed solution canbe modulated by a simple equation based on the individualHP-pH progress (Figure 3)
The pressure-induced changes in pH of a 120573-Lg solutionat its natural pH was determined by the pressure-induceddissociation and unfolding of120573-Lg and the concurrent degreeof accessibility of titratable side groups in this case the twohistidine side chains The importance of the HP effect on thestructural changes of the protein and the rearrangement ofthe protein-water system on solvent pH was supported bythe pressure effect on pH in a 120573-CN solution 120573-CN lacks athree-dimensional structure hence the pressure-induced pHchanges were explained purely by the shifts in the equilibriaof the histidine and phosphoserine residues as affected bypressure and waterrsquos self-ionisation The HP-induced pHchanges in a mixed 120573-Lg and 120573-CN solution were found tobe a simple mix of the effects from the individual pH profilesunder pressure
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
6 Journal of Spectroscopy
References
[1] F Salaun B Mietton and F Gaucheron ldquoBuffering capacity ofdairy productsrdquo International Dairy Journal vol 15 no 2 pp95ndash109 2005
[2] M H Famelart F Gaucheron F Mariette Y Le Graet KRaulot and E Boyaval ldquoAcidification of pressure-treated milkrdquoInternational Dairy Journal vol 7 no 5 pp 325ndash330 1997
[3] V Orlien L Boserup and K Olsen ldquoCasein micelle dissoci-ation in skim milk during high-pressure treatment effects ofpressure pH and temperaturerdquo Journal of Dairy Science vol93 no 1 pp 12ndash18 2010
[4] M Arias R Lopez-Fandino and A Olano ldquoInfluence of pH onthe effects of high pressure onmilk proteinsrdquoMilchwissenschaftvol 55 no 4 pp 191ndash194 2000
[5] T Huppertz P F Fox and A L Kelly ldquoHigh pressure treatmentof bovine milk effects on casein micelles and whey proteinsrdquoJournal of Dairy Research vol 71 no 1 pp 97ndash106 2004
[6] S KMin P S Chaminda and SK Sastry ldquoIn situmeasurementof reaction volume and calculation of pH of weak acid buffersolutions under high pressurerdquo Journal of Physical Chemistry Bvol 115 no 20 pp 6564ndash6571 2011
[7] VOrlien KOlsen and LH Skibsted ldquoIn situmeasurements ofpH changes in 120573-lactoglobulin solutions under high hydrostaticpressurerdquo Journal of Agricultural and Food Chemistry vol 55no 11 pp 4422ndash4428 2007
[8] V M Stippl A Delgado and T M Becker ldquoDevelopment of amethod for the optical in-situ determination of pH value duringhigh-pressure treatment of fluid foodrdquo Innovative Food Scienceamp Emerging Technologies vol 5 no 3 pp 285ndash292 2004
[9] M Hayert J-M Perrier-Cornet and P Gervais ldquoA simplemethod for measuring the pH of acid solutions under highpressurerdquo The Journal of Physical Chemistry A vol 103 no 12pp 1785ndash1789 1999
[10] T K Hitchens and R G Bryant ldquoPressure dependence of weakacid ionization in deuterium oxide solutionsrdquo The Journal ofPhysical Chemistry B vol 102 no 6 pp 1002ndash1004 1998
[11] R C Neuman Jr W Kauzmann and A Zipp ldquoPressuredependence of weak acid lonization in aqueous buffersrdquo TheJournal of Physical Chemistry vol 77 no 22 pp 2687ndash26911973
[12] K R Kristiansen J Otte R Ipsen and K B Qvist ldquoLarge-scalepreparation of 120573-lactoglobulin A and B by ultrafiltration andion-exchange chromatographyrdquo International Dairy Journalvol 8 no 2 pp 113ndash118 1998
[13] F Fogolari L Ragona S Licciardi et al ldquoElectrostatic prop-erties of bovine 120573-lactoglobulinrdquo Proteins Structure Functionand Genetics vol 39 no 4 pp 317ndash330 2000
[14] B Y QinM C Bewley L K Creamer HM Baker E N Bakerand G B Jameson ldquoStructural basis of the tanford transition ofbovine 120573-lactoglobulinrdquo Biochemistry vol 37 no 40 pp 14014ndash14023 1998
[15] Y H Yong and E A Foegeding ldquoEffects of caseins on thermalstability of bovine 120573-lactoglobulinrdquo Journal of Agricultural andFood Chemistry vol 56 no 21 pp 10352ndash10358 2008
[16] S G Anema R Stockmann and E K Lowe ldquoDenaturationof 120573-lactoglobulin in pressure-treated skim milkrdquo Journal ofAgricultural and Food Chemistry vol 53 no 20 pp 7783ndash77912005
[17] H Stapelfeldt and L H Skibsted ldquoPressure denaturation andaggregation of 120573-lactoglobulin studied by intrinsic fluorescence
depolarization Rayleigh scattering radiationless energy trans-fer and hydrophobic fluoroprobingrdquo Journal of Dairy Researchvol 66 no 4 pp 545ndash558 1999
