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CHAPTER 11
In luence o pH and ype o acid anion on he
linea iscoelas ic p ope ies o Egg Yolk
Aguila , J.M., Co dobés, F. , De la Fuen e, J., and Fe nández-Espada, L.
1 Depa amen o de Ingenie ía Química, Facul ad de Química, Uni e sidad de Se illa (Spain)
In oduc ion
The na i e con o ma ion o a p o ein can be explained in e ms o a ne balance
be ween a ac i e and epulsi e o ces. P o eins p esen a maximum s abili y a
he isoelec ic poin . Thus, any change in he en i onmen al condi ions (as pH o
chao opic sal s) can modi y and modula e he unc ional p ope ies o p o eins o
a la ge ex en .
Ex eme pH alues cause a high ne cha ge leading o elec os a ic epulsion
be ween neighbou molecules and modi ying he cha ge dis ibu ion among he
amino acid side chains. As a consequence, changing pH o ex eme alues is a
well known me hod o p o ein dena u a ion a low empe a u e [1].
Ion e ec s in biological sys ems we e i s ly in es iga ed by Ho meis e and co-
wo ke s. They published a se ies o pape s in which hey showed ha sal s could
be classi ied acco ding o hei sal ing-in and sal ing-ou beha iou agains
p o eins p ecipi a ion [2]. Nowadays, his speci ic ion e ec s make up he
Ho meis e phenomena and he se ies o ca ions and anions a e usually gi en in
e ms o he abili y o he ions o s abilize he s uc u e o p o eins [3]. Howe e ,
he e is no a single and unique Ho meis e se ies, and, in gene al, ca ion e ec s
a e less p onounced han speci ic anion e ec s [4].
The objec i e o his s udy is o assess he in luence o pH, as well as he
con ibu ion o each anion o he linea iscoelas ic p ope ies o egg yolk.
Expe imen al
Ma e ials
Egg Yolk (EY) was ob ained om g ade A, ype L (63-73 g) esh chicken eggs
acco ding o EU speci ica ions. Any c acked o damaged egg was disca ded and
Pe spec i es in Fundamen al and Applied Rheology
144
he yolk was ca e ully ex ac ed om he eggs by he Ha ison & Cunningham
p epa a ion me hod [5]. Analy ical g ade Hyd ochlo ic acid (37%) was ob ained
om Fishe Scien i ic (F ance). Chemically pu e g ade (pu issimus) Ci ic acid
anhyd ous and O hophospho ic acid (85%) we e ob ained om Pan eac (Spain).
Me hods
EY dispe sions we e ob ained by dilu ion o na i e EY (50-52 w % in solid con en ).
The na i e pH o he sys em (ca. pH 6) was modi ied using solu ions (1-5 M) o
ci ic, o hyd ochlo ic, o o hophospho ic acid. Samples wi h pH alues 2-6 we e
p epa ed by adding he sui able acidulan and demine alised wa e in o de o
each he desi ed pH (± 0.2 pH uni s) in a EY dispe sion wi h a 45 w % in solids
con en . Samples we e e ige a ed o e nigh a 4ºC.
F equency sweep es s o egg yolk dispe sions con aining 45 w % in solids a
di e en pH alues we e pe o med by means o an AR-2000 (TA Ins umen s, UK)
using a low-ine ia 60 mm aluminium pla e-pla e geome y wi h 1 mm gap. As he
iscoelas ic esponse is di e en in he expe imen al equency ange (10-2 – 102
ad/s), i was subdi ided in se e al in e als o e lapping a he ends o he ange.
Any measu emen was ca ied ou a 20 ºC and a a s ess clea ly lowe han he
c i ical alue o linea iscoelas ici y. All samples we e placed a oom
empe a u e o 30 min be o e hei loading on he senso . A e wa ds, hey we e
sealed wi h aseline oil o p e en hei d ying du ing he es o samples. A leas
wo eplica es we e pe o med o each heological es .
Resul s and Discussion
Figu e 1 shows s ess sweep es s o EY (45 w %) a 1 ad/s. I can be clea ly
seen ha he h ee dispe sions exhibi a p edominan ly iscous beha iou a pH 4.