[18] T Considine H Singh H A Patel and L K CreamerldquoInfluence of binding of sodium dodecyl sulfate all-trans-retinol and 8-anilino-1-naphthalenesulfonate on the high-pressure-induced unfolding and aggregation of 120573-lactoglobulinBrdquo Journal of Agricultural and Food Chemistry vol 53 no 20pp 8010ndash8018 2005
[19] J-J Baumy P Guenot S Sinbandhit and G Brule ldquoStudyof calcium binding to phosphoserine residues of beta-caseinand its phosphopeptide (1ndash25) by 13P NMRrdquo Journal of DairyResearch vol 56 no 3 pp 403ndash409 1989
[20] J-S He S Zhu T-H Mu Y Yu J Li and N Azumaldquo120572s-casein inhibits the pressure-induced aggregation of 120573-lactoglobulin through its molecular chaperone-like propertiesrdquoFood Hydrocolloids vol 25 no 6 pp 1581ndash1586 2011
[21] C Mazri L Sanchez S J Ramos M Calvo and M D PerezldquoEffect of high-pressure treatment on denaturation of bovine 120573-lactoglobulin and 120572-lactalbuminrdquo European Food Research andTechnology vol 234 no 5 pp 813ndash819 2012
Figure 2 Pressure induced changes in the visible absorption spectraof bromothymol blue in an aqueous solution of120573-Lg B (100mgmL)and of120573-LgB+120573-CN (1 1mgmL) at the partially transformed format pH 70 Pressure level in MPa is given by the legend The abruptdecrease in absorbance at 650 nm is due to the deuterium lamp inthe spectrophotometer
(2) the change in pH was calculated according to (1) and theresults are presented in Figure 3
The major factors that control the impact of HP on themolecular structure is the electrostriction of charged andpolar groups elimination of packing defects and the solva-tion of hydrophobic groups Pressure treatment was foundto induce changes in the pH of the 120573-Lg aqueous solutionsdependent on the working pressure and the minimum in pHwas found to be around 150MPa (Figure 3) The pH profileobserved for 120573-Lg with an initial pH 70 (Figure 3) is oppositeto the observed pressure dependency for 120573-Lg with an initialpH of 40 [7] and reflects the difference in the titratable sitesat the respective pH (histidine versus aspartic and glutamicacid) and in the titration behaviour during pressurizationdue to conformational changes (extent of residues beingexposed to solvent versus buried) From Figure 3 it is seenthat high pressure of an unbuffered aqueous solution of 120573-Lgwith an initial pH of 7 induces an immediate decrease inpH up to 150MPa followed by an abrupt increase in pHbeyond the initial pH following a gradual increase to a pHaround 73 at 500MPa Several investigations of high pressureeffects on 120573-Lg have been carried out and several modelsfor the resulting conformational changes have accordinglybeen suggested The study of denaturation of 120573-Lg in skimmilk leads to the overall reaction scheme dissociation ofthe dimer to monomers unfolding of the monomeric (still)native structure and irreversible aggregation with 120573-Lg orcaseins [16] The pressure denaturation of purified 120573-Lgwas earlier described as a three-step process including an
0 50 100 150 200 250 300 350 400 450 500
minus06
minus04
minus02
00
02
04
ΔpH
120573-Lg 1mgmL120573-Lg 3mgmL120573-CN 1mgmL
120573-Lg + 120573-CN (1 1)Calculated mix
P (MPa)
Figure 3 Pressure dependence of pH of aqueous solutions of 120573-LgB at 100mgmL (◼) and 300mgmL (e) 120573-CN 100mgmL (998787)and 120573-Lg B + 120573-CN (1 1mgmL) (995333) all with an initial pH of 70The change in pH ΔpH(119875) = pH(01MPa) minus pH(119875) is calculatedfrom spectral data according to (1) The dashed line represents theΔpH calculated from the changes of 120573-Lg (100mgmL) and 120573-CN(100mgmL) according to (3)
initial pressure-melted state for pressure up to 50MPa areversible denaturation up to 200MPa and an irreversibledenaturation above 200MPa [17] Likewise a three-stagedenaturation model for purified 120573-Lg was suggested withthree discernible structural stages stage I (up to 150MPa)is the native stable structure in stage II (200ndash450MPa) thenative monomers are reversibly interchanged with nonnativemonomers and disulfide-bonded dimer and in stage III (over500MPa) unfolded monomers and dimers interact to formaggregates [18] The pressure-induced structural changes areas emphasized by Anema [16] a complex series of moreconsecutive andor concurrent pathways than the three men-tioned general steps dependent on conditions like solventpressure duration and temperature Thus the HP effect onthe structure of 120573-Lg could be brought