1E-4 1E-3 0,01 0,1 1 10 100
10-2
10-1
100
101
102
HCl Ci ic O-Phosp
0.1Hz 1 ad/s 1 ad/s
G''
G"
G' , G" / (Pa)
s ain
Figu e 1. S ess Sweep Tes s a 1 ad/s and 2 ºC o EY (45 w %) acidi ied un il pH 4
using di e en acids.
Pa II: Food, Cosme ics and Pha maceu ical P oduc s
145
A wide LVR is always ound, al hough he c i ical s ain depends on equency, pH
and he ype o acid (i.e. a sligh ly smalle alue may be obse ed in Fig. 1 when
o ho-phospho ic acid is used).
Figu e 2 and 3 compa e mechanical spec a o EY dispe sions o he h ee acids
s udied, a pH 4 and 2, espec i ely. The na i e EY (45 w % a pH 6) is also
plo ed in bo h igu es as no acid. As may be app ecia ed he o ho-phospho ic
acid leads o mode a e changes in he iscoelas ici y beha iou o na i e EY, ha
become mo e appa en a low pH.
0,1 1 10 100
1E-3
0,01
0,1
1
10
100
1000
G' G''
No Acid
HCl
Ci ic ' '
O-Phpos '
G', G" / (Pa)
/ ( ad/s)
Figu e 2. Mechanical Spec a a 20 ºC o EY dispe sions (45 w % solids con en ) acidi ied
un il pH4 using di e en acids.
All he mechanical spec a plo ed in Fig. 2 co espond o a p edominan ly iscous
beha iou in which G‘‘ is always highe han G‘ wi hin he expe imen al equency
ange. This beha iou is ypical o ela i ely dilu e dispe sions such as ha
co esponding o he na i e EY. Only a ce ain endency o ind a c osso e
be ween bo h moduli a high equency may be no iced o he HCl-acidi ied
dispe sion.
On he o he hand, he beha iou shown by EY a pH 2 (acidi ied by HCl o ci ic
acids) is comple ely di e en o ha one exhibi ed a pH 4 o 6. Bo h acids lead o
a gel beha iou cha ac e ized by alues o G‘ being much highe han G‘‘ whe e
bo h unc ions show almos no dependence on equency. This beha iou
con as s wi h he luid-like beha iou displayed by he dispe sion acidi ied wi h
o ho-phospho ic acid.
Pe spec i es in Fundamen al and Applied Rheology
146
0,1 1 10 100
10-3
10-2
10-1
100
101
102
103
104
105
G' G''
No Acid
HCl
Ci ic ' '
O-Phpos ' '
G', G" / (Pa)
/ ( ad/s)
Figu e 3. Mechanical Spec a a 20 ºC o EY dispe sions (45 w % solids con en ) acidi ied
un il pH 2 using di e en acids.
Figu e 4 shows he e olu ion o he complex modulus a 1 ad/s (G1*) as a unc ion
o pH o EY dispe sions (45 w % solids con en ) using he h ee acids unde s udy.
The highe alues o his pa ame e co espond o he use o hyd ochlo ic acid,
whe eas he use o o-phospho ic acid p oduced he lowe alues. In any case, all
s udied acids exhibi an inc ease o his pa ame e as he pH dec ease. Howe e ,
a highe slope can be obse ed when he pH dec ease om mode a e pH alues
(3 o 4) o pH 2. This e ec is s ill mo e p onounced o ci ic and hyd ochlo ic
acids, o which he complex modulus, G1*, inc eases in se e al o de s o
magni ude. Howe e , i should be no ed ha his e olu ion doesn‘ co espond o
he o iginal sequence es ablished by Ho meis e , acco ding o which, ci ic and o-
phospho ic acid should exhibi a highe s uc u e-making e ec , hus limi ing he
amoun o a ailable wa e o hyd a e p o ein molecules. This di e ence may be
ela ed o he ac ha unde he expe imen al condi ions, p o eins p esen a
posi i e su ace cha ge, whe eas in he o iginal sequence he p o ein su ace was
nega i ely cha ged. This di e ence may be ega ded as esponsible o he
in e sion o he sequence, which would explain he highe alues (excep o pH 2)
co esponding o hyd ochlo ic acid.