together dissociationinto monomers various molten globule structures denatu-ration and aggregation It was shown that upon these HP-induced conformational changes charged residues undergoa change from buried to exposed leading to an unexpectedpH variation of a 120573-Lg solution under pressure [7] The pHprofiles in Figure 3 show that the initial step where dimers aredissociated into monomers at low pressure of 50MPa resultsin no (1mgmL) or minor (3mgmL) changes in pH of thesolution The following transformation into molten globulesis identical to the changes in the 120573-Lg structure in aqueoussolution with initial pH of 40 under increasing pressure [7]despite beingwith a different effect on solution pHThe initialdissociation increases the accessible surface area resulting inan increase in hydration of the protein molecules resultingin the contraction of solvent water (due to electrostriction)and leading to volume decrease and disruption of ion pairs
Journal of Spectroscopy 5
in the protein molecule The p119870119886for histidine residue 146
is 75 and 68 for the dimer and monomer respectivelywhile p119870
119886for histidine residue 161 is 65 and 58 for the
dimer and monomer respectively [13] Hence the pH ofthe 120573-Lg solution under pressure depends on whether theimidazole rings are protonated or deprotonated Accordingto the equilibrium HisH+ + H2O 999448999471 His + H3O
+ at theinitial pH before pressurisation most of the His146 wasprotonated and most of the His161 was deprotonated 120573-Lgdissociated from dimer to monomer at the initial increaseof pressure and the His146 became available for solventand shifted to the deprotonated state (due to lower p119870
119886
for the monomer) resulting in a decrease in pH Duringpressurisation up to 150MPa the dimers are dissociated andprovide pathways for water to penetrate into the interiorof the monomers leading to the molten globule states Inparticular the dissociation at the dimerization area resultedin an increased accessibility of His161 When the remainingprotonated His161 experiences the solvent pH (still higherthan the p119870
119886for the monomer) it became deprotonated
resulting in further decrease of pH The pressure and solventpH effects are optimally balanced at 150MPa where themaximum decrease in pH occurs (Figure 3) At furtherincrease in pressure the tertiary structure of 120573-Lg is disruptedleading to a denatured proteinmaking both histidine residuesavailable for the solvent water As a result of gradual waterpenetration and the accompanying electrostriction of waterthe deprotonated His residues will reassociate the protonfrom water which will lead to an excess amount of OHminus inthe solution corresponding to an increase in pH as seen inFigure 3 As seen in Figure 3 the extent of the pH change isdependent on the concentration of 120573-Lg and increasing con-centration from 1 to 3mgmLbrings about a higher amount ofhistidine residues in effect resulting in a greater pH changeA pH reduction of the observed magnitude (034 units for120573-Lg at 3mgmL at 500MPa) can be of major importancefrom a technological point of view Adjusting the pH ofmilk by 05ndash07 units prior to HP-treatment at 250ndash600MPaat 20∘C was found to reduce the extent of denaturation of120573-Lg considerably compared to milk at normal pH [4 5]Interestingly after reaching a minimum in pH at 150MPa thepH increased abruptly for both concentrations of 120573-Lg andincreased gradually at further pressurizing
The nonglobular 120573-CN is therefore insensitive forpressure-induced conformational changes At the same timeit is highly charged and may therefore give electrostrictiona significant role in the pH behaviour under pressure Theamphiphilic nature of120573-CNusually results in self-associationinto large oligomersmicelle upon dissolving in aqueousmedia but the concentration used and the preparation of thesolution in this study ensure a solution of 120573-CN monomersAt 01MPa and pH 70 both the 5 histidine residues (p119870
119886asymp
65 [1]) and the 5 phosphoserine residues (p1198701198862asymp 63ndash
68 for phosphoserine residues in 120573-CN [19]) are shiftedto the deprotonated states Hence no specific structuralrearrangement of the protein will affect solvent pH onlythe three ldquosimplerdquo ionisation equilibria related to histidinephosphoserine and water self-ionisation will govern the pHchange of the 120573-CN solution under pressure The underlying
electrostrictive effect is from the water self-ionisation whichis increasingly promoted at increasing pressure thereby gen-erating charged hydronium and hydroxide ions It is acceptedthat exposure andor generation of polar and charged groupswill lead to a decrease in volume due to electrostrictivepacking effects Thus the H
3O+ ions are reassociated with
the deprotonated His and Ser-PO3
2minus in effect shifting thecorresponding equilibria to the protonated states This willpromote a further dissociation of water which will lead to anexcess amount of OHminus in the solution corresponding to anincrease in pH as seen in Figure 3
Mixing equal amounts of 120573-Lg and 120573-CN resulted in adepressing effect of the individual pressure effects on bothproteins as seen in Figure 3The initial pHdecrease caused bythe dimer dissociation and deprotonation of His146 in 120573-Lgwas counterbalanced by the increasing concentration of OHminusdue to the electrostrictive effect of the 120573-CN-water systemThis finding indicates that the stability of120573-Lg under pressuredepends on the treatment media and is in agreement withother reports [20 21] It was found that 120572
119904-casein suppressed
the pressure-induced aggregation of 120573-Lg because of thechaperone property of casein [20] Whether 120573-CN acted in asimilar chaperone manner thereby reducing the dissociationand denaturation effect or it was a simple balancing of the pHdue to release of H
3O+ and OHminus from the 120573-Lg and 120573-CN
respectively cannot be deduced from this study Howeverthe subsequent pH increase in the solution of the mixedproteins seemed to be an equal contribution of the effect onpH by the individual protein-water systems Moreover thepressure course of the pH changes of the mixed solution canbe modulated by a simple equation based on the individualHP-pH progress (Figure 3)
The pressure-induced changes in pH of a 120573-Lg solutionat its natural pH was determined by the pressure-induceddissociation and unfolding of120573-Lg and the concurrent degreeof accessibility of titratable side groups in this case the twohistidine side chains The importance of the HP effect on thestructural changes of the protein and the rearrangement ofthe protein-water system on solvent pH was supported bythe pressure effect on pH in a 120573-CN solution 120573-CN lacks athree-dimensional structure hence the pressure-induced pHchanges were explained purely by the shifts in the equilibriaof the histidine and phosphoserine residues as affected bypressure and waterrsquos self-ionisation The HP-induced pHchanges in a mixed 120573-Lg and 120573-CN solution were found tobe a simple mix of the effects from the individual pH profilesunder pressure
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
6 Journal of Spectroscopy
References
[1] F Salaun B Mietton and F Gaucheron ldquoBuffering capacity ofdairy productsrdquo International Dairy Journal vol 15 no 2 pp95ndash109 2005
[2] M H Famelart F Gaucheron F Mariette Y Le Graet KRaulot and E Boyaval ldquoAcidification of pressure-treated milkrdquoInternational Dairy Journal vol 7 no 5 pp 325ndash330 1997
[3] V Orlien L Boserup and K Olsen ldquoCasein micelle dissoci-ation in skim milk during high-pressure treatment effects ofpressure pH and temperaturerdquo Journal of Dairy Science vol93 no 1 pp 12ndash18 2010
[4] M Arias R Lopez-Fandino and A Olano ldquoInfluence of pH onthe effects of high pressure onmilk proteinsrdquoMilchwissenschaftvol 55 no 4 pp 191ndash194 2000
[5] T Huppertz P F Fox and A L Kelly ldquoHigh pressure treatmentof bovine milk effects on casein micelles and whey proteinsrdquoJournal of Dairy Research vol 71 no 1 pp 97ndash106 2004
[6] S KMin P S Chaminda and SK Sastry ldquoIn situmeasurementof reaction volume and calculation of pH of weak acid buffersolutions under high pressurerdquo Journal of Physical Chemistry Bvol 115 no 20 pp 6564ndash6571 2011
[7] VOrlien KOlsen and LH Skibsted ldquoIn situmeasurements ofpH changes in 120573-lactoglobulin solutions under high hydrostaticpressurerdquo Journal of Agricultural and Food Chemistry vol 55no 11 pp 4422ndash4428 2007
[8] V M Stippl A Delgado and T M Becker ldquoDevelopment of amethod for the optical in-situ determination of pH value duringhigh-pressure treatment of fluid foodrdquo Innovative Food Scienceamp Emerging Technologies vol 5 no 3 pp 285ndash292 2004
[9] M Hayert J-M Perrier-Cornet and P Gervais ldquoA simplemethod for measuring the pH of acid solutions under highpressurerdquo The Journal of Physical Chemistry A vol 103 no 12pp 1785ndash1789 1999