2 3 4 5 6
10-1
100
101
102
103
104
G1* / (Pa)
pH
Hyd ochlo ic
Ci ic
O-Phospho ic
Fig. 4 E olu ion o complex modulus alues a 1 ad/s o EY (45 w %) dispe sions as
unc ion o pH and ype o acidulan .
Pa II: Food, Cosme ics and Pha maceu ical P oduc s
147
Fu he mo e, ci ic acid is ha dly dissocia ed a pH 2 and p esen low abili y o
neu alize he cha ged p o ein esidues, whe eas hyd ochlo ic, being a s ong acid,
is ully dissocia ed a all pH alues. As o he o ho-phospho ic acid, i exhibi s a
unique anionic species in he expe imen al pH ange. The sha ply G1* p o ile
ound o ci ic acidi ied EY has o be ela ed o a highe amoun o anionic species
wi h inc easing pH.
The e olu ion o loss angen a 1 ad/s as a unc ion o pH is plo ed in Figu e 5.
As shown, mos o he sys ems unde s udy exhibi a clea ly p edominan iscous
componen o e he elas ic one, wi h loss angen alues be ween 3 and 9.
Howe e , when he pH alue dec eases o 2, he alues o his pa ame e all o
alues less han one, leading o a p edominan ly elas ic beha iou .
2 3 4 5 6
0,1
1
10
g1 (-)
pH
Hyd ochlo ic
Ci ic
O-Phospho ic
Fig. 5 E olu ion o loss angen alues a 1 ad/s o EY dispe sions (45 w %) as a unc ion
o pH and ype o acid.
Conclusions
The educ ion o pH media leads o an inc ease on he iscoelas ic p ope ies o
EY dispe sions o he h ee acids unde s udy. This inc ease depends on he pH
alue and he ype o acid used.
The addi ion o Hyd ochlo ic o Ci ic acid showed a simila beha iou leading o a
gel o ma ion when he pH 2 is eached. Bu , his change is mo e sha ply when
he ci ic acid is used, which may be ela ed o he di e en dissocia ion pa e n o
i s ionic species.
On he o he hand, he addi ion o o ho-Phospho ic acid showed a mo e
mode a e dependency o he iscoelas ic p ope ies on he pH alues, wi hou any
sol-gel ansi ion aking place wi hin he expe imen al pH ange. These esul s
sugges ha dihyd ogen-phospha e anion exe s a ce ain inhibi ion e ec on he
hyd ophobic in e ac ions and hyd ogen bonds be ween p o ein segmen s.

Pe spec i es in Fundamen al and Applied Rheology
148
Acknowledgemen s
Au ho s acknowledge inancial suppo o his wo k by he Andalousian
Go e nmen h ough he p ojec TEP-6134-2010.
Re e ences
1. Damoda an, S. (2010). In: Fennema Química de los alimen os (Damoda an, S., Pa kin,
K.L., and Fennema, O.R.), Vol. 5, pp. 215-325, 3 d edn, Ac ibia, Za agoza.
2. Kunz, W., Henle, J., and Ninham, B.W. (2004). Cu Opin Colloid & In e ace Sci. 9,
19-37.
3. V ka, L., Jungwi h, P., Bauduin, P., Tou aud, D., and Kunz, W. (2006). J. Phys. Chem.
B 110, 7036-7043.
4. Kunz, W. Cu . Opin. in Colloid & In e ace Sci. 15, 34-39.
5. Ha ison, L.J., and Cunningham, F.E. (1986). Poul y Sci. 65, 915-921.
Con ac Add ess:
J.M. Aguila ([email p o ec ed])
Depa men o Chemical Enginee ing, Facul y o Chemis y
Uni e si y o Se ille
Campus de Reina Me cedes, c / P o eso Ga cía González, s/n, 41012 - Se illa
Tel .: +34 954557179 ; Fax: +34 954556447