[10] T K Hitchens and R G Bryant ldquoPressure dependence of weakacid ionization in deuterium oxide solutionsrdquo The Journal ofPhysical Chemistry B vol 102 no 6 pp 1002ndash1004 1998
[11] R C Neuman Jr W Kauzmann and A Zipp ldquoPressuredependence of weak acid lonization in aqueous buffersrdquo TheJournal of Physical Chemistry vol 77 no 22 pp 2687ndash26911973
[12] K R Kristiansen J Otte R Ipsen and K B Qvist ldquoLarge-scalepreparation of 120573-lactoglobulin A and B by ultrafiltration andion-exchange chromatographyrdquo International Dairy Journalvol 8 no 2 pp 113ndash118 1998
[13] F Fogolari L Ragona S Licciardi et al ldquoElectrostatic prop-erties of bovine 120573-lactoglobulinrdquo Proteins Structure Functionand Genetics vol 39 no 4 pp 317ndash330 2000
[14] B Y QinM C Bewley L K Creamer HM Baker E N Bakerand G B Jameson ldquoStructural basis of the tanford transition ofbovine 120573-lactoglobulinrdquo Biochemistry vol 37 no 40 pp 14014ndash14023 1998
[15] Y H Yong and E A Foegeding ldquoEffects of caseins on thermalstability of bovine 120573-lactoglobulinrdquo Journal of Agricultural andFood Chemistry vol 56 no 21 pp 10352ndash10358 2008
[16] S G Anema R Stockmann and E K Lowe ldquoDenaturationof 120573-lactoglobulin in pressure-treated skim milkrdquo Journal ofAgricultural and Food Chemistry vol 53 no 20 pp 7783ndash77912005
[17] H Stapelfeldt and L H Skibsted ldquoPressure denaturation andaggregation of 120573-lactoglobulin studied by intrinsic fluorescence
depolarization Rayleigh scattering radiationless energy trans-fer and hydrophobic fluoroprobingrdquo Journal of Dairy Researchvol 66 no 4 pp 545ndash558 1999
[18] T Considine H Singh H A Patel and L K CreamerldquoInfluence of binding of sodium dodecyl sulfate all-trans-retinol and 8-anilino-1-naphthalenesulfonate on the high-pressure-induced unfolding and aggregation of 120573-lactoglobulinBrdquo Journal of Agricultural and Food Chemistry vol 53 no 20pp 8010ndash8018 2005
[19] J-J Baumy P Guenot S Sinbandhit and G Brule ldquoStudyof calcium binding to phosphoserine residues of beta-caseinand its phosphopeptide (1ndash25) by 13P NMRrdquo Journal of DairyResearch vol 56 no 3 pp 403ndash409 1989
[20] J-S He S Zhu T-H Mu Y Yu J Li and N Azumaldquo120572s-casein inhibits the pressure-induced aggregation of 120573-lactoglobulin through its molecular chaperone-like propertiesrdquoFood Hydrocolloids vol 25 no 6 pp 1581ndash1586 2011
[21] C Mazri L Sanchez S J Ramos M Calvo and M D PerezldquoEffect of high-pressure treatment on denaturation of bovine 120573-lactoglobulin and 120572-lactalbuminrdquo European Food Research andTechnology vol 234 no 5 pp 813ndash819 2012
in the protein molecule The p119870119886for histidine residue 146
is 75 and 68 for the dimer and monomer respectivelywhile p119870
119886for histidine residue 161 is 65 and 58 for the
dimer and monomer respectively [13] Hence the pH ofthe 120573-Lg solution under pressure depends on whether theimidazole rings are protonated or deprotonated Accordingto the equilibrium HisH+ + H2O 999448999471 His + H3O
+ at theinitial pH before pressurisation most of the His146 wasprotonated and most of the His161 was deprotonated 120573-Lgdissociated from dimer to monomer at the initial increaseof pressure and the His146 became available for solventand shifted to the deprotonated state (due to lower p119870
119886
for the monomer) resulting in a decrease in pH Duringpressurisation up to 150MPa the dimers are dissociated andprovide pathways for water to penetrate into the interiorof the monomers leading to the molten globule states Inparticular the dissociation at the dimerization area resultedin an increased accessibility of His161 When the remainingprotonated His161 experiences the solvent pH (still higherthan the p119870
119886for the monomer) it became deprotonated
resulting in further decrease of pH The pressure and solventpH effects are optimally balanced at 150MPa where themaximum decrease in pH occurs (Figure 3) At furtherincrease in pressure the tertiary structure of 120573-Lg is disruptedleading to a denatured proteinmaking both histidine residuesavailable for the solvent water As a result of gradual waterpenetration and the accompanying electrostriction of waterthe deprotonated His residues will reassociate the protonfrom water which will lead to an excess amount of OHminus inthe solution corresponding to an increase in pH as seen inFigure 3 As seen in Figure 3 the extent of the pH change isdependent on the concentration of 120573-Lg and increasing con-centration from 1 to 3mgmLbrings about a higher amount ofhistidine residues in effect resulting in a greater pH changeA pH reduction of the observed magnitude (034 units for120573-Lg at 3mgmL at 500MPa) can be of major importancefrom a technological point of view Adjusting the pH ofmilk by 05ndash07 units prior to HP-treatment at 250ndash600MPaat 20∘C was found to reduce the extent of denaturation of120573-Lg considerably compared to milk at normal pH [4 5]Interestingly after reaching a minimum in pH at 150MPa thepH increased abruptly for both concentrations of 120573-Lg andincreased gradually at further pressurizing
The nonglobular 120573-CN is therefore insensitive forpressure-induced conformational changes At the same timeit is highly charged and may therefore give electrostrictiona significant role in the pH behaviour under pressure Theamphiphilic nature of120573-CNusually results in self-associationinto large oligomersmicelle upon dissolving in aqueousmedia but the concentration used and the preparation of thesolution in this study ensure a solution of 120573-CN monomersAt 01MPa and pH 70 both the 5 histidine residues (p119870
119886asymp
65 [1]) and the 5 phosphoserine residues (p1198701198862asymp 63ndash
68 for phosphoserine residues in 120573-CN [19]) are shiftedto the deprotonated states Hence no specific structuralrearrangement of the protein will affect solvent pH onlythe three ldquosimplerdquo ionisation equilibria related to histidinephosphoserine and water self-ionisation will govern the pHchange of the 120573-CN solution under pressure The underlying
electrostrictive effect is from the water self-ionisation whichis increasingly promoted at increasing pressure thereby gen-erating charged hydronium and hydroxide ions It is acceptedthat exposure andor generation of polar and charged groupswill lead to a decrease in volume due to electrostrictivepacking effects Thus the H
3O+ ions are reassociated with
the deprotonated His and Ser-PO3
2minus in effect shifting thecorresponding equilibria to the protonated states This willpromote a further dissociation of water which will lead to anexcess amount of OHminus in the solution corresponding to anincrease in pH as seen in Figure 3
Mixing equal amounts of 120573-Lg and 120573-CN resulted in adepressing effect of the individual pressure effects on bothproteins as seen in Figure 3The initial pHdecrease caused bythe dimer dissociation and deprotonation of His146 in 120573-Lgwas counterbalanced by the increasing concentration of OHminusdue to the electrostrictive effect of the 120573-CN-water systemThis finding indicates that the stability of120573-Lg under pressuredepends on the treatment media and is in agreement withother reports [20 21] It was found that 120572
119904-casein suppressed
the pressure-induced aggregation of 120573-Lg because of thechaperone property of casein [20] Whether 120573-CN acted in asimilar chaperone manner thereby reducing the dissociationand denaturation effect or it was a simple balancing of the pHdue to release of H
3O+ and OHminus from the 120573-Lg and 120573-CN
respectively cannot be deduced from this study Howeverthe subsequent pH increase in the solution of the mixedproteins seemed to be an equal contribution of the effect onpH by the individual protein-water systems Moreover thepressure course of the pH changes of the mixed solution canbe modulated by a simple equation based on the individualHP-pH progress (Figure 3)
The pressure-induced changes in pH of a 120573-Lg solutionat its natural pH was determined by the pressure-induceddissociation and unfolding of120573-Lg and the concurrent degreeof accessibility of titratable side groups in this case the twohistidine side chains The importance of the HP effect on thestructural changes of the protein and the rearrangement ofthe protein-water system on solvent pH was supported bythe pressure effect on pH in a 120573-CN solution 120573-CN lacks athree-dimensional structure hence the pressure-induced pHchanges were explained purely by the shifts in the equilibriaof the histidine and phosphoserine residues as affected bypressure and waterrsquos self-ionisation The HP-induced pHchanges in a mixed 120573-Lg and 120573-CN solution were found tobe a simple mix of the effects from the individual pH profilesunder pressure
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
6 Journal of Spectroscopy
References
[1] F Salaun B Mietton and F Gaucheron ldquoBuffering capacity ofdairy productsrdquo International Dairy Journal vol 15 no 2 pp95ndash109 2005
[2] M H Famelart F Gaucheron F Mariette Y Le Graet KRaulot and E Boyaval ldquoAcidification of pressure-treated milkrdquoInternational Dairy Journal vol 7 no 5 pp 325ndash330 1997
[3] V Orlien L Boserup and K Olsen ldquoCasein micelle dissoci-ation in skim milk during high-pressure treatment effects ofpressure pH and temperaturerdquo Journal of Dairy Science vol93 no 1 pp 12ndash18 2010
[4] M Arias R Lopez-Fandino and A Olano ldquoInfluence of pH onthe effects of high pressure onmilk proteinsrdquoMilchwissenschaftvol 55 no 4 pp 191ndash194 2000
[5] T Huppertz P F Fox and A L Kelly ldquoHigh pressure treatmentof bovine milk effects on casein micelles and whey proteinsrdquoJournal of Dairy Research vol 71 no 1 pp 97ndash106 2004
[6] S KMin P S Chaminda and SK Sastry ldquoIn situmeasurementof reaction volume and calculation of pH of weak acid buffersolutions under high pressurerdquo Journal of Physical Chemistry Bvol 115 no 20 pp 6564ndash6571 2011
[7] VOrlien KOlsen and LH Skibsted ldquoIn situmeasurements ofpH changes in 120573-lactoglobulin solutions under high hydrostaticpressurerdquo Journal of Agricultural and Food Chemistry vol 55no 11 pp 4422ndash4428 2007
[8] V M Stippl A Delgado and T M Becker ldquoDevelopment of amethod for the optical in-situ determination of pH value duringhigh-pressure treatment of fluid foodrdquo Innovative Food Scienceamp Emerging Technologies vol 5 no 3 pp 285ndash292 2004
[9] M Hayert J-M Perrier-Cornet and P Gervais ldquoA simplemethod for measuring the pH of acid solutions under highpressurerdquo The Journal of Physical Chemistry A vol 103 no 12pp 1785ndash1789 1999
[10] T K Hitchens and R G Bryant ldquoPressure dependence of weakacid ionization in deuterium oxide solutionsrdquo The Journal ofPhysical Chemistry B vol 102 no 6 pp 1002ndash1004 1998
[11] R C Neuman Jr W Kauzmann and A Zipp ldquoPressuredependence of weak acid lonization in aqueous buffersrdquo TheJournal of Physical Chemistry vol 77 no 22 pp 2687ndash26911973
[12] K R Kristiansen J Otte R Ipsen and K B Qvist ldquoLarge-scalepreparation of 120573-lactoglobulin A and B by ultrafiltration andion-exchange chromatographyrdquo International Dairy Journalvol 8 no 2 pp 113ndash118 1998
[13] F Fogolari L Ragona S Licciardi et al ldquoElectrostatic prop-erties of bovine 120573-lactoglobulinrdquo Proteins Structure Functionand Genetics vol 39 no 4 pp 317ndash330 2000
[14] B Y QinM C Bewley L K Creamer HM Baker E N Bakerand G B Jameson ldquoStructural basis of the tanford transition ofbovine 120573-lactoglobulinrdquo Biochemistry vol 37 no 40 pp 14014ndash14023 1998
[15] Y H Yong and E A Foegeding ldquoEffects of caseins on thermalstability of bovine 120573-lactoglobulinrdquo Journal of Agricultural andFood Chemistry vol 56 no 21 pp 10352ndash10358 2008
[16] S G Anema R Stockmann and E K Lowe ldquoDenaturationof 120573-lactoglobulin in pressure-treated skim milkrdquo Journal ofAgricultural and Food Chemistry vol 53 no 20 pp 7783ndash77912005
[17] H Stapelfeldt and L H Skibsted ldquoPressure denaturation andaggregation of 120573-lactoglobulin studied by intrinsic fluorescence
depolarization Rayleigh scattering radiationless energy trans-fer and hydrophobic fluoroprobingrdquo Journal of Dairy Researchvol 66 no 4 pp 545ndash558 1999
[18] T Considine H Singh H A Patel and L K CreamerldquoInfluence of binding of sodium dodecyl sulfate all-trans-retinol and 8-anilino-1-naphthalenesulfonate on the high-pressure-induced unfolding and aggregation of 120573-lactoglobulinBrdquo Journal of Agricultural and Food Chemistry vol 53 no 20pp 8010ndash8018 2005
[19] J-J Baumy P Guenot S Sinbandhit and G Brule ldquoStudyof calcium binding to phosphoserine residues of beta-caseinand its phosphopeptide (1ndash25) by 13P NMRrdquo Journal of DairyResearch vol 56 no 3 pp 403ndash409 1989
[20] J-S He S Zhu T-H Mu Y Yu J Li and N Azumaldquo120572s-casein inhibits the pressure-induced aggregation of 120573-lactoglobulin through its molecular chaperone-like propertiesrdquoFood Hydrocolloids vol 25 no 6 pp 1581ndash1586 2011
[21] C Mazri L Sanchez S J Ramos M Calvo and M D PerezldquoEffect of high-pressure treatment on denaturation of bovine 120573-lactoglobulin and 120572-lactalbuminrdquo European Food Research andTechnology vol 234 no 5 pp 813ndash819 2012
[1] F Salaun B Mietton and F Gaucheron ldquoBuffering capacity ofdairy productsrdquo International Dairy Journal vol 15 no 2 pp95ndash109 2005
[2] M H Famelart F Gaucheron F Mariette Y Le Graet KRaulot and E Boyaval ldquoAcidification of pressure-treated milkrdquoInternational Dairy Journal vol 7 no 5 pp 325ndash330 1997
[3] V Orlien L Boserup and K Olsen ldquoCasein micelle dissoci-ation in skim milk during high-pressure treatment effects ofpressure pH and temperaturerdquo Journal of Dairy Science vol93 no 1 pp 12ndash18 2010
[4] M Arias R Lopez-Fandino and A Olano ldquoInfluence of pH onthe effects of high pressure onmilk proteinsrdquoMilchwissenschaftvol 55 no 4 pp 191ndash194 2000
[5] T Huppertz P F Fox and A L Kelly ldquoHigh pressure treatmentof bovine milk effects on casein micelles and whey proteinsrdquoJournal of Dairy Research vol 71 no 1 pp 97ndash106 2004
[6] S KMin P S Chaminda and SK Sastry ldquoIn situmeasurementof reaction volume and calculation of pH of weak acid buffersolutions under high pressurerdquo Journal of Physical Chemistry Bvol 115 no 20 pp 6564ndash6571 2011
[7] VOrlien KOlsen and LH Skibsted ldquoIn situmeasurements ofpH changes in 120573-lactoglobulin solutions under high hydrostaticpressurerdquo Journal of Agricultural and Food Chemistry vol 55no 11 pp 4422ndash4428 2007
[8] V M Stippl A Delgado and T M Becker ldquoDevelopment of amethod for the optical in-situ determination of pH value duringhigh-pressure treatment of fluid foodrdquo Innovative Food Scienceamp Emerging Technologies vol 5 no 3 pp 285ndash292 2004
[9] M Hayert J-M Perrier-Cornet and P Gervais ldquoA simplemethod for measuring the pH of acid solutions under highpressurerdquo The Journal of Physical Chemistry A vol 103 no 12pp 1785ndash1789 1999
[10] T K Hitchens and R G Bryant ldquoPressure dependence of weakacid ionization in deuterium oxide solutionsrdquo The Journal ofPhysical Chemistry B vol 102 no 6 pp 1002ndash1004 1998
[11] R C Neuman Jr W Kauzmann and A Zipp ldquoPressuredependence of weak acid lonization in aqueous buffersrdquo TheJournal of Physical Chemistry vol 77 no 22 pp 2687ndash26911973
[12] K R Kristiansen J Otte R Ipsen and K B Qvist ldquoLarge-scalepreparation of 120573-lactoglobulin A and B by ultrafiltration andion-exchange chromatographyrdquo International Dairy Journalvol 8 no 2 pp 113ndash118 1998
[13] F Fogolari L Ragona S Licciardi et al ldquoElectrostatic prop-erties of bovine 120573-lactoglobulinrdquo Proteins Structure Functionand Genetics vol 39 no 4 pp 317ndash330 2000
[14] B Y QinM C Bewley L K Creamer HM Baker E N Bakerand G B Jameson ldquoStructural basis of the tanford transition ofbovine 120573-lactoglobulinrdquo Biochemistry vol 37 no 40 pp 14014ndash14023 1998
[15] Y H Yong and E A Foegeding ldquoEffects of caseins on thermalstability of bovine 120573-lactoglobulinrdquo Journal of Agricultural andFood Chemistry vol 56 no 21 pp 10352ndash10358 2008
[16] S G Anema R Stockmann and E K Lowe ldquoDenaturationof 120573-lactoglobulin in pressure-treated skim milkrdquo Journal ofAgricultural and Food Chemistry vol 53 no 20 pp 7783ndash77912005
[17] H Stapelfeldt and L H Skibsted ldquoPressure denaturation andaggregation of 120573-lactoglobulin studied by intrinsic fluorescence
depolarization Rayleigh scattering radiationless energy trans-fer and hydrophobic fluoroprobingrdquo Journal of Dairy Researchvol 66 no 4 pp 545ndash558 1999
[18] T Considine H Singh H A Patel and L K CreamerldquoInfluence of binding of sodium dodecyl sulfate all-trans-retinol and 8-anilino-1-naphthalenesulfonate on the high-pressure-induced unfolding and aggregation of 120573-lactoglobulinBrdquo Journal of Agricultural and Food Chemistry vol 53 no 20pp 8010ndash8018 2005
[19] J-J Baumy P Guenot S Sinbandhit and G Brule ldquoStudyof calcium binding to phosphoserine residues of beta-caseinand its phosphopeptide (1ndash25) by 13P NMRrdquo Journal of DairyResearch vol 56 no 3 pp 403ndash409 1989
[20] J-S He S Zhu T-H Mu Y Yu J Li and N Azumaldquo120572s-casein inhibits the pressure-induced aggregation of 120573-lactoglobulin through its molecular chaperone-like propertiesrdquoFood Hydrocolloids vol 25 no 6 pp 1581ndash1586 2011
[21] C Mazri L Sanchez S J Ramos M Calvo and M D PerezldquoEffect of high-pressure treatment on denaturation of bovine 120573-lactoglobulin and 120572-lactalbuminrdquo European Food Research andTechnology vol 234 no 5 pp 813ndash819 2